Gastrointestinal Tract Detection Methods, Devices And Systems Patent Application (2024)

U.S. patent application number 16/827807 was filed with the patent office on 2020-07-16 for gastrointestinal tract detection methods, devices and systems. The applicant listed for this patent is Progenity, Inc.. Invention is credited to Mitchell Lawrence Jones, Sharat Singh, Christopher Loren Wahl.

View All Diagrams

GASTROINTESTINAL TRACT DETECTION METHODS, DEVICES AND SYSTEMS

Abstract

The present disclosure relates to gastrointestinal (GI) tractdetection methods, devices and systems.

Related U.S. Patent Documents

Claims

1.-95. (canceled)

96. A device, comprising: a housing comprising a chamber; acomposition in the chamber, the composition comprising: a dye; anda reagent capable of selectively lysing eukaryotic cells; and asystem in the housing, wherein: the system is configured to detectfluorescence from a product of a reaction of the composition and asample collected from an exterior of the housing; and the device isan ingestible device.

97. The device of claim 96, wherein the system comprises a lightsource and a light detector.

98. The device of claim 97, wherein the light source is configuredto emit light that interacts with the product so that the productfluoresces.

99. The device of claim 98, wherein the system further comprises aplurality of lenses disposed along a path of the light from thelight source to the light detector.

100. The device of claim 99, wherein the system further comprises amirror disposed along the path of the light from the light sourceto the light detector.

101. The device of claim 98, wherein the light detector isconfigured to detect the fluorescence from the product.

102. The device of claim 96, wherein: the system comprises a lightsource, a first light detector, and a second light detector. thelight source is configured to emit light that interacts with theproduct so that the product fluoresces; the first light detector isconfigured to detect fluorescence from the product; and the secondlight detector is configured to detect fluorescence from theproduct.

103. The device of claim 96, wherein the dye is capable of bindingto or reacting with a target component of a viable cell.

104. The device of claim 103, wherein the dye exhibits fluorescencethat is measurably altered when the dye is bound to or reacted withthe target component of the viable cell.

105. The device of claim 96, wherein the dye exhibits fluorescencewhen bound to a nucleic acid.

106. The device of claim 96, wherein the dye is a lipophilic dyethat exhibits fluorescence when metabolized by a cell.

107. The device of claim 96, wherein the dye exhibits fluorescencewhen reduced by a cell or a cell component.

108. The device of claim 96, wherein the dye comprises a memberselected from the group consisting of resazurin,C.sup.12-resazurin, 7-hydroxy-9H-(1,3dichloro-9,9-dimethylacridin-2-ol)N-oxide,6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine, and a tetrazoliumsalt.

109. The device of claim 96, wherein the dye exhibits fluorescencewhen oxidized by a cell or a cell component.

110. The device of claim 96, wherein the dye exhibits fluorescencewhen de-acetylated and/or oxidized by a cell or a cellcomponent.

111. The device of claim 96, wherein the dye exhibits fluorescencewhen reacted with a peptidase.

112. The device of claim 96, wherein the dye comprises achemiluminescent dye that exhibits chemiluminescence whenmetabolized by a viable cell.

113. The device of claim 96, wherein the dye comprises luminol.

114. The device of claim 96, wherein the reagent comprises adetergent.

115. The device of claim 96, wherein the reagent comprises anon-ionic detergent.

116. The device of claim 96, further comprising a second reagentcapable of selectively lysing eukaryotic cells.

117. The device of claim 116, further comprising anelectrolyte.

118. The device of claim 96, further comprising an electrolyte.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The application claims priority to the following co-pendingU.S. Patent Applications: U.S. Ser. No. 62/431,297, entitled"Compositions, Methods, and Devices for Bacteria Detection andQuantitation," filed on Dec. 7, 2016; U.S. Ser. No. 62/434,320,entitled "An Ingestible Device for Sampling, Diluting and Culturinga Biological Sample," filed on Dec. 14, 2016; U.S. Ser. No.62/502,383, entitled "Devices for Analyte Detection" filed on May5, 2017; U.S. Ser. No. 62/560,618, entitled "Ingestible Devices andRelated Systems and Methods," filed on Sep. 19, 2017; U.S. Ser. No.62/478,753, entitled "Treatment of a Disease of theGastrointestinal Tract with an IL-6R Inhibitor," filed on Mar. 30,2017; U.S. Ser. No. 62/545,157, entitled "Treatment of a Disease ofthe Gastrointestinal Tract with an Immunosuppressant," filed onAug. 14, 2017; and U.S. Ser. No. 62/583,768, entitled "Treatment ofa Disease of the Gastrointestinal Tract with a TNF Inhibitor,"filed on Nov. 9, 2017.

INCORPORATION BY REFERENCE

[0002] The application incorporates by reference the followingco-pending U.S. patent applications: U.S. Ser. No. 62/431,297,entitled "Compositions, Methods, and Devices for Bacteria Detectionand Quantitation," filed on Dec. 7, 2016; U.S. Ser. No. 62/434,320,entitled "An Ingestible Device for Sampling, Diluting and Culturinga Biological Sample," filed on Dec. 14, 2016; U.S. Ser. No.62/502,383, entitled "Devices for Analyte Detection" filed on May5, 2017; U.S. Ser. No. 62/560,618, entitled "Ingestible Devices andRelated Systems and Methods," filed on Sep. 19, 2017; U.S. Ser. No.62/478,753, entitled "Treatment of a Disease of theGastrointestinal Tract with an IL-6R Inihibitor," filed on Mar. 30,2017; U.S. Ser. No. 62/545,157, entitled "Treatment of a Disease ofthe Gastrointestinal Tract with an Immunosuppressant," filed onAug. 14, 2017; U.S. Ser. No. 62/583,768, entitled "Treatment of aDisease of the Gastrointestinal Tract with a TNF Inihibitor," filedon Nov. 9, 2017; U.S. Ser. No. 14/460,893, entitled "IngestibleMedical Device," and filed Aug. 15, 2014; U.S. Ser. No. 15/514,413,entitled "Electromechanical Pill Device with LocalizationCapabilities," and filed Mar. 24, 2017; U.S. Ser. No. 15/680,400,entitled "Systems and Methods for Obtaining Samples usingIngestible Devices," filed on Aug. 18, 2017; U.S. Ser. No.15/680,430, entitled "Sampling Systems and Related Materials andMethods," filed on Aug. 18, 2017; U.S. Ser. No. 15/699,848,entitled "Electromechanical Ingestible Delivery of a DispensableSubstance," filed on Sep. 8, 2017; U.S. Ser. No. 62/480,187,entitled "Localization Systems and Methods for anOptoelectromechanical Pill Device," filed on Mar. 31, 2017; andU.S. Ser. No. 62/540,873, entitled "Localization Systems andMethods for an Ingestible Device," filed on Aug. 3, 2017.

FIELD

[0003] The disclosure relates to gastrointestinal (GI) tractdetection methods, devices and systems.

BACKGROUND

[0004] The GI tract can contain information regarding anindividual's body.

SUMMARY

[0005] The disclosure relates to gastrointestinal (GI) tractdetection methods, devices and systems.

[0006] The technology disclosed herein allows for rapid, real timeassessment of information relating to a subject (e.g., informationrelating to the subject's GI tract). In some embodiments, theinformation can relate to the presence and/or quantity of ananalyte of interest (e.g., an analyte of interest in the GI tractof a subject). In certain embodiments, the technology can beimplemented using an ingestible device that may be used to take oneor more samples of a subject (e.g., one or more samples in one ormore locations of the GI tract of the subject). Such a device canbe implemented in an autonomous fashion. For example, informationcan be exchanged between the ingestible device when present in thesubject (in vivo) and outside the subject (ex vivo). In someembodiments, the information can be exchanged in real time. Incertain embodiments, the technology can be used to help determinewhether a subject has a given GI disorder. In some embodiments, thetechnology can be used to help determine a treatment protocoland/or to monitor or assess efficacy of a treatment protocol for asubject (e.g., a GI disorder treatment protocol for the subject).The detection techniques disclosed herein can be used individuallyor in any combination, as desired. In some embodiments, aningestible device is configured such that different detectiontechniques are performed in different chambers (e.g., samplechambers) of an ingestible device. Optionally, multiple differentdetection methods may be used to provide complementary informationregarding a subject (e.g., provide information relating to thesubject's GI tract) and/or supplementary information regarding asubject e.g., provide information relating to the subject's GI.

[0007] In one aspect, provided herein a method, comprisingtransferring a fluid sample from the gastrointestinal (GI) tract orfrom the reproductive tract of a subject into a first dilutionchamber of a device in vivo; and combining the fluid sample and afirst dilution fluid in the first dilution chamber to produce afirst diluted sample. In some embodiments, the device comprises aplurality of dilution chambers; and for each at least some of theplurality of dilution chambers, the method comprises: transferringa fluid sample into the dilution chamber; and combining the fluidsample and the first dilution fluid in the first dilution chamberto produce a diluted sample. In some embodiments, the methodfurther comprises combining diluted samples from at least twodifferent dilution chambers to provide a further dilutedsample.

[0008] In some embodiments, the device is an ingestible device. Insome embodiments, the method further comprises orally administeringthe device to the subject. In some embodiments, the method furthercomprises introducing the device into the reproductive tract of thesubject.

[0009] In some embodiments, the first dilution fluid comprises asterile medium.

[0010] In some embodiments, the method further comprises culturingthe diluted sample to produce a cultured sample. In someembodiments, the culturing is performed in vivo. In someembodiments, the culturing is performed ex vivo.

[0011] In some embodiments, the method further comprises recoveringthe device ex vivo. In some embodiments, the method furthercomprises removing the sample from the device.

[0012] In some embodiments, the method further comprises detectingan analyte in the sample. In some embodiments, the detecting occursin vivo. In some embodiments, the analyte comprises a cell. In someembodiments, the cell comprises a bacteria. In some embodiments,the cell comprises a eukaryotic cell. In some embodiments, theeukaryotic cell is selected from the group consisting of anepithelial cell and a peripheral blood mononuclear cell (PBMC).

[0013] In some embodiments, the device comprises a port, a valveand/or a pump; and transferring the fluid sample to the dilutionfirst chamber comprises controlling the port, valve and/orpump.

[0014] In some embodiments, the device comprises a port having anopen position and a first position; in the open position, the portis in fluid communication with the GI tract or with thereproductive tract of the subject, and the fluid sample enters theport; and in the first position, the port is in fluid communicationwith the first dilution chamber and the fluid sample combines withthe first dilution fluid. In some embodiments, when the port is inits open position, the fluid sample enters the port; and when theport is in its first position, the fluid sample combines with thefirst dilution fluid to provide a first dilution. In someembodiments, the port has a second position in which the port is influid communication with a second dilution chamber comprising afluid; and the method further comprises moving the port from itsfirst position to its second position so that the first dilutioncombines with the fluid in the second chamber to provide a seconddilution. In some embodiments, before the port moves from its firstposition to its second position, the fluid in the second dilutionchamber comprises a sterile medium; and the second dilutioncomprises the sterile medium. In some embodiments, the methodcomprises moving in the port sequentially from its open position toits first and then second positions, to sequentially provide thefirst and then second dilutions. In some embodiments, the port hasa third position in which the port is in fluid communication with athird dilution chamber which comprises a fluid; and the methodfurther comprises moving the port from its second position to itsthird position so that the second dilution combines with the fluidin the third chamber to provide a third dilution. In someembodiments, the method comprises moving in the port sequentiallyfrom its open position to its first, second and then thirdpositions, to sequentially provide the first, second and then thirddilutions. In some embodiments, the port has a fourth position inwhich the port is in fluid communication with a fourth dilutionchamber which comprises a fluid; and the method further comprisesmoving the port from its third position to its fourth position sothat the third dilution combines with the fluid in the fourthchamber to provide a fourth dilution. In some embodiments, themethod comprises moving in the port sequentially from its openposition to its first, second, third and then fourth positions, tosequentially provide the first, second, third and fourthdilutions.

[0015] In some embodiments, the device further comprises amicrocontroller configured to control an actuator configured torotate the port. In some embodiments, the microcontroller isconfigured to control a rotatable element which is configured tomove the port.

[0016] In some embodiments, a ratio of a volume of the fluid sampleto a volume of the dilution fluid is from about 1:1 and about1:1000. In some embodiments, the ratio is from about 1:1 and toabout 1:100. In some embodiments, the ratio is from about 1:1 toabout 1:20. In some embodiments, the ratio is from about 1:1 toabout 1:10.

[0017] In some embodiments, the first dilution fluid comprises ananti-fungal agent. In some embodiments, the anti-fungal agentcomprises amphotericin B.

[0018] In some embodiments, the first dilution fluid comprises asterile medium. In some embodiments, the first dilution mediumcomprises a preservative. In some embodiments, the sterile mediumcomprise an agent that inhibits growth of a cell and/or an agentthat promotes the growth of a cell. In some embodiments, the cellcomprises a bacterium. In some embodiments, the sterile medium isselective for one or more types of bacteria. In some embodiments,the medium is selective for Gram-negative bacteria.

[0019] In some embodiments, the first dilution fluid comprisessterile media, and the sterile media comprise an antibiotic.

[0020] In some embodiments, the method further comprises culturingthe first diluted sample to produce a cultured sample. In someembodiments, the method further comprises detecting the presence orabsence of bacterial growth within the cultured sample. In someembodiments, the presence of bacterial growth indicates thepresence of bacteria that are resistant to the antibiotic in thefluid sample.

[0021] In some embodiments, the first dilution fluid comprises anindicator media. In some embodiments, the method further comprisesdetecting an analyte in the first dilution at a plurality of timepoints. In some embodiments, the analyte comprises a cell.

[0022] In some embodiments, the method further comprises detectingan analyte in one or more of the first dilution, the seconddilution, the third dilution and/or the fourth dilution at a firsttime point and at a second time point. In some embodiments, theanalyte comprises a cell. In some embodiments, the first time pointrepresents a control. In some embodiments, the second time point isbetween about 1 hour and about 6 hours after the first time point.In some embodiments, the second point in time is between about 1hour and 4 hours after the first time point.

[0023] In some embodiments, the method further comprises culturingthe one or more diluted samples to produce one or more culturedsamples, and detecting the presence or absence of an analyte in theone or more cultured samples. In some embodiments, the analytecomprises a cell. In some embodiments, the cell is a bacterium, andthe method comprises detecting the presence or absence of bacterialgrowth in the one or more cultured samples.

[0024] In some embodiments, the volume of the fluid sample is about5 .mu.L, the dilution of the fluid sample is a dilution of about1:10000 and detecting the presence of bacterial growth in thedilution is indicative of a bacterial concentration of 10.sup.5 orgreater colony forming units (CFU)/mL in the fluid sample. In someembodiments, the fluid sample is jejunal fluid and a bacterialconcentration of 10.sup.5 CFU/mL or greater in the jejunal fluid isindicative that the subject has Small Intestinal BacterialOvergrowth (SIBO).

[0025] In some embodiments, the method further comprises detectinga level of bacteria in the one or more diluted or cultured samples,wherein the fluid sample is jejunal fluid and a bacterialconcentration of 10.sup.5 CFU/mL or greater in the jejunal fluid isindicative that the subject has SIBO. In some embodiments, themethod comprises detecting the level of bacteria at three or moretime points to generate one or more growth curves for the one ormore cultured samples. In some embodiments, the method furthercomprises comparing the one or more growth curves to one or morestandard growth curves. In some embodiments, the standard growthcurves are representative of fluid samples with a known totalbacterial count. In some embodiments, the standard growth curvesare representative of samples from subjects with SIBO.

[0026] In some embodiments, the method comprises detecting thelevel of an analyte in the one or more diluted samples or culturedsamples in the one or more dilution chambers.

[0027] In some embodiments, the method further comprisestransferring the diluted sample or cultured sample to a detectionchamber, and detecting the level of an analyte in the dilutedsample or cultured sample in the detection chamber. In someembodiments, the analyte comprises a cell.

[0028] In some embodiments, detecting comprises using a Coultercounter.

[0029] In some embodiments, detecting comprises using a lightsource and a photodetector. In some embodiments, detectingcomprises measuring an absorbance of the one or more dilutedsamples or cultured samples at a wavelength. In some embodiments,the wavelength is between about 400 and 1000 nm. In someembodiments, the wavelength is between about 500 and 700 nm. Insome embodiments, the wavelength is about 600 nm.

[0030] In some embodiments, the device comprises an environmentalsensor. In some embodiments, the method further comprises measuringenvironmental data of the GI tract or reproductive tract externalto the device in the subject. In some embodiments, the methodfurther comprises measuring environmental data of the GI tractexternal to the device in the subject at a plurality of time pointsas the device passes through the GI tract of the subject. In someembodiments, the method comprises measuring at least one parameterselected from the group consisting of capacitance, temperature,impedance, pH, and reflectance. In some embodiments, the methodfurther comprises using the environmental data to determine alocation of the device within the GI tract of the subject.

[0031] In some embodiments, the transferring the fluid sample intothe first dilution chamber happens when the device is in the smallintestine of the subject.

[0032] In some embodiments, transferring the fluid sample into thefirst dilution chamber happens when the device is in the jejunum ofthe subject.

[0033] In some embodiments, the method further comprisesdetermining the total bacterial count (TBC) of the fluid samplebased on the level of bacteria within the one or more dilutedsamples or cultured samples. In some embodiments, the fluid sampleis jejunal fluid, and the method comprises diagnosing the subjectas having SIBO if the TBC of the fluid sample is greater than10.sup.5 CFU/mL.

[0034] In some embodiments, the method further comprisesidentifying one or more characteristics of a cell within the one ormore diluted samples or cultured samples. In some embodiments, thecell is a bacterium and the method comprises identifying thebacterium as Gram-positive or Gram-negative. In some embodiments,the dilution fluid comprises conjugated bile acids, and the methodcomprises measuring bile salt hydrolase activity in the one or morediluted samples or cultured samples. In some embodiments, the cellis a eukaryotic cell and the method comprises detecting one or morebiomarkers associated with cancer or inflammation.

[0035] In some embodiments, the method further comprisestransmitting data from the device to an external base stationand/or receiving operating parameters from an external basestation. In some embodiments, the data comprises a measure of theconcentration of an analyte in the fluid sample. In someembodiments, the analyte comprises a cell. In some embodiments, theoperating parameters comprise timing instructions for transferringall or part of the fluid sample from the GI tract or from thereproductive tract into the one or more dilution chambers.

[0036] In one aspect, provided herein is a device, comprising achamber configured to dilute a fluid sample from the GI tract orthe reproductive tract of a subject; and a dilution chamberconfigured to house dilution fluid to dilute the fluid sample inthe dilution chamber, wherein the device is an ingestibledevice.

[0037] In some embodiments, the device comprises one or more ports,valves and/or pumps configured to control transfer of fluid fromthe GI tract or from the reproductive tract into the dilutionchamber. In some embodiments, the device comprises a plurality ofdilution chambers and one or more ports, valves and/or pumpsconfigured to control transfer of fluid between the dilutionchambers. In some embodiments, the device further comprises amicrocontroller configured to control the one or more ports, valvesand/or pumps. In some embodiments, the device is configured tocombine fluid sample with dilution fluid in the plurality ofdilution chambers to produce a dilution series. In someembodiments, the device comprises a port configured to receive thefluid sample from the GI tract or reproductive tract. In someembodiments, the port is movable between an open position and afirst position; in the open position, the port is exposed on anexternal surface of the device; and in the first position, the portis in fluid communication with a first dilution chamber of thedevice. In some embodiments, the port is movable between its firstposition and a second position; in its second position, the port isin fluid communication with a second dilution chamber of thedevice. In some embodiments, the port is movable between its secondposition and a third position; and in its third position, the portis in fluid communication with a dilution incubation chamber of thedevice. In some embodiments, the port is movable between its thirdposition and a fourth position; and in its fourth position, theport is in fluid communication with a fourth dilution chamber ofthe device. In some embodiments, the device further comprises anactuator configured to move the port. In some embodiments, theactuator is coupled to a rotatable element, and the rotatableelement is configured to rotate the port. In some embodiments, theport has a fluid volume of about 1 .mu.L to about 50 .mu.L. In someembodiments, the port is a depression on a surface of the rotatableelement. In some embodiments, the one or more dilution chambers arepositioned circumferentially around an axis of rotation of therotatable element.

[0038] In some embodiments, the device further comprises thedilution fluid. In some embodiments, the dilution fluid comprisesan anti-fungal agent. In some embodiments, the anti-fungal agentcomprises amphotericin B. In some embodiments, the dilution fluidcomprises sterile media. In some embodiments, the sterile mediacomprises at least one member selected from the group consisting ofan agent that promotes growth of a cell, and an agent that inhibitsgrowth of a cell. In some embodiments, the sterile media comprisesan antibiotic. In some embodiments, the sterile media is selectivefor the growth of one or more types of cells. In some embodiments,the sterile media is selective for the growth of a eukaryoticcell.

[0039] In some embodiments, the device further comprises adetection system configured to detect an analyte in the fluidsample or dilution thereof. In some embodiments, the analytecomprises a cell. In some embodiments, the device further comprisesa detection chamber in fluid communication with the one or moredilution incubation chambers. In some embodiments, fluidcommunication between the detection chamber and the one or moredilution chambers is controlled by one or more ports, valves and/orpumps. In some embodiments, the detection system is configured todetect the analyte in the fluid sample or dilution thereof at aplurality of time points. In some embodiments, the detection systemis configured to detect the analyte at a first time point and at asecond time point. In some embodiments, the first time pointrepresents a control. In some embodiments, the second time point isbetween 1 hour and 6 hours after the first time point.

[0040] In some embodiments, the detection system is configured todetect the presence or absence of bacterial growth in the one ormore dilution chambers or in the one or more detectionchambers.

[0041] In some embodiments, the volume of the fluid sample is about5 .mu.L.

[0042] In some embodiments, the device further comprises adetection system configured to detect a level of bacteria in theone or more dilution chambers or in the one or more detectionchambers. In some embodiments, the detection system is configuredto detect the level of bacteria at three or more time points toproduce a growth curve.

[0043] In some embodiments, the device comprises a Coultercounter.

[0044] In some embodiments, the device comprises a light source anda photodetector. In some embodiments, the light source andphotodetector are operable to define a light path through the oneor more dilution chambers or through the one or more detectionchambers.

[0045] In some embodiments, the device comprises a detection systemconfigured to detect an analyte in the fluid sample or dilutionthereof. In some embodiments, the analyte is a byproduct from abacterium.

[0046] In some embodiments, the device further comprises anenvironmental sensor configured to measure environmental data ofthe GI tract or of the reproductive tract external to the device inthe subject. In some embodiments, the environmental sensorcomprises at least one member selected from the group consisting ofa capacitance sensor, a temperature sensor, an impedance sensor, apH level sensor, and a light sensor. In some embodiments, theenvironmental data is usable to determine a location of the devicewithin the GI tract of the subject.

[0047] In some embodiments, the device further comprises amicrocontroller configured to control operation of the device. Insome embodiments, the microcontroller is configured to controltransfer of the fluid sample from the GI tract to the one or moredilution chambers based on the location of the device within the GItract. In some embodiments, the microcontroller controls one ormore ports, valves and/or pumps.

[0048] In some embodiments, the device further comprises a sensorconfigured to identify the types of cells or the characteristics ofthe cells within the one or more dilution chambers.

[0049] In some embodiments, the device further comprises acommunication sub-unit that is configured to receive operatingparameters from an external base station and/or transmit data to anexternal base station. In some embodiments, the operatingparameters comprise timing instructions for obtaining a fluidsample from the GI tract or from the reproductive tract andtransferring the fluid sample into one or more dilution chambers.In some embodiments, the data is indicative of the presence and/orabsence of bacterial growth in the one or more dilutionchambers.

[0050] In one aspect, provided herein is a device, comprising anelement having a port on a wall of the element; and a shellsurrounding the element to define a first dilution chamber betweenthe element and the shell, wherein the device is configured toallow relative movement between the element and the shell; theshell has an aperture configured to expose a portion of the wall ofthe element to an exterior of the device; and the device is aningestible device. In some embodiments, the device is configured toallow relative rotational movement between the element and theshell. In some embodiments, the element is rotatable. In someembodiments, the element is cylindrical. In some embodiments, theshell is cylindrical.

[0051] In some embodiments, the device is configured so thatrelative movement between the element and the shell aligns the portwith the aperture so that an exterior of the device is in fluidcommunication with the port via the aperture.

[0052] In some embodiments, the element and the shell define afirst dilution chamber; and the device is configured so thatrelative movement between the element results in fluidcommunication between the port and the first dilution chamber. Insome embodiments, the shell and the element define a seconddilution chamber that is separate from the first dilution chamber;and the device is configured so that relative movement between theelement results in fluid communication between the port and asecond dilution chamber. In some embodiments, the first dilutionchamber contains a first dilution fluid, and the second dilutionchamber contains a second dilution fluid. In some embodiments, thedevice is configured so that, during use of the device, the firstdilution fluid is pumped into the first dilution chamber from areservoir of the ingestible device when the ingestible devicearrives at a target location of the GI tract.

[0053] In some embodiments, the wall of the element comprises anyof one or more ports, valves and pumps configured to transfer fluidfrom an exterior of the device to the first dilution chamber.

[0054] In some embodiments, the shell and the element define aplurality of dilution chambers, and one or more ports, valvesand/or pumps are configured to control transfer of fluid betweenthe dilution chambers.

[0055] In some embodiments, the device comprises an actuatorcoupled to the element to move the port.

[0056] In some embodiments, the port is a depression on the wall ofthe rotatable element. In some embodiments, the first dilutionchamber and the second dilution chamber are positionedcircumferentially about the element.

[0057] In some embodiments, the first dilution fluid comprises amedia to culture a GI fluid sample. In some embodiments, the deviceis configured so that the dilution and culturing of the GI fluidsample are performed in vivo. In some embodiments, the device isconfigured so that culturing of the GI fluid sample is performed exvivo after the ingestible device has been evacuated and recoveredfrom the subject.

[0058] In some embodiments, the device further comprises amicrocontroller configured to control a movement of theelement.

[0059] In some embodiments, the device further comprises a sensorconfigured to identify types of cells and/or characteristics of thecells.

[0060] In some embodiments, the device further comprises acommunication sub-unit that is configured to receive operatingparameters from an external base station and/or transmit data to anexternal base station. In some embodiments, the operatingparameters include timing instructions for obtaining a fluid samplefrom the GI tract or from the reproductive tract and transferringthe fluid sample into one or more dilution chambers. In someembodiments, the data is indicative of the presence and/or absenceof bacterial growth in the one or more dilution chambers.

[0061] In one aspect, provided herein is a method comprising usingthe device to obtain a fluid sample in the GI tract of a subject.In some embodiments, the method further comprises serially rotatingthe element to sequentially align the port with a series ofdilution chambers.

[0062] In one aspect, provided herein is a composition, comprisinga dye; and a reagent capable of selectively lysing eukaryoticcells. In some embodiments, the dye is capable of binding to orreacting with a target component of a viable cell. In someembodiments, the dye exhibits fluorescence that is measurablyaltered when the dye is bound to or reacted with the targetcomponent of the viable cell. In some embodiments, the dye isinternalizable by the viable cell.

[0063] In some embodiments, the target component of the viable cellcomprises a member selected from the group consisting of a nucleicacid, actin, tubulin, an enzyme, a nucleotide-binding protein, anion-transport protein, mitochondria, a cytoplasmic component, and amembrane component.

[0064] In some embodiments, the dye exhibits fluorescence whenbound to a nucleic acid. In some embodiments, the dye comprises amember selected from the group consisting of acridine orange,calcein-AM, DAPI, Hoechst 33342, Hoechst 33258, PicoGreen, SYTO 16,SYBR Green I, Texas Red, Redmond Red, a Bodipy dye, Oregon Green,ethidium bromide, and propidium iodide.

[0065] In some embodiments, the dye is a fluorogenic dye thatexhibits fluorescence when metabolized by the viable cell.

[0066] In some embodiments, the dye is a lipophilic dye thatexhibits fluorescence when metabolized by a cell.

[0067] In some embodiments, the dye exhibits fluorescence whenreduced by a cell or a cell component.

[0068] In some embodiments, the dye comprises a member selectedfrom the group consisting of resazurin, C.sup.12-resazurin,7-hydroxy-9H-(1,3 dichloro-9,9-dimethylacridin-2-ol) N-oxide,6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine, and a tetrazoliumsalt.

[0069] In some embodiments, the dye exhibits fluorescence whenoxidized by a cell or a cell component. In some embodiments, thedye comprises a member selected from the group consisting ofdihydrocalcein AM, dihydrorhodamine 123, dihydroethidium;2,3,4,5,6-pentafluorotetramethyldihydrorosamine, and3'-(p-aminophenyl) fluorescein.

[0070] In some embodiments, the dye exhibits fluorescence whende-acetylated and/or oxidized by a cell or a cell component.

[0071] In some embodiments, the dye comprises a member selectedfrom the group consisting of dihydrorhodamines,dihydrofluoresceins, 2',7'-dichlorodihydrofluorescein diacetate;5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate, andchloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetylester.

[0072] In some embodiments, the dye exhibits fluorescence whenreacted with a peptidase. In some embodiments, the dye comprises amember selected from the group consisting of:(CBZ-Ala-Ala-Ala-Ala)2-R110 elastase 2; (CBZ-Ala-Ala-Asp)2-R110granzyme B; and 7-amino-4-methylcoumarin; andN-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide.

[0073] In some embodiments, the dye comprises a chemiluminescentdye that exhibits chemiluminescence when metabolized by a viablecell.

[0074] In some embodiments, the dye comprises luminol.

[0075] In some embodiments, the reagent comprises a detergent. Insome embodiments, the reagent comprises a non-ionic detergent. Insome embodiments, the reagent comprises a member selected from thegroup consisting of Nonidet P40, deoxycholate, Igepal CA 630,Triton-X 100, Zwittergent, SDS, and Tween 20.

[0076] In some embodiments, the reagent comprises deoxycholate. Insome embodiments, the composition comprises deoxycholate at aconcentration of from 0.0001 wt % to 1 wt %. In some embodiments,the composition comprises deoxycholate at a concentration of 0.005wt %.

[0077] In some embodiments, the composition further comprises asecond reagent capable of selectively lysing eukaryotic cells. Insome embodiments, the second reagent comprises a detergent. In someembodiments, the second reagent comprises a member selected thegroup consisting of Nonidet P40, deoxycholate, Igepal CA 630,Triton-X 100, Zwittergent, sodium dodecyl sulfate (SDS), and Tween20. In some embodiments, the second reagent is Triton X-100. Insome embodiments, the composition comprises Triton X-100 at aconcentration of from 0.1 wt % to 0.05 wt %.

[0078] In some embodiments, the composition further comprises anelectrolyte. In some embodiments, the electrolyte is a divalentelectrolyte. In some embodiments, the electrolyte is MgCl.sub.2. Insome embodiments, the composition comprises MgCl.sub.2 at aconcentration of from 0.1 mM to 100 mM. In some embodiments, thecomposition comprises MgCl.sub.2 at a concentration of from 0.5 mMto 50 mM.

[0079] In some embodiments, the composition further compriseswater.

[0080] In some embodiments, the composition is an aqueoussolution.

[0081] In some embodiments, the composition has a pH of from 5 to8. In some embodiments, the composition has a pH of from 6 to7.8.

[0082] In some embodiments, the composition is a solid orsemi-solid.

[0083] In some embodiments, the viable cell is a bacterialcell.

[0084] In one aspect, provided herein is an article comprising amember comprising an absorptive material; and a compositiondescribed herein, wherein the composition is at least partiallyabsorbed in the absorptive material. In some embodiments, theabsorptive material comprises a sponge. In some embodiments, thesponge comprises a hydrophilic sponge. In some embodiments, theabsorptive material comprises a material selected from the groupconsisting of cotton, rayon, glass, polyester, polyethylene,polyurethane, and nitrocellulose.

[0085] In one aspect, provided herein is a device comprising amember comprising an absorptive material; and a compositionprovided herein, wherein the composition is at least partiallyabsorbed in the absorptive material, and the device is aningestible device. In some embodiments, the device furthercomprises a housing with an opening configured, wherein theabsorptive material is disposed within the housing such that theabsorptive material is in fluid communication with an exterior ofthe device via the opening in the housing.

[0086] In some embodiments, the ingestible device, comprises ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0087] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system has first, second andthird states; the first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem; the second state of the multi-stage valve system isdifferent from the first and third states of the multi-stage valvesystem; when the multi-stage valve system is in its first state,the opening prevents fluid communication between the interior ofthe ingestible device and the exterior of the ingestible device;when the multi-stage valve system is in its second state, theopening allows fluid communication between the interior of theingestible device and the exterior of the ingestible device; andwhen the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

[0088] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system comprises: anactuator system comprising a first member; a trigger comprising afirst peg and a first lip; a gate comprising a protrusion, and agate leg having an opening; and a biasing system comprising firstand second biasing members; when the multi-stage valve system is ina first stage: the first biasing member applies a force to thetrigger so that the first peg contacts the first member; the firstmember opposes the force applied to the trigger by the firstbiasing member; the second biasing member applies a force to thegate so that the protrusion contacts the first lip; the first lipopposes the force applied to the gate by the second biasing member;and the opening in the gate leg is not aligned with the opening inthe ingestible device.

[0089] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible device,wherein: the sampling system comprises: a first member comprisingabsorptive material; and a second member comprising a secondabsorptive material different from the first absorptive material;and the sampling system is configured so that fluid that flows fromthe exterior of the ingestible device to the interior of theingestible device enters the first absorptive material; and thesampling system is configured to allow fluid to flow from the firstabsorptive material to the second absorptive material.

[0090] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible deviceconfigured to absorb a fluid that enters the interior of theingestible device via the opening, the sampling system comprisingthe absorptive material and at least one preservative at leastpartially absorbed in the absorptive material.

[0091] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0092] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing, wherein the sampling chamber contains an absorptivematerial; an inlet port connecting the opening in the housing tothe sampling chamber; a single use sealing device positioned withinthe inlet port that seals the inlet port; and a heating elementproximate to the single use sealing device, wherein: the heatingelement is configured to apply heat to the single use sealingdevice to unseal the inlet port and open the sampling chamber, andat least a portion of the absorptive material proximate to theinlet port is configured to expand when in contact with a sampleand reseal the inlet port.

[0093] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing having an entry port and an exit port on an oppositeend of the sampling chamber from the entry port, wherein the exitport is configured to allow gas to exit the chamber and prevent atleast a portion of a sample from exiting the chamber; an inletregion connecting the opening in the housing to the entry port ofthe sampling chamber; and a moveable valve positioned to open andclose the inlet region, wherein: the moveable valve in an openposition allows the sample to enter the sampling chamber; and themoveable valve in a closed position prevents the sample fromentering the sampling chamber.

[0094] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to determine a location of theingestible device in a portion of a gastrointestinal (GI) tract ofa subject to an accuracy of at least 85%.

[0095] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to determine that the ingestible deviceis in the cecum of a subject to an accuracy of at least 70%.

[0096] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to transmit data to a device capable ofimplementing the data to determine a location of the ingestibledevice in a portion of a GI tract of a subject to an accuracy of atleast 85%.

[0097] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to transmit data to an external devicecapable of implementing the data to determine that the ingestibledevice is in the cecum of subject to an accuracy of at least70%.

[0098] In some embodiments, the device further comprises first andsecond light sources, wherein the first light source is configuredto emit light at a first wavelength, and the second light source isconfigured to emit light at a second wavelength different from thefirst wavelength. In some embodiments, the device further comprisesfirst and second detectors, wherein the first detector isconfigured to detect light at the first wavelength, and the seconddetector is configured to detect light at the secondwavelength.

[0099] In one aspect, provided herein is a kit, comprising: amember comprising an absorptive material; and a compositiondescribed herein, wherein the composition is at least partiallyabsorbed in the absorptive material.

[0100] In one aspect, provided herein is a kit, comprising anarticle described herein or a device described herein.

[0101] In one aspect, provided herein is a method, comprising:contacting a sample with either a composition described herein, anarticle described herein, or a device described herein, to yield aproduct; and measuring fluorescence of the product to detect viablebacterial cells in the sample.

[0102] In some embodiments, the method comprises measuring thetotal fluorescence of the product to detect viable bacterial cellsin the sample. In some embodiments, the method further comprisescomparing the measured total fluorescence of the product to a totalfluorescence produced by a control, to detect viable bacterialcells in the sample. In some embodiments, the method furthercomprises correlating the comparative total fluorescence to thenumber of viable bacterial cells in the sample.

[0103] In some embodiments, the method comprises measuring a changein fluorescence of the product as a function of time to detectviable bacterial cells in the sample. In some embodiments, themethod further comprises comparing a measured rate of change offluorescence of the product as a function of time to a rate ofchange of fluorescence as a function of time produced by a control,to detect viable bacterial cells in the sample. In someembodiments, the method further comprises correlating thecomparative rate of change of fluorescence as a function of time tothe number of viable bacterial cells in the sample.

[0104] In some embodiments, the control comprises a compositionidentical to the sample but that does not comprise viable bacterialcells.

[0105] In some embodiments, the control comprises a compositionidentical to the sample but comprises a known number of viablebacterial cells.

[0106] In some embodiments, the sample comprises a biologicalsample. In some embodiments, the sample comprises an environmentalsample. In some embodiments, the sample comprises a human sample.In some embodiments, the sample comprises a human GI tractsample.

[0107] In some embodiments, the viable bacterial cells comprisebacterial cells selected from the group consisting of Escherichiacoli, Bacillus anthraces, Bacillus cereus, Clostridium botulinum,Yersinia pestis, Yersinia enterocolitica, Brucella species,Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Staphylococcus species, Mycobacterium species, GroupA Streptococcus, Group B Streptococcus, Streptococcus pneumoniae,Helicobacter pylori, Francisella tularensis, Salmonellaenteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasmasalivarium, Mycoplasma fermentans, Mycoplasma pneumoniae,Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacteriumavium, Mycobacterium leprae, Rickettsia rickettsia, Rickettsiaakari, Rickettsia prowazekii, Rickettsia canada, Bacillus subtilis,Bacillus subtilis niger, Bacillus thuringiensis, Coxiella burnetti,Faecalibacterium prausnitzii, Roseburia hominis, Eubacteriumrectale, Dialister invisus, Ruminococcus albus, Ruminococcuscallidus, and Ruminococcus bromii.

[0108] In one aspect, provided herein is a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the GI tract, the methodcomprising: contacting a sample from the GI tract of the subjectwith a composition described herein to provide a product; measuringa parameter selected from: i) total fluorescence of the product; orii) rate of change of fluorescence of the product as a function oftime; and correlating the parameter to a number of viable bacterialcells in the sample. In some embodiments, the method furthercomprises using the correlation to determine whether the subject issuffering from or at risk of overgrowth of bacterial cells in theGI tract. In some embodiments, the method further comprisesdetermining that when the number of the viable bacterial cells inthe sample is greater than about 10.sup.5 colony forming units(CFU)/mL, the subject needs treatment for overgrowth of bacterialcells in the GI tract. In some embodiments, the parameter comprisesthe total fluorescence of the product. In some embodiments, theparameter comprises the rate of change of fluorescence of theproduct as a function of time.

[0109] In some embodiments, the method comprises obtaining thesample from the GI tract of the subject; measuring the totalfluorescence of the product; comparing the measured totalfluorescence to a total fluorescence produced by a control; andcorrelating the comparative fluorescence to the number of viablebacterial cells present in the sample. In some embodiments, themethod further comprises determining that when the number of theviable bacterial cells in the sample is greater than about 10.sup.5CFU/mL, the subject needs treatment for overgrowth of bacterialcells in the GI tract.

[0110] In some embodiments, the method comprises obtaining thesample from the GI tract of the subject; measuring the totalfluorescence of the product; comparing a rate of change offluorescence of the product as a function of time to a rate ofchange of fluorescence as a function of time produced by a control;and correlating the comparative rate of change of fluorescence as afunction of time to the number of viable bacterial cells in thesample. In some embodiments, the control comprises a compositionidentical to the sample that does not comprise viable bacterialcells. In some embodiments, the control comprises a compositionidentical to the sample but that comprises a known number of viablebacterial cells.

[0111] In one aspect, provided herein is a method, comprising:disposing a sample in an article described herein, therebyproducing a product; and measuring a parameter selected from totalfluorescence of the product in the article, and a rate of change offluorescence as a function of time of the product in thearticle.

[0112] In some embodiments, the sample comprises an aqueoussolution. In some embodiments, the method further comprisesremoving water from the product.

[0113] In some embodiments, the method further comprises heatingthe product. In some embodiments, the product is heated to atemperature above 0.degree. C. In some embodiments, the product isheated to a temperature of at most 100.degree. C.

[0114] In some embodiments, the method comprises reducing a totalwater content of the product by at least 50%.

[0115] In some embodiments, the parameter is total fluorescence ofthe product in the article.

[0116] In some embodiments, the method further comprises comparingthe measured total fluorescence detected in the product to a totalfluorescence produced by a control, and correlating the comparativefluorescence to detect viable bacterial cells in the sample. Insome embodiments, the method further comprises correlating thecomparative total fluorescence detected in the product to thenumber of viable bacterial cells in the sample.

[0117] In some embodiments, the parameter is the rate of change offluorescence as a function of time of the product in the article,and the method further comprises comparing the rate of change offluorescence as a function of time to a rate of change offluorescence as a function of time produced by a control to detectthe viable bacterial cells in the sample. In some embodiments, themethod further comprises correlating the comparative rate of changeof fluorescence as a function of time to the number of viablebacterial cells in the sample.

[0118] In some embodiments, the control comprises a productidentical to the product but that is devoid of viable bacterialcells. In some embodiments, the control comprises a productidentical to the product but comprises a known number of viablebacterial cells.

[0119] In some embodiments, the method comprises continuouslymeasuring for up to 330 minutes.

[0120] In some embodiments, the sample comprises a biologicalsample. In some embodiments, the sample comprises an environmentalsample. In some embodiments, the sample comprises a human sample.In some embodiments, the sample comprises a human GI tractsample.

[0121] In some embodiments, the viable bacterial cells comprisebacterial cells selected from the group consisting of Escherichiacoli, Bacillus anthraces, Bacillus cereus, Clostridium botulinum,Yersinia pestis, Yersinia enterocolitica, Francisella tularensis,Brucella species, Clostridium perfringens, Burkholderia mallei,Burkholderia pseudomallei, Staphylococcus species, Mycobacteriumspecies, Group A Streptococcus, Group B Streptococcus,Streptococcus pneumoniae, Helicobacter pylori, Salmonellaenteritidis, Mycoplasma hominis, Mycoplasma orale, Mycoplasmasalivarium, Mycoplasma fermentans, Mycoplasma pneumoniae,Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacteriumavium, Mycobacterium leprae, Rickettsia rickettsia, Rickettsiaakari, Rickettsia prowazekii, Rickettsia canada, Bacillus subtilis,Bacillus subtilis niger, Bacillus thuringiensis, Coxiella burnetti,Faecalibacterium prausnitzii, Roseburia hominis, Eubacteriumrectale, Dialister invisus, Ruminococcus albus, Ruminococcuscallidus, and Ruminococcus bromii.

[0122] In one aspect, provided herein is a method of assessing ormonitoring the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the GI tract, the methodcomprising: obtaining a sample from the gastrointestinal tract ofthe subject; disposing the sample in an article described herein toprovide a product; measuring a parameter selected from a totalfluorescence of the product; and a rate of change of fluorescenceof the product as a function of time; correlating the measuredparameter to a number of viable bacterial cells in the sample; anddetermining that the subject is in need of treatment for or at riskof overgrowth of bacterial cells in the gastrointestinal tract,when the number of viable bacterial cells is greater than about10.sup.5 CFU/mL.

[0123] In some embodiments, the parameter comprises the totalfluorescence of the product, and the method further comprises:comparing the measured total fluorescence to a total fluorescenceproduced by a control; and correlating the comparative totalfluorescence to the number of viable bacterial cells in thesample.

[0124] In some embodiments, the parameter comprises the rate ofchange of fluorescence of the product as a function of time, andthe method further comprises: comparing the measured rate of changeof fluorescence of the product as a function of time to a rate ofchange of fluorescence as a function of time produced by a control;correlating the comparative rate of change of fluorescence as afunction of time to the number of viable bacterial cells in thesample. In some embodiments, the control comprises a compositionidentical to the sample but does not comprise viable bacterialcells. In some embodiments, the control comprises a compositionidentical to the sample but comprises a known number of viablebacterial cells.

[0125] In some embodiments, the method comprises collecting thesample from the GI tract of a subject. In some embodiments, themethod comprises disposing the sample into an ingestible devicewhile the ingestible device is in the GI tract of the subject.

[0126] In some embodiments, the method is performed within the bodyof the subject.

[0127] In some embodiments, the method is partially performedoutside the body of the subject.

[0128] In some embodiments, the ingestible device, comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0129] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system has first, second andthird states; the first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem; the second state of the multi-stage valve system isdifferent from the first and third states of the multi-stage valvesystem; when the multi-stage valve system is in its first state,the opening prevents fluid communication between the interior ofthe ingestible device and the exterior of the ingestible device;when the multi-stage valve system is in its second state, theopening allows fluid communication between the interior of theingestible device and the exterior of the ingestible device; andwhen the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

[0130] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system comprises: anactuator system comprising a first member; a trigger comprising afirst peg and a first lip; a gate comprising a protrusion, and agate leg having an opening; and a biasing system comprising firstand second biasing members; when the multi-stage valve system is ina first stage: the first biasing member applies a force to thetrigger so that the first peg contacts the first member; the firstmember opposes the force applied to the trigger by the firstbiasing member; the second biasing member applies a force to thegate so that the protrusion contacts the first lip; the first lipopposes the force applied to the gate by the second biasing member;and the opening in the gate leg is not aligned with the opening inthe ingestible device.

[0131] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible device,wherein: the sampling system comprises: a first member comprising afirst absorptive material; and a second member comprising a secondabsorbent member different from the first absorptive material; andthe sampling system is configured so that fluid that flows from theexterior of the ingestible device to the interior of the ingestibledevice enters the first absorptive material; and the samplingsystem is configured to allow fluid to flow from the firstabsorptive material to the second absorptive material.

[0132] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible deviceconfigured to absorb a fluid that enters the interior of theingestible device via the opening, the sampling system comprising amember which comprises an absorptive material and at least onepreservative at least partially absorbed in the absorptivematerial.

[0133] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0134] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing, wherein the sampling chamber contains an absorptivematerial; an inlet port connecting the opening in the housing tothe sampling chamber; a single use sealing device positioned withinthe inlet port that seals the inlet port; and a heating elementproximate to the single use sealing device, wherein: the heatingelement is configured to apply heat to the single use sealingdevice to unseal the inlet port and open the sampling chamber, andat least a portion of the absorptive material proximate to theinlet port is configured to expand when in contact with a sampleand reseal the inlet port.

[0135] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing having an entry port and an exit port on an oppositeend of the sampling chamber from the entry port, wherein the exitport is configured to allow gas to exit the chamber and prevent atleast a portion of a sample from exiting the chamber; an inletregion connecting the opening in the housing to the entry port ofthe sampling chamber; and a moveable valve positioned to open andclose the inlet region, wherein: the moveable valve in an openposition allows the sample to enter the sampling chamber; and themoveable valve in a closed position prevents the sample fromentering the sampling chamber.

[0136] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to determine alocation of the ingestible device in a portion of a GI tract of asubject to an accuracy of at least 85%.

[0137] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to determine thatthe ingestible device is in the cecum of a subject to an accuracyof at least 70%.

[0138] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to transmit datato a device capable of implementing the data to determine alocation of the ingestible device in a portion of a GI tract of asubject to an accuracy of at least 85%.

[0139] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to transmit datato an external device capable of implementing the data to determinethat the ingestible device is in the cecum of subject to anaccuracy of at least 70%.

[0140] In some embodiments, the ingestible device further comprisesfirst and second light sources, wherein the first light source isconfigured to emit light at a first wavelength, and the secondlight source is configured to emit light at a second wavelengthdifferent from the first wavelength. In some embodiments, theingestible device further comprises first and second detectors,wherein the first detector is configured to detect light at thefirst wavelength, and the second detector is configured to detectlight at the second wavelength.

[0141] In one aspect, provided herein is a device, comprising: asampling chamber; and a composition in the sampling chamber,wherein: the composition comprises a plurality of donor particlesand a plurality of acceptor particles, each donor particlecomprises a photosensitizer coupled to a first analyte-bindingagent that binds to an analyte, in an excited state, thephotosensitizer generates singlet oxygen; each acceptor particlecomprises a chemiluminescent compound coupled to a secondanalyte-binding agent that binds to the analyte; thechemiluminescent compound reacts with singlet oxygen to emitluminescence; and the device is an ingestible device.

[0142] In some embodiments, the composition further comprises anaqueous medium comprising the donor and acceptor particles. In someembodiments, the donor and acceptor particles are suspended in theaqueous medium.

[0143] In some embodiments, the acceptor particles compriseparticles selected from the group consisting of latex particles,lipid bilayers, oil droplets, silica particles, and metal sols.

[0144] In some embodiments, the acceptor particles comprise latexparticles.

[0145] In some embodiments, the chemiluminescent compound comprisesa compound selected from the group consisting of Chemiluminescer,Thioxene+Diphenyl anthracence, Thioxene+Umbelliferone derivative,Thioxene+Europium chelate, Thioxene+Samarium Chelate,Thioxene+terbium Chelate, N-Phenyl Oxazine+Umbelliferonederivative, N-Phenyl Oxazine+Europium chelate, N-phenylOxazine+Samarium Chelate, N-phenyl Oxazine+terbium Chelate,Dioxene+Umbelliferone derivative, Dioxene+Europium chelate,Dioxene+Samarium Chelate, and N-phenyl Oxazine+terbium Chelate.

[0146] In some embodiments, the donor particles comprise particlesselected from the group consisting of latex particles, lipidbilayers, oil droplets, silica particles, and metal sols.

[0147] In some embodiments, the donor particles comprise latexparticles. In some embodiments, the donor particles furthercomprise streptavidin. In some embodiments, the streptavidin iscoated on the latex particles.

[0148] In some embodiments, the photosensitizer comprises amaterial selected from the group consisting of a dye, an aromaticcompound, an enzyme, and a metal salt.

[0149] In some embodiments, a ratio of a number of the donorparticles to a number of the acceptor particles in the compositionis between 10:1 to 10:1.

[0150] In one aspect, provided herein is a device, comprising: asampling chamber; and a composition in the sampling chamber,wherein the composition comprises: a first analyte-binding agentcomprising a first fluorescent dye, wherein the firstanalyte-binding agent is capable of binding to an analyte; and asecond analyte-binding agent comprising a second fluorescent dye,wherein the second analyte-binding agent is capable of binding tothe analyte, and wherein the second fluorescent dye exhibitsincreased fluorescence when spatially proximal to the firstfluorescent dye; and wherein the device is an ingestible device. Insome embodiments, the spatial proximity between the firstfluorescent dye and the second fluorescent dye results in energytransfer from the first fluorescent dye to the second fluorescentdye.

[0151] In one aspect, provided herein is a device, comprising: asampling chamber; and a composition in the sampling chamber,wherein the composition comprises: a first analyte-binding agentcomprising a photosensitizer, wherein the first analyte-bindingagent is capable of binding to an analyte, and wherein thephotosensitizer generates singlet oxygen in an excited state; and asecond analyte-binding agent comprising a fluorogenic dye, whereinthe fluorogenic dye emits fluorescence upon reacting with singletoxygen; and wherein the device is an ingestible device.

[0152] In some embodiments, the composition comprises an aqueousmedium. In some embodiments, the aqueous medium comprises apreservative.

[0153] In some embodiments, the first analyte-binding agent and/orthe second analyte-binding agent is an antigen-binding agent.

[0154] In some embodiments, the first analyte-binding agent and/orthe second analyte-binding agent is an antibody.

[0155] In some embodiments, the device is configured detect theanalyte in vivo.

[0156] In some embodiments, the sampling chamber is configured tohouse an absorptive material. In some embodiments, the absorptivematerial is configured to at least partially absorb thecomposition. In some embodiments, the absorptive material comprisesa sponge.

[0157] In some embodiments, the analyte comprises a biomolecule, amicroorganism, a therapeutic agent, a drug, a biomarker, apesticide, a pollutant, a fragment thereof, or a metabolitethereof.

[0158] In some embodiments, the analyte comprises a protein, anaptamer, a nucleic acid, a steroid, a polysaccharide, or ametabolite.

[0159] In some embodiments, the protein is selected from the groupconsisting of an antibody, an affimer, a cytokine, a chemokine, anenzyme, a hormone, a cancer antigen, a tissue-specific antigen, ahistone, an albumin, a globulin, a scleroprotein, a phosphoprotein,a mucoprotein, a chromoprotein, a lipoprotein, a nucleoprotein, aglycoprotein, a receptor, a membrane-anchored protein, atransmembrane protein, a secreted protein, a human leukocyteantigen (HLA), a blood clotting factor, a microbial protein, andfragments thereof.

[0160] In some embodiments, the metabolite is selected from thegroup consisting of serotonin (5-HT), 5-hydroxyindole acetic acid(5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenicacid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid(3-HAA), quinolinic acid, anthranilic acid, and combinationsthereof.

[0161] In some embodiments, the microorganism is a bacterium, avirus, a prion, a protozoan, a fungus, or a parasite.

[0162] In some embodiments, the bacterium is selected from thegroup consisting of Escherichia coli, Bacillus anthraces, Bacilluscereus, Clostridium botulinum, Clostridium difficile, Yersiniapestis, Yersinia enterocolitica, Francisella tularensis, Brucellaspecies, Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Staphylococcus species, Mycobacterium species, GroupA Streptococcus, Group B Streptococcus, Streptococcus pneumoniae,Helicobacter pylori, Salmonella enteritidis, Mycoplasma hominis,Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans,Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsiarickettsia, Rickettsia akari, Rickettsia prowazekii, Rickettsiacanada, Bacillus subtilis, Bacillus subtilis niger, Bacillusthuringiensis, Coxiella burnetti, Faecalibacterium prausnitzii,Roseburia hominis, Eubacterium rectale, Dialister invisus,Ruminococcus albus, Ruminococcus callidus, and Ruminococcusbromii.

[0163] In some embodiments, the therapeutic agent is selected fromthe group consisting of a TNF.alpha. inhibitor, an IL-12/IL-23inhibitor, an IL-6 receptor inhibitor, an integrin inhibitor, atoll-like receptor (TLR) agonist, a TLR antagonist, a SMAD7inhibitor, a JAK inhibitor, an immunosuppressant, a livebiotherapeutic, a carbohydrate sulfotransferase 15 (CHST15)inhibitor, an IL-1 inhibitor, an IL-13 inhibitor, an IL-10 receptoragonist, glatiramer acetate, a CD40/CD40L inhibitor, a CD3inhibitor, a CD14 inhibitor, a CD20 inhibitor, a CD25 inhibitor, aCD28 inhibitor, a CD49 inhibitor, a CD89 inhibitor, and achemokine/chemokine receptor inhibitor.

[0164] In some embodiments, the analyte is a bile acid or a bileacid salt. In some embodiments, the analyte is an antibiotic. Insome embodiments, the analyte is associated with a disease, adisorder, or a pathogen.

[0165] In some embodiments, the analyte comprises TNF.alpha.,lipoteichoic acid (LTA), lipopolysaccharide (LPS),lipopolysaccharide binding protein (LBP), a cytokine, a chemokine,IL12/23, IL-6, IL-10, MADCAM, .alpha.4.beta.7 integrin, hepatocytegrowth factor (HGF), epidermal growth factor (EGF), heparin-bindingepidermal growth factor (HB-EGF), TGF.beta., adalimumab,infliximab, certolizumab pegol, vedolizumab, natalizumab,golimumab, bevacizumab, or cetuximab.

[0166] In some embodiments, the first analyte-binding agentcomprises an agent selected from the group consisting of anantibody, an affimer, an antigen, a small molecule, a nucleic acid,a receptor, an aptamer, a receptor ligand, biotin, streptavidin,avidin, protein A, protein G, protein L, and derivativesthereof.

[0167] In some embodiments, the second analyte-binding agent arecomprises an agent selected from the group consisting of anantibody, an affimer, an antigen, a small molecule, a nucleic acid,a receptor, an aptamer, a receptor ligand, biotin, streptavidin,avidin, protein A, protein G, protein L, and derivativesthereof.

[0168] In some embodiments, the first analyte-binding agent isdifferent from the second analyte-binding agent. In someembodiments, the first analyte-binding agent is the same as thesecond analyte-binding agent.

[0169] In some embodiments, the first analyte-binding agentcomprises an antibody. In some embodiments, the secondanalyte-binding agent comprises an antibody. In some embodiments,the first analyte-binding agent comprises a biotinylated antibody.In some embodiments, the antibody comprises an anti-bacterialantibody. In some embodiments, the antibody comprises an antibodyselected from the group consisting of an anti-Gram-positivebacteria antibody, an anti-Gram-negative bacteria antibody, ananti-lipoteichoic acid (LTA) antibody, an anti-E. coli antibody, ananti-lipid A antibody, an anti-TNF.alpha. antibody, and derivativesthereof. In some embodiments, the antibody comprises an antibodyselected from the group consisting of MA1-7401 antibody, MA1-40134antibody, ab127996 antibody, ab35654 antibody, ab35654 antibody,ab137967 antibody, ab8467 antibody, and derivatives or fragmentsthereof.

[0170] In some embodiments, the first analyte-binding agentcomprises a biotinylated antibody, and the donor particles comprisea coating which comprises streptavidin. In some embodiments, thesecond analyte-binding agent comprises an antibody covalentlyconjugated to the acceptor particles.

[0171] In some embodiments, the composition further comprisescyclodextrin having a concentration range of 25-50 nM.

[0172] In some embodiments, the device further comprises aninternal calibrator.

[0173] In some embodiments, the device further comprises a lightsource. In some embodiments, the light source is configured toprovide light having at least one wavelength selected from thegroup consisting of 678 nm, 633 nm, and 780 nm. In someembodiments, the light source is configured to irradiate thecomposition with light.

[0174] In some embodiments, the device further comprises a detectorconfigured to detect luminescence emitted by the chemiluminescentcompound. In some embodiments, the detector comprises a photodiodeconfigured to detect luminescence emitted by the chemiluminescentcompound. In some embodiments, the detector comprises a photodiodeconfigured to detect luminescence emitted by the chemiluminescentcompound at at least one wavelength selected from the groupconsisting of 613 nm and 660 nm.

[0175] In one aspect, provided herein is a kit comprising a devicedescribed herein.

[0176] In one aspect, provided herein is a method comprising usinga device described herein to detect the analyte.

[0177] In some embodiments, the method further comprises disposinga sample from a subject into the sampling chamber. In someembodiments, the sample is disposed in the sampling chamber invivo. In some embodiments, the method further comprises irradiatingthe sample, and detecting luminescence emitted from the sample.

[0178] In some embodiments, detecting luminescence comprisesmeasuring an amount of luminescence. In some embodiments, detectingluminescence comprises measuring a total amount of luminescence. Insome embodiments, detecting luminescence comprises measuring a rateof change of luminescence as a function of time.

[0179] In some embodiments, the fluid sample is taken from thegastrointestinal (GI) tract of the subject.

[0180] In some embodiments, the method further comprisesquantifying an amount of the analyte based on measured totalluminescence. In some embodiments, the method further comprisesquantifying an amount of the analyte based a rate of change ofluminescence.

[0181] In some embodiments, the analyte comprises a biomolecule, amicroorganism, a therapeutic agent, a drug, a biomarker, apesticide, a pollutant, a fragment thereof, or a metabolitethereof.

[0182] In some embodiments, the analyte comprises a protein, anaptamer, nucleic acid, a steroid, a polysaccharide, or ametabolite.

[0183] In some embodiments, the protein is selected from the groupconsisting of an antibody, an affimer, a cytokine, a chemokine, anenzyme, a hormone, a cancer antigen, a tissue-specific antigen, ahistone, an albumin, a globulin, a scleroprotein, a phosphoprotein,a mucoprotein, a chromoprotein, a lipoprotein, a nucleoprotein, aglycoprotein, a receptor, a membrane-anchored protein, atransmembrane protein, a secreted protein, a human leukocyteantigen (HLA), a blood clotting factor, a microbial protein, andfragments thereof.

[0184] In some embodiments, the metabolite is selected from thegroup consisting of serotonin (5-HT), 5-hydroxyindole acetic acid(5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenicacid (KA), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid(3-HAA), quinolinic acid, anthranilic acid, and combinationsthereof.

[0185] In some embodiments, the microorganism is a bacterium, avirus, a prion, a protozoan, a fungus, or a parasite.

[0186] In some embodiments, the bacterium is selected from thegroup consisting of Escherichia coli, Bacillus anthraces, Bacilluscereus, Clostridium botulinum, Clostridium difficile, Yersiniapestis, Yersinia enterocolitica, Francisella tularensis, Brucellaspecies, Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Staphylococcus species, Mycobacterium species, GroupA Streptococcus, Group B Streptococcus, Streptococcus pneumoniae,Helicobacter pylori, Salmonella enteritidis, Mycoplasma hominis,Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans,Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsiarickettsia, Rickettsia akari, Rickettsia prowazekii, Rickettsiacanada, Bacillus subtilis, Bacillus subtilis niger, Bacillusthuringiensis, Coxiella burnetti, Faecalibacterium prausnitzii,Roseburia hominis, Eubacterium rectale, Dialister invisus,Ruminococcus albus, Ruminococcus callidus, and Ruminococcusbromii.

[0187] In some embodiments, the therapeutic agent is selected fromthe group consisting of a TNF.alpha. inhibitor, an IL-12/IL-23inhibitor, an IL-6 receptor inhibitor, an integrin inhibitor, atoll-like receptor (TLR) agonist, a TLR antagonist, a SMAD7inhibitor, a JAK inhibitor, an immunosuppressant, a livebiotherapeutic, a carbohydrate sulfotransferase 15 (CHST15)inhibitor, an IL-1 inhibitor, an IL-13 inhibitor, an IL-10 receptoragonist, glatiramer acetate, a CD40/CD40L inhibitor, a CD3inhibitor, a CD14 inhibitor, a CD20 inhibitor, a CD25 inhibitor, aCD28 inhibitor, a CD49 inhibitor, a CD89 inhibitor, and achemokine/chemokine receptor inhibitor.

[0188] In some embodiments, the analyte is a bile acid or a bileacid salt. In some embodiments, the analyte is an antibiotic. Insome embodiments, the analyte is associated with a disease, adisorder, or a pathogen.

[0189] In some embodiments, the analyte comprises TNF.alpha.,lipoteichoic acid (LTA), lipopolysaccharide (LPS),lipopolysaccharide binding protein (LBP), a cytokine, a chemokine,IL12/23, IL-6, IL-10, MADCAM, .alpha.4.beta.7 integrin, hepatocytegrowth factor (HGF), epidermal growth factor (EGF), heparin-bindingepidermal growth factor (HB-EGF), TGF.beta., adalimumab,infliximab, certolizumab pegol, vedolizumab, natalizumab,golimumab, bevacizumab, or cetuximab.

[0190] In some embodiments, the method further comprisesdetermining, based on the detected luminescence, that the subjectis suffering from or at risk of overgrowth of bacterial cells inthe GI tract. In some embodiments, the method further comprisescorrelating a total luminescence and/or a rate of change ofluminescence as a function of time measured in the sample to theamount of the analyte in the sample. In some embodiments, themethod further comprises correlating the amount of the analyte inthe sample to the number of viable bacterial cells in the sample.In some embodiments, determining that the determined number of theviable bacterial cells is greater than about 10.sup.5 CFU/mLindicates a need for treatment.

[0191] In some embodiments, the method further comprisesdetermining, based on the detected luminescence, that the subjectis suffering from or at risk of overgrowth of bacterial cells inthe gastrointestinal tract.

[0192] In some embodiments, the subject is suffering from or atrisk of overgrowth of bacterial cells in the gastrointestinaltract.

[0193] In some embodiments, the device comprises a plurality ofsampling chambers, and the method further comprises disposingdifferent samples in different sampling chambers. In someembodiments, the method comprises taking different samples atdifferent times. In some embodiments, the method comprises takingdifferent samples at different locations within thegastrointestinal tract. In some embodiments, the differentlocations comprise locations selected from the group consisting ofthe mouth, the throat, the esophagus, the stomach, the smallintestine, the large intestine, the duodenum, the jejunum, theileum, the ascending colon, the transverse colon, and thedescending colon. In some embodiments, the method further comprisescreating a molecular map that maps each location from the number ofdifferent locations within the GI tract to a respective measurementof the analyte.

[0194] In one aspect, provided herein is a device, comprising adiffractive optics sensor, wherein the device is an ingestibledevice. In some embodiments, the diffractive optics sensor isconfigured to detect an analyte present in the device. In someembodiments, the diffractive optics sensor comprises: a diffractiongrating; an analyte-binding agent linked to the diffractiongrating, wherein the analyte-binding agent is capable of binding tothe analyte; and a detector configured to detect light diffractedby the diffraction grating, wherein the device is configured sothat, when the analyte is bound to the analyte-binding agent, adiffraction pattern of light diffracted by the diffraction gratingchanges. In some embodiments, the change in the diffraction patterncomprises a change in an intensity of light diffracted by thediffraction grating. In some embodiments, a magnitude of the changein the intensity of light diffracted by the diffraction grating isindicative of the concentration of the analyte in the sample.

[0195] In some embodiments, the device further comprises a lightsource configured so that light emitted by the light sourceimpinges on the diffraction grating with an angle of incidence60.degree. measured from surface. In some embodiments, the lightsource is configured to generate light having a wavelength of 670nm.

[0196] In some embodiments, the diffraction grating has a period of15 .mu.m. In some embodiments, the diffraction grating comprises aseries of grooves comprising adjacent recessed portions and whereinraised portions of the grooves have a depth from about 1 nm toabout 1000 nm.

[0197] In some embodiments, the diffraction pattern comprises lightin a plurality of diffraction orders, and the detector detects anintensity of light in one or more of the diffraction orders.

[0198] In some embodiments, the diffraction optics are configuredfor total internal reflection.

[0199] In some embodiments, the analyte comprises a member selectedfrom the group consisting of a biomolecule, a microorganism, atherapeutic agent, a drug, a biomarker, a pesticide, a pollutant,fragments thereof, and metabolites thereof.

[0200] In some embodiments, the analyte comprises a member selectedfrom the group consisting of a protein, a nucleic acid, a steroid,a polysaccharide, and a metabolite. In some embodiments, theanalyte comprises a protein selected from the group consisting ofan antibody, an aptamer, an affimer, a cytokine, a chemokine, anenzyme, a hormone, a cancer antigen, a tissue-specific antigen, ahistone, an albumin, a globulin, a scleroprotein, a phosphoprotein,a mucoprotein, a chromoprotein, a lipoprotein, a nucleoprotein, aglycoprotein, a receptor, a membrane-anchored protein, atransmembrane protein, a secreted protein, a human leukocyteantigen (HLA), a blood clotting factor, a microbial protein, andfragments thereof.

[0201] In some embodiments, the analyte comprises a metaboliteselected from the group consisting of serotonin (5-HT),5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP),kynurenine (K), kynurenic acid (KA), 3-hydroxykynurenine (3-HK),3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilicacid, and combinations thereof.

[0202] In some embodiments, the analyte comprises a bile acid or abile acid salt.

[0203] In some embodiments, the analyte comprises anantibiotic.

[0204] In some embodiments, the analyte comprises a microorganismselected from the group consisting of a bacterium, a virus, aprion, a protozoan, a fungus, and a parasite.

[0205] In some embodiments, the bacterium comprises a memberselected from the group consisting of Escherichia coli, Bacillusanthraces, Bacillus cereus, Clostridium botulinum, Clostridiumdifficile, Yersinia pestis, Yersinia enterocolitica, Francisellatularensis, Brucella species, Clostridium perfringens, Burkholderiamallei, Burkholderia pseudomallei, Staphylococcus species,Mycobacterium species, Group A Streptococcus, Group BStreptococcus, Streptococcus pneumoniae, Helicobacter pylori,Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale,Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasmapneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia,Rickettsia akari, Rickettsia prowazekii, Rickettsia canada,Bacillus subtilis, Bacillus subtilis niger, Bacillus thuringiensis,Coxiella burnetti, Faecalibacterium prausnitzii, Roseburia hominis,Eubacterium rectale, Dialister invisus, Ruminococcus albus,Ruminococcus callidus, and Ruminococcus bromii.

[0206] In some embodiments, the therapeutic agent comprises amember selected from the group consisting of a TNF.alpha.inhibitor, an IL-12/IL-23 inhibitor, an IL-6 receptor inhibitor, anintegrin inhibitor, a toll-like receptor (TLR) agonist, a TLRantagonist, a SMAD7 inhibitor, a JAK inhibitor, animmunosuppressant, a live biotherapeutic, a carbohydratesulfotransferase 15 (CHST15) inhibitor, an IL-1 inhibitor, an IL-13inhibitor, an IL-10 receptor agonist, glatiramer acetate, aCD40/CD40L inhibitor, a CD3 inhibitor, a CD14 inhibitor, a CD20inhibitor, a CD25 inhibitor, a CD28 inhibitor, a CD49 inhibitor, aCD89 inhibitor, and a chemokine/chemokine receptor inhibitor.

[0207] In some embodiments, the analyte is associated with adisease, a disorder, or a pathogen. In some embodiments, theanalyte-binding agent comprises an antibody, an affimer, anantigen, a small molecule, a nucleic acid, a receptor, or anaptamer.

[0208] In some embodiments, the analyte-binding agent specificallybinds to an analyte present in a particular genus, species orstrain of microorganism.

[0209] In some embodiments, the analyte-binding agent is covalentlylinked to the substrate. In some embodiments, the analyte-bindingagent is non-covalently linked to the substrate. In someembodiments, the analyte-binding agent is directly linked to thesubstrate. In some embodiments, the analyte-binding agent isindirectly linked to the substrate. In some embodiments, theanalyte-binding agent is indirectly linked to the substrate througha spacer.

[0210] In some embodiments, the analyte-binding agent comprises anantibody which comprises an Fc region, and the analyte-bindingagent is directly or indirectly linked to the substrate through theFc region.

[0211] In some embodiments, the diffraction grating comprises aseries of grooves comprising adjacent recessed portions and raisedportions, and the analyte-binding agent is linked to the raisedportions. In some embodiments, the diffraction grating comprises aseries of grooves comprising adjacent recessed portions and raisedportions, and the analyte-binding agent is linked to the recessedportions.

[0212] In some embodiments, the device further comprises a firstchamber configured to contain a sample. In some embodiments, thefirst chamber has a volume of at most 1000 pt. In some embodiments,the diffractive optics sensor is configured to analyze the samplewhen the sample is contained in the first chamber.

[0213] In some embodiments, the device further comprises an openingand a cover, wherein: the cover has a first position and a secondposition; in the first position, the cover prevents fluid fromentering the first chamber from an exterior of the device and alsoprevents fluid from exiting the first chamber to the exterior ofthe device; and in the second position, the cover allows fluid toenter the first chamber from the exterior of the device.

[0214] In some embodiments, the device further comprises a secondchamber configured so that the sample can move from the firstchamber to the second chamber, wherein the second chamber isconfigured to incubate the sample when the sample is in the secondchamber. In some embodiments, the second chamber has a volume of atmost 1000 .mu.L. In some embodiments, the diffractive optics sensoris configured to analyze the sample when the sample is contained inthe second chamber.

[0215] In some embodiments, the device further comprises at leastone member selected from the group consisting of a port, a valveand a pump, wherein the at least one member is configured to movethe sample when the sample is in the device. In some embodiments,the device is configured so that the sample movement in the devicedoes not substantially disrupt binding of the analyte to theanalyte-binding agent.

[0216] In some embodiments, the device is configured so that flowof the sample through the incubation chamber is less than 500.mu.L/min. In some embodiments, the diffractive optics sensorcomprises a plurality of diffraction gratings, wherein eachdiffraction grating comprises an analyte-binding agent capable ofbinding to a different analyte.

[0217] In some embodiments, the device is configured to detect theanalyte at a location within the gastrointestinal (GI) tract of asubject. In some embodiments, the location within the GI tract ofthe subject comprises a member selected from the group consistingof the mouth, the throat, the esophagus, the stomach, the smallintestine, the large intestine, the rectum, the anus, thesphincter, the duodenum, the jejunum, the ileum, and the colon.

[0218] In some embodiments, the device further comprises a systemconfigured to determine a location of the device within the GItract of a subject.

[0219] In some embodiments, the system comprises at least onemember selected from the group consisting of a spectrometer, acapacitance sensor, a temperature sensor, an impedance sensor, a pHsensor, a heart rate sensor, an acoustic sensor, a reflected lightsensor, an image sensor, and a movement sensor.

[0220] In some embodiments, the device further comprises a unitconfigured to: a) transmit data to a base station; and/or b)receive data from the base station. In some embodiments, the basestation is ex vivo.

[0221] In some embodiments, the device further comprises aprocessing unit configured to determine a presence and/or an amountof an analyte in a sample contained in the device based on a signalgenerated by the diffractive optics sensor. In some embodiments,the processing unit is configured to determine the presence and/orthe level of the analyte by comparing a signal generated by thediffractive optics sensor to one or more control levels.

[0222] In some embodiments, the device further comprises asecondary detection agent that binds to the analyte and increases arefractive index of a complex comprising the analyte bound to theanalyte-binding agent when bound to the complex. In someembodiments, the secondary detection agent comprises ananoparticle.

[0223] In some embodiments, the ingestible device, comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0224] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system has first, second andthird states; the first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem; the second state of the multi-stage valve system isdifferent from the first and third states of the multi-stage valvesystem; when the multi-stage valve system is in its first state,the opening prevents fluid communication between the interior ofthe ingestible device and the exterior of the ingestible device;when the multi-stage valve system is in its second state, theopening allows fluid communication between the interior of theingestible device and the exterior of the ingestible device; andwhen the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

[0225] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system comprises: anactuator system comprising a first member; a trigger comprising afirst peg and a first lip; a gate comprising a protrusion, and agate leg having an opening; and a biasing system comprising firstand second biasing members; when the multi-stage valve system is ina first stage: the first biasing member applies a force to thetrigger so that the first peg contacts the first member; the firstmember opposes the force applied to the trigger by the firstbiasing member; the second biasing member applies a force to thegate so that the protrusion contacts the first lip; the first lipopposes the force applied to the gate by the second biasing member;and the opening in the gate leg is not aligned with the opening inthe ingestible device.

[0226] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible device,wherein: the sampling system comprises: a first member comprising afirst absorptive material; and a second member comprising a secondabsorptive material different from the first absorptive material;and the sampling system is configured so that fluid that flows fromthe exterior of the ingestible device to the interior of theingestible device enters the first absorptive material; and thesampling system is configured to allow fluid to flow from the firstabsorptive material to the second absorptive material.

[0227] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible deviceconfigured to absorb a fluid that enters the interior of theingestible device via the opening, the sampling system comprising amember which comprises an absorptive material and at least onepreservative at least partially absorbed in the absorptivematerial.

[0228] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0229] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing, wherein the sampling chamber contains a membercomprising an absorptive material; an inlet port connecting theopening in the housing to the sampling chamber; a single usesealing device positioned within the inlet port that seals theinlet port; and a heating element proximate to the single usesealing device, wherein: the heating element is configured to applyheat to the single use sealing device to unseal the inlet port andopen the sampling chamber, and at least a portion of the absorptivematerial proximate to the inlet port is configured to expand whenin contact with a sample and reseal the inlet port.

[0230] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing having an entry port and an exit port on an oppositeend of the sampling chamber from the entry port, wherein the exitport is configured to allow gas to exit the chamber and prevent atleast a portion of a sample from exiting the chamber; an inletregion connecting the opening in the housing to the entry port ofthe sampling chamber; and a moveable valve positioned to open andclose the inlet region, wherein: the moveable valve in an openposition allows the sample to enter the sampling chamber; and themoveable valve in a closed position prevents the sample fromentering the sampling chamber.

[0231] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to determine a location of theingestible device in a portion of a GI tract of a subject to anaccuracy of at least 85%.

[0232] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to determine that the ingestible deviceis in the cecum of a subject to an accuracy of at least 70%.

[0233] In some embodiments, the device further comprises: one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to transmit data to a device capable ofimplementing the data to determine a location of the medical devicein a portion of a GI tract of a subject to an accuracy of at least85%.

[0234] In some embodiments, the device further comprises one ormore processing devices; and one or more machine readable hardwarestorage devices storing instructions that are executable by the oneor more processing devices to transmit data to an external devicecapable of implementing the data to determine that the ingestibledevice is in the cecum of subject to an accuracy of at least70%.

[0235] In some embodiments, the device further comprises first andsecond light sources, wherein the first light source is configuredto emit light at a first wavelength, and the second light source isconfigured to emit light at a second wavelength different from thefirst wavelength.

[0236] In some embodiments, the device further comprises first andsecond detectors, wherein the first detector is configured todetect light at the first wavelength, and the second detector isconfigured to detect light at the second wavelength.

[0237] In one aspect, provided herein is a system, comprising aningestible device described herein; and a processing unitconfigured to determine a presence and/or a level of an analyte ina sample based on a signal generated by the diffractive opticssensor, wherein the processing unit is external to the ingestibledevice.

[0238] In some embodiments, the processing unit is configured todetermine the presence and/or the level of the analyte by comparinga signal generated by the diffractive optics sensor to one or morecontrol levels. In some embodiments, the processing unit is locatedex vivo, and the ingestible device comprises a communications unitfor transmitting the signal to the processing unit.

[0239] In one aspect, provided herein is a method comprisingoperating an ingestible device within the GI tract of a subject todetect an analyte, wherein the ingestible device is a devicedescribed herein.

[0240] In some embodiments, the method further comprises:collecting a sample from the GI tract of the subject; aftercollecting the sample, using the diffractive optics sensor tomeasure a diffraction pattern; and using the diffraction pattern todetect a presence and/or a level of the analyte in the sample. Insome embodiments, the method further comprises measuring thediffraction pattern at more than one point in time.

[0241] In some embodiments, the method further comprises using asecondary detection agent to bind to the analyte, therebyincreasing a refractive index of a complex comprising the analytebound to the analyte-binding agent. In some embodiments, thesecondary detection agent comprises a nanoparticle.

[0242] In some embodiments, the method further comprises incubatingthe sample.

[0243] In some embodiments, the method further comprises, beforeadministering the device to the subject, determining the locationwithin the GI tract of the subject.

[0244] In some embodiments, the method further comprisestransmitting data from the device to a base station and/ortransmitting data from the base station to the device, wherein thebase station is external to the subject. In some embodiments, thedata is representative of a signal generated by the diffractiveoptics biosensor.

[0245] In one aspect, provided herein is a method, comprising:using an ingestible device to obtain a sample within a GI tract ofa subject; and using diffractive optics to analyze the sample. Insome embodiments, the ingestible device comprises the diffractiveoptics. In some embodiments, the sample is analyzed in vivo.

[0246] In some embodiments, the ingestible device, comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0247] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system has first, second andthird states; the first state of the multi-stage valve system isdifferent from the second and third states of the multi-stage valvesystem; the second state of the multi-stage valve system isdifferent from the first and third states of the multi-stage valvesystem; when the multi-stage valve system is in its first state,the opening prevents fluid communication between the interior ofthe ingestible device and the exterior of the ingestible device;when the multi-stage valve system is in its second state, theopening allows fluid communication between the interior of theingestible device and the exterior of the ingestible device; andwhen the multi-stage valve system is in its third state, theopening prevents fluid communication between the interior of theingestible device and the exterior of the ingestible device.

[0248] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a multi-stage valve system in the interior of the ingestibledevice, wherein: the multi-stage valve system comprises: anactuator system comprising a first member; a trigger comprising afirst peg and a first lip; a gate comprising a protrusion, and agate leg having an opening; and a biasing system comprising firstand second biasing members; when the multi-stage valve system is ina first stage: the first biasing member applies a force to thetrigger so that the first peg contacts the first member; the firstmember opposes the force applied to the trigger by the firstbiasing member; the second biasing member applies a force to thegate so that the protrusion contacts the first lip; the first lipopposes the force applied to the gate by the second biasing member;and the opening in the gate leg is not aligned with the opening inthe ingestible device.

[0249] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible device,wherein: the sampling system comprises: a first member comprising afirst absorptive material; and a second member comprising a secondabsorptive material different from the first absorptive material;and the sampling system is configured so that fluid that flows fromthe exterior of the ingestible device to the interior of theingestible device enters the first absorptive material; and thesampling system is configured to allow fluid to flow from the firstabsorptive material to the second absorptive material.

[0250] In some embodiments, the ingestible device has an openingbetween an interior of the ingestible device and an exterior of theingestible device, and the ingestible device comprises: a chamber;and a sampling system in the interior of the ingestible deviceconfigured to absorb a fluid that enters the interior of theingestible device via the opening, the sampling system comprising amember which comprises an absorptive material and at least onepreservative at least partially absorbed in the absorptivematerial.

[0251] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, and a wall extending longitudinally from thefirst end to the second end; a first opening in the wall of thehousing; a second opening in the first end of the housing, thesecond opening being oriented substantially perpendicular to thefirst opening; and a curved chamber connecting the first openingand the second opening, wherein at least a portion of the curvedchamber forms a sampling chamber within the ingestible device.

[0252] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing, wherein the sampling chamber contains an absorptivematerial; an inlet port connecting the opening in the housing tothe sampling chamber; a single use sealing device positioned withinthe inlet port that seals the inlet port; and a heating elementproximate to the single use sealing device, wherein: the heatingelement is configured to apply heat to the single use sealingdevice to unseal the inlet port and open the sampling chamber, andat least a portion of the absorptive material proximate to theinlet port is configured to expand when in contact with a sampleand reseal the inlet port.

[0253] In some embodiments, the ingestible device comprises: ahousing defined by a first end, a second end substantially oppositefrom the first end, a wall extending longitudinally from the firstend to the second end, and an opening; a sampling chamber withinthe housing having an entry port and an exit port on an oppositeend of the sampling chamber from the entry port, wherein the exitport is configured to allow gas to exit the chamber and prevent atleast a portion of a sample from exiting the chamber; an inletregion connecting the opening in the housing to the entry port ofthe sampling chamber; and a moveable valve positioned to open andclose the inlet region, wherein: the moveable valve in an openposition allows the sample to enter the sampling chamber; and themoveable valve in a closed position prevents the sample fromentering the sampling chamber.

[0254] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to determine alocation of the ingestible device in a portion of a GI tract of asubject to an accuracy of at least 85%.

[0255] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to determine thatthe ingestible device is in the cecum of a subject to an accuracyof at least 70%.

[0256] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to transmit datato a device capable of implementing the data to determine alocation of the medical device in a portion of a GI tract of asubject to an accuracy of at least 85%.

[0257] In some embodiments, the ingestible device furthercomprises: one or more processing devices; and one or more machinereadable hardware storage devices storing instructions that areexecutable by the one or more processing devices to transmit datato an external device capable of implementing the data to determinethat the ingestible device is in the cecum of subject to anaccuracy of at least 70%.

[0258] In some embodiments, the ingestible device further comprisesfirst and second light sources, wherein the first light source isconfigured to emit light at a first wavelength, and the secondlight source is configured to emit light at a second wavelengthdifferent from the first wavelength.

[0259] In some embodiments, the ingestible device further comprisesfirst and second detectors, wherein the first detector isconfigured to detect light at the first wavelength, and the seconddetector is configured to detect light at the secondwavelength.

BRIEF DESCRIPTION OF THE DRAWINGS

[0260] Exemplary embodiments of the disclosure are provided belowwith reference to the drawings.

[0261] FIG. 1 shows an ingestible device.

[0262] FIG. 2 shows an ingestible device.

[0263] FIG. 3 shows a valve.

[0264] FIGS. 4 and 5 illustrate operation of a valve.

[0265] FIG. 6 shows an ingestible device.

[0266] FIG. 7 shows valve designs.

[0267] FIG. 8 shows a sampling chamber.

[0268] FIG. 9 shows a pumping mechanism.

[0269] FIG. 10 shows an ingestible device.

[0270] FIG. 11 shows an ingestible device.

[0271] FIG. 12 illustrates a valve system.

[0272] FIGS. 13A and 13B illustrate a portion of a two-stage valvesystem in its first and second stages, respectively.

[0273] FIGS. 14A and 14B illustrate a portion of a two-stage valvesystem in its first and second stages, respectively.

[0274] FIGS. 15A and 15B illustrate a portion of a two-stage valvesystem in its first and second stages, respectively.

[0275] FIG. 16 illustrates a more detailed view of an ingestibledevice.

[0276] FIGS. 17A-17C illustrate a portion of a three-stage valvesystem in its first, second and third stages, respectively.

[0277] FIGS. 18A-18C illustrate a portion of a three-stage valvesystem in its first, second and third stages, respectively.

[0278] FIGS. 19A-19C illustrate a portion of a three-stage valvesystem in its first, second and third stages, respectively.

[0279] FIG. 20 illustrates a three-stage valve system in its firststage.

[0280] FIG. 21A illustrates a portion of an ingestible device.

[0281] FIG. 21B illustrates a portion of an ingestible device.

[0282] FIG. 22 illustrates an ingestible device.

[0283] FIG. 23 illustrates an ingestible device.

[0284] FIG. 24 illustrates an ingestible device.

[0285] FIG. 25 illustrates an ingestible device.

[0286] FIG. 26 is an exploded view of an ingestible device.

[0287] FIG. 27 illustrates a portion of an ingestible device.

[0288] FIG. 28 illustrates a portion of an ingestible device.

[0289] FIG. 29 illustrates a member forming part of a set of fiveincubation chambers suitable for an ingestible device.

[0290] FIG. 30 illustrates a partial cross-sectional view of opticsin an ingestible device.

[0291] FIG. 31 illustrates components of the optics and flowchamber systems in an ingestible device.

[0292] FIG. 32 shows a partial view of an ingestible device FIGS.33A, 33B and 33C illustrate operation of ingestible device.

[0293] FIG. 34 illustrates an exploded view of the components ofingestible device.

[0294] FIG. 35 illustrates an ingestible device.

[0295] FIG. 36 illustrates aspects of a mechanism for an ingestibledevice.

[0296] FIG. 37 illustrates an ingestible device.

[0297] FIG. 38 illustrates an ingestible device.

[0298] FIG. 39 illustrates an ingestible device.

[0299] FIGS. 40, 41 and 42 illustrate exemplary anchoringmechanisms of an ingestible device.

[0300] FIG. 43 illustrates an ingestible device.

[0301] FIG. 44A illustrates a portion of an ingestible device.

[0302] FIG. 44B illustrates a partial sectional view of a burstdisc holder.

[0303] FIG. 45 illustrates an ingestible device.

[0304] FIG. 46 illustrates an ingestible device.

[0305] FIG. 47 illustrates an ingestible device.

[0306] FIG. 48 illustrates an ingestible device.

[0307] FIG. 49 illustrates an ingestible device.

[0308] FIG. 50 illustrates an ingestible device.

[0309] FIG. 51 illustrates an ingestible device.

[0310] FIG. 52 illustrates an ingestible device.

[0311] FIG. 53 illustrates an ingestible device.

[0312] FIG. 54 illustrates an ingestible device.

[0313] FIG. 55 illustrates an ingestible device.

[0314] FIG. 56 is a view of an ingestible device.

[0315] FIG. 57 is an exploded view of an ingestible device.

[0316] FIG. 58 is a diagram of an ingestible device during anexample transit through a GI tract.

[0317] FIG. 59 is a diagram of an ingestible device during anexample transit through a jejunum.

[0318] FIG. 60 is a flowchart of illustrative steps for determininga location of an ingestible device as it transits through a GItract.

[0319] FIG. 61 is a flowchart of illustrative steps for detectingtransitions from a stomach to a duodenum and from a duodenum backto a stomach.

[0320] FIG. 62 is a plot illustrating data collected during anexample operation of an ingestible device.

[0321] FIG. 63 is another plot illustrating data collected duringan example operation of an ingestible device.

[0322] FIG. 64 is a flowchart of illustrative steps for detecting atransition from a duodenum to a jejunum.

[0323] FIG. 65 is a plot illustrating data collected during anexample operation of an ingestible device.

[0324] FIG. 66 is a plot illustrating muscle contractions detectedby an ingestible device over time.

[0325] FIG. 67 is a flowchart of illustrative steps for detecting atransition from a jejunum to an ileum.

[0326] FIG. 68 is a flowchart of illustrative steps for detecting atransition from a jejunum to an ileum.

[0327] FIG. 69 is a flowchart of illustrative steps for detecting atransition from an ileum to a cecum.

[0328] FIG. 70 is a flowchart of illustrative steps for detecting atransition from a cecum to a colon.

[0329] FIG. 71 illustrates an exemplary system for collecting,communicating and/or analyzing data about a subject

[0330] FIG. 72A shows the use of a Thorlabs FESH0550 shortpassfilter for filtering excitation wavelength.

[0331] FIG. 72B shows the use of a Thorlabs FB580-10 bandpassfilter for filtering emission wavelength.

[0332] FIG. 72C shows a cross sectional view of an exemplaryfluorescent assay test fixture depicting collimating, focusing, andfiltering lenses.

[0333] FIG. 73A shows a first proximity assay, where abacteria-specific antibody to Linker of T cell activation (LTA) orlipopolysaccharide (LPS) is labeled with F2 dye. F1 dye has ahydrophobic chain, which enables it to incorporate in bacterialmembranes. F1 dye becomes fluorescent upon binding to the bacterialmembranes. Binding of the anti-LPS or anti-LTA antibody labeledwith F2 to the bacterial surface would result in close proximity ofF1 and F2 dyes, leading to an energy transfer from F1 to F2 (i.e.,F1 fluorescence decreases and F2 fluorescence increases).

[0334] FIG. 73B shows a second proximity assay, where F1 dye isattached to a first antibody against LTA (or a specific antigen ona bacteria), and F2 dye is attached to a second antibody againstLTA (or a specific antigen on a bacteria). Binding of bothantibodies to the bacterial surface (e.g., to LTA or the specificantigen) would result in close proximity of F1 and F2dyes, leadingto an energy transfer from F1 to F2 (i.e., F1 fluorescencedecreases and F2 fluorescence increases).

[0335] FIG. 74 shows a forest plot showing the results of 11studies that compared the results of glucose breath test andendoscopy aspirate culture.

[0336] FIG. 75A shows kinetic analysis of Resazurin when added tovarying concentrations of E. coli ATCC 25922. Four replicates wererun in 4 different plates on the same day, where FU=relativefluorescence units.

[0337] FIG. 75B shows an expanded view of the kinetic analysis ofResazurin when added to 10.sup.4 and 10.sup.5 CFU/mL of E. coliATCC 25922.

[0338] FIG. 75C shows a sample challenge plate lay out. Each platerepresents 1 replicate (4 replicates performed).

[0339] FIG. 76A shows the wells in the presence or absence of anabsorptive sponge.

[0340] FIG. 76B shows fluorescence detection plotted over time (120min to 240 min) in the presence of RSS Sponge, which is saturatedwith a solution containing a dye, and 0.01% Mucin/0.05% Triton.

[0341] FIG. 76C shows fluorescence detection plotted over time (120min to 240 min) in the presence of a solution containing a dye and0.01% Mucin/0.05% Triton (no sponge was present).

[0342] FIG. 76D shows fluorescence detection plotted over time (120min to 240 min) in the presence of RSS sponge, which is saturatedwith a solution containing a dye and 0.01% Mucin/0.1% Triton.

[0343] FIG. 76E shows fluorescence detection plotted over time (120min to 240 min) in the presence of a solution containing a dye and0.01% Mucin/0.1% Triton (no sponge was present).

[0344] FIG. 76F shows fluorescence detection plotted over time (120min to 240 min) in the presence of a solution containing a dye and1% Mucin/0.1% Triton (no sponge was present).

[0345] FIG. 76G shows fluorescence detection plotted over time (120min to 240 min) in the presence of RSS sponge, which is saturatedwith a solution containing a dye and 0.01% Mucin/0.1% Triton.

[0346] FIG. 76H summarizes the mean slopes presented in thefluorescence detection over time in the presence of sponge.

[0347] FIG. 76I summarizes the mean slopes presented in thefluorescence detection over time in the absence of sponge.

[0348] FIG. 77A shows the effect of pH on detection of livebacterial cells in a sample. The data demonstrates that the pHvariation within the pH range of 6.5-8 does not impact the abilityto accurately discern positive and negative calls.

[0349] FIG. 77B shows the effect of bile on detection of livebacterial cells in a sample. The use of deoxycholate buffers theeffects of bile concentration. The presence of bile in the testedconcentration ranges showed no impact on the ability to accuratelydiscern positive and negative calls.

[0350] FIG. 77C shows the effect of mucin on detection of livebacterial cells in a sample. With both E. coli and S. aureus, therewas a decrease in mean slope which correlated to an increase inmucin concentration. This, however, did not impact the ability toaccurately discern positive and negative calls. The effect of mucincan be further mitigated using higher mucin concentrations in thedye formulation.

[0351] FIG. 77D shows the effect of yeast on detection of livebacterial cells in a sample. Use of amphotericin B buffered theeffects of increased yeast concentration. Yeast at testedconcentrations showed no impact on the ability to accuratelydiscern positive and negative calls.

[0352] FIG. 78A shows simulated data demonstrating Failure Mode #1where an ingestible device of this disclosure (e.g., a capsule)samples early in the stomach. The low pH of the stomach acid (pH1-4) reduces the baseline fluorescence (sampled at activation)rapidly (within 5 minutes after sample acquisition). Capsulereports: ERROR, DX data not valid.

[0353] FIG. 78B shows simulated data demonstrating Failure Mode #2where a capsule samples late in the colon. The high levels ofbacteria (>10.sup.12 CFU/mL rapidly convert Resazurin toResorufin (within 1 minute during sample acquisition). Rapid autoquenching reduces the signal quickly below 3,000 RFU within 5minutes. Capsule reports: ERROR, DX data not valid.

[0354] FIG. 78C shows simulated data demonstrating early detectionof a SIBO+Ve case presenting with >10.sup.7 CFU/mL. The highlevels of bacteria rapidly convert Resazurin to Resorufin (within60 minutes after Sample acquisition). Rapid auto quenching reducesthe signal quickly below 20,000 RFU within 240 minutes. Capsulereports: Positive SIBO call within 60 minutes.

[0355] FIG. 78D shows simulated data demonstrating early detectionof a SIBO+Ve case presenting with >10.sup.5 CFU/mL. The lowlevels of bacteria slowly convert Resazurin to Resorufin (within240 minutes after Sample acquisition). Capsule reports: PositiveSIBO call with 240 minutes.

[0356] FIG. 78E shows simulated data demonstrating early detectionof a SIBO+Ve case presenting with .ltoreq.10.sup.4 CFU/mL. The lowlevels of bacteria slowly convert Resazurin to Resorufin (within240 minutes after Sample acquisition). Slope<10. Capsulereports: Negative SIBO call with 240 minutes;

[0357] FIG. 79A shows an exemplary sample challenge plate intriplicate by using a Sterilin 96 well round bottom microtitreplate (P/N H511A), where the plate was loaded with 100 .mu.L of adiluted dynamic range of bacteria or failure modes.

[0358] FIG. 79B shows fluorescence detection plotted over time induodenal aspirate spiked with various concentrations of E. coli.The data demonstrated that there is a strong, discernable signalresponse from spiked duodenal samples in good agreement with thesimulated data.

[0359] FIG. 80A shows a simulated performance with jejunal samples,where Pe=(PP+PN).times.(PP+NP)/N{circumflex over ()}2+(PN+NN).times.(NP+NN)/N{circumflex over ( )}2. A kappastatistic equal to zero indicates that agreement is no better thanchance, a kappa of 1.0 indicates perfect agreement, 0-0.4 indicatespoor agreement, 0.4-0.75 indicates fair to good agreement andgreater than 0.75 indicates excellent agreement (Fleiss 1981).

[0360] FIG. 80B shows a simulated performance with Human DuodenalSamples, where Pe=(PP+PN).times.(PP+NP)/N{circumflex over ()}2+(PN+NN).times.(NP+NN)/N{circumflex over ( )}2. A kappastatistic equal to zero indicates that agreement is no better thanchance, a kappa of 1.0 indicates perfect agreement, 0-0.4 indicatespoor agreement, 0.4-0.75 indicates fair to good agreement andgreater than 0.75 indicates excellent agreement (Fleiss 1981).

[0361] FIG. 81A shows results from testing E. coli DH5-Alpha, usingSpectrophotometer 1, Absorbance (600 nm) Y-axis, plotted overactual mean log 10 CFU/mL.

[0362] FIG. 81B shows the % transmittance over actual mean log 10CFU/mL.

[0363] FIG. 82A shows results from testing E. coli ATCC 25922,using Spectrophotometer 1, Absorbance (600 nm) Y-axis, plotted overactual mean log 10 CFU/mL.

[0364] FIG. 82B shows the % transmittance over actual mean log 10CFU/mL.

[0365] FIG. 83A shows results from testing S. epidermidis ATCC12228, using Spectrophotometer 1, Absorbance (600 nm) Y-axis,plotted over actual mean log 10 CFU/mL.

[0366] FIG. 83B shows the % transmittance over actual mean log 10CFU/mL.

[0367] FIGS. 84A and 84B show a graphical representation of ODprediction of CFU/mL at time=4 hours for E. coli ATCC 25922 (84A)and S. epidermidis ATCC 12228 (84B). Bars represent actual meanLog.sub.10 CFU/mL recovered after a 4 hour incubation at 37.degree.C. The line represents the Mean OD 600 measurement for each initialinoculum density. Initial inoculum densities sampled cover adynamic range of 10.sup.4, 10.sup.5 and 10.sup.6 CFU/mL.

[0368] FIGS. 85A and 85B show a graphical representation of OD (A600 nm) (85A) and % Transmittance (85B) data over a 5 hour timecourse assay for E. coli ATCC 25922 using a 50 .mu.L samplevolume.

[0369] FIGS. 86A and 86B show a graphical representation of OD (A600 nm) (86A) and % Transmittance (86B) data over a 5 hour timecourse assay for E. coli ATCC 25922 using a 200 .mu.L samplevolume.

[0370] FIGS. 87A and 87B show a graphical representation of OD (A600 nm) (87A) and % Transmittance (87B) data over a 5 hour timecourse assay for S. epidermidis ATCC 12228 using a 50 .mu.L samplevolume.

[0371] FIGS. 88A and 88B show a graphical representation of OD (A600 nm) (88A) and % Transmittance (88B) data over a 5 hour timecourse assay for S. epidermidis ATCC 12228 using a 200 .mu.L samplevolume.

[0372] FIG. 89 shows results from a bile acid concentration testwith E. coli ATCC 25922 Optical Density (A 600 nm) plotted at t=4hours over a dynamic range of initial inoculum densities in a 50.mu.L sample volume. Growth control (GC) data is also plotted forreference.

[0373] FIG. 90 shows results from a mucin concentration test withE. coli ATCC 25922 Optical Density (A 600 nm) plotted at t=4 hoursover a dynamic range of initial inoculum densities in a 50 .mu.Lsample volume. Growth control (GC) data is also plotted forreference.

[0374] FIG. 91 shows results from a pH range test with E. coli ATCC25922 Optical Density (A 600 nm) plotted at t=4 hours over adynamic range of initial inoculum densities in a 50 .mu.L samplevolume. Growth control (GC) data is also plotted for reference.

[0375] FIG. 92 shows results from a fungal interference test withE. coli ATCC 25922 Optical Density (A 600 nm) plotted at t=4 hoursover a dynamic range of initial inoculum densities in a 50 .mu.LSample volume. Growth control (GC) data is also plotted forreference. A=amphotericin B.

[0376] FIG. 93 shows results from a bile acid concentration testwith S. epidermidis ATCC 12228 Optical Density (A 600 nm) plottedat t=4 hours over a dynamic range of initial inoculum densities ina 50 .mu.L sample volume. Growth Control (GC) data is also plottedfor reference.

[0377] FIG. 94 shows results from a mucin concentration test withS. epidermidis ATCC 12228 Optical Density (A 600 nm) plotted at t=4hours over a dynamic range of initial inoculum densities in a 50.mu.L sample volume. Growth Control (GC) data is also plotted forreference.

[0378] FIG. 95 shows results from a pH range test with S.epidermidis ATCC 12228 Optical Density (A 600 nm) plotted at t=4hours over a dynamic range of initial inoculum densities in a 50.mu.L sample volume. Growth Control (GC) data is also plotted forreference.

[0379] FIG. 96 shows results from a fungal interference test withS. epidermidis ATCC 12228 Optical Density (A 600 nm) plotted at t=4hours over a dynamic range of initial inoculum densities in a 50.mu.L sample volume. Growth Control (GC) data is also plotted forreference. A=amphotericin B.

[0380] FIG. 97 shows the results from dynamic range testing of aminiature OD reader (SCDBS OD) compared to a lab spectrometer (LabSpec) using Coomassie R-250. Primary vertical axis is %Transmittance (Lab Spec); Secondary vertical axis is voltage (SCDBSOD output). Comparative data outputs plotted against the dyeconcentration range (% dye in 0.9% Saline).

[0381] FIG. 98 shows the results from dynamic range testing of aminiature OD reader (SCDBS OD) compared to a lab spectrometer (LabSpec) using bacterial samples of E. coli ATCC 25922. Primaryvertical axis is % Transmittance (Lab Spec); Secondary verticalaxis is voltage (SCDBS OD output). Comparative data outputs plottedagainst the bacterial concentration range (CFU/mL in 0.9%Saline).

[0382] FIG. 99 shows the visual appearance of a plate incubated for16 hours with serial dilutions of a 5 .mu.l sample of bacterialculture of S. aureus ATCC 29213 having initial bacterialconcentrations from 0 (control) to 10.sup.8 CFU/ml. Wells withoutbacterial growth have a clear appearance and are clearlydistinguished from wells with bacterial growth that have a cloudyappearance.

[0383] FIG. 100 shows results for detection of TNF.alpha. withvarying concentrations of Acceptor and Donor Beads. This testmatrix was composed of varying concentrations of both Donor andAcceptor Beads with a constant concentration of BiotinylatedAntibody. Each varying bead concentration was tested against threedifferent TNF.alpha. concentrations and compared against a control.Retest of this matrix narrowed down which combination ofDonor/Acceptor Beads resulted in the best assay data.

[0384] FIG. 101 shows results for detection of TNF.alpha. withvarying concentrations of Acceptor and Donor Beads. This testmatrix was composed of varying concentrations of both Donor andAcceptor Beads being tested in a 5:1 and 10:1 ratio, with aconstant concentration of Biotinylated Antibody. Each varyingconcentration was tested against three different TNF.alpha.concentrations and compared against a control. Retest of thismatrix narrowed down which ratio of Donor: Acceptor beads resultedin the best assay data.

[0385] FIG. 102 shows results for detection of TNF.alpha. withvarying concentrations of biotinylated antibody. This matrix testedan integrated test method (no intermediate incubations) againstvarious concentrations of Donor: Acceptor Beads. Each varyingconcentration was tested against three different concentrations ofTNF.alpha. and compared against a control. The donor and acceptorbeads concentration may be varied, e.g., the donor beadconcentration is 10 and 5 ugs/ml and the acceptor beadconcentration is 1 and 2 ugs/ml, respectively.

[0386] FIG. 103A shows the upper range of TNF.alpha. concentrationswith varying cyclodextrin addition. Hydroxy propyl cyclodextrin isused to overcome sample interference, especially bile acidinterferences; bile acids bind to hydroxy propyl cyclodextrin.

[0387] FIG. 103B shows the lower range of TNF.alpha. concentrationswith varying cyclodextrin concentrations.

[0388] FIG. 104 shows samples of absorptive sponge material (M13:Ahlstrom (6613H)) and (O3: Whatman (Grade F/F) (29009411) cut tofit the 96 well microtitire plate configuration using a whole punchand trimmed with sterile scissors.

[0389] FIG. 105 shows results for detection of TNF.alpha. insamples on an absorptive sample pad. This test matrix consisted ofrunning the optimized bead concentration on a sponge, with an n=3.The limit of detection for this assay is shown to be around 10pg/ml for 03 and 100 pgs/ml for M3. Inset graph showing higherranges of TNF.alpha. concentrations.

[0390] FIG. 106A shows the results of repeat TNF.alpha. detectionin the same assay mixture over time. TNF.alpha. was added to thewell containing the assay mixture after 15 minute incubations.

[0391] FIG. 106B shows the results of repeat TNF.alpha. detectionin the same assay mixture over time. The test matrices consisted ofexemplary bead concentrations (2 .mu.L Acceptor Beads, 2.5 .mu.LBiotinylated Antibody and 10 .mu.L Donor Beads) tested in wells,with continuous reads on a lower instrument setting. Every 15minutes, 5 .mu.L of TNF.alpha. was added to the well, and the testwell was re-read.

[0392] FIG. 107A shows results for TNF.alpha. detection andquantification on absorptive sample pads, where the assay wasprepared with 50 mg of cyclodextrin.

[0393] FIG. 107B shows results for TNF.alpha. detection andquantification on absorptive sample pads, where the assay wasprepared with 25 mg of cyclodextrin.

[0394] FIG. 108 shows assay signal readouts over the testeddilution range of OMNI beads. The OMNI beads 5 .mu.g/mL stocksolution was added into PE Buffer to make a 0.5 .mu.g/mL solution,which was subsequently serially diluted down 1:10 from Row A to RowG, and read on the plate reader at 680/615 nm. The OMNI beads areused to calibrate the capsule and to characterize signal uniformityand reliability of the capsule. The OMNI beads may be loaded withNapthalo-silicon pthalocyanine (Excitation: 780 nm and emission 615nm).

[0395] FIG. 109A shows results of preliminary antibody specificityinvestigation. Specificity of antibodies Ab11, Ab12 and Ab2 weretested using the antibody screening protocol. The assay wasperformed in 50 .mu.L volume. Graph bars represent mean andstandard deviation (SD) from triplicates determination.

[0396] FIG. 109B shows results for antibody screening of Gramnegative bacteria. Specificity of the anti-Gram-negative antibodiesAb11 and Ab12, and of the anti-Gram-positive antibody Ab2 using theantibody screening protocol. Two separate batches of bacteria wereused for each condition, as indicated (N=new batch; 0=old batch).The assay was performed in 50 .mu.L. Graph bars represent mean andSD from triplicates determination.

[0397] FIG. 109C shows results for antibody screening of Grampositive bacteria. The assay was performed in 25 .mu.L volume usingthe AlphaLISA buffer. Two lots of biotin-Ab were tested for ab #2and 4 and only one lot for ab #6. Biotin-Ab and high concentrationacceptor-Ab beads were tested at 1 nM and 10 .mu.g/mL,respectively. The Streptavidin-Donor (SA-Donor) beads were used at20 .mu.g/mL.

[0398] FIG. 109D shows results for the detection of a dynamic rangeof S. aureus. High conjugation (HC) acceptor beads or normalacceptor beads (AB10) were used at 40 .mu.g/mL final and theBiotin-Ab at 0.3 nM final for S. aureus (Ab2, Ab6) using differentdilutions of bacteria. The SA-Donor beads were used at 10 .mu.g/mL.Bacteria were washed twice in PBS before final resuspension inBuffer B. Assay protocol is given below each graph along withsignal-to-background ratio (S/B) obtained for each dilution ofbacteria tested (no bacteria condition as background). Graph barsrepresent mean and SD from triplicate determination.

[0399] FIG. 109E shows detection of a dynamic range of E. coli.High conjugation (HC) acceptor beads or normal acceptor beads(AB10) were used at 40 .mu.g/mL final and the Biotin-Ab at 3 nMfinal for E. coli (Ab10) using different dilutions of bacteria. TheSA-Donor beads were used at 10 .mu.g/mL. Bacteria were washed twicein PBS before final resuspension in Buffer B. Assay protocol isgiven below each graph along with S/B obtained for each dilution ofbacteria tested (no bacteria condition as background). Graph barsrepresent mean and SD from triplicate determination.

[0400] FIG. 109F shows interference of simulated intestinal fluidand bile. FASSIF-V2, a complex of taurocholate and lecithin, whichis used as an example substitute for gas trointestinal fluids, andOxgall, which can be usually obtained from cows, and is mixed withalcohol, were tested using TruHits, where TruHits assay principleand protocols were used. The Oxgall is a greenish-brown liquidmixture containing cholesterol, lecithin, taurocholic acid, andglycocholic acid, which is used as an example subsititute for GIfluids.

[0401] FIG. 109G shows interference of simulated intestinal fluidand bile. FASSIF-V2 and Oxgall were tested using TruHits, whereincreasing concentrations (percentages) of FASSIF-V2 and Oxgallwere tested using the standard protocol shown in Panel A, e.g.,St-Av Donar beads and biotin labeled acceptor beads.

[0402] FIG. 109H shows results of LBP-based assays using freshbacteria. A fixed dilution of S. aureus and E. coli (washed twicein PBS before final resuspension in Buffer B) was tested in withincreasing concentrations of tagged LBP. The detection involved anequimolar mix of His-LBP and Bio-LBP.

[0403] FIG. 1091 shows results of LBP-based assays using freshbacteria. A fixed dilution of S. aureus and E. coli (washed twicein PBS before final resuspension in Buffer B) was tested withincreasing concentrations of tagged LBP. The assay involved His-LBPonly.

[0404] FIG. 110 is a cross-sectional view of an exemplarydiffraction grating.

[0405] FIG. 111 depicts exemplary diffraction signals at differentsteps in a process.

[0406] FIG. 112 shows diffraction intensity data and beaddistribution data.

[0407] FIG. 113 shows diffraction intensity data and beaddistribution data.

[0408] FIG. 114 shows bead distribution data.

[0409] FIG. 115 shows data relating to incubation flow rate andbinding signal.

[0410] FIG. 116 shows data relating to binding.

[0411] FIG. 117 shows data relating to binding signal forincubation without flow.

[0412] FIG. 118 shows binding signal data.

[0413] FIG. 119 shows diffraction intensity data.

[0414] FIG. 120 shows diffraction intensity data.

[0415] FIG. 121 shows diffraction intensity data.

[0416] FIG. 122 shows diffraction intensity data.

[0417] FIG. 123 shows diffraction intensity data.

[0418] FIG. 124 shows diffraction intensity data.

[0419] FIG. 125 shows diffraction intensity data.

[0420] FIG. 126 shows diffraction intensity data.

[0421] FIG. 127 shows gold nanoparticle amplification-relateddata.

[0422] FIG. 128 shows exemplary data for the impact of gratingdesign on diffraction efficiency.

[0423] FIG. 129 shows exemplary data for the impact of angle ofincidence on diffraction efficiency.

[0424] FIG. 130 is a bar graph showing the calculated regressionslopes for total bacterial count determinations using aresazurin-based assay with samples comprising either anaerobicallyenriched fecal or duodenal aspirate clinical samples plated at adynamic dilution range using a liquid format (10.sup.4-10.sup.6CFU/mL dynamic range) or in pad format (1.times.10.sup.6 CFU/mL.)The assay was read after 330 minutes or 22 hours. RFU: relativefluorescence units; Control: samples diluted in PBS.

[0425] FIGS. 131A and 131B show the quantitation of anaerobicbacterial strains using a resazurin-based assay in liquid format.S/D=standard deviation; Mean max signal shown as relativefluorescence units (RFU); diagonal from upper right to lowerleft=<6 CFU; diagonal from upper left to lower right=<5 CFU;cross-hatch=regression slope>3 standard deviations of blankcontrol (3.10+(3.times.0.438))=4.41); 1:10=dilution of exponentialphase culture in cell above in SJFA.

[0426] FIG. 132A shows the quantitation of anaerobic bacterialstrains using a resazurin-based assay in liquid format performedunder microaerophilic conditions and read over 330 minutes. Meanmax signal shown as relative fluorescence units (RFU); diagonalfrom upper left to lower right=regression slope>20; diagonalfrom upper right to lower left=regression slope<10; F 1:100O/N=overnight control read; CONT=PBS control.

[0427] FIG. 132B shows the quantitation of anaerobic bacterialstrains using a resazurin-based assay in liquid format performedunder microaerophilic conditions and read over 20 hours. Mean maxsignal shown as relative fluorescence units (RFU); diagonal fromupper left to lower right=regression slope>20; diagonal fromupper right to lower left=regression slope<10; F 1:100O/N=overnight control read; CONT=PBS control.

[0428] FIG. 132C shows the quantitation of anaerobic bacterialstrains using a resazurin-based assay in liquid format performedunder strict aerobic conditions and read over 24 hours. Mean maxsignal shown as relative fluorescence units (RFU); diagonal fromupper left to lower right=regression slope>20; diagonal fromupper right to lower left=regression slope<10; F 1:100O/N=overnight control read; CONT=PBS control.

[0429] FIGS. 133A-133H are regression plots showing the relationbetween the number of bacterial colony forming units (CFU)/mL andthe time to reach maximum signal detection in resazurin-basedassays using samples comprising the aerobic bacteria Escherichiacoli (FIG. 133A), Staphylococcus aureus (FIG. 133B), Klebsiellapneumoniae (FIG. 133C), Pseudomonas aeruginosa (FIG. 133D),Enterobacter aerogenes (FIG. 133E), Streptococcus mutans (FIG.133F), Enterococcus faecalis (FIG. 133G), and Proteus mirabilis(FIG. 133H). Charted data are mean (n=3) regression slopes tomaximum signal detection.

[0430] FIG. 134A is a bar graph showing the calculated regressionslopes for total bacterial count determinations using aresazurin-based assay with samples comprising a dynamic dilutionrange of Escherichia coli, Staphylococcus aureus, Klebsiellapneumoniae, Pseudomonas aeruginosa, or negative control ("CTRL").Charted data are mean (n=3) regression slopes to maximum signaldetection.

[0431] FIG. 134B is a bar graph showing the calculated regressionslopes for total bacterial count determinations using aresazurin-based assay with samples comprising a dynamic dilutionrange of Enterobacter aerogenes, Streptococcus mutans, Enterococcusfaecalis, Proteus mirabilis or negative control ("CTRL").

[0432] FIGS. 135A-135D are line graphs showing the relativefluorescent units (RFU) as a function of time for resazurin-basedassay with samples comprising a dynamic dilution range ofEscherichia coli, Staphylococcus aureus, Klebsiella pneumoniae,Pseudomonas aeruginosa, or negative control ("CTRL"). FIG. 135Acorresponds to 10.sup.3 CFU/mL; FIG. 135B corresponds to 10.sup.4CFU/mL; FIG. 135C corresponds to 10.sup.5 CFU/mL; and FIG. 135Dcorresponds to 10.sup.6 CFU/mL.

[0433] FIGS. 136A-136D are line graphs showing the relativefluorescent units (RFU) as a function of time for resazurin-basedassay with samples comprising a dynamic dilution range ofEnterobacter aerogenes, Streptococcus mutans, Enterococcusfaecalis, Proteus mirabilis, or negative control ("CTRL"). FIG.136A corresponds to 10.sup.3 CFU/mL; FIG. 136B corresponds to10.sup.4 CFU/mL; FIG. 136C corresponds to 10.sup.5 CFU/mL; and FIG.136D corresponds to 10.sup.6 CFU/mL.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0434] Various apparatuses, systems, devices, components and/orprocesses will be described below to provide illustrative andnon-limiting examples. No embodiment described below limits thesubject matter covered by any claim, and any claim may coverprocesses or apparatuses that differ from those described below. Asan example, the subject matter covered by the claims is not limitedto apparatuses, systems, devices, components and/or processeshaving all of the features of any one apparatus, system, device,component and/or process described below or to features common tomultiple or all of the apparatuses or processes described below. Itis possible that a given apparatus, system, device, componentand/or or process described below is not covered by a given claim.Any embodiment disclosed herein that is not covered by one or moreclaims in this document may be coveed by one or more claims in oneor more other protective instruments, such as, for example, one ormore continuing patent applications and/or one or more divisionalpatent applications. The Applicants, inventors and/or owners do notnecessarily intend to abandon, disclaim or dedicate to the publicany subject matter disclosed herein but not covered by a claimherein.

[0435] Furthermore, it will be appreciated that for simplicity andclarity of illustration, where considered appropriate, referencenumerals may be repeated among the figures to indicatecorresponding or analogous elements. In addition, numerous specificdetails are set forth in order to provide a thorough understandingof the embodiments described herein. However, it is to beunderstood that the embodiments described herein may be practicedwithout these specific details. In other instances, well-knownmethods, procedures and components may have not been described indetail so as not to obscure the embodiments described herein. Also,the description is not to be considered as limiting the scope ofthe embodiments described herein.

Definitions

[0436] Unless otherwise defined herein, scientific and technicalterms used in this disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art.Generally, nomenclature used in connection with, and techniques of,chemistry, cell and tissue culture, molecular biology, cell andcancer biology, neurobiology, neurochemistry, virology, immunology,microbiology, pharmacology, genetics and protein and nucleic acidchemistry, described herein, are those well-known and commonly usedin the art.

[0437] The methods and techniques of the present disclosure aregenerally performed, unless otherwise indicated, according toconventional methods well known in the art and as described invarious general and more specific references that are cited anddiscussed throughout this specification.

[0438] Chemistry terms used herein are used according toconventional usage in the art, as exemplified by "The McGraw-HillDictionary of Chemical Terms," Parker S., Ed., McGraw-Hill, SanFrancisco, C.A. (1985).

[0439] All of the publications, patents and published patentdisclosures referred to in this disclosure are specificallyincorporated by reference herein. In case of conflict, the presentspecification, including its specific definitions, willcontrol.

[0440] The term "reproductive tract" as used herein refers to allportions of an organ system responsible for sexual reproduction ina woman, including but not limited to, the ovaries, Fallopian tube,uterus, cervix and vagina.

[0441] A "patient," "subject," or "individual" are usedinterchangeably and refer to either a human or a non-human animal.These terms include mammals, such as humans, primates, livestockanimals (including bovine, porcine, etc.), companion animals (e.g.,canine, feline, etc.) and rodents (e.g., mice and rats). The term"animal" refers to humans (male or female), companion animals(e.g., dogs, cats and horses), food-source animals, zoo animals,marine animals, birds and other similar animal species. "Edibleanimals" refers to food-source animals such as cows, pigs, sheepand poultry.

[0442] The terms "treating," "treat," or "treatment" embrace bothpreventative, i.e., prophylactic, and palliative treatment. In someembodiments, the methods described herein include the use of aningestible device for detecting a GI disorder in a subject who hasor is at risk of developing a GI disorder. In some embodiments, thesubject has been previously identified as having a GI disorder.Some embodiments of any of the methods provided herein furtherinclude, prior to the providing an ingestible device step,determining that the subject has a GI disorder. Some embodiments ofany of the methods can further include identifying or diagnosing asubject as having a GI disorder.

[0443] "Eukaryotic" as recited herein relates to any type ofeukaryotic organism excluding fungi, such as animals, in particularanimals containing blood, and includes invertebrate animals such ascrustaceans and vertebrates. Vertebrates include both cold-blooded(fish, reptiles, amphibians) and warm blooded animal (birds andmammals). Mammals include in particular primates and moreparticularly humans.

[0444] "Selective lysis" as used in the present disclosure isobtained in a sample when a certain type of cell (e.g., a bacterialcell (e.g., a Gram-positive or a Gram-negative bacterial cell) or aeukaryotic cell) is preferentially lysed over a different type ofcell in the sample (e.g., eukaryotic cell or a bacterial cell). Insome embodiments cells of a particular genera, species or strainare preferentially lysed over cells of a different genera, speciesor strain. In some embodiments, the percentage of cells of a firstgenera, species, or strain in the sample that remain intact issignificantly higher (e.g. 2, 5, 10, 20, 50, 100, 250, 500, or1,000 times more) than the percentage of cells of a second genera,species, or strain in the sample that remain intact, upon treatmentof or contact with a composition or device as described herein. Insome embodiments, the percentage of the bacterial cell in thesample is significantly lower (e.g., 2, 5, 10, 20, 50, 100, 250,500, or 1,000 times less) than the percentage of the eukaryoticcells in the sample that remain intact, upon treatment of orcontact with a composition or device described herein. In someembodiments, the percentage of bacterial cells in the sample thatremain intact is significantly higher (e.g. 2, 5, 10, 20, 50, 100,250, 500, or 1,000 times more) than the percentage of theeukaryotic cells in the sample that remain intact, upon treatmentof or contact with a composition or device as described herein. Insome embodiments, the percentage of Gram-positive bacterial cell inthe sample that remain intact is significantly higher (e.g. 2, 5,10, 20, 50, 100, 250, 500, or 1,000 times more) than the percentageof the Gram-negative bacterial cells in the sample that remainintact, upon treatment of or contact with a composition or deviceas described herein. In some embodiments, the percentage ofGram-negative bacterial cell in the sample that remain intact issignificantly higher (e.g. 2, 5, 10, 20, 50, 100, 250, 500, or1,000 times more) than the percentage of the Gram-positivebacterial cells in the sample that remain intact, upon treatment ofor contact with a composition or device as described herein.

[0445] A "sample" as used in the present disclosure may be abiological sample or an environmental sample. Such samples may beobtained from any organism or environmental site desired. Forexample, the compositions, methods and devices of this disclosuremay be used for detecting and quantifying bacterial cells in asample obtained from, without limitation, soil, rock, plants,animals, cell or tissue culture, biofilms, organic debris, orwater. In some embodiments, samples are obtained from mammals suchas humans. In some embodiments, samples are obtained from a human'sGI tract. In some embodiments, samples are body fluid samplesincluding, but not limited to urine, blood, plasma, serum, saliva,semen, stool, sputum, cerebral spinal fluid, tears, mucus, and thelike. In some embodiments, a single device collects multiplesamples, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100or more samples. In some embodiments, the sample is between 1-2000.mu.L (e.g., 1-1500 .mu.L, 1-1900 .mu.L, 1-1000 .mu.L, 1-500 ul,1-250 ul, 1-100 .mu.l-50 .mu.l-10 and 1-5 .mu.l).

[0446] A "colony-forming unit" or "CFU" refers to a unit used toestimate the number of viable bacteria or fungal cells in a sample.Viable is defined by the cell's ability to divide and form apopulation (or colony). In some embodiments, the viable bacterialcells in a sample may be derived from bacteria selected from thegroup consisting of: Escherichia coli (or E. coli), Bacillusanthraces, Bacillus cereus, Bacteroides vulgatus, Clostridiumbotulinum, Clostridium butyricum, Yersinia pestis, Yersiniaenterocolitica, Francisella tularensis, Brucella species,Clostridium perfringens, Clostridium sporogenes, Klebsiellapneumoniae, Enterobacter aerogenes, Burkholderia mallei,Burkholderia pseudomallei, Staphylococcus species, Staphylococcusaureus, Mycobacterium species, Enterococcus faecalis, Group AStreptococcus, Group B Streptococcus, Streptococcus pneumoniae,Streptococcus mutans, Proteus mirabilis, Helicobacter pylori,Francisella tularensis, Salmonella enteritidis, Mycoplasma hominis,Mycoplasma orale, Mycoplasma salivarium, Mycoplasma fermentans,Mycoplasma pneumoniae, Mycobacterium bovis, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium leprae, Rickettsiarickettsia, Rickettsia akari, Rickettsia prowazekii, Rickettsiacanada, Bacillus subtilis, Bacillus subtilis niger, Bacillusthuringiensis and Coxiella burnetti.

[0447] As used herein, the term "coupled" indicates that twoelements can be directly coupled to one another or coupled to oneanother through one or more intermediate elements.

[0448] The term "saturate" means to permeate or be permeated with aliquid. In some embodiments, an absorptive sponge of the presentdisclosure may be fully saturated with an amount of a liquid suchthat no more liquid can be held. In some embodiments, an absorptivesponge of the present disclosure may be partially saturated with aliquid at an amount that is less than the maximum amount of theliquid that can be held by the sponge. For instance, in someembodiments, a sponge is half-saturated with a liquid at half ofthe maximum amount of the liquid that can be held by thesponge.

[0449] The term "semi-solid" means a material that is neither solid(elastic behavior) nor liquid (viscous behavior) and possesses thecharacteristics of both viscosity and elasticity. Examples ofsemi-solid materials include gels, ointments, creams, and highlyviscous liquids.

[0450] As used herein "culturing" refers to maintaining cells in anenvironment that allows a population of one or more cells toincrease in number through cell division. For example, in someembodiments "culturing" may include combining the cells with mediain a dilution chamber at a temperature that permits cell growth,optionally a temperature found in vivo within the GI tract orreproductive tract of a subject. In some embodiments, the cells arecultured at a temperature between about 35.degree. C. and42.degree. C. In some embodiments, the cells are cultured at atemperature of about 37.degree. C.

[0451] As used herein "dilution fluid" refers to a fluid within thedevice for diluting a fluid sample from the GI tract orreproductive tract. In some embodiments, the dilution fluid is anaqueous solution. In some embodiments, the dilution fluid includesone or more agents that promote or inhibit the growth of anorganism, such as a fungus or bacteria. In some embodiments, thedilution fluid includes one or more agents that facilitate thedetection of an analyte, such as dyes or binding agents foranalytes.

[0452] In some embodiments, a dilution fluid is a sterile media. Asused herein, "sterile media" refers to media that does not containany viable bacteria or other cells that would grow and increase innumber through cell division. Media may be rendered sterile byvarious techniques known in the art such as, but not limited to,autoclaving and/or preparing the media using asceptic techniques.In some embodiments, the media is a liquid media. Examples of mediasuitable for culturing bacteria include nutrient broth, LysogenyBroth (LB) (also known as Luria Broth), Wilkins chalgren, andTryptic Soy Broth (TSB). Other growth or culture media known in theart may also be used in the methods and devices described herein.In some embodiments, the media has a carbon source, such as glucoseor glycerol, a nitrogen source such as ammonium salts or nitratesor amino acids, as well as salts and/or trace elements and vitaminsfor microbial growth. In some embodiments, the media is suitablefor maintaining eukaryotic cells. In some embodiments, the mediaincludes one or more agents that promote or inhibit the growth ofbacteria, optionally agents that promote or inhibit the growth ofspecific types of bacteria.

[0453] In some embodiments, the media is a selective media. As usedherein, "selective media" refers to a media that allows certaintypes of cells to grow and inhibits the growth of other organisms.Accordingly, the growth of cells in a selective media indicates thepresence of certain types of cells within the cultured sample. Forexample, in some embodiments the media is selective forGram-positive or Gram-negative bacteria. In some embodiments, themedia contains crystal violet and bile salts (such as found inMacConkey agar) that inhibit the growth of Gram-positive organismsand allows for the selection and isolation of Gram-negativebacteria. In another embodiment, the media contains a highconcentration of salt (e.g., NaCl) (such as found in Mannitol saltagar) and is selective for Gram-positive bacteria. In someembodiments, the media selectively kills eukaryotic cells or onlygrows prokaryotic cells. In another embodiment, the mediaselectively kills prokaryotic cells (or alternatively only growseukaryotic cells), for example, using a media that includesantibiotics.

[0454] In some embodiments, the media is an indicator media. Asused herein, "indicator media" refers to a media that containsspecific nutrients or indicators (such as, but not limited toneutral red, phenol red, eosin y, or methylene blue) that produce adetectable signal when a certain type of cells are cultured in theindicator media.

[0455] As used herein, "detecting bacteria" refers to determiningthe presence or absence of bacteria within a sample or estimatingthe concentration of bacteria within a sample. For example, in someembodiments, bacterial growth can be determined based on theconcentration of bacteria within a sample. In some embodiments, thedetection system detects and/or quantitates a particular bacterialgenus, species or strain within a sample. In some embodiments, thedetection system detects the products of bacterial growth withinthe cultured and/or diluted sample or a change in concentration ofcertain components within the media due to bacterial growth. Insome embodiments, products of bacterial growth include analytesproduced and/or secreted by the bacteria that are present in themedia, including, but not limited to, bacterial toxins, exosomes,secreted proteins, and metabolites.

[0456] A "photosensitizer" as used herein refers to a sensitizerfor generation of singlet oxygen usually by excitation with light.Exemplary photosensitizers suitable for use in the presentapplication include those described in U.S. Pat. Nos. 6,251,581,5,516,636, 8,907,081, 6,545,012, 6,331,530, 8,247,180, 5,763,602,5,705,622, 5,516,636, 7,217,531, and U.S. Patent Publication No.2007/0059316, all of which are herein expressly incorporated byreference in their entireties. The photosensitizer can bephotoactivatable (e.g., dyes and aromatic compounds) orchemiactivated (e.g., enzymes and metal salts). When excited bylight, the photosensitizer is usually a compound included ofcovalently bonded atoms, usually with multiple conjugated double ortriple bonds. The compound should absorb light in the wavelengthrange of 200-1100 nm, usually 300-1000 nm, e.g., 450-950 nm, withan extinction coefficient at its absorbance maximum greater than500 M.sup.-1cm.sup.-1, e.g., at least 5000 M.sup.-1cm.sup.-1, or atleast 50,000 M.sup.-1cm.sup.-1 at the excitation wavelength. Thelifetime of an excited state produced following absorption of lightin the absence of oxygen will usually be at least 100 nsec, e.g.,at least 1 .mu.sec. In general, the lifetime is desirablysufficiently long to permit energy transfer to oxygen, which willnormally be present at concentrations in the range of 10.sup.-5 to10.sup.-13M depending on the medium. The sensitizer excited statewill usually have a different spin quantum number (S) than itsground state and will usually be a triplet (S=1) when, as isusually the case, the ground state is a singlet (S.dbd.O). In someembodiments, the sensitizer will have a high intersystem crossingyield. That is, photoexcitation of a sensitizer will produce thelong lived state (usually triplet) with an efficiency of at least10%, at least 40%, e.g., greater than 80%. The photosensitizer willusually be at most weakly fluorescent under the assay conditions(quantum yield usually less that 0.5, or less that 0.1).

GI Tract

[0457] As used herein, the term "gastrointestinal tract" or "GItract" refers to all portions of an organ system responsible forconsuming and digesting foodstuffs, absorbing nutrients, andexpelling waste. This includes orifices and organs such as themouth, throat, esophagus, stomach, small intestine, largeintestine, rectum, anus, and the like, as well as the variouspassageways and sphincters connecting the aforementioned parts. Thedevice may be used to detect, analyze and/or quantify an analyte,e.g., bacterial cells, in a sample from the GI tract (e.g., in oneor more of the mouth, throat, esophagus, stomach, small intestine,large intestine, rectum, anus, sphincter, duodenum, jejunum, ileum,ascending colon, transverse colon, and descending colon) of asubject. The device may also be used to detect or quantifybacterial cells from outside the GI tract, including the femalereproductive tract. In some embodiments, the samples from thesubject are environmental samples that do not contain eukaryoticcells.

[0458] The GI tract is a large organ that extends from the buccalcavity to the anus. The primary function of the GI tract is todigest food, absorb nutrients and eliminated any waste. The GItract is composed of the esophagus, the stomach, and theintestines. The different segments of the GI tract are generallyassociated with different characteristics. Chewed food flowsthrough the esophagus, and into the stomach where it is temporarilystored and mixed with gastric acid. Involuntary musclecontractions, termed peristalsis, push the food out of the stomachand into the small intestine. The small intestine can be dividedinto the duodenum, the jejunum and the ileum. The majority of fooddigestion and absorption occurs in the ileum. Waste and unwantedproducts are passed into the colon, or large intestine. Typically,food resides for 10 to 14 seconds in the esophagus, and travelswithin the small intestine for 2 to 4 hours. Half of the contentsof the stomach is emptied within 60 to 90 minutes (Khutoryanskiy(2015) Nature Materials 14: 963-964). While food enters theesophagus at approximately pH 7.0, foods are acidified within thestomach (pH 1-5). The pH in the proximal small intestine is between6.8 and 7.88; between 5.26 and 6.72 in the distal small intestine,between 5.26-6.72 in the ascending colon, and between 5.20 and 7.02in the descending colon (Khutoryanskiy (2015) Nature Materials 14:963-964).

[0459] Over 1000 different microbial species have been identifiedthat can live in the human GI tract, e.g., Actinobacteria,Bifidobacterium spp., Coriobacteriales, Eggerthella, Slackia spp.,Actinomycetales, Bacteroidetes, Firmicutes, Gemella, Clostridia,Lachnospiraceae, Negativicutes, Fusobacteria, and fungi (e.g.,Eukarya). See, e.g., Rajilic-Stojanovic and de Vos (2014) FEMSMicrobiol. Rev. 38(5): 996-1047; and Carroll et al. (2015) Mamm.Genome 20(7): 395-403. Whereas the small intestine contains veryfew bacteria, the colon comprises between 10.sup.13 and 10.sup.14commensal bacteria (Johansson et al. (2013) Nat. Rev.Gastroenterol. Hepatol. 10(6): 352-361).

[0460] The intestinal fluid can contain a variety of digestiveenzymes (e.g., pepsin, lipase, amylase, enterokinase, sucrose,maltase, lactase, secretin, motilin). See, e.g., Ulleberg et al.(2011) Food Dig. 2(1-3): 52-61.

Diseases or Disorders

[0461] The detection and/or analysis of an analyte described hereinmay be used to determine whether the subject has or is at risk ofdeveloping a disease or disorder (e.g., a GI disorder). Thesediseases and disorders are not limited to diseases and disoderspresent in the GI tract of the subject, and can include diseases ordisoders at sites other than the GI tract of the subject. Forexample, in some embodiments, analytes present in the GI tract maybe indicative of a systemic disease or disorder. In someembodiments, the analytes are associated with a systemic disease ordisorder. In some embodiments, analytes present in the GI tract maybe indicative of a disease or disorder described herein, including,but not limited to an infectious disease, IBD, Crohn's disease, andcancer.

[0462] In some embodiments of any of the methods described herein,the subject has a GI disorder. In some embodiments, the analytesdisclosed herein may be indicative of a GI disorder in a subject.Examples of such GI disorders include inflammatory bowel disease(IBD), Crohn's disease (e.g., active Crohn's disease, refractoryCrohn's disease, or fistulizing Crohn's disease), ulcerativecolitis, indeterminate colitis, infectious colitis, microscopiccolitis, drug or chemical-induced colitis, diverticulitis, ischemiccolitis, pseudomembranous colitis, hemorrhagic colitis,hemolytic-uremic syndrome colitis, collagenous colitis, colitisassociated with disorders of innate immunity as in leukocyteadhesion deficiency-1, diversion colitis, gastritis, peptic ulcers,stress ulcers, bleeding ulcers, gastric hyperacidity, dyspepsia,gastroparesis, Zollinger-Ellison syndrome, gastroesophageal refluxdisease, short-bowel (anastomosis) syndrome, mucositis (e.g., oralmucositis, gastrointestinal mucositis, nasal mucositis andproctitis), necrotizing enterocolitis, esophagitis, ahypersecretory state associated with systemic mastocytosis,basophilic leukemia, hyperhistaminemia, Celiac disease (e.g.,nontropical Sprue), enteropathy associated with seronegativearthropathies, eosinophilic gastroenteritis, colitis associatedwith radiotherapy or chemotherapy (such as checkpoint inhibitorchemotherapy), colitis associated with disorders of innate immunitysuch as leukocyte adhesion deficiency-1, gastritis, chronicgranulomatous disease, food allergies, infectious gastritis orenterocolitis (e.g., Helicobacter pylori-infected chronic activegastritis), other forms of gastrointestinal inflammation caused byan infectious agent, irritable colon syndrome, small intestinalbacterial overgrowth (SIBO) and pouchitis.

[0463] "Inflammatory Bowel Disease" or "IBD" is a chronicinflammatory autoimmune condition of the GI tract. Although thecause of IBD remains unknown, several factors such as genetic,infectious and immunologic susceptibility have been implicated. IBDis much more common in Caucasians, especially those of Jewishdescent.

[0464] A chronic inflammatory autoimmune condition of the GI tractpresents clinically as either ulcerative colitis (UC) or Crohn'sdisease (CD). Both IBD conditions are associated with an increasedrisk for malignancy of the GI tract. "Crohn's disease" ("CD") is achronic transmural inflammatory disease with the potential toaffect any part of the entire GI tract, and UC is a mucosalinflammation of the colon. Both conditions are characterizedclinically by frequent bowel motions, malnutrition, anddehydration, with disruption in the activities of daily living. CDis frequently complicated by the development of malabsorption,strictures, and fistulae and may require repeated surgery. UC, lessfrequently, may be complicated by severe bloody diarrhea and toxicmegacolon, also requiring surgery. The most prominent feature ofCrohn's disease is the granular, reddish-purple edematousthickening of the bowel wall. With the development of inflammation,these granulomas often lose their circumscribed borders andintegrate with the surrounding tissue. Diarrhea and obstruction ofthe bowel are the predominant clinical features. As with ulcerativecolitis, the course of Crohn's disease may be continuous orrelapsing, mild or severe, but unlike ulcerative colitis, Crohn'sdisease is not curable by resection of the involved segment ofbowel. Most patients with Crohn's disease require surgery at somepoint, but subsequent relapse is common and continuous medicaltreatment is usual. Crohn's disease may involve any part of thealimentary tract from the mouth to the anus, although typically itappears in the ileocolic, small-intestinal or colonic-anorectalregions. Histopathologically, the disease manifests bydiscontinuous granulomatomas, crypt abscesses, fissures andaphthous ulcers. The inflammatory infiltrate is mixed, consistingof lymphocytes (both T and B cells), plasma cells, macrophages, andneutrophils. There is a disproportionate increase in IgM- andIgG-secreting plasma cells, macrophages and neutrophils.

[0465] "Ulcerative colitis (UC)" afflicts the large intestine. Thecourse of the disease may be continuous or relapsing, mild orsevere. The earliest lesion is an inflammatory infiltration withabscess formation at the base of the crypts of Lieberkuhn.Coalescence of these distended and ruptured crypts tends toseparate the overlying mucosa from its blood supply, leading toulceration. Symptoms of the disease include cramping, lowerabdominal pain, rectal bleeding, and frequent, loose dischargesconsisting mainly of blood, pus and mucus with scanty fecalparticles. A total colectomy may be required for acute, severe orchronic, unremitting ulcerative colitis.

[0466] A "symptom" of a disease or disorder (e.g., an inflammatorybowel disease, e.g., ulcerative colitis or Crohn's disease) is anymorbid phenomenon or departure from the normal in structure,function, or sensation, experienced by a subject and indicative ofdisease.

[0467] In certain embodiments, the subject has small intestinalbacterial overgrowth (SIBO). The small intestine houses less than10.sup.3 bacteria/mL under healthy conditions. When the homeostasisof the gut microbiome is disrupted or aberrant, various functionsof the gut microbiota are uncontrolled. See, e.g., Shreiner et al.(2016) Curr. Opin. Gastroenterol. 31(1): 69-75; Bures et al. (2010)World J. Gastroenterol. 16(24): 2978-2990. Excessive levels ofbacteria (over 10.sup.5 bacteria/mL) and abnormal types of bacteriain the small intestine leads to the development of SIBO. SIBO isassociated with chronic diarrhea, abdominal discomfort, bloating,malabsorption, flatulence, and unintentional weight loss. WhileGram-positive bacteria are typically found in the small intestine,subjects suffering from SIBO have a variety of bacteria in thesmall intestine including Gram-negative bacteria, which arenormally only present in very small numbers or not at all withinthe small intestine. For example, bacteria present in SIBO maysecrete mucosal damaging toxins or metabolize bile salts, which canlead to malabsorption and bloating. A study comparing theprevalence of SIBO in subjects aged 24 to 50 and in subjects aged61 or older found that SIBO was more prevalent in older subjects ascompared to younger subjects (15.6% and 5.9% respectively)(Parlesak et al. (2003) J. Am. Geriatr. Soc. 51(6): 768-773). SIBOwas also seen more frequently in subjects with reduced body weight.Risk factors for developing SIBO include: metabolic disorders(e.g., diabetes, hypochloryhydria), malnutrition, irritable bowelsyndrome (IBS), Celiac disease, Crohn's disease, cirrhosis, renalfailure, gastroparesis, small bowel dysmotility, structuralabnormalities of the GI tract (e.g., jejunal diverticula), gastricresection and immuno-deficiency. Additional risk factors includethe use of certain medications (e.g., antibiotics, gastric acidsecretion inhibitors). See, e.g., Dukowicz et al. (2007)Gastronenterol. Hepatol. 3(2): 112-122. In some embodiments,subjects having SIBO have delayed intestinal transit times (Cuocoet al. (2002) Hepatogastroenterology 49: 1582-1586). In someembodiments, subjects having SIBO have accelerated intestinaltransit times (Van Citters and Lin (2006) Clin. Nutrition inGastrointestinal Disease. Thorofare: Slack Inc; 2006; 271-280).

[0468] As used herein, a subject has or is at risk of having SIBOif the subject has intestinal bacteria levels that are greater than10.sup.3 colony forming units (CFU)/mL, e.g., greater than 10.sup.4CFU/mL, greater than 10.sup.5 CFU/mL, greater than 10.sup.6 CFU/mL,greater than 10.sup.7 CFU/mL, greater than 10.sup.8 CFU/mL, greaterthan 10.sup.9 CFU/mL, greater than 10.sup.10 CFU/mL. In someembodiments, the bacteria are both Gram-positive and Gram-negativebacteria. In some embodiments, the bacteria are Gram-positivebacteria. In some embodiments, the bacteria are Gram-negativebacteria.

[0469] The prevalence of SIBO in healthy individuals varies fromabout 0-20% (see, e.g., Lombardo et at (2010) Clin. Gastroenterol.Hepatol. 8: 504-8; Sabate et al. (2008) Obes. Surg. 18: 371-7;Posserud et al. (2007) Gut 56: 802-8; Teo (2004) J. Gastroenterol.Hepatol. 19: 904-9; Lewis et al. (1999) Age Ageing 28: 181-5;Pimentel et al. (2003) Am. J. Gastroenterol. 98: 412-9; Rana et al.(2011) Diabetes Technol. Ther. 13: 1115-20; Bratten et al. (2008)Am. J. Gastroenterol. 103: 958-63; and Scarpellini et al. (2009) J.Pediatr. 155: 416-20). Several clinical conditions are associatedwith SIBO and are referred to herein as "SIBO-related conditions."Exemplary SIBO-related conditions include, but are not limited to,coeliac disease (see, e.g., Rana et al. (2007) Trop. Gastroenterol.28: 159-61; Rubio-Tapia et al. (2009) J. Clin. Gastroenterol. 43:157-61; and Tursi et al. (2003) Am. J. Gastroenterol. 98: 839-43),connective tissue diseases such as scleroderma (see, e.g., Levesqueet al. (2009) Rheumatology 48: 1314-9; and Parodi et al. (2008) Am.J. Gastroenterol. 103: 1257-62), Crohn's disease (see, e.g.,Fukushima et al. (1999) Dis. Colon Rectum 42: 1072-7; Klaus et al.(2009) Gastroenterol. 9: 61; and U.S. Publication No.2002/0039599), diabetes mellitus (see, e.g., Rana et al. (2011)Diabetes Technol Ther 13: 1115-20, and Zaccardi et al. (2009) Eur.Rev. Med. Pharmacol. Sci. 13: 419-23), hypothyroidism (see, e.g.,Lauritano et al. (2007) J. Clin. Endocr. Metab. 92: 4180-4),nonspecific dysmotility (see, e.g., Jacobs et al. (2013) Aliment.Pharmacol. Ther. 37: 1103-11), radiation enteropathy (see, e.g.,Wedlake et al. (2008) Eur. J Cancer 44: 2212-7), ulcerative colitis(see, e.g., Ibanez et al. (2008) Gastroenterology 134: A-350),chronic fatigue syndrome (see, e.g., Ojetti et al. (2009) Eur. Rev.Med. Pharmacol. Sci. 13: 419-23), chronic pancreatitis (see, e.g.,Mancilla et al. (2008) 136: 976-80; and Trespi et al (1999) Curr.Med. Res. Opin. 15: 47-52), drug-induced inhibition of acidsecretion (see, e.g., Jacobs (2013) Aliment. Pharmacol. Ther. 37:1103-11; Compare et al. (2010) Eur. J Clin. Invest. 41: 380-6; andLombardo et al. (2010) Clin. Gastroenterol. Hepatol. 8: 504-8),end-stage renal failure (see, e.g., Strid et al. (2003) Digestion67: 129-37), fibromyalgia (see, e.g., U.S. Publication No.2002/0039599), irritable bowel syndrome (Posserud et al. (2007) Gut56: 802-8; Bratten et al. (2008) Am. Gastroenterol. 103: 958-63;30. Pimentel et al. (2000) Am. J. Gastroenterol. 95: 3503-6; Nuceraet al. (2005) Aliment. Pharmacol. Ther. 21: 1391-5; Lupascu et al.(2005) Aliment. Pharmacol. Ther. 22: 1157-60; and Grover et al.(2008) Neurogastroenterol. Motil. 20: 998-1008), immunodeficiencysyndromes such as HIV-infection and chronic lymphocytic leukaemia(see, e.g., Chave et al. Am. J. Gastroenterol. 89: 2168-71; andSmith et al. (1990) J. Clin. Pathol. 43: 57-9), liver cirrhosis(see, e.g., Yang et al. (1998) Scand. J. Gastroenterol. 33: 867-71;and Gunnarsdottir (2003) Am. J. Gastroenterol. 98: 1362-70),obesity (see, e.g., Sabate et al. (2008) Obes. Surg. 18: 371-7; andMadrid et al. (2011) Dig. Dis. Sci. 56: 155-60), parenteralnutrition (see, e.g., Gutierrez et al. (2012) J. Pediatr. Surg. 47:1150-4), rosacea (Parodi et al. Clin. Gastroenterol. Hepatol. 6:759-64), muscular dystrophy (see, e.g., Tarnopolsky et al. (2010)Muscle Nerve 42: 853-5), and Parkinson's disease (see, e.g.,Gabrielli (2011) Movement Disord. 26: 889-92). Thus, in someembodiments of any of the methods described herein, the subject hasa SIBO-related condition selected from the group consisting ofcoeliac disease, a connective tissue disease (e.g., scleroderma),Crohn's disease, diabetes mellitus, hypothyroidism, nonspecificdysmotility, radiation enteropathy, ulcerative colitis, chronicfatigue syndrome, chronic pancreatitis, drug-induced inhibition ofacid secretion, end-stage renal failure, fibromyalgia, irritablebowel syndrome, an immunodeficiency syndrome (e.g., HIV-infectionand chronic lymphocytic leukaemia), obesity, parenteral nutrition,rosacea, muscular dystrophy, and Parkinson's disease. For example,the methods described herein may be used to detect SIBO in asubject having a SIBO-related condition.

[0470] In some embodiment of any of the methods described herein,the subject is suspected of having SIBO or a SIBO-relatedcondition. In some embodiments of any of the methods describedherein, the subject has one or more symptoms selected from thegroup consisting of bloating, diarrhea, flatulence, abdominal pain,constipation, weight loss, fever, abdominal tenderness, nausea,gastric stasis, and steatorrhea.

[0471] In some embodiments of any of the methods described herein,the subject has been subjected to a surgical intervention. Forexample, SIBO is prevalent in subjects that have undergoneabdominal surgery, bilateral vagotomy, gastrectomy, ileocaecalvalve resection, and roux-en-Y reconstruction (see, e.g., Grace etal. (2013) Aliment. Pharmacol. Ther. 38(7):674-88, the entirecontents of which are expressly incorporated herein by reference).In some embodiment of any of the methods described herein, thesubject has been subjected to a surgical intervention selected fromthe group consisting of abdominal surgery, bilateral vagotomy,gastrectomy, ileocaecal valve resection, and roux-en-Yreconstruction.

[0472] In some embodiments, detection of analytes disclosed hereinare indicative of disorders of the gastrointestinal tractassociated with anomalous bacterial populations. The bacteria mayinclude, but are not limited to, the types of bacteria present inthe fluid sample or the concentration of bacteria in specificregions of the GI tract. Data obtained using the methods describedherein may be used to determine whether a subject has an infection,such as Small Intestinal Bacterial Overgrowth (SIBO), or tocharacterize bacterial populations within the GI tract fordiagnostic or other purposes. In some embodiments, detection of ananalyte disclosed herein in a subject may be indicative of adisease or condition originating from the endoderm in a subject. Insome embodiments of any of the methods described herein, thesubject has a disease or condition orginating from the endodermselected from the group of: gastritis, Celiac disease, hepatitis,alcoholic lever disease, fatty liver disease (hepatic steatosis),non-alcoholic fatty liver disease (NASH), cirrhosis, primaryschlerosing cholangitis, pancreatitis, insterstitial cystitits,asthma, chronic obstructic pulmonary disease, pulmonary fibrosis,pharyngitis, thyroiditis, hyperthyroidism, parathyroiditis,nephritis, Hashimoto's disease, Addison's disease, Graves' disease,Sjogren syndrome, type 1 diabetes, pelvic inflammatory disease,auditory canal inflammation, tinnitus, vestibular neuritis, otitismedia, auditory canal inflammation, tracheitis, cholestatic liverdisease, primary biliary schlerosis, liver parenchyma, an inheritedmetabolic disorder of the liver, Byler syndrome, cerebrotendinous,xanthomatosis, Zellweger's syndrome, neonatal hepatitis, cysticfibrosis, ALGS (Alagilles syndrome), PFIC (progressive familialintrahepatic cholestasis), autoimmune hepatitis, primary biliarycirrhosis (PBC), liver fibrosis, NAFLD, portal hypertension,general cholestasis, such as in jaundice due to drugs or duringpregnancy, intra- and extrahepatic cholestasis, such as hereditaryforms of cholestasis, such as PFIC1, gall stones andcholedocholithiasis, malignancy causing obstruction of the biliarytree, symptoms (scratching, pruritus) due to cholestasis/jaundice,chronic autoimmune liver disease leading to progressivecholestasis, and pruritus of cholestatic liver disease, duodenalulcers, enteritis (radiation-, chemotherapy-, or infection-inducedenteritis), diverticulitis, pouchitis, cholecystitis, andcholangitis. In some embodiments of any of the methods describedherein, the inflammatory disease or condition that arises in atissue originating from the endoderm is inflammation of theliver.

[0473] In some embodiments, the detection of analytes disclosedherein is indicative of diseases or disorders of the liver. In someembodiments, detection of an analyte disclosed herein in a subjectmay be indicative of a liver disease or disorder in a subject. Forexample, the methods, devices, and compositions described hereinmay be used to determine whether a subject has or is at risk ofdeveloping a liver disease or disorder, and/or to determine ormonitor a course of treatment for a liver disease or disorder. Anon-exhaustive list of liver diseases and disorders, include, butare not limited to fibrosis, cirrhosis, alcoholic lever disease,fatty liver disease (hepatic steatosis), non-alcoholic fatty liverdisease (NASH), cholestatic liver disease, liver parenchyma, aninherited metabolic disorder of the liver, PFIC (progressivefamilial intrahepatic cholestasis), autoimmune hepatitis, primarybiliary cirrhosis (PBC), liver fibrosis, NAFLD, chronic autoimmuneliver disease leading to progressive cholestasis, pruritus ofcholestatic liver disease, inflammation of the liver, and liverfibrosis.

Methods of Selecting and Optimizing Treatment

[0474] In some embodiments, the methods described herein includethe administration of one or more treatments, e.g., antibiotics, toa subject identified as having or being at risk of developing a GIdisorder (e.g., SIBO). The methods can also include selecting atreatment for a subject who has a GI disorder or is determined tobe at risk for developing a GI disorder, based upon the presence orabsence of an analyte, or based upon the amount of an analyte. Themethods can also include administering a treatment selected by amethod described hereinto a subject who has or is at risk ofdeveloping a GI disorder to treat, delay disease progression, orreduce the risk of developing of the disease. For example, in someembodiments, the methods described herein can include theadministration of an antibiotic (e.g., rifaximin) to a subjectidentified as having or being at risk of developing SIBO. In someembodiments, the methods can also include selecting a subjecthaving SIBO or who is at risk of developing SIBO (e.g., a subjecthaving a SIBO-related condition), and treating the subject with anantibiotic (e.g., rifaximin) to treat, delay disease progression,or reduce the risk of developing SIBO.

[0475] In some embodiments of any of the methods described herein,the method can further include the step of monitoring a subject,e.g., for an increase or decrease in one or more analytes, or anyother parameter associated with clinical outcome. In someembodiments, the step of monitoring includes providing the subjectwith an ingestible device to determining the presence or absence ofan analyte and/or the levels or amount of an analyte. In someembodiments, the step of monitoring occurs prior to administering atreatment, during the course of a treatment, or after treatment. Insome embodiments, the step of monitoring includes an additionalstep of ingesting an ingestible device that was previously providedto the subject to determine the presence or absence of an analyteand/or the levels or amounts of an analyte.

[0476] Also provided herein are methods of determining the efficacyof a GI disorder treatment. In some embodiments, providing aningestible device can determine successful treatment of a GIdisorder in a subject (e.g., the presence or absence of an analyteis determined; the levels of an analyte is decreased as compared tothe levels of the analyte determined in the subject at an earlyperiod of time; the levels of an analyte is decreased as comparedto the levels of the analyte determined in a control subject (e.g.,a subject that does not have a GI disorder, or is not at risk ofdeveloping a GI disorder); the levels of an analyte is increased ascompared to the levels of the analyte determined in the subject atan early period of time). In some embodiments, prior to theproviding an ingestible device step, the subject received treatmentfor a GI disorder (e.g., any of the treatment described herein).For example, in some embodiments, the level of an analyte (e.g.,any of the analytes described herein) is decreased as compared tothe level of the analyte described herein prior to treatment for aGI disorder, and further treatment is discontinued. For example, insome embodiments, the level of an analyte (e.g., any of theanalytes described herein) is increased as compared to the level ofthe analyte described herein prior to treatment for a GI disorder,and a different treatment is administered.

[0477] Non-limiting examples of such agents for treating orpreventing a gastrointestinal disorder (e.g., Crohn's disease,ulcerative colitis) include substances that suppress cytokineproduction, downregulate or suppress self-antigen expression, ormask MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.4,665,077); non-steroidal anti-inflammatory drugs (NSAIDs);ganciclovir; tacrolimus; glucocorticoids such as Cortisol oraldosterone; anti-inflammatory agents such as a cyclooxygenaseinhibitor; a 5-lipoxygenase inhibitor; or a leukotriene receptorantagonist; purine antagonists such as azathioprine ormycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde(which masks the MHC antigens, as described in U.S. Pat. No.4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporine; 6-mercaptopurine; steroids such ascorticosteroids or glucocorticosteroids or glucocorticoid analogs,e.g., prednisone, methylprednisolone, including SOLU-MEDROL.RTM.,methylprednisolone sodium succinate, and dexamethasone;dihydrofolate reductase inhibitors such as methotrexate (oral orsubcutaneous); anti-malarial agents such as chloroquine andhydroxychloroquine; sulfasalazine; leflunomide; cytokine orcytokine receptor antibodies or 5 antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumornecrosis factor(TNF)-alpha antibodies (infliximab (REMICADE.RTM.)or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),anti-TNF-beta antibodies, antiinterleukin-2 (IL-2) antibodies andanti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6)receptor antibodies and antagonists; anti-LFA-1 antibodies,including anti-CD 1 la and anti-CD 18 antibodies; anti-L3T4antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26,1990); streptokinase; transforming growth factor-beta (TGF-beta);streptodomase; RNA or DNA from the host; FK506; RS-61443;chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen etal, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner etal. Science, 251: 430-432 (1991); WO90/11294; Janeway, Nature, 341:482 (1989); and WO 91/01133); BAFF antagonists such as BAFF or BR3antibodies or immunoadhesins and zTNF4 antagonists (for review, seeMackay and Mackay, Trends Immunol, 23: 113-5 (2002); biologicagents that interfere with T cell helper signals, such as anti-CD40receptor or anti-CD40 ligand (CD 154), including blockingantibodies to CD40-CD40 ligand. (e.g., Dune et al, Science, 261:1328-30 (1993); Mohan et al, J. Immunol, 154: 1470-80 (1995)) andCTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T-cellreceptor antibodies (EP340,109) such as T10B9. Non-limitingexamples of adjunct agents also include the following: budenoside;epidermal growth factor; aminosalicylates; metronidazole;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonalantibodies; growth factors; elastase inhibitors; pyridinylimidazolecompounds; TNF antagonists; IL-4, IL-10, IL-13 and/or TGF.beta.cytokines or agonists thereof (e.g., agonist antibodies); IL-11;glucuronide- or dextran-conjugated prodrugs of prednisolone,dexamethasone or budesonide; ICAM-I antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.);soluble complement receptor 1 (TPlO; T Cell Sciences, Inc.);slow-release mesalazine; antagonists of platelet activating factor(PAF); ciprofloxacin; and lignocaine. In some embodiments, theagents for treating or preventing a gastrointestinal disorder(e.g., SIBO) include any antibiotic described herein (e.g.,rifaximin). Examples of agents for UC are sulfasalazine and relatedsalicylate-containing drugs for mild cases and corticosteroid drugsin severe cases.

[0478] Topical administration of either salicylates orcorticosteroids is sometimes effective, particularly when thedisease is limited to the distal bowel, and is associated withdecreased side effects compared with systemic use. Supportivemeasures such as administration of iron and antidiarrheal agentsare sometimes indicated. Azathioprine, 6-mercaptopurine andmethotrexate are sometimes also prescribed for use in refractorycorticosteroid-dependent cases.

[0479] In some embodiments, the antibiotic selected for treatmentis selected from the group consisting of: beta-lactam antibiotics,aminoglycosides, ansa-type antibiotics, anthraquinones, antibioticazoles, antibiotic glycopeptides, macrolides, antibioticnucleosides, antibiotic peptides, antibiotic polyenes, antibioticpolyethers, quinolones, antibiotic steroids, sulfonamides,tetracycline, dicarboxylic acids, antibiotic metals, oxidizingagents, substances that release free radicals and/or active oxygen,cationic antimicrobial agents, quaternary ammonium compounds,biguanides, triguanides, bisbiguanides and analogs and polymersthereof and naturally occurring antibiotic compounds.

[0480] Beta-lactam antibiotics include, but are not limited to,2-(3-alanyl)clavam, 2-hydroxymethylclavam, 8-epi-thienamycin,acetyl-thienamycin, amoxicillin, amoxicillin sodium, amoxicillintrihydrate, amoxicillin-potassium clavulanate combination,ampicillin, ampicillin sodium, ampicillin trihydrate,ampicillin-sulbactam, apalcillin, aspoxicillin, azidocillin,azlocillin, aztreonam, bacampicillin, biapenem, carbenicillin,carbenicillin disodium, carfecillin, carindacillin, carpetimycin,cefacetril, cefaclor, cefadroxil, cefalexin, cefaloridine,cefalotin, cefamandole, cefamandole, cefapirin, cefatrizine,cefatrizine propylene glycol, cefazedone, cefazolin, cefbuperazone,cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren,cefditoren pivoxil, cefepime, cefetamet, cefetamet pivoxil,cefixime, cefinenoxime, cefinetazole, cefminox, cefminox,cefmolexin, cefodizime, cefonicid, cefoperazone, ceforanide,cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran,cefpiramide, cefpirome, cefpodoxime, cefpodoxime proxetil,cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin,ceftazidime, cefteram, cefteram pivoxil, ceftezole, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, cefuroxime axetil,cephalosporin, cephamycin, chitinovorin, ciclacillin, clavulanicacid, clometocillin, cloxacillin, cycloserine, deoxypluracidomycin, dicloxacillin, dihydro pluracidomycin, epicillin,epithienamycin, ertapenem, faropenem, flomoxef, flucloxacillin,hetacillin, imipenem, lenampicillin, loracarbef, mecillinam,meropenem, metampicillin, meticillin, mezlocillin, moxalactam,nafcillin, northienamycin, oxacillin, panipenem, penamecillin,penicillin, phenethicillin, piperacillin, tazobactam,pivampicillin, pivcefalexin, pivmecillinam, pivmecillinamhydrochloride, pluracidomycin, propicillin, sarmoxicillin,sulbactam, sulbenicillin, talampicillin, temocillin, terconazole,thienamycin, ticarcillin and analogs, salts and derivativesthereof.

[0481] Aminoglycosides include, but are not limited to,1,2'-N-DL-isoseryl-3',4'dideoxykanamycin B,1,2'-N-DL-isoseryl-kanamycin B,1,2'-N--[(S)-4-amino-2-hydroxybutyryl]-3',4'-dideoxykanamycinB,1,2'-N-[(S)-4-amino-2-hydroxybutyryl]kanamycin B,1-N-(2-Aminobutanesulfonyl) kanamycin A,1-N-(2-aminoethanesulfonyl)3',4'-dideoxyribostamycin,l-N-(2-aminoethanesulfonyl)3'deoxyribostamycin,1-N-(2-aminoethanesulfonyl)3'4'-dideoxykanamycin B,1-N-(2-aminoethanesulfonyl) kanamycin A,1-N-(2aminoethanesulfonyl)kanamycin B,1-N-(2-aminoethanesulfonyl)ribostamycin,1-N-(2-aminopropanesulfonyl)3'-deoxykanamycin B,l-N-(2-aminopropanesulfonyl)3'4'-dideoxy kanamycin B,1-N-(2-aminopropanesulfonyl) kanamycin A,1-N-(2-aminopropanesulfonyl) kanamycin B,l-N-(L-4-amino-2-hydroxy-butyryl)2,'3'-dideoxy-2'-fluorokanamycinA,1-N-(L-4-amino-2-hydroxy-propionyl)2,'3'-dideoxy-2'-fluorokanamycinA, 1-N-DL-3',4'-dideoxy-isoserylkanamycinB,1-N-DL-isoserylkanamycin, 1-N-DL-isoserylkanamycin B,1-N-[L-(-)-(alpha-hydroxygamma-aminobutyryl)]-XK-62-2,2',3'-dideoxy-2'-fl-uorokanamycin A,2-hydroxygentamycin A 3,2-hydroxygentamycin B,2-hydroxygentamycin Bl, 2-hydroxygentamycin JI-20A,2-hydroxygentamycin JI-20B, 3''-N-methyl-4''-C-methyl-3',4'-dodeoxykanamycin A, 3''-N-methyl-4''-C-methyl-3',4'-dodeoxy kanamycin B,3''-N-methyl-4''-C-methyl-3',4'-dodeoxy-6'methyl kanamycin B,3',4'-Dideoxy-3'-eno-ribostamycin,3',4'-dideoxyneamine,3',4'dideoxyribost-amycin, 3'-deoxy-6'-N-methyl-kanamycinB,3'-deoxyneamine,3'deoxyribostamycin,3'-oxysaccharocin,3,3'-nepotrehalosadiamine,3-demethoxy-2''-Nformimidoylistamycin B disulfate tetrahydrate,3-demethoxyistamycin B,3-0-demethyl-2-N-formimidoylistamycin B,3-0-demethylistamycin B,3-trehalosamine,411,6 11-dideoxydibekacin,4-N-glycyl-KA-6606VI, 5''-Amino-3',4',5''-trideoxy-butirosin A,611-deoxydibekacin,61-epifortimicin A, 6-deoxy-neomycin (structure6-deoxy-neomycin B),6-deoxy-neomycin B, 6-deoxy-neomycin C,6-deoxy-paromomycin, acmimycin, AHB-3',4'-dideoxyribostamycin,AHB-3'-deoxykanamycin B, AHB-3'-deoxyneamine,AHB-3'-deoxyribostamycin, AHB-411-611-dideoxydibekacin,AHB-611-deoxydibekacin, AHB-dideoxyneamine, AHB-kanamycin B,AHB-methyl-3'-deoxykanamycin B, amikacin, amikacin sulfate,apramycin, arbekacin, astromicin, astromicin sulfate, bekanamycin,bluensomycin, boholmycin, butirosin, butirosin B, catenulin,coumamidine gammal, coumamidinegamma2,D,L-1-N-(alpha-hydroxy-betaaminopropionyl)-XK-62-2,dactimicin, de-O-methyl-4-N-glycyl-KA-6606VI, de-O-methyl-KA-66061,de-O-methyl-KA-70381, destomycin A, destomycin B,di-N6',03-demethylistamycin A, dibekacin, dibekacin sulfate,dihydrostreptomycin, dihydrostreptomycin sulfate,epi-formamidoylglycidylfortimicin B, epihygromycin,formimidoyl-istamycin A, formimidoyl-istamycin B, fortimicin B,fortimicin C, fortimicin D, fortimicin KE, fortimicin KF,fortimicin KG, fortimicin KG1 (stereoisomer KG1/KG2), fortimicinKG2 (stereoisomer KG1/KG2), fortimicin KG3, framycetin, framycetinsulphate, gentamicin, gentamycin sulfate, globeomycin, hybrimycinA1, hybrimycin A2, hybrimycin B1, hybrimycin B2, hybrimycin C1,hybrimycin C2, hydroxystreptomycin, hygromycin, hygromycin B,isepamicin, isepamicin sulfate, istamycin, kanamycin, kanamycinsulphate, kasugamycin, lividomycin, marcomycin, micronomicin,micronomicin sulfate, mutamicin, myomycin, N-demethy1-7-0-demethylcelesticetin, demethylcelesticetin, methanesulfonicacid derivative of istamycin, nebramycin, nebramycin, neomycin,netilmicin, oligostatin, paromomycin, quintomycin, ribostamycin,saccharocin, seldomycin, sisomicin, sorbistin, spectinomycin,streptomycin, tobramycin, trehalosmaine, trestatin, validamycin,verdamycin, xylostasin, zygomycin and analogs, salts andderivatives thereof.

[0482] Antibiotic anthraquinones include, but are not limited to,auramycin, cinerubin, ditrisarubicin, ditrisarubicin C, figaroicacid fragilomycin, minomycin, rabelomycin, rudolfomycin,sulfurmycin and analogs, salts and derivatives thereof.

[0483] Antibiotic azoles include, but are not limited to,azanidazole, bifonazole, butoconazol, chlormidazole, chlormidazolehydrochloride, cloconazole, cloconazole monohydrochloride,clotrimazol, dimetridazole, econazole, econazole nitrate,enilconazole, fenticonazole, fenticonazole nitrate, fezatione,fluconazole, flutrimazole, isoconazole, isoconazole nitrate,itraconazole, ketoconazole, lanoconazole, metronidazole,metronidazole benzoate, miconazole, miconazole nitrate,neticonazole, nimorazole, niridazole, omoconazol, omidazole,oxiconazole, oxiconazole nitrate, propenidazole, secnidazol,sertaconazole, sertaconazole nitrate, sulconazole, sulconazolenitrate, tinidazole, tioconazole, voriconazol and analogs, saltsand derivatives thereof.

[0484] Antibiotic glycopeptides include, but are not limited to,acanthomycin, actaplanin, avoparcin, balhimycin, bleomycin B(copper bleomycin), chloroorienticin, chloropolysporin,demethylvancomycin, enduracidin, galacardin, guanidylfungin,hachimycin, demethylvancomycin, N-nonanoyl-teicoplanin, phleomycin,platomycin, ristocetin, staphylocidin, talisomycin, teicoplanin,vancomycin, victomycin, xylocandin, zorbamycin and analogs, saltsand derivatives thereof.

[0485] Macrolides include, but are not limited to,acetylleucomycin, acetylkitasamycin, angolamycin, azithromycin,bafilomycin, brefeldin, carbomycin, chalcomycin, cirramycin,clarithromycin, concanamycin, deisovaleryl-niddamycin,demycinosyl-mycinamycin, Di-O-methyltiacumicidin, dirithromycin,erythromycin, erythromycin estolate, erythromycin ethyl succinate,erythromycin lactobionate, erythromycin stearate, flurithromycin,focusin, foromacidin, haterumalide, haterumalide, josamycin,josamycin ropionate, juvenimycin, juvenimycin, kitasamycin,ketotiacumicin, lankavacidin, lankavamycin, leucomycin, machecin,maridomycin, megalomicin, methylleucomycin, methymycin,midecamycin, miocamycin, mycaminosyltylactone, mycinomycin,neutramycin, niddamycin, nonactin, oleandomycin,phenylacetyideltamycin, pamamycin, picromycin, rokitamycin,rosaramicin, roxithromycin, sedecamycin, shincomycin, spiramycin,swalpamycin, tacrolimus, telithromycin, tiacumicin, tilmicosin,treponemycin, troleandomycin, tylosin, venturicidin and analogs,salts and derivatives thereof.

[0486] Antibiotic nucleosides include, but are not limited to,amicetin, angustmycin, azathymidine, blasticidin S, epiroprim,flucytosine, gougerotin, mildiomycin, nikkomycin, nucleocidin,oxanosine, oxanosine, puromycin, pyrazomycin, showdomycin,sinefungin, sparsogenin, spicamycin, tunicamycin, uracil polyoxin,vengicide and analogs, salts and derivatives thereof.

[0487] Antibiotic peptides include, but are not limited to,actinomycin, aculeacin, alazopeptin, arnfomycin, amythiamycin,antifungal from Zalerion arboricola, antrimycin, apid, apidaecin,aspartocin, auromomycin, bacileucin, bacillomycin, bacillopeptin,bacitracin, bagacidin, beminamycin, beta-alanyl-L-tyrosine,bottromycin, capreomycin, caspofungine, cepacidine, cerexin,cilofungin, circulin, colistin, cyclodepsipeptide, cytophagin,dactinomycin, daptomycin, decapeptide, desoxymulundocandin,echanomycin, echinocandin B, echinomycin, ecomycin, enniatin,etamycin, fabatin, ferrimycin, ferrimycin, ficellomycin,fluoronocathiacin, fusaricidin, gardimycin, gatavalin, globopeptin,glyphomycin, gramicidin, herbicolin, iomycin, iturin, iyomycin,izupeptin, janiemycin, janthinocin, jolipeptin, katanosin,killertoxin, lipopeptide antibiotic, lipopeptide from Zalerion sp.,lysobactin, lysozyme, macromomycin, magainin, melittin, mersacidin,mikamycin, mureidomycin, mycoplanecin, mycosubtilin, neopeptifluorin, neoviri dogrisein, netropsin, nisin, nocathiacin,nocathiacin 6-deoxyglycoside, nosiheptide, octapeptin, pacidamycin,pentadecapeptide, peptifluorin, permetin, phytoactin,phytostreptin, planothiocin, plusbacin, polcillin, polymyxinantibiotic complex, polymyxin B, polymyxin Bl, polymyxin F,preneocarzinostatin, quinomycin, quinupristin-dalfopristin,safracin, salmycin, salmycin, salmycin, sandramycin, saramycetin,siomycin, sperabillin, sporamycin, a Streptomyces compound,subtilin, teicoplanin aglycone, telomycin, thermothiocin,thiopeptin, thiostrepton, tridecaptin, tsushimycin,tuberactinomycin, tuberactinomycin, tyrothricin, valinomycin,viomycin, virginiamycin, zervacin and analogs, salts andderivatives thereof.

[0488] In some embodiments, the antibiotic peptide is anaturally-occurring peptide that possesses an antibacterial and/oran antifungal activity. Such peptide can be obtained from an herbalor a vertebrate source.

[0489] Polyenes include, but are not limited to, amphotericin,amphotericin, aureofungin, ayfactin, azalomycin, blasticidin,candicidin, candicidin methyl ester, candimycin, candimycin methylester, chinopricin, filipin, flavofungin, fradicin, hamycin,hydropricin, levorin, lucensomycin, lucknomycin, mediocidin,mediocidin methyl ester, mepartricin, methylamphotericin,natamycin, niphimycin, nystatin, nystatin methyl ester, oxypricin,partricin, pentamycin, perimycin, pimaricin, primycin, proticin,rimocidin, sistomycosin, sorangicin, trichomycin and analogs, saltsand derivatives thereof.

[0490] Polyethers include, but are not limited to,20-deoxy-epi-narasin, 20-deoxysalinomycin, carriomycin, dianemycin,dihydrolonomycin, etheromycin, ionomycin, iso-lasalocid, lasalocid,lenoremycin, lonomycin, lysocellin, monensin, narasin,oxolonomycin, a polycyclic ether antibiotic, salinomycin andanalogs, salts and derivatives thereof.

[0491] Quinolones include, but are not limited to, analkyl-methylendioxy-4(1H)-2 5 oxocinnoline-3-carboxylic acid,alatrofloxacin, cinoxacin, ciprofloxacin, ciprofloxacinhydrochloride, danofloxacin, dermofongin A, enoxacin, enrofloxacin,fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin,levofloxacin, lomefloxacin, lomefloxacin, hydrochloride, miloxacin,moxifloxacin, nadifloxacin, nalidixic acid, nifuroquine,norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pazufloxacine,pefloxacin, pefloxacin mesylate, pipemidic acid, piromidic acid,premafloxacin, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin and analogs, salts and derivativesthereof.

[0492] Antibiotic steroids include, but are not limited to,aminosterol, ascosteroside, cladosporide A, dihydrofusidic acid,dehydro-dihydrofusidic acid, dehydrofusidic acid, fusidic acid,squalamine and analogs, salts and derivatives thereof.

[0493] Sulfonamides include, but are not limited to, chloramine,dapsone, mafenide, phthalylsulfathiazole, succinylsulfathiazole,sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfadiazine,sulfadiazine silver, sulfadicramide, sulfadimethoxine, sulfadoxine,sulfaguanidine, sulfalene, sulfamazone, sulfamerazine,sulfamethazine, sulfamethizole, sulfamethoxazole,sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxol,sulfanilamide, sulfaperine, sulfaphenazol, sulfapyridine,sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea,sulfatolamide, sulfatriazin, sulfisomidine, sulfisoxazole,sulfisoxazole acetyl, sulfacarbamide and analogs, salts andderivatives thereof.

[0494] Tetracyclines include, but are not limited to,dihydrosteffimycin, demethyltetracycline, aclacinomycin,akrobomycin, baumycin, bromotetracycline, cetocyclin,chlortetracycline, clomocycline, daunorubicin, demeclocycline,doxorubicin, doxorubicin hydrochloride, doxycycline, lymecyclin,marcellomycin, meclocycline, meclocycline sulfosalicylate,methacycline, minocycline, minocycline hydrochloride, musettamycin,oxytetracycline, rhodirubin, rolitetracycline, rubomycin,serirubicin, steffimycin, tetracycline and analogs, salts andderivatives thereof.

Analytes

[0495] The compositions and methods described herein can be used todetect, analyze, and/or quantitate a variety of analytes in a humansubject. "Analyte" as used in the present application refers to acompound or composition to be detected in a sample. Exemplaryanalytes suitable for use in the present application include thosedescribed in U.S. Pat. No. 6,251,581, which is incorporated byreference herein in its entirety. Broadly speaking, an analyte canbe any substance (e.g., a substance with one or more antigens)capable of being detected. An exemplary and non-limiting list ofanalytes includes ligands, proteins and fragments thereof, bloodclotting factors, hormones, cytokines, polysaccharides, nucleicacids, carbohydrates, mucopolysaccharides, lipids, fatty acids,microorganisms (e.g., bacteria), microbial antigens, andtherapeutic agents (including fragments and metabolitesthereof).

[0496] For instance, the analyte may be a substance that binds toan analyte-binding agent (e.g., a biomolecule) and forms a complex.In some embodiments, the analyte may bemonovalent (monoepitopic) orpolyvalent (polyepitopic), usually antigenic or haptenic. In someembodiments, the analyte is a single compound or plurality ofcompounds. In some embodiments, the analyte is a plurality ofcompounds which share at least one common epitopic or determinantsite. The analyte can be a part of a cell such as bacteria or acell bearing a blood group antigen such as A, B, D, etc., a humanleukocyte antigen (HLA), or other cell surface antigen. The analytecan also be a microorganism (e.g., bacterium (e.g. a pathogenicbacterium), a fungus, protozoan, or a virus), a protein, a nucleicacid, a lipid, or a hormone. In some embodiments, the analyte canbe an exosome or a part of an exosome (e.g., a bacterial exosome).In some embodiments, the analyte is derived from a subject (e.g., ahuman subject). In some embodiments, the analyte is derived from amicroorganism present in the subject. In some embodiments, theanalyte is a nucleic acid (e.g., a DNA molecule or a RNA molecule),a protein (e.g., a soluble protein, a cell surface protein), or afragment thereof, that can be detected using any of the devices andmethods provided herein.

[0497] The polyvalent ligand analytes will normally be poly(aminoacids), i.e., a polypeptide (i.e., protein) or a peptide,polysaccharides, nucleic acids (e.g., DNA or RNA), and combinationsthereof. Such combinations include components of bacteria, viruses,chromosomes, genes, mitochondria, nuclei, cell membranes, and thelike.

[0498] In some embodiments, the polyepitopic ligand analytes have amolecular weight of at least about 5,000 Da, more usually at leastabout 10,000 Da. In the poly(amino acid) category, the poly(aminoacids) of interest may generally have a molecular weight from about5,000 Da to about 5,000,000 Da, more usually from about 20,000 Dato 1,000,000 Da; among the hormones of interest, the molecularweights will usually range from about 5,000 Da to 60,000 Da.

[0499] In some embodiments, the monoepitopic ligand analytesgenerally have a molecular weight of from about 100 to 2,000 Da,more usually from 125 to 1,000 Da.

[0500] A wide variety of proteins may be considered as to thefamily of proteins having similar structural features, proteinshaving particular biological functions, proteins related tospecific microorganisms, particularly disease causingmicroorganisms, etc. Such proteins include, for example,immunoglobulins, cytokines, enzymes, hormones, cancer antigens,nutritional markers, tissue specific antigens, etc.

[0501] In some embodiments, the analyte is a protein. In someembodiments, the analyte is a protein, e.g., an enzyme (e.g., ahemolysin, a protease, a phospholipase), a soluble protein, amembrane-bound protein, or an exotoxin. In some embodiments, theanalyte is a fragment of a protein, a peptide, or an antigen. Insome embodiments, the analyte is a peptide of at least 5 aminoacids (e.g., at least 6, at least 7, at least 8, at least 9, atleast 10, at least 25, at least, 50, or at least 100 amino acids).Exemplary lengths include 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, or100 amino acids. Exemplary classes of protein analytes include, butare not limited to: protamines, histones, albumins, globulins,scleroproteins, phosphoproteins, antibodies, affimers,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, cell surfacereceptors, membrane-anchored proteins, transmembrane proteins,secreted proteins, HLA, and unclassified proteins. In someembodiments, the analyte is an affimer (see, e.g., Tiede et al.(2017) eLife 6: e24903, which is expressly incorporated herein byreference).

[0502] Exemplary analytes include: Prealbumin, Albumin,.alpha..sub.1-Lipoprotein, .alpha..sub.1-Antitrypsin,.alpha..sub.1-Glycoprotein, Transcortin, 4.6S-Postalbumin,.alpha..sub.1-glycoprotein, .alpha..sub.1X-Glycoprotein,Thyroxin-binding globulin, Inter-.alpha.-trypsin-inhibitor,Gc-globulin (Gc 1-1, Gc 2-1, Gc 2-2), Haptoglobin (Hp 1-1, Hp 2-1,Hp 2-2), Ceruloplasmin, Cholinesterase,.alpha..sub.2-Lipoprotein(s), Myoglobin, C-Reactive Protein,.alpha..sub.2-Macroglobulin, .alpha..sub.2-HS-glycoprotein,Zn-.alpha..sub.2-glycoprotein,.alpha..sub.2-Neuramino-glycoprotein, Erythropoietin,.beta.-lipoprotein, Transferrin, Hemopexin, Fibrinogen,Plasminogen, .beta..sub.2-glycoprotein I, .beta..sub.2-glycoproteinII, Immunoglobulin G (IgG) or .gamma.G-globulin, Immunoglobulin A(IgA) or .gamma.A-globulin, Immunoglobulin M (IgM) or.gamma.M-globulin, Immunoglobulin D (IgD) or .gamma.D-Globulin(.gamma.D), Immunoglobulin E (IgE) or .gamma.E-Globulin (.gamma.E),Free .kappa. and .lamda. light chains, and Complement factors: C'1,(C'1q, C'1r, C'1s, C'2, C'3 (.beta..sub.1A, .alpha..sub.2D), C'4,C'5, C'6, C'7, C'8, C'9.

[0503] Additional examples of analytes include tumor necrosisfactor-.alpha. (TNF.alpha.), interleukin-12 (IL-12), IL-23, IL-6,.alpha.2.beta.1 integrin, .alpha.1.beta.1 integrin, .alpha.4.beta.7integrin, integrin .alpha.4.beta.1 (VLA-4), E-selectin, ICAM-1,.alpha.5.beta.1 integrin, .alpha.4.beta.1 integrin, VLA-4,.alpha.2.beta.1 integrin, .alpha.5.beta.3 integrin, .alpha.5.beta.5integrin, .alpha.IIb.beta.3 integrin, MAdCAM-1, SMAD7, JAK1, JAK2,JAK3, TYK-2, CHST15, IL-1, IL-1.alpha., IL-1.beta., IL-18,IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-13,CD40L, CD40, CD3.gamma., CD3.epsilon., CD3.epsilon., CD3.zeta.,TCR, TCR.alpha., TCR.beta., TCR.delta., TCR.gamma., CD14, CD20,CD25, IL-2, IL-2 .beta. chain, IL-2 .gamma. chain, CD28, CD80,CD86, CD49, MMP1, CD89, IgA, CXCL10, CCL11, an ELR chemokine, CCR2,CCR9, CXCR3, CCR3, CCR5, CCL2, CCL8, CCL16, CCL25, CXCR1m CXCR2mCXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and CXCL8, and anucleic acid (e.g., mRNA) encoding any of the same.

[0504] In some embodiments, the analyte is a blood clotting factor.Exemplary blood dotting factors include, but are not limitedto:

TABLE-US-00001 International designation Name I Fibrinogen IIProthrombin IIa Thrombin III Tissue thromboplastin V and VIProaccelerin, accelerator globulin VII Proconvertin VIIIAntihemophilic globulin (AHG) IX Christmas factor plasmathromboplastin component (PTC) X Stuart-Prower factor,autoprothrombin III XI Plasma thromboplastin antecedent (PTA) XIIHagemann factor XIII Fibrin-stabilizing factor

[0505] In some embodiments, the analyte is a hormone. Exemplaryhormones include, but are not limited to: Peptide and ProteinHormones, Parathyroid hormone, (parathromone), Thyrocalcitonin,Insulin, Glucagon, Relaxin, Erythropoietin, Melanotropin(melancyte-stimulating hormone; intermedin), Somatotropin (growthhormone), Corticotropin (adrenocorticotropic hormone), Thyrotropin,Follicle-stimulating hormone, Luteinizing hormone (interstitialcell-stimulating hormone), Luteomammotropic hormone (luteotropin,prolactin), Gonadotropin (chorionic gonadotropin), Secretin,Gastrin, Angiotensin I and II, Bradykinin, and Human placentallactogen, thyroxine, cortisol, triiodothyronine, testosterone,estradiol, estrone, progestrone, luteinizing hormone-releasinghormone (LHRH), and immunosuppressants such as cyclosporin, FK506,mycophenolic acid, and so forth.

[0506] In some embodiments, the analyte is a peptide hormone (e.g.,a peptide hormone from the neurohypophysis). Exemplary peptidehormones from the neurohypophysis include, but are not limited to:Oxytocin, Vasopressin, and releasing factors (RF) (e.g.,corticotropin releasing factor (CRF), luteinizing hormone releasingfactor (LRF), thyrotropin releasing factor (TRF), Somatotropin-RF,growth hormone releasing factor (GRF), follicle stimulatinghormone-releasing factor (FSH-RF), prolactin inhibiting factor(PIF), and melanocyte stimulating hormone inhibiting factor(MIF)).

[0507] In some embodiments, the analyte is a cytokine or achemokine. Exemplary cytokines include, but are not limited to:interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),epidermal growth factor (EGF), tumor necrosis factor (TNF, e.g.,TNF-.alpha. or TNF-.beta.), and nerve growth factor (NGF).

[0508] In some embodiments, the analyte is a cancer antigen.Exemplary cancer antigens include, but are not limited to:prostate-specific antigen (PSA), carcinoembryonic antigen (CEA),.alpha.-fetoprotein, Acid phosphatase, CA19.9, CA125, CD19, WT-1,CD22, L1-CAM, ROR-1, CD30, CD125, AFP, CEA, ETA, MAGE, andMUC16.

[0509] In some embodiments, the analyte is a tissue-specificantigen. Exemplary tissue specific antigens include, but are notlimited to: alkaline phosphatase, myoglobin, CPK-MB, calcitonin,and myelin basic protein.

[0510] In some embodiments, the analyte is a mucopolysaccharide ora polysaccharide.

[0511] In some embodiments, the analyte is a microorganism, or amolecule derived from or produced by a microorganism (e.g., abacteria, a virus, prion, or a protozoan). For example, in someembodiments, the analyte is a molecule (e.g., a protein or anucleic acid) that is specific for a particular microbial genus,species, or strain (e.g., a specific bacterial genus, species, orstrain). In some embodiments, the microorganism is pathogenic(i.e., causes disease). In some embodiments, the microorganism isnon-pathogenic (e.g., a commensal microorganism). Exemplarymicroorganisms include, but are not limited to:

TABLE-US-00002 Corynebacteria Corynebacterium diphtheriaPneumococci Diplococcus pneumoniae Streptococci Streptococcuspyrogenes Streptococcus salivarus Staphylococci Staphylococcusaureus Staphylococcus albus Neisseria Neisseria meningitidisNeisseria gonorrhea Enterobacteriaciae Escherichia coli Aerobacteraerogenes The coliform Klebsiella pneumoniae bacteria Salmonellatyphosa Salmonella choleraesuis The Salmonellae Salmonellatyphimurium Shigella dysenteria Shigella schmitzii Shigellaarabinotarda The Shigellae Shigella flexneri Shigella boydiiShigella sonnei Other enteric bacilli Proteus vulgaris Proteusmirabilis Peoteus species Proteus morgani Pseudomonas aeruginosaAlcaligenes faecalis Vibrio cholerae Hemophilus-Bordetella groupRhizopus oryzae Hemophilus influenza, H. ducryi Rhizopus arrhizuaPhycomycetes Hemophilus hemophilus Rhizopus nigricans Hemophilusaegypticus Sporotrichum schenkii Hemophilus parainfluenzaFlonsecaea pedrosoi Bordetella pertussis Fonsecacea compactPasteurellae Fonsecacea dermatidis Pasteurella pestis Cladosporiumcarrionii Pusteurella tulareusis Phialophora verrucosa BrucellaeAspergillus nidulans Brucella meltensis Madurella mycetomi Brucellaabortus Madurella grisea Brucella suis Allescheria boydii AerobicSpore-forming Bacilli Phialophora jeanselmei Bacillus anthracisMicrosporum gypseum Bacillus subtilis Trichophyton mentagrophytesBacillus megaterium Keratinomyces ajelloi Bacillus cereusMicrosporum canis Anaerobic Spore-forming Bacilli Trichophytonrubrum Clostridium botulinum Microsporum adouini Clostridium tetaniViruses Clostridium perfringens Adenoviruses Clostridium novyiHerpes Viruses Clostridium septicum Herpes simplex Clostridiumhistoyticum Varicella (Chicken pox) Clostridium tertium HerpesZoster (Shingles) Clostridium bifermentans Virus B Clostridiumsporogenes Cytomegalovirus Mycobacteria Pox Viruses Mycobacteriumtuberculosis hominis Variola (smallpox) Mycobacterium bovisVaccinia Mycobacterium avium Poxvirus bovis Mycobacterium lepraeParavaccinia Mycobacterium paratuberculosis Molluscum contagiosumActinomycetes (fungus-ike bacteria) Picornaviruses ActinomycesIsaeli Poliovirus Actinomyces bovis Coxsackievirus Actinomycesnaesiundii Echoviruses Nocardia asteroides Rhinoviruses Nocardiabrasiliensis Myxoviruses The Spirochetes Influenza(A, R, and C)Treponema pallidum Parainfluenza (1-4) Treponema pertenue MumpsVirus Spirillum minus Streptobacillus monoiliformis NewcastleDisease Virus Treponema carateum Measles Virus Borrelia recurrentisRinderpest Virus Leptospira icterohemorrhagiae Canine DistemperVirus Leptospira canicola Respiratory Syncytial Virus TrypanasomesRubella Virus Mycoplasmas Arboviruses Myroplasma pneumoniae Otherpathogens Eastern Equine Encephalitis Virus Listeria monocytogenesWestern Equine Encephalitis Virus Erysipeothrix rhusiopathiaeSindbis Virus Streptobacillus moniliformis Chikugunya VirusDonvania granulomatis Semliki Forest Virus Entamoeba histolyticaMayora Virus Plasmodium falciparum St. Louis EncephalitisPlasmodium japonicum California Encephalitis Virus Bartonellabacilliformis Colorado Tick Fever Virus Rickettsia (bacteria-likeparasites) Yellow Fever Virus Rickettsia prowazekii Dengue VirusRickettsia mooseri Reoviruses Rickettsia rickettsii Reovirus Types1-3 Rickettsia conori Retroviruses Rickettsia australis HumanImmunodeficiency Rickettsia sibiricus Viruses I and II (HTLV)Rickettsia akari Human T-cell Lymphotrophic Rickettsiatsutsugamushi Virus I & H (HIV) Rickettsia burnetti HepatitisRickettsia quintana Hepatitis A Virus Chlamydia (unclassifiableparasites Hepatitis B Virus bacterial/viral) Hepatitis C VirusChlamydia agents (naming uncertain) Tumor Viruses Chlamydiatrachomatis Fungi Rauscher Leukemia Virus Cryptococcus neoformansGross Virus Blastomyces dermatidis Maloney Leukemia VirusHistoplasma capsulatum Coccidioides immitis Human Papilloma VirusParacoccidioides brasliensis Candida albicans Aspergillus fumigatusMucor corymbifer (Absidia corymbifera)

[0512] In some embodiments, the analyte is a bacterium. Exemplarybacteria include, but are not limited to: Escherichia coli (or E.coli), Bacillus anthraces, Bacillus cereus, Clostridium botulinum,Clostridium difficile, Yersinia pestis, Yersinia enterocolitica,Francisella tularensis, Brucella species, Clostridium perfringens,Burkholderia mallei, Burkholderia pseudomallei, Staphylococcusspecies, Mycobacterium species, Group A Streptococcus, Group BStreptococcus, Streptococcus pneumoniae, Helicobacter pylori,Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale,Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasmapneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia,Rickettsia akari, Rickettsia prowazekii, Rickettsia canada,Bacillus subtilis, Bacillus subtilis niger, Bacillus thuringiensis,Coxiella burnetti, Faecalibacterium prausnitzii (also known asBacteroides praussnitzii), Roseburia hominis, Eubacterium rectale,Dialister invisus, Ruminococcus albus, Ruminococcus callidus, andRuminococcus bromii. Additional exemplary bacteria include bacteriaof the phyla Firmicutes (e.g., Clostridium clusters XIVa and IV),bacteria of the phyla Bacteroidetes (e.g., Bacteroides fragilis orBacteroides vulgatus), and bacteria of the phyla Actinobacteria(e.g., Coriobacteriaceae spp. or Bifidobacterium adolescentis).Bacteria of the Clostridium cluster XIVa includes species belongingto, for example, the Clostridium, Ruminococcus, Lachnospira,Roseburia, Eubacterium, Coprococcus, Dorea, and Butyrivibriogenera. Bacteria of the Clostridium cluster IV includes speciesbelonging to, for example, the Clostridium, Ruminococcus,Eubacterium and Anaerofilum genera. In some embodiments, theanalyte is Candida, e.g., Candida albicans. In some embodiments,the analyte is a byproduct from a bacterium or other microorganism,e.g., helminth ova, enterotoxin (Clostridium difficile toxin A;TcdA) or cytotoxin (Clostridium difficile toxin B; TcdB).

[0513] In some embodiments, the bacterium is a pathogenicbacterium. Non-limiting examples of pathogenic bacteria belong tothe genera Bacillus, Bordetella, Borrelia, Brucella, Campylobacter,Chlamydia, Chlamydophila, Clostridium, Corynebacterium,Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus,Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium,Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella,Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, andYersinia. Non-limiting examples of specific pathogenic bacterialspecies include a strain of Bacillus anthracis, a strain of astrain of Bordetella pertussis, a strain of a strain of Borreliaburgdorferi, a strain of a strain of Brucella abortus, a strain ofa strain of Brucella canis, a strain of a strain of Brucellamelitensis, a strain of a strain of Brucella suis, a strain of astrain of Campylobacter jejuni, a strain of Chlamydia pneumoniae, astrain of Chlamydia trachomatis, a strain of Chlamyclophilapsittaci, a strain of Clostridium botulinum, a strain ofClostridium difficile, a strain of Clostridium perfringens, astrain of Clostridium tetani, a strain of Corynebacteriumdiphtheria, a strain of Enterobacter sakazakii, a strain ofEnterococcus faecalis, a strain of Enterococcus faecium, a strainof Escherichia con (e.g., E. coli O157 H7), a strain of Francisellatularensis, a strain of Haemophilus influenza, a strain ofHelicobacter pylori, a strain of Legionella pneumophila, a strainof Leptospira interrogans, a strain of Listeria monocytogenes, astrain of Mycobacterium leprae, a strain of Mycobacteriumtuberculosis, a strain of Mycobacterium ulcerans, a strain ofMycoplasma pneumonia, a strain of Neisseria gonorrhoeae, a strainof Neisseria meningitides, a strain of Pseudomonas aeruginosa, astrain of Rickettsia rickettsia, a strain of Salmonella typhi andSalmonella typhimurium, a strain of Shigella sonnei, a strain ofStaphylococcus aureus, strain of Staphylococcus epidermidis, astrain of Staphylococcus saprophyticus , a strain of Streptococcusagalactiae, a strain of Streptococcus pneumonia, a strain ofStreptococcus pyogenes, a strain of Treponema palliating, a strainof Vibrio cholera, a strain of Yersinia enterocolitica, and, astrain of Yersinia pestis.

[0514] In some embodiments, the bacterium is a commensal bacterium(e.g., a probiotic). In some embodiments, the bacterium has beenpreviously administered to a subject, e.g., as a livebiotherapeutic agent. Exemplary commensal bacteria include, but arenot limited to, Faecalibacterium prausnitzii (also referred to asBacteroides praussnitzii), Roseburia hominis, Eubacterium rectale,Dialister invisus, Ruminococcus albus, Ruminococcus gnavus,Ruminococcus torques, Ruminococcus callidus, and Ruminococcusbromii.

[0515] In some embodiments, the analyte is a virus. In someembodiments, the virus is a pathogenic virus. Non-limiting examplesof pathogenic viruses belong to the families Adenoviridae,Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae,Polyomavirus, Rhabdoviridae, and Togaviridae.

[0516] In some embodiments, the analyte is a fungus. In someembodiments, the fungi is a pathogenic fungus. Non-limitingexamples of pathogenic fungi belong to the genera Asperfillus,Canidia, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys.Non-limiting examples of specific pathogenic fungi species includea strain of Aspergillus clavatus, Aspergillus fumigatus;Aspergillus flavus, Canidia albicans, Cryptococcus albidus,Cryptococcus gattii, Cryptococcus laurentii, Cryptococcusneoformans, Histoplasma capsulation, Pneumocystis jirovecii,Pneumocystis carinii, and Stachybotrys chartarum.

[0517] In some embodiments, the analyte is a protozoan. In someembodiments, the analyte is a pathogenic protozoan. Non-limitingexamples of pathogenic protozoa belong to the genera Acanthamoeba,Balamuthia, Cryptosporidium, Dientamoeba, Endolimax, Entamoeba,Giardia, Iodamoeba, Leishmania, Naegleria, Plasmodium, Sappinia,Toxoplasma, Trichomonas, and Trypanosoma. Non-limiting examples ofspecific pathogenic protozoa species include a strain ofAcanthamoeba spp. Balamuthia mandrillaris, Cryptosporidium canis,Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidiummeleagridis, Cryptosporidium muris, Cryptosporidium parvum,Dientamoeba fragilis, Endolimax nana, Entamoeba dispar, Entamoebahartmanni, Entamoeba histolytica, Entamoeba coli, Entamoebamoshkovskii, Giardia lamblia, Iodamoeba butschlii, Leishmaniaaethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmaniadonovani, Leishmania infantum, Leishmania major, Leishmaniamexicana, Leishmania tropica, Naegleriafowleri, Plasmodiumfalciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodiumovale, Plasmodium vivax, Sappinia diploidea, Toxoplasma gondii,Trichomonas vaginalis, Trypanosoma brucei, and Trypanosomacruzi.

[0518] In some embodiments, the analyte is secreted by or expressedon the cell surface of a microorganism (e.g., a bacterium, acolonic bacterium, a viable bacterium, a dead bacterium, a parasite(e.g., Giardia lamblia, Cryptosporidium, Cystoisosporiasis belli,and Balantidium coli), a virus (e.g., a herpes virus, acytomegalovirus, a herpes simplex virus, an Epstein-Barr virus, ahuman papilloma virus, a rotavirus, a human herpesvirus-8; Goodgame(1999) Curr. Gastroenterol. Rep. 1(4): 292-300). In someembodiments, the analyte is secreted by or expressed on the cellsurface of a Gram-negative bacterium (e.g., E. coli, Helicobacterpylori). In some embodiments, the analyte is secreted by orexpressed on the cell surface (e.g., a bacterial surface epitope)of a Gram-positive bacterium (e.g., Staphylococcus aureus,Clostridium botulinum, Clostridium difficile).

[0519] In some embodiments, the analyte is a molecule expressed onthe surface of a bacterial cell (e.g., a bacterial cell surfaceprotein). In some embodiments, the analyte is a bacterial toxin(e.g., TcdA and/or TcdB from Clostridium difficile). In someembodiments, the analyte is CFA/I fimbriae, flagella,lipopolysaccharide (LPS), lipoteichoic acid, or a peptidoglycan.Non-limiting examples of bacterium that may express an analyte thatcan be detected using any of the devices and methods describedherein include: Bacillus anthraces, Bacillus cereus, Clostridiumbotulinum, Clostridium difficile, Escherichia coli, Yersiniapestis, Yersinia enterocolitica, Francisella tularensis, Brucellaspecies, Clostridium perfringens, Burkholderia mallei, Burkholderiapseudomallei, Helicobacter pylori, Staphylococcus species,Mycobacterium species, Group A Streptococcus, Group BStreptococcus, Streptococcus pneumoniae, Francisella tularensis,Salmonella enteritidis, Mycoplasma hominis, Mycoplasma orale,Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasmapneumoniae, Mycobacterium bovis, Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium leprae, Rickettsia rickettsia,Rickettsia akari, Rickettsia prowazekii, Rickettsia canada,Bacillus subtilis, Bacillus subtilis niger, Bacillus thuringiensis,Coxiella bumetti, Candida albicans, Bacteroides fragilis,Leptospira interrogans, Listeria monocytogenes, Pasteurellamultocida, Salmonella typhi, Salmonella typhimurium, Shigelladysenteriae, Shigella flexneria, Shigella sonnei, Vibrio cholera,and Vibrio parahaemolyticus.

[0520] In some embodiments, the analyte is a byproduct from abacterium or another microorganism, e.g., helminth ova, enterotoxin(Clostridium difficile toxin A; TcdA), cytotoxin (Clostridiumdifficile toxin B; TcdB), and ammonia. In some embodiments, theanalyte is an antigen from a microorganism (e.g., a bacteria,virus, prion, fungus, protozoan or a parasite).

[0521] In some embodiments, the analytes include drugs,metabolites, pesticides, pollutants, and the like. Included amongdrugs of interest are the alkaloids. Among the alkaloids aremorphine alkaloids, which includes morphine, codeine, heroin,dextromethorphan, their derivatives and metabolites; cocainealkaloids, which include cocaine and benzyl ecgonine, theirderivatives and metabolites; ergot alkaloids, which include thediethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinolinealkaloids, which include quinine and quinidine; diterpenealkaloids, their derivatives and metabolites.

[0522] In some embodiments, the analyte is a steroid selected fromthe estrogens, androgens, andreocortical steroids, bile acids,cardiotonic glycosides and aglycones, which includes digoxin anddigoxigenin, saponins and sapogenins, their derivatives andmetabolites. Also included are the steroid mimetic substances, suchas diethylstilbestrol.

[0523] In some embodiments, the analyte is a bile acid or a bilesalt (also known as a conjugated bile acid). Bile acids areproducts of cholesterol synthesis that are synthesized in theliver, conjugated to taurine or glycine, and stored in thegallbladder until released into the small intestine. The primarybile acids are cholic acid, and chenodeoxycholic acid, which aredeconjugated and dehydroxylated by instestinal bacteria to form thesecondary bile acids deoxycholic acid and lithocholic acid,respectively. The majority of bile acids (about 95%) are reabsorbedin the distal ileum and returned to the liver (see, e.g., U.S.Publication No. 2017/0343535, incorporated herein by reference).Impaired absorption of bile acids in the ileum can lead to excessbile acids in the colon which can cause symptoms of bile acidmalabsorption (BAM; also known as bile acid diarrhea), includingwatery stool and fecal incontinence. Interestingly, up to 50% ofpatients with irritable bowel syndrome with diarrhea (IBS-D) alsohave BAM (see, e.g., Camilleri et al. (2009) Neurogastroeterol.Motil. 21(7): 734-43). In some embodiments, the presence, absence,and/or a specific level of one or more bile acids or bile salts inthe GI tract of a subject is indicative of a condition or diseasestate (e.g., a GI disorder and/or a non-GI disorder (e.g., asystemic disorder or a liver disease)). In some embodiments, thecompositions, devices, and methods described herein may be used todetect, analyze and/or quantify at least one bile acid or bile saltin the GI tract of the subject to diagnose a GI disorder such asBAM or IBS (e.g., IBS-D). In some embodiments, the devices, methodsand compositions described herein can be used to detect,quantitate, and/or analyze a bile acid or a bile salt in the GItract of a subject. For instance, the presence and/or absence,and/or the concentration of a bile acid, a bile salt, or acombination thereof, may be determined at a specific region of theGI tract of a subject (e.g., one or more of the duodenum, jejunum,ileum, ascending colon, transverse colon or descending colon) todetermine whether the subject has or is at risk of developing a GIdisorder, such as BAM or IBS-D. In some embodiments, the devices,methods and compositions described herein can be used to determinethe ratio of two or more bile acids or bile acid salts in the GItract of a subject (e.g., a specific region of the GI tract of asubject including one or more of the duodenum, jejunum, ileum,ascending colon, transverse colon or descending colon). In someembodiments, the presence and/or absence, and/or the concentrationof a bile acid, a bile salt, or a combination thereof, isdetermined in the ileum of a subject. In some embodiments, thepresence and/or absence, and/or the concentration of a bile acid, abile salt, or a combination thereof, is determined in the colon ofa subject. In some embodiments, the concentration of a bile acid, abile salt, or a combination thereof, is determined in specificregions of the GI tract of the subject, and for example, comparedto determine where along the GI tract the compounds areaccumulating. In some embodiments, the detection of a concentrationof a bile acid, bile salt, or a combination thereof, in a specificregion of the GI tract of the subject (e.g., the colon or theileum) that is above a reference level of a bile acid, bile salt,or a combination thereof (e.g., the average level of a bile acid inhealthy subjects) may be indicative of BAM and/or IBS-D in asubject. In some embodiments, the bile acid is selected from thegroup consisting of chenodeoxycholic acid, cholic acid,deoxycholate, lithocholate, and ursodeoxycholic acid. In someembodiments, the bile acid comprises cholesten-3-one or astructural variant thereof. In some embodiments, the bile acid ischolesten-3-one or a structural variant thereof. In someembodiments, the bile acid is cholesten-3-one. In some embodiments,the bile acid is a structural variant of cholesten-3-one. In someembodiments, the bile salt is selected from the group consisting ofglycocholic acid, taurocholic acid, glycodeoxycholic acid,glycochenodeoxycholic acid, taurodeoxycholic acid,taurochenodeoxycholic acid, glycolithocholic acid, andtaurolithocholic acid.

[0524] In some embodiments, the analyte is7.alpha.-hydroxy-4-cholesten-3-one (7.alpha.C4). The measurement of7.alpha.C4 allows for the monitoring of the enzymatic activity ofhepatic cholesterol 7.alpha.-hydroxylase, the rate limiting enzymein the synthesis of bile acids and can be used as a surrogate todetect BAM (see, e.g., Galman et al. (2003) J. Lipid. Res. 44:859-66; and Camilleri et al. (2009) Neurogastroeterol. Motil.21(7): 734-43, incorporated herein by reference in theirentirety).

[0525] In some embodiments, the analyte comprises cholesterol, alipid, a fat soluble vitamin (e.g., ascorbic acid, cholecalciferol,ergocalciferol, a tocopherol, a tocotrienol, phylloquinone, and amenaquinone), bilirubin, fibroblast growth factor 19 (FGF19), TGR5(also known as GP-BAR1 or M-BAR), glycine, taurine, orcholecystokinin (CCK or CCK-PZ). In some embodiments, the analytecomprises cholecystokinin. Cholecystokinin is a peptide hormonethat contributes to control intestinal motility (see Rehfeld (2017)Front. Endocrinol. (Lausanne) 8: 47). In some embodiments, theanalyte comprises secretin. Secretin is a peptide hormone thatregulates the pH of the duodenal content by controlling gastricacid secretion, regulates bile acid and bicarbonate secretion inthe duodenum, and regulates water homeostasis (see, e.g., Afroze etal. (2013) Ann. Transl. Med. 1(3): 29). In some embodiments, asubject has been administered cholecystokinin or secretin to inducethe release of an analyte (e.g., from the liver and/or gall bladderinto the GI tract).

[0526] In some embodiments, the analyte is a metabolite in theserotonin, tryptophan and/or kynurenine pathways, including but notlimited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA),5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA),3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA),quinolinic acid, anthranilic acid, and combinations thereof 5-HT isa molecule that plays a role in the regulation of gastrointestinalmotility, secretion, and sensation. Imbalances in the levels of5-HT are associated with several diseases including inflammatorybowel syndrome (IBS), autism, gastric ulcer formation, non-cardiacchest pain, and functional dyspepsia (see, e.g., Faure et al.(2010) Gastroenterology 139(1): 249-58 and Muller et al. (2016)Neuroscience 321: 24-41, and International Publication No. WO2014/188377, each of which are incorporated herein by reference).Conversion of metabolites within the serotonin, tryptophan and/orkynurenine pathways affects the levels of 5-HT in a subject.Therefore, measuring the levels of one or more of the metabolitesin this pathway may be used for the diagnosis, management andtreatment of a disease or disorder associated with 5-HT imbalanceincluding but not limited to IBS, autism, carcinoid syndrome,depression, hypertension, Alzheimer's disease, constipation,migraine, and serotonin syndrome. One or more analytes in theserotonin, tryptophan and/or kynurenine pathways can be detectedand/or quantitated using, for example, methods and analyte-bindingagents that bind to these metabolites including, e.g., antibodies,known in the art (see, e.g., International Publication No.WO2014/188377, the entire contents of which are expresslyincorporated herein by reference).

[0527] In some embodiments, the analyte is a lactam having from 5to 6 annular members selected from barbituates, e.g., phenobarbitaland secobarbital, diphenylhydantonin, primidone, ethosuximide, andmetabolites thereof.

[0528] In some embodiments, the analyte is an aminoalkylbenzene,with alkyl of from 2 to 3 carbon atoms, selected from theamphetamines; catecholamines, which includes ephedrine, L-dopa,epinephrine; narceine; papaverine; and metabolites thereof.

[0529] In some embodiments, the analyte is a benzheterocyclicselected from oxazepam, chlorpromazine, tegretol, their derivativesand metabolites, the heterocyclic rings being azepines, diazepinesand phenothiazines.

[0530] In some embodiments, the analyte is a purine selected fromtheophylline, caffeine, their metabolites and derivatives.

[0531] In some embodiments, the analyte is marijuana, cannabinol ortetrahydrocannabinol.

[0532] In some embodiments, the analyte is a vitamin such asvitamin A, vitamin B, e.g. vitamin B.sub.12, vitamin C, vitamin D,vitamin E and vitamin K, folic acid, thiamine.

[0533] In some embodiments, the analyte is selected fromprostaglandins, which differ by the degree and sites ofhydroxylation and unsaturation.

[0534] In some embodiments, the analyte is a tricyclicantidepressant selected from imipramine, dismethylimipramine,amitriptyline, nortriptyline, protriptyline, trimipramine,chlomipramine, doxepine, and desmethyldoxepin.

[0535] In some embodiments, the analyte is selected fromanti-neoplastics, including methotrexate.

[0536] In some embodiments, the analyte is an antibiotic asdescribed herein, including, but not limited to, penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, andmetabolites and derivatives.

[0537] In some embodiments, the analyte is a nucleoside ornucleotide selected from ATP, NAD, FMN, adenosine, guanosine,thymidine, and cytidine with their appropriate sugar and phosphatesubstituents.

[0538] In some embodiments, the analyte is selected from methadone,meprobamate, serotonin, meperidine, lidocaine, procainamide,acetylprocainamide, propranolol, griseofulvin, valproic acid,butyrophenones, antihistamines, chloramphenicol, anticholinergicdrugs, such as atropine, their metabolites and derivatives.

[0539] In some embodiments, the analyte is a metabolite related toa diseased state. Such metabolites include, but are not limited tospermine, galactose, phenylpyruvic acid, and porphyrin Type 1.

[0540] In some embodiments, the analyte is an aminoglycoside, suchas gentamicin, kanamicin, tobramycin, or amikacin.

[0541] In some embodiments, the analyte is a pesticide. Amongpesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sulfenamides,their metabolites and derivatives.

[0542] In some embodiments, the analyte has a molecular weight ofabout 500 Da to about 1,000,000 Da (e.g., about 500 to about500,000 Da, about 1,000 to about 100,000 Da).

[0543] In some embodiments, the analyte is a receptor, with amolecular weight ranging from 10,000 to 2.times.10.sup.8 Da, moreusually from 10,000 to 10.sup.6 Da. For immunoglobulins, IgA, IgG,IgE and IgM, the molecular weights will generally vary from about160,000 Da to about 10.sup.6 Da. Enzymes will normally range inmolecular weight from about 10,000 Da to about 1,000,000 Da.Natural receptors vary widely, generally having a molecular weightof at least about 25,000 Da and may be 10.sup.6 or higher Da,including such materials as avidin, DNA, RNA, thyroxine bindingglobulin, thyroxine binding prealbumin, transcortin, etc.

[0544] In some embodiments, the term "analyte" further includespolynucleotide analytes such as those polynucleotides definedbelow. These include m-RNA, r-RNA, t-RNA, DNA, DNA-DNA duplexes,DNA-RNA duplexes, nucleic acid molecules comprising modified bases,locked nucleic acid molecules (LNA molecules), antagomirs, peptidenucleic acid molecules (PNA molecules), antisense RNA or DNAmolecules (e.g., antisense molecules including modifications to thesugars, bases, backbone linkages that allow for specificdetection), chimeric antisense oligonucleotides, antisenseoligonucleotides comprising modified linkages, interference RNA(RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA(shRNA); small RNA-induced gene activation (RNAa); small activatingRNAs (saRNAs), etc. The term analyte also includespolynucleotide-binding agents, such as, for example, restrictionenzymes, trascription factors, transcription activators,transcription repressors, nucleases, polymerases, histones, DNArepair enzymes, intercalating gagents, chemotherapeutic agents, andthe like.

[0545] In some embodiments, the analyte may be a molecule founddirectly in a sample such as a body fluid from a host. The samplecan be examined directly or may be pretreated to render the analytemore readily detectible. Furthermore, the analyte of interest maybe determined by detecting an agent probative of the analyte ofinterest (i.e., an analyte-binding agent), such as a specificbinding pair member complementary to the analyte of interest, whosepresence will be detected only when the analyte of interest ispresent in a sample. Thus, the agent probative of the analytebecomes the analyte that is detected in an assay.

[0546] In some embodiments, the analyte a nucleic acid (e.g., abacterial DNA molecule or a bacterial RNA molecule (e.g., abacterial tRNA, a transfer-messenger RNA (tmRNA)). See, e.g.,Sjostrom et al. (2015) Scientific Reports 5: 15329; Ghosal (2017)Microbial Pathogenesis 104: 161-163; Shen et al. (2012) Cell HostMicrobe. 12(4): 509-520.

[0547] In some embodiments, the analyte is a component of an outermembrane vesicle (OMV) (e.g., an OmpU protein, Elluri et al. (2014)PloS One 9: e106731). See, e.g., Kulp and Kuehn (2010) AnnualReview of microbiology 64: 163-184; Berleman and Auer (2013)Environmental microbiology 15: 347-354; Wai et al. (1995)Microbiology and immunology 39: 451-456; Lindmark et al. (2009) BMCmicrobiology 9: 220; Sjostrom et al. (2015) Scientific Reports 5:15329.

[0548] In some embodiments, the analyte is G-CSF, which canstimulate the bone marrow to produce granulocytes and stem cellsand release them into the bloodstream.

[0549] In some embodiments, the analyte is an enzyme such asglutathione S-transferase. For example, the ingestible device caninclude P28GST, a 28 kDa helminth protein from Schistosoma withpotent immunogenic and antioxidant properties. P28GST preventsintestinal inflammation in experimental colitis through a Th2-typeresponse with mucosal eosinophils and can be recombinantly produced(e.g., in S cerevisiae). See, for example, U.S. Pat. No. 9,593,313,Driss et al., Mucosal Immunology, 2016 9, 322-335; and Capron etal., Gastroenterology, 146(5):S-638.

[0550] In some embodiments, the analyte is a metabolite in theserotonin, tryptophan and/or kynurenine pathways, including but notlimited to, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA),5-hydroxytryptophan (5-HTP), kynurenine (K), kynurenic acid (KA),3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA),quinolinic acid, anthranilic acid, and combinations thereof.

[0551] In some embodiments, analytes are therapeutic agents,fragments thereof, and metabolites thereof (e.g., antibiotics). Insome embodiments, analytes are biomarkers. In some embodiments, theanalytes are antibodies. In some embodiments, the analytes areantibiotics. Additional exemplary analytes (e.g., therapeuticagents (e.g., drugs), antibodies, antibiotics and biomarkers) areprovided below.

[0552] A. Antibodies

[0553] In some embodiments, the analyte or the analyte-bindingagent is an antibody. An "antibody" is an immunoglobulin moleculecapable of specific binding to a target, such as a carbohydrate,polynucleotide, lipid, polypeptide, etc., through at least oneantigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term encompasses notonly intact polyclonal or monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) anddomain antibodies), and fusion proteins including an antibodyportion, and any other modified configuration of the immunoglobulinmolecule that includes an antigen recognition site. The termantibody includes antibody fragments (e.g., antigen-bindingfragments) such as an Fv fragment, a Fab fragment, a F(ab')2fragment, and a Fab' fragment. Additional examples ofantigen-binding fragments include an antigen-binding fragment of anIgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, orIgG4) (e.g., an antigen-binding fragment of a human or humanizedIgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); anantigen-binding fragment of an IgA (e.g., an antigen-bindingfragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of ahuman or humanized IgA, e.g., a human or humanized IgA1 or IgA2);an antigen-binding fragment of an IgD (e.g., an antigen-bindingfragment of a human or humanized IgD); an antigen-binding fragmentof an IgE (e.g., an antigen-binding fragment of a human orhumanized IgE); or an antigen-binding fragment of an IgM (e.g., anantigen-binding fragment of a human or humanized IgM). An antibodyincludes an antibody of any class, such as IgG, IgA, or IgM (orsub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of theconstant domain of its heavy chains, immunoglobulins can beassigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domainsthat correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. Thesubunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known.

[0554] As used herein, "monoclonal antibody" refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies including the population areidentical except for possible naturally-occurring mutations thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site.Furthermore, in contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen. The modifier"monoclonal" indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies,and is not to be construed as requiring production of the antibodyby any particular method. For example, the monoclonal antibodies tobe used in accordance with the present invention may be made by thehybridoma method first described by Kohler and Milstein, 1975,Nature 256:495, or may be made by recombinant DNA methods such asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies mayalso be isolated from phage libraries generated using thetechniques described in McCafferty et al., 1990, Nature348:552-554, for example.

[0555] A "variable region" of an antibody refers to the variableregion of the antibody light chain or the variable region of theantibody heavy chain, either alone or in combination. As known inthe art, the variable regions of the heavy and light chain eachconsist of four framework regions (FR) connected by threecomplementarity determining regions (CDRs) that containhypervariable regions. The CDRs in each chain are held together inclose proximity by the FRs and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site ofantibodies. There are at least two techniques for determining CDRs:(1) an approach based on cross-species sequence variability (i.e.,Kabat et al. Sequences of Proteins of Immunological Interest, (5thed., 1991, National Institutes of Health, Bethesda Md.)); and (2)an approach based on crystallographic studies of antigen-antibodycomplexes (Al-Lazikani et al, 1997, J. Molec. Biol. 273:927-948).As used herein, a CDR may refer to CDRs defined by either approachor by a combination of both approaches.

[0556] As known in the art, a "constant region" of an antibodyrefers to the constant region of the antibody light chain or theconstant region of the antibody heavy chain, either alone or incombination.

[0557] A "derivative" refers to any polypeptide (e.g., an antibody)having a substantially identical amino acid sequence to thenaturally occurring polypeptide, in which one or more amino acidshave been modified at side groups of the amino acids (e.g., anbiotinylated protein or antibody). The term "derivative" shall alsoinclude any polypeptide (e.g., an antibody) which has one or moreamino acids deleted from, added to, or substituted from the naturalpolypeptide sequence, but which retains a substantial amino acidsequence homology to the natural sequence. A substantial sequencehomology is any homology greater than 50 percent.

[0558] In some embodiments, the antibody can be a humanizedantibody, a chimeric antibody, a multivalent antibody, or afragment thereof. In some embodiments, an antibody can be a scFv-Fc(Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050,2017), a VHH domain (Li et al., Immunol. Lett. 188:89-95, 2017), aVNAR domain (Hasler et al., Mol. Immunol. 75:28-37, 2016), a(scFv).sub.2, a minibody (Kim et al., PLoS One 10(1):e113442,2014), or a BiTE. In some embodiments, an antibody can be a DVD-Ig(Wu et al., Nat. Biotechnol. 25(11):1290-1297, 2007; WO 08/024188;WO 07/024715), and a dual-affinity re-targeting antibody (DART)(Tsai et al., Mol. Ther. Oncolytics 3:15024, 2016), a triomab(Chelius et al., MAbs 2(3):309-319, 2010), kih IgG with a common LC(Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), acrossmab (Regula et al., EMBO Mol. Med. 9(7):985, 2017), anortho-Fab IgG (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), a 2-in-1-IgG (Kontermann et al., DrugDiscovery Today 20(7):838-847, 2015), IgG-scFv (Cheat et al., Mol.Cancer Ther. 13(7):1803-1812, 2014), scFv2-Fc (Natsume et al., J.Biochem. 140(3):359-368, 2006), a bi-nanobody (Kontermann et al.,Drug Discovery Today 20(7):838-847, 2015), tanden antibody(Kontermann et al., Drug Discovery Today 20(7):838-847, 2015), aDART-Fc (Kontermann et al., Drug Discovery Today 20(7):838-847,2015), a scFv-HSA-scFv (Kontermann et al., Drug Discovery Today20(7):838-847, 2015), DNL-Fab3 (Kontermann et al., Drug DiscoveryToday 20(7):838-847, 2015), DAF (two-in-one or four-in-one),DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly,charge pair antibody, Fab-arm exchange antibody, SEEDbody, Triomab,LUZ-Y, Fcab, ka-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv,scFv-(H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG (L,H)-Fc, IgG(H)-V,V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv,scFv4-Ig, Zybody, DVI-IgG, nanobody (e.g., antibodies derived fromCamelus bactriamus, Calelus dromaderius, or Lama paccos) (U.S. Pat.No. 5,759,808; Stijlemans et al., J. Biol. Chem. 279:1256-1261,2004; Dumoulin et al., Nature 424:783-788, 2003; and Pleschbergeret al., Bioconjugate Chem. 14:440-448, 2003), nanobody-HSA, adiabody (e.g., Poljak, Structure 2(12):1121-1123, 1994; Hudson etal., J. Immunol. Methods 23(1-2):177-189, 1999), a TandAb (Reuschet al., mAbs 6(3):727-738, 2014), scDiabody (Cuesta et al., Trendsin Biotechnol. 28(7):355-362, 2010), scDiabody-CH3 (Sanz et al.,Trends in Immunol. 25(2):85-91, 2004), Diabody-CH3 (Guo et al.),Triple Body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH,Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFV2, scFv-KIH, Fab-scFv-Fc,tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc,intrabody (Huston et al., Human Antibodies 10(3-4):127-142, 2001;Wheeler et al., Mol. Ther. 8(3):355-366, 2003; Stocks, Drug Discov.Today 9(22):960-966, 2004), dock and lock bispecific antibody,ImmTAC, HSAbody, scDiabody-HSA, tandem scFv, IgG-IgG, Cov-X-Body,and scFvl-PEG-scFv2.

[0559] In some embodiments, an antibody can be an IgNAR, abispecific antibody (Milstein and Cuello, Nature 305:537-539, 1983;Suresh et al., Methods in Enzymology 121:210, 1986; WO 96/27011;Brennan et al., Science 229:81, 1985; Shalaby et al., J. Exp. Med.175:217-225, 1992; Kolstelny et al., J. Immunol. 148(5):1547-1553,1992; Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448,1993; Gruber et al., J. Immunol. 152:5368, 1994; Tuft et al., J.Immunol. 147:60, 1991), a bispecific diabody, a triabody(Schoonooghe et al., BMC Biotechnol. 9:70, 2009), a tetrabody,scFv-Fc knobs-into-holes, a scFv-Fc-scFv, a (Fab'scFv).sub.2, aV-IgG, a IvG-V, a dual V domain IgG, a heavy chain immunoglobulinor a camelid (Holt et al., Trends Biotechnol. 21(11):484-490,2003), an intrabody, a monoclonal antibody (e.g., a human orhumanized monoclonal antibody), a heteroconjugate antibody (e.g.,U.S. Pat. No. 4,676,980), a linear antibody (Zapata et al., ProteinEng. 8(10:1057-1062, 1995), a trispecific antibody (Tutt et al., J.Immunol. 147:60, 1991), a Fabs-in-Tandem immunoglobulin (WO15/103072), or a humanized camelid antibody.

[0560] In some embodiments, the antibody binds specifically to ametabolite in the serotonin, tryptophan and/or kynurenine pathways,including but not limited to, serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), kynurenine (K),kynurenic acid (KA), 3-hydroxykynurenine (3-HK),3-hydroxyanthranilic acid (3-HAA), quinolinic acid, anthranilicacid. Exemplary antibodies that bind to metabolites in thesepathways are disclosed, for example, in International PublicationNo. WO2014/188377, the entire contents of which are incorporatedherein by reference.

[0561] In some embodiments, the antibody is specific for aparticular genus, species, or strain of a microorganism, and maytherefore be used for the detection, analysis and/or quantitationof the microorganism using the detection methods described below.In some embodiments, the antibody specifically binds to asurface-specific biomolecule (e.g., a pilus subunit or a flagellaprotein) present in a particular genus, species or strain ofmicroorganism, and does not cross-react with other microorganisms.In some embodiments, these antibodies may be used in the methodsdescribed herein to diagnose a subject with a particular infectionor disease, or to monitor an infection (e.g., during or aftertreatment). In some embodiments, the antibody specifically binds toan antigen present in a particular genera, species or strain of amicroorganism. Exemplary antigens, the corresponding microorganismthat can be detected, and the disease caused by the microorganism(in parentheticals) include: outer membrane protein A OmpA(Acinetobacter baumannii, Acinetobacter infections)); HIV p24antigen, HIV Eenvelope proteins (Gp120, Gp41, Gp160) (HIV (Humanimmunodeficiency virus), AIDS (Acquired immunodeficiencysyndrome)); galactose-inhibitable adherence protein GIAP, 29 kDaantigen Eh29, GaVGaINAc lectin, protein CRT, 125 kDa immunodominantantigen, protein M17, adhesin ADH112, protein STIRP (Entamoebahistolytica, Amoebiasis); protective Antigen PA, edema factor EF,lethal facotor LF, the S-layer homology proteins SLH (Bacillusanthraces, Anthrax); nucleocapsid protein NP, glycoproteinprecursor GPC, glycoprotein GP1, glycoprotein GP2 (Junin virus,Argentine hemorrhagic fever); 41 kDa allergen Asp v13, allergen Aspf3, major conidial surface protein rodlet A, protease Peplp,GPI-anchored protein Gellp, GPI-anchored protein Crflp (Aspergillusgenus, Aspergillosis); outer surface protein A OspA, outer surfaceprotein OspB, outer surface protein OspC, decorin binding protein ADbpA, flagellar filament 41 kDa core protein Fla, basic membraneprotein A precursor BmpA (Immunodominant antigen P39), outersurface 22 kDa lipoprotein precursor (antigen IPLA7), variablesurface lipoprotein vIsE (Borrelia genus, Borrelia infection);OmpA-like transmembrane domain-containing protein Omp31,immunogenic 39-kDa protein M5 P39, 25 kDa outer-membraneimmunogenic protein precursor Omp25, outer membrane protein MotYOmp16, conserved outer membrane protein D15, malate dehydrogenaseMdh, component of the Type-IV secretion system (T4SS) VirJ,lipoprotein of unknown function BAB1-0187 (Brucella genus,Brucellosis); major outer membrane protein PorA, flagellin FIaA,surface antigen CjaA, fibronectin binding protein CadF,aspartate/glutamate-binding ABC transporter protein Peb1A, proteinFspA1, protein FspA2 (Campylobacter genus, Campylobacteriosis);glycolytic enzyme enolase, secreted aspartyl proteinases SAP1-10,glycophosphatidylinositol (GPI)-linked cell wall protein, adhesinAls3p, cell surface hydrophobicity protein CSH (usually Candidaalbicans and other Candida species, Candidiasis); envelopeglycoproteins (gB, gC, gE, gH, gI, gK, gL) (Varicella zoster virus(VZV), Chickenpox); major outer membrane protein MOMP, probableouter membrane protein PMPC, outer membrane complex protein B OmcB(Chlamydia trachomatis, Chlamydia); major outer membrane proteinMOMP, outer membrane protein 2 Omp2, (Chlamydophila pneumoniae,Chlamydophila pneumoniae infection); outer membrane protein U PorinompU, (Vibrio cholerae, Cholera); surface layer proteins SLPs, CellWall Protein CwpV, flagellar protein FliC, flagellar protein FliD(Clostridium difficile, Clostridium difficile infection); acidicribosomal protein P2 CpP2, mucin antigens Muc1, Muc2, Muc3 Muc4,Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherenceprotein CP23, surface protein CP12, surface protein CP21, surfaceprotein CP40, surface protein CP60, surface protein CP15,surface-associated glycopeptides gp40, surface-associatedglycopeptides gp15, oocyst wall protein AB, profilin PRF, apyrase(Cryptosporidium genus, Cryptosporidiosis); membrane protein pp15,capsid-proximal tegument protein pp150 (Cytomegalovirus,Cytomegalovirus infection); prion protein (vCJD prion, VariantCreutzfeldt-Jakob disease (vCJD, nvCJD)); cyst wall proteins CWP1,CWP2, CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4,VSP5, VSP6, 56 kDa antigen (Giardia intestinalis, Giardiasis);minor pilin-associated subunit pilC, major pilin subunit andvariants pilE, pilS (Neisseria gonorrhoeae, Gonorrhea); outermembrane protein A OmpA, outer membrane protein C OmpC, outermembrane protein K17 OmpK17 (Klebsiella granulomatis, Granulomainguinale (Donovanosis)); fibronectin-binding protein Sfb(Streptococcus pyogenes, Group A streptococcal infection); outermembrane protein P6 (Haemophilus influenzae, Haemophilus influenzaeinfection); integral membrane proteins, aggregation-prone proteins,O-antigen, toxin-antigens Stx2B, toxin-antigen Stx1B,adhesion-antigen fragment Int28, protein EspA, protein EspB,Intimin, protein Tir, protein IntC300, protein Eae (Escherichiacoli O157:H7, O111 and O104:H4, Hemolytic-uremic syndrome (HUS));hepatitis A surface antigen HBAg (Hepatitis A Virus, Hepatitis A);hepatitis B surface antigen HBsAg (Hepatitis B Virus, Hepatitis B);envelope glycoprotein E1 gp32 gp35, envelope glycoprotein E2 NS1gp68 gp70, capsid protein C, (Hepatitis C Virus, Hepatitis C); typeIV pilin PilE, outer membrane protein MIP, major outer membraneprotein MompS (Legionella pneumophila, Legionellosis (Legionnaires'disease, Pontiac fever)); minor pilin-associated subunit pilC,major pilin subunit and variants pilE, pilS (Neisseriameningitidis, Meningococcal disease); adhesin P1, adhesion P30(Mycoplasma pneumoniae, Mycoplasma pneumonia); F1 capsule antigen,outer membrane protease Pla, (Yersinia pestis, Plague); surfaceadhesin PsaA, cell wall surface anchored protein psrP(Streptococcus pneumoniae, Pneumococcal infection); flagellin FliC,invasion protein SipC, glycoprotein gp43, outer membrane proteinLamB, outer membrane protein PagC, outer membrane protein TolC,outer membrane protein NmpC, outer membrane protein FadL, transportprotein SadA (Salmonella genus, Salmonellosis); collagen adhesinCna, fibronectin-binding protein A FnbA, secretory antigen SssA(Staphylococcus genus, Staphylococcal food poisoning); collagenadhesin Can (Staphylococcus genus, Staphylococcal infection);fibronectin-binding protein A FbpA (Ag85A), fibronectin-bindingprotein D FbpD, fibronectin-binding protein C FbpC1, heat-shockprotein HSP65, protein PST-S(Mycobacterium tuberculosis,Tuberculosis); and outer membrane protein FobA, outer membraneprotein FobB, type IV pili glycosylation protein, outer membraneprotein tolC, protein TolQ (Francisella tularensis, Tularemia).Additional exemplary microorganisms and corresponding antigens aredisclosed, e.g., in U.S. Publication No. 2015/0118264, the entirecontents of which are expressly incorporated herein byreference.

[0562] In some embodiments, a plurality of antibodies (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more antibodies) are usedas analyte-binding agents in any of the methods described herein(e.g., to detect the presence of one or more analytes in a sample).In some embodiments, the plurality of antibodies bind to the sameanalyte (e.g., an antigen). In some embodiments, the plurality ofantibodes bind to the same epitope present on the analyte (e.g., anantigen). In some embodiments, the plurality of antibodies bind todifferent epitopes present on the same analyte. In someembodiments, the plurality of antibodies bind to overlappingepitopes present on the same analyte. In some embodiments, theplurality of antibodies bind to non-overlapping epitopes present onthe same analyte.

[0563] B. Antibiotics

[0564] In some embodiments, the analyte or analyte-binding agent isan antibiotic. An "antibiotic" or "antibiotic agent" refers to asubstance that has the capacity to inhibit or slow down the growthof, or to destroy bacteria and/or other microorganisms. In someembodiments, the antibiotic agent is a bacteriostatic antibioticagent. In some embodiments, the antibiotic is a bacteriolyticantibiotic agent. Exemplary antibiotic agents are set forth in theU.S. Patent Publication US 2006/0269485, which is herebyincorporated by reference herein in its entirety.

[0565] In some embodiments, the antibiotic agent is selected fromthe classes consisting of beta-lactam antibiotics, aminoglycosides,ansa-type antibiotics, anthraquinones, antibiotic azoles,antibiotic glycopeptides, macrolides, antibiotic nucleosides,antibiotic peptides, antibiotic polyenes, antibiotic polyethers,quinolones, antibiotic steroids, sulfonamides, tetracycline,dicarboxylic acids, antibiotic metals, oxidizing agents, substancesthat release free radicals and/or active oxygen, cationicantimicrobial agents, quaternary ammonium compounds, biguanides,triguanides, bisbiguanides and analogs and polymers thereof andnaturally occurring antibiotic compounds. In some embodiments, theantibiotic is rifaximin.

[0566] Beta-lactam antibiotics include, but are not limited to,2-(3-alanyl)clavam, 2-hydroxymethylclavam, 8-epi-thienamycin,acetyl-thienamycin, amoxicillin, amoxicillin sodium, amoxicillintrihydrate, amoxicillin-potassium clavulanate combination,ampicillin, ampicillin sodium, ampicillin trihydrate,ampicillin-sulbactam, apalcillin, aspoxicillin, azidocillin,azlocillin, aztreonam, bacampicillin, biapenem, carbenicillin,carbenicillin disodium, carfecillin, carindacillin, carpetimycin,cefacetril, cefaclor, cefadroxil, cefalexin, cefaloridine,cefalotin, cefamandole, cefamandole, cefapirin, cefatrizine,cefatrizine propylene glycol, cefazedone, cefazolin, cefbuperazone,cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren,cefditoren pivoxil, cefepime, cefetamet, cefetamet pivoxil,cefixime, cefinenoxime, cefinetazole, cefminox, cefminox,cefmolexin, cefodizime, cefonicid, cefoperazone, ceforanide,cefoselis, cefotaxime, cefotetan, cefotiam, cefoxitin, cefozopran,cefpiramide, cefpirome, cefpodoxime, cefpodoxime proxetil,cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin,ceftazidime, cefteram, cefteram pivoxil, ceftezole, ceftibuten,ceftizoxime, ceftriaxone, cefuroxime, cefuroxime axetil,cephalosporin, cephamycin, chitinovorin, ciclacillin, clavulanicacid, clometocillin, cloxacillin, cycloserine, deoxypluracidomycin, dicloxacillin, dihydro pluracidomycin, epicillin,epithienamycin, ertapenem, faropenem, flomoxef, flucloxacillin,hetacillin, imipenem, lenampicillin, loracarbef, mecillinam,meropenem, metampicillin, meticillin, mezlocillin, moxalactam,nafcillin, northienamycin, oxacillin, panipenem, penamecillin,penicillin, phenethicillin, piperacillin, tazobactam,pivampicillin, pivcefalexin, pivmecillinam, pivmecillinamhydrochloride, pluracidomycin, propicillin, sarmoxicillin,sulbactam, sulbenicillin, talampicillin, temocillin, terconazole,thienamycin, ticarcillin and analogs, salts and derivativesthereof.

[0567] Aminoglycosides include, but are not limited to,1,2'-N-DL-isoseryl-3',4'-dideoxykanamycin B,1,2'-N-DL-isoseryl-kanamycin B,1,2'-N-[(S)-4-amino-2-hydroxybutyryl]-3',4'-dideoxykanamycin B,1,2'-N-[(S)-4-amino-2-hydroxybutyryl]-kanamycin B,1-N-(2-Aminobutanesulfonyl) kanamycin A,1-N-(2-aminoethanesulfonyl)3',4'-dideoxyribostamycin,1-N-(2-Aminoethanesulfonyl)3'-deoxyribostamycin,1-N-(2-aminoethanesulfonyl)3'4'-dideoxykanamycin B,1-N-(2-aminoethanesulfonyl)kanamycin A,1-N-(2-aminoethanesulfonyl)kanamycin B,1-N-(2-aminoethanesulfonyl)ribostamycin,1-N-(2-aminopropanesulfonyl)3'-deoxykanamycin B,1-N-(2-aminopropanesulfonyl)3'4'-dideoxykanamycin B,1-N-(2-aminopropanesulfonyl)kanamycin A,1-N-(2-aminopropanesulfonyl)kanamycin B,1-N-(L-4-amino-2-hydroxy-butyryl)2,'3'-dideoxy-2'-fluorokanamycinA,1-N-(L-4-amino-2-hydroxy-propionyl)2,'3'-dideoxy-2'-fluorokanamycinA, 1-N-DL-3',4'-dideoxy-isoserylkanamycin B,1-N-DL-isoserylkanamycin, 1-N-DL-isoserylkanamycin B,1-N-[L-(-)-(alpha-hydroxy-gamma-aminobutyryl)]-XK-62-2,2',3'-dideoxy-2'-f-luorokanamycin A,2-hydroxygentamycin A3,2-hydroxygentamycin B,2-hydroxygentamycin B1, 2-hydroxygentamycin JI-20A,2-hydroxygentamycin 3''-N-methyl-4''-C-methyl-3',4'-dodeoxykanamycin A, 3''-N-methyl-4''-C-methyl-3',4'-dodeoxy kanamycin B,3''-N-methyl-4''-C-methyl-3',4'-dodeoxy-6'-methyl kanamycin B,3',4'-Dideoxy-3'-eno-ribostamycin,3',4'-dideoxyneamine,3',4'-dideoxyribos-tamycin, 3'-deoxy-6'-N-methyl-kanamycinB,3'-deoxyneamine,3'-deoxyribostamycin,3'-oxysaccharocin,3,3'-nepotrehalosadiamine,3-demethoxy-2''-N-formimidoylistamycin B disulfate tetrahydrate,3-demethoxyistamycin B,3-O-demethyl-2-N-formimidoylistamycin B,3-O-demethylistamycin B,3-trehalosamine,4'',6''-dideoxydibekacin,4-N-glycyl-KA-6606VI, 5''-Amino-3',4',5''-trideoxy-butirosin A,6''-deoxydibekacin,6'-epifortimicin A, 6-deoxy-neomycin (structure6-deoxy-neomycin B),6-deoxy-neomycin B, 6-deoxy-neomycin C,6-deoxy-paromomycin, acmimycin, AHB-3',4'-dideoxyribostamycin,AHB-3'-deoxykanamycin B, AHB-3'-deoxyneamine,AHB-3'-deoxyribostamycin, AHB-4''-6''-dideoxydibekacin,AHB-6''-deoxydibekacin, AHB-dideoxyneamine, AHB-kanamycin B,AHB-methyl-3'-deoxykanamycin B, amikacin, amikacin sulfate,apramycin, arbekacin, astromicin, astromicin sulfate, bekanamycin,bluensomycin, boholmycin, butirosin, butirosin B, catenulin,coumamidine gammal, coumamidinegamma2,D,L-1-N-(alpha-hydroxy-beta-aminopropionyl)-XK-62-2,dactimicin, de-O-methyl-4-N-glycyl-KA-6606VI, de-O-methyl-KA-6606I,de-O-methyl-KA-7038I, destomycin A, destomycin B,di-N6',O3-demethylistamycin A, dibekacin, dibekacin sulfate,dihydrostreptomycin, dihydrostreptomycin sulfate,epi-formamidoylglycidylfortimicin B, epihygromycin,formimidoyl-istamycin A, formimidoyl-istamycin B, fortimicin B,fortimicin C, fortimicin D, fortimicin KE, fortimicin KF,fortimicin KG, fortimicin KG1 (stereoisomer KG1/KG2), fortimicinKG2 (stereoisomer KG1/KG2), fortimicin KG3, framycetin, framycetinsulphate, gentamicin, gentamycin sulfate, globeomycin, hybrimycinA1, hybrimycin A2, hybrimycin B1, hybrimycin B2, hybrimycin C1,hybrimycin C2, hydroxystreptomycin, hygromycin, hygromycin B,isepamicin, isepamicin sulfate, istamycin, kanamycin, kanamycinsulphate, kasugamycin, lividomycin, marcomycin, micronomicin,micronomicin sulfate, mutamicin, myomycin,N-demethyl-7-O-demethylcelesticetin, demethylcelesticetin,methanesulfonic acid derivative of istamycin, nebramycin,nebramycin, neomycin, netilmicin, oligostatin, paromomycin,quintomycin, ribostamycin, saccharocin, seldomycin, sisomicin,sorbistin, spectinomycin, streptomycin, tobramycin, trehalosmaine,trestatin, validamycin, verdamycin, xylostasin, zygomycin andanalogs, salts and derivatives thereof.

[0568] Ansa-type antibiotics include, but are not limited to,21-hydroxy-25-demethyl-25-methylth ioprotostreptovaricin,3-methylth iorifamycin, ansamitocin, atropisostreptovaricin,awamycin, halomicin, maytansine, naphthomycin, rifabutin, rifamide,rifampicin, rifamycin, rifapentine, rifaximin (e.g., Xifaxan.RTM.),rubradirin, streptovaricin, tolypomycin and analogs, salts andderivatives thereof.

[0569] Antibiotic anthraquinones include, but are not limited to,auramycin, cinerubin, ditrisarubicin, ditrisarubicin C, figaroicacid fragilomycin, minomycin, rabelomycin, rudolfomycin,sulfurmycin and analogs, salts and derivatives thereof.

[0570] Antibiotic azoles include, but are not limited to,azanidazole, bifonazole, butoconazol, chlormidazole, chlormidazolehydrochloride, cloconazole, cloconazole monohydrochloride,clotrimazol, dimetridazole, econazole, econazole nitrate,enilconazole, fenticonazole, fenticonazole nitrate, fezatione,fluconazole, flutrimazole, isoconazole, isoconazole nitrate,itraconazole, ketoconazole, lanoconazole, metronidazole,metronidazole benzoate, miconazole, miconazole nitrate,neticonazole, nimorazole, niridazole, omoconazol, omidazole,oxiconazole, oxiconazole nitrate, propenidazole, secnidazol,sertaconazole, sertaconazole nitrate, sulconazole, sulconazolenitrate, tinidazole, tioconazole, voriconazol and analogs, saltsand derivatives thereof.

[0571] Antibiotic glycopeptides include, but are not limited to,acanthomycin, actaplanin, avoparcin, balhimycin, bleomycin B(copper bleomycin), chloroorienticin, chloropolysporin,demethylvancomycin, enduracidin, galacardin, guanidylfungin,hachimycin, demethylvancomycin, N-nonanoyl-teicoplanin, phleomycin,platomycin, ristocetin, staphylocidin, talisomycin, teicoplanin,vancomycin, victomycin, xylocandin, zorbamycin and analogs, saltsand derivatives thereof.

[0572] Macrolides include, but are not limited to,acetylleucomycin, acetylkitasamycin, angolamycin, azithromycin,bafilomycin, brefeldin, carbomycin, chalcomycin, cirramycin,clarithromycin, concanamycin, deisovaleryl-niddamycin,demycinosyl-mycinamycin, Di-O-methyltiacumicidin, dirithromycin,erythromycin, erythromycin estolate, erythromycin ethyl succinate,erythromycin lactobionate, erythromycin stearate, flurithromycin,focusin, foromacidin, haterumalide, haterumalide, josamycin,josamycin ropionate, juvenimycin, juvenimycin, kitasamycin,ketotiacumicin, lankavacidin, lankavamycin, leucomycin, machecin,maridomycin, megalomicin, methylleucomycin, methymycin,midecamycin, miocamycin, mycaminosyltylactone, mycinomycin,neutramycin, niddamycin, nonactin, oleandomycin,phenylacetyideltamycin, pamamycin, picromycin, rokitamycin,rosaramicin, roxithromycin, sedecamycin, shincomycin, spiramycin,swalpamycin, tacrolimus, telithromycin, tiacumicin, tilmicosin,treponemycin, troleandomycin, tylosin, venturicidin and analogs,salts and derivatives thereof.

[0573] Antibiotic nucleosides include, but are not limited to,amicetin, angustmycin, azathymidine, blasticidin S, epiroprim,flucytosine, gougerotin, mildiomycin, nikkomycin, nucleocidin,oxanosine, oxanosine, puromycin, pyrazomycin, showdomycin,sinefungin, sparsogenin, spicamycin, tunicamycin, uracil polyoxin,vengicide and analogs, salts and derivatives thereof.

[0574] Antibiotic peptides include, but are not limited to,actinomycin, aculeacin, alazopeptin, amfomycin, amythiamycin,antifungal from Zalerion arboricola, antrimycin, apid, apidaecin,aspartocin, auromomycin, bacileucin, bacillomycin, bacillopeptin,bacitracin, bagacidin, beminamycin, beta-alanyl-L-tyrosine,bottromycin, capreomycin, caspofungine, cepacidine, cerexin,cilofungin, circulin, colistin, cyclodepsipeptide, cytophagin,dactinomycin, daptomycin, decapeptide, desoxymulundocandin,echanomycin, echinocandin B, echinomycin, ecomycin, enniatin,etamycin, fabatin, ferrimycin, ferrimycin, ficellomycin,fluoronocathiacin, fusaricidin, gardimycin, gatavalin, globopeptin,glyphomycin, gramicidin, herbicolin, iomycin, iturin, iyomycin,izupeptin, janiemycin, janthinocin, jolipeptin, katanosin,killertoxin, lipopeptide antibiotic, lipopeptide from Zalerion sp.,lysobactin, lysozyme, macromomycin, magainin, melittin, mersacidin,mikamycin, mureidomycin, mycoplanecin, mycosubtilin,neopeptifluorin, neoviridogrisein, netropsin, nisin, nocathiacin,nocathiacin 6-deoxyglycoside, nosiheptide, octapeptin, pacidamycin,pentadecapeptide, peptifluorin, permetin, phytoactin,phytostreptin, planothiocin, plusbacin, polcillin, polymyxinantibiotic complex, polymyxin B, polymyxin B1, polymyxin F,preneocarzinostatin, quinomycin, quinupristin-dalfopristin,safracin, salmycin, salmycin, salmycin, sandramycin, saramycetin,siomycin, sperabillin, sporamycin, a Streptomyces compound,subtilin, teicoplanin aglycone, telomycin, thermothiocin,thiopeptin, thiostrepton, tridecaptin, tsushimycin,tuberactinomycin, tuberactinomycin, tyrothricin, valinomycin,viomycin, virginiamycin, zervacin and analogs, salts andderivatives thereof.

[0575] In some embodiments, the antibiotic peptide is anaturally-occurring peptide that possesses an antibacterial and/oran antifungal activity. Such peptide can be obtained from an herbalor a vertebrate source.

[0576] Polyenes include, but are not limited to, amphotericin,amphotericin, aureofungin, ayfactin, azalomycin, blasticidin,candicidin, candicidin methyl ester, candimycin, candimycin methylester, chinopricin, filipin, flavofungin, fradicin, hamycin,hydropricin, levorin, lucensomycin, lucknomycin, mediocidin,mediocidin methyl ester, mepartricin, methylamphotericin,natamycin, niphimycin, nystatin, nystatin methyl ester, oxypricin,partricin, pentamycin, perimycin, pimaricin, primycin, proticin,rimocidin, sistomycosin, sorangicin, trichomycin and analogs, saltsand derivatives thereof.

[0577] Polyethers include, but are not limited to,20-deoxy-epi-narasin, 20-deoxysalinomycin, carriomycin, dianemycin,dihydrolonomycin, etheromycin, ionomycin, iso-lasalocid, lasalocid,lenoremycin, lonomycin, lysocellin, monensin, narasin,oxolonomycin, a polycyclic ether antibiotic, salinomycin andanalogs, salts and derivatives thereof.

[0578] Quinolones include, but are not limited to, analkyl-methylendioxy-4(1H)-oxocinnoline-3-carboxylic acid,alatrofloxacin, cinoxacin, ciprofloxacin, ciprofloxacinhydrochloride, danofloxacin, dermofongin A, enoxacin, enrofloxacin,fleroxacin, flumequine, gatifloxacin, gemifloxacin, grepafloxacin,levofloxacin, lomefloxacin, lomefloxacin, hydrochloride, miloxacin,moxifloxacin, nadifloxacin, nalidixic acid, nifuroquine,norfloxacin, ofloxacin, orbifloxacin, oxolinic acid, pazufloxacine,pefloxacin, pefloxacin mesylate, pipemidic acid, piromidic acid,premafloxacin, rosoxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, trovafloxacin and analogs, salts and derivativesthereof.

[0579] Antibiotic steroids include, but are not limited to,aminosterol, ascosteroside, cladosporide A, dihydrofusidic acid,dehydro-dihydrofusidic acid, dehydrofusidic acid, fusidic acid,squalamine and analogs, salts and derivatives thereof.

[0580] Sulfonamides include, but are not limited to, chloramine,dapsone, mafenide, phthalylsulfathiazole, succinylsulfathiazole,sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfadiazine,sulfadiazine silver, sulfadicramide, sulfadimethoxine, sulfadoxine,sulfaguanidine, sulfalene, sulfamazone, sulfamerazine,sulfamethazine, sulfamethizole, sulfamethoxazole,sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxol,sulfanilamide, sulfaperine, sulfaphenazol, sulfapyridine,sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea,sulfatolamide, sulfatriazin, sulfisomidine, sulfisoxazole,sulfisoxazole acetyl, sulfacarbamide and analogs, salts andderivatives thereof.

[0581] Tetracyclines include, but are not limited to,dihydrosteffimycin, demethyltetracycline, aclacinomycin,akrobomycin, baumycin, bromotetracycline, cetocyclin,chlortetracycline, clomocycline, daunorubicin, demeclocycline,doxorubicin, doxorubicin hydrochloride, doxycycline, lymecyclin,marcellomycin, meclocycline, meclocycline sulfosalicylate,methacycline, minocycline, minocycline hydrochloride, musettamycin,oxytetracycline, rhodirubin, rolitetracycline, rubomycin,serirubicin, steffimycin, tetracycline and analogs, salts andderivatives thereof.

[0582] Dicarboxylic acids, having between about 6 and about 14carbon atoms in their carbon atom skeleton are particularly usefulin the treatment of disorders of the skin and mucosal membranesthat involve microbial. Suitable dicarboxylic acid moietiesinclude, but are not limited to, adipic acid, pimelic acid, subericacid, azelaic acid, sebacic acid, 1,11-undecanedioic acid,1,12-dodecanedioic acid, 1,13-tridecanedioic acid and1,14-tetradecanedioic acid. Thus, in one or more embodiments of thepresent disclosure, dicarboxylic acids, having between about 6 andabout 14 carbon atoms in their carbon atom skeleton, as well astheir salts and derivatives (e.g., esters, amides,mercapto-derivatives, anhydraides), are useful immunomodulators inthe treatment of disorders of the skin and mucosal membranes thatinvolve inflammation. Azelaic acid and its salts and derivativesare preferred. It has antibacterial effects on both aerobic andanaerobic organisms, particularly Propionibacterium acnes andStaphylococcus epidermidis, normalizes keratinization, and has acytotoxic effect on malignant or hyperactive melanocytes. In apreferred embodiment, the dicarboxylic acid is azelaic acid in aconcentration greater than 10%. Preferably, the concentration ofazelaic acid is between about 10% and about 25%. In suchconcentrates, azelaic acid is suitable for the treatment of avariety of skin disorders, such as acne, rosacea andhyperpigmentation.

[0583] In some embodiments, the antibiotic agent is an antibioticmetal. A number of metals ions have been shown to possessantibiotic activity, including silver, copper, zinc, mercury, tin,lead, bismutin, cadmium, chromium and ions thereof. It has beentheorized that these antibiotic metal ions exert their effects bydisrupting respiration and electron transport systems uponabsorption into bacterial or fungal cells. Anti-microbial metalions of silver, copper, zinc, and gold, in particular, areconsidered safe for in vivo use. Anti-microbial silver and silverions are particularly useful due to the fact that they are notsubstantially absorbed into the body. Thus, in one or moreembodiment, the antibiotic metal consists of an elemental metal,selected from the group consisting of silver, copper, zinc,mercury, tin, lead, bismutin, cadmium, chromium and gold, which issuspended in the composition as particles, microparticles,nanoparticles or colloidal particles. The antibiotic metal canfurther be intercalated in a chelating substrate.

[0584] In further embodiments, the antibiotic metal is ionic. Theionic antibiotic metal can be presented as an inorganic or organicsalt (coupled with a counterion), an organometallic complex or anintercalate. Non-binding examples of counter inorganic and organicions are sulfadiazine, acetate, benzoate, carbonate, iodate,iodide, lactate, laurate, nitrate, oxide, and palmitate, anegatively charged protein. In preferred embodiments, theantibiotic metal salt is a silver salt, such as silver acetate,silver benzoate, silver carbonate, silver iodate, silver iodide,silver lactate, silver laurate, silver nitrate, silver oxide,silver palmitate, silver protein, and silver sulfadiazine.

[0585] In one or more embodiments, the antibiotic metal or metalion is embedded into a substrate, such as a polymer, or a mineral(such as zeolite, clay and silica).

[0586] In one or more embodiments, the antibiotic agent includesstrong oxidants and free radical liberating compounds, such asoxygen, hydrogen peroxide, benzoyl peroxide, elemental halogenspecies, as well as oxygenated halogen species, bleaching agents(e.g., sodium, calcium or magnesium hypochloride and the like),perchlorite species, iodine, iodate, and benzoyl peroxide. Organicoxidizing agents, such as quinones, are also included. Such agentspossess a potent broad-spectrum activity.

[0587] In one or more embodiments, the antibiotic agent is acationic antimicrobial agent. The outermost surface of bacterialcells universally carries a net negative charge, making themsensitive to cationic substances. Examples of cationic antibioticagents include: quaternary ammonium compounds (QAC's)--QAC's aresurfactants, generally containing one quaternary nitrogenassociated with at least one major hydrophobic moiety;alkyltrimethyl ammonium bromides are mixtures of where the alkylgroup is between 8 and 18 carbons long, such as cetrimide(tetradecyltrimethylammonium bromide); benzalkonium chloride, whichis a mixture of n-alkyldimethylbenzyl ammonium chloride where thealkyl groups (the hydrophobic moiety) can be of variable length;dialkylmethyl ammonium halides; dialkylbenzyl ammonium halides; andQAC dimmers, which bear bi-polar positive charges in conjunctionwith interstitial hydrophobic regions.

[0588] In one or more embodiments, the cationic antimicrobial agentis a polymer. Cationic antimicrobial polymers include, for example,guanide polymers, biguanide polymers, or polymers having sidechains containing biguanide moieties or other cationic functionalgroups, such as benzalkonium groups or quarternium groups (e.g.,quaternary amine groups). It is understood that the term "polymer"as used herein includes any organic material including three ormore repeating units, and includes oligomers, polymers, copolymers,block copolymers, terpolymers, etc. The polymer backbone may be,for example a polyethylene, ploypropylene or polysilanepolymer.

[0589] In one or more embodiments, the cationic antimicrobialpolymer is a polymeric biguanide compound. When applied to asubstrate, such a polymer is known to form a barrier film that canengage and disrupt a microorganism. An exemplary polymericbiguanide compound is polyhexamethylene biguanide (PHMB) salts.Other exemplary biguanide polymers include, but are not limited topoly(hexamethylenebiguanide), poly(hexamethylenebiguanide)hydrochloride, poly(hexamethylenebiguanide) gluconate,poly(hexamethylenebiguanide) stearate, or a derivative thereof. Inone or more embodiments, the antimicrobial material issubstantially water-insoluble.

[0590] In some embodiments, the antibiotic agent is selected fromthe group of biguanides, triguanides, bisbiguanides and analogsthereof.

[0591] Guanides, biguanides, biguanidines and triguanides areunsaturated nitrogen containing molecules that readily obtain oneor more positive charges, which make them effective antimicrobialagents. The basic structures a guanide, a biguanide, a biguanidineand a triguanide are provided below.

##STR00001##

In some embodiments, the guanide, biguanide, biguanidine ortriguanide, provide bi-polar configurations of cationic andhydrophobic domains within a single molecule.

[0592] Examples of guanides, biguanides, biguanidines andtriguanides that are currently been used as antibacterial agentsinclude chlorhexidine and chlorohexidine salts, analogs andderivatives, such as chlorhexidine acetate, chlorhexidine gluconateand chlorhexidine hydrochloride, picloxydine, alexidine andpolihexanide. Other examples of guanides, biguanides, biguanidinesand triguanides that can conceivably be used according to thepresent disclosure are chlorproguanil hydrochloride, proguanilhydrochloride (currently used as antimalarial agents), mefforminhydrochloride, phenformin and buformin hydrochloride (currentlyused as antidiabetic agents).

[0593] Yet, in one or more embodiments, the antibiotic is anon-classified antibiotic agent, including, without limitation,aabomycin, acetomycin, acetoxycycloheximide, acetylnanaomycin, anActinoplanes sp. compound, actinopyrone, aflastatin, albacarcin,albacarcin, albofungin, albofungin, alisamycin,alpha-R,S-methoxycarbonylbenzylmonate, altromycin, amicetin,amycin, amycin demanoyl compound, amycine, amycomycin, anandimycin,anisomycin, anthramycin, anti-syphilis immune substance,anti-tuberculosis immune substance, an antibiotic from Escherichiacoli, an antibiotic from Streptomyces refuineus, anticapsin,antimycin, aplasmomycin, aranorosin, aranorosinol, arugomycin,ascofuranone, ascomycin, ascosin, Aspergillus flavus antibiotic,asukamycin, aurantinin, an Aureolic acid antibiotic substance,aurodox, avilamycin, azidamfenicol, azidimycin, bacillaene, aBacillus larvae antibiotic, bactobolin, benanomycin, benzanthrin,benzylmonate, bicozamycin, bravomicin, brodimoprim, butalactin,calcimycin, calvatic acid, candiplanecin, carumonam, carzinophilin,celesticetin, cepacin, cerulenin, cervinomycin, chartreusin,chloramphenicol, chloramphenicol palmitate, chloramphenicolsuccinate sodium, chlorflavonin, chlorobiocin, chlorocarcin,chromomycin, ciclopirox, ciclopirox olamine, citreamicin,cladosporin, clazamycin, clecarmycin, clindamycin, coliformin,collinomycin, copiamycin, corallopyronin, corynecandin,coumermycin, culpin, cuprimyxin, cyclamidomycin, cycloheximide,dactylomycin, danomycin, danubomycin, delaminomycin,demethoxyrapamycin, demethylscytophycin, dermadin, desdamethine,dexylosyl-benanomycin, pseudoaglycone, dihydromocimycin,dihydronancimycin, diumycin, dnacin, dorrigocin, dynemycin,dynemycin triacetate, ecteinascidin, efrotomycin, endomycin,ensanchomycin, equisetin, ericamycin, esperamicin, ethylmonate,everninomicin, feldamycin, flambamycin, flavensomycin, florfenicol,fluvomycin, fosfomycin, fosfonochlorin, fredericamycin, frenolicin,fumagillin, fumifungin, funginon, fusacandin, fusafungin,gelbecidine, glidobactin, grahamimycin, granaticin, griseofulvin,griseoviridin, grisonomycin, hayumicin, hayumicin, hazymicin,hedamycin, heneicomycin, heptelicid acid, holomycin, humidin,isohematinic acid, karnatakin, kazusamycin, kristenin,L-dihydrophenylalanine, aL-isoleucyl-L-2-amino-4-(4'-amino-2',5'-cyclohexadienyl)derivative, lanomycin, leinamycin, leptomycin, libanomycin,lincomycin, lomofungin, lysolipin, magnesidin, manumycin,melanomycin, methoxycarbonylmethylmonate,methoxycarbonylethylmonate, methoxycarbonylphenylmonate, methylpseudomonate, methylmonate, microcin, mitomalcin, mocimycin,moenomycin, monoacetyl cladosporin, monomethyl cladosporin,mupirocin, mupirocin calcium, mycobacidin, myriocin, myxopyronin,pseudoaglycone, nanaomycin, nancimycin, nargenicin,neocarcinostatin, neoenactin, neothramycin, nifurtoinol,nocardicin, nogalamycin, novobiocin, octylmonate, olivomycin,orthosomycin, oudemansin, oxirapentyn, oxoglaucine methiodide,pactacin, pactamycin, papulacandin, paulomycin, phaeoramulariafungicide, phenelfamycin, phenyl, cerulenin, phenylmonate,pholipomycin, pirlimycin, pleuromutilin, a polylactone derivative,polynitroxin, polyoxin, porfiromycin, pradimicin, prenomycin,prop-2-enylmonate, protomycin, Pseudomonas antibiotic, pseudomonicacid, purpuromycin, pyrinodemin, pyrrolnitrin, pyrrolomycin, amino,chloro pentenedioic acid, rapamycin, rebeccamycin, resistomycin,reuterin, reveromycin, rhizocticin, roridin, rubiflavin,naphthyridinomycin, saframycin, saphenamycin, sarkomycin,sarkomycin, sclopularin, selenomycin, siccanin, spartanamicin,spectinomycin, spongistatin, stravidin, streptolydigin,Streptomyces arenae antibiotic complex, streptonigrin,streptothricins, streptovitacin, streptozotocine, a strobilurinderivative, stubomycin, sulfamethoxazol-trimethoprim, sakamycin,tejeramycin, terpentecin, tetrocarcin, thermorubin,thermozymocidin, thiamphenicol, thioaurin, thiolutin, thiomarinol,thiomarinol, tirandamycin, tolytoxin, trichodermin, trienomycin,trimethoprim, trioxacarcin, tyrissamycin, umbrinomycin,unphenelfamycin, urauchimycin, usnic acid, uredolysin, variotin,vermisporin, verrucarin and analogs, salts and derivativesthereof.

[0594] In one or more embodiments, the antibiotic agent is anaturally occurring antibiotic compound. As used herein, the term"naturally-occurring antibiotic agent" includes all antibioticsthat are obtained, derived or extracted from plant or vertebratesources. Non-limiting examples of families of naturally-occurringantibiotic agents include phenol, resorcinol, antibioticaminoglycosides, anamycin, quinines, anthraquinones, antibioticglycopeptides, azoles, macrolides, avilamycin, agropyrene, cnicin,aucubin antibioticsaponin fractions, berberine (isoquinolinealkaloid), arctiopicrin (sesquiterpene lactone), lupulone, humulone(bitter acids), allicin, hyperforin, echinacoside, coniosetin,tetramic acid, imanine and novoimanine.

[0595] Ciclopirox and ciclopiroxolamine possess fungicidal,fungistatic and sporicidal activity. They are active against abroad spectrum of dermatophytes, yeasts, moulds and other fungi,such as Trichophytons species, Microsporum species, Epidermophytonspecies and yeasts (Candida albicans, Candida glabrata, othercandida species and Cryptococcus neoformans). Some Aspergillusspecies are sensitive to ciclopirox as are some Penicillium.Likewise, ciclopirox is effective against many Gram-positive andGram-negative bacteria (e.g., Escherichia coli, Proteus mirabilis,Pseudomonas aeruginosa, Staphylococcus and Streptococcus species),as well as Mycoplasma species, Trichomonas vaginalis andActinomyces.

[0596] Plant oils and extracts which contain antibiotic agents arealso useful. Non-limiting examples of plants that contain agentsinclude thyme, Perilla, lavender, tea tree, Terfezia clayeryi,Micromonospora, Putterlickia verrucosa, Putterlickia pyracantha,Putterlickia retrospinosa, Maytenus ilicifolia, Maytenusevonymoides, Maytenus aquifolia, Faenia interjecta, Cordycepssinensis, couchgrass, holy thistle, plantain, burdock, hops,echinacea, buchu, chaparral, myrrh, red clover and yellow dock,garlic, and St. John's wort.Mixtures of the antibiotic agents asdescribed herein may also be employed.

[0597] C. Biomarkers

[0598] In some embodiments, the analyte or analyte-binding agent isa biomarker. In general, biomarkers of diseases and disorders maybe detected, analyzed and/or quantitated using the devices,compositions and methods described herein. The detection, analysisand quantification of a biomarker using the devices, methods andcompositions described herein is particular useful in determiningand monitoring the course of treatment that could be used to treata condition in a subject (e.g., a human subject). Biomarkers can bedetected and analyzed locally in the GI tract of a subject todetermine whether the subject has or is at risk of developing adisease or disorder. In addition, biomarkers can be monitored usingthe compositions and methods described herein to determine whethera particular course of treatment in a subject diagnosed with adisease or disorder is effective or should be altered. For example,in some embodiments, inflammatory biomarker(s) is/are detected andanalyzed in a subject using the ingestible devices described hereinto determine whether a subject has or is at risk of developing IBD.As necessary, the subject can then be administered one or morecourses of treatment (e.g., an anti-TNF.alpha. antibody) and thelevel of such inflammatory biomarker(s) can be monitored to assessefficacy of treatment.

[0599] In some embodiments, biomarkers are detected and analyzed ina subject to determine whether the subject has or is at risk ofdeveloping a disease or disorder. These diseases and disorders mayoccur in the GI tract of the subject or at a non-GI tract site inthe subject. For example, biomarkers present in the GI tract may beindicative of a systemic disease or disorder. In some embodiments,the biomarkers are associated with a systemic disease or disorder.In some embodiments, the biomarkers are associated with one or moreof a GI disorder, inflammation, cancer, an infectious disease, aliver disease, and an inflammatory disease. Exemplary clases ofbiomarkers include antibodies (e.g., therapeutic antibodies),antigens (e.g., bacterial antigens), and cytokines). In someembodiments, the analyte or the analyte-binding agent is abiomarker, e.g., a biomarker of a GI disorder. An illustrative listof examples of biomarkers for detection, diagnosis or monitoring oftreatment efficacy for GI disorders includes interferon-.gamma.,IL-1.beta., IL-6, IL-22, IL-17A, TNF.alpha., IL-2, memory cells(CD44.sup.+CD45RB.sup.-CD4.sup.+ cells); .alpha.4.beta.7; VEGF;ICAM; VCAM; SAA; Calprotectin; lactoferrin; FGF2; TGFb; ANG-1;ANG-2; PLGF; a biologic (e.g., infliximab (REMICADE); adalimumab(HUMIRA); ustekinumab (STELARA); vedolizumab (ENTYVIO); golimumab(SIMPONI); Jak inhibitors; and others); EGF; IL12/23p40; GMCSF; A4B7; AeB7; CRP; SAA; ICAM; VCAM; AREG; EREG; HB-EGF; HRG; BTC;TGF.alpha.; SCF; TWEAK; MMP-9; MMP-6; Ceacam CD66; IL10; ADA;Madcam-1; CD166 (AL CAM); FGF2; FGF7; FGF9; FGF19; Anti-neutrophilcytoplasmic antibody (ANCA); Anti-Saccharomyces cerevisiae AntibodyIgA (ASCAA); Anti-Saccharomyces cerevisiae Antibody IgG (ASCAG);Anti-Clostridium cluster XIVa flagellin CBirl antibody (CBirl);Anti-Clostridium cluster XIVa flagellin 2 antibody (A4-Fla2);Anti-Clostridium cluster XIVa flagellin X antibody (FlaX);Anti-Escherichia coli Outer Membrane Protein C (OmpC); PerinuclearAntiNeutrophil Cytoplasmic Antibody (ANCA); Amphiregulin Protein(AREG); Betacellulin Protein (BTC); Epidermal Growth Factor (EGF);Epiregulin Protein (EREG); Heparin Binding Epidermal Growth Factors(HBEGF); Hepatocyte Growth Factor (HGF); Neuregulin-1 (HRG);Transforming Growth Factor alpha (TGFA); C-Reactive Protein (CRP);Serum Amyloid A (SAA); Intercellular Adhesion Molecule 1 (ICAM-1);Vascular Cell Adhesion Molecule 1 (VCAM-1); and fibroblastsunderlying the intestinal epithelium.

[0600] In some embodiments, a biomarker is an IBD biomarker, suchas, for example: anti-glycan; anti-Saccharomyces cerevisiae (ASCA);anti-laminaribioside (ALCA); anti-chitobioside (ACCA);anti-mannobioside (AMCA); anti-laminarin (anti-L); anti-chitin(anti-C) antibodies: anti-outer membrane porin C (anti-OmpC),anti-Cbirl flagellin; anti-I2 antibody; autoantibodies targetingthe exocrine pancreas (PAB); and perinuclear anti-neutrophilantibody (pANCA); and calprotectin.

[0601] In some embodiments, a biomarker is associated with membranerepair, fibrosis, angiogenesis. In certain embodiments, a biomarkeris an inflammatory biomarker, an anti-inflammatory biomarker, anMMP biomarker, an immune marker, or a TNF pathway biomarker. Insome embodiments, a biomarker is gut-specific.

[0602] For tissue samples, HER2 can be used as a biomarker relatingto cytotoxic T cells. Additionally, other cytokine levels can beused as biomarkers in tissue (e.g., phospho STAT 1, STAT 3 and STAT5), in plasma (e.g., VEGF, VCAM, ICAM, IL-6), or both.

[0603] In some embodiments, the biomarker include one or moreimmunoglobulins, such as, for example, immunoglobulin M (IgM),immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin E(IgE) and/or immunoglobulin A (IgA). In some embodiments, IgM is abiomarker of infection and/or inflammation. In some embodiments,IgD is a biomarker of autoimmune disease. In some embodiments, IgGis a biomarker of Alzheimer's disease and/or for cancer. In someembodiments, IgE is a biomarker of asthma and/or allergenimmunotherapy. In some embodiments, IgA is a biomarker of kidneydisease.

[0604] In some embodiments, the biomarker is High SensitivityC-reactive Protein (hsCRP); 7.alpha.-hydroxy-4-cholesten-3-one(7.alpha.C4); Anti-Endomysial IgA (EMA IgA); Anti-Human TissueTransglutaminase IgA (tTG IgA); Total Serum IgA by Nephelometry;Fecal Calprotectin; or Fecal Gastrointestinal Pathogens.

[0605] In some embodiments, the biomarker is:

[0606] a) an anti-gliadin IgA antibody, an anti-gliadin IgGantibody, an anti-tissue transglutaminase (tTG) antibody, ananti-endomysial antibody;

[0607] b)i) a serological biomarker that is ASCA-A, ASCA-G, ANCA,pANCA, anti-OmpC antibody, anti-CBir1 antibody, anti-FlaX antibody,or anti-A4-Fla2 antibody;

[0608] b)ii) an inflammation biomarker that is VEGF, ICAM, VCAM,SAA, or CRP;

[0609] b)iii) the genotype of the genetic biomarkers ATG16L1, ECM1,NKX2-3, or STAT3;

[0610] c) a bacterial antigen antibody biomarker;

[0611] d) a mast cell biomarker;

[0612] e) an inflammatory cell biomarker;

[0613] f) a bile acid malabsorption (BAM) biomarker;

[0614] g) a kynurenine biomarker;

[0615] or

[0616] h) a serotonin biomarker.

[0617] In some embodiments, the biomarker is a bacterial antigenantibody biomarker selected from the group consisting of ananti-Fla1 antibody, anti-Fla2 antibody, anti-FlaA antibody,anti-FliC antibody, anti-FliC2 antibody, anti-FliC3 antibody,anti-YBaN1 antibody, anti-ECFliC antibody, anti-Ec0FliC antibody,anti-SeFljB antibody, anti-CjFlaA antibody, anti-CjFlaB antibody,anti-SfFliC antibody, anti-CjCgtA antibody, anti-Cjdmh antibody,anti-CjGT-A antibody, anti-EcYidX antibody, anti-EcEra antibody,anti-EcFrvX antibody, anti-EcGabT antibody, anti-EcYedK antibody,anti-EcYbaN antibody, anti-EcYhgN antibody, anti-RtMaga antibody,anti-RbCpaF antibody, anti-RgPilD antibody, anti-LaFrc antibody,anti-LaEno antibody, anti-LjEFTu antibody, anti-BfOmpa antibody,anti-PrOmpA antibody, anti-Cp10bA antibody, anti-CpSpA antibody,anti-EfSant antibody, anti-LmOsp antibody, anti-SfET-2 antibody,anti-Cpatox antibody, anti-Cpbtox antibody, anti-EcSta2 antibody,anti-EcOStx2A antibody, anti-CjcdtB/C antibody, anti-CdTcdA/Bantibody, and combinations thereof.

[0618] In some embodiments, the biomarker is a mast cell biomarkerselected from the group consisting of beta-tryptase, histamine,prostaglandin E2 (PGE2), and combinations thereof.

[0619] In some embodiments, the biomarker is an inflammatorybiomarker is selected from the group consisting of CRP, ICAM, VCAM,SAA, GRO.alpha., and combinations thereof.

[0620] In some embodiments, the biomarker is a bile acidmalabsorption biomarker selected from the group consisting of7.alpha.-hydroxy-4-cholesten-3-one, FGF19, and a combinationthereof.

[0621] In some embodiments, the biomarker is a kynurenine biomarkerselected from the group consisting of kynurenine (K), kynurenicacid (KyA), anthranilic acid (AA), 3-hydroxykynurenine (3-HK),3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA),quinolinic acid (QA), tryptophan, 5-hydroxytryptophan (5-HTP), andcombinations thereof.

[0622] In some embodiments, the biomarker is a serotonin biomarkerselected from the group consisting of serotonin (5-HT),5-hydroxyindoleacetic acid (5-HIAA), serotonin-O-sulfate,serotonin-O-phosphate, and combinations thereof.

[0623] Additional biomarkers are disclosed, e.g., at U.S. Pat. No.9,739,786, the entire contents of which are incorporated herein byreference.

[0624] In some embodiments, the biomarker is associated with aliver disease or disorder. In some embodiments, the analyte oranalyte-binding agent is a biomarker of a liver disease or a liverdisorder. In some embodiments, the devices, compositions andmethods disclosed herein may be used to detect, analyze and/orquantitate a biomarker associated with a liver disease or disorder,e.g., to determine whether a subject has or is at risk ofdeveloping a liver disease or disorder. In some embodiments, thedevices, compositions, and methods described herein can be used todetect an analyte (e.g., a biomarker) in a sample from thegastrointestinal tract of the subject to determine whether thesubject has or is at risk of developing a liver disease or disorder(e.g., NASH). In some embodiments, the detection, analysis andquantification of a liver disease biomarker using the devices,methods and compositions described herein may be used indetermining and monitoring the course of treatment that could beused to treat a liver disease or disorder in a subject (e.g., ahuman subject). An illustrative list of examples of biomarkers thatmay be used for the detection, diagnosis, or monitoring oftreatment efficacy for a liver disease or disorder includesbilirubin, gamma-glutamyl transferase (GGT), haptoglobin,apolipoprotein A1, alpha2-macroglobulin, cholesterol,triglycerides, alanine aminotransferase (ALT), aspartateaminotransferase (AST), glucose, cytokeratin-18 (CK18) fragment,hyaluronic acid, TGF-.beta., fatty acid binding protein,hydroxysteroid 17-beta dehydrogenase 13 (17.beta.-HSD13), glutamyldipeptides, glutamyl valine, glutamyl leucine, glutamylphenylalanine, glutamyl tyrosine, carnitine, butylcarnitine,lysine, tyrosine, isoleucine, glycerophosphatidylcholine,glycerylphsphorylethanolamine, taurine, glycine conjugates,taurocholic acid, taurodeoxycholic acid, lactate, glutamate,cysteine-gluthatione disulfide, caprate, 10-undecenoate,oleoyl-lysophosphatidylcholine, oxidized and reduced gluthatione,glutamate, andenosine triphosphate, creatine, cholic acid, andglycodeoxycholic acid. Additional biomarkers, as well astherapeutic agents, for liver diseases and disorders are known inthe art (see, e.g., Hirsova and Gores (2015) Cell. Mol.Gastroenterol. Hepatol. 1(1): 17-27; Willebrords et al. (2015)Progress in Lipid Research 59: 106-125; Alkhouri et al. (2011)Expert Rev. Gastroenterol. Hepatol. 5(2): 201-12; Wang (2014) CellDeath and Disease 5: e996; and Alonso et al. (2017)Gastroenterology 152: 1449-61, incorporated herein byreference).

[0625] D. Therapeutic Agents

[0626] In some embodiments, the analyte or analyte-binding agent isa therapeutic agent, a fragment of a therapeutic agent and/or ametabolite of a therapeutic agent. The compositions and methodsprovided below may also be used to detect, analyze and/orquantitate a therapeutic agent, a fragment of a therapeutic agent,and/or a metabolite of a therapeutic agent. Exemplary therapeuticagents include antibodies, nucleic acids (e.g., inhibitory nucleicacids), small molecules, and live biotherapeutics such asprobiotics. In some embodiments, the analyte or the analyte-bindingagent used in any of the detection methods described herein is adrug or a therapeutic agent. In some embodiments, the drug ortherapeutic agent is used for the treatment of inflammatory boweldisease (IBD), for example, Crohn's Disease or Ulcerative Colitis(UC). Nonlimiting examples of such agents for treating orpreventing inflammatory bowel disease include substances thatsuppress cytokine production, down-regulate or suppressself-antigen expression, or mask the MHC antigens. Examples of suchagents include CHST15 inhibitors (e.g., STNM01); IL-6 receptorinhibitora (e.g., tocilizumab); IL-12/IL-23 inhibitors (e.g.,ustekinumab and brazikumab); integrin inhibitors (e.g., vedolizumaband natalizumab); JAK inhibitors (e.g., tofacitinib); SMAD7inhibitors (e.g., Mongersen); IL-13 inhibitors; IL-1 receptorinhibitors; TLR agonists (e.g., Kappaproct); stem cells (e.g.,Cx601); 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs);ganciclovir; tacrolimus; glucocorticoids such as Cortisol oraldosterone; anti-inflammatory agents such as a cyclooxygenaseinhibitor; a 5-lipoxygenase inhibitor; or a leukotriene receptorantagonist; purine antagonists such as azathioprine ormycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde(which masks the MHC antigens, as described in U.S. Pat. No.4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporine; 6-mercaptopurine; steroids such ascorticosteroids or glucocorticosteroids or glucocorticoid analogs,e.g., prednisone, methylprednisolone, including SOLU-MEDROL.RTM.,methylprednisolone sodium succinate, and dexamethasone;dihydrofolate reductase inhibitors such as methotrexate (oral orsubcutaneous); anti-malarial agents such as chloroquine andhydroxychloroquine; sulfasalazine; leflunomide; cytokine orcytokine receptor antibodies or antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumornecrosis factor(TNF)-alpha antibodies (infliximab (REMICADE.RTM.)or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),anti-TNF-beta antibodies, antiinterleukin-2 (IL-2) antibodies andanti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6)receptor antibodies and antagonists; anti-LFA-1 antibodies,including anti-CD 1 la and anti-CD 18 antibodies; anti-L3T4antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26,1990); streptokinase; transforming growth factor-beta (TGF-beta);streptodomase; RNA or DNA from the host; FK506; RS-61443;chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen etal, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner etal, Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature,341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF orBR3 antibodies or immunoadhesins and zTNF4 antagonists (for review,see Mackay and Mackay, Trends Immunol, 23: 113-5 (2002) and seealso definition below); 10 biologic agents that interfere with Tcell helper signals, such as anti-CD40 receptor or anti-CD40 ligand(CD 154), including blocking antibodies to CD40-CD40 ligand. (e.g.,Durie et al, Science, 261: 1328-30 (1993); Mohan et al, J. Immunol,154: 1470-80 (1995)) and CTLA4-Ig (Finck et al, Science, 265:1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such asT10B9. Non-limiting examples of agents also include the following:budenoside; epidermal growth factor; aminosalicylates;metronidazole; mesalamine; olsalazine; balsalazide; antioxidants;thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-1monoclonal antibodies; growth factors; elastase inhibitors;pyridinylimidazole compounds; TNF antagonists; IL-4, IL-10, IL-13and/or TGF.beta. cytokines or agonists thereof (e.g., agonistantibodies); IL-11; glucuronide- or dextran-conjugated prodrugs ofprednisolone, dexamethasone or budesonide; ICAM-I antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; IsisPharmaceuticals, Inc.); soluble complement receptor 1 (TPlO; T CellSciences, Inc.); slow-release mesalazine; antagonists of plateletactivating factor (PAF); ciprofloxacin; and lignocaine. Examples ofagents for UC are sulfasalazine and related salicylate-containingdrugs for mild cases and corticosteroid drugs in severe cases.Exemplary therapeutic agents that may be used for the treatment ofa liver disease or disorder (e.g., liver fibrosis or NASH) includeelafibranor (GFT 505; Genfit Corp.), obeticholic acid (OCA;Intercept Pharmaceuticals, Inc.), cenicriviroc (CVC; Allergan plc),selonsertib (formerly GS-4997; Gilead Sciences, Inc.), ananti-LOXL2 antibody (simtuzumab (formerly GS 6624; Gilead Sciences,Inc.)), GS-9450 (Gilead Sciences, Inc.), GS-9674 (Gilead Sciences,Inc.), GS-0976 (formerly NDI-010976; Gilead Sciences, Inc.),Emricasan (Conatus Pharmaceuticals, Inc.), Arachidyl-amidocholanoic acid (Aramchol.TM.; Galmed Pharmaceuticals Ltd.), AKN-083(Allergan plc (Akarna Therapeutics Ltd.)), TGFTX4 (Genfit Corp.),TGFTX5 (Genfit Corp.), TGFTX1 (Genfit Corp.), a RoRy agonist (e.g.,LYC-55716; Lycera Corp.), an ileal bile acid transporter (iBAT)inhibitor (e.g., elobixibat, Albireo Pharma, Inc.; GSK2330672,GlaxoSmithKline plc; and A4250; Albireo Pharma, Inc.), stem cells,a CCR2 inhibitor, bardoxolone methyl (Reata Pharmaceuticals, Inc.),a bone morphogenetic protein-7 (BMP-7) mimetic (e.g., THR-123 (see,e.g., Sugimoto et al. (2012) Nature Medicine 18: 396-404)), ananti-TGF-.beta. antibody (e.g., fresolimumab; see also U.S. Pat.Nos. 7,527,791 and 8,383,780, incorporated herein by reference),pirfenidone (Esbriet.RTM., Genentech USA Inc.), an anti-integrin.alpha.v.beta.6 antibody, an anti-connective tissue growth factor(CTGF) antibody (e.g., pamrevlumab; FibroGen Inc.), pentoxifylline,vascular endothelial growth factor (VEGF), a renin angiotensinaldosterone system (RAAS) inhibitor (e.g., a rennin inhibitor (e.g.pepstatin, CGP2928, aliskiren), or an ACE inhibitor (e.g.,captopril, zofenopril, enalapril, ramipril, quinapril, perindopril,lisinopril, benazepril, imidapril, fosinopril, and trandolapril)),thrombospondin, a statin, bardoxolone, a PDES inhibitor (e.g.,sidenafil, vardenafil, and tadalafil), a NADPH oxidase-1 (NOX1)inhibitor (see, e.g., U.S. Publication No. 2011/0178082,incorporated herein by reference), a NADPH oxidase-4 (NOX4)inhibitor (see, e.g., U.S. Publication No. 2014/0323500,incorporated herein by reference), an ETA antagonist (e.g.,sitaxentan, ambrisentan, atrasentan, BQ-123, and zibotentan),nintedanib (Boehringer Ingelheim), INT-767 (InterceptPharmaceuticals, Inc.), VBY-376 (Virobay Inc.),PF-04634817(Pfizer), EXC 001 (Pfizer), GM-CT-01 (GalectinTherapeutics), GCS-100 (La Jolla Pharmaceuticals), hepatocytegrowth factor mimetic (Refanalin.RTM.; Angion Biomedica), SAR156597(Sanofi), tralokinumab (AstraZeneca), pomalidomide (Celgene),STX-100 (Biogen IDEC), CC-930 (Celgene), anti-miR-21 (RegulusTherapeutics), PRM-151 (Promedior), BOT191 (BiOrion), Palomid 529(Paloma Pharamaceuticals), IMD1041 (IMMD, Japan), serelaxin(Novartis), PEG-relaxin (Ambrx and Bristol-Myers Squibb), ANG-4011(Angion Biomedica), FT011 (Fibrotech Therapeutics), pirfenidone(InterMune), F351 (pirfenidone derivative (GNI Pharma), vitamin E(e.g., tocotrienol (alpha, beta, gamma, and delta) and tocopherol(alpha, beta, gamma, and delta)), pentoxifylline, an insulinsensitizer (e.g., rosiglitazone and pioglitazone), cathepsin Binhibitor R-3020, etanercept and biosimilars thereof, peptides thatblock the activation of Fas (see, e.g., International PublicationNo. WO 2005/117940, incorporated herein by reference), caspaseinhibitor VX-166, caspase inhibitor Z-VAD-fmk, fasudil, belnacasan(VX-765), and pralnacasan (VX-740).

[0627] Exemplary additional therapeutic agents are provided belowand include exemplary drug classes, and exemplary embodiments foreach, that may be detected and analyzed using the methodsherein.

1. TNF Inhibitors

[0628] The term "TNF.alpha. inhibitor" refers to an agent whichdirectly or indirectly inhibits, impairs, reduces, down-regulates,or blocks TNF.alpha. activity and/or expression. In someembodiments, a TNF.alpha. inhibitor is an inhibitory nucleic acid,an antibody or an antigen-binding fragment thereof, a fusionprotein, a soluble TNF.alpha. receptor (a soluble TNFR1 or asoluble TNFR2), or a small molecule TNF.alpha. antagonist. In someembodiments, the inhibitory nucleic acid is a ribozyme, smallhairpin RNA, a small interfering RNA, an antisense nucleic acid, oran aptamer.

[0629] Exemplary TNF.alpha. inhibitors that directly inhibit,impair, reduce, down-regulate, or block TNF.alpha. activity and/orexpression can, e.g., inhibit or reduce binding of TNF.alpha. toits receptor (TNFR1 and/or TNFR2) and/or inhibit or decrease theexpression level of TNF.alpha. or a receptor of TNF.alpha. (TNFR1or TNFR2) in a cell (e.g., a mammalian cell). Non-limiting examplesof TNF.alpha. inhibitors that directly inhibit, impair, reduce,down-regulate, or block TNF.alpha. activity and/or expressioninclude inhibitory nucleic acids (e.g., any of the examples ofinhibitory nucleic acids described herein), an antibody or fragmentthereof, a fusion protein, a soluble TNF.alpha. receptor (e.g., asoluble TNFR1 or soluble TNFR2), and a small molecule TNF.alpha.antagonist.

[0630] Exemplary TNF.alpha. inhibitors that can indirectly inhibit,impair, reduce, down-regulate, or block TNF.alpha. activity and/orexpression can, e.g., inhibit or decrease the level of downstreamsignaling of a TNF.alpha. receptor (e.g., TNFR1 or TNFR2) in amammalian cell (e.g., decrease the level and/or activity of one ormore of the following signaling proteins: TRADD, TRAF2, MEKK1/4,MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK, andNF-.kappa.B in a mammalian cell), and/or decrease the level ofTNF.alpha.-induced gene expression in a mammalian cell (e.g.,decrease the transcription of genes regulated by, e.g., one or moretranscription factors selected from the group of NF-.kappa.B,c-Jun, and ATF2). A description of downstream signaling of aTNF.alpha. receptor is provided in Waj ant et al., Cell DeathDifferentiation 10:45-65, 2003 (incorporated herein by reference).For example, such indirect TNF.alpha. inhibitors can be aninhibitory nucleic acid that targets (decreases the expression) asignaling component downstream of a TNF.alpha. receptor (e.g., anyone or more of the signaling components downstream of a TNF.alpha.receptor described herein or known in the art), aTNF.alpha.-induced gene (e.g., any TNF.alpha.-induced gene known inthe art), or a transcription factor selected from the group ofNF-.kappa.B, c-Jun, and ATF2.

[0631] In other examples, such indirect TNF.alpha. inhibitors canbe a small molecule inhibitor of a signaling component downstreamof a TNF.alpha. receptor (e.g., any of the signaling componentsdownstream of a TNF.alpha. receptor described herein or known inthe art), a small molecule inhibitor of a protein encoded by aTNF.alpha.-induced gene (e.g., any protein encoded by aTNF.alpha.-induced gene known in the art), and a small moleculeinhibitor of a transcription factor selected from the group ofNF-.kappa.B, c-Jun, and ATF2.

[0632] In other embodiments, TNF.alpha. inhibitors that canindirectly inhibit, impair, reduce, down-regulate, or block one ormore components in a mammalian cell (e.g., a macrophage, a CD4+lymphocyte, a NK cell, a neutrophil, a mast cell, a eosinophil, ora neuron) that are involved in the signaling pathway that resultsin TNF.alpha. mRNA transcription, TNF.alpha. mRNA stabilization,and TNF.alpha. mRNA translation (e.g., one or more componentsselected from the group of CD14, MyD88, IRAK, lipopolysaccharidebinding protein (LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK,I.kappa.B, NF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR,TTP, and MK2). For example, such indirect TNF.alpha. inhibitors canbe an inhibitory nucleic acid that targets (decreases theexpression) of a component in a mammalian cell that is involved inthe signaling pathway that results in TNF.alpha. mRNAtranscription, TNF.alpha. mRNA stabilization, and TNF.alpha. mRNAtranslation (e.g., a component selected from the group of CD14,MyD88, IRAK, lipopolysaccharide binding protein (LBP), TRAF6, ras,raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7,JNK, c-jun, MEK3/6, p38, PKR, TTP, and MK2). In other examples, anindirect TNF.alpha. inhibitors is a small molecule inhibitor of acomponent in a mammalian cell that is involved in the signalingpathway that results in TNF.alpha. mRNA transcription, TNF.alpha.mRNA stabilization, and TNF.alpha. mRNA translation (e.g., acomponent selected from the group of CD14, MyD88, IRAK,lipopolysaccharide binding protein (LBP), TRAF6, ras, raf, MEK1/2,ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7, JNK, c-jun,MEK3/6, p38, PKR, TTP, and MK2).

Inhibitory Nucleic Acids

[0633] Inhibitory nucleic acids that can decrease the expression ofTNF.alpha., TNFR1, TNFR2, TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK,AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK, NF-.kappa.B, CD14,MyD88, IRAK, lipopolysaccharide binding protein (LBP), TRAF6, ras,raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7,JNK, c-jun, MEK3/6, p38, PKR, TTP, or MK2 mRNA expression in amammalian cell include antisense nucleic acid molecules, i.e.,nucleic acid molecules whose nucleotide sequence is complementaryto all or part of a TNF.alpha., TNFR1, TNFR2, TRADD, TRAF2,MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK,NF-.kappa.B, CD14, MyD88, IRAK, lipopolysaccharide binding protein(LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.BNF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR, TTP, or MK2mRNA.

[0634] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding a TNF.alpha., TNFR1, TNFR2, TRADD,TRAF2, MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK,IKK, NF-.kappa.B, CD14, MyD88, IRAK, lipopolysaccharide bindingprotein (LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK,I.kappa.B, NF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR,TTP, or MK2 protein. Non-coding regions (5' and 3' untranslatedregions) are the 5' and 3' sequences that flank the coding regionin a gene and are not translated into amino acids.

[0635] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a TNF.alpha.,TNFR1, TNFR2, TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP,MEKK 3/6, MAPK, NIK, IKK, I.kappa.B, NF-.kappa.B, CD14, MyD88,IRAK, lipopolysaccharide binding protein (LBP), TRAF6, ras, raf,MEK1/2, ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7, JNK,c-jun, MEK3/6, p38, PKR, TTP, or MK2 protein (e.g., specificity fora TNF.alpha., TNFR1, TNFR2, TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK,AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK, NF-.kappa.B, CD14,MyD88, IRAK, lipopolysaccharide binding protein (LBP), TRAF6, ras,raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7,JNK, c-jun, MEK3/6, p38, PKR, TTP, or MK2 mRNA.

[0636] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a TNF.alpha., TNFR1, TNFR2, TRADD, TRAF2, MEKK1/4,MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK,NF-.kappa.B, CD14, MyD88, IRAK, lipopolysaccharide binding protein(LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B,NF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR, TTP, or MK2polypeptide can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding theTNF.alpha., TNFR1, TNFR2, TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK,AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK, NF-.kappa.B, CD14,MyD88, IRAK, lipopolysaccharide binding protein (LBP), TRAF6, ras,raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B, NF-.kappa.B, rac, MEK4/7,JNK, c-jun, MEK3/6, p38, PKR, TTP, or MK2 polypeptide (e.g., thepromoter and/or enhancer, e.g., a sequence that is at least 1 kb, 2kb, 3 kb, 4 kb, or 5 kb upstream of the transcription initiationstart state) to form triple helical structures that preventtranscription of the gene in target cells.

[0637] In some embodiments, a TNF.alpha. inhibitor can be a siRNAmolecule used to decrease expression of a TNF.alpha., TNFR1, TNFR2,TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6,MAPK, NIK, IKK, NF-.kappa.B, CD14, MyD88, IRAK, lipopolysaccharidebinding protein (LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK,I.kappa.B, NF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR,TTP, or MK2 mRNA.

[0638] Exemplary TNF.alpha. inhibitors that are inhibitory nucleicacids targeting TNF.alpha. include, e.g., antisense DNA (e.g.,Myers et al., J Pharmacol Exp Ther. 304(1):411-424, 2003; Wasmuthet al., Invest. Opthalmol. Vis. Sci, 2003; Dong et al., J. Orthop.Res. 26(8):1114-1120, 2008; U.S. Patent Application Serial Nos.2003/0083275, 2003/0022848, and 2004/0770970; ISIS 104838; U.S.Pat. Nos. 6,180,403, 6,080,580, and 6,228,642; Kobzik et al.,Inhibition of TNF Synthesis by Antisense Oligonucleotides, inManual of Antisense Methodology, Kluwer Academic Publishers, Vol.4, pp. 107-123, 1999; Taylor et al., Antisense Nucleic Acid DrugDevelop. 8(3):199-205, 1998; Mayne et al., Stroke 32:240-248, 2001;Mochizuki et al., J. Controlled Release 151(2):155-161, 2011; Donget al., J. Orthopaedic Res. 26(8):1114-1120, 2008; Dong et al.,Pharm. Res. 28(6):1349-1356, 2011; and Pampfer et al., Biol.Reproduction 52(6):1316-1326, 1995), antisense RNA, shortinterfering RNA (siRNA) (e.g., Taishi et al., Brain Research1156:125-132, 2007; Presumey et al., Eur. J. Pharm. Biopharm.82(3):457-467, 2012; Laroui et al., J. Controlled Release186:41-53, 2014; D'Amore et al., Int. J Immunopathology Pharmacol.21:1045-1047, 2008; Choi et al., J. Dermatol. Sci. 52:87-97, 2008;Qin et al., Artificial Organs 35:706-714, 2011; McCarthy et al., J.Controlled Release 168: 28-34, 2013; Khoury et al., Current Opin.Mol. Therapeutics 9(5):483-489, 2007; Lu et al., RNA InterferenceTechnology From Basic Science to Drug Development 303, 2005; Xie etal., PharmaGenomics 4(6):28-34, 2004; Aldawsari et al., CurrentPharmaceutical Design 21(31):4594-4605, 2015; Zheng et al., Arch.Med. Sci. 11:1296-1302, 2015; Peng et al., Chinese J. Surgery47(5):377-380, 2009; Aldayel et al., Molecular Therapy. NucleicAcids 5(7):e340, 2016; Bai et al., Current Drug Targets16:1531-1539, 2015; U.S. Patent Application Publications Nos.2008/0097091, 2009/0306356, and 2005/0227935; and WO 14/168264),short hairpin RNA (shRNA) (e.g., Jakobsen et al., Mol. Ther.17(10): 1743-1753, 2009; Ogawa et al., PLoS One 9(3): e92073, 2014;Ding et al., Bone Joint 94-6(Suppl. 11):44, 2014; andHernandez-Alejandro et al., J. Surgical Res. 176(2):614-620, 2012),and microRNAs (see, e.g., WO 15/26249). In some embodiments, theinhibitory nucleic acid blocks pre-mRNA splicing of TNF.alpha.(e.g., Chiu et al., Mol. Pharmacol. 71(6): 1640-1645, 2007).

[0639] In some embodiments, the inhibitory nucleic acid, e.g., anaptamer (e.g., Orava et al., ACS Chem Biol. 2013; 8(1): 170-178,2013), can block the binding of a TNF.alpha. protein with itsreceptor (TNFR1 and/or TNFR2).

[0640] In some embodiments, the inhibitory nucleic acid candown-regulate the expression of a TNF.alpha.-induced downstreammediator (e.g., TRADD, TRAF2, MEKK1/4, MEKK4/7, JNK, AP-1, ASK1,RIP, MEKK 3/6, MAPK, NIK, IKK, NF-.kappa.B, p38, JNK,I.kappa.B-.alpha., or CCL2). Further teachings of downstreamTNF.alpha.-induced mediators can be found in, e.g., Schwamborn etal., BMC Genomics 4:46, 2003; and Zhou et al., Oncogene 22:2034-2044, 2003, incorporated by reference herein. Additionalaspects of inhibitory nucleic acids are described in Aagaard etal., Adv. Drug Delivery Rev. 59(2):75-86, 2007, and Burnett et al.,Biotechnol. J. 6(9):1130-1146, 2011.

[0641] In certain embodiments, the inhibitory nucleic acid targetsa nucleic acid encoding a TNF.alpha., TNFR1, TNFR2, TRADD, TRAF2,MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK,NF-.kappa.B, CD14, MyD88, IRAK, lipopolysaccharide binding protein(LBP), TRAF6, ras, raf, MEK1/2, ERK1/2, NIK, IKK, I.kappa.B,NF-.kappa.B, rac, MEK4/7, JNK, c-jun, MEK3/6, p38, PKR, TTP, orMK2.

Antibodies

[0642] In some embodiments, the TNF.alpha. inhibitor is an antibodyor an antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, an antibody or antigen-binding fragment describedherein binds specifically to any one of TNF.alpha., TNFR1, orTNFR2. In some embodiments, an antibody or antigen-binding fragmentof an antibody described herein can bind specifically toTNF.alpha.. In some embodiments, an antibody or antigen-bindingfragment of an antibody described herein can bind specifically to aTNF.alpha. receptor (TNFR1 or TNFR2).

[0643] Non-limiting examples of TNF inhibitors that are antibodiesthat specifically bind to TNF.alpha. are described in Elliott etal., Lancet 1994; 344: 1125-1127, 1994; Rankin et al., Br. J.Rheumatol. 2:334-342, 1995; Butler et al., Eur. Cytokine Network6(4):225-230, 1994; Lorenz et al., J Immunol. 156(4):1646-1653,1996; Hinshaw et al., Circulatory Shock 30(3):279-292, 1990; Wanneret al., Shock 11(6):391-395, 1999; Bongartz et al., JAMA295(19):2275-2285, 2006; Knight et al., Molecular Immunol.30(16):1443-1453, 1993; Feldman, Nature Reviews Immunol.2(5):364-371, 2002; Taylor et al., Nature Reviews Rheumatol.5(10):578-582, 2009; Garces et al., Annals Rheumatic Dis.72(12):1947-1955, 2013; Palladino et al., Nature Rev. DrugDiscovery 2(9):736-746, 2003; Sandborn et al., Inflammatory BowelDiseases 5(2):119-133, 1999; Atzeni et al., Autoimmunity Reviews12(7):703-708, 2013; Maini et al., Immunol. Rev. 144(1):195-223,1995; Ordas et al., Clin. Pharmacol. Therapeutics 91(4):635-646,2012; Cohen et al., Canadian J. Gastroenterol. Hepatol.15(6):376-384, 2001; Feldmann et al., Ann. Rev. Immunol.19(1):163-196, 2001; Ben-Horin et al., Autoimmunity Rev.13(1):24-30, 2014; and U.S. Pat. Nos. 6,090,382; 6,258,562; and6,509,015).

[0644] In certain embodiments, the TNF.alpha. inhibitor can includeor is infliximab (Remicade.TM.), CDP571, CDP 870, golimumab(Golimumab.TM.), adalimumab (Humira.TM.), or certolizumab pegol(Cimzia.TM.). In certain embodiments, the TNF.alpha. inhibitor canbe a TNF.alpha. inhibitor biosimilar. Examples of approved andlate-phase TNF.alpha. inhibitor biosimilars include, but are notlimited to, infliximab biosimilars such as Remsima.TM. andInflectra.RTM. (CT-P13) from Celltrion/Pfizer, GS071 from Aprogen,Flixabi.TM. (SB2) from Samsung Bioepis, PF-06438179 fromPfizer/Sandoz, NI-071 from Nichi-Iko Pharmaceutical Co., and ABP710 from Amgen; adalimumab biosimilars such as Exemptia.TM.(ZRC3197) from Zydus Cadila, India, Solymbic.RTM. and Amgevita.RTM.(ABP 501) from Amgen, Imraldi (SB5) from Samsung Bioepis, GP-2017from Sandoz, Switzerland, ONS-3010 from Oncobiologics,M923/Viropro, U.S.A., from Momenta Pharmaceuticals/Baxalta (Baxterspinoff USA), PF-06410293 from Pfizer, BMO-2 or MYL-1401-A fromBiocon/Mylan, CHS-1420 from Coherus, FKB327 from Fujifilm/KyowaHakko Kirin (Fujifilm Kyowa Kirin Biologics), and Cyltezo (BI695501) from Boehringer Ingelheim, CT-P17 from Celltrion, BAX 923from Baxalta (now a part of Shire), MSB11022 from Fresenius Kabi(bought from Merck kGaA (Merck Group) in 2017), LBAL from LG LifeSciences/Mochida Pharmaceutical, South Korea/Japan, PBP1502 fromPrestige Biopharma, Adfrar from Torrent Pharmaceuticals, India, abiosimilar of adalimumab in development by Adello Biologics, abiosimilar of adalimumab in development by AET Biotech/BioXpressTherapeutics, Germany/Switzerland, a biosimilar of adalimumab frommAbxience, Spain, a biosimilar of adalimumab in development byPlantForm, Canada; and etanercept biosimilars such as Erelzi.TM.from Sandoz/Novartis, Brenzys.TM. (SB4) from Samsung Bioepis,GP2015 from Sandoz, TuNEX.RTM. from Mycenax, LBEC0101 from LG Life,and CHS-0214 from Coherus.

[0645] In some embodiments, a biosimilar is an antibody orantigen-binding fragment thereof that has a light chain that hasthe same primary amino acid sequence as compared to a referenceantibody (e.g., adalimumab) and a heavy chain that has the sameprimary amino acid sequence as compared to the reference antibody.In some examples, a biosimilar is an antibody or antigen-bindingfragment thereof that has a light chain that includes the samelight chain variable domain sequence as a reference antibody (e.g.,adalimumab) and a heavy chain that includes the same heavy chainvariable domain sequence as a reference antibody. In someembodiments, a biosimilar can have a similar glycosylation patternas compared to the reference antibody (e.g., adalimumab). In otherembodiments, a biosimilar can have a different glycosylationpattern as compared to the reference antibody (e.g., adalimumab).Changes in the N-linked glycosylation profile of a biosimilar ascompared to a reference antibody (e.g., adalimumab) can be detectedusing 2-anthranilic acid (AA)-derivatization and normal phaseliquid chromatography with fluorescence detection, as generallydescribed in Kamoda et al., J. Chromatography J. 1133:332-339,2006. For example, a biosimilar can have changes in one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, oreleven) of the following types of N-glycosylation as compared tothe reference antibody (e.g., adalimumab): neutrally-chargedoligosaccharides; monosialylated fucose-containingoligosaccharides; monosialylated oligosaccharides; bisialylatedfucose-containing oligosaccharide; bisialylated oligosaccharides;triantennary, trisiaylated oligosaccharides of form 1;triantennary, trisialylated oligosaccharides of form 2;mannose-6-phosphate oligosaccharides; monophosphorylatedoligosaccharides; tetrasialylated oligosaccharides; monosialylatedand monophosphorylated oligosaccharides; andbis-mannose-6-phosphate oligosaccharides.

[0646] In some embodiments, the biosimilar can have a change inone, two, or three of: the percentage of species having oneC-terminal lysine, the percentage of species having two C-terminallysines, and the percentage of species having three C-terminallysines as compared to the reference antibody (e.g.,adalimumab).

[0647] In some embodiments, the biosimilar can have a change in thelevel of one, two, or three of acidic species, neutral species, andbasic species in the composition as compared to the referenceantibody (e.g., adalimumab).

[0648] In some embodiments, the biosimilar can have a change in thelevel of sulfation as compared to the reference antibody.

[0649] In some embodiments, the TNF.alpha. inhibitor can beSAR252067 (e.g., a monoclonal antibody that specifically binds toTNFSF14, described in U.S. Patent Application Publication No.2013/0315913) or MDGN-002 (described in U.S. Patent ApplicationPublication No. 2015/0337046). In some embodiments, the TNF.alpha.inhibitor can be PF-06480605, which binds specifically to TNFSF15(e.g., described in U.S. Patent Application Publication No.2015/0132311). Additional examples of TNF.alpha. inhibitors includeDLCX105 (described in Tsianakas et al., Exp. Dermatol. 25:428-433,2016) and PF-06480605, which binds specifically to TNFSF15(described in U.S. Patent Application Publication No.2015/0132311).

Fusion Proteins

[0650] In some embodiments, the TNF.alpha. inhibitory agent is afusion protein (e.g., an extracellular domain of a TNFR fused to apartner peptide, e.g., an Fc region of an immunoglobulin, e.g.,human IgG) (see, e.g., Peppel et al., J. Exp. Med.174(6):1483-1489, 1991; Deeg et al., Leukemia 16(2):162, 2002) or asoluble TNFR (e.g., TNFR1 or TNFR2) that binds specifically toTNF.alpha.. In some embodiments, the TNF.alpha. inhibitor includesor is etanercept (Enbrel.TM.) (see, e.g., WO 91/03553 and WO09/406,476, incorporated by reference herein). In some embodiments,the TNF.alpha. inhibitor includes or is r-TBP-I (e.g., Gradstein etal., J. Acquir. Immune Defic. Syndr. 26(2): 111-117, 2001). In someembodiments, the TNF.alpha. inhibitor includes or is a solubleTNF.alpha. receptor (e.g., Watt et al., J Leukoc Biol.66(6):1005-1013, 1999; Tsao et al., Eur Respir J. 14(3):490-495,1999; Kozak et al., Am. J. Physiol. Reg. Integrative ComparativePhysiol. 269(1):R23-R29, 1995; Mohler et al., J Immunol.151(3):1548-1561, 1993; Nophar et al., EMBO J 9(10):3269, 1990;Bjornberg et al., Lymphokine Cytokine Res. 13(3):203-211, 1994;Piguet et al., Eur. Respiratory J. 7(3):515-518, 1994; and Gray etal., Proc. Natl. Acad. Sci. U.S.A. 87(19):7380-7384, 1990).

Small Molecules

[0651] In some embodiments, the TNF.alpha. inhibitor is a smallmolecule. In some embodiments, the TNF.alpha. inhibitor is C87 (Maet al., J. Biol. Chem. 289(18):12457-66, 2014). In someembodiments, the small molecule is LMP-420 (e.g., Haraguchi et al.,AIDS Res. Ther. 3:8, 2006). In some embodiments, the small moleculeis a tumor necrosis factor-converting enzyme (TACE) inhibitor(e.g., Moss et al., Nature Clinical Practice Rheumatology 4:300-309, 2008). In some embodiments, the TACE inhibitor is TMI-005and BMS-561392. Additional examples of small molecule inhibitorsare described in, e.g., He et al., Science 310(5750):1022-1025,2005.

[0652] In some examples, the TNF.alpha. inhibitor is a smallmolecule that inhibits the activity of one of TRADD, TRAF2,MEKK1/4, MEKK4/7, JNK, AP-1, ASK1, RIP, MEKK 3/6, MAPK, NIK, IKK,and NF-.kappa.B, in a mammalian cell.

[0653] In some examples, the TNF.alpha. inhibitor is a smallmolecule that inhibits the activity of one of CD14, MyD88 (see,e.g., Olson et al., Scientific Reports 5:14246, 2015), IRAK(Chaudhary et al., J. Med. Chem. 58(1):96-110, 2015),lipopolysaccharide binding protein (LBP) (see, e.g., U.S. Pat. No.5,705,398), TRAF6 (e.g.,3-[(2,5-Dimethylphenyl)amino]-1-phenyl-2-propen-1-one), ras (e.g.,Baker et al., Nature 497:577-578, 2013), raf (e.g., vemurafenib(PLX4032, RG7204), sorafenib tosylate, PLX-4720, dabrafenib(GSK2118436), GDC-0879, RAF265 (CHIR-265), AZ 628, NVP-BHG712,SB590885, ZM 336372, sorafenib, GW5074, TAK-632, CEP-32496,encorafenib (LGX818), CCT196969, LY3009120, RO5126766 (CH5126766),PLX7904, and MLN2480), MEK1/2 (e.g., Facciorusso et al., ExpertReview Gastroentrol. Hepatol. 9:993-1003, 2015), ERK1/2 (e.g.,Mandal et al., Oncogene 35:2547-2561, 2016), NIK (e.g., Mortier etal., Bioorg. Med. Chem. Lett. 20:4515-4520, 2010), IKK (e.g.,Reilly et al., Nature Med. 19:313-321, 2013), I.kappa.B (e.g.,Suzuki et al., Expert. Opin. Invest. Drugs 20:395-405, 2011),NF-.kappa.B (e.g., Gupta et al., Biochim. Biophys. Acta1799(10-12):775-787, 2010), rac (e.g., U.S. Pat. No. 9,278,956),MEK4/7, JNK (e.g., AEG 3482, BI 78D3, CEP 1347, c-JUN peptide, IQ1S, JIP-1 (153-163), SP600125, SU 3327, and TCS JNK6o), c-jun(e.g., AEG 3482, BI 78D3, CEP 1347, c-JUN peptide, IQ 1S, JIP-1(153-163), SP600125, SU 3327, and TCS JNK6o), MEK3/6 (e.g.,Akinleye et al., I Hematol. Oncol. 6:27, 2013), p38 (e.g., AL 8697,AMG 548, BIRB 796, CMPD-1, DBM 1285 dihydrochloride, EO 1428, JX401, ML 3403, Org 48762-0, PH 797804, RWJ 67657, SB 202190, SB203580, SB 239063, SB 706504, SCIO 469, SKF 86002, SX 011, TA 01,TA 02, TAK 715, VX 702, and VX 745), PKR (e.g., 2-aminopurine orCAS 608512-97-6), TTP (e.g., CAS 329907-28-0), and MK2 (PF 3644022and PHA 767491).

2. IL-12/IL-23 Inhibitors

[0654] The term "IL-12/IL-23 inhibitors" refers to an agent whichdecreases IL-12 or IL-23 expression and/or the ability of IL-12 tobind to an IL-12 receptor or the ability of IL-23 to bind to anIL-23 receptor. IL-12 is a heterodimeric cytokine that includesboth IL-12A (p35) and IL-12B (p40) polypeptides. IL-23 is aheterodimeric cytokine that includes both IL-23 (p19) and IL-12B(p40) polypeptides. The receptor for IL-12 is a heterodimericreceptor includes IL-12R .beta.1 and IL-12R .beta.2. The receptorfor IL-23 receptor is a heterodimeric receptor that includes bothIL-12R .beta.1 and IL-23R.

[0655] In some embodiments, the IL-12/IL-23 inhibitor can decreasethe binding of IL-12 to the receptor for IL-12. In someembodiments, the IL-12/IL-23 inhibitor can decrease the binding ofIL-23 to the receptor for IL-23. In some embodiments, theIL-12/IL-23 inhibitor decreases the expression of IL-12 or IL-23.In some embodiments, the IL-12/IL-23 inhibitor decreases theexpression of a receptor for IL-12. In some embodiments, theIL-12/IL-23 inhibitor decreases the expression of a receptor forIL-23.

[0656] In some embodiments, the IL-12/IL-23 inhibitory agenttargets IL-12B (p40) subunit. In some embodiments, the IL-12/IL-23inhibitory agent targets IL-12A (p35). In some embodiments, theIL-12/IL-23 inhibitory agent targets IL-23 (p19). In someembodiments, the IL-12/IL-23 inhibitory agent targets the receptorfor IL-12 (one or both of IL-12R .beta.1 or IL-12R .beta.2). Insome embodiments, the IL-12/IL-23 inhibitory agent targets thereceptor for IL-23 (one or both of IL-12R .beta.1 and IL-23R).

[0657] In some embodiments, an IL-12/IL-23 inhibitor can be aninhibitory nucleic acid. In some embodiments, the inhibitorynucleic acid can be an antisense nucleic acid, a ribozyme, and asmall interfering RNA (siRNA).

[0658] Inhibitory nucleic acids that can decrease the expression ofIL-12A (p35), IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R.beta.2, or IL-23R mRNA expression in a mammalian cell includeantisense nucleic acid molecules, i.e., nucleic acid moleculeswhose nucleotide sequence is complementary to all or part of anIL-12A (p35), IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R.beta.2, or IL-23R mRNA. An antisense nucleic acid molecule can becomplementary to all or part of a non-coding region of the codingstrand of a nucleotide sequence encoding an IL-12A (p35), IL-12B(p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2, or IL-23Rprotein. Non-coding regions (5' and 3' untranslated regions) arethe 5' and 3' sequences that flank the coding region in a gene andare not translated into amino acids.

[0659] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding an IL-12A (p35),IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2, orIL-23R protein (e.g., specificity for an IL-12A (p35), IL-12B(p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2, or IL-23RmRNA).

[0660] An inhibitor nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of an IL-12A (p35), IL-12B (p40), IL-23 (p19), IL-12R.beta.1, IL-12R .beta.2, or IL-23R protein can be inhibited bytargeting nucleotide sequences complementary to the regulatoryregion of the gene encoding the IL-12A (p35), IL-12B (p40), IL-23(p19), IL-12R .beta.1, IL-12R .beta.2, or IL-23R protein (e.g., thepromoter and/or enhancer, e.g., a sequence that is at least 1 kb, 2kb, 3 kb, 4 kb, or 5 kb upstream of the transcription initiationstart state) to form triple helical structures that preventtranscription of the gene in target cells.

[0661] Other examples of a IL-12/IL-23 inhibitor include siRNA thatdecrease the level of IL-12A (p35), IL-12B (p40), IL-23 (p19),IL-12R .beta.1, IL-12R .beta.2, or IL-23R mRNA.

[0662] Non-limiting examples of siRNAs targeting IL-12A (p35),IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2, orIL-23R are described in Tan et al., J. Alzheimers Dis. 38(3):633-646, 2014; Niimi et al., J. Neuroimmunol. 254(1-2):39-45, 2013.Non-limiting examples of short hairpin RNA (shRNA) targeting IL-12A(p35), IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2,or IL-23R are described in Bak et al., BMC Dermatol. 11:5,2011.

[0663] Non-limiting examples of inhibitory nucleic acids aremicroRNAs (e.g., microRNA-29 (Brain et al., Immunity 39(3):521-536,2013), miR-10a (Xue et al., J. Immunol. 187(11):5879-5886, 2011),microRNA-155 (Podsiad et al., Am. J. Physiol. Lung Cell Mol.Physiol. 310(5):L465-75, 2016).

Antibodies

[0664] In some embodiments, the IL-12/IL-23 inhibitor is anantibody or an antigen-binding fragment thereof (e.g., a Fab or ascFv). In some embodiments, an antibody or antigen-binding fragmentdescribed herein binds specifically to any one of IL-12A (p35),IL-12B (p40), IL-23 (p19), IL-12R .beta.1, IL-12R .beta.2, orIL-23R, or a combination thereof.

[0665] In some embodiments, the antibody is ustekinumab (CNTO 1275,Stelara.RTM.) or a variant thereof (Krueger et al., N Engl. J. Med.356(6):580-592, 2007; Kauffman et al., J. Invest. Dermatol.123(6):1037-1044, 2004; Gottlieb et al., Curr. Med. Res. Opin.23(5):1081-1092, 2007; Leonardi et al., Lancet 371(9625):1665-1674,2008; Papp et al., Lancet 371(9625):1675-1684, 2008). In someembodiments, the antibody is briakinumab (ABT-874, J-695) or avariant thereof (Gordon et al., J. Invest. Dermatol.132(2):304-314, 2012; Kimball et al., Arch Dermatol. 144(2):200-207, 2008).

[0666] In some embodiments, the antibody is guselkumab (CNTO-1959)(Callis-Duffin et al., J. Am. Acad. Dermatol. 70(5 Suppl 1), 2014);AB162 (Sofen et al., J. Allergy Clin. Immunol. 133: 1032-40, 2014);tildrakizumab (MK-3222, SCH900222) (Papp et al. (2015) Br. J.Dermatol. 2015); Langley et al., Oral Presentation at: AmericanAcademy of Dermatology, March 21-25, Denver Colo., 2014); AMG 139(MEDI2070, brazikumab) (Gomollon, Gastroenterol. Hepatol. 38(Suppl.1):13-19, 2015; Kock et al., Br. J. Pharmacol. 172(1):159-172,2015); FM-202 (Tang et al., Immunology 135(2):112-124, 2012);FM-303 (Tang et al., Immunology 135(2):112-124, 2012); ADC-1012(Tang et al., Immunology 135(2):112-124, 2012); LY-2525623 (Gaffenet al., Nat. Rev. Immunol. 14:585-600, 2014; Sands, Gastroenterol.Hepatol. 12(12):784-786, 2016), LY-3074828 (Coskun et al., TrendsPharmacol. Sci. 38(2):127-142, 2017), BI-655066 (risankizumab)(Singh et al., MAbs 7(4):778-791, 2015; Krueger et al., J. AllergyClin. Immunol. 136(1):116-124, 2015) or a variant thereof.

[0667] See e.g., Tang et al., Immunology 135(2):112-124, 2012.Further teachings of IL-12/IL-23 antibodies and antigen-bindingfragments thereof are described in U.S. Pat. Nos. 6,902,734;7,247,711; 7,252,971; and 7,491,391; US 2012/0288494; and US2013/0302343, each of which is incorporated by reference in itsentirety.

[0668] In some embodiments, the IL-12/IL-23 inhibitor is PTG-200,an IL-23R inhibitor currently in preclinical development byProtagonist Therapeutics.

[0669] In some embodiments, the IL-12/IL-23 inhibitor isMirikizumab (LY 3074828), an IL-23R inhibitor currently in clinicaldevelopment (Phase II) by Eli Lilly.

Fusion Proteins

[0670] In some embodiments, the IL-12/IL-23 inhibitor is a fusionprotein, a soluble antagonist, or an antimicrobial peptide. In someembodiments, the fusion protein comprises a soluble fragment of areceptor of IL-12 or a soluble fragment of a receptor of IL-23. Insome embodiments, the fusion protein comprises an extracellulardomain of a receptor of IL-12 or an extracellular domain of areceptor of IL-23.

[0671] In some embodiments, the fusion protein is adnectin or avariant thereof (Tang et al., Immunology 135(2):112-124, 2012). Insome embodiments, the soluble antagonist is a human IL-23Ra-chainmRNA transcript (Raymond et al., J. Immunol. 185(12):7302-7308,2010). In some embodiments, the IL-12/IL-23 is an antimicrobialpeptide (e.g., MP-196 (Wenzel et al., PNAS 111(14):E1409-E1418,2014)).

Small Molecules

[0672] In some embodiments, the IL-12/IL-23 inhibitor is a smallmolecule. In some embodiments, the small molecule is STA-5326(apilimod) or a variant thereof (Keino et al., Arthritis Res. Ther.10: R122, 2008; Wada et al., Blood 109(3):1156-1164, 2007; Sands etal., Inflamm. Bowel Dis. 16(7):1209-1218, 2010).

3. IL-6 Receptor Inhibitors

[0673] The term "IL-6 receptor inhibitor" refers to an agent whichdecreases IL-6 receptor expression and/or the ability of IL-6 tobind to an IL-6 receptor. In some embodiments, the IL-6 receptorinhibitor targets the IL-6 receptor .beta.-subunit, glycoprotein130 (sIL6gp130). In other embodiments, the IL-6 receptor inhibitortargets the IL-6 receptor subunit (IL6R). In other embodiments, theIL-6 receptor inhibitor targets the complex consisting of both theIL-6 receptor subunit (IL6R) and the IL-6 receptor .beta.-subunit,glycoprotein 130 (sIL6gp130). In some embodiments, the IL-6receptor inhibitor targets IL-6.

[0674] In some embodiments, an IL-6 receptor inhibitor is aninhibitory nucleic acid, an antibody or an antigen-binding fragmentthereof, a fusion protein, a IL-6 receptor antagonist, or a smallmolecule. In some embodiments, the inhibitory nucleic acid is asmall interfering RNA, an antisense nucleic acid, an aptamer, or amicroRNA. Exemplary IL-6 receptor inhibitors are described herein.Additional examples of IL-6 receptor inhibitors are known in theart.

[0675] Exemplary aspects of different inhibitory nucleic acids aredescribed below. Any of the examples of inhibitory nucleic acidsthat can decrease expression of an IL6R, sIL6gp130, or IL-6 mRNA.Inhibitory nucleic acids that can decrease the expression of IL6R,sIL6gp130, or IL-6 mRNA in a mammalian cell include antisensenucleic acid molecules, i.e., nucleic acid molecules whosenucleotide sequence is complementary to all or part of an IL6R,sIL6gp130, or IL-6 mRNA.

Inhibitory Nucleic Acids

[0676] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding an IL6R, sIL6gp130, or IL-6 protein.Non-coding regions (5' and 3' untranslated regions) are the 5' and3' sequences that flank the coding region in a gene and are nottranslated into amino acids. Exemplary antisense nucleic acids thatare IL-6 receptor inhibitors are described in Keller et al., J.Immunol. 154(8):4091-4098, 1995; and Jiang et al., Anticancer Res.31(9): 2899-2906, 2011.

[0677] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding an IL6R,sIL6gp130, or IL-6 protein (e.g., specificity for an IL6R,sIL6gp130, or IL-6 mRNA).

[0678] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of an IL6R, sIL6gp130, or IL-6 polypeptide can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the gene encoding the IL6R, sIL6gp130, or IL-6polypeptide (e.g., the promoter and/or enhancer, e.g., a sequencethat is at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of thetranscription initiation start state) to form triple helicalstructures that prevent transcription of the gene in targetcells.

[0679] Additional examples of IL-6 receptor inhibitors includesiRNA that decrease the level of IL6R, sIL6gp130, or IL-6 mRNA.Non-limiting examples of short interfering RNA (siRNA) that areIL-6 receptor inhibitors are described in Yi et al., Int. J. Oncol.41(1):310-316, 2012; and Shinriki et al., Clin. Can. Res.15(17):5426-5434, 2009). Non-limiting examples of microRNAs thatare IL-6 receptor inhibitors are described in miR34a (Li et al.,Int. J. Clin. Exp. Pathol. 8(2):1364-1373, 2015) and miR-451 (Liuet al., Cancer Epidemiol. 38(1):85-92, 2014).

[0680] Non-limiting examples of aptamers that are IL-6 receptorinhibitors are described in Meyer et al., RNA Biol. 11(1):57-65,2014; Meyer et al., RNA Biol. 9(1):67-80, 2012; and Mittelberger etal., RNA Biol. 12(9):1043-1053, 2015. Additional examples ofinhibitory nucleic acids that are IL-6 receptor inhibitors aredescribed in, e.g., WO 96/040157.

Antibodies

[0681] In some embodiments, the IL-6 receptor inhibitor is anantibody or an antigen-binding fragment thereof (e.g., a Fab or ascFv). In some embodiments, an antibody or antigen-binding fragmentdescribed herein binds specifically to IL-6. In some embodiments,an antibody or antigen-binding fragment described herein bindsspecifically to IL-6 receptor (e.g., one or both of IL6R andsIL6gp130).

[0682] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of tocilizumab(artlizumab, Actemra.RTM.; Sebba, Am. J. Health Syst. Pharm.65(15):1413-1418, 2008; Tanaka et al., FEBS Letters585(23):3699-3709, 2011; Nishimoto et al., Arthritis Rheum.50:1761-1769, 2004; Yokota et al., Lancet 371(9617):998-1006, 2008;Emery et al., Ann. Rheum. Dis. 67(11):1516-1523, 2008; Roll et al.,Arthritis Rheum. 63(5):1255-1264, 2011); clazakizumab (BMS945429;ALD518, a humanized monoclonal antibody that binds circulating IL-6cytokine rather than the IL-6 receptor, blocking both classicsignaling and trans-signaling (Weinblatt, Michael E., et al. "TheEfficacy and Safety of Subcutaneous Clazakizumab in Patients WithModerate-to-Severe Rheumatoid Arthritis and an Inadequate Responseto Methotrexate: Results From a Multinational, Phase IIb,Randomized, Double-Blind, Placebo/Active-Controlled, Dose-RangingStudy." Arthritis & Rheumatology 67.10 (2015): 2591-2600.));sarilumab (REGN88 or SAR153191; Huizinga et al., Ann. Rheum. Dis.73(9):1626-1634, 2014; Sieper et al., Ann. Rheum. Dis.74(6):1051-1057, 2014; Cooper, Immunotherapy 8(3): 249-250, 2016);MR-16 (Hartman et al., PLosOne 11(12):e0167195, 2016; Fujita etal., Biochim. Biophys. Acta. 10:3170-80, 2014; Okazaki et al.,Immunol. Lett. 84(3):231-40, 2002; Noguchi-Sasaki et al., BMCCancer 16:270, 2016; Ueda et al., Sci. Rep. 3:1196, 2013); rhPM-1(MRA; Nishimoto et al., Blood 95: 56-61, 2000; Nishimoto et al.,Blood 106: 2627-2632, 2005; Nakahara et al., Arthritis Rheum.48(6): 1521-1529, 2003); NI-1201 (Lacroix et al., J. Biol. Chem.290(45):26943-26953, 2015); EBI-029 (Schmidt et al., ElevenBiotherapeutics Poster # B0200, 2014). In some embodiments, theantibody is a nanobody (e.g., ALX-0061 (Van Roy et al., ArthritisRes. Ther. 17: 135, 2015; Kim et al., Arch. Pharm. Res.38(5):575-584, 2015)). In some embodiments, the antibody is NRI ora variant thereof (Adachi et al., Mol. Ther. 11(1):5262-263, 2005;Hoshino et al., Can. Res. 67(3): 871-875, 2007). In someembodiments, the antibody is PF-04236921 (Pfizer) (Wallace et al.,Ann. Rheum. Dis. 76(3):534-542, 2017).

[0683] In some embodiments, the antibody is siltuximab(Sylvant.RTM.), also known as CNTO 328, a chimeric, human-murine,immunoglobulin (Ig) G.kappa. mAb that binds and neutralizes humanIL-6 with high affinity and specificity. The variable region ofsiltuximab is derived from a murine anti-IL-6 antibody, CLB8, andthe constant region is derived from a human IgG1.kappa. molecule.Sylvant.RTM. is approved for the treatment of patients withmulticentric Castleman's disease (MCD).

[0684] In some embodiments, the IL-6R inhibitor is AMG220, alsoknown as C326, an avimer that displays bi-specificity to itsinterleukin target, as well as binding to the Fc domain of IgG(resulting in reduced renal clearance and FcRn recycling). Thecompound has subpicomolar affinity for IL-6 and displays a moderateserum half-life (.about.30 h). Phase I clinical trials of AMG220 inCrohn's disease revealed dose-dependent reduction in serumC-reactive protein, an inflammation biomarker synthesized byhepatocytes in response to IL-6. Despite its apparent efficacy,Amgen has suspended the clinical development of the compound.

Fusion Proteins

[0685] In some embodiments, the IL-6 receptor inhibitor is a fusionprotein, a soluble receptor, or a peptide (see e.g., U.S. Pat. No.5,591,827). In some embodiments, the IL-6 receptor fusion proteincomprises or consists of soluble gp130 (Jostock et al., Eur. J.Biochem. 268(1):160-167, 2001; Richards et al., Arthritis Rheum.54(5):1662-1672, 2006; Rose-John et al., Exp. Opin. Ther. Targets11(5):613-624, 2007).

[0686] In some embodiments, the IL-6 receptor fusion proteincomprises or consists of FE999301 (Jostock et al., Eur. J. Biochem.268(1):160-167, 2001) or sgp130Fc protein (Jones et al., J. Clin.Invest. 121(9):3375-3383, 2011). In some embodiments, the IL-6receptor inhibitor is a peptide (e.g., S7 (Su et al., Cancer Res.65(11):4827-4835, 2005). In some embodiments, the IL-6 receptorinhibitor is a triterpenoid saponin (e.g., chikusetsuaponin IVabutyl ester (CS-Iva-Be) (Yang et al., Mol. Cancer. Ther.15(6):1190-200, 2016).

Small Molecules

[0687] In some embodiments, the IL-6 receptor inhibitor is a smallmolecule (see, e.g., U.S. Pat. No. 9,409,990). In some embodiments,the small molecule is LMT-28 (Hong et al., J. Immunol. 195(1):237-245, 2015); ERBA (Enomoto et al., Biochem. Biophys. Res.Commun. 323:1096-1102, 2004; Boos et al., J. Nat. Prod.75(4):661-668, 2012), ERBF (TB-2-081) (Hayashi et al., J.Pharmacol. Exp. Ther. 303:104-109, 2002; Vardanyan et al., Pain151(2):257-265, 2010; Kino et al., J. Allergy Clin. Immunol.120(2):437-444, 2007), or a variant thereof

4. Integrin Inhibitors

[0688] The term "integrin inhibitor" refers to an agent whichdecreases the expression of one or more integrins and/or decreasesthe binding of an integrin ligand to one or more integrins thatplay a role in the recruitment, extravasation, and/or activation ofa leukocyte. In some embodiments, the integrin inhibitorspecifically binds to at least a portion of a ligand binding siteon a target integrin. In some embodiments, the integrin inhibitorspecifically binds to a target integrin at the same site as anendogenous ligand. In some embodiments, the integrin inhibitordecreases the level of expression of the target integrin in amammalian cell. In some embodiments, the integrin inhibitorspecifically binds to an integrin ligand.

[0689] Non-limiting examples of integrins that can be targeted byany of the integrin inhibitors described herein include:.alpha.2.beta.1 integrin, .alpha.1.beta.1 integrin, .alpha.4.beta.7integrin, integrin .alpha.4.beta.1 (VLA-4), E-selectin, ICAM-1,.alpha.5.beta.1 integrin, .alpha.4.beta.1 integrin, VLA-4,.alpha.2.beta.1 integrin, .alpha.5.beta.3 integrin, .alpha.5.beta.5integrin, .alpha.IIb.beta.3 integrin, and MAdCAM-1. A non-limitingexample of integrin inhibitor that can decrease the expressionand/or activity of .alpha.4.beta.7 integrin is FTY720. Anon-limiting example of an integrin inhibitor that specificallytargets MAdCAM is PF-547659 (Pfizer). Non-limiting examples of anintegrin inhibitor that specifically targets .alpha.4.beta.7 isAJM300 (Ajinomoto), etrolizumab (Genentech), and vedolizumab(Millenium/Takeda).

[0690] In some embodiments, the integrin inhibitor is an.alpha.IIb.beta.3 integrin inhibitor. In some embodiments, the.alpha.IIb.beta.3 integrin inhibitor is abciximab (ReoPro.RTM.,c7E3; Kononczuk et al., Curr. Drug Targets 16(13):1429-1437, 2015;Jiang et al., Appl. Microbiol. Biotechnol. 98(1):105-114, 2014),eptifibatide (Integrilin.RTM.; Scarborough et al., J. Biol. Chem.268:1066-1073, 1993; Tcheng et al., Circulation 91:2151-2157, 1995)or tirofiban (Aggrastat.RTM.; Hartman et al., J. Med. Chem.35:4640-4642, 1992; Pierro et al., Eur. J. Ophthalmol.26(4):e74-76, 2016; Guan et al., Eur. J. Pharmacol 761:144-152,2015). In some embodiments, the integrin inhibitor is an.alpha.L-selective integrin inhibitor. In some embodiments, theintegrin inhibitor is a .beta.2 integrin inhibitor.

[0691] In some embodiments, the integrin inhibitor is an .alpha.4integrin (e.g., an .alpha.4.beta.1 integrin (e.g., Very LateAntigen-4 (VLA-4), CD49d, or CD29)) inhibitor, an .alpha.4.beta.7integrin inhibitor. In some embodiments, the integrin inhibitortargets endothelial VCAM1, fibronectin, mucosal addressin cellularadhesion molecule-1 (MAdCAM-1), vitronectin, tenascin-C,osteopontin (OPN), nephronectin, agiostatin, tissue-typetransglutaminase, factor XIII, Von Willebrand factor (VWF), an ADAMprotein, an ICAM protein, collagen, e-cadherin, laminin, fibulin-5,or TGF.beta.. In some embodiments, the .alpha.4 integrin inhibitoris natalizumab (Tysabri.RTM.; Targan et al., Gastroenterology132(5):1672-1683, 2007; Sandbom et al., N Engl. J. Med.353(18):1912-1925, 2005; Nakamura et al., Intern. Med.56(2):211-214, 2017; and Singh et al., J. Pediatr. Gastroenterol.Nutr. 62(6):863-866, 2016). In some embodiments, the integrininhibitor is an endogenous integrin inhibitor (e.g., SHARPIN(Rantala et al., Nat. Cell. Biol. 13(11):1315-1324, 2011).

[0692] In some embodiments, the integrin inhibitor is an .alpha.vintegrin (e.g., an .alpha.5.beta.1 integrin, an .alpha.5.beta.3integrin, an .alpha.5.beta.5 integrin inhibitor, and/or an.alpha.5.beta.6 integrin) inhibitor.

[0693] In some embodiments, the integrin inhibitor is an.alpha.5.beta.1 integrin inhibitor.

[0694] In some embodiments, an integrin inhibitor is an inhibitorynucleic acid, an antibody or antigen-binding fragment thereof, afusion protein, an integrin antagonist, a cyclic peptide, adisintegrin, a peptidomimetic, or a small molecule. In someembodiments, the inhibitory nucleic acid is a small hairpin RNA, asmall interfering RNA, an antisense, an aptamer, or a microRNA.

Inhibitory Nucleic Acids

[0695] In some embodiments, the inhibitory nucleic acid can be anantisense nucleic acid, a ribozyme, a small interfering RNA, asmall hairpin RNA, or a microRNA. Inhibitory nucleic acids that candecrease the expression of target integrin mRNA or a targetintegrin ligand mRNA (e.g., any of the exemplary integrinsdescribed herein or any of the exemplary integrin ligands describedherein) in a mammalian cell include antisense nucleic acidmolecules, i.e., nucleic acid molecules whose nucleotide sequenceis complementary to all or part of target integrin mRNA or a targetintegrin ligand mRNA. An antisense nucleic acid molecule can becomplementary to all or part of a non-coding region of the codingstrand of a nucleotide sequence encoding a target integrin or atarget integrin ligand (e.g., any of the exemplary target integrinsor any of the exemplary integrin ligands described herein).Non-coding regions (5' and 3' untranslated regions) are the 5' and3' sequences that flank the coding region in a gene and are nottranslated into amino acids. Exemplary integrin inhibitors that areantisense nucleic acids include ATL1102 (e.g., Limmroth et al.,Neurology 83(20):1780-1788, 2014; Li et al., Dig. Liver Dis.39(6):557-565, 2007; Goto et al., Inflamm. Bowel Dis.12(8):758-765, 2006).

[0696] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a target integrin(e.g., any of the exemplary target integrins described herein) oran integrin ligand (e.g., any of the exemplary integrin ligandsdescribed herein).

[0697] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a target integrin (e.g., any of the exemplary targetintegrins described herein) or an integrin ligand (e.g., any of theexemplary integrin ligands described herein) can be inhibited bytargeting nucleotide sequences complementary to the regulatoryregion of the gene encoding the target integrin (e.g., any of theexemplary target integrins described herein) or the integrin ligand(e.g., any of the exemplary integrin ligands described herein)(e.g., the promoter and/or enhancer, e.g., a sequence that is atleast 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of the transcriptioninitiation start state) to form triple helical structures thatprevent transcription of the gene in target cells.

[0698] In some embodiments, an integrin inhibitor is a siRNA thatdecreases the level of a target integrin (e.g., any of theexemplary target integrins described herein) mRNA or an integrinligand (e.g., any of the exemplary integrin ligands describedherein) mRNA. Non-limiting examples of integrin inhibitors that areshort interfering RNAs (siRNAs) are described in Wang et al.,Cancer Cell Int 16:90, 2016). In some embodiments, the integrininhibitor is a short hairpin RNA (shRNA).

[0699] Non-limiting examples of integrin inhibitors that aremicroRNA include miR-124 (Cai et al., Sci. Rep. 7:40733, 2017),miR-134 (Qin et al., Oncol. Rep. 37(2):823-830, 2017), miR-92b (Maet al., Oncotarget 8(4):6681-6690, 2007), miR-17 (Gong et al.,Oncol. Rep. 36(4), 2016), miR-338 (Chen et al., Oncol. Rep.36(3):1467-74, 2016), and miR-30a-5p (Li et al., Int. J. Oncol.48(3):1155-1164, 2016).

Antibodies

[0700] In some embodiments, the integrin inhibitor is an antibodyor an antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, the antibody can be a humanized antibody, achimeric antibody, a multivalent antibody, or a fragmentthereof.

[0701] In some embodiments, the antibody is a pan-.beta.1 antibody(e.g., OS2966 (Carbonell et al., Cancer Res. 73(10):3145-3154,2013). In some embodiments, the integrin antibody is a monoclonalantibody (e.g., 17E6 (Castel et al., Eur. J. Cell. Biol.79(7):502-512, 2000); Mitjans et al., Int. J. Cancer 87(5):716-723,2000)). In some embodiments, the monoclonal antibody is vedolizumab(e.g., Entyvio.RTM.) or a variant thereof (Feagan et al., N Engl.J. Med 369:699-710, 2013; Sandborn et al., N. Engl. J. Med.369:711-721, 2013; Sands et al., Gastroenterology 147:618-627,2014; and Milch et al., Neuroimmunol. 264:123-126, 2013; Wyant etal., J. Crohns Colitis 10(12):1437-1444, 2016; and Feagan et al.,Gastroenterology 142(5):5160-5161, 2012).

[0702] In some embodiments, the antibody can be a Fab fragment of amonoclonal chimeric mouse-human antibody (e.g., abciximab (ReoPro,c7E3), Kononczuk et al., Curr. Drug Targets 16(13):1429-1437, 2015;Jiang et al., Appl. Microbiol. Biotechnol. 98(1):105-114, 2014), ora variant thereof. In some embodiments, the integrin antibody is ahumanized monoclonal antibody. In some embodiments, the humanizedmonoclonal antibody is natalizumab (Tysabri.RTM.) (Targan et al.,Gastroenterology 132(5):1672-1683, 2007; Sandborn et al., N. Engl.J. Med. 353(18):1912-1925, 2005; Nakamura et al., Intern Med56(2):211-214, 2017; Singh et al., J. Pediatr. Gastroenterol. Nutr.62(6):863-866, 2016). In some embodiments, the humanized monoclonalantibody is vitaxin (MEDI-523) or a variant thereof (Huveneers etal., Int, J. Radiat. Biol. 81(11-12):743-751, 2007; Coleman et al.,Circ. Res. 84(11):1268-1276, 1999). In some embodiments, thehumanized monoclonal antibody is etaracizumab (Abegrin.RTM.,MEDI-522, LM609) or a variant thereof (Hersey et al., Cancer116(6):1526-1534, 2010; Delbaldo et al., Invest New Drugs26(1):35-43, 2008). In some embodiments, the humanized monoclonalantibody is CNTO95 (Intetumumab.RTM.) or a variant thereof (Jia etal., Anticancer Drugs 24(3):237-250, 2013; Heidenreich et al., Ann.Oncol. 24(2):329-336, 2013; Wu et al., J. Neurooncol. 110(1):27-36,2012). In some embodiments, the humanized monoclonal antibody isefalizumab (Raptiva.RTM.) or a variant thereof (Krueger et al., J.Invest. Dermatol. 128(11):2615-2624, 2008; Li et al., PNAS106(11):4349-4354, 2009; Woolacott et al., Health Technol. Assess10:1-233, 2006). In some embodiments, the humanized monoclonalantibody is STX-100 (Stromedix.RTM.) or a variant thereof (vanAarsen et al., Cancer Res. 68:561-570, 2008; Lo et al., Am. J.Transplant. 13(12):3085-3093, 2013). In some embodiments, thehumanized monoclonal antibody is 264RAD or a variant thereof(Eberlein et al., Oncogene 32(37):4406-4417, 2013).

[0703] In some embodiments, the humanized monoclonal antibody isrovelizumab or a variant thereof (Goodman et al., Trends Pharmacol.Sci 33:405-412, 2012). In some embodiments, the humanizedmonoclonal antibody is Cytolin.RTM. or a variant thereof (Rychertet al., Virology J. 10:120, 2013). In some embodiments, thehumanized monoclonal antibody is etrolizumab or a variant thereof(Vermeire et al., Lancet 384:309-318, 2014; Rutgeerts et al., Gut62:1122-1130, 2013; Lin et al., Gastroenterology 146:307-309, 2014;Ludviksson et al., J. Immunol. 162(8):4975-4982, 1999; Stefanich etal., Br. J. Pharmacol. 162(8):1855-1870, 2011). In someembodiments, the humanized monoclonal antibody is abrilumab (AMG181; MEDI-7183) or a variant thereof (Pan et al., Br. J. Pharmacol.169(1):51-68, 2013; Pan et al., Br. J. Clin. Pharmacol.78(6):1315-1333, 2014). In some embodiments, the humanizedmonoclonal antibody is PF-00547659 (SHP647) or a variant thereof(Vermeire et al., Gut 60(8):1068-1075, 2011; Sandborn et al.,Gastroenterology 1448(4):S-162, 2015). In some embodiments, thehumanized monoclonal antibody is SAN-300 (hAQC2) or a variantthereof (Karpusas et al., J. Mol. Biol. 327:1031-1041, 2003). Insome embodiments, the humanized monoclonal antibody is DI176E6 (EMD5257) or a variant thereof (Goodman et al., Trends Pharmacol. Sci33:405-412, 2012; and Sheridan et al., Nat. Biotech. 32:205-207,2014).

[0704] In some embodiments, the integrin antibody is a chimericmonoclonal antibody. In some embodiments, the chimeric monoclonalantibody is volociximab or a variant thereof (Kuwada et al., Curr.Opin. Mol. Ther. 9(1):92-98, 2007; Ricart et al., Clin. Cancer Res.14(23):7924-7929, 2008; Ramakrishnan et al., I Exp. Ther. Oncol.5(4):273-86, 2006; Bell-McGuinn et al., Gynecol. Oncol.121:273-279, 2011; Almokadem et al., Exp. Opin. Biol. Ther.12:251-7, 2012).

[0705] In some embodiments, the antibody specifically binds one ormore (e.g., 1, 2, 3, 4, or 5) integrin. In some embodiments, theantibody specifically binds an integrin dimer (e.g., MLN-00002,MLNO2 (Feagan et al., Clin. Gastroenterol. Hepatol.6(12):1370-1377, 2008; Feagan et al., N Engl. J. Med.352(24):2499-2507, 2005). In certain embodiments, the antibodycomprises or consists of an antigen-binding fragment of abciximab(Reopro.TM.) (Straub et al., Eur. I Cardiothorac Surg.27(4):617-621, 2005; Kim et al., Korean I Intern. Med.19(4):220-229, 2004). In some embodiments, the integrin inhibitoris an antibody-drug conjugate (e.g., IMGN388 (Bendell et al., EJCSuppl 8(7):152, 2010).

[0706] Further examples of antibodies and antigen-binding fragmentsthereof are described in U.S. Pat. Nos. 5,919,792; 6,214,834;7,074,408; 6,833,373; 7,655,624; 7,465,449; 9,558,899; 7,659,374;8,562,986; 8,398,975; and 8,853,149; US 2007/0117849; US2009/0180951; US 2014/0349944; US 2004/0018192; WO 11/137418; andWO 01/068586; each of which is incorporated by reference in itsentirety.

Fusion Proteins

[0707] In some embodiments, the integrin inhibitor is a fusionprotein (e.g., an Fc fusion protein of an extracellular domain ofan integrin or an integrin receptor), a soluble receptor (e.g., theextracellular domain of an integrin or an integrin receptor), or arecombinant integrin binding protein (e.g., an integrin ligand).See, e.g., Lode et al., PNAS 96(4):1591-1596, 1999; Stephens etal., Cell Adhesion Comm. 7:377-390, 2000; and US 2008/0739003;incorporated by reference herein). Non-limiting examples of fusionproteins that are integrin inhibitors include Ag25426(Proteintech).

Small Molecules Antagonists

[0708] In some embodiments, the integrin inhibitor is a smallmolecule. In some embodiments, the small molecule is a non-peptidesmall molecule. In some embodiments, the non-peptide small moleculeis a RGD (ArgGlyAsp)-mimetic antagonist (e.g., tirofiban(Aggrastat.RTM.); Pierro et al., Eur. J. Ophthalmol. 26(4):e74-76,2016; Guan et al., Eur. J. Pharmacol 761:144-152, 2015. In someembodiments, the small molecule is .alpha.4 antagonist (e.g.,firategrast (Miller et al., Lancet Neurol. 11(2):131-139, 2012)AJM300 (Yoshimura et al., Gastroenterology 149(7):1775-1783, 2015;Takazoe et al., Gastroenterology 136(5):A-181, 2009; Sugiura etal., J. Crohns Colitis 7(11):e533-542, 2013)). In some embodiments,the small molecule is .alpha.4.beta.1 antagonist (e.g., IVL745(Norris et al., J. Allergy Clin. Immunol. 116(4):761-767, 2005; Coxet al., Nat. Rev. Drug Discov. 9(10):804-820, 2010)), BIO-1211(Abraham et al., Am. J. Respir. Crit. Care Med. 162:603-611, 2000;Ramroodi et al., Immunol. Invest. 44(7):694-712, 2015; Lin et al.,J. Med. Chem. 42(5):920-934, 1999), HMR 1031 (Diamant et al., Clin.Exp. Allergy 35(8):1080-1087, 2005); valategrast (R411) (Cox etal., Nat. Rev. Drug Discov. 9(10):804-820, 2010), GW559090X(Ravensberg et al., Allergy 61(9):1097-1103, 2006), TR14035 (Sircaret al., Bioorg. Med. Chem. 10(6):2051-2066, 2002; Cortijo et al.,Br. J. Pharmacol. 147(6):661-670, 2006)). In some embodiments, thesmall molecule is .alpha.v.beta.3 antagonist (e.g., L0000845704,SB273005). In some embodiments, the small molecule is.alpha.5.beta.1 antagonist (e.g., JSM6427). In some embodiments,the small molecule is GLPG0974 (Vermeire et al., J. Crohns ColitisSuppl. 1:S39, 2015). In some embodiments, the small molecule isMK-0429 (Pickarksi et al., Oncol. Rep. 33(6):2737-45, 2015;Rosenthal et al., Asia Paci Clin. Oncol. 6:42-8, 2010). In someembodiments, the small molecule is JSM-6427 or a variant thereof(Zahn et al., Arch. Ophthalmol. 127(10):1329-1335, 2009; Stragieset al., J. Med. Chem. 50:3786-94, 2007).

[0709] In some embodiments, the small molecule targets a .beta.2integrin. In some embodiments, the small molecule is SAR-118(SAR1118) or a variant thereof (Zhong et al., ACS Med. Chem. Lett.3(3):203-206, 2012; Suchard et al., J. Immunol. 184:3917-3926,2010; Yandrapu et al., J. Ocul. Pharmacol. Ther. 29(2):236-248,2013; Semba et al., Am. J. Ophthalmol. 153:1050-60, 2012). In someembodiments, the small molecule is BMS-587101 or a variant thereof(Suchard et al., J. Immunol. 184(7):3917-3926, 2010; Potin et al.,J. Med. Chem. 49:6946-6949, 2006). See e.g., Shimaoka et al.,Immunity 19(3):391-402, 2003; U.S. Pat. Nos. 7,138,417; 7,928,113;7,943,660; and 9,216,174; US 2008/0242710; and US 2008/0300237.

[0710] In some embodiments, the small molecule integrin inhibitorcan be PTG-100, which is described in, e.g., Shames et al.,"Pharmakokinetics and Pharmacodynamics of the Novel Oral PeptideTherapeutic PTG-100 (.alpha.4.beta.7 Integrin Antagonist) in NormalHealthy Volunteers," 24th United European Gastroentrology Week,October 15-19, Vienna, Austria, 2016.

Cyclic Peptides

[0711] In some embodiments, the integrin inhibitor is a cyclicpeptide. In some embodiments, the cyclic peptide comprises orconsists of an amino acid sequence as set forth in the amino acidsequence of a ligand recognition sequence of an endogenous integrinligand. In some embodiments, the cyclic peptide competes for atarget integrin ligand binding site with an endogenous integrinligand. In some embodiments, the cyclic peptide includes one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8) D-amino acids. In someembodiments, the cyclic peptide is a synthetic cyclic peptide. Insome embodiments, the synthetic cyclic peptide is a heptapeptide.In some embodiments, the synthetic cyclic peptide is eptifabitide(Integrilin.TM.), or a variant thereof. In some embodiments, thecyclic peptide comprises a heterocyclic nucleic (e.g., abenzodiazepinone, a piperazine, a benzoazepinone, a nitroaryl, anisoxazoline, an indazole, or a phenol; Spalluto et al., Curr. Med.Chem. 12:51-70, 2005). In some embodiments, the cyclic peptide is amacrocycle (see, e.g., Halland et al., ACS Med. Chem. Lett.5(2):193-198, 2014). In some embodiments, the peptide is ALG-1001or a variant thereof (Mathis et al., Retin. Phys. 9:70, 2012). Insome embodiments, the cyclic peptide is animidazolone-phenylalanine derivative, a heteroaryl, hetrocyclic,and aryl derivative, a bicyclic-aromatic amino acid derivative, acyclohexane-carboxylic acid derivative, a di-aryl substituted ureaderivative, a multimeric L-alanine derivative, a L-alaninederivative, or a pyrimidyl-sulfonamide derivative (see, e.g., U.S.Pat. Nos. 6,630,492; 6,794,506; 7,049,306; 7,371,854; 7,759,387;8,030,328; 8,129,366; 7,820,687; 8,350,010; and 9,345,793).

Peptidomimetics

[0712] In some embodiments, the integrin inhibitor is apeptidomimetic. In some embodiments, the peptidomimetic has anintegrin-ligand recognition motif (e.g., RGD, KTS, or MLD). See,e.g., Carron et al., Cancer Research 58:1930-1935, 1998; Fanelli etal., Vascular Cell 6:11, 2014; and De Marco et al., Curr. Top. Med.Chem. 16(3):343-359, 2016.

[0713] In some embodiments, the peptidomimetic is anRGD(ArgGlyAsp)-based peptide (U.S. Pat. No. 8,809,338, incorporatedby reference in its entirety herein). In some embodiments, theRGD-based peptide can be cilengitide or a variant thereof (EMD12974) (Mas-Moruno et al., Anticancer Agents Med. Chem. 10:753-768,2010; Reardon et al., Future Oncol. 7(3):339-354, 2011; Beekman etal., Clin. Genitourin Cancer 4(4):299-302, 2006; SC56631 (e.g.,Engleman et al., Am Soc. Clin. Invest. 99(9):2284-2292, 1997; Penget al., Nature Chem Biol. 2:381-389, 2006). In some embodiments,the peptidomimetic can be a Lys-Gly-Asp (KGD)-based peptide. Insome embodiments, the peptidomimetic can be vipegitide or a variantthereof (Momic et al., Drug Design Devel. Therapy 9:291-304, 2015).In some embodiments, the peptidomimetic can be a peptide conjugatedwith an antimicrobial synthetic peptide. (e.g., ACDCRGDCFCconjugated with (KLAKLAK).sub.2 (Ellerby et al., Nat. Med.5(9):1032-1038, 1999). See, e.g., U.S. Pat. No. 8,636,977.

Disintegrins

[0714] In some embodiments, the integrin inhibitor can be adisintegrin. The term "disintegrin" as used herein refers to a lowmolecular weight peptide integrin inhibitor derived from a snakevenom (e.g., pit viper venom). In some embodiments, the disintegrinis a RGD(ArgGlyAsp)-, a KTS- or an MLD-based disintegrin.

[0715] Non-limiting examples of disintegrins include accutin,accurhagin-C, albolabrin, altemagin-c, barbourin, basilicin,bitisgabonin-1, bitisgabonin-2, bitistatin, cerastin, cereberin,cumanastatin 1, contortrostatin, cotiarin, crotatroxin,dendroaspin, disba-01, durissin, echistatin, EC3, elegantin,eristicophin, eristostatin, EMS11, EO4, EO5, flavoridin,flavostatin, insularin, jarastatin, jerdonin, jerdostatin,lachesin, lebein (e.g., lebein-1, lebein-2), leberagin-C,lebestatin, lutosin, molossin, obtustatin, ocellatusin, rhodocetin,rhodostomin, R-mojastin 1, salmosin, saxatilin, schistatin,tablysin-15, tergeminin, triflavin, trigramin, trimestatin, VA6,vicrostatin, viridin, viperstatin, VB7, VLO4, and VLO5, or avariant thereof. See, e.g., Arruda Macedo et al., Curr. Protein.Pept. Sci. 16(6):532-548, 2015; Hsu et al., Sci. Rep. 6:23387,2016; Kele et al. Curr. Protein Pept. Sci. 6:532-548, 2015; Koh etal., Toxicon 59(4):497-506, 2012; Scarborough et al., J. Biol.Chem. 268:1058-1065, 1993; Kisiel et al., FEBSLett. 577:478-482,2004; Souza et al., Arch. Biochem. Biophys. 384:341-350, 2000; Ebleet al., J. Biol. Chem. 278:26488-26496, 2003; Marcinkiewicz et al.,J. Biol. Chem. 274:12468-12473, 1999; Calvete et al., I ProteomeRes. 6:326-336, 2007; Scibelli et al., FEMS Microbiol. Lett.247:51-57, 2005; Oliva et al., Toxicon 50:1053-1063, 2007; Minea etal., Toxicon 59:472-486, 2012; Smith et al., FEBSLett. 512:111-115,2002; Tselepis et al., J. Biol. Chem. 272:21341-21348, 1997; DaSilva et al., Tromb. Res. 123:731-739, 2009; Thibault et al., Mol.Pharmacol. 58:1137-1145, 2000; Lu et al., Biochem. J. 304:818-825,1994; Yeh et al., Biochim. Biophys. Acta. 1425:493-504, 1998; Huanget al., Exp. Hematol. 36:1704-1713, 2008; Shih et al., Matrix Biol.32:152-159, 2013; Wang et al., Br. I Pharmacol. 160:1338-1351,2010; Della-Casa et al., Toxicon 57:125-133, 2011; Sheu et al.,Biochim. Biophys. Acta. 1336:445-454, 1997; Fujii et al., I Mol.Biol. 332:115-122, 2003; Bilgrami et al., I Mol. Biol. 341:829-837,2004; Zhou et al., Toxicon 43:69-75, 2004; Scarborough et al., J.Biol. Chem. 268:1066-1073, 1993; Shebuski et al., J. Biol. Chem.264:21550-21556, 1989; Lu et al., Biochem. J. 304:929-936, 1994;McLane et al., Biochem. J. 301:429-436, 1994; Juarez et al.,Toxicon 56:1052-1058, 2010; Olfa et al., Lab. Invest. 85:1507-1516,2005; Elbe et al., Matrix Biol. 21:547-558, 2002; Bazan-Socha etal., Biochemistry 43:1639-1647, 2004; Danen et al., Exp. Cell. Res.238:188-196, 1998; Marcinkiewicz et al., Biochemistry38(40):13302-13309, 1999; Calvete et al., Biochem. I 372:725-734,2003; Swenson et al., Pathophysiol. Haemost. Thromb. 34:169-176,2005; Kwon et al., PLoS One 8; e81165, 2013; Yang et al., Toxicon45:661-669, 2005; Limam et al., Matrix Biol. 29:117-126, 2010; Ganet al., J. Biol. Chem. 263:19827-19832, 1988; Ma et al., Thromb.Haemost. 105(6):1032-1045, 2011; and U.S. Pat. No. 7,074,408,incorporated in their entirety herein.

5. TLR Agonists/Antagonists

[0716] The term "TLR agonist" is an agent that binds to andactivates a toll-like receptor (TLR) expressed in a mammalian cell(e.g., a human cell). In some embodiments, the TLR agonist binds toand activates TLR1. In some embodiments, the TLR agonist binds toand activates TLR2. In some embodiments, the TLR agonist binds toand activates TLR3. In some embodiments, the TLR agonist binds toand activates TLR4. In some embodiments, the TLR agonist binds toand activates TLR5. In some embodiments, the TLR agonist binds toand activates TLR6. In some embodiments, the TLR agonist binds toand activates TLR7. In some embodiments, the TLR agonist binds toand activates TLR8. In some embodiments, the TLR agonist binds toand activates TLR9. In some embodiments, the TLR agonist binds toand activates TLR10. In some embodiments, the TLR agonist binds toand activates TLR11. In some embodiments, the TLR agonist binds toand activates two or more (e.g., three, four, five, six, seven,eight, nine, ten, or eleven) TLRs (e.g., two or more of any ofTLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, andTLR11 (in any combination)).

[0717] In some embodiments, the TLR agonist is a synthetic TLRagonist, a TLR mimic, or a small molecule. Non-limiting examples ofTLR agonists are described in Bhardwaj et al., Cancer J.16(4):382-391, 2010; Meyer et al., Exp. Opin. Investig. Drugs17(7):1051-1065, 2008; Adams, Immunotherapy 1(6):949-964, 2009;Hennessy et al., Nat. Rev. Drug Discov. 9:293-307, 2010; and U.S.Pat. Nos. 7,498,409; 9,421,254; 8,409,813; 8,361,986; 8,795,678;8,728,486; 8,636,979; 8,999,946; 9,359,360; 9,050,376; and9,556,167; US 2014/0322271; US 2016/0206690; US 2009/0253622; US2011/0135669; US 2011/0250175; US 2014/0220074; and US2012/0219615; each incorporated in its entirety herein. In someembodiments, the TLR agonist is a peptide or a fusion protein(Huleatt et al., Vaccine 25: 763-775, 2007).

[0718] In some embodiments, a TLR agonist specifically binds to andactivates a single TLR (e.g., TLR4, TLR7, TLR8, or TLR9; Zhu etal., J. Clin. Invest. 120:607-616, 2010; Zhu et al., PNAS105:16260-16265, 2008; Wang et al., J. Virol. 79(22):14355-14370,2005). In some embodiments, the TLR agonist binds to and activatesmore than one TLR (e.g., Bacillus of Calmette-Guerin, Myobacteriumbovis (BCG); Morton et al., Ann. Surg. 180(4):635-643, 1974;Mortoon et al., J. Clin. Oncol. ASCO Ann. Meeting Proceedings PartI 25(18 Suppl), 2007). In some embodiments, the TLR agonist is aTLR2/TLR6 agonist (e.g., Pam2CSK4 or MALP-2 (Agnihotri et al., J.Med. Chem. 54: 8148-8160, 2011; Wu et al., J. Med. Chem. 53:3198-3213, 2010)).

[0719] In some embodiments, the TLR agonist is an endogenousmolecule released from dead cells (e.g., a heat shock protein (HSP)and mobility group box 1 (HMGB1); Asea et al., J. Biol. Chem.277:15028-15034, 2002; Kepp et al., Cancer Metastasis 30: 61-69,2011).

TLR3 Agonists

[0720] In some embodiments, the TLR agonist specifically binds andactivates TLR3 (e.g., a synthetic agonist). Non-limiting examplesof TLR agonists that bind and activate TLR3 are described inNicodemus et al., Immunotherapy 2:137-140, 2010. In someembodiments, the TLR3 agonist is a synthetic double-stranded RNA(dsRNA) complex (e.g., polyribosinic: polyribocytidic acid(polyI:C); Sivori et al., PNAS 101:10116-10121, 2004; Sloat et al.,Pharmaceutical Res. 23:1217-1226, 2006; Ichinohe et al., Microbesand infection/Institut Pasteur 9:1333-1340, 2007; Robinson et al.,J. Natl. Cancer Inst. 57(3):599-602, 1976). In some embodiments,the TLR3 agonist is a TLR3 mimic (e.g., polyadenosine-polyuridylicacid (poly A:U) (Veyrat et al., Oncotarget 7(50):82580-82593, 2016;Alizadeh et al., Iran J. Allergy Asthma Immunol. 12(2):161-167,2013); rintatolimod (polyI: polyCU, Ampligen.RTM.) (Steinman etal., Nature 449: 419-426, 2007; Jasani et al., Vaccine27(25-26):3401-3404, 2009; Strayer et al., PLoS One 7(3): e31334,2012). In some embodiments, the TLR3 mimic ispolyionisinic-polycytidylic acid stabilized with poly-L-lysine andcarboxymethylcellulose (Poly-ICLC, Hiltonol.RTM.; Hawkins et al.,J. Biol. Resp. Mod. 4:664-668, 1985; Butowski et al., J.Neurooncol. 91:175-182, 2009; Jeong et al., J. Neurochem.doi.10.1111, 2015). In some embodiments, the TLR3 agonist is RGC100(Naumann et al., Clin. Dev. Immunol. 283649, 2013), IPH-3102(Basith et al., Exp. Opin. Ther. Pat. 21: 927-944, 2011), or avariant thereof. In some embodiments, the TLR3 agonist is CQ-07001(Clinquest). In some embodiments, the TLR3 agonist is Ampligenpoly(I):poly(C12U) (Hemispherx Biopharma). In some embodiments, theTLR3 agonist is IPH-31XX (Innate Pharma). In some embodiments, theTLR3 agonist is MCT-465-dsRNA (MultiCell Technologies).

TLR4 Agonists

[0721] In some embodiments, the TLR agonist specifically binds toand activates TLR4 (Peri et al., J. Med. Chem. 57(9):3612-3622,2014). In some embodiments, the TLR4 agonist is bacteriallipopolysaccharide (LPS) or a variant thereof. In some embodiments,the TLR4 agonist is monophosphoryl lipid A (MPL, MPLA, GLA, GLA-SE)(Ribi et al., J. Immunol. 6:567-572, 1984; Okemoto et al., J.Immunol. 176:1203-1208, 2006; Matzner et al., Int. J. Cancer138:1754-1764, 2016; Cauwelaert et al., PLoS One 11(1):e0146372,2016). In some embodiments, the TLR agonist is AS15 or AS02b(Brichard et al., Vaccine 25(Suppl. 2):B61-B71, 2007; Kruit et al.,J. Clin. Oncol. 26(Suppl): Abstract 9065, 2008). In someembodiments, the TLR agonist is an aminoalkyl glucosaminide4-phosphate (e.g., RC-529, Ribi.529, E6020) or a variant thereof(Baldridge et al., J. Endotoxin Res. 8:453-458, 2002; Morefield etal., Clin. Vaccine Immunol. 14: 1499-1504, 2007). In someembodiments, the TLR agonist is picibanil (OK-432) (Hazim et al.,Med. J. Malaysia 71(6):328-330, 2016; Tian et al., Asian Pac J.Cancer Prev. 16(11):4537-4542, 2015; Rebuffini et al., Dent Rese.J. 9(Suppl. 2):S192-S196, 2012). In some embodiments, the TLR4agonist is Spirulina complex polysaccharide (Kwanishi et al.,Microbiol. Immunol. 57:63-73, 2013). In some embodiments, the TLR4agonist is chitohexaose or a variant thereof (Panda et al.,8:e1002717, 2012; Barman et al., Cell Death Dis. 7:e2224, 2016). Insome embodiments, the TLR4 agonist is E5564 (Eritoran) (Eisai). Insome embodiments, the TLR4 agonist is CRX-675 or CRX-527 (GSK).

TLR5 Agonists

[0722] In some embodiments, the TLR agonist binds and activatesTLR5. In some embodiments, the TLR5 agonist is flagellin or avariant thereof (e.g., entolimod (CBLB502)) (Yoon et al., Science335: 859-864, 2012; Fukuzawa et al., J. Immunol. 187:3831-3839,2011; Brackett et al., PNAS 113(7):E874-E883, 2015; Leigh et al.,PLoS One 9(1):e85587, 2014; Hossain et al., Blood 120:255, 2012).In some embodiments, the TLR5 agonist is flagellin HuHa (Vaxinate)or flagellin HuM2e (Vaxinate).

TLR7/8 Agonists

[0723] In some embodiments, the TLR agonist binds and activatesTLR7/8 (e.g., TLR7 agonist, TLR8 agonist, or a TLR7 and TLR8agonist). In some embodiments, the TLR7/8 agonist is ANA975(isotorabine) (Anadys/Novartis), ANA773 (Anadys/Novartis),

[0724] In some embodiments, the TLR7/8 agonist is animidazoquinoline or a variant thereof (e.g., imiquimod (Aldara.TM.;Kaspari et al., British J. Dermatology 147: 757-759, 2002; Smorlesiet al., Gene Therapy 12: 1324-133, 2005; Prins et al., J. Immunol.176: 157-164, 2006; Shackleton et al., Cancer Immun. 4:9, 2004;Green et al., Br. J. Dermatol. 156(2):337-345, 2007; Geisse et al.,Am. Acad. Dermatol. 50(5):722-733, 2004; Wolf et al., Arch.Dermatol. 139(3):273-276, 2003), resiquimod (R848; Hemmi et al.,Nat. Immunol. 3:196-200, 2002; Jurk et al., Nat. Immunol. 3:49,2002; Rook et al., Blood 126(12):1452-1461, 2015; Dovedi et al.,Blood 121: 251-259, 2013). In some embodiments, the TLR agonist isa synthetic imiadzoquinoline mimicking viral single stranded RNA(ssRNA) (852A) or a variant thereof (Dudek et al., Clin. CancerRes. 13(23):7119-7125, 2007; Dummer et al., Clin. Cancer Res.14(3):856-864, 2008; Weigel et al., Am. J. Hematol. 87(10):953-956,2012; Geller et al., Cancer Immunol. Immunother. 59(12):1877-1884,2010; Inglefield et al., J. Interferon Cytokine Res. 28(4):253-263,2008). In some embodiments, the TLR agonist is a small molecule. Insome embodiments, the small molecule mimics viral ssRNA (e.g.,motolimod (VTX-2337)) or a variant thereof (Dietsch et al., Clin.Cancer Res. 21(24):5445-5452, 2015; Northfelt et al., Clin. CancerRes. 20(14):3683-3691, 2014; Lu et al., Clin. Cancer Res.18(2):499-509, 2012). In some embodiments, the small molecule isGS-9620 or a variant thereof (Bam et al., Antimicrob AgentsChemother. 61(1):e01369, 2016; Rebbapragada et al., PLoS One11(1):e0146835, 2016; Gane et al., J. Hepatol. 63(2): 320-328,2015; Fosdick et al., J. Med. Chem. 56(18):7324-7333, 2013). Insome embodiments, the small molecule is SC1 (Wiedemann et al.,Oncoimmunology 5(7):e1189051, 2016; Hamm et al., J. Immunol.6(4):257-265, 2009). In some embodiments, the small molecule isgardiquimod (Ma et al., Cell. Mol. Immunol. 7:381-388, 2010; Hjelmet al., Hum. Vaccin. Immunother. 10(2): 410-416, 2014; Buitendijket al., AIDS Res. Hum. Retroviruses 29(6):907-918, 2013), CL075(Philbin et al., J. Allergy Clin. Immunol. 130:195-204, 2012;Dowling et al., PLoS One 8(3): e58164, 2013), CL097 (Gorden et al.,J. Immunol. 174:1259-1268, 2005; Gorski et al., Int.Immunol.18:1115, 2006; Levy et al., Blood 108:1284-1289, 2006;Wille-Reece et al., J. Exp. Med. 203: 1249-1258, 2006), loxoribine(Pope et al., Cell Immunol. 162:333, 1995; Heil et al., Eur. J.Immunol. 33:2987-2997, 2003; Lee et al., PNAS 100:6646-6651, 2003),or VTX-294 (Dowling et al., PLoS One 8(3):e58164, 2013). In someembodiments, the TLR7/8 agonist is IMO-9200. In some embodiments,the TLR7 agaonist is IPH-32XX (Innate Pharma).

TLR9 Agonists

[0725] In some embodiments, the TLR agonist binds and activatesTLR9. In some embodiments, the TLR9 agonist is a syntheticoligonucleotide. In some embodiments, the synthetic oligonucleotidecontains unmethylated CpG dinucleotide (CpG-ODN) (Krieg, J. Clin.Invest. 117:1184-1194, 2007; Carpentier et al., Neuro-oncol.8(1):60-66, 2006; Link et al., J. Immunother. 29(5): 558-568, 2006;Pashenkov et al., J. Clin. Oncol. 24(36): 5716-5724, 2006; Meng etal., BMC Biotechnol. 11:88, 2011). In some embodiments, the TLR9agonist is PF-3512676 or a variant thereof (Hofmann et al., J.Immunother. 31(5):520-527, 2008; Molenkamp et al., Clin. Caner.Res. 14(14):4532-4542, 2008). In some embodiments, the TLR9 agonistis IMO-2055 (EMD1201801) or a variant thereof (Machiels et al.,Investig. New Drugs 31:1207-1216, 2013). In some embodiments, theTLR9 agonist is DIMS0150 (Atreya et al., J. Crohns Colitis10(11):1294-1302, 2016). In some embodiments, the TLR9 agonist isCpG7909 (Vaximmune) (Coley, GSK, Novartis, DARPA). In someembodiments, the TLR9 agonist is IMO-9200. In some embodiments, theTLR9 agonist is AVE0675 (Coley, Sanofi Aventis). In someembodiments, the TLR9 agonist is Amplivax (Idera).

Microbial Products as TLR Agonists

[0726] In some embodiments, the TLR agonist is a bacterial or viralcomponent. In some embodiments, the TLR agonist is derived from thecell wall Mycobacterium bovis (BCG). In some embodiments, theMycobacterium bovis cell wall component is a TLR2 and/or TLR4agonist (e.g., SMP105 (Murata et al., Cancer Sci. 99:1435-1440,2008; Miyauchi et al., Drug Discov. Ther. 6: 218-225, 2013; Tsujiet al., Infect Immun. 68: 6883-6890, 2000; Smith et al., CancerImmunol. Immunother. 63(8):787-796, 2014). Additional examples ofTLR agonists are known in the art.

TLR Antagonists

[0727] By the term "TLR antagonist" means an agent that decreasesthe binding of a TLR agonist to TLR4 or TLR9 expressed in amammalian cell (e.g., a human cell). For example, a TLR antagonistcan be a TLR4 antagonist. In other examples, a TLR antagonist is aTLR9 antagonist. Non-limiting examples of TLR antagonists aredescribed in Fukata et al., Mucosal Immunity 6:451-463, 2013.

[0728] A non-limiting example of a TLR4 antagonist is 1A6 (Ungaroet al., Am. J. Physiol. Gastrointest. Liver Physiol.296:G1167-G1179, 2009) or CRX-526 (Fort et al., J. Immunol.174:6416-6423, 2005). Additional examples of TLR4 antagonistsinclude eritoran tetrasodium (E5564) (Sun et al., InvestigativeOphthalmol. Visual Sci. 50(3):1247-1254, 2009), small heat shockprotein B8 (HSP22) (Roelofs et al., J. Immunol. 176(11):7021-7027,2006), CRX-527 (Bazin et al., Bioorganic Med Chem. Letters18(2):5350-5354, 2008), E5564 (Kitazawa et al., J. Gastroentrol.Hepatol. 25(5):1009-1012, 2010), IAXO-102 (Huggins et al.,Atherosclerosis 242(2):563-570, 2015), AG-411 (Kondo et al., TrendsImmunol. 33(9):449-458, 2012), CRX-52624 (Alderson et al., J.Endotoxin Res. 12(5):313-319, 2006), E5531 (Becker et al., Toxicol.Appl. Pharmacol. 207(2):269-275, 2005).

[0729] A non-limiting example of a TLR9 antagonist is adenoviraloligodeoxynucleotides (AV-ODN) (Obermeier et al., Gastroenterology129:913-927, 2005). Additional examples of TLR9 antagonists includeODN 2088, ODN 4084-F, ODN INH-1, ODN INH-18, ODN TTAGGG (A151), andG-ODN (each commercially available from InvivoGen). In someembodiments, the TLR9 antagonist is CpG-ODN c41 (Li et al., Vaccine29:2193-2198, 2011). In some embodiments, the TLR9 antagonist isCOV08-0064 (Shaker et al., Biochemical Pharmacol. 112:90-101, 2016;Hoque et al., J. Immunol. 190(8):4297-4304, 2013); ODN 1585, ODN1826, ODN 2395, and ODN 2088 (Boivin et al., Antiviral Res.96(3):414-421, 2012); IMO-8400 (Zhu et al., J. Immunol. 188(1):119,2012); IRS869 (Mandl et al., Nature Med. 14(10:1077-1087, 2008);IMO-3100 (Hennessy et al., Nature Rev. Drug Discov. 9(4):293-307,2010); TTAGGG (Carvalho et al., PLoS One 6(11):e28256, 2011); andCpG ODN 2088 (David et al., J. Neurotrauma 31(21):1800-1806,2014).

6. SMAD7 Inhibitors

[0730] The term "SMAD7 inhibitor" refers to an agent whichdecreases SMAD7 expression, decreases SMAD7's ability to decreaseformation of Smad2/Smad4 complexes, and/or decreases the ability ofSMAD7 to bind to TGF-.beta. type I receptor. In some embodiments,the SMAD7 inhibitor decreases SMAD7 expression in a mammalian cell.In some embodiments, the SMAD7 inhibitor decreases SMAD7's abilityto decrease formation of Smad2/Smad4 complexes in a mammalian cell.In some embodiments, the SMAD7 inhibitor decreases the ability ofSMAD7 to bind to a TGF-.beta. type I receptor in a mammalian cell.In some embodiments, the SMAD7 inhibitor decreases SMAD7 expressionin a mammalian cell.

[0731] In some embodiments, a SMAD7 inhibitory agent is aninhibitory nucleic acid. In some embodiments, the inhibitorynucleic acid is an antisense nucleic acid, a small interfering RNA,or a microRNA. Examples of aspects of these different inhibitorynucleic acids are described below.

[0732] Inhibitory nucleic acids that can decrease the expression ofSMAD7 expression in a mammalian cell include antisense nucleic acidmolecules, i.e., nucleic acid molecules whose nucleotide sequenceis complementary to all or part of SMAD7 mRNA. An antisense nucleicacid molecule can be complementary to all or part of a non-codingregion of the coding strand of a nucleotide sequence encoding aSMAD7 protein. Non-coding regions (5' and 3' untranslated regions)are the 5' and 3' sequences that flank the coding region in a geneand are not translated into amino acids. Non-limiting examples ofSMAD7 inhibitors that are antisense nucleic acids include mongersen(GED0301) (Monteleon et al., N Engl. J. Med. 372:1104-1113, 2015)and Smad7-as (Kleiter et al., J. Neuroimmunol. 187(1-2):61-73,2007; and Boirivant et al., Gastroenterology 131(6):1786-1798,2006).

[0733] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a SMAD7 protein(e.g., specificity for a SMAD7 mRNA).

[0734] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a SMAD7 polypeptide can be inhibited by targetingnucleotide sequences complementary to the regulatory region of thegene encoding the SMAD7 polypeptide (e.g., the promoter and/orenhancer, e.g., a sequence that is at least 1 kb, 2 kb, 3 kb, 4 kb,or 5 kb upstream of the transcription initiation start state) toform triple helical structures that prevent transcription of thegene in target cells.

[0735] An inhibitory nucleic acid can be a siRNA that decreases thelevel of a SMAD7 mRNA. Non-limiting examples of short interferingRNA (siRNA) that target nucleic acid that encodes SMAD7 aredescribed in, e.g., Su et al., Mol. Vis. 18:1881-1884, 2012.

[0736] Inhibitory nucleic acids targeting SMAD7 also includemicroRNAs (e.g., miR-497 (Hu et al., Am. J. Transl. Res. 8(7):3023-3031, 2016; Liu et al., DNA Cell Biol. 35(9): 521-529, 2016),miR-21 (Lin et al., Cell Physiol. Biochem. 38(6): 2152-2162, 2016;He et al., Heart Vessels 31(10):1696-1708, 2016).

7. Inhibitory Agents of Janus Kinase (JAK) Activity and/orExpression

[0737] The term "JAK inhibitor" refers to an agent which decreasesthe expression of Janus kinase 1 (JAK1), JAK2, JAK3, ornon-receptor protein tyrosine kinase 2 (TYK-2) and/or the kinaseactivity of at least one of JAK1, JAK2, JAK3, and TYK-2. In someembodiments, the JAK inhibitor decreases the expression of JAK1. Insome embodiments, the JAK inhibitor decreases the expression ofJAK2. In some embodiments, the JAK inhibitor decreases theexpression of JAK3. In some embodiments, the JAK inhibitordecreases the expression of TYK-2.

[0738] In some embodiments, the JAK inhibitor decreases the kinaseactivity of JAK1. In some embodiments, the JAK inhibitor decreasesthe kinase activity of JAK2. In some embodiments, the JAK inhibitordecreases the kinase activity of JAK3. In some embodiments, the JAKinhibitor decreases the kinase activity of TYK-2. In someembodiments, the JAK inhibitor is a decreases the kinase activityof JAK1, JAK2, JAK3, and TYK2. In some embodiments, the JAKinhibitor decreases the kinase activity of two or more (e.g., 3 or4) of: JAK1, JAK2, JAK3 and TYK2. In some embodiments, the JAKinhibitor decreases the kinase activity of a single JAK isoform(e.g., JAK1, JAK2, JAK3, or TYK2).

[0739] In some embodiments, the JAK inhibitor decreases the kinaseactivity of JAK1 and JAK2. In some embodiments, the JAK inhibitordecreases the kinase activity of JAK1 and JAK3. In someembodiments, the JAK inhibitor decreases the kinase activity ofJAK2 and JAK3. In some embodiments, the JAK inhibitor decreases thekinase activity of JAK1, JAK2 and JAK3.

[0740] In some embodiments, a JAK inhibitory agent is an inhibitorynucleic acid or a small molecule. In some embodiments, theinhibitory nucleic acid is an antisense nucleic acid, a ribozyme, asmall interfering RNA, a small hairpin RNA, or a microRNA. Examplesof aspects of these different inhibitory nucleic acids aredescribed below.

[0741] Inhibitory nucleic acids that can decrease the expression ofJAK1, JAK2, JAK3, or TYK2 mRNA expression in a mammalian cellinclude antisense nucleic acid molecules, i.e., nucleic acidmolecules whose nucleotide sequence is complementary to all or partof a JAK1, JAK2, JAK3, or TYK2 mRNA.

Inhibitory Nucleic Acids

[0742] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding a JAK1, JAK2, JAK3, or TYK2 protein.Non-coding regions (5' and 3' untranslated regions) are the 5' and3' sequences that flank the coding region in a gene and are nottranslated into amino acids.

[0743] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a JAK1, JAK2,JAK3, or TYK2 protein (e.g., specificity for a JAK1, JAK2, JAK3, orTYK2 mRNA).

[0744] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a JAK1, JAK2, JAK3, or JAK4 polypeptide can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the gene encoding the JAK1, JAK2, JAK3, orTYK2 polypeptide (e.g., the promoter and/or enhancer, e.g., asequence that is at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstreamof the transcription initiation start state) to form triple helicalstructures that prevent transcription of the gene in targetcells.

[0745] An inhibitory nucleic acid can also be a siRNA thatdecreases the level of a JAK1, JAK2, JAK3, or TYK2 mRNA.Non-limiting examples of JAK inhibitors that are short interferingRNAs (siRNAs) are described in Cook et al., Blood 123:2826-2837,2014. Non-limiting examples of JAK inhibitors that are shorthairpin RNAs (shRNAs) are described in Koppikar et al., Nature489(7414):155-159, 2012).

Small Molecules

[0746] In some embodiments, the JAK inhibitor is a small molecule.In some embodiments, the JAK inhibitory agent is a pan-JAKinhibitor (e.g., 3-O-methylthespesilactam (Li et al., Biochem.Pharmacol. 86(10):1411-8, 2013)).

[0747] In some embodiments, the JAK inhibitor is a JAK1 and JAK2inhibitor. In some embodiments, the JAK1 and JAK2 inhibitor isruxolitinib (Jakafi.RTM., Jakavi.RTM., INCB018424) (Harrison etal., N. Engl. J Med. 366:787-798, 2012; Pieri et al., Am. JHematol. 92(2):187-195, 2017; Mackay-Wiggan et al., JCI Insight1(15):e89790, 2016; Rudolph et al., Leukemia 30(10):2119-2123,2016; Furqan et al., Biomark Res. 1(1):5, 2013), baricitinib(INCB028050, LY3009104) (Gras, Drugs Today (Barc) 52(10):543-550,2016; Smolen et al., Ann. Rheum. Dis. 76(4):694-700, 2016; Kubo etal., Expert. Rev. Clin. Immunol. 12(9):911-919, 2016; Fridman etal., J. Immunol. 84(9):5298-5307, 2010), AZD1480 (Guschin et al.,EMBO J. 14:1421-1429, 1995; Ioannidis et al., J. Med. Chem. 54:262-276, 2011; Moisan et al., Nat. Cell Biol. 17(1):57-67, 2015;Qin et al., J. Neurosci. 36(18):5144059, 2016; Jiang et al.,Biochem. Biophys. Res. Commun. 458(4):908-912, 2015; Verstovsek etal., Leuk. Res. 39(2):157-163, 2015; Plimack et al., Oncologist18(7): 819-820, 2013; Yan et al., Oncotarget 4(3):433-445, 2013),filgotinib (GLPG0634, G146034) (Vermeire et al., Lancet389(10066):266-275, 2017; Menet et al., J Med. Chem.57(22):9323-9342, 2014; Van Rompaey et al., J Immunol.191(7):3568-3577, 2013; Namour et al., Clin. Pharmacokinet.54(8):859-874, 2015), momelotinib (GS-0387, CYT387) (Pardanani etal., Leukemia 23: 1441-1445, 2009; Gupta et al., Haematologica102(1):94-102, 2017; Hu et al., Mol. Pharm. 13(2):689-697, 2016;Abubaker et al., BMC Cancer 14: 317, 2014; Durmus et al.,Pharmacol. Res. 76:9-16, 2013; Pardanani et al., Leukemia 27(6):1322-1327, 2013; Monaghan et al., Leukemia 25(12):1891-1899, 2011;Tyner et al., Blood 115(25):5232-5240, 2010).

[0748] In some embodiments, the JAK inhibitory agent is a JAK1inhibitor (e.g., GSK2586184 (Kahl et al., Lupus 25(13): 1420-1430,2016; Ludbrook et al., Br. J. Dermatol. 174(5):985-995, 2016; vanVollenhoven et al., Lupus 24(6): 648-649, 2015), oclacitinib(PF03394197, Apoquel.RTM.) (Gonzales et al., J. Vet. Pharmacol.Ther. 37(4):317-324, 2014; Collard et al., J. Vet. Pharmacol. Ther.37(3):279-285, 2014; Cosgrove et al., Vet. Dermatol. 24(6):587-597,2013), upadacitinib (ABT494) (Kremer et al., Arthritis Rheumatol.68(12):2867-2877, 2016; Mohamed et al., Clin. Pharmaco. 55(12):1547-1558, 2016), GLG0778 (O'Shea et al., Ann. Rev. Med.66(1):311-28, 2015; Schwartz et al., Nat. Rev. Rheum. 12: 25-36,2016), INCB039110 (Mascarenhas et al., Haematologica102(2):327-335, 2017; Bissonnette et al., J. Dermatolog. Treat.27(4):332-338, 2016; Rosenthal et al., Exp. Opin. Pharmacother.15(9):1265-1276, 2014), PF04965842 (Gadina et al., Curr. Opin.Rheumatol. 26(2):237-243, 2014; Degryset et al., J. Hematol. Oncol.8:91, 2015); SAR-20347 (Works et al., J. Immunol. 193(7):3278-3287,2014)).

[0749] In some embodiments, the JAK inhibitory agent is a JAK2inhibitor (e.g., CEP-33779 (Dugan et al., J. Med. Chem.55(11):5243-5254, 2012; Seavey et al., Mol. Cancer Ther.11(4):984-993, 2012; Stump et al., Arthritis Res. Ther. 13(2):R68,2011), fedratinib (TG101348, SAR302503) (Pardanani et al., J. Clin.Oncol. 29:789-796, 2011; Jamieson et al., J. Transl. Med. 13:294,2015; Zhang et al., Oncotarget 6(16):14329-14343, 2015; Wernig etal., Blood 105:4508-4515, 2008); lestaurtinib (CEP-701) (Hexnet etal., Blood 111:5663-5671, 2008; Santos et al., Blood 115:1131-1136, 2010; Smith et al., Blood 103: 3669-3676, 2004; Hexneret al., Leuk. Lymphoma. 56(9):2543, 2015; Geyer et al., Hematology17(Supp11):5129-132, 2012; Diaz et al., PLoS One 6(4):e18856, 2011;Minturn et al., Cancer Chemother. Pharmacol. 68(4):1057-1065,2011), AC-430 (O'Shea et al., Immunity 36(4):542-550, 2012;Patterson et al., Clin. Exp. Immunol. 176:1-10, 2014), pacritinib(SB1518) (Deeg et al., J. Clin. Oncol. 29: Abstract 6515, 2011;Verstovsek et al., J. Hematol. Oncol. 9(1):137, 2016; Chow et al.,Onco Targets. Ther. 9:2655-2665, 2016; Komrokji et al., Blood125(17):2649-2655, 2015; Jayaraman et al., Drug Metab. Lett.9(1):28-47, 2015), BMS-911543 (Mace et al., Oncotarget6(42):44509-44522, 2015; Wan et al., ACS Med. Chem. Lett.6(8):850-855, 2015; Purandare et al., Leukemia 26(2):280-288,2012), XL019 (Verstovsek et al., Leuk. Res. 38(3):316-322, 2014;Forsyth et al., Bioorg. Med. Chem. Lett. 22(24):7653-7658, 2012),INCB039110 (Mascarenhas et al., Haematologica 102(2):327-335, 2017;Bissonnette et al., J. Dermatol. Treat. 27(4):332-338, 2016),Gandotinib.RTM. (LY-2784544) (Ma et al., Blood Cancer J. 3:e109,2013; Verstovsek et al., Blood 122: 665, 2013; Mitchell et al.,Org. Process Res. Dev. 16(1):70-81. 2012); R723 (Shide et al.,Blood 117(25): 6866-6875, 2011)); Z3 (Sayyah et al., Mol. Cancer.Ther. 7(8):2308-2318, 2008)) or a variant thereof.

[0750] In some embodiments, the JAK inhibitory agent is a JAK3inhibitor (e.g., decernotinib (VX-509) (Elwood et al., J.Pharmacol. Exp. Ther. 2017; Genovese et al., Ann Rheum Dis.75(11):1979-1983, 2016; Gadina et al., Arthritis Rheumatol.68(1):31-34, 2016; Farmer et al., J. Med. Chem. 58(18):7195-7216,2015; Fleischmann et al., Arthritis Rheumatol. 67(2):334-343, 2015;Mahajan et al., J. Pharmacol. 353(2):405-414, 2015), R348 or avariant thereof (Velotta et al., Transplantation 87(5):653-659,2009; Deuse et al., Transplantation 85(6):885-892, 2008)). In someembodiments, the small molecule is R256 or a variant thereof(Ashino et al., J. Allergy Clin. Immunol. 133(4):1162-1174, 2014).In some embodiments, the small molecule is R333 or a variantthereof. In some embodiments, the small molecule is INCB047986 or avariant thereof (Norman, Exp. Opin. Investig. Drugs23(8):1067-1077, 2014). In some embodiments, the small molecule isINCB16562 or a variant thereof (Koppikar et al., Blood115(4):2919-2927, 2010; Li et al., Neoplasia 12(1):28-38, 2010). Insome embodiments, the small molecule is NVP-BSK805 or a variantthereof (Ringel et al., Acta Haematol. 132(1):75-86, 2014; Baffertet al., Mol. Cancer. Ther. 9(7):1945-1955, 2010). In someembodiments, the small molecule is peficitinib (ASP015K,JNJ-54781532) or a variant thereof (Genovese et al., ArthritisRheumatol., 2017; Ito et al., J. Pharmacol. Sci. 133(1):25-33,2017; Cao et al. (2016) Clin. Pharmacol. Drug Dev. 5(6):435-449,2016; Takeuchi et al., Ann. Rheum. Dis. 75(6):1057-1064, 2016). Insome embodiments, the small molecule is tofacitinib (Xeljanz.RTM.,Jakvinus.RTM., CP-690, 500) or a variant thereof (Ghoreschi et al.,J. Immunol. 186(7):4234-4243, 2011; Yoshida et al., Biochem.Biophys. Res. Commun 418(2):234-240, 2012; Calama et al., Pulm.Pharmacol. Ther. S1094-5539(16):30060-30068, 2017; Cutolo et al.,J. Inflamm. Res. 6:129-137, 2013). In some embodiments, the smallmolecule is cucurbitacin I (JSI-124) or a variant thereof (Oi etal., Int. J. Oncol. 49(6):2275-2284, 2016; Qi et al., Am. J. Chin.Med. 43(2):337-347, 2015; Seo et al., Food Chem. Toxicol.64:217-224, 2014). In some embodiments, the small molecule isCHZ868 or a variant thereof (Wu et al., Cancer Cell 28(1):29-41,2015; Meyer et al., Cancer Cell 28(1):15-28, 2015).

[0751] In some embodiments, the small molecule is a TYK2 inhibitor(e.g., Masse et al., J. Immunol. 194(1):67, 2015; Menet, Pharm.Pat. Anal. 3(4):449-466, 2014; Liang et al., Euro. J. Med. Chem.67: 175-187, 2013; Jang et al., Bioorg. Med. Chem. Lett.25(18):3947-3952, 2015); U.S. Pat. Nos. 9,296,725 and 9,309,240; US2013/0231340; and US 2016/0251376). In some embodiments, the TYK2inhibitor is Ndi-031301 (Akahane et al., Blood 128:1596, 2016);BMS-986165 (Gillooly et al., 2016 ACR/ARHP Annual Meeting, Abstract11L, 2016); SAR-20347 (Works et al., J. Immunol. 193(7):3278-3287,2014); tyrphostin A1 (Ishizaki et al., Int. Immunol. 26(5):257-267,2014); a triazolopyridine (US 2013/0143915); or a variantthereof.

[0752] Additional examples of JAK inhibitors that are smallmolecules are described in, e.g., Furomoto et al., BioDrugs27(5):431-438, 2013; 0' Shea et al., Ann. Rheum. Dis.72(2):ii111-ii-115, 2013; Sonbol et al., Ther. Adv. Hematol.4(1):15-35, 2013; and Tanaka et al. (2015) J. Biochem. 158(3):173-179, 2015.

[0753] In some embodiments, the JAK inhibitor is a pan-JAKinhibitor. As used herein, the term "pan-JAK inhibitor" is an agentthat has an IC.sub.50 of about 500 nM to 4 .mu.M (e.g., about 500nM to about 2 .mu.M) for each of human JAK1, human JAK2, and humanJAK3 isoforms, when the IC.sub.50 is determined for each ofwildtype human JAK1, wildtype human JAK2, and wildtype human JAK3using similar assay conditions (e.g., the same assay conditions).In some embodiments, a pan-JAK inhibitor can be an agent that hasan IC.sub.50 for wildtype human JAK1, wildtype human JAK2, andwildtype human JAK3 that are within .+-.10% of each other, wheneach of the IC.sub.50 values is assays under similar assayconditions (e.g., the same assay, e.g., the human wildtype JAK1,wildtype human JAK2, and wildtype human JAK3 assay described in Kimet al., J. Med. Chem. 58(18):7596-5602, 2015).

[0754] In some embodiments, the pan-JAK inhibitor is tofacitinib(Xeljanz.RTM., Jakvinus.RTM., tasocitinib, CP-690550; Yokoyama etal., J. Clin. Immunol. 33(3):586-594, 2013; and Thoma et al., IMed. Chem. 54(1):284-288, 2011); cerdulatinib (PRT2070; Coffey etal. (2014) J. Pharmacol. Exp. Ther. 351(3):538-548, 2014; and Ma etal., Oncotarget 6(41):43881-43896, 2015); Pyridone 6 (P6; Nakagawaet al., J. Immunol. 187(9): 4611-4620, 2011; and Pedranzini et al.,Cancer Res. 66(19):9714-9721, 2006); PF-06263276 (Jones et al."Design and Synthesis of a Pan-Janus Kinase Inhibitor ClinicalCandidate (PF-06263276) Suitable for Inhaled and Topical Deliveryfor the Treatment of Inflammatory Diseases of the Lungs and Skin"J. Med. Chem., 2017, 60 (2), pp 767-786); JAK inhibitor 1 (CAS457081-03-07; JAKi; Wang et al., Antimicrob. Agents Chemother.60(5):2834-48, 2016; Bordonaro et al., PLoS One 9:e115068, 2014;and Osorio et al., PLoS Pathogens 10(6):e1004165, 2014); orbaricitinib (Olumiant; LY3009104; INCB-28050; and Hsu andArmstrong, I Immunol. Res. Article ID 283617, 2014).

[0755] In some embodiments, the JAK inhibitor is a selectiveJAK1/JAK3 inhibitor. As used herein, the term "selective JAK1/JAK3inhibitor" means an agent that has an IC.sub.50 for wildtype humanJAK1 and wildtype human JAK3, that are each at least 5-fold (e.g.,at least 10-fold or at least 20-fold) lowerthan the IC.sub.50 forwildtype human JAK2, when the IC.sub.50 is determined for each ofwildtype human JAK1, wildtype human JAK2, and wildtype human JAK3using similar assay conditions (e.g., the same assay, e.g., thehuman wildtype JAK1, wildtype human JAK2, and wildtype human JAK3assay described in Kim et al., J. Med. Chem. 58(18):7596-5602,2015).

[0756] In some embodiments, the JAK inhibitor is a selective JAK1inhibitor. As used herein, the term "selective JAK1 inhibitor"means an agent that has an IC.sub.50 for wildtype human JAK1 thatis at least 10-fold (e.g., at least 20-fold) lower than each of theIC.sub.50 for wildtype human JAK2 and the IC.sub.50 for wildtypehuman JAK3 when measured using similar assay conditions (e.g., thesame assay, e.g., the human wildtype JAK1, wildtype human JAK2, andwildtype human JAK3 assay described in Kim et al., J. Med. Chem.58(18):7596-5602, 2015). In some embodiments, the JAK1 inhibitor is(3lS,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2--trifluoroethyl)pyrrolidine-1-carboxamide as disclosed ininternational patent application PCT/US2014/062145, incorporated byreference herein in its entirety.

[0757] In some embodiments, the JAK inhibitor is a selective JAK3inhibitor. As used herein, the term "selective JAK3 inhibitor"means an agent that has an IC.sub.50 for wildtype human JAK3 thatis at least 10-fold (e.g., at least 20-fold) lower than each of theIC50 for wildtype human JAK2 and the IC.sub.50 for wildtype humanJAK1 when measured using similar assay conditions (e.g., the sameassay, e.g., the human wildtype JAK1, wildtype human JAK2, andwildtype human JAK3 assay described in Kim et al., J. Med. Chem.58(18):7596-5602, 2015).

[0758] In some embodiments, the JAK inhibitor is a JAK1 and JAK3inhibitor (e.g., a selective JAK1/JAK3 inhibitor). In someembodiments, the selective JAK1/JAK3 inhibitor is ZM 39923 (Brownet al., Bioorg. Med. Chem. Lett. 10(6):575-579, 2000; and Lai etal., Chem. Biol. 15(9):969-978, 2008); or peficitinib (ASP015K;JNJ-54781532; Ito et al., J. Pharmacol. Sci. 133(1):25-33, 2017;Cao et al., Clin. Pharmacol. Drug Dev. 5(6):435-449, 2016; Takeuchiet al., Ann. Rheum. Dis. 75(6):1057-1064, 2016); and Papp et al.,Br. J. Dermatol. 173(3):767-776, 2015).

[0759] In some embodiments, the kinase inhibitor is TOP-1288 fromTopiVert Pharma Ltd., which is described in "The PharmacologicalProfile of TOP1288, a Narrow Spectrum Kinase Inhibitor (NSKI) inClinical Development as an Anti-Inflammatory Treatment forUlcerative Colitis" Foster, Martyn et al. Gastroenterology, Volume152, Issue 5, 5766.

8. Immunosuppressants

[0760] An "immunosuppressant" as disclosed is a low molecularweight immunosuppressants, with low molecular weight defined as<1500 Da, such as <1000 Da. The term "immunosuppressant"refers to a corticosteroid, a direct calcineurin inhibitor, acytostatic, or a direct mTOR inhibitor that can suppress, restrict,or reduce the response of the immune system of a subject (e.g., oneor both of the innate and adaptive immune system). In someexamples, an immunosuppressant drug can decrease the level ofactivation and/or migration of a leukocyte (e.g., a T lymphocyte ora B lymphocyte, a macrophage, a mononcyte, a natural killer cell, aneutrophil, an eosinophil, or a basophil).

[0761] In some embodiments, the immunosuppressant is methotrexate,sulfasalazine, minocycline, or leflunomide) (Zink et al., Annals ofthe Rheumatic Diseases 64: 1274-1279, 2005).

[0762] Non-limiting examples of FDA-approved immunosuppressantdrugs include: CellCept.RTM., Rapamune.RTM., Velcade.RTM.,Protopic.RTM., Afinitor.RTM., Arava.RTM., Zenapax.RTM.,Sandimmune.RTM., Advagraf.RTM., Protopic.RTM., Prograf.RTM.,Astagraf XL.RTM., Elidel.RTM., Myfortic.RTM., Imuran.RTM., andAzasan.RTM..

[0763] Non-limiting examples of immunosuppressants are describedin: Bakr et al., Exp. Clin. Transplant 15(Suppl. 1):16-23, 2017;Palmer et al., Am. J. Kidney Dis. S0272-6386(17):30036-7, 2017;Moran et al., Semin Hematol 49(3):270-276, 2012; Kamel et al.,World J. Transplant 6(4):697-702, 2016; Shrestha et al., Exp. Clin.Transplant. 15(1):1-9, 2017; Liu et al., PLoS One 12(1):e0170246,2017; Chon and Josephson, Expert Rev. Clin. Immunol. 7(3): 273-281,2011; Sollinger et al., Transplantation 60: 225-232, 1995;Salvardori et al., Am. J. Transplant 4: 231-236, 2004; Webster etal., Cochrane Database Syst. Rev. 19(2): CD004290, 2006; Nashan etal., Transplantation 78: 1332-1340, 2004; and Hardinger et al., Am.J Transplant 2: 867-871, 2002.

[0764] Exemplary corticosteroids, cytostatics, calcineurininhibitors, and mTOR inhibitors, are described below.

Corticosteroids

[0765] In some embodiments, the immunosuppressant drug is acorticosteroid. In some embodiments, the immunosuppressant drug canbe a glucocorticosteroid (Coutinho et al., Mol. Cell. Endocrinol.335(1): 2-13, 2011; van Staa et al., QJM 93: 105-111, 2000; Wust etal., J. Immunol. 180: 8434-8443, 2008) or glucocorticoid.Non-limiting examples of corticosteroids include:11-dehydrocorticosterone (also called 11-oxocorticosterone and17-deoxycortisone); 11-deoxycorticosterone (also calleddeoxycortone, desoxycortone, and 21-hydroxyprogesterone);11-deoxycortisol (also called cortodoxone and cortexolone);11-ketoprogesterone (also called 11-oxoprogesterone andketogestin); 110-hydroxypregnenolone; 11.beta.-hydroxyprogesterone(also known as 21-deoxycorticosterone);11.beta.,17.alpha.,21-trihydroxypregnenolone;17.alpha.,21-dihydroxypregnenolone; 17.alpha.-hydroxypregnenolone;17.alpha.-hydroxyprogesterone; 18-hydroxy-11-deoxycorticosterone;18-hydroxycorticosterone; 18-hydroxyprogesterone; 21-deoxycortisol;21-doxycortisone; 21-hydroxypregnenolone (also known asprebediolone); aldosterone; corticosterone (also known as17-deoxycortisol); cortisol (also known as hydrocortisone);cortisone; pregnenolone; progesterone; flugestone (also known asflurogestone); fluorometholone; medrysone (also known ashydroxymethylprogesterone); prebediolone acetate (also known as21-acetoxypregnenolone); chlormadinone acetate; cyproteroneacetate; medrogestone; medroxyprogesterone acetate; megastrolacetate; segesterone acetate; chloropredisone; cloprednol;difluprednate; fludrocortisone; fluocinolone; fluperolone;fluprednisolone; loteprednol; methylprednisolone; prednicarbate;prednisolone; prednisone; tixocortol; triamcinolone; methasone;alclometasone; beclomethasone; betamethasone; clobetasol;clobetasone; clocortolone; desoximetasone; dexamethasone;diflorasone; difluocortolone; fluclorolone; flumetasone;fluocortin; fluocortolone; fluprednidene; fluticasone; fluticasonefuroate; halometasone; mepredisone; mometasone; mometasone furoate;paramethasone; prednylidene; rimexolone; ulobetasol (also known ashalobetasol); amcinonide; budesonide; ciclesonide; deflazacort;desonide; formocortal (also known as fluoroformylone); flucloroloneacetonide (also known as flucloronide); fludroxycortide (also knownas flurandrenolone and flurandrenolide); flunisolide; fluocinoloneacetonide; fluocinonide; halcinonide; triamcinolone acetonide;cortivazol; and RU-28362. In some embodiments, the corticosteroidcan be budesonide (e.g., Entocort.RTM.), dexamethasone,hydrocortisone (e.g., Cortef.RTM., Cortenema.RTM., andProctofoam.RTM.), methylprednisolone, prednisolone (e.g.,Orapred.RTM.), and prednisone. Additional examples ofcorticosteroids are known in the art.

Cytostatics

[0766] In some embodiments, the immunosuppressant drug is acytostatic (e.g., an alkylating agent or an antimetabolite) (Mor etal., BioDrugs 8(6): 469-88, 1997). In some embodiments, thecytostatic is an antimetabolite drug (e.g., a folic acid analogue,(e.g., methotrexate), a purine analogue (e.g., azathioprine ormercaptopurine), a pyrimidine analogue (e.g., fluorouracil), aprotein synthesis inhibitors, and cytotoxic antibiotics (e.g.,dactinomycin, an anthracycline, mitomycin C, bleomycin, andmithramycin).

[0767] In some embodiments, the cytostatic can be an inhibitor ofde novo purine synthesis (e.g., azathioprine (AZA, Imuran.RTM., orAzasan.RTM.), mycophenolate mofetil (MMF, CellCept.RTM.),mycophenolate acid (MPA, Myfortic.RTM.), mizoribin, ormethotrexate). In some embodiments, the cytostatic is an inhibitorof de novo pyrimidine synthesis (e.g., leflunomide, brequinar, ormethotrexate).

[0768] In some embodiments, the cytostatic is an alkylating agent.In some embodiments, the alkylating agent is cyclophosphamide(Luznik et al., Blood 115(16): 3224-330, 2010). In someembodiments, the cytostatic is chlorambucil (Chen et al., Clin. J.Am. Soc. Nephrol. 8(5):787-796, 2013). In some embodiments, thecytostatic is mycophenolate mofetil (MMF, CellCept.RTM.) (Mor etal., BioDrugs 8(6):469-88, 1997). In some embodiments, thecytostatic is mycophenolate sodium (Albano et al., Ann Transplant21: 250-261, 2016). In some embodiments, the cytostatic isazathioprine (Imuran.RTM.) (Maley et al., J. Am. Acad Dermatol73(3): 439-43, 2015). In some embodiments, the immunosuppressantdrug is 6-mercaptopurine (e.g., Purinethol.RTM.) (Kombluth et al.,Gastroenterologist 2(3): 239-46, 1994). In some embodiments, thecytostatic is an inhibitor of inosine monophosphate dehydrogenase(e.g., VX-148; Jain et al., J. Pharmacol Exper Ther 302(2):1272-1277, 2002).

[0769] In some embodiments, the cytostatic is a vitamin D analog(e.g., MC1288). See, e.g., Binderup et al., Biochem. Pharmacol.42:1569-1575, 1991; and Johnsson et al., Transplant Int 7:392-397,1994).

[0770] In some embodiments, the cytostatic is brequinar (Crramer etal., Transplantation 53:303-308, 1992; Xu et al., J. Immunol.160(2):846-53, 1998). In some embodiments, the cytostatic ismizoribine (Bredinin) (Aikawa et al., Transplant. Proc.37(7):2947-50, 2005). In some embodiments, the cytostatic isgusperimus (Perenyei et al., Rheumatology (Oxford)53(10):1732-1741, 2014).

Calcineurin Inhibitors

[0771] In some embodiments, the immunosuppressant is a calcineurininhibitor. See, e.g., Beland et al., Transpl. Int doi: 10.1111/tri12934, 2017. In some embodiments, the calcineurin inhibitor isvoclosporin (Luveniq.RTM.) (Busque et al., Am. J. Transplant11(12):2675-2684, 2011). Voclosporin is a structural analog ofcyclosporine A, with an additional single carbon extension that hasa double-bond on one side chain. The binding affinities ofvoclosporin and cyclosporine A for cyclophilin are comparable;however, upon binding, the ethynyl side chain of voclosporininduces structural changes in calcineurin that may result inincreased immunosuppressive activity relative to cyclosporine A. Insome embodiments, the calcineurin inhibitor is cyclosporin A (e.g.,gengraf, Neural.RTM., or Sandimmune.RTM.) (Canafax and Ascher,Clin. Pharm. 2(6):515-524, 1983; Goring et al., Curr. Med. Res.Opin. 30(8): 1473-87, 2014), a cyclosporin analogue (see, e.g.,Wenger et al., Transplant Proc. 18:213-218, 1986; Jeffery, Clin.Biochem. 24:15-21, 1991; Wenger, Angewandte Chem. 24:77-85, 1985;Lazarova et al., Expert Opin. Ther. Patents 13(9):1327-1332, 2003;Thomson, Lancet 338:195, 1991; U.S. Pat. Nos. 4,885,276, 7,511,013,8,367,053, 8,481,483, 9,175,042, 9,200,038, and 9,226,927; US2011/0092669, US 2006/0069016, US 2010/0708671, US 2012/0088734, WO12/051193, WO 15/31381, WO 12/51194, and WO 12/051193), or acyclosporin analogue (see, e.g., Rothbard et al., Nature6(11):1253-1257, 2000; Cho et al., Arch. Pharm. Res. 27:662, 2004;US 2012/0157385; and U.S. Pat. No. 6,316,405). In some embodiments,the calcineurin inhibitor is tacrolimus, also called FK-506 orfujimycin (e.g., Hecoria.RTM., Prograf.RTM., Astagraf XL.RTM., orProtopic.RTM.) (Helmschrott et al., Drug Des. Devel. Ther.9:1217-1224, 2015; Bloom et al., Clin. Transplant 27(6):E685-93,2013; Riva et al., Fam. Hosp. 41(2):150-168, 2017; McCormack, Drugs74917, 2014); Cryan et al., Biochem. Biophys. Res. Commun. 180(2):846-852, 1991; and Graf et al., J. Clin. Rheumatol. 9(5):310-315,2003). In some embodiments, the calcineurin inhibitor ispimecrolimus (Elidel.RTM.) (Malachowski et al., Pediatr. Dermatol.33(6): e360-e361, 2016; Eichenfiled and Eichenfield, J. Pediatr.167(5):1171-1172, 2015). In some embodiments, the calcineurininhibitor is Sanglifehrin A (SFA) (see, e.g., Hartel et al., Scand.J. Immunol. 63(1):26-34, 2006; Zhang et al., J. Immunol.166(9):5611-5618, 2001; and Woltman et al., J. Immunol. 172(10):6482-6489, 2004). Additional examples of calcineurin inhibitors aredescribed in U.S. Pat. No. 7,041,283.

mTOR Inhibitors

[0772] In some embodiments, an mTOR inhibitor can be rapamycin(mTOR) inhibitor (e.g., sirolimus (Rapamune.RTM.), everolimus)(Forster et al., Transplantation 100(11):2461-2470, 2016; Opelz etal., Nephrol. Dial. Transplant. 31(8): 1360-1367, 2016; andBaroja-Mazo et al., World J. Transplant. 6(1): 183-92, 2016.Another example of an mTOR inhibitor is everolimus (e.g.,Afinitor.RTM. or Zortress.RTM.). Another example of an mTORinhibitor is dactolisib (also called BEZ235 or NVP-BEZ235). Anotherexample of an mTOR inhibitor is temsirolimus (also called CCI-779)(e.g., Torisel.RTM.).

[0773] In some embodiments, the low molecular weightimmunosuppressant is selected from (molecular weights are shown inparenthesis): [0774] a. Cyclosporine (1202 Da); [0775] b.Tacrolimus (804 Da); [0776] c. Methotrexate (454 Da); [0777] d.Sirolimus (914 Da); [0778] e. Everolimus (958 Da); [0779] f.Corticosteroids (360-430 Da); [0780] g. Voclosporin (1214 Da);[0781] h. Azathioprine (277 Da); and [0782] i. Purinethol or 6-MP(6-mercaptopurine) (152 Da).

9. Live Biotherapeutics

[0783] In some embodiments, a live biotherapeutic (also can bereferred to as a live cell therapy) can be detected and analyzed bythe methods herein.

[0784] In some embodiments, the live biotherapeutic includespopulations of live bacteria and/or yeast, optionally incombination with a prebiotic such as a non-digestible carbohydrate,oligosaccharide, or short polysaccharide (e.g., one or more ofinulin, oligofructose, galactofructose, a galacto-oligosaccharides,or a xylo-oligosaccharide) and/or an antibiotic or antifungalagent, or both an antibiotic and antifungal agent. The bacteria orthe yeast can be recombinant. The populations of live bacteriaand/or yeast can be used to selectively alter beneficial specieswithin the GI tract and/or to reduce detrimental species within theGI tract of the subject. See, for example, U.S. Patent PublicationNo. 20070258953; U.S. Patent Publication No. 20080003207;WO2007076534; WO2007136719; and WO2010099824.

[0785] In some embodiments, the live biotherapeutic includes one ormore species of bacteria (e.g., two or more, three or more, four ormore, five or more, six or more, or seven or more species) that areunderrepresented in patients with IBD. The microbiotas of Crohn'sdisease (CD) and ulcerative colitis (UC) patients havestatistically significant differences from those ofnon-inflammatory bowel disease controls, including a reduction inbeneficial commensal bacteria in IBD patients relative tonon-inflammatory bowel disease patients. For example, members ofthe phyla Firmicutes (e.g., Clostridium clusters XIVa and IV),Bacteroidetes (e.g., Bacteroides fragilis or Bacteroides vulgatus),and Actinobacteria (e.g., Coriobacteriaceae spp. or Bifidobacteriumadolescentis) are reduced in CD and UC patients. See, e.g., Frank,et al., Proc Natl Acad Sci USA, 2007, 104:13780-13785; Forbes, etal., Front Microbiol., 2016; 7: 1081, and Nagao-Kitamoto andKamada, Immune Netw. 2017 17(1): 1-12. Clostridium cluster XIVaincludes species belonging to, for example, the Clostridium,Ruminococcus, Lachnospira, Roseburia, Eubacterium, Coprococcus,Dorea, and Butyrivibrio genera. Clostridium cluster IV includesspecies belonging to, for example, the Clostridium, Ruminococcus,Eubacterium and Anaerofilum genera. For example, Faecalibacteriumprausnitzii (also referred to as Bacteroides praussnitzii),Roseburia hominis, Eubacterium rectale, Dialister invisus,Ruminococcus albus, Ruminococcus callidus, and Ruminococcus bromiiare less abundant in CD or UC patients. See, e.g., Nagao-Kitamotoand Kamada, 2017, supra.

[0786] In some embodiments, the live biotherapeutic includes one ormore species of bacteria (e.g., two or more, three or more, four ormore, five or more, six or more, or seven or more species) thatproduce a desired product such as a short chain fatty acid (SCFA)(e.g., butyrate, acetate, or propionate) or induce production(e.g., Clostridium butyricum or F. prausnitzii) of ananti-inflammatory agent such as interleukin-10 in host cells. See,e.g., Hayashi, et al., Cell Host Microbe (2013) 13:711-722.

[0787] In some embodiments, the live biotherapeutic includes one ormore species of bacteria (e.g., two or more, three or more, four ormore, five or more, six or more, or seven or more species) that areunderrepresented in patients with IBD and one or more probiotics(e.g., two or more, three or more, four or more, five or more, sixor more, seven or more, or eight or more probiotics).

[0788] In some embodiments, the live biotherapeutic is FIN-524(Finch Therapeutics, Somerville, Mass.), a cocktail of culturedmicrobial strains that are linked to positive outcomes among IBDpatients.

[0789] In some embodiments, the live biotherapeutic includes one ormore species of bacteria from a healthy donor (e.g., as collectedfrom a stool sample). See, e.g., Vermeire, J Crohns Colitis, 2016,10(4): 387-394. For example, the live biotherapeutic can be FIN-403(Finch Therapeutics, Somerville, Mass.), a candidate forClostridium difficile treatment.

[0790] In some embodiments, the live biotherapeutic includes one ormore agents for inhibiting the growth of a fungus (e.g., a yeastsuch as a species of Candida). In some subjects with Crohn'sdisease, the bacterial species of E. coli and Serratia marcescensand the yeast species Candida tropicalis are found at higherconcentrations versus that of healthy relatives, indicating thatthe bacteria and fungus may interact in the intestines. In someembodiments, the agent inhibiting the growth of a fungus (i.e., ananti-fungal agent) is amphotericin B, an echinocandin such asCaspofungin, Micafungin, or Anidulafungin, or an extended-spectrumtriazole. In some embodiments, the therapeutic includes about 2.5mg/L of Amphotericin B.

[0791] In some embodiments, the live biotherapeutic is abacteriophage or prophage (i.e., the genetic material of abacteriophage incorporated into the genome of a bacterium orexisting as an extrachromosomal plasmid of the bacterium, and ableto produce phages if specifically activated). The bacteriophage canbe lytic or lysogenic. In some embodiments, the bacteriophage caninfect bacteria commonly found in the GI tract. For example, thebacteriophage can infect one or more, two or more, three or more,four or more, five or more, six or more, seven or more, eight ormore, nine or more, or ten or more species of bacteria within theGI tract. See, for example, Wang, et al., Inflamm Bowel Dis., 2015;21(6): 1419-1427. In some embodiments, the bacteriophage can belytic bacteriophage and infect one or more detrimental bacterialspecies in the GI tract to reduce the detrimental species in the GItract. For example, the bacteriophage can infect two or more, threeor more, four or more, five or more, six or more, or seven or moredetrimental bacterial species. In some embodiments, bacteriophagecan be a member of the families from the order Caudovirales such asSiphoviridae, Myroviridae, Podoviridae, or Microviridae. See, e.g.,Babickova and Gardlik, World J. Gastroentrol. 2015;21(40):11321-11330. In some embodiments, the bacteriophage caninclude one or more of bacteriophage K (such as ATCC strain 19685-B1), bacteriophage 17 (such as ATCC strain 23361-B 1), and Stab8.See, e.g., WO2016172380A1. In some embodiments, the livebiotherapeutic includes one or more bacteriophages, and one or moreprobiotics or prebiotics, optionally in combination with anantibiotic.

[0792] In some embodiments, the live biotherapeutic can includebacteriophage or prophage that are genetically modified to produceone or more products that are anti-inflammatory and/or that canenhance intestinal barrier function.

[0793] In some embodiments, the live biotherapeutic includesregulatory T cells (Treg cells). Autologous Treg cells can beprepared by isolating peripheral blood mononuclear cells (PBMCs)from the subject's blood and then expanding ova-specific T cells byculturing the PBMCs in the presence of ovalbumin using Drosophiladerived artificial antigen presenting cells transfected withspecific stimulatory molecules. See, e.g., Brun, et al., IntImmunopharmacol., 2009, 9(5):609-13. T cells can be cloned andOva-Treg clones can be selected based on an ovalbumin-specificIL-10 production. A phase 1/2a study in 20 patients showed that asingle injection of antigen-specific (ovalbumin) Treg cells wassafe in CD and about 40% of the patients show a clinical responseafter treatment. See, e.g., Neurath, 2014, supra; and Desreumaux,et al., Gastroenterology, 2012, 143:1207-1217.

[0794] In some embodiments, the live biotherapeutic can bebacteriophage or bacteria carrying plasmids that encode a targetedantimicrobial. A targeted antimicrobial can include RNA-guidednucleases (RGNs) targeting specific DNA sequences within a targetbacteria. For example, a targeted antimicrobial can couple a phagevector with the CRISPR (clustered regularly interspaced shortpalindromic repeats)/Cas system (e.g., the biological nanobots fromEligo Bioscience (Eligobiotics)). The biological nanobots can becomposed of a capsid from a bacteriophage virus (modified to notmultiply) that infect targeted bacteria and deliver the CRISPR/Cas9system into the targeted bacteria, resulting in the targetedbacteria being killed by cleavage of the bacterial genome by Cas9enzyme within a predetermined pathogenic sequence. See, forexample, WO2017/009399A1 and Citorik, et al., Nat Biotechnol.,2014, 32(11): 1141-1145.

[0795] In some embodiments, the live biotherapeutic can comprisestem cells. The term "stem cell" is used herein to refer to a cellthat is capable of differentiating into a two or more differentcell types. As used herein, the term "a stem cell" may refer to oneor more stem cells.

[0796] In some embodiments, the stem cells can be hematopoieticstem cells (HSC) capable of differentiating into different types ofblood cells, including myeloid and lymphoid lineages of bloodcells. HSC can be obtained from bone marrow, cord blood, orperipheral blood, and are commonly used for bone marrowtransfusions in combination with chemotherapy to restart the immunesystem. HSC are CD34.sup.+ cells. Cell-surface markers can beidentified by any suitable conventional technique, including, forexample, positive selection using monoclonal antibodies againstcell-surface markers.

[0797] In some embodiments, the stem cells are capable ofdifferentiating into two or more different cell types other thanblood cells. In some embodiments, the stem cells are capable ofdifferentiating into cells of each of the three embryonic germlayers (i.e., endoderm, ectoderm, and mesoderm). As used herein,"capable of differentiating" means that a given cell, or itsprogeny, can proceed to a differentiated phenotype under theappropriate culture conditions. The capacity of the cells todifferentiate into at least two cell types can be assayed bymethods known in the art.

[0798] Non-limiting examples of stem cells include embryonic stemcells or adult stem cells such as mesenchymal stem cells (MSC)(also can be referred to as mesenchymal stromal cells) or othermultipotent stem cells; endothelial progenitor cells; stem cellsfrom a particular tissue or organ such as intestinal stem cells,adipose stem cells, or testes stem cells; or induced pluripotentstem cells (iPSC). In some embodiments, stem cells from aparticular tissue also can be classified as MSC.

[0799] In some embodiments, the stem cells are MSC, which candifferentiate into bone, muscle, cartilage, or adipose type cells.MSC can down-regulate inflammation and have a strongimmunoregulatory potential. MSC can be obtained from varioustissues, including from, for example, bone marrow, placenta,amniotic fluid, Wharton's jelly, amniotic membrane, chorionicvilli, umbilical cord, umbilical cord blood, adipose tissue, dentalpulp, synovial membrane, or peripheral blood. Depending on thesource of MSC and the sternness (i.e., multipotency), the MSC canexpress a variety of different markers, including, for example, oneor more of CD105, CD73, CD90, CD13, CD29, CD44, CD10, Stro-1,CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3, SISD2,Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1 (e.g., 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, or 10 or more of such markers), and lackexpression of one or more of CD45, CD34, CD14, CD19, and HLA-DR(e.g., lack expression of two or more, three or more, four or more,or five or more such markers). In some embodiments, MSC can expressCD105, CD73, and CD90. In some embodiments, MSC can express CD105,CD73, CD90, CD13, CD29, CD44, and CD10. In some embodiments, MSCcan express CD105, CD73, and CD90 and one or more sternness markerssuch as Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5,SSEA-3. SISD2, Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, orTM4SF1. In some embodiments, MSC can express CD105, CD73, CD90,CD13, CD29, CD44, and CD10 and one or more sternness markers suchas Stro-1, CD271, SSEA-4, CD146, CD49f, CD349, GD2, 3G5, SSEA-3.SISD2, Stro-4, MSCA-1, CD56, CD200, PODX1, Sox11, or TM4SF1. See,e.g., Lv, et al., Stem Cells, 2014, 32:1408-1419.

[0800] Intestinal stem cells (ISC) can be positive for one or morebiomarkers such as Musashi-1 (Msi-1), Asc12, Bmi-1, Doublecortinand Ca2+/calmodulin-dependent kinase-like 1 (DCAMKL1), andLeucin-rich repeat-containing G-protein-coupled receptor 5 (Lgr5).See, e.g., Mohamed, et al., Cytotechnology, 2015 67(2):177-189.

[0801] In some embodiments, MSCs are commercially available. See,e.g. Prochymal.RTM. from Osiris Therapeutics.

[0802] In some embodiments, the stem cells can be PF-05285401 cells(Multistem.RTM. cells), which are human stem cells obtained fromadult bone marrow or other nonembryonic tissue sources.Multistem.RTM. cells are commercially available from AthersysInc.

[0803] In some embodiments, the stem cells can be autologousadipose derived stem cells such as Cx401cells.

[0804] In some embodiments, the stem cells can be human iPSCs,which can be generated from adult somatic cells (e.g., fibroblasts,keratinocytes, dental pulp cells, cord blood, or peripheral bloodmononuclear cells) or MSC. iPSCs can be generated using retroviralor non-retroviral methods. See, for example, Loh, et al., Blood2009, 113:5476-5479, Okita, et al., Nat Methods. 2011, 8(5):409-12,or Okita, et al., Stem Cells, 2013, 31(3): 458-466. In someembodiments, p53 suppression and nontransforming L-Myc can be usedto generate human induced pluripotent stem cells (iPSCs) withepisomal plasmid vectors encoding OCT3/4, SOX2, KLF4, and LIN28. Insome embodiments, adult somatic cells can be transduced withretroviruses encoding four pluripotency factors (SOX2, KLF4, c-MYC,and OCT4). Fully reprogrammed iPSCs have similar properties toembryonic stem cells (ESCs). Patient's cells can be used to deriveiPSCs, which can then be induced to undergo differentiation intovarious types of somatic cells, all with the same geneticinformation as the patient. See, Azizeh-Mitra, et al., Stem CellsInt 2016; 6180487. In other embodiments, allogenic cells are usedto derive iPSCs.

[0805] In some embodiments, the stem cells can be intestinal stemcells (ISC), which can differentiate into intestinal cell subtypessuch as globet cells, Paneth cells, and enterocytes. ISC arelocated at the crypt base within the intestine and can be positivefor one or more markers such as Musashi-1 (Msi-1), Asc12, Bmi-1,Doublecortin and Ca.sup.2+/calmodulin-dependent kinase-like 1(DCAMKL1), and Leucin-rich repeat-containing G-protein-coupledreceptor 5 (Lgr5). See, e.g., Mohamed, et al., Cytotechnology, 201567(2): 177-189. In addition, ISC or crypts can be used to produceintestinal organoids using a biodegradable scaffold (e.g.,poly-glycolic acid), growth factors such as epidermal growth factor(EGF), R-s pondin, Jagged-1 peptide, or Noggin, and extracellularmatrix. In some embodiments, mesenchymal cells are included in theculture to support the growth. The intestinal organoid can includea central lumen lined by a villus-like epithelium. See, e.g.,US20160287670A1 and WO2015183920A2. Pre-clinical studies havedemonstrated the intestinal organoid efficacy in differentiatinginto all GI cell lineages and regrowing parts of the intestine,muscle layer included. See, Agopian, et al., J Gastrointest Surg.,2009, 13(5):971-82; Kuratnik and Giardina, Biochem Pharmacol.,2013, 85:1721-1726; and Belchior et al., Semin Pediatr Surg., 2014,23:141-149.

[0806] In some embodiments, the stem cells can be allogeneicadipose-derived stem cells (ASC) such as ALLO-ASC cells or expandedASC (eASC) (e.g., Cx601 cells). See, for example, Panes et al.,Lancet; 2016, 388: 1281-90; and U.S. Patent Publication No.20120020930. Cx601 cells are commercially available from TiGenix.Cx601 cells have been used for treating complex perianal fistulasin Crohn's disease patients. ALLO-ASC cells are commerciallyavailable from Anterogen Co., Ltd., and have been used for treatingCrohn's disease.

[0807] In some embodiments, the stem cells can be human placentalderived stem cells such as PDA-001 cells from Celgene. PDA-001cells are a culture-expanded, plastic adherent, undifferentiated invitro cell population that express the nominal phenotype CD34-,CD10+, CD105+ and CD200+. PDA-001 cells constitutively expressmoderate levels of HLA Class I and undetectable levels of HLA ClassII, and they do not express the co-stimulatory molecules CD80 andCD86. PDA-001 is genetically stable, displaying a normal diploidchromosome count, normal karyotype and exhibit normal senescenceafter prolonged in vitro culture. See, e.g., U.S. Pat. No.8,916,146.

10. Carbohydrate Sulfotransferase 15 (CHST15) Inhibitor

[0808] The term "CHST15 inhibitor" refers to an agent whichdecreases CHST15 activity and/or expression. A non-limiting exampleof CHST15 activity is the transfer of sulfate from3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 hydroxylgroup of the GalNAc 4-sulfate residue of chondroitin sulfate A.

[0809] In some embodiments, a CHST15 inhibitor can be an inhibitorynucleic acid. In some embodiments, the inhibitory nucleic acid canbe an antisense nucleic acid, a ribozyme, and a small interferingRNA (siRNA). Examples of aspects of these differentoligonucleotides are described below. Any of the examples ofinhibitory nucleic acids that can decrease expression of CHST15mRNA in a mammalian cell can be synthesized in vitro.

[0810] Inhibitory nucleic acids that can decrease the expression ofCHST15 mRNA expression in a mammalian cell include antisensenucleic acid molecules, i.e., nucleic acid molecules whosenucleotide sequence is complementary to all or part of an CHST15mRNA.

[0811] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding a CHST15 protein. Non-coding regions(5' and 3' untranslated regions) are the 5' and 3' sequences thatflank the coding region in a gene and are not translated into aminoacids.

[0812] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a CHST15 protein(e.g., specificity for a CHST15 mRNA).

[0813] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a CHST15 polypeptide can be inhibited by targetingnucleotide sequences complementary to the regulatory region of thegene encoding the CHST15 polypeptide (e.g., the promoter and/orenhancer, e.g., a sequence that is at least 1 kb, 2 kb, 3 kb, 4 kb,or 5 kb upstream of the transcription initiation start state) toform triple helical structures that prevent transcription of thegene in target cells.

[0814] An inhibitory nucleic acid is a siRNA molecule thatdecreases the level of a CHST15 mRNA. Non-limiting examples ofsiRNAs targeting CHST15 are described in Takakura et al., PLosOne10(12):e0142981, 2015; Watanabe et al., Cell Signal.27(7):1517-1524, 2015; Suzuki et al., PLos One 11(7):e0158967,2016; Kai et al., Mol. Ther. Nucl. Acids 6: 163-172, 2017). In someembodiments, the siRNA targeting CHST15 is STNM01 or a variantthereof (Suzuki et al., J. Crohns Colitis 11(2):221-228, 2017;Atreya et al., Eur. Crohn's Colitis Organisation, Congress AbstractDOP073, 2017; US 2016/0355818; US 2017/0067058; US2016/0348118).

[0815] Additional examples of CHST15 inhibitory nucleic acids aredescribed in US 2015/0337313 and US 2016/0348118, which areincorporated by reference in its entirety.

11. IL-1 Inhibitors

[0816] The term "IL-1 inhibitor" refers to an agent that decreasesthe expression of an IL-1 cytokine or an IL-1 receptor and/ordecreases the ability of an IL-1 cytokine to bind specifically toan IL-1 receptor. Non-limiting examples of IL-1 cytokines includeIL-1.alpha., IL-1.beta., IL-18, IL-36.alpha., IL-36.beta.,IL-36.gamma., IL-38, and IL-33. In some examples, an IL-1 cytokineis IL-1.alpha.. In some examples, an IL-1 cytokine isIL-1.beta..

[0817] As is known in the art, IL-1.alpha. and IL-1(3 each binds toa complex of IL-1R1 and IL1RAP proteins; IL-18 binds to IL-18Ra;IL-36.alpha., IL-36.beta., and IL-36.gamma. each binds to a complexof IL-1RL2 and IL-1RAP proteins; and IL-33 binds to a complex ofIL1RL1 and IL1RAP proteins. IL-1Ra is an endogenous soluble proteinthat decreases the ability of IL-1.alpha. and IL-1.beta. to bind totheir receptor (e.g., a complex of IL-1R1 and IL1RAP proteins).IL-36Ra is an endogenous soluble protein that decreases the abilityof IL-36.alpha., IL-36.beta., and IL-36.gamma. to bind to theirreceptor (e.g., a complex of IL-1RL2 and IL-1RAP proteins).

[0818] In some embodiments, the IL-1 inhibitor mimicks native humaninterleukin 1 receptor antagonist (IL1-Ra).

[0819] In some embodiments, the IL-1 inhibitor targets IL-1.alpha..In some embodiments, the IL-1 inhibitor targets IL-1.beta.. In someembodiments, the IL-1 inhibitor targets one or both of IL-1R1 andIL1RAP. For example, an IL-1 inhibitor can decrease the expressionof IL-1.alpha. and/or decrease the ability of IL-1.alpha. to bindto its receptor (e.g., a complex of IL-1R1 and IL1RAP proteins). Inanother example, an IL-1 inhibitor can decrease the expression ofIL-1.beta. and/or decrease the ability of IL-1.beta. to binds toits receptor (e.g., a complex of IL-1R1 and IL1RAP proteins). Insome embodiments, an IL-1 inhibitor can decrease the expression ofone or both of IL-1R1 and IL1RAP.

[0820] In some embodiments, the IL-1 inhibitor targets IL-18. Insome embodiments, the IL-1 inhibitor targets IL-18Ra. In someembodiments, the IL-1 inhibitor decreases the ability of IL-18 tobind to its receptor (e.g., IL-18R.alpha.). In some embodiments,the IL-1 inhibitor decreases the expression of IL-18. In someembodiments, the IL-1 inhibitor decreases the expression ofIL-18R.alpha..

[0821] In some embodiments, the IL-1 inhibitor targets one or more(e.g., two or three) of IL-36.alpha., IL-36.beta., andIL-36.gamma.. In some embodiments, the IL-1 inhibitor targets oneor both of IL-1RL2 and IL-1RAP. In some embodiments, the IL-1inhibitor decreases the expression of one or more (e.g., two orthree) of IL-36.alpha., IL-36.beta., and IL-36.gamma.. In someembodiments, the IL-1 inhibitor decreases the expression of one orboth of IL-1RL2 and IL-1RAP proteins. In some embodiments, the IL-1inhibitor decreases the ability of IL-36a to bind to its receptor(e.g., a complex including IL-1RL2 and IL-1RAP). In some examples,the IL-1 inhibitor decreases the ability of IL-36.beta. to bind toits receptor (e.g., a complex including IL-1RL2 and IL-1RAP). Insome examples, the IL-1 inhibitor decreases the ability ofIL-36.gamma. to bind to its receptor (e.g., a complex includingIL-1RL2 and IL-1RAP).

[0822] In some embodiments, the IL-1 inhibitor targets IL-33. Insome embodiments, the IL-1 inhibitor targets one or both of IL1RL1and IL1RAP. In some embodiments, the IL-1 inhibitor decreases theexpression of IL-33. In some embodiments, the IL-1 inhibitordecreases the expression of one or both of IL1RL1 and IL1RAP. Insome embodiments, the IL-1 inhibitor decreases the ability of IL-33to bind to its receptor (e.g., a complex of IL1RL1 and IL1RAPproteins).

[0823] In some embodiments, an IL-1 inhibitory agent is aninhibitory nucleic acid, an antibody or fragment thereof, or afusion protein. In some embodiments, the inhibitory nucleic acid isan antisense nucleic acid, a ribozyme, or a small interferingRNA.

Inhibitory Nucleic Acids

[0824] Inhibitory nucleic acids that can decrease the expression ofIL-1.alpha., IL-1.beta., IL-18, IL-36.alpha., IL-36.beta.,IL-36.gamma., IL-38, IL-33, IL-1R1, IL1RAP, IL-18R.alpha., IL-1RL2,or IL1RL1 mRNA in a mammalian cell include antisense nucleic acidmolecules, i.e., nucleic acid molecules whose nucleotide sequenceis complementary to all or part of an IL-1.alpha., IL-1.beta.,IL-18, IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, IL-33,IL-1R1, IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 mRNA.

[0825] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding an IL-1.alpha., IL-1.beta., IL-18,IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-1R1,IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 protein. Non-codingregions (5' and 3' untranslated regions) are the 5' and 3'sequences that flank the coding region in a gene and are nottranslated into amino acids.

[0826] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding an IL-1.alpha.,IL-1.beta., IL-18, IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38,IL-33, IL-1R1, IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 protein(e.g., specificity for an IL-1.alpha., IL-1.beta., IL-18,IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-1R1,IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 mRNA).

[0827] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of an IL-1.alpha., IL-1.beta., IL-18, IL-36.alpha.,IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-1R1, IL1RAP,IL-18R.alpha., IL-1RL2, or IL1RL1 polypeptide can be inhibited bytargeting nucleotide sequences complementary to the regulatoryregion of the gene encoding the IL-1.alpha., IL-1.beta., IL-18,IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-1R1,IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 polypeptide (e.g., thepromoter and/or enhancer, e.g., a sequence that is at least 1 kb, 2kb, 3 kb, 4 kb, or 5 kb upstream of the transcription initiationstart state) to form triple helical structures that preventtranscription of the gene in target cells.

[0828] An inhibitory nucleic acid can be a siRNA that decreases theexpression of an IL-1.alpha., IL-1.beta., IL-18, IL-36.alpha.,IL-36.beta., IL-36.gamma., IL-38, IL-33, IL-1R1, IL1RAP,IL-18R.alpha., IL-1RL2, or IL1RL1 mRNA.

[0829] As described herein, inhibitory nucleic acids preferentiallybind (e.g., hybridize) to a nucleic acid encoding IL-1.alpha.,IL-1.beta., IL-18, IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38,IL-33, IL-1R1, IL1RAP, IL-18R.alpha., IL-1RL2, or IL1RL1 protein totreat allergic diseases (e.g., asthma (Corren et al., N. Engl. J.Med. 365: 1088-1098, 2011)), radiation lung injury (Chung et al.,Sci. Rep. 6: 39714, 2016), ulcerative colitis (Hua et al., Br. J.Clin. Pharmacol. 80:101-109, 2015), dermatitis (Guttman-Yassky etal., Exp. Opin. Biol. Ther. 13(4):1517, 2013), and chronicobstructive pulmonary disease (COPD) (Walsh et al. (2010) Curr.Opin. Investig Drugs 11(11):1305-1312, 2010).

[0830] Exemplary IL-1 inhibitors that are antisense nucleic acidsare described in Yilmaz-Elis et al., Mol. Ther. Nucleic Acids 2(1):e66, 2013; Lu et al., J. Immunol. 190(12): 6570-6578, 2013), smallinterfering RNA (siRNA) (e.g., Ma et al., Ann. Hepatol. 15(2):260-270, 2016), or combinations thereof.

Antibodies

[0831] In some embodiments, the IL-1 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, an antibody or antigen-binding fragment describedherein binds specifically to any one of IL-1.alpha., IL-1.beta.,IL-18, IL-36.alpha., IL-36.beta., IL-36.gamma., IL-38, and IL-33.In some embodiments, an antibody or antigen-binding fragment of anantibody described herein can bind specifically to one or both ofIL-1R1 and IL1RAP. In some embodiments, an antibody orantigen-binding fragment of an antibody described herein can bindspecifically to IL-18R.alpha.. In some embodiments, an antibody orantigen-binding fragment of an antibody described herein can bindspecifically to one or both of IL1RL1 and IL1RAP. In someembodiments, an antibody or antigen-binding fragment of an antibodydescribed herein can bind to one or both of IL-1RL2 andIL-1RAP.

[0832] In some embodiments, the IL-1 inhibitor is canakinumab(ACZ885, Ilaris.RTM. (Dhimolea, MAbs 2(1): 3-13, 2010; Yokota etal., Clin. Exp. Rheumatol. 2016; Torene et al., Ann. Rheum. Dis.76(1):303-309, 2017; Gram, Curr. Opin. Chem. Biol. 32:1-9, 2016;Kontzias et al., Semin. Arthritis Rheum 42(2):201-205, 2012). Insome embodiments, the IL-1 inhibitor is anakinra (Kineret.RTM.;Beynon et al., J. Clin. Rheumatol. 23(3):181-183, 2017; Stanam etal., Oncotarget 7(46):76087-76100, 2016; Nayki et al., J. ObstetGynaecol. Res. 42(11):1525-1533, 2016; Greenhalgh et al., Dis.Model Mech. 5(6):823-833, 2012), or a variant thereof. In someembodiments, the IL-1 inhibitor is gevokizumab (XOMA 052;Knicklebein et al., Am. J. Ophthalmol. 172:104-110, 2016; Roubilleet al., Atherosclerosis 236(2):277-285, 2014; Issafras et al., J.Pharmacol. Exp. Ther. 348(1):202-215, 2014; Handa et al., Obesity21(2):306-309, 2013; Geiler et al., Curr. Opin. Mol. Ther.12(6):755-769, 2010), LY2189102 (Bihorel et al., AAPS J.16(5):1009-1117, 2014; Sloan-Lancaster et al., Diabetes Care36(8):2239-2246, 2013), MABp1 (Hickish et al., Lancey Oncol.18(2):192-201, 2017; Timper et al., J. Diabetes Complications29(7):955-960, 2015), CDP-484 (Braddock et al., Drug Discov.3:330-339, 2004), or a variant thereof (Dinarello et al., Nat. Rev.Drug Discov. 11(8): 633-652, 2012).

[0833] Further teachings of IL-1 inhibitors that are antibodies orantigen-binding fragments thereof are described in U.S. Pat. Nos.5,075,222; 7,446,175; 7,531,166; 7,744,865; 7,829,093; and8,273,350; US 2016/0326243; US 2016/0194392, and US 2009/0191187,each of which is incorporated by reference in its entirety.

Fusion Proteins or Soluble Receptors

[0834] In some embodiments, the IL-1 inhibitor is a fusion proteinor a soluble receptor. For example, a fusion can include anextracellular domain of any one of IL-1R1, IL1RAP, IL-18Ra,IL-1RL2, and IL1RL1 fused to a partner amino acid sequence (e.g., astabilizing domain, e.g., an IgG Fc region, e.g., a human IgG Fcregion). In some embodiments, the IL-1 inhibitor is a solubleversion of one or both of IL-1RL1 and IL1RAP. In some embodiments,the IL-1 inhibitor is a soluble version of IL-18R.alpha.. In someembodiments, the IL-1 inhibitor is a soluble version of one or bothof IL-1RL2 and IL-1RAP.

[0835] In some embodiments, the IL-1 inhibitor is a fusion proteincomprising or consisting of rilonacept (IL-1 Trap, Arcalyst.RTM.)(see, e.g., Kapur & Bonk, P. T. 34(3):138-141, 2009; Church etal., Biologics 2(4):733-742, 2008; McDermott, Drugs Today (Barc)45(6):423-430, 2009). In some embodiments, the IL-1 inhibitor is afusion protein that is chimeric (e.g., EBI-005 (Isunakinra.RTM.)(Furfine et al., Invest. Ophthalmol. Vis. Sci. 53(14):2340-2340,2012; Goldstein et al., Eye Contact Lens 41(3):145-155, 2015;Goldstein et al., Eye Contact Lens, 2016)).

[0836] In some embodiments, the IL-1 inhibitor is a solublereceptor that comprises or consists of sIL-1RI and/or sIL-1RII(Svenson et al., Eur. J. Immunol. 25(10): 2842-2850, 1995).

Endogenous IL-I Inhibitor Peptides

[0837] In some embodiments, the IL-1 inhibitor can be an endogenousligand or an active fragment thereof, e.g., IL-1Ra or IL-36Ra.IL-1Ra is an endogenous soluble protein that decreases the abilityof IL-1.alpha. and IL-1.beta. to bind to their receptor (e.g., acomplex of IL-1R1 and IL1RAP proteins). IL-36Ra is an endogenoussoluble protein that decreases the ability of IL-36.alpha.,IL-36.beta., and IL-36.gamma. to bind to their receptor (e.g., acomplex of IL-1RL2 and IL-1RAP proteins).

12. IL-13 Inhibitors

[0838] The term "IL-13 inhibitor" refers to an agent whichdecreases IL-13 expression and/or decreases the binding of IL-13 toan IL-13 receptor. In some embodiments, the IL-13 inhibitordecreases the ability of IL-13 to bind an IL-13 receptor (e.g., acomplex including IL-4R.alpha. and IL-13R.alpha.1, or a complexincluding IL-13R.alpha.1 and IL-13R.alpha.2).

[0839] In some embodiments, the IL-13 inhibitor targets theIL-4R.alpha. subunit. In some embodiments, the IL-13 inhibitortargets the IL-13R.alpha.1. In some embodiments, the IL-13inhibitor targets IL-13R.alpha.2. In some embodiments, the IL-13inhibitor targets an IL-13 receptor including IL-4R.alpha. andIL-13R.alpha.1. In some embodiments, the IL-13 inhibitor targets anIL-13 receptor including IL-13R.alpha.1 and IL-13R.alpha.2. In someembodiments, the IL-13 inhibitor targets IL-13.

[0840] In some embodiments, an IL-13 inhibitor is an inhibitorynucleic acid, an antibody or an antigen-binding fragment thereof,or a fusion protein. In some embodiments, the inhibitory nucleicacid can be an antisense nucleic acid, a ribozyme, a smallinterfering RNA, a small hairpin RNA, or a microRNA. Examples ofaspects of these different inhibitory nucleic acids are describedbelow.

[0841] Inhibitory nucleic acids that can decrease the expression ofIL-13, IL-13R.alpha.1, IL-13R.alpha.2, or IL-4R.alpha. mRNAexpression in a mammalian cell include antisense nucleic acidmolecules, i.e., nucleic acid molecules whose nucleotide sequenceis complementary to all or part of an IL-13, IL-13R.alpha.1,IL-13R.alpha.2, or IL-Ra mRNA.

[0842] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding an IL-13, IL-13R.alpha.1,IL-13R.alpha.2, or IL-4R.alpha. protein. Non-coding regions (5' and3' untranslated regions) are the 5' and the 3' sequences that flankthe coding region in a gene and are not translated into aminoacids. Non-limiting examples of an inhibitors that are antisensenucleic acids are described in Kim et al., J. Gene Med.11(1):26-37, 2009; and Mousavi et al., Iran J. Allergy AsthmaImmunol. 2(3):131-137, 2003.

[0843] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding an IL-13,IL-13R.alpha.1, IL-13R.alpha.2, or IL-4R.alpha. (e.g., specificityfor an IL-13, IL-13R.alpha.1, IL-13R.alpha.2, or IL-4R.alpha.mRNA).

[0844] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of an IL-13, IL-13R.alpha.1, IL-13R.alpha.2, orIL-4R.alpha. polypeptide can be inhibiting by targeting nucleotidesequences complementary to the regulatory region of the geneencoding the IL-13, IL-13R.alpha.1, IL-13R.alpha.2, or IL-4R.alpha.polypeptide (e.g., the promoter and/or enhancer, e.g., a sequencethat is at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of thetranscription initiation start site) to form triple helicalstructures that prevent transcription of the gene in targetcells.

[0845] As described herein, inhibitory nucleic acid preferentiallybind (e.g., hybridize) to a nucleic acid encoding IL-13,IL-13R.alpha.1, IL-13R.alpha.2, or IL-4R.alpha. protein to treatallergic diseases (e.g., asthma (Corren et al., N. Engl. J. Med.365:1088-1098, 2011), radiation lung injury (Chung et al., Sci.Rep. 6:39714, 2016), ulcerative colitis (Hua et al., Br. J. Clin.Pharmacol. 80:101-109, 2015), dermatitis (Guttman-Yassky et al.,Exp. Opin. Biol. Ther. 13(4):1517, 2013), and chronic obstructivepulmonary disease (COPD) (Walsh et al., Curr. Opin. Investig. Drugs11(11):1305-1312, 2010)).

[0846] An inhibitory nucleic acid can be a siRNA molecule thatdecreases the level of an IL-13, IL-13R.alpha.1, IL-13R.alpha.2, orIL-4R.alpha. mRNA. Non-limiting examples of siRNAs that are IL-13inhibitors are described in Lively et al., J. Allergy Clin.Immunol. 121(1):88-94, 2008. Non-limiting examples of short hairpinRNA (shRHA) that are IL-13 inhibitors are described in Lee et al.,Hum. Gene Ther. 22(5):577-586, 2011, and Shilovskiy et al., Eur.Resp. J. 42:P523, 2013.

[0847] In some embodiments, an inhibitory nucleic acid can be amicroRNA. Non-limiting examples of microRNAs that are IL-13inhibitors are let-7 (Kumar et al., I Allergy Clin. Immunol.128(5):1077-1085, 2011).

Antibodies

[0848] In some embodiments, the IL-13 inhibitor is an antibody oran antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, an antibody or antigen-binding fragment describedherein binds specifically to any one of IL-13, IL-13R.alpha.1,IL-13R.alpha.2, or IL-4R.alpha., or a combination thereof. In someembodiments, an antibody or antigen-binding fragment of an antibodydescribed herein can bind specifically to IL-13. In someembodiments, an antibody or antigen-binding fragment of an antibodydescribed herein can bind specifically to an IL-13 receptor (e.g.,a complex including IL-4R.alpha. and IL-13R.alpha.1, or a complexincluding IL-13R.alpha.1 and IL-13R.alpha.2).

[0849] In some embodiments, the IL-13 inhibitor is a monoclonalantibody (Bagnasco et al., Int. Arch. Allergy Immunol. 170:122-131,2016). In some embodiments, the IL-13 inhibitor is QAX576(Novartis) or an antigen-binding fragment thereof (see, e.g.,Kariyawasam et al., B92 New Treatment Approaches for Asthma andAllergery San Diego, 2009; Rothenberg et al., J. Allergy Clin.Immunol. 135:500-507, 2015). In some embodiments, the IL-13inhibitor is ABT-308 (Abbott) or an antigen-binding fragmentthereof (see, e.g., Ying et al., American Thoracic Society 2010International Conference, May 14-19, 2010, New Orleans; AbstractA6644). In some embodiments, the IL-13 inhibitor is CNTO-5825(Centrocore) or an antigen-binding fragment thereof (see, e.g., vanHartingsveldt et al., British J. Clin. Pharmacol. 75:1289-1298,2013). In some embodiments, the IL-13 inhibitor is dupilumab(REGN668/SAR231893) or an antigen-binding fragment thereof (see,e.g., Simpson et al., N Eng. J. Med. 375:2335-2348, 2016; Thaci etal., Lancet 387:40-52, 2016). In some embodiments, the IL-13inhibitor is AMG317 (Amgen) or an antigen-binding fragment thereof(Polosa et al., Drug Discovery Today 17:591-599, 2012; Holgate,British J. Clinical Pharmacol. 76:277-291, 2013). In someembodiments, the IL-13 inhibitor is an antibody that specificallybinds to IL-13R.alpha.1 (see, e.g., U.S. Pat. No. 7,807,158; WO96/29417; WO 97/15663; and WO 03/080675).

[0850] In some embodiments, the IL-13 inhibitor is a humanizedmonoclonal antibody (e.g., lebrikizumab (TNX-650) (Thomson et al.,Biologics 6:329-335, 2012; and Hanania et al., Thorax70(8):748-756, 2015). In some embodiments, the IL-13 inhibitor isan anti-IL-13 antibody, e.g., GSK679586 or a variant thereof(Hodsman et al., Br. J. Clin. Pharmacol. 75(1):118-128, 2013; andDe Boever et al., I Allergy Clin. Immunol. 133(4):989-996, 2014).In some embodiments, the IL-13 inhibitor is tralokinumab (CAT-354)or a variant thereof (Brightling et al., Lancet 3(9): 692-701,2015; Walsh et al. (2010) Curr. Opin. Investig. Drugs11(11):1305-1312, 2010; Piper et al., Euro. Resp. 1 41:330-338,2013; May et al., Br. J. Pharmacol. 166(1): 177-193, 2012; Singh etal., BMC Pulm Med. 10:3, 2010; Blanchard et al., Clin. Exp. Allergy35(8): 1096-1103, 2005). In some embodiments, the 11-13 inhibitoris anrukinzumab (IMA-638) (Hua et al., Br. J. Clin. Pharmacol. 80:101-109, 2015; Reinisch et al., Gut 64(6): 894-900, 2015; Gauvreauet al., Am. J. Respir. Crit. Care Med. 183(8):1007-1014, 2011; Breeet al., I Allergy Clin. Immunol. 119(5):1251-1257, 2007). Furtherteachings of IL-13 inhibitors that are antibodies orantigen-binding fragments thereof are described in U.S. Pat. Nos.8,067,199; 7,910,708; 8,221,752; 8,388,965; 8,399,630; and8,734,801; US 2014/0341913; US 2015/0259411; US 2016/0075777; US2016/0130339, US 2011/0243928, and US 2014/0105897 each of which isincorporated by reference in its entirety.

Fusion Proteins

[0851] In some embodiments, the IL-13 inhibitor is a fusion proteinor a soluble antagonist. In some embodiments, the fusion proteincomprises a soluble fragment of a receptor of IL-13 (e.g., asoluble fragment of a complex including IL-13R.alpha.1 andIL-4R.alpha., a soluble fragment of a complex includingIL-13R.alpha.1 and IL-13R.alpha.2, a soluble fragment ofIL-13R.alpha.1, a soluble fragment of IL-13R.alpha.2, or solublefragment of IL-4R.alpha.). In some embodiments, the fusion proteincomprises an extracellular domain of a receptor of IL-13 (e.g., afusion protein including an extracellular domain of bothIL-13R.alpha.1 and IL-4R.alpha., a fusion protein including anextracellular domain of both IL-13R.alpha.1 and IL-13R.alpha.2, afusion protein including an extracellular domain of IL-13R.alpha.1,a fusion protein including an extracellular domain ofIL-13R.alpha.2, or a fusion protein including an extracellulardomain of IL-4R.alpha.).

[0852] In some embodiments, the fusion protein comprises orconsists of sIL-13R.alpha.2-Fc (see, e.g., Chiaramonte et al., J.Clin. Invest. 104(6):777-785, 1999; Kasaian et al., Am. IRespir.Cell. Mol. Biol. 36(3):368-376, 2007; Miyahara et al., I AllergyClin. Immunol. 118(5):1110-1116, 2006; Rahaman et al., Cancer Res.62(4):1103-1109, 2002; incorporated by reference herein). In someembodiments, the fusion protein comprises or consists of an IL-13fusion cytotoxin (e.g., IL-13/diphtheria toxin fusion protein (Liet al., Protein Eng. 15(5):419-427, 2002), IL-13-PE38QQR (IL-13-PE)(Blease et al. (2001) J. Immunol. 167(11):6583-6592, 2001; andHusain et al., J. Neuro-Oncol. 65(1):37-48, 2003)).

13. IL-10 and IL-10 Receptor Agonists

[0853] The term "IL-10 receptor agonist" is any molecule that bindsto and activates a receptor for IL-10 expressed on a mammalian cellor a nucleic acid that encodes any such molecule. A receptor forIL-10 can include, e.g., a complex of two IL-10 receptor-1(IL-10R1) proteins and two IL-10 receptor 2 (IL-10R2) proteins. Insome examples, an IL-10 receptor agonist is an antibody or anantigen-binding antibody fragment that specifically binds to andactivates a receptor for IL-10 (e.g., a human receptor for IL-10).In some examples, an IL-10 receptor agonist is a recombinant IL-10(e.g., human recombinant IL-10). In some examples, an IL-10receptor agonist is a pegylated recombinant IL-10 (e.g., pegylatedrecombinant human IL-10). In some examples, an IL-10 receptoragonist is a fusion protein. In some examples, an IL-10 receptoragonist is an IL-10 peptide mimetic.

[0854] Further teachings of IL-1 inhibitors that are antibodies orantigen-binding fragments thereof are described in U.S. Pat. Nos.5,075,222; 7,446,175; 7,531,166; 7,744,865; 7,829,093; and8,273,350; US 2016/0326243; US 2016/0194392, and US 2009/0191187,each of which is incorporated by reference in its entirety.

Recombinant IL-10

[0855] In some examples, an IL-10 receptor agonist is a recombinantIL-10 protein. In some examples, a recombinant IL-10 protein has anamino acid sequence that is identical to a human IL-10 protein.Non-limiting commercial sources of recombinant human IL-10 proteinare available from Peprotech (Rocky Hill, N.J.), Novus Biologicals(Littleton, Colo.), Stemcell.TM. Technologies (Cambridge, Mass.),Millipore Sigma (Billerica, Mass.), and R&D Systems(Minneapolis, Minn.). In some examples, a recombinant human IL-10protein can be Tenovil.TM. (Schering Corporation).

[0856] In some examples, a recombinant IL-10 protein is afunctional fragment of human IL-10 protein. In some examples, afunctional fragment of human IL-10 is a fragment of a human IL-10protein that is able to specifically bind to and activate a humanreceptor of IL-10. A functional fragment of a human IL-10 proteincan have one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, or twenty amino acids removed from the N-and/or C-terminus of the wildtype mature human IL-10 protein. Insome embodiments, the recombinant human IL-10 can include asequence that is at least 80% identical (e.g., at least 82%identical, at least 84% identical, at least 86% identical, at least88% identical, at least 90% identical, at least 92% identical, atleast 94% identical, at least 95% identical, at least 96%identical, at least 98% identical, or at least 99% identical) tothe sequence of wildtype, mature human IL-10, and is able tospecifically bind to and activate a human receptor of IL-10.Mutation of amino acids that are not conserved between differentmammalian species is less likely to have a negative effect on theactivity of a recombinant IL-10 protein.

[0857] In some embodiments, the IL-10 receptor agonist is rhuIL-10(Tenovil) or a variant thereof. See, e.g., McHutchison et al., IInterferon Cytokine Res. 1:1265-1270, 1999; Rosenblum et al.,Regul. Toxicol. Pharmacol. 35:56-71, 2002; Schreiber et al.,Gastroenterology 119(6):1461-1472, 2000; Maini et al., ArthritisRheum. 40(Suppl):224, 1997.

[0858] Exemplary methods of making a recombinant human IL-10 aredescribed in Pajkrt et al., J. Immunol. 158: 3971-3977, 1997).Additional exemplary methods of making recombinant IL-10 aredescribed herein and are known in the art.

[0859] In some embodiments, a recombinant IL-10 is a pegylatedrecombinant IL-10 (e.g., pegylated recombinant human IL-10) (e.g.,a 5 kDa N-terminally PEGylated form of IL-10; AM0010) (Infante etal., ASCO Meeting Abstracts 33(15_suppl):3017, 2015; Chan et al.,PLoS One 11(6):e0156229, 2016; Mumm et al., Cancer Cell20(6):781-796, 2011; Teng et al., Cancer Cell 20(6):691-693, 2011;U.S. Pat. Nos. 8,691,205; 8,865,652; 9,259,478; and 9,364,517; andU.S. Patent Application Publication Nos. 2008/0081031;2009/0214471; 2011/0250163; 2011/0091419; 2014/0227223;2015/0079031; 2015/0086505; 2016/0193352; 2016/0367689;2016/0375101; and 2016/0166647).

[0860] In some embodiments, a recombinant IL-10 is a stabilizedisoform of a recombinant IL-10. In some embodiments, the stabilizedisoform of a recombinant IL-10 is a viral IL-10 protein (e.g., ahuman cytomegalovirus IL10 (e.g., cmv-IL10, LA-cmv-IL-10 (e.g., Linet al., Virus Res. 131(2):213-223, 2008; Jenkins et al., J. Virol.78(3):1440-1447, 2004; Kotenko et al., Proc. Natl. Acad. Sci.U.S.A. 97(4):1695-1700, 2000; Jones et al., Proc. Natl. Acad. Sci.U.S.A. 99(14):9404-9409, 2002) or a latency-associated viral IL-10protein (e.g., Poole et al., J. Virol. 88(24):13947-13955,2014).

[0861] In some embodiments, the recombinant IL-10 is a mammalianIL-10 homolog (see, e.g., WO 00/073457). In some embodiments, amammalian IL-10 homolog is BCRF1, an EBV homolog of human IL-10,also known as viral IL-10, or a variant thereof (Liu et al., J.Immunol. 158(2):604-613, 1997).

Fusion Proteins

[0862] In some embodiments, the IL-10 receptor agonist is a fusionprotein. In some embodiments, the fusion protein comprises theamino acid sequence of an IL-10 protein (or a functional fragmentthereof) and a fusion partner (e.g., an Fc region (e.g., human IgGFc) or human serum albumin). In some embodiments the fusion partnercan be an antibody or an antigen-binding antibody fragment (e.g.,an scFv) that targets IL-10 receptor agonist to an inflamed tissue.In some embodiments, the antibody or antigen-binding fragment thatis a fusion partner can bind specifically, or preferentially, toinflamed gastrointestinal cells by, e.g., CD69. In someembodiments, an IL-10 receptor agonist that is a fusion protein canbe, e.g., F8-IL-10, such as Dekavil (Philogen).

[0863] In some embodiments, the fusion protein is a L19-IL-10fusion protein, a HyHEL10-IL-10 fusion protein, or a variantthereof. See, e.g., Trachsel et al., Arthritis Res. Ther. 9(1):R9,2007, and Walmsley et al., Arthritis Rheum. 39: 495-503, 1996.

IL-10 Peptide Mimetic

[0864] In some embodiments, the IL-10 receptor agonist is an IL-10peptide mimetic. A non-limiting example of an IL-10 peptide mimeticis IT 9302 or a variant thereof (Osman et al., Surgery124(3):584-92, 1998; Lopez et al., Immunobiology 216(10):1117-1126,2011). Additional examples of IL-10 peptide mimetics are describedin DeWitt, Nature Biotech. 17:214, 1999, and Reineke et al., NatureBiotech. 17:271-275, 1999.

Antibodies

[0865] In some embodiments, the IL-10 receptor agonist is anantibody or an antigen-binding antibody fragment that binds to andactivates an IL-10 receptor (e.g., a human IL-10 receptor). In someembodiments, the antibody or antigen-binding antibody fragment thatspecifically binds to an epitope on IL-10R-1 protein (e.g., humanIL-10R-1 protein). In some embodiments, the antibody orantigen-binding antibody fragment that specifically binds to anepitope on IL-10R-2 protein (e.g., a human IL-10R-2 protein). Insome embodiments, the antibody or the antigen-binding antibodyfragment that specifically binds to an epitope on IL-10R-1 andIL-10R-2 proteins (e.g., human IL-10R-1 and human IL-10R-2proteins).

[0866] In some embodiments, the IL-10 receptor agonist is anantibody (e.g., F8-IL10 (also known as DEKAVIL) or a variantthereof (see, e.g., Schwager et al., Arthritis Res. Ther.11(5):R142, 2009; Franz et al., Int. J. Cardiol. 195:311-322, 2015;Galeazzi et al., Isr. Med. Assoc. J. 16(10):666, 2014).

Cells Producing a Recombinant IL-10

[0867] In some embodiments, a recombinant cell (e.g., a recombinantmammalian cell) secretes a recombinant IL-10 (e.g., any of therecombinant IL-10 proteins described herein). In some embodiments,a cell (e.g., a mammalian cell) secretes IL-10 (e.g., human IL-10).In some embodiments, the mammalian cell can be a mammalian cellobtained from the subject, after the introduction of a nucleic acidencoding the recombinant IL-10 (e.g., any of the recombinant IL-10proteins described herein) into the cell obtained from thesubject.

[0868] In some examples, the recombinant mammalian cell can be aChinese Hamster Ovary (CHO) cell, a B cell, a CD8.sup.+ T cell, adendritic cell, a keratinocyte or an epithelial cell. See, e.g.,Mosser et al., Immunol. Rev. 226:205-218, 2009; Fillatreau et al.,Nat. Rev. Immunol. 8:391-397, 2008; Ryan et al., Crit. Rev.Immunol. 27:15-32, 2007; Moore et al., Annu. Rev. Immunol.19:683-765, 2001. In some embodiments, the recombinant mammaliancell can be a mesenchymal stem cell (e.g., Gupte et al., Biomed. J.40(1):49-54, 2017).

Nucleic Acids and Vectors the Encode an IL-10 Receptor Agonist

[0869] In some examples, an IL-10 receptor agonist can be a nucleicacid (e.g., a vector) that includes a sequence encoding an IL-10receptor agonist (e.g., any of the IL-10 proteins describedherein). In some embodiments, the nucleic acid includes a sequenceencoding IL-10 (e.g., human IL-10). In some embodiments, thenucleic acid includes a sequence encoding a recombinant IL-10(e.g., a recombinant human IL-10).

[0870] The nucleic acid can be, e.g., a vector. In someembodiments, a vector can be a viral vector (e.g., an adenovirusvector, a herpes virus vector, a baculovirus vector, or aretrovirus vector). A vector can also be, e.g., a plasmid or acosmid. Additional examples of vectors are known in the art. Avector can include a promoter sequence operably linked to thesequence encoding an IL-10 receptor agonist (e.g., any of therecombinant IL-10 proteins described herein).

[0871] A non-limiting example of a composition including a nucleicacid that encodes an IL-10 receptor agonist is XT-150 (XaludTherapeutics).

Additional Examples of IL-10 Receptor Agonists

[0872] In some embodiments, the recombinant cell is a recombinantGram-positive bacterial cell (e.g., a genetically modifiedLactococcus lactis (LL-Thy12) (see, e.g., Steidler et al., Science289:1352-1355, 2000; Braat et al., Clin. Gastroenterol. Heptal.4:754-759, 2006). In some embodiments, the recombinant cell is arecombinant Gram-negative bacterial cell (e.g., a Shigella flexnericell) that secretes an IL-10 receptor agonist (e.g., a recombinantIL-10 protein) (Chamekh et al., J. Immunol. 180(6): 4292-4298,2008).

[0873] In some embodiments, the IL-10 receptor agonist is a cell(e.g., a Clostridium butyricum cell) that induces IL-10 productionand secretion by a different cell (e.g., a macrophage) (e.g.,Hayashi et al., Cell Host Microbe 13:711-722, 2013). In someembodiments, the IL-10 receptor agonist is a recombinant bacterialcell (e.g., a Lactobacillus acidophilus cell) that is deficient inlipoteichoic acid and induces IL-10 production and secretion by adifferent cell (e.g., a dendritic cell) (e.g., Mohamadzadeh et al.,Proc. Natl. Acad. Sci. U.S.A. 108(Suppl. 1):4623-4630, 2011;Konstantinov et al., Proc. Natl. Acad. Sci. U.S.A. 105(49):19474-9,2008). In some embodiments, the IL-10 receptor agonist is abacterial cell or a fragment of a bacterial cell that is maintainedin the supernatant that induces IL-10 secretion in a different cell(e.g., an immune cell) (e.g., a Faecalibacterium prausnitzii cellor a Faecalibacterium prausnitzii supernatant) (see, e.g., Sokol etal., Proc. Natl. Acad. Sci. U.S.A. 105(43):16731-16736, 2008).

[0874] Additional examples of other IL-10 receptor agonists aredescribed in, e.g., U.S. Pat. No. 6,936,586; WO 96/01318; WO91/00349; WO 13/130913; each incorporated in its entiretyherein.

14. Glatiramer Acetate

[0875] Glatiramer acetate, formerly known as copolymer-1, consistsof the acetate salts of synthetic polypeptides, containing fournaturally occurring amino acids: L-glutamic acid, L-alanine,L-tyrosine, and L-lysine with an average molar fraction of 0.141,0.427, 0.095, and 0.338, respectively. The average molecule weightof glatiramer acetate is 4,700-11,000 daltons.

[0876] Chemically, glatiramer acetate is designated L-glutamic acidpolymer with L-alanine, L-lysine and L-tyrosine, acetate (salt).The CAS number for glatiramer acetate is CAS-147245-92-9. The IUPACname for glatiramer acetate is acetic acid;(2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2S)-2-aminopentanedioic acid; (2S)-2-aminopropanoic acid;(2S)-2,6-diaminohexanoic acid.

[0877] Glatiramer acetate is marketed as the active ingredient ofCopaxone.RTM. by Teva Pharmaceuticals Ltd., Israel. Copaxone.RTM.is a clear, colorless to slightly yellow, sterile, nonpyrogenicsolution. Each 1 mL of Copaxone.RTM. solution contains 20 mg or 40mg of glatiramer acetate and 40 mg of mannitol. The pH ofCopaxone.RTM. solution is approximately 5.5 to 7.0. Copaxone.RTM.20 mg/mL is an FDA-approved product. Copaxone.RTM. 40 mg/mL in aprefilled syringe was developed as a newer formulation of theactive ingredient glatiramer acetate.

[0878] Glatiramer acetate is known as being useful for thetreatment of inflammatory and autoimmune diseases, in addition toits uses for treating multiple sclerosis, see, e.g., U.S. Pat. Nos.7,033,582, 7,053,043, 7,074,580, 7,279,172, and 7,425,332, herebyincorporated by reference in their entirety. Glatiramer acetate hasbeen shown to therapeutically reduce inflammation and amelioratethe pathological manifestations of inflammatory bowel disease (IBD)in numerous murine models (see, e.g., Aharoni et al., J. ofPharmacology and Experimental Therapeutics 318:68-78, 2006; Yao etal., Eur. J. Immunol. 43:125-136, 2013; and Yablecovitch et al., Iof Pharmacology and Experimental Therapeutics 337:391-399, 2011,each of which is hereby incorporated by reference in itsentirety).

[0879] Various glatiramer acetate formulations and methods ofpreparing glatiramer acetate and glatiramer acetate formulationshave been described in, for example, U.S. Pat. Nos. 8,399,413,8,859,489, 8,920,373, 8,921,116, 8,969,302, 8,993,722, 9,018,170,9,029,507, 9,155,775, and 9,402,874, which are hereby incorporatedby reference in their entirety.

15. CD40/CD40L Inhibitors

[0880] The term "CD40/CD40L inhibitors" refers to an agent whichdecreases CD40 or CD40L (CD154) expression and/or the ability ofCD40 to bind to CD40L (CD154). CD40 is a costimulatory receptorthat binds to its ligand, CD40L (CD154).

[0881] In some embodiments, the CD40/CD40L inhibitor can decreasethe binding between CD40 and CD40L by blocking the ability of CD40to interact with CD40L. In some embodiments, the CD40/CD40Linhibitor can decrease the binding between CD40 and CD40L byblocking the ability of CD40L to interact with CD40. In someembodiments, the CD40/CD40L inhibitor decreases the expression ofCD40 or CD40L. In some embodiments, the CD40/CD40L inhibitordecreases the expression of CD40. In some embodiments, theCD40/CD40L inhibitor decreases the expression of CD40L.

[0882] In some embodiments, the CD40/CD40L inhibitor is aninhibitory nucleic acid, an antibody or an antigen-binding fragmentthereof, a fusion protein, or a small molecule. In someembodiments, the inhibitory nucleic acid is a small interferingRNA, an antisense nucleic acid, an aptamer, or a microRNA.Exemplary CD40/CD40L inhibitors are described herein. Additionalexamples of CD40/CD40L inhibitors are known in the art.

[0883] Exemplary aspects of different inhibitory nucleic acids aredescribed below. Any of the examples of inhibitory nucleic acidsthat can decrease expression of CD40 or CD40L mRNA in a mammaliancell can be synthesized in vitro. Inhibitory nucleic acids that candecrease the expression of CD40 or CD40L mRNA in a mammalian cellinclude antisense nucleic acid molecules, i.e., nucleic acidmolecules whose nucleotide sequence is complementary to all or partof a CD40 or CD40L mRNA.

Inhibitory Nucleic Acids

[0884] An antisense nucleic acid molecule can be complementary toall or part of a non-coding region of the coding strand of anucleotide sequence encoding a CD40 or CD40L protein. Non-codingregions (5' and 3' untranslated regions) are the 5' and 3'sequences that flank the coding region in a gene and are nottranslated into amino acids.

[0885] Some exemplary antisense nucleic acids that are CD40 orCD40L inhibitors are described, e.g., in U.S. Pat. Nos. 6,197,584and 7,745,609; Gao et al., Gut 54(1):70-77, 2005; Arranz et al., J.Control Release 165(3):163-172, 2012; Donner et al., Mol. Ther.Nucleic Acids 4:e265, 2015.

[0886] Another example of an inhibitory nucleic acid is a ribozymethat has specificity for a nucleic acid encoding a CD40 or CD40Lprotein (e.g., specificity for a CD40 or CD40L mRNA).

[0887] An inhibitory nucleic acid can also be a nucleic acidmolecule that forms triple helical structures. For example,expression of a CD40 or CD40L polypeptide can be inhibited bytargeting nucleotide sequences complementary to the regulatoryregion of the gene encoding the CD40 or CD40L polypeptide (e.g.,the promoter and/or enhancer, e.g., a sequence that is at least 1kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of the transcriptioninitiation start state) to form triple helical structures thatprevent transcription of the gene in target cells.

[0888] An inhibitory nucleic acid can be a siRNA molecule thatdecreases the level of a CD40 or CD40L mRNA. Non-limiting examplesof short interfering RNA (siRNA) that are CD40/CD40L inhibitors aredescribed in, e.g., Pluvinet et al., Blood 104:3642-3646, 2004;Karimi et al., Cell Immunol. 259(1):74-81, 2009; and Zheng et al.,Arthritis Res. Ther. 12(1):R13, 2010. Non-limiting examples ofshort hairpin RNA (shRNA) targeting CD40/CD40L are described inZhang et al., Gene Therapy 21:709-714, 2014. Non-limiting examplesof microRNAs that are CD40/CD40L inhibitors include, for example,miR146a (Chen et al., FEBS Letters 585(3):567-573, 2011), miR-424,and miR-503 (Lee et al., Sci. Rep. 7:2528, 2017).

[0889] Non-limiting examples of aptamers that are CD40/CD40Linhibitors are described in Soldevilla et al., Biomaterials67:274-285, 2015.

Antibodies

[0890] In some embodiments, the CD40/CD40L inhibitor is an antibodyor an antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, an antibody or antigen-binding fragment describedherein binds specifically to CD40 or CD40L, or to both CD40 andCD40L.

[0891] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of PG102 (Pangenetics)(Bankert et al., J. Immunol. 194(9):4319-4327, 2015); 2C10 (Lowe etal., Am. J. Transplant 12(8):2079-2087, 2012); ASKP1240(Bleselumab) (Watanabe et al., Am. J. Transplant 13(8):1976-1988,2013); 4D11 (Imai et al., Transplantation 84(8):1020-1028, 2007);BI 655064 (Boehringer Ingelheim) (Visvanathan et al., 2016 AmericanCollege of Rheumatology Annual Meeting, Abstract 1588, Sep. 28,2016); 5D12 (Kasran et al., Aliment. Pharmacol. Ther.,22(2):111-122, 2005; Boon et al., Toxicology 174(1):53-65, 2002);ruplizumab (hu5c8) (Kirk et al., Nat. Med. 5(6):686-693, 1999);CHIR12.12 (HCD122) (Weng et al., Blood 104(11):3279, 2004; Tai etal., Cancer Res. 65(13):5898-5906, 2005); CDP7657 (Shock et al.,Arthritis Res. Ther. 17(1):234, 2015); BMS-986004 domain antibody(dAb) (Kim et al., Am. J. Transplant. 17(5):1182-1192, 2017); 5c8(Xie et al., J. Immunol. 192(9):4083-4092, 2014); dacetuzumab(SGN-40) (Lewis et al., Leukemia 25(6):1007-1016, 2011; andKhubchandani et al., Curr. Opin. Investig. Drugs 10(6):579-587,2009); lucatumumab (HCD122) (Bensinger et al., Br. J. Haematol.159: 58-66, 2012; and Byrd et al., Leuk. Lymphoma 53(11):10.3109/10428194.2012.681655, 2012); PG102 (FFP104) (Bankert etal., J. Immunol. 194(9):4319-4327, 2015); Chi Lob 7/4 (Johnson etal., J. Clin. Oncol. 28:2507, 2019); and ASKP1240 (Okimura et al.,Am. J. Transplant. 14(6): 1290-1299, 2014; and Ma et al.,Transplantation 97(4): 397-404, 2014).

[0892] Further teachings of CD40/CD40L antibodies andantigen-binding fragments thereof are described in, for example,U.S. Pat. Nos. 5,874,082; 7,169,389; 7,271,152; 7,288,252;7,445,780; 7,537,763, 8,277,810; 8,293,237, 8,551,485; 8,591,900;8,647,625; 8,784,823; 8,852,597; 8,961,976; 9,023,360, 9,028,826;9,090,696, 9,221,913; US2014/0093497; and US2015/0017155, each ofwhich is incorporated by reference in its entirety.

Fusion and Truncated Proteins and Peptides

[0893] In some embodiments, the CD40/CD40L inhibitor is a fusionprotein, a truncated protein (e.g., a soluble receptor) or apeptide. In some embodiments, the CD40/CD40L inhibitor is atruncated protein as disclosed in, for example, WO 01/096397. Insome embodiments, the CD40/CD40L inhibitor is a peptide, such as acyclic peptide (see, e.g., U.S. Pat. No. 8,802,634; Bianco et al.,Org. Biomol. Chem. 4:1461-1463, 2006; Deambrosis et al., J. Mol.Med. 87(2):181-197, 2009; Vaitaitis et al., Diabetologia57(11):2366-2373, 2014). In some embodiments, the CD40/CD40Linhibitor is a CD40 ligand binder, for example, a Tumor NecrosisFactor Receptor-associated Factor (TRAF): TRAF2, TRAF3, TRAF6,TRAF5 and TTRAP, or E3 ubiquitin-protein ligase RNF128.

Small Molecules

[0894] In some embodiments, the CD40/CD40L inhibitor is a smallmolecule (see, e.g., U.S. Pat. No. 7,173,046, U.S. PatentApplication No. 2011/0065675). In some embodiments, the smallmolecule is Bio8898 (Silvian et al., ACS Chem. Biol. 6(6):636-647,2011); Suramin (Margolles-Clark et al., Biochem. Pharmacol.77(7):1236-1245, 2009); a small-molecule organic dye(Margolles-Clark et al., J. Mol. Med. 87(11):1133-1143, 2009;Buchwald et al., J. Mol. Recognit. 23(1):65-73, 2010), anaphthalenesulphonic acid derivative (Margolles-Clark et al., Chem.Biol. Drug Des. 76(4):305-313, 2010), or a variant thereof.

16. CD3 Inhibitors

[0895] The term "CD3 inhibitor" refers to an agent which decreasesthe ability of one or more of CD3.gamma., CD3.delta., CD3.epsilon.,and CD3.zeta. to associate with one or more of TCR-.alpha.,TCR-.beta., TCR-.delta., and TCR-.gamma.. In some embodiments, theCD3 inhibitor can decrease the association between one or more ofCD3.gamma., CD38, CD3.epsilon., and CD3 and one or more ofTCR-.alpha., TCR-.beta., TCR-.delta., and TCR-.gamma. by blockingthe ability of one or more of CD3.gamma., CD3.delta., CD3.epsilon.,and CD3.zeta. to interact with one or more of TCR-.alpha.,TCR-.beta., TCR-.delta., and TCR-.gamma..

[0896] In some embodiments, the CD3 inhibitor is an antibody or anantigen-binding fragment thereof, a fusion protein, or a smallmolecule. Exemplary CD3 inhibitors are described herein. Additionalexamples of CD3 inhibitors are known in the art.

Antibodies

[0897] In some embodiments, the CD3 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, the CD3 inhibitor is an antibody or antigen-bindingfragment that binds specifically to CD3.gamma.. In someembodiments, the CD3 inhibitor is an antibody or antigen-bindingfragment that binds specifically to CD3.delta.. In somemebodiments, the CD3 inhibitor is an antibody or antigen-bindingfragment that binds specifically to CD3.epsilon.. In someembodiments, the CD3 inhibitor is an antibody or antigen-bindingfragment that binds specifically to CD3. In some embodiments, theCD3 inhibitor is an antibody or an antigen-binding fragment thatcan bind to two or more (e.g., two, three, or four) of CD3.gamma.,CD3.delta., CD3.epsilon., and CD3.zeta..

[0898] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of visiluzumab (Nuvion;HuM-291; M291; SMART anti-CD3 antibody) (Carpenter et al., Biol.Blood Marrow Transplant 11(6): 465-471, 2005; Trajkovic Curr. Opin.Investig. Drugs 3(3): 411-414, 2002; Malviya et al., J. Nucl. Med.50(10): 1683-1691, 2009); muromonab-CD3 (orthoclone OKT3) (Hori etal., Surg. Today 41(4): 585-590, 2011; Norman Ther. Drug Monit17(6): 615-620, 1995; and Gramatzki et al., Leukemia 9(3): 382-390,19); otelixizumab (TRX4) (Vossenkamper et al., Gastroenterology147(1): 172-183, 2014; and Wiczling et al., J. Clin. Pharmacol.50(5): 494-506, 2010); foralumab (NI-0401) (Ogura et al., Clin.Immunol. 183: 240-246; and van der Woude et al., Inflamm. BowelDis. 16: 1708-1716, 2010); ChAgly CD3; teplizumab (MGA031)(Waldron-Lynch et al., Sci. Transl. Med. 4(118): 118ra12, 2012; andSkelley et al., Ann. Pharmacother. 46(10): 1405-1412, 2012); orcatumaxomab (Removab.RTM.) (Linke et al., Mabs 2(2): 129-136, 2010;and Bokemeyer et al., Gastric Cancer 18(4): 833-842, 2015).

[0899] Additional examples of CD3 inhibitors that are antibodies orantibody fragments are described in, e.g., U.S. Patent ApplicationPublication Nos. 2017/0204194, 2017/0137519, 2016/0368988,2016/0333095, 2016/0194399, 2016/0168247, 2015/0166661,2015/0118252, 2014/0193399, 2014/0099318, 2014/0088295,2014/0080147, 2013/0115213, 2013/0078238, 2012/0269826,2011/0217790, 2010/0209437, 2010/0183554, 2008/0025975,2007/0190045, 2007/0190052, 2007/0154477, 2007/0134241,2007/0065437, 2006/0275292, 2006/0269547, 2006/0233787,2006/0177896, 2006/0165693, 2006/0088526, 2004/0253237,2004/0202657, 2004/0052783, 2003/0216551, and 2002/0142000, each ofwhich is herein incorporated by reference in its entirety (e.g.,the sections describing the CD3 inhibitors). Additional CD3inhibitors that are antibodies or antigen-binding antibodyfragments are described in, e.g., Smith et al., J. Exp. Med.185(8):1413-1422, 1997; Chatenaud et al., Nature 7:622-632,2007.

[0900] In some embodiments, the CD3 inhibitor comprises or consistsof a bispecific antibody (e.g., JNJ-63709178) (Gaudet et al., Blood128(22): 2824, 2016); JNJ-64007957 (Girgis et al., Blood 128: 5668,2016); MGD009 (Tolcher et al., J. Clin. Oncol. 34:15, 2016); ERY974(Ishiguro et al., Sci. Transl. Med. 9(410): pii.eaa14291, 2017);AMV564 (Hoseini and Cheung Blood Cancer J. 7:e522, 2017); AFM11(Reusch et al., MAbs 7(3): 584-604, 2015); duvortuxizumab (JNJ64052781); R06958688; blinatumomab (Blincyto.RTM.; AMG103) (RiberaExpert Rev. Hematol. 1:1-11, 2017; and Mori et al., N Engl. J. Med.376(23):e49, 2017); XmAb13676; or REGN1979 (Bannerji et al., Blood128: 621, 2016; and Smith et al., Sci. Rep. 5:17943, 2015)).

[0901] In some embodiments, the CD3 inhibitor comprises or consistsof a trispecific antibody (e.g., ertumaxomab (Kiewe and Thiel,Expert Opin. Investig. Drugs 17(10): 1553-1558, 2008; and Haense etal., BMC Cancer 16:420, 2016); or FBTA05 (Bi20; Lymphomun) (Buhmannet al., J. Transl. Med. 11:160, 2013; and Schuster et al., Br. J.Haematol. 169(1): 90-102, 2015)).

Fusion and Truncated Proteins and Peptides

[0902] In some embodiments, the CD3 inhibitor is a fusion protein,a truncated protein (e.g., a soluble receptor), or a peptide. Insome embodiments, the CD3 inhibitor can be a fusion protein (see,e.g., Lee et al., Oncol. Rep. 15(5): 1211-1216, 2006).

Small Molecules

[0903] In some embodiments, the CD3 inhibitor comprises or consistsof a bispecific small molecule-antibody conjugate (see, e.g., Kimet al., PNAS 110(44): 17796-17801, 2013; Viola et al., Eur. J.Immunol. 27(11):3080-3083, 1997).

17. CD14 Inhibitors

[0904] The term "CD14 inhibitors" refers to an agent whichdecreases the ability of CD14 to bind to lipopolysaccharide (LPS).CD14 acts as a co-receptor with Toll-like receptor 4 (TLR4) thatbinds LPS in the presence of lipopolysaccharide-binding protein(LBP).

[0905] In some embodiments, the CD14 inhibitor can decrease thebinding between CD14 and LPS by blocking the ability of CD14 tointeract with LPS.

[0906] In some embodiments, the CD14 inhibitor is an antibody or anantigen-binding fragment thereof. In some embodiments, the CD14inhibitor is a small molecule. Exemplary CD14 inhibitors aredescribed herein. Additional examples of CD14 inhibitors are knownin the art.

Antibodies

[0907] In some embodiments, the CD14 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, the CD14 inhibitor is an antibody or antigen-bindingfragment that binds specifically to CD14.

[0908] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of IC14 (Axtelle andPribble, J. Endotoxin Res. 7(4): 310-314, 2001; Reinhart et al.,Crit. Care Med. 32(5): 1100-1108, 2004; Spek et al., J. Clin.Immunol. 23(2): 132-140, 2003). Additional examples of anti-CD14antibodies and CD14 inhibitors can be found, e.g., in WO2015/140591 and WO 2014/122660, incorporated in its entiretyherein.

[0909] Additional examples of CD14 inhibitors that are antibodiesor antibody fragments are described in, e.g., U.S. PatentApplication Serial No. 2017/0107294, 2014/0050727, 2012/0227412,2009/0203052, 2009/0029396, 2008/0286290, 2007/0106067,2006/0257411, 2006/0073145, 2006/0068445, 2004/0092712,2004/0091478, and 2002/0150882, each of which is hereinincorporated by reference (e.g., the sections that describe CD14inhibitors).

Small Molecules

[0910] In some embodiments, the CD14 inhibitor is a small molecule.Non-limiting examples of CD14 inhibitors that are small moleculesare described in, e.g., methyl6-deoxy-6-N-dimethyl-N-cyclopentylammonium-2,3-di-O-tetradecyl-.alpha.-D-glucopyranoside iodide (IAXO-101);methyl6-Deoxy-6-amino-2,3-di-O-tetradecyl-.alpha.-D-glucopyranoside(IAXO-102);N-(3,4-bis-tetradecyloxy-benzyl)-N-cyclopentyl-N,N-dimethylammoniumiodide (IAXO-103); and IMO-9200.

[0911] Additional examples of CD14 inhibitors that are smallmolecules are known in the art.

18. CD20 Inhibitors

[0912] The term "CD20 inhibitors" refers to an agent that bindsspecifically to CD20 expressed on the surface of a mammaliancell.

[0913] In some embodiments, the CD20 inhibitor is an antibody or anantigen-binding fragment thereof, or a fusion protein or peptide.Exemplary CD20 inhibitors are described herein.

[0914] Additional examples of CD20 inhibitors are known in theart.

Antibodies

[0915] In some embodiments, the CD20 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv).

[0916] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of rituximab(Rituxan.RTM., MabThera.RTM., MK-8808) (Ji et al., Indian J.Hematol. Blood Transfus. 33(4): 525-533, 2017; and Calderon-Gomezand Panes Gastroenterology 142(1): 1741-76, 2012); -PF-05280586;ocrelizumab (Ocrevus.TM.) (Sharp N. Engl. J. Med. 376(17): 1692,2017); ofatumumab (Arzerra.RTM.; HuMax-CD20) (AlDallal Ther. Clin.Risk Manag. 13:905-907, 2017; and Furman et al., Lancet Haematol.4(1): e24-e34, 2017); PF-05280586 (Williams et al., Br. J. Clin.Pharmacol. 82(6): 1568-1579, 2016; and Cohen et al., Br. J. Clin.Pharmacol. 82(1): 129-138, 2016); obinutuzumab (Gazyva.RTM.) (Reddyet al., Rheumatology 56(7): 1227-1237, 2017; and Marcus et al., N.Engl. J. Med. 377(14): 1331-1344, 2017); ocaratuzumab (AME-133v;LY2469298) (Cheney et al., Mabs 6(3): 749-755, 2014; and Tobinai etal., Cancer Sci. 102(2): 432-8, 2011); GP2013 (Jurczak et al.,Lancet Haenatol. 4(8): e350-e361, 2017); IBI301; HLX01; veltuzumab(hA20) (Kalaycio et al., Leuk. Lymphoma 57(4): 803-811, 2016; andEllebrecht et al., JAMA Dermatol. 150(12): 1331-1335, 2014); SCT400(Gui et al., Chin. J Cancer Res. 28(2): 197-208); ibritumomabtiuxetan (Zevalin.RTM.) (Philippe et al., Bone Marrow Transplant51(8): 1140-1142, 2016; and Lossos et al., Leuk. Lymphoma 56(6):1750-1755, 2015); ublituximab (TG1101) (Sharman et al., Blood 124:4679, 2014; and Sawas et al., Br. J. Haematol. 177(2): 243-253,2017); LFB-R603 (Esteves et al., Blood 118: 1660, 2011; andBaritaki et al., Int. J. Oncol. 38(6): 1683-1694, 2011); ortositumomab (Bexxar) (Buchegger et al., J. Nucl. Med. 52(6):896-900, 2011; and William and Bierman Expert Opin. Biol. Ther.10(8): 1271-1278, 2010). Additional examples of CD20 antibodies areknown in the art (see, e.g., WO 2008/156713).

[0917] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of a bispecific antibody(e.g., XmAb13676; REGN1979 (Bannerji et al., Blood 128: 621, 2016;and Smith et al., Sci. Rep. 5: 17943, 2015); PRO131921 (Casulo etal., Clin. Immnol. 154(1): 37-46, 2014; and Robak and RobakBioDrugs 25(1): 13-25, 2011); or Acellbia).

[0918] In some embodiments, the CD20 inhibitor comprises orconsists of a trispecific antibody (e.g., FBTA05 (Bi20; Lymphomun)(Buhmann et al., J. Transl. Med. 11:160, 2013; and Schuster et al.,Br. J. Haematol. 169(1): 90-102, 2015)).

[0919] Additional examples of CD20 inhibitors that are antibodiesor antigen-binding fragments are described in, e.g., U.S. PatentApplication Publication Nos. 2017/0304441, 2017/0128587,2017/0088625, 2017/0037139, 2017/0002084, 2016/0362472,2016/0347852, 2016/0333106, 2016/0271249, 2016/0243226,2016/0115238, 2016/0108126, 2016/0017050, 2016/0017047,2016/0000912, 2016/0000911, 2015/0344585, 2015/0290317,2015/0274834, 2015/0265703, 2015/0259428, 2015/0218280,2015/0125446, 2015/0093376, 2015/0079073, 2015/0071911,2015/0056186, 2015/0010540, 2014/0363424, 2014/0356352,2014/0328843, 2014/0322200, 2014/0294807, 2014/0248262,2014/0234298, 2014/0093454, 2014/0065134, 2014/0044705,2014/0004104, 2014/0004037, 2013/0280243, 2013/0273041,2013/0251706, 2013/0195846, 2013/0183290, 2013/0089540,2013/0004480, 2012/0315268, 2012/0301459, 2012/0276085,2012/0263713, 2012/0258102, 2012/0258101, 2012/0251534,2012/0219549, 2012/0183545, 2012/0100133, 2012/0034185,2011/0287006, 2011/0263825, 2011/0243931, 2011/0217298,2011/0200598, 2011/0195022, 2011/0195021, 2011/0177067,2011/0165159, 2011/0165152, 2011/0165151, 2011/0129412,2011/0086025, 2011/0081681, 2011/0020322, 2010/0330089,2010/0310581, 2010/0303808, 2010/0183601, 2010/0080769,2009/0285795, 2009/0203886, 2009/0197330, 2009/0196879,2009/0191195, 2009/0175854, 2009/0155253, 2009/0136516,2009/0130089, 2009/0110688, 2009/0098118, 2009/0074760,2009/0060913, 2009/0035322, 2008/0260641, 2008/0213273,2008/0089885, 2008/0044421, 2008/0038261, 2007/0280882,2007/0231324, 2007/0224189, 2007/0059306, 2007/0020259,2007/0014785, 2007/0014720, 2006/0121032, 2005/0180972,2005/0112060, 2005/0069545, 2005/0025764, 2004/0213784,2004/0167319, 2004/0093621, 2003/0219433, 2003/0206903,2003/0180292, 2003/0026804, 2002/0039557, 2002/0012665, and2001/0018041, each herein incorporated by reference in theirentirety (e.g., sections describing CD20 inhibitors).

Peptides and Fusion Proteins

[0920] In some embodiments, the CD20 inhibitor is an immunotoxin(e.g., MT-3724 (Hamlin Blood 128: 4200, 2016).

[0921] In some embodiments, the CD20 inhibitor is a fusion protein(e.g., TRU-015 (Rubbert-Roth Curr. Opin. Mol. Ther. 12(1): 115-123,2010). Additional examples of CD20 inhibitors that are fusionproteins are described in, e.g., U.S. Patent ApplicationPublication Nos. 2012/0195895, 2012/0034185, 2009/0155253,2007/0020259, and 2003/0219433, each of which are hereinincorporated by reference in their entirety (e.g., sectionsdescribing CD20 inhibitors).

19. CD25 Inhibitors

[0922] The term "CD25 inhibitors" refers to an agent whichdecreases the ability of CD25 (also called interleukin-2 receptoralpha chain) to bind to interleukin-2. CD25 forms a complex withinterleukin-2 receptor beta chain and interleukin-2 common gammachain.

[0923] In some embodiments, the CD25 inhibitor is an antibody or anantigen-binding fragment thereof, or a fusion protein. ExemplaryCD25 inhibitors are described herein. Additional examples of CD25inhibitors are known in the art.

Antibodies

[0924] In some embodiments, the CD25 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, a CD25 inhibitor is an antibody or an antigen-bindingfragment thereof that specifically binds to CD25. In someembodiments, a CD25 inhibitor is an antibody that specificallybinds to IL-2.

[0925] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of basiliximab(Simulect.TM.) (Wang et al., Clin. Exp. Immunol. 155(3): 496-503,2009; and Kircher et al., Clin. Exp. Immunol. 134(3): 426-430,2003); daclizumab (Zenapax; Zinbryta.RTM.) (Berkowitz et al., Clin.Immunol. 155(2): 176-187, 2014; and Bielekova et al., Arch Neurol.66(4): 483-489, 2009); or IMTOX-25.

[0926] In some embodiments, the CD25 inhibitor is anantibody-drug-conjugate (e.g., ADCT-301 (Flynn et al., Blood 124:4491, 2014)).

[0927] Additional examples of CD25 inhibitors that are antibodiesare known in the art (see, e.g., WO 2004/045512). Additionalexamples of CD25 inhibitors that are antibodies or antigen-bindingfragments are described in, e.g., U.S. Patent ApplicationPublication Nos. 2017/0240640, 2017/0233481, 2015/0259424,2015/0010539, 2015/0010538, 2012/0244069, 2009/0081219,2009/0041775, 2008/0286281, 2008/0171017, 2004/0170626,2001/0041179, and 2010/0055098, each of which is incorporatedherein by reference (e.g., sections that describe CD25inhibitors).

Fusion Proteins

[0928] In some embodiments, the CD25 inhibitor is a fusion protein.See, e.g., Zhang et al., PNAS 100(4): 1891-1895, 2003.

20. CD28 Inhibitors

[0929] The term "CD28 inhibitors" refers to an agent whichdecreases the ability of CD28 to bind to one or both of CD80 andCD86. CD28 is a receptor that binds to its ligands, CD80 (alsocalled B7.1) and CD86 (called B7.2).

[0930] In some embodiments, the CD28 inhibitor can decrease thebinding between CD28 and CD80 by blocking the ability of CD28 tointeract with CD80. In some embodiments, the CD28 inhibitor candecrease the binding between CD28 and CD86 by blocking the abilityof CD28 to interact with CD86. In some embodiments, the CD28inhibitor can decrease the binding of CD28 to each of CD80 andCD86.

[0931] In some embodiments, the CD28 inhibitor is an antibody or anantigen-binding fragment thereof, a fusion protein, or peptide.Exemplary CD28 inhibitors are described herein. Additional examplesof CD28 inhibitors are known in the art.

Antibodies

[0932] In some embodiments, the CD28 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv).

[0933] In some embodiments, the CD28 inhibitor is a monovalent Fab'antibody (e.g., CFR104) (Poirier et al., Am. J. Transplant 15(1):88-100, 2015).

[0934] Additional examples of CD28 inhibitors that are antibodiesor antigen-binding fragments are described in, e.g., U.S. PatentApplication Publication Nos. 2017/0240636, 2017/0114136,2016/0017039, 2015/0376278, 2015/0299321, 2015/0232558,2015/0150968, 2015/0071916, 2013/0266577, 2013/0230540,2013/0109846, 2013/0078257, 2013/0078236, 2013/0058933,2012/0201814, 2011/0097339, 2011/0059071, 2011/0009602,2010/0266605, 2010/0028354, 2009/0246204, 2009/0117135,2009/0117108, 2008/0095774, 2008/0038273, 2007/0154468,2007/0134240, 2007/0122410, 2006/0188493, 2006/0165690,2006/0039909, 2006/0009382, 2006/0008457, 2004/0116675,2004/0092718, 2003/0170232, 2003/0086932, 2002/0006403,2013/0197202, 2007/0065436, 2003/0180290, 2017/0015747,2012/0100139, and 2007/0148162, each of which is incorporated byreference in its entirety (e.g., sections that described CD28inhibitors).

Fusion Proteins and Peptides

[0935] In some embodiments, the CD28 inhibitor is a fusion protein(see, e.g., U.S. Pat. No. 5,521,288; and US 2002/0018783). In someembodiments, the CD28 inhibitor is abatacept (Orencia.RTM.)(Herrero-Beaumont et al., Rheumatol. Clin. 8: 78-83, 2012; andKorhonen and Moilanen Basic Clin. Pharmacol. Toxicol. 104(4):276-284, 2009).

[0936] In some embodiments, the CD28 inhibitor is a peptide mimetic(e.g., AB103) (see, e.g., Bulger et al., JAMA Surg. 149(6):528-536, 2014), or a synthetical peptoid (see, e.g., Li et al.,Cell Mol. Immunol. 7(2): 133-142, 2010).

21. CD49 Inhibitors

[0937] The term "CD49 inhibitors" refers to an agent whichdecreases the ability of CD49 to bind to one of its ligands (e.g.,MMP1). In some embodiments, the CD49 inhibitor is an antibody or anantigen-binding fragment thereof. Exemplary CD49 inhibitors aredescribed herein. Additional examples of CD49 inhibitors are knownin the art.

Antibodies

[0938] In some embodiments, the CD49 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv).

[0939] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of natalizumab(Tysabri.RTM.; Antegren.RTM.) (see, e.g., Pagnini et al., ExpertOpin. Biol. Ther. 17(11): 1433-1438, 2017; and Chataway and MillerNeurotherapeutics 10(1): 19-28, 2013; or vatelizumab(ELND-004)).

22. CD89 Inhibitors

[0940] The term "CD89 inhibitors" refers to an agent whichdecreases the ability of CD89 to bind to IgA. CD89 is atransmembrane glycoprotein that binds to the heavy-chain constantregion of IgA. In some embodiments, the CD89 inhibitor can decreasethe binding between CD89 and IgA by blocking the ability of CD89 tointeract with IgA. In some embodiments, the CD89 inhibitor is anantibody or an antigen-binding fragment thereof. Exemplary CD89inhibitors are described herein. Additional examples of CD89inhibitors are known in the art.

Antibodies

[0941] In some embodiments, the CD89 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv).

[0942] In certain embodiments, the antibody comprises or consistsof an antigen-binding fragment or portion of HF-1020. Additionalexamples of CD89 antibodies are known in the art (see, e.g., WO2002/064634).

23. Chemokine/Chemokine Receptor Inhibitors

[0943] The term "chemokine/chemokine receptor inhibitors" refers toan agent which decreases the ability of a chemokine to bind to itsreceptor, where the chemokine is one of CXCL10 (IL-10), CCL11, oran ELR chemokine, or the chemokine receptor is CCR2 or CCR9.

CXCL10 (IP-10) Inhibitors

[0944] As used herein "CXCL10", "interferon gamma-induced protein10" and "IP-10" can be used interchangeably. CXCL10 binds to theCXCR3 receptor (e.g., CXCR3-A or CXCR3-B).

[0945] The term "CXCL10 inhibitor" refers to an agent whichdecreases the ability of CXCL10 to bind to a CXCR3 receptor (e.g.,CXCR3-A and/or CXCR3-B).

[0946] In some embodiments, the CXCL10 inhibitor can decrease thebinding between CXCL10 and CXCR3-A by blocking the ability ofCXCL10 to interact with CXCR3-A. In some embodiments, the CXCL10inhibitor can decrease the binding between CXCL10 and CXCR3-B byblocking the ability of CXCL10 to interact with CXCR3-B.

[0947] In some instances, the CXCL10 inhibitor that decreases thebinding between CXCL10 and a CXCR3 (e.g., CXCR3-A and/or CXCR3-B)is a small molecule. In some instances, the CXCL10 inhibitor thatdecreases the binding between CXCL10 and a CXCR3 (e.g., CXCR3-Aand/or CXCR3-B) is an antibody or an antigen-binding antibodyfragment. In some instances, the CXCL10 inhibitor that decreasesthe binding between CXCL10 and a CXCR3 (e.g., CXCR3-A and/orCXCR3-B) is a peptide (e.g., a peptide antagonist of a CXCR3receptor, e.g., one or both of CXCR-A and/or CXCR-B).

CXCL10 Inhibitors--Antibodies

[0948] In some embodiments, the CXCL10 inhibitor is an antibody oran antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, an antibody or antigen-binding fragment describedherein binds specifically to CXCL10 or a CXCR3 receptor (e.g.,CXCR3-A and/or CXCR3-B), or both a CXCL10 and a CXCR3 receptor(e.g., CXCR3-A and/or CXCR3-B). In some embodiments, a CXCL10inhibitor can bind to both CXCR3-A and CXCR3-B.

[0949] In other instances, the CXCL10 inhibitor is a monoclonalantibody (mAb) (see, e.g., WO 05/58815). For example, the CXCL10inhibitor can be Eldelumab.RTM. (MDX-1100 or BMS-936557),BMS-986184 (Bristol-Meyers Squibb), or NI-0801 (Novlmmune). See,e.g., Kuhne et al., J. Immunol. 178(1):5241, 2007; Sandborn et al.,J. Crohns Colitis 11(7):811-819, 2017; and Danese et al.,Gastroenterology 147(5):981-989, 2014. Additional examples ofCXCL10 inhibitors that are antibodies are described in U.S. PatentApplication Publication Nos. 2017/0158757, 2017/0081413,2016/0009808, 2015/0266951, 2015/0104866, 2014/0127229,2014/0065164, 2013/0216549, 2010/0330094, 2010/0322941,2010/0077497, 2010/0021463, 2009/0285835, 2009/0169561,2008/0063646, 2005/0191293, 2005/0112119, 2003/0158392,2003/0031645, and 2002/0018776; and WO 98/11218, each of which isincorporated by reference in its entirety (e.g., the description ofCXCL10 inhibitors).

CCL11 Inhibitors

[0950] The term "CCL11 inhibitor" refers to an agent whichdecreases the ability of CCL11 to bind to one or more of CCR2,CCR3, and CCR5.

[0951] In some embodiments, the CCL11 inhibitor can decrease thebinding between CCL11 and CCR2 by blocking the ability of CCL11 tointeract with CCR2. In some embodiments, the CCL11 inhibitor candecrease the binding between CCL11 and CCR3 by blocking the abilityof CCL11 to interact with CCR3. In some embodiments, the CCL11inhibitor can decrease the binding between CCL11 and CCR5 byblocking the ability of CCL11 to interact with CCR5.

[0952] In some embodiments, a CCL11 inhibitor is an antibody or anantigen-binding fragment thereof.

CCL11 Inhibitors--Antibodies

[0953] In some embodiments, the CCL11 inhibitor is an antibody oran antigen-binding fragment thereof (e.g., a Fab or a scFv). Insome embodiments, an antibody or antigen-binding fragment describedherein binds specifically to CCL11, CCR2, CCR3, or CCR5, or canspecifically bind to two or more of CCL11, CCR2, CCR3, and CCR5. Insome embodiments, a CCL11 inhibitor can bind to two or more ofCCR2, CCR3, and CCR5.

[0954] In some examples the chemokine/chemokine receptor inhibitoris bertilimumab (Immune Pharmaceuticals), an anti-eotaxin-1monoclonal antibody that targets CCL11, and is currently in a PhaseII clinical study for ulcerative colitis. Additional examples ofCCL11 inhibitors are described in U.S. Patent ApplicationPublication Nos. 2016/0289329, 2015/0086546, 2014/0342450,2014/0178367, 2013/0344070, 2013/0071381, 2011/0274696,2011/0038871, 2010/0074886, 2009/0297502, 2009/0191192,2009/0169541, 2009/0142339, 2008/0268536, 2008/0241923,2008/0241136, 2005/0260139, 2005/0048052, 2004/0265303,2004/0132980, 2004/0126851, 2003/0165494, 2002/0150576,2002/0150570, 2002/0051782, 2002/0051781, 2002/0037285,2002/0028436, 2002/0015700, 2002/0012664, 2017/0131282,2016/0368979, 2016/0208011, 2011/0268723, 2009/0123375,2007/0190055, 2017/0049884, 2011/0165182, 2009/0226434,2009/0110686, 2009/0047735, 2009/0028881, 2008/0107647,2008/0107595, 2008/0015348, 2007/0274986, 2007/0231327,2007/0036796, 2007/0031408, 2006/0229336, 2003/0228306,2003/0166870, 2003/0003440, 2002/0019345, and 2001/0000241, each ofwhich is incorporated by reference in its entirety (e.g., thedescription of CCL11 inhibitors).

CXCL10 Inhibitors--Small Molecules and Peptides

[0955] In some instances, the CXCL10 inhibitor is a small molecule.For example, the CXCL10 inhibitor can be ganodermycin (see, e.g.,Jung et al., J. Antiobiotics 64:683-686, 2011). Additionalexemplary small molecule CXCL10 inhibitors are described in: U.S.Patent Application Publication No. 2005/0075333; U.S. PatentApplication Publication No. 2004/0242498; U.S. Patent ApplicationPublication No. 2003/0069234; U.S. Patent Application PublicationNo. 2003/0055054; U.S. Patent Application Publication No.2002/0169159; WO 97/24325; WO 98/38167; WO 97/44329; WO 98/04554;WO 98/27815; WO 98/25604; WO 98/25605; WO 98/25617; WO 98/31364;Hesselgesser et al., J. Biol. Chem. 273(25):15687-15692 (1998); andHoward et al., J. Med. Chem. 41(13):2184-2193 (1998).

[0956] In some examples, the CXCL10 inhibitor is a peptideantagonist of a CXCR3 receptor (e.g., as described in U.S. PatentApplication Publication No. 2007/0116669, 2006/0204498, and WO98/09642). In some examples, the CXCL10 inhibitor is a chemokinemutant or analogue, e.g., those described in U.S. Pat. No.5,739,103, WO 96/38559, and WO 98/06751. Additional examples ofCXCL10 inhibitors that are small molecules or peptides are known inthe art.

CCR2 Inhibitors

[0957] As used herein "CCR2," "CC chemokine receptor 2," or "MCP-1"can be used interchangeably. CCL2, CCL8, and CCL16 eachindividually bind to CCR2.

[0958] The term "CCR2 inhibitor" refers to an agent which decreasesthe ability of CCR2 to bind to one or more (e.g., two, or three) ofCCL2, CCL8, and CCL16.

[0959] In some embodiments, the CCR2 inhibitor can decrease thebinding between CCL2 and CCR2 by blocking the ability of CCL2 tointeract with CCR2. In some embodiments, the CCR2 inhibitor candecrease the binding between CCL8 and CCR2 by blocking the abilityof CCL8 to interact with CCR2. In some embodiments, the CCR2inhibitor can decrease the binding between CCL16 and CCR2 byblocking the ability of CCL16 to interact with CCR2.

[0960] In some embodiments, the CCR2 inhibitor decreases theability of CCR2 to bind to each of CCL2 and CCL8. In someembodiments, the CCR2 inhibitor decreases the ability of CCR2 tobind to each of CCL2 and CCL16. In some embodiments, the CCR2inhibitor decreases the ability of CCR2 to bind to each of CCL8 andCCL16. In some embodiments, the CCRS inhibitor decreases theability of CCR2 to bind to each of CCL2, CCL8, and CCL16.

[0961] In some instances, the CCR2 inhibitor is a small molecule.In some instances, the CCR2 inhibitor is an antibody or anantigen-binding antibody fragment. In some instances, the CCR2inhibitor is a peptide.

CCR2 Inhibitors--Antibodies

[0962] In some embodiments, the CCR2 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, an antibody or antigen-binding fragment describedherein binds specifically to CCR2. In some embodiments, an antibodyor antigen-binding fragment described herein binds specifically toCCL2. In some embodiments, an antibody or antigen-binding fragmentdescribed herein binds specifically to CCL8. In some embodiments,an antibody or antigen-binding fragment described herein bindsspecifically to CCL16. In some embodiments, an antibody orantigen-binding fragment described herein binds specifically toCCR2 and one or more of (e.g., one, two, or three) of CCL2, CCL8,and CCL16.

[0963] In some embodiments, the CCR2 inhibitor is a monoclonalantibody. For example, the CCR2 inhibitor can be MLN1202(Millennium Pharmaceuticals), C775, STI-B0201, STI-B0211,STI-B0221, STI-B0232, carlumab (CNTO 888; Centocor, Inc.), orSTI-B0234, or an antigen-binding fragment thereof. See also, e.g.,Vergunst et al., Arthritis Rheum. 58(7):1931-1939, 2008. Additionalexamples of CCR2 inhibitors that are antibodies or antigen-bindingantibody fragments are described in, e.g., U.S. Patent ApplicationPublication Nos. 2015/0086546, 2016/0272702, 2016/0289329,2016/0083482, 2015/0361167; 2014/0342450, 2014/0178367,2013/0344070, 2013/0071381, 2011/0274696, 2011/0059107,2011/0038871, 2009/0068109, 2009/0297502, 2009/0142339,2008/0268536, 2008/0241923, 2008/0241136, 2007/0128112,2007/0116708, 2007/0111259, 2006/0246069, 2006/0039913,2005/0232923, 2005/0260139, 2005/0058639, 2004/0265303,2004/0132980, 2004/0126851, 2004/0219644, 2004/0047860,2003/0165494, 2003/0211105, 2002/0150576, 2002/0051782,2002/0042370, and 2002/0015700; and U.S. Pat. Nos. 6,312,689,6,084,075, 6,406,694, 6,406,865, 6,696,550, 6,727,349, 7,442,775,7,858,318, 5,859,205, 5,693,762, and 6,075,181, each of which isincorporated by reference (e.g., the description of the CCR2inhibitors). Additional examples of CCR2 inhibitors are describedin, e.g., WO 00/05265. Additional examples of CCR2 inhibitors thatare antibodies or antigen-binding antibodies fragments aredescribed in, e.g., Loberg et al., Cancer Res. 67(19):9417,2007.

CCR2 Inhibitors--Small Molecules and Peptides

[0964] In some examples, the CCR2 inhibitor is a small molecule.For example, the CCR2 inhibitor can be elubrixin, PF-04634817,BMS-741672, or CCX872. See, e.g., U.S. Pat. No. 9,434,766; U.S.Patent Application Publication No. 20070021466; Deerberg et al.,Org. Process Rev. Dev. 20(11):1949-1966, 2016; and Morganti et al.,J. Neurosci. 35(2):748-760, 2015.

[0965] Additional non-limiting examples of CCR2 inhibitors that aresmall molecules include, e.g., the phenylamino substitutedquaternary salt compounds described in U.S. Patent ApplicationPublication No. 2009/0112004; the biaryl derivatives described inU.S. Patent Application Publication No. 2009/0048238; the pyrazolderivatives described in U.S. Patent Application Publication No.2009/0029963; the heterocyclic compounds described in U.S. PatentApplication Publication No. 2009/0023713; the imidazole derivativesdescribed in U.S. Patent Application Publication No. 2009/0012063;the aminopyrrolidines described in U.S. Patent ApplicationPublication No. 2008/0176883; the heterocyclic cyclopentyltetrahydroisoquinolones and tetrahydropyridopyridines described inU.S. Patent Application Publication No. 2008/0081803; theheteroaryl sulfonamides described in U.S. Patent ApplicationPublication No. 2010/0056509; the triazolyl pyridylbenzenesulfonamides described in U.S. Patent ApplicationPublication No. 2010/0152186; the bicyclic and bridged nitrogenheterocycles described in U.S. Patent Application Publication No.2006/0074121; the fused heteroaryl pyridyl and phenylbenzenesulfonamides described in WO 09/009740; and the3-aminopyrrolidene derivatives described in WO 04/050024.

[0966] Additional non-limiting examples of CCR2 inhibitors include:N-((1R,3S)-3-isopropyl-3-{[3-(trifluoromethyl)-7,8-dihydro-1,6-naph-thyri--din-6(5H)-yl]carbonyl}cyclopentyl)-N-[(3S,4S)-3-methoxytetrahydro-2H-pyra-n-4-yl]amine;3[(3S,4R)-1-((1R,3S)-3-isopropyl-2-oxo-3-{[6-(trifluoromethyl)-2H-1,3-ben--z-oxazin-3(4H)-yl]methyl}cyclopentyl)-3-methylpiperidin-4-yl]benzoicacid;(3S,48)-N-((1R,3S)-3-isopropyl-3-{[7-(trifluoromethyl)-3,4-dihydroi-soquin-olin-2(1B)-yl]carbonyl}cyclopentyl)-3-methyltetrahydro-2H-p-yran-4--aminium; 3-[(3S,4R or3R,4S)-1-((1R,3S)-3-Isopropyl-3-{[6-(trifluoromethyl)-2H-1,3-benzoxazin-3--(4H)-yl]carbonyl}cyclopentyl)-3-methylpiperidin-4-yl]benzoic acid;INCB3284; Eotaxin-3; PF-04178903 (Pfizer), and pharmaceuticallyacceptable salts thereof.

[0967] Additional non-limiting examples of CCR2 inhibitors include:bindarit (2-((1-benzyl-1H-indazol-3-yl)methoxy)-2-methylpropionicacid); AZD2423 (AstraZeneca); the indole describes described inU.S. Pat. Nos. 7,297,696, 6,962,926, 6,737,435, and 6,569,888; thebicyclic pyrrole derivatives described in U.S. Pat. Nos. 6,441,004and 6,479,527; the CCR2 inhibitors described in U.S. PatentApplication Publications Nos. 2005/0054668, 2005/0026975,2004/0198719, and 2004/0047860, and Howard et al., Expert Opin.Ther. Patents 11(7):1147-1151 (2001).

[0968] Additional non-limiting examples of CCR2 inhibitors that aresmall molecules are described in, e.g., WO 97/24325; WO 98/38167;WO 97/44329; WO 98/04554; WO 98/27815; WO 98/25604; WO 98/25605; WO98/25617; WO 98/31364; Hesselgesser et al., J. Biol. Chem.273(25):15687-15692, 1998; and Howard et al., J. Med. Chem.41(13):2184-2193, 1998.

[0969] In some embodiments, the CCR2 inhibitor is a small nucleicacid, e.g., NOX-E36 (a 40-nucleotide L-RNA oligonucleotide that islinked to a 40-kDa PEG; NOXXON Pharma AG).

[0970] In some embodiments, the CCR2 inhibitor is a peptide, e.g.,a dominant negative peptide described in, e.g., Kiyota et al., Mol.Ther. 17(5):803-809, 2009, and U.S. Patent Application PublicationNo. 20070004906, or an antagonistic peptide, e.g., the antagonisticpeptides described in WO 05/037305 and Jiang-Hong Gong, et al., J.Exp. Med. 186:131, 1997. Additional examples of CCR2 inhibitorsthat are peptides are described in, e.g., U.S. Pat. No. 5,739,103;WO 96/38559; WO 98/06751; and WO 98/09642. In some embodiments, aCCR2 inhibitor is a CCR2 mutein (e.g., U.S. Patent ApplicationPublication No. 2004/0185450).

[0971] Additional examples of CCR2 inhibitors that are smallmolecules and peptides are known in the art.

CCR9 Inhibitors

[0972] As used herein "CCR9" or "CC chemokine receptor 9" can beused interchangeably. CCR9 specifically binds to CCL25.

[0973] The term "CCR9 inhibitor" refers to an agent which decreasesthe ability of CCR9 to bind to CCL25.

[0974] In some embodiments, the CCR9 inhibitor can decrease thebinding between CCL25 and CCR9 by blocking the ability of CCL25 tointeract with CCR9. In some instances, the CCR9 inhibitor is asmall molecule. In some instances, the CCR9 inhibitor is anantibody or an antigen-binding antibody fragment.

CCR9 Inhibitors--Antibodies

[0975] In some embodiments, the CCR9 inhibitor is an antibody or anantigen-binding fragment thereof (e.g., a Fab or a scFv). In someembodiments, an antibody or antigen-binding fragment describedherein binds specifically to CCR9. In some embodiments, an antibodyor antigen-binding fragment described herein binds specifically toCCL25. In some embodiments, an antibody or antigen-binding fragmentdescribed herein binds specifically to both CCR9 and CCL25.

[0976] In other instances, the CCR9 inhibitor is a monoclonalantibody. For example, the CCR9 antibody can be 91R, see, e.g.,Chamorro et al., MAbs 6(4): 1000-1012, 2014. Additionalnon-limiting examples of CCR9 inhibitors are described in, e.g.,U.S. Patent Application Publication Nos. 2012/0100554,2012/0100154, 2011/0123603, 2009/0028866, and 2005/0181501.

CCR9 Inhibitors--Small Molecules

[0977] In some instances, the CCR9 inhibitor is a small molecule.For example, the CCR9 inhibitor can be Traficet-EN.RTM. (alsocalled Vercirnon, CCX282, and GSK1605786) or Tu1652 CCX507. See,e.g., Eksteen et al., IDrugs 13(7):472-481, 2010; and Walters etal., Gastroenterology 144(5): S-815, 2013.

[0978] Additional examples of CCR9 inhibitors that are smallmolecules are known in the art.

ELR Chemokine Inhibitors

[0979] ELR chemokines are CXC chemokines that have a glutamicacid-leucine-arginine (ELR) motif. See, e.g., Strieter et al., J.Biol. Chem. 270:27348-27357, 1995.

[0980] The term "ELR chemokine inhibitor" refers to an agent whichdecreases the ability of CXCR1 and/or CXCR2 to bind to one or more(e.g., two, three, four, five, six, seven, or eight) of CXCL1,CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, and CXCL8.

[0981] In some embodiments, the ELR chemokine inhibitor candecrease the binding between CXCR1 and CXCL8 by blocking theability of CXCR1 to interact with CXCL8. In some embodiments, theELR chemokine inhibitor can decrease the binding between CXCR1 andCXCL6 by blocking the ability of CXCR1 to interact with CXCL6. Insome embodiments, the ELR chemokine inhibitor can decrease thebinding between CXCR1 and each of CXCL8 and CXCL6.

[0982] In some embodiments, the ELR chemokine inhibitor candecrease the binding between CXCR2 and CXCL1 by blocking theability of CXCR2 to interact with CXCL1. In some embodiments, theELR chemokine inhibitor can decrease the binding between CXCR2 andCXCL2 by blocking the ability of CXCR2 to interact with CXCL2. Insome embodiments, the ELR chemokine inhibitor can decrease thebinding between CXCR2 and CXCL3 by blocking the ability of CXCR2 tointeract with CXCL3. In some embodiments, the ELR chemokineinhibitor can decrease the binding between CXCR2 and CXCL4 byblocking the ability of CXCR2 to interact with CXCL4. In someembodiments, the ELR chemokine inhibitor can decrease the bindingbetween CXCR2 and CXCL5 by blocking the ability of CXCR2 tointeract with CXCL5. In some embodiments, the ELR chemokineinhibitor can decrease the binding between CXCR2 and CXCL6 byblocking the ability of CXCR2 to interact with CXCL6. In someembodiments, the ELR chemokine inhibitor can decrease the bindingbetween CXCR2 and CXCL7 by blocking the ability of CXCR2 tointeract with CXCL7. In some embodiments, the ELR chemokineinhibitor can decrease the binding between CXCR2 and one or more(e.g., two, three, four, five, six, or seven) of CXCL1, CXCL2,CXCL3, CXCL4, CXCL5, CXCL6, and CXCL7.

[0983] In some embodiments, the ELR chemokine inhibitor candecrease the binding of CXCR1 to one or both of CXCL6 and CXCL8,and can decrease the binding to CXCR2 to one or more (e.g., two,three, four, five, six, or seven) of CXCL1, CXCL2, CXCL3, CXCL4,CXCL5, CXCL6, and CXCL7.

[0984] In some instances, the ELR chemokine inhibitor is a smallmolecule. In some instances, the ELR chemokine inhibitor is anantibody or an antigen-binding antibody fragment.

ELR Chemokine Inhibitors--Antibodies

[0985] In some embodiments, the ELR chemokine inhibitor is anantibody or an antigen-binding fragment thereof (e.g., a Fab or ascFv). In some embodiments, an antibody or antigen-binding fragmentdescribed herein binds specifically to CXCR1 and/or CXCR2. In someembodiments, an antibody or antigen-binding fragment describedherein binds specifically to one or more (e.g., two, three, four,five, six, seven, or eight) of: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, and CXCL8 (IL-8).

[0986] An ELR chemokine inhibitor can be, e.g., a monoclonalantibody. A non-limiting example of an ELR inhibitor is TAB-099MZ.Additional examples of ELR chemokine inhibitors that are antibodiesor antigen-binding antibody fragments are described in, e.g., U.S.Pat. No. 9,290,570; and U.S. Patent Application Publication Nos.2004/0170628, 2010/0136031, 2015/0160227, 2015/0224190,2016/0060347, 2016/0152699, 2016/0108117, 2017/0131282,2016/0060347, 2014/0271647, 2014/0170156, 2012/0164143,2010/0254941, 2009/0130110, 2008/0118517, 2004/0208873,2003/0021790, 2002/0082396, and 2001/0006637, each of which isherein incorporated by reference (e.g., the portions describing ELRchemokine inhibitors).

ELR Chemokine Inhibitors--Small Molecules

[0987] In some instances, the ELR chemokine inhibitor is, e.g., asmall molecule. For example, the ELR chemokine inhibitor can be,e.g., LY-3041658 or repertaxin (Reparixin; DF 1681Y). Additionalnon-limiting examples of ELR chemokine inhibitors that are smallmolecules are described in, e.g., U.S. Patent ApplicationPublication Nos. 2007/0248594, 2006/0014794, 2004/0063709,2004/0034229, 2003/0204085, 2003/0097004, 2004/0186142,2004/0235908, 2006/0025453, 2017/0224679, 2017/0190681,2017/0144996, and 2017/0128474, each of which are incorporated byreference (e.g., the portions describing the ELR chemokineinhibitors).

[0988] In some embodiments, the ELR chemokine inhibitor is apeptide, e.g., any of the peptides described in U.S. PatentApplication Publication Nos. 2009/0270318, 2009/0118469, and2007/0160574, 2007/0021593, 2003/0077705, and 2007/0181987, each ofwhich is incorporated by reference (e.g., the portions describingthe ELR chemokine inhibitors).

Combination Detection

[0989] Any combination of the analytes, e.g., bacteria, biomarkers,and/or drugs disclosed herein can be detected using any of themethods described herein. For example, the methods and devicesdisclosed herein can be used to detect combinations of analytessuch as a biomarker indicative of a GI disorder and a drug used totreat the GI disorder. The methods and devices can be used todetect a drug disclosed above and another drug, e.g., another drugused in combination with the first drug. Examples of such drugsinclude 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.4,665,077); non-steroidal antiinflammatory drugs (NSAIDs);ganciclovir; tacrolimus; lucocorticoids such as Cortisol oraldosterone; anti-inflammatory agents such as a cyclooxygenaseinhibitor; a 5-lipoxygenase inhibitor; or a leukotriene receptorantagonist; purine antagonists such as azathioprine ormycophenolate mofetil (MMF); alkylating agents such ascyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde(which masks the MHC antigens, as described in U.S. Pat. No.4,120,649); anti-idiotypic antibodies for MHC antigens and MHCfragments; cyclosporine; 6-mercaptopurine; steroids such ascorticosteroids or glucocorticosteroids or glucocorticoid analogs,e.g., prednisone, methylprednisolone, including SOLU-MEDROL.RTM.,methylprednisolone sodium succinate, and dexamethasone;dihydrofolate reductase inhibitors such as methotrexate (oral orsubcutaneous); anti-malarial agents such as chloroquine andhydroxychloroquine; sulfasalazine; leflunomide; cytokine orcytokine receptor antibodies or antagonists includinganti-interferon-alpha, -beta, or -gamma antibodies, anti-tumornecrosis factor(TNF)-alpha antibodies (infliximab (REMICADE.RTM.)or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies andanti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6)receptor antibodies and antagonists; anti-LFA-1 antibodies,including anti-CD 1 la and anti-CD 18 antibodies; anti-L3T4antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, anti-CD3 or anti-CD4/CD4a antibodies; soluble peptidecontaining a LFA-3 binding domain (WO 90/08187 published Jul. 26,1990); streptokinase; transforming growth factor-beta (TGF-beta);streptodomase; RNA or DNA from the host; FK506; RS-61443;chlorambucil; deoxyspergualin; rapamycin; T-cell receptor (Cohen etal, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner etal, Science, 251: 430-432 (1991); WO 90/11294; Ianeway, Nature,341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF orBR3 antibodies or immunoadhesins and zTNF4 antagonists (for review,see Mackay and Mackay, Trends Immunol, 23: 113-5 (2002) and seealso definition below); biologic agents that interfere with T cellhelper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154), including blocking antibodies to CD4O-CD40 ligand. (e.g.,Durie et al, Science, 261: 1328-30 (1993); Mohan et al, J. Immunol,154: 1470-80 (1995)) and CTLA4-Ig (Finck et al, Science, 265:1225-7 (1994)); and T-cell receptor antibodies (EP 340,109) such asT10B9. Non-limiting examples of drugs that may be detected usingany of the methods described herein also include: budenoside;epidermal growth factor; aminosalicylates; metronidazole;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonalantibodies; growth factors; elastase inhibitors;pyridinyl-imidazole compounds; TNF antagonists; IL-4, IL-10, IL-13and/or TGF.beta. cytokines or agonists thereof (e.g., agonistantibodies); IL-11; glucuronide- or dextran-conjugated prodrugs ofprednisolone, dexamethasone or budesonide; ICAM-I antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; IsisPharmaceuticals, Inc.); soluble complement receptor 1 (TPlO; T CellSciences, Inc.); slow-release mesalazine; antagonists of plateletactivating factor (PAF); ciprofloxacin; and lignocaine. Examples ofdrugs that can be detected using the presently claimed methodsinclude sulfasalazine, related salicylate-containing drugs, andcorticosteroids. In some embodiments, the methods described hereincan be used to detect iron, antidiarrheal agents, azathioprine,6-mercaptopurine, and/or methotrexate.

[0990] In other embodiments, the methods described herein canprovide for detection of a TNF inhibitor as described herein andone or more of: a CHST15 inhibitor, a IL-6 receptor inhibitor, anIL-12/IL-23 inhibitor, an integrin inhibitor, a JAK inhibitor, aSMAD7 inhibitor, a IL-13 inhibitor, an IL-1 receptor inhibitor, aTLR agonist, an immunosuppressant, a live biotherapeutic (e.g.,bacteria of the species Roseburia hominis, Eubacterium rectale,Dialister invisus, Ruminococcus albus, Ruminococcus callidus, andRuminococcus bromii), or a stem cell.

Analyte-Binding Agents

[0991] Certain detection methods described below can utilize atleast one analyte-binding agent in order to detect an analyte in asample. An "analyte-binding agent" is a molecule that binds to aspecific analyte. Some analyte-binding agents may comprise analytes(e.g., the analytes described above) in accordance with the abilityof the analyte to bind to another molecule to be detected using themethods described below. For example, in some embodiments, theanalyte-binding agent comprises an antibody when used as a reagentto detect and/or quantify an antigen that the antibody specificallybinds to. However, in some embodiments, the antibody is an analyte(e.g., an antibody which is a drug, such as a TNF.alpha. antibody)and the analyte-binding agent comprises an antigen to which theantibody specifically binds, thereby allowing for its use as areagent to detect and/or quantify the antibody. In someembodiments, the analyte-binding agent binds to analyte that isspecific to a particular genus, species, or strain of amicroorganism (e.g., a pathogenic bacteria). In some embodiments,an analyte-binding agent has an area on the surface or in a cavitywhich specifically binds to and is thereby defined as complementarywith a particular spatial and polar organization of the analyte. Insome embodiments, the analyte-binding agent and the correspondinganalyte form a binding pair, such as, but not limited to, animmunological pair (such as antigen-antibody), a biotin-avidinpair, a hormone-hormone receptor pair, a nucleic acid duplex,IgG-protein A pair, a polynucleotide pair such as DNA-DNA, DNA-RNA,and the like. In some embodiments, the analyte-binding agentcomprises an antibody (e.g., a monoclonal antibody), an affimer, anaptamer, an antigen, a receptor, a small molecule, and a nucleicacid (e.g., a DNA molecule or an RNA molecule). In someembodiments, either member of the binding pair (e.g., theanalyte-binding agent and/or the analyte) can be detectably labeledas described herein.

[0992] In some embodiments, the analyte-binding agent comprises aportion of a nucleic acid that is complementary to the nucleic acidsequence of the target analyte. As used herein, "complementary"refers to the capacity for pairing through hydrogen binding betweentwo nucleic acid sequences. For example, if a nucleic acid base atone position of the target analyte is capable of hydrogen bondingwith a nucleic acid base at a corresponding position of ananalyte-binding agent, then the bases are considered to becomplementary to each other at that position. In some embodiments,100% complementarity is not required. In some embodiments, 100%complementarity is required. Routine methods can be used to designan analyte-binding agent that binds to a nucleic acid sequence of atarget analyte. In some embodiments, the analyte-binding agentcomprises a nucleic acid sequence that is complementary to at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 45, 50, 55, 60, 65 or more contiguous nucleotides ornucleosides present in the nucleic acid sequence of the targetanalyte (e.g., a DNA molecule or an RNA molecule). In general, theanalyte-binding agents useful in the devices and methods describedherein have at least 80% sequence complementarity to a nucleic acidsequence of a target analyte, e.g., at least 85%, at least 90%, atlast 92%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or are 100% complementary to a nucleicacid sequence of a target analyte).

[0993] In some embodiments, the analyte-binding agent comprises adetectable moiety such as a photosensitizer, a fluorescentcompound, and/or chemiluminescent compound descibed herein. In someembodiments, the analyte-binding agent is capable of being detectedby a detection system of a device described herein, e.g., anoptical detection system.

Ingestible Devices

[0994] Ingestible devices and their use are described, for example,in the following U.S. patent applications, each of which is herebyincorporated by reference: U.S. Ser. No. 14/460,893, entitled"Ingestible Medical Device," and filed Aug. 15, 2014; U.S. Ser. No.15/514,413, entitled "Electromechanical Pill Device withLocalization Capabilities," and filed Mar. 24, 2017; U.S. Ser. No.15/680,400, entitled "Systems and Methods for Obtaining Samplesusing Ingestible Devices," filed on Aug. 18, 2017; U.S. Ser. No.15/680,430, entitled "Sampling Systems and Related Materials andMethods," filed on Aug. 18, 2017; U.S. Ser. No. 15/699,848,entitled "Electromechanical Ingestible Delivery of a DispensableSubstance," filed on Sep. 8, 2017; U.S. Ser. No. 62/480,187,entitled "Localization Systems and Methods for anOptoelectromechanical Pill Device," filed on Mar. 31, 2017; andU.S. Ser. No. 62/540,873, entitled "Localization Systems andMethods for an Ingestible Device," filed on Aug. 3, 2017.

[0995] In general, an ingestible device is configured to be able toenter the GI tract (e.g., via the mouth) and collect one or moresamples while passing through one or more regions of the GI tract.Optionally, the device can include one or more additionalfunctionalities, including the ability to analyze the sample whilein the GI tract of the subject (in vivo), the ability to deliver asubstance (e.g., a therapeutic agent) while in the GI tract of thesubject (in vivo) and/or the ability to locate the device outsidethe GI tract of the subject (ex vivo).

[0996] The ingestible device described herein may generally be inthe shape of a capsule, like a conventional pill. In someembodiments, the device is an ingestible device. In someembodiment, the device is for insertion and removal from thereproductive tract. Accordingly, the shape of the device providesfor easier ingestion, or insertion and removal, and is alsofamiliar to healthcare practitioners and patients.

[0997] Unlike a conventional pill, the device is designed towithstand the chemical and mechanical environment of the G1 tract(e.g., effects of muscle contractile forces and concentratedhydrochloric acid in the stomach) or reproductive tract. However,unlike other devices that are intended to stay inside a patient'sbody (e.g., medical implants), the ingestible device is designed(in general) to only temporarily travel within the body, or to beselectively inserted and removed from the body in the case of thefemale reproductive tract. Accordingly, the regulatory rulesgoverning the materials and manufacture of the ingestible devicemay be less strict than for the devices that are intended to stayinside the body. Nevertheless, since the ingestible device stillenters the body, the material(s) used to manufacture the ingestibledevice are generally selected to at least comply with the standardsfor biocompatibility (e.g., ISO 10993). Furthermore, componentsinside the ingestible device are free of any restricted and/ortoxic metals and are lead-free pursuant to the Directive2002/95/EC, which is also known as the Restriction of HazardousSubstances (RoHS).

[0998] There is a broad range of materials that may be used formanufacturing the ingestible device. Different materials may beused for each of the different components of the ingestible device.Examples of these materials include, but are not limited to,thermoplastics, fluoropolymers, elastomers, stainless steel andglass complying with ISO 10993 and USP Class VI specifications forbiocompatibility. In certain embodiments, these materials mayfurther include liquid silicone rubber material with a hardnesslevel of 10 to 90 as determined using a durometer (e.g.,MED-4942.TM. manufactured by NuSil.TM.), a soft biocompatiblepolymer material such as, but not limited to, polyvinyl chloride(PVC), polyethersulfone (PES), polyethylene (PE), polyurethane (PU)or polytetrafluoroethylene (PTFE), and a rigid polymer materialcoated with a biocompatible material that is soft or pliable (e.g.,a poly(methyl methacrylate) (PMMA) material coated with siliconepolymer). Use of different materials for different components mayenable functionalization of certain surfaces for interaction withproteins, antibodies, and other biomarkers. For example,Teflon.RTM. may be used as a material in the ingestible device forany movable components in order to reduce friction between thesecomponents. Other example materials may include other materialscommonly used in micro-fabrication, such as polydimethylsiloxane(PDMS), borosilicate glass, and/or silicon.

[0999] Generally, an enclosure of the ingestible device may bemanufactured from a type of plastic, such as a photosensitiveacrylic polymer material. The enclosure may be formed by couplingtwo enclosure ends together. The enclosure, in effect, protects theinterior of the ingestible device from its external environment andalso protects the external environment (e.g., the G1 tract orreproductive tract) from components inside the device.

[1000] Furthermore, the device may include one or more additionallayers of protection. The additional protection may protect thepatient against any adverse effects arising from any structuralproblems associated with the enclosure (e.g., the two enclosureends falling apart or a fracture developing in the enclosure). Forexample, a power supply inside the device may be coated with aninert and pliable material (e.g., a thin layer of silicone polymer)so that only electrical contacts on the power supply are exposed.This additional protection to the power supply may preventchemicals inside the device from seeping into the patient'sbody.

[1001] Also, a surface of the device and surfaces of the differentcomponents in the device may receive different treatments that varyaccording to their intended use. For example, the surface of thedevice may receive plasma activation for increasing hydrophilicbehavior. Dilution chambers, storage components, ports, valves,pumps and/or conduits that are intended to come into contact with afluid such as biological fluid or dilution fluid during normaloperation of the device may also receive hydrophilic treatmentwhile certain other components may receive hydrophobictreatments.

[1002] FIG. 1 illustrates an example ingestible device 100 withmultiple openings in the housing. The ingestible device 100 has anouter housing with a first end 102A, a second end 102B, and a wall104 extending longitudinally from the first end 102A to the secondend 102B. Ingestible device 100 has a first opening 106 in thehousing, which is connected to a second opening 108 in the housing.The first opening 106 of the ingestible device 100 is orientedsubstantially perpendicular to the second opening 108, and theconnection between the first opening 106 and the second opening 108forms a curved chamber 110 within the ingestible device 100.

[1003] The overall shape of the ingestible device 100, or any ofthe other ingestible devices discussed in this disclosure, may besimilar to an elongated pill or capsule. This may make theingestible device 100 easy to consume, and allow it to traveleasily through the GI tract. In certain portions of the GI tract,such as the stomach, the ingestible device 100 may be free to moveor rotate in any direction. In other portions of the GI tract, themovement of the ingestible device 100 may be restricted. Forexample, in the relatively narrow confines of the small intestine,the walls of the small intestine may squeeze down on the ingestibledevice, forcing the ingestible device 100 to orient itselflongitudinally along the length of the small intestine. In thiscase, the walls of the small intestine wrap around thelongitudinally extending wall 104 of the ingestible device 100, andthe ingestible device 100 travels through the small intestine withone of the ends 102A or 102B in front.

[1004] For illustrative purposes, the ingestible device 100 of FIG.1 shows the first opening 106 located in a portion of the wall 104and oriented radially, and the second opening 108 located near thefirst end 102A and oriented longitudinally. However, in someembodiments, the exact location and orientation of the firstopening 106 and the second opening 108 may be different from thatshown in FIG. 1. During transit through the GI Tract, naturalcontractions within the small intestine may apply pressure radiallyto different portions of the wall 104 of the ingestible device 100,which may force solids or fluids into the first opening 106. As newmaterial (e.g., fluid and solid particulates from the smallintestine or other portions of the GI tract) enters the curvedchamber 110 through the first opening 106, older material alreadylocated in the curved chamber 110 may be naturally forced out ofthe curved chamber 110 through the second opening 108.

[1005] In some embodiments, a portion of the curved chamber 110 maybe used as a sampling chamber, which may hold samples obtained fromthe GI tract. In some embodiments the curved chamber 110 issubdivided into sub-chambers, each of which may be separated by aseries of one or more valves or interlocks. For example,sub-chambers may be used to retain multiple samples withindifferent portions of the curved chamber 110. In some embodiments,the curved chamber 110 is connected to other chambers within theingestible device 100, or other openings located on the housing ofthe ingestible device 100. This may allow new samples to beacquired in the curved chamber 110 while older samples of interestare still stored within the ingestible device 100. In someembodiments, the ingestible device 100 is equipped with sensors todetect the properties a sample contained in the sampling chamber,or the results of an assay technique applied to the sample. In someembodiments, the ingestible device 100 is configured to obtain andretain a sample within the sampling chamber, which may be retrievedat a later time.

[1006] In some embodiments, the first opening 106, the secondopening 108, or the curved chamber 110 include one or more of ahydrophilic or hydrophobic material, a sponge, a valve, or an airpermeable membrane. For example, a one-way valve may preventmaterial from entering the curved chamber 110 through the secondopening 108. As an alternate example, placing an air permeablemembrane within the curved chamber 110 near the second opening 108may allow unwanted gasses and air bubbles to pass through the airpermeable membrane and exit the curved chamber 110, while solid orliquid samples may be prevented from passing through the airpermeable membrane, and are retained within the curved chamber 110.The air permeable membrane may also prevent solid or liquid samplesfrom entering the curved chamber 110 through the second opening108.

[1007] The use of a hydrophilic material or sponge may allowsamples to be retained within the curved chamber 110, and mayreduce the amount of pressure needed for fluid to enter through thefirst opening 106 and dislodge air or gas in the curved chamber110. Examples of hydrophilic materials that may be incorporatedinto the ingestible device 100 include hydrophilic polymers such aspolyvinyl alcohol, polyvinyl pyrrolidone, and the like. Similarly,materials that have undergone various types of treatments, such asplasma treatments, may have suitable hydrophilic properties, andmay be incorporated into the investible device 100. Sponges may bemade of any suitable material or combination of materials, such asfibers of cotton, rayon, glass, polyester, polyethylene,polyurethane, and the like. Sponges generally may be made fromcommercially available materials, such as those produced byPorex.RTM..

[1008] In some embodiments, the sponges may be treated in order tochange their absorbency or to help preserve samples. Examples ofmaterials which may be used to treat the sponges, alone or incombination, include sorbic acid, propyl parabene, citric acid,surfactants such as Tween.RTM. (polysorbate), DNA inhibitors andstabilizers, RNA inhibitors and stabilizers, protein inhibitors andstabilizers, and the like. In some embodiments, the sponges may becut or abraded to change their absorbency or other physicalproperties.

[1009] Hydrophobic materials located near the second opening 108may repel liquids, discouraging liquid samples from entering orexiting the curved chamber 110 through the second opening 108. Thismay serve a similar function as an air permeable membrane. Examplesof hydrophobic materials which may be incorporated into theingestible device 100 include polycarbonate, acrylics,fluorocarbons, styrenes, certain forms of vinyl, and the like.

[1010] The various materials listed above are provided as examples,and are not limiting. In practice, any type of suitablehydrophilic, hydrophobic, or sample preserving material may be usedin the ingestible device 100, and the teachings discussed inrelation to ingestible device 100 may be incorporated into any ofthe other ingestible devices described in this disclosure. Variousmethods for taking samples, controlling the movement of samples, orremoving unwanted gasses, are discussed in detail in relation toFIGS. 2-9, and any of the various structures or techniquesdescribed in connection with FIGS. 2-9 may be incorporated into theingestible device 100.

[1011] FIG. 2 illustrates an example ingestible device 200 withmultiple openings in the housing and various modifications that maybe made to the ingestible device 100 (FIG. 1). Similar to theingestible device 100, the ingestible device 200 has an outerhousing with a first end 202A, a second end 202B, and a wall 204extending longitudinally from the first end 202A to the second end202B. Also similar to the ingestible device 100, the ingestibledevice 200 has a first opening 206 in the housing, which isconnected to a second opening 208 in the housing. The connectionbetween the first opening 206 and the second opening 208 forms acurved chamber 210 within the ingestible device 200.

[1012] In the ingestible device 200, a portion of the curvedchamber 210 forms a sampling chamber 212. In some embodiments, theingestible device 200 may include a sensor (not shown) within orproximate to the sampling chamber. This sensor may be used todetect a property of the sample. In some embodiments, an assaytechnique is applied to a sample within the sampling chamber, andthe sensor may be used to detect the results of the assaytechnique. A first valve 214 is located between the first opening206 and the sampling chamber 212. Similarly, a second valve 216 islocated between the second opening 208 and the sampling chamber212. In some embodiments, the valves 214 and 216 prevent a fluidfrom entering or exiting the sampling chamber 212, or may be usedto isolate a sample within the sampling chamber 212.

[1013] The ingestible device 200 includes a mechanical actuator 218coupled to the valves 214 and 216. In some embodiments, themechanical actuator 218 is used to move one or both of the valves214 and 216 between an open and a closed position. In someembodiments, the mechanical actuator 218 is controlled by amicrocontroller, microprocessor, or other circuitry inside theingestible device 200. In an open position, the first valve 214 mayallow a sample to pass in and out of the sampling chamber 212through the portion of the curved chamber 210 connected to thefirst opening 206. Similarly, in an open position, the second valve216 may allow a sample to pass in and out of the sampling chamber212 from the portion of the curved chamber 210 connected to thesecond opening 208. When the valves 214 and 216 are in the closedpositions, they may not allow a sample to pass into or out of thesampling chamber 212.

[1014] In some embodiments, the valves 214 and 216 are rotaryvalves, pin valves, flap valves, butterfly valves, ball valves,plug valves, or any other suitable type of one-way or two-wayvalves, and may be the same or different types of valves. In someembodiments, one or both of the valves 214 and 216 are automaticvalves that reseal themselves after a sample has been obtained,similar to the osmotic valve mechanism discussed in relation toFIG. 3. In some embodiments, one or both of the valves 214 and 216include a pumping mechanism, such as the pumping mechanismdiscussed in relation to FIG. 9. For illustrative purposes, theingestible device 200 is depicted with both of the valves 214 and216 as moveable two-way valves coupled to the mechanical actuator218. However, in some embodiments, the mechanical actuator 218 iscoupled to only one of the valves, and the other valve may bereplaced with a passive one-way valve. For example, the mechanicalactuator 218 may be coupled to only the first valve 214, and thesecond valve 216 may be replaced with a passive one-way valve thatallows gases, fluids, or solids to exit the sampling chamber 212through the portion of the curved chamber 210 connected to thesecond opening 208. This may restrict fluid from entering thesampling chamber 212 from the second opening 208, but allowunwanted material to be removed from the sampling chamber 212 asthe sample is obtained.

[1015] In some embodiments, the ingestible device 200 may be ableto detect the approximate location of the ingestible device 200within the GI tract. For example, it may be possible to use variouscombinations of light emitting diodes and sensors positioned alongthe ingestible device 200 to determine whether the device is in thestomach, small intestine, or large intestine. Methods fordetermining the location of an ingestible device within a GI tractare described in greater detail elsewhere herein. In theseembodiments, the ingestible device 200 may be configured to use themechanical actuator 218 to move the valves 214 and 216 into an openposition in response to determining that the ingestible device 200has reached a predetermined location within the GI tract. Forexample, a microcontroller on board the ingestible device 200 maybe configured to open the valves 214 and 216 only when theingestible device 200 is within the small intestine, therebyobtaining a sample from within the small intestine.

[1016] For illustrative purposes, the ingestible device 200 isdepicted with the mechanical actuator 218, the first valve 214, andthe second valve 216 oriented in a substantially straight line,with a single shaft 220 being used to couple the mechanicalactuator 218 to the valves 214 and 216. However, in someembodiments, the orientation and/or positioning of the valves 214and 216 relative to the position of the mechanical actuator 218 maybe different than that shown, and the coupling of the mechanicalactuator 218 to the valves 214 and 216 may also be different. Insome embodiments, the mechanical actuator 218 simultaneously movesthe valves 214 and 216. For example, in some embodiments the valves214 and 216 are rotary valves, and they may be simultaneouslyopened and closed by rotating the shaft 220 that extends from themechanical actuator 218 along the length of the ingestible device200. As an alternate example, the valves 214 and 216 may be pinvalves, and the pins may be attached to the shaft 220 that extendsfrom the mechanical actuator 218 along the length of the ingestibledevice 200. In this case, the mechanical actuator 218 may open andclose the valves by moving the shaft 220 linearly. This may beaccomplished either by configuring mechanical actuator 218 to be alinear actuator, such as a solenoid. Alternately, the mechanicalactuator 218 may be a rotary actuator, and the rotation may beconverted into a linear motion. One skilled in the art willunderstand that this may be done any number of ways, for example,by coupling the mechanical actuator 218 to a ball screw mechanism,a threaded lead nut and lead screw mechanism, a rack and pinionmechanism, or the like.

[1017] In some embodiments, the ingestible device 200 does notinclude the second valve 216 at all. In this case, fluids andsolids contained within the sampling chamber 212 may be free toexit through the second opening 208. Alternately, the second valve216 near the second opening 208 may be replaced by an air-permeablemembrane, which may allow gasses and unwanted air bubbles to exitthe sampling chamber 212 through the second opening 208, whilestill retaining fluids and/or solids within the sampling chamber212. Alternately, the second valve 216 near the second opening 208may be replaced with a hydrophobic material. Similar to an airpermeable membrane, an appropriately positioned hydrophobicmaterial may be used to line the walls of the curved chamber 210proximate to the second opening 208, which may allow gasses orunwanted air bubbles to exit the sampling chamber 212 through thesecond opening 208, while restricting some fluids from entering orexiting the sampling chamber 212 through the second opening 208. Insome embodiments, one or more of the above described mechanisms maybe combined in the same ingestible device. For example, theingestible device 200 may implement the second valve 216 as atwo-way valve, and also have hydrophobic material and anair-permeable membrane located near the second opening 208.

[1018] In some embodiments, the curved chamber 210 is connected toone or more sub-chambers (not shown). Each of these sub-chambersmay be configured to hold one or more samples, and isolate thesamples from both the sampling chamber 212, and the othersub-chambers. For example, each sub-chamber may be connected to thecurved chamber 210 through a one-way valve, allowing samples toenter the sub-chamber from the curved chamber 210, but preventingthe obtained samples from exiting the sub-chamber and re-enteringeither the curved chamber 210 or the sampling chamber 212. Ingeneral, any type of valve or other suitable mechanism may be usedto isolate samples contained in the sub-chambers. In someembodiments, the ingestible device 200 distributes differentsamples into different sub-chambers at different times, or fromdifferent locations of the GI tract. For example, the ingestibledevice 200 may obtain a sample from the duodenum and distribute itinto a first sub-chamber, and the ingestible device 200 may laterobtain a sample from the ileum and distribute it into a secondsub-chamber. In some embodiments, different types of assaytechniques or diagnostics are applied to some of the samplescontained in the different sub-chambers.

[1019] FIG. 3 illustrates an example of an osmotic valve mechanism300, which may be incorporated into an ingestible device in orderto obtain samples. The osmotic valve mechanism 300 may be used inan ingestible device that features a first end, a second end, and awall extending longitudinally between the first end and the secondend, similar to the shape of the ingestible devices 100 (FIG. 1)and 200 (FIG. 2).

[1020] The osmotic valve mechanism 300 includes an inlet port 302,which is connected to a sampling chamber 304. In some embodiments,the inlet port 302 connects sampling chamber 304 directly orindirectly to an opening in the housing of an ingestibledevice.

[1021] The initial state of the osmotic valve mechanism 300 isshown in diagram 300A. As shown in diagram 300A, the inlet port 302of the osmotic valve mechanism 300 is sealed using a single usesealing device 306 positioned within the inlet port 302. The singleuse sealing device 306 is positioned adjacent to a heating element308. When it is time for the osmotic valve mechanism 300 to beopened (which may be determined by a localization mechanism thatdetermines the ingestible device is located in a desirable portionof the GI tract), the heating element 308 applies heat to thesealing device 306, causing the sealing device 306 to deform andunseal the inlet port 302.

[1022] In some embodiments, the sealing device 306 may be a plugmade out of a material that is meltable, deformable, and/ordestroyable through the use of the heating element 308, such aswax. For example, in some embodiments, the heating element 308 maybe a resistive heater that undergoes ohmic heating as an electricalcurrent is passed through it, and the sealing device 306 is a waxplug. In some embodiments, the type of wax used to form the waxplug has a melting point between 38 degrees and 80 degrees Celsius,which is above the ambient temperature of a human body, but whichmay be easily achieved using the heating element 308. Someembodiments of the osmotic valve mechanism 300 may use a sealingdevice 306 that is melted or deformed at temperatures outside ofthe range described above, but practical considerations may be madeto ensure that the osmotic valve mechanism 300 does not causeunwanted damage or burning to the GI tract. In some embodiments, amicroprocessor is configured to control the heating element 308,causing it to generate heat. For example, the microprocessor may beconfigured to activate the heating element 308 once the ingestibledevice reaches a particular location within the GI tract. Anexample mechanism for unsealing the inlet port 302 is described ingreater detail in relation to FIGS. 4 and 5. Although FIGS. 3, 4,and 5 depict the sealing device 306 as a type of plug, any type ofsuitable sealing device may be used. For example, in someembodiments, the sealing device includes a breakable membrane,which may be destroyed when heat is applied to the membrane. Insome embodiments, the osmotic valve mechanism 300 does not includea heating element 308, and the sealing device 306 is melted,deformed, destroyed, or dislodged from the inlet port 302 by amechanical actuator, or through electromagnetic fields. Forexample, the sealing device 306 may be a membrane that will rupturewhen a sufficiently large electrical current or magnetic field isapplied to the membrane.

[1023] Inside the sampling chamber 304 of the osmotic valvemechanism 300 is made of a member including an absorptive material310, and at least a portion of the absorptive material 310 islocated near the inlet port 302. The absorptive material 310 mayinclude any suitable sponge material or hydrophilic material, suchas any of the materials described in relation to FIG. 1. Theportion of the absorptive material 310 located near the inlet port302 may have a tendency to expand when it comes into contact withfluids. The osmotic valve mechanism 300 has a barrier 312 insidethe sampling chamber 304, which is divided into three portions. Thefirst portion of the barrier 312 is a flexible membrane 314, thesecond portion of the barrier 312 adjacent to the flexible membrane314 is a rigid portion 316, and the third portion of the barrier312 adjacent to the rigid portion 316 is a semi-permeable membrane318.

[1024] The barrier 312 within the sampling chamber 304 ispositioned between the inlet port 302 and the absorptive material310, covering a surface of the absorptive material 310. When theinlet port 302 is unsealed, a sample (e.g., a fluid samplecontaining solid particulates taken from the GI tract) enters thesampling chamber 304 through the inlet port 302, and begins to fillthe sampling chamber 304. The absorptive material 310 may have anatural tendency to expand when it comes into contact with a fluidsample. However, by covering a surface of the absorptive material310, the barrier 312 may allow only certain portions of absorptivematerial 310 to expand. The barrier 312 may also direct the flow ofa fluid sample as it enters the sampling chamber 304, and allow thefluid sample to come into contact with only certain parts of theabsorptive material 310.

[1025] Diagram 300B shows the osmotic valve mechanism 300 shortlyafter the inlet port 302 is unsealed. Once the inlet port 302 isunsealed, the sampling chamber 304 may be opened, and a sample mayenter the sampling chamber 304 through the inlet port 302. In someembodiments, the sample cannot cross the flexible membrane 314 andcontact the absorptive material 310. As a result, the flexiblemembrane 314 may be used to guide the sample as it enters thesampling chamber 304. Similarly, in some embodiments the samplecannot cross the rigid portion 316 of the barrier 312, and therigid portion 316 may also be used to guide the sample as it entersthe sampling chamber 304. The semi-permeable membrane 318 allows atleast a portion of the sample to pass through the semi-permeablemembrane and contact the absorptive material 310. This may allowthe sample to be absorbed by the absorptive material 310 after thesample has filled the top portion of the sampling chamber 304,which in turn may cause the absorptive material 310 to begin toexpand.

[1026] Diagram 300C shows the state of the osmotic valve mechanism300 after the absorptive material 310 has absorbed a portion of thesample. The portion of the absorptive material 310 under theflexible membrane 314 expands when the absorptive material 310absorbs the sample. As the absorptive material 310 expands, theflexible membrane 314 is forced up against the inlet port 302,effectively sealing the inlet port 302 from the sampling chamber304. In some embodiments, the rigid portion 316 prevents theportion of the absorptive material 310 under the rigid portion 316from expanding. In some embodiments, the semi-permeable membrane318 may be rigid, and prevent the portion of the absorptivematerial 310 adjacent to the semi-permeable membrane 318 fromexpanding.

[1027] After the absorptive material 310 expands, causing the inletport 302 to be resealed, a portion of the sample may be confinedwithin the sampling chamber 304. Once a sample has been properlyconfined, it may be possible to apply a wide range of assaytechniques or diagnostics to the sample. In some embodiments, theportion of the sampling chamber 304 between the rigid portion 316and the wall of the sampling chamber forms a testing area. Forexample, a sensor may be placed within or proximate to the samplingchamber 304 in order to study the portion of the sample containedwithin the testing area located above the rigid portion 316. Thissensor may be used to study properties of the sample, or it may beused to detect the results of an assay technique applied to thesample.

[1028] Diagram 300C is shown for illustrative purposes only, and isnot limiting. In some embodiments, the osmotic valve mechanism 300does not include the barrier 312, or one or more portions of thebarrier 312 are excluded or rearranged within the sampling chamber304. For example, the location of the rigid portion 316 and thesemi-permeable membrane 318 may be reversed, or the rigid portion316 may be removed and the semi-permeable membrane 318 extended sothat it connects directly with the flexible membrane 314. When theosmotic valve mechanism 300 does not include a barrier 312 or doesnot include the flexible membrane 314, a portion of the absorptivematerial 310 near the inlet port 302 may expand and clog the inletport 302, effectively resealing the inlet port 302.

[1029] In some embodiments, the material used to form theabsorptive material 310 expands at a controlled rate, which mayensure that sufficient time has passed for the sample to enter thesampling chamber 304 and for the sampling chamber 304 to be filledbefore the inlet port 302 is resealed. This may be particularlyuseful for embodiments where the osmotic valve mechanism 300 doesnot include a flexible membrane 314 and/or the semi-permeablemembrane 318. In some embodiments, a portion of the absorptivematerial 310 is covered by a dissolvable film or membrane, whichmay prevent the absorptive material 310 from expanding until asufficient amount of time has passed for the film to dissolve.

[1030] In some embodiments, the sampling chamber 304 is connectedto one or more sub-chambers (not shown). Each of these sub-chambersmay be configured to hold samples, and isolate the samples fromboth the sampling chamber 304, and the other sub-chambers. Forexample, each sub-chamber may be connected to the sampling chamber304 through a one-way valve, allowing samples to enter thesub-chamber from the sampling chamber, but preventing the obtainedsamples from exiting the sub-chamber. As an alternate example, eachof the sub-chambers may employ a sealing device, heating element,and member made of absorptive material arranged similar to osmoticvalve mechanism 300. In these embodiments, each of the sub-chambersmay be opened by activating their respective heating elements, andmay be automatically sealed off from the sampling chamber 304 aftera sufficient amount of the sample has been obtained. In general,any type of valve or other suitable mechanism may be used toisolate samples contained in the sub-chambers. In some embodiments,similar to ingestible device 200, an ingestible device employingmultiple sub-chambers in conjunction with the osmotic valvemechanism 300 may distribute different samples into differentsub-chambers at different times, or from different locations of theGI tract.

[1031] It will be understood by one skilled in the art thatvariations of the osmotic valve mechanism 300 may be combined withany of the other ingestible devices described in this disclosure.For example, in some embodiments of the ingestible device 200 shownand described in relation to FIG. 2, one or both of the valves 214and 216 may be replaced with certain embodiments of the osmoticvalve mechanism 300. One or both of the valves 214 and 216 mayinclude a sealing device that can be destroyed or deformed (e.g.,by the mechanical actuator 218 or through a heating element), andone or both of the valves 214 and 216 may be automatically resealedby the expansion of absorptive material located within the samplingchamber 212.

[1032] FIGS. 4 and 5 illustrate in detail how some embodiments ofthe osmotic valve mechanism 300 (FIG. 3) may be operated in orderto obtain a sample.

[1033] FIG. 4 shows a detailed view of an inlet port 400, which maybe incorporated into osmotic valve mechanism 300, prior to beingunsealed. The inlet port 400 features an exterior portion 402,which is separated by a middle portion 404 from an interior portion406. The middle portion 404 of the inlet port 400 contains asealing device 408, which may be the same as sealing device 306shown and described in relation to FIG. 3. A heating element 410 islocated near the middle portion 404, and adjacent to the sealingdevice 408. The sides of the inlet port 412A and 412B form theshape of the inlet port 400, and may be constructed from aninsulating material, such as insulating ceramic, or polymers suchas polyamide-imide, polyphenylene sulfide, polyphenylene oxide, andthe like. For illustrative purposes, the exterior portion 402 ofthe inlet port 400 is depicted as being filled with a sample 414,which may be a fluid sample obtained from the GI tract. However, insome embodiments, the inlet port 400 may be operated regardless ofwhether a sample 414 is actually contained in the exterior portion402. The exterior portion 402 and the interior portion 406 arewider than the middle portion 404. A sloped wall 416 graduallyreduces the width of the exterior portion 402, to transition fromthe wider width of the exterior portion 402 to the narrower widthof the middle portion 404. This configuration may reduce theoverall volume of the sealing device 408 (compared to aconfiguration with a wider middle portion 404), and reduce thesurface area of the sealing device 408 exposed to the sample 414,which may reduce the amount of heat lost from the sealing device408 to the sample 414. In turn, this may make it easier to raisethe temperature of the sealing device 408 using the heating element410. In some embodiments, the geometry of the inlet port 400 mayallow an air pocket (not shown) to form in the exterior portion402, separating the sealing device 408 from fluid contained withinthe GI tract. This may act as an insulating barrier around thesealing device 408, and also make it easier to raise thetemperature of the sealing device 408 using the heating element410. Moreover, the larger width of the interior portion 406relative to the middle portion 404 forms a remnant capture area418, which may hold the remnants of the sealing device 408 afterthe inlet port 400 is unsealed.

[1034] In some embodiments, the exterior portion 402 of the inletport 400 may be connected directly or indirectly to an opening inthe housing of an ingestible device. In some embodiments, there isnothing to restrict a sample from entering the opening, and, at anygiven time, the exterior portion 402 of the inlet port 400 may befilled with a fluid sample 414 gathered from whatever portion ofthe GI tract the ingestible device is located within.

[1035] Sealing device 408 prevents the fluid sample 414 containedwithin the exterior portion 402 of the inlet port 400 from enteringthe interior portion 406 of the inlet port 400. For simplicity,FIGS. 4 and 5 depict the sealing device 408 as a plug, which formsa seal that may be broken by using a heating element 410. However,in some embodiments the sealing device 408 may be any other type ofbreakable seal or valve used within the middle portion 404 toseparate the exterior portion 402 of the inlet port 400 and theinterior portion 406 of the inlet port 400.

[1036] In some embodiments, the heating element 410 may be operatedby a microcontroller. For example, the microcontroller may beconfigured to operate the heating element 410 and unseal the inletport 400 when the ingestible device is in a certain portion of theGI tract. The sides of the inlet port 412A and 412B may be formedfrom an insulating material, which may shield the ingestible deviceand the fluid sample 414 from the heat generated by the heatingelement 410. This may also help to focus the heat produced byheating element 410 in the direction of the sealing device 408, andmay reduce the total amount of power to drive the heating element410 to melt, deform, or destroy the sealing device 408.

[1037] In some embodiments, the dimensions of the inlet port 400are chosen such that a fluid sample 414 is naturally drawn into theexterior portion 402, and ultimately through the middle portion 404into the interior portion 406, through capillary action. Typically,the cross-section of the exterior portion 402, the middle portion404, and the interior portion 406 will be square, circular, orrectangular, but any type of cross-section may be used. The overallcross-sectional area of the exterior portion 402, the middleportion 404, and the interior portion 406 of the inlet port 400 istypically less than 50 square millimeters given the sizeconstraints of the ingestible device, with 0.2 to 2 squaremillimeters being common. However, the cross-sectional areas listedabove are only examples, and any cross-sectional area may be chosenin order to better draw in samples from the different portions ofthe GI tract. One skilled in the art will understand that the exactshape and dimensions will depend on the physical properties of thesample to be acquired, and some embodiments may use cross-sectionsother than the ones mentioned above.

[1038] FIG. 5, shows a detailed view of an inlet port 500, whichmay be incorporated into osmotic valve mechanism 300, after it hasbeen unsealed.

[1039] After the heating element 510 has heated the sealing device508 sufficiently, the sealing device 508 may deform, melt, orotherwise be destroyed, effectively unsealing the inlet port 500.Once the inlet port 500 is unsealed, the fluid sample 514 is ableto flow naturally from the exterior portion 502 of the inlet port500 to the interior portion 506 of the inlet port 500 through themiddle portion 504. Similar to the embodiments described inrelation to FIG. 4, the sides 512A and 512B of the inlet port maybe made of an appropriate insulating material, and form the shapeof the inlet port 500, the exterior portion 502 with the slopedwall 516, the middle portion 504, and the interior portion 506along with the remnant capture area 518. As the fluid sample 514enters the interior portion 506 of the inlet port 500, the naturalflow of the fluid sample 514 may carry any of the remnants of thesealing device 508 into the remnant capture area 518 located withinthe interior portion 506. In some embodiments, once the melted ordeformed remnants of the sealing device 508 cease to be in contactwith the heating element 510 and instead come into contact with theinsulating material that make up the walls of the remnant capturearea 518, the remnants of the sealing device 508 re-solidifies orre-forms along the walls of the remnant capture area 518. As aresult, the remnant capture area 518 may provide a location for there-solidified remnants of the sealing device 508 to be stored, andmay prevent the remnants of the sealing device 508 from impedingthe flow of the sample 514.

[1040] In some embodiments, electromagnetic forces are used toattract the remnants of the sealing device 508 to the remnantcapture area 518. For example, the sealing device (e.g., thesealing device 408) may be made from a magnetic material, and aninduced or permanent magnetic field may be used to attract theremnants of the sealing device 508 to the remnant capture area 518.This magnetic field may be applied after the heating element 510 isactivated, and until the remnants of the sealing device 508re-solidify or re-form within the remnant capture area 518.

[1041] It will be understood that the embodiments described byFIGS. 3, 4, and 5, are merely illustrative, and they may bemodified and combined with other techniques for drawing in orpumping fluid samples without departing from the spirit and scopeof this disclosure. For example, to encourage samples to be drawninto the sampling chamber 304, the sampling chamber 304 may containa low-pressure vacuum, and samples may be forcibly drawn into thesampling chamber 304 when the inlet port 302 is unsealed. A similareffect may also be produced by connecting the sampling chamber 304to a sub-chamber containing a low-pressure vacuum, or by using byusing a mechanical actuator to either pump the fluid samples or toincrease the volume of the sampling chamber 304. In someembodiments, the geometry and relative size of the exteriorportions 402 and 502, the middle portions 404 and 504, and interiorportions 406 and 506, may be different from those depicted in FIGS.4 and 5. For example, the different portions 402, 404, 406, 502,504, and 506 may have a uniform width, and the sloped walls 416 and516 and/or the remnant capture areas 418 and 518 are not included.As another example, a sloped wall may be used to form the remnantcapture areas 418 and 518.

[1042] FIG. 6 illustrates another example of an ingestible device600 with a sampling chamber that includes an exit port. Similar tothe ingestible devices 100 and 200, the ingestible device 600 isdesigned to have an outer housing with a first end 602A, a secondend 602B, and a wall 604 extending longitudinally from the firstend 602A to the second end 602B. The ingestible device 600 has anopening 606 in the housing, which allows samples to enter theingestible device 600 from the surrounding environment. Theingestible device 600 has an inlet region 608 connected to theopening 606. The inlet region 608 is connected to an entry port 610of a sampling chamber 612. The inlet region 608 is divided intothree portions. A first portion 608A of the inlet region 608 isconnected to the opening 606 and a second portion 608B, and a thirdportion 608C is connected to the entry port 610 of the samplingchamber 612. The second portion 608B connects the first portion608A to the third portion 608C, and may contain a moveable valve614 that is used to prevent samples from flowing through the inletregion 608, and isolate the first portion 608A of the inlet region608 from the third portion 608C of the inlet region 608.

[1043] The ingestible device 600 has a mechanical actuator 624coupled to the moveable valve 614. In some embodiments, amicroprocessor or microcontroller is configured to control themechanical actuator 624 and move the moveable valve 614 between anopen and a closed position. For example, the microcontroller may beconfigured to move the moveable valve 614 into an open positionafter the ingestible device reaches a particular location withinthe GI tract. In some embodiments, the mechanical actuator may bedriven by a set of batteries or other power source located withinthe ingestible device 600. When the moveable valve 614 is movedinto an open position, a sample may be allowed to flow through theinlet region 608, and enter the sampling chamber 612 through theentry port 610. When the moveable valve 614 is in a closedposition, the sample is prevented from flowing through the inletregion 608 and reaching the sampling chamber 612 from the opening606.

[1044] For illustrative purposes, FIG. 6 depicts the moveable valve614 as a diaphragm valve, which uses a mechanical actuator 624 tomove a flexible diaphragm in order to seal or unseal an aperture inthe second portion 608B of the inlet region 608, which mayeffectively block or unblock the inlet region 608. However, it willbe understood that, in some embodiments, the moveable valve 614 maybe a different type of valve. For example, in some embodiments themoveable valve 614 may be replaced by a pumping mechanism, such asthe pumping mechanism described in relation to FIG. 9. As anotherexample, in some embodiments the moveable valve 614 is replacedwith an osmotic valve, similar to the embodiments described inrelation to FIGS. 3, 4, and 5. Several examples of other differentvalve types are described in relation to FIG. 7.

[1045] The sampling chamber 612 of the ingestible device 600 has anexit port 616 located on the opposite end of the sampling chamber612 from the entry port 610. In general, the exit port 616 may belocated anywhere within the sampling chamber 612. The exit port 616is configured to allow air or gas 618 to exit the sampling chamber612, while preventing at least a portion of the sample obtained bythe ingestible device 600 from exiting the sampling chamber 612.For example, the exit port 616 may include a gas-permeablemembrane, which allows the gas 618 to exit the sampling chamber612, but which would prevent a liquid or solid sample from leavingthe sampling chamber 612 through the exit port 616. Allowing thegas 618 to exit the sampling chamber 612 may prevent pressure frombuilding up within the sampling chamber 612 as the sample entersthrough the entry port 610. This may result in the sample beingdrawn into the sampling chamber 612 more easily, and result inincreasing the overall volume of the sample able to be collected bythe ingestible device 600, and increasing the ease with which thesample is brought into the sampling chamber 612.

[1046] The ingestible device 600 includes a one-way valve 620 aspart of the exit port 616. This valve may prevent the gas 618 fromre-entering the sampling chamber 612. However, in some embodimentsthe one-way valve 620 may be excluded from the ingestible device600. In some embodiments, the exit port 616 includes a gaspermeable membrane. This gas permeable membrane may lose itspermeability when it is placed in contact with the sample. Forexample, the gas permeable membrane may include a spongy materialthat allows the gas 618 to exit the sampling chamber 612 throughthe exit port 616. Once the spongy material becomes moist throughcontact with the sample, it may become no longer gas permeable, orthe permeability may be greatly reduced, thereby preventing the gas618 from reentering the sampling chamber 612. In some embodiments,the gas permeable membrane may include expandedpolytetrafluorethylene, polypropylene, or the like. In someembodiments, the material used to make the gas permeable membranemay be filter-like, as opposed to sponge-like materials. Generally,the gas permeable membrane may be made of any material that allowgas to permeate, but which prevents liquid from flowing through themembrane due to sufficient resistance or surface tensioneffects.

[1047] In the ingestible device 600, the exit port 616 is connectedto a volume within the housing of ingestible device 600 outside ofthe sampling chamber. Depending on the manufacturing process usedto produce the ingestible device 600, the volume within the housingof the ingestible device 600 may contain air or some other type ofgas.

[1048] The ingestible device 600 includes an outlet port 622, whichis connected to the volume within housing of the ingestible device600. The outlet port 622 may provide a path for the gas 618 to exitthe ingestible device 600 and be released into the environmentsurrounding the ingestible device 600. This may be advantageouswhen the volume of gas 618 is relatively large, since it mayprevent pressure from building up within the housing of theingestible device 600. In some embodiments, the ingestible device600 does not include an outlet port 622, and the gas 618 staysinside the volume of the ingestible device 600. In someembodiments, the outlet port 622 is directly or indirectlyconnected to the exit port 616, for example, by a tube or channel.In some embodiments, the exit port 616 leads directly from thesampling chamber 612 to an opening in the ingestible device 600,and the exit port 616 may effectively replace the outlet port 622.In some embodiments, the outlet port 622 may contain a gaspermeable membrane, a one-way valve, a hydrophobic channel, or someother mechanism to avoid unwanted material, (e.g., fluids and solidparticulates from within the GI tract), from entering theingestible device 600 through the outlet port 622.

[1049] In some embodiments, the ingestible device 600 may include asensor within or proximate to the sampling chamber 612. Forexample, this sensor may be used to detect various properties of asample contained within the sampling chamber 612, or this sensormay be used to detect the results of an assay technique applied tothe sample contained within the sampling chamber 612.

[1050] In some embodiments, a hydrophilic sponge is located withinthe sampling chamber 612, and the hydrophilic sponge may beconfigured to absorb the sample as the sample enters the samplingchamber 612. In some embodiments, the hydrophilic sponge fills asubstantial portion of the sampling chamber 612, and holds thesample for an extended period of time. This may be particularlyadvantageous if the sample is collected from the ingestible device600 after the ingestible device 600 exits the body. In someembodiments, the hydrophilic sponge is placed on only certainsurfaces or fills only certain portions of the sampling chamber612. For example, it may be possible to line certain walls (or allwalls) of the sampling chamber 612 with a hydrophilic sponge toassist in drawing in the sample, while leaving some (or none) ofthe walls of the sampling chamber 612 uncovered. Leaving wallsuncovered may allow the use of diagnostics or assay techniques thatinvolve a relatively un-obscured optical path. An example of suchan embodiment is described in detail in relation to FIG. 8. In someembodiments, the sponge material may be placed on all walls of thesampling chamber 612. This may prevent unwanted ambient light fromentering the sampling chamber 612, which may be useful for certaintypes of low light detection assays. In some embodiments, an opaquematerial is used to cover some or all sides of the sampling chamber612. This may also prevent unwanted ambient light from entering thesampling chamber 612.

[1051] In some embodiments, the ingestible device 600 may include asealed vacuum chamber connected to the exit port 616, or connecteddirectly or indirectly to the sampling chamber 612. The sealedvacuum chamber may have an internal pressure that is substantiallylower than ambient pressure of the sampling chamber 612 and/or theinlet region 608. In these embodiments, the ingestible device 600unseals the vacuum chamber in order to reduce the pressure withinthe sampling chamber. This change in pressure may force the sampleto be sucked into the sampling chamber, or allow the sample to bedrawn into the sampling chamber quickly.

[1052] For simplicity, FIG. 6 depicts only a single samplingchamber 612, but it will be understood that the inlet region 608may be connected to multiple sampling chambers arranged throughoutthe device, each of which may be controlled independently throughthe use of one or more valves. For example, in some embodimentsthere may be one or more sub-chambers connected to the inlet region608. Each of the sub-chambers may be configured to hold samplesgathered from within the GI tract, and keep those samples isolated.In general, any type of valve or other suitable mechanism may beused to isolate samples contained in the sub-chambers, includingany of the valves or mechanisms described in relation to FIGS. 1-5.In some embodiments, the ingestible device 600 distributesdifferent samples into each of the different sub-chambers atdifferent times, or from different locations within the GI tract.For example, the ingestible device 600 may accomplish this byopening up a valve to connect the interior of inlet region 608 tothe appropriate sub-chamber before opening up the inlet region 608to draw in the sample from the opening 606 in the housing.

[1053] FIG. 7 depicts different types of moveable valves that maybe incorporated into an ingestible device, such as the ingestibledevices 100, 200 or 600. The ingestible device 702 illustrates howa pin valve may be used as a moveable valve (e.g., as moveablevalve 614 of ingestible device 600 (FIG. 6)), with diagram 702Ashowing the pin valve in a closed position, and diagram 702Bshowing the pin valve in an open position. In the ingestible device702, a mechanical actuator may be configured to move the pin valvelinearly in order to switch between an open position and a closedposition. For example, in diagram 702A, the ingestible device 702has a pin inserted into the inlet port, thereby preventing thesample from flowing into the sampling chamber from the opening inthe ingestible device 702. In diagram 702B, the ingestible device702 has a pin that has been removed from the inlet port, allowingthe sample to flow freely into the sampling chamber from theopening in the ingestible device 702. In order to generate linearmotion, the mechanical actuator may be a linear actuator, such as asolenoid. Alternately, the mechanical actuator may be a rotatoryactuator, and the rotation may be converted into a linear motion.One skilled in the art will understand that this may be done anynumber of ways, for example, by coupling the mechanical actuator toa ball screw mechanism, a threaded lead nut and lead screwmechanism, a rack and pinion mechanism, or the like.

[1054] Ingestible device 704 illustrates how a rotary valve may beused as a moveable valve (e.g., as moveable valve 614 of ingestibledevice 600 (FIG. 6)), with diagram 704A showing the rotary valve ina closed position, and diagram 704B showing the rotary valve in anopen position. In diagram 704A, the ingestible device 704 has arotary pin oriented such that the sample is prevented from enteringthe sampling chamber from the opening in the ingestible device 704.In diagram 704B, the ingestible device 704 has a rotary pin thathas been rotated into an orientation where the sample is free toflow into the sampling chamber from the opening in the ingestibledevice 704. In order to operate the rotary valve, the mechanicalactuator in ingestible device 704 may be a rotatory actuator, whichis capable of rotating the rotary pin to switch between the openposition and the closed position.

[1055] Ingestible device 706 illustrates how a flexible diaphragm,or diaphragm valve, may be used as a moveable valve (e.g., asmoveable valve 614 of ingestible device 600 (FIG. 6)), with diagram706A showing the diaphragm valve in a closed position, and diagram706B showing the diaphragm valve in an open position. In diagram706A, the ingestible device 706 has a diaphragm valve in a closedposition, with the flexible diaphragm being pressed against anaperture in the inlet region due to the pressure generated by themechanical actuator against the flexible diaphragm. This mayeffectively block a sample from flowing through the inlet region,and thereby preventing a sample from entering the sampling chamberfrom the opening in the ingestible device 706. In diagram 706B, theingestible device 706 has a diaphragm valve in an open position,with the pressure removed from the flexible diaphragm. Thediaphragm returns to a position away from the aperture in the inletregion, allowing a sample to flow freely into the sampling chamberfrom the opening the in ingestible device 706.

[1056] In some embodiments, ingestible device 706 has a springmechanism near the diaphragm or in direct contact with thediaphragm. The spring mechanism may apply pressure to the diaphragmto oppose the pressure applied by the mechanical actuator, whichmay cause the flexible diaphragm to be moved into an open positionwhen the mechanical actuator is not applying pressure to theflexible diaphragm. Additionally, this may ensure that thediaphragm valve remains open when the mechanical actuator is notapplying pressure across the flexible diaphragm.

[1057] In some embodiments, moving the mechanical actuator from aclosed position to an open position causes a volume of the inletregion within the ingestible device to increase. This may cause thepressure within the inlet region to be reduced, generating suctionto draw a sample into the inlet region. Similarly, moving themechanical actuator from an open position to a closed position maycause the volume of the inlet region to be reduced. This may causethe pressure within the inlet region to be increased, pushing thesample out of the inlet region. Depending on the design of theinlet region, the mechanical actuator, and the moveable valve, thismay push the sample into the sampling chamber rather than pushingthe sample back through the opening in the ingestible device. Anexample of such a design is described in greater detail in relationto FIG. 9.

[1058] FIG. 8 illustrates an example of a sampling mechanism thatmay be incorporated into an ingestible device, such as theingestible devices 100, 200, 600, and 702-706. The samplingmechanism 800 is partially lined with hydrophilic sponges 802A and802B. In between the hydrophilic sponges 802A and 802B is a testingregion 804 within the sampling mechanism 800. The hydrophilicsponges 802A and 802B attract a liquid or fluid sample 806, and maydraw the sample 806 into the sampling mechanism 800. As thehydrophilic sponges 802A and 802B are saturated with the sample806, a meniscus 808 is formed at the end of the sample 806, betweenthe hydrophilic sponges 802A and 802B. This system may be usefulfor acquiring particularly viscous samples, which may havedifficulty flowing into the sampling mechanism 800 naturally.

[1059] The sampling mechanism 800 includes an exit port 810connected to a channel 812. As the sample 806 is drawn into thesampling mechanism 800, air or gas contained in the samplingmechanism 800 may be pushed out of the sampling mechanism 800through the exit port 810 and into the channel 812. This may avoidgas being trapped within the sampling mechanism 800, which in turnmay avoid pressure building inside of the sampling mechanism 800and preventing the sample 806 from being drawn into the testingregion 804.

[1060] In some embodiments, the sampling mechanism 800 may notinclude an exit port 810 or a channel 812, and any air or gas inthe sampling mechanism 800 may be allowed to remain within thesampling mechanism 800. In some embodiments, the sampling mechanism800 may be filled with a low pressure vacuum, attached to a pump orother mechanism to create a vacuum, or attached to a sealed chambercontaining a low pressure vacuum that may be unsealed. The use of avacuum may allow the sampling mechanism 800 to forcibly draw in asample.

[1061] In some embodiments, an ingestible device may includesensors or diagnostics to study the sample 806 contained within thesampling mechanism 800. Because there is no sponge material on thefront and back walls of the testing region 804, information aboutthe sample 806 contained within the testing region 804 may begathered by using sensors and/or assay techniques that involve aclear optical path, which would otherwise be obscured by a sponge(e.g., the hydrophilic sponges 802A and 802B). For example, lightsources and/or optical sensors may be placed near the front and/orback walls in order to test optical properties of the sample, or todetect the results of certain assay techniques.

[1062] It will be understood by those skilled in the art that thesampling mechanism 800 depicted in FIG. 8 is merely illustrative,and the general techniques described in relation to FIG. 8 may beapplied to a wide range of different chambers, channels, and fluidpathways, and incorporated into a wide range of differentingestible devices. Furthermore, in some embodiments, the overallgeometry of FIG. 8 and the positioning of the sponges and thetesting area may be altered. For example, the sponge may be formedin the shape of hollow tubes, with testing areas located in themiddle of each tube. In this case, there would be a clear opticalpath from one end of the tube to the other.

[1063] FIG. 9 illustrates a pumping mechanism 900 that may beincorporated into an ingestible device, including certainembodiments of ingestible devices 100, 200, 600, and 702-706. Forillustrative purposes, the pumping mechanism 900 may be describedin the context of an ingestible device similar to ingestible device600 (FIG. 6). When it is incorporated into an ingestible devicesimilar to ingestible device 600, the pumping mechanism 900 mayfunction as a moveable valve (e.g., moveable valve 614 ofingestible device 600), and control the ability of samples to flowbetween the opening 606 in the housing and the entry port 610 ofthe sampling chamber 612. Additionally, the pumping chamber 904 ofthe pumping mechanism 900 may form part of the second portion 608Bof the inlet region 608. However, the general structure andprinciples of pumping mechanism 900 are not limited to theingestible devices described in this disclosure, and they may beapplied to a wide range of ingestible devices.

[1064] Pumping mechanism 900 is designed to draw in a samplethrough a first opening 902 into a pumping chamber 904, and push aportion of the sample out of the pumping chamber 904 through asecond opening 906. In some embodiments, the first opening 902 maybe connected directly or indirectly to an opening in the housing ofan ingestible device. For example, an inlet region (e.g., the firstportion 608A of the inlet region 608 of the ingestible device 600(FIG. 6)) may connect an opening in the housing of an ingestibledevice (e.g., the opening 606 in the housing of ingestible device600 (FIG. 6)) to the first opening 902. In some embodiments, thesecond opening 906 is connected directly or indirectly to asampling chamber of an ingestible device. For example, the secondopening 906 may be connected to an entry port of a sampling chamber(e.g., connected via the third portion 608C of the inlet region 608to the entry port 610 of the sampling chamber 612 of the ingestibledevice 600 (FIG. 6)).

[1065] The pumping mechanism 900 features a moveable pump head 908contained within the pumping chamber 904. The protrusion 908A ofthe moveable pump head 908 is shaped to fit within the firstopening 902, or otherwise block the first opening 902. The base908B of the moveable pump head 908 is able to cover the secondopening 906 or otherwise block the second opening 906. Moreover,the protrusion 908A and the base 908B of the moveable pump head 908are sized and oriented from each other in such a manner such thatwhen the protrusion 908A blocks the first opening 902, the base908B may simultaneously block the second opening 906 or leave thesecond opening 906 unblocked. Furthermore, when the base 908Bblocks the second opening 906, the protrusion 908A may always beconfigured to also block the first opening 902.

[1066] As the moveable pump head 908 is moved up and down, theopenings 902 and 906 may be sealed or unsealed, switching thepumping mechanism 900 across an open position, a partially closedposition, and a closed position. In the open position (as is shownin the diagram 912), both the first opening 902 and the secondopening 906 are unsealed or open. In the partially closed position(as is shown in the diagram 914, the moveable pump head 908 ispositioned to only seal the first opening 902, while leaving thesecond opening 906 open. Finally, in the closed position (as isshown in the diagrams 910 and 918), both the first opening 902 andthe second opening 906 are sealed.

[1067] In some embodiments, the moveable pump head 908 may beconnected to a mechanical actuator (e.g., the mechanical actuator624 of the ingestible device 600 (FIG. 6)), which may be configuredto move the moveable pump head 908 linearly up and down. Forexample, the moveable pump head 908 may be located on the end of ashaft that is attached to the mechanical actuator. In someembodiments, the mechanical actuator and the positioning of themoveable pump head 908 may be controlled by a microcontroller ormicroprocessor located within the ingestible device. For example, amicrocontroller may be configured to move the pump head 908 andbegin pumping a sample through the pumping chamber 904 only afterthe ingestible device has reached a particular location within theGI tract.

[1068] Diagram 910 depicts the pumping mechanism 900 in a fullyclosed position. When the pumping mechanism 900 is in the fullyclosed position, the protrusion 908A of the moveable pump head 908may be positioned within the first opening 902, and the base 908Bof the moveable pump head 908 may be positioned adjacent to thesecond opening 906. In the fully closed position, the positioningof the moveable pump head 908 may effectively prevent a sample fromentering or exiting the pumping chamber 904 from the openings 902or 906.

[1069] Diagram 912 depicts the pumping mechanism 900 in an openposition. When the pumping mechanism 900 is in the open position,the moveable pump head 908 is moved away from the first opening902, moving the protrusion 908A of the moveable pump head 908 outof the first opening 902, and moving the base 908B of the moveablepump away from the second opening 906. In this position, thepumping mechanism 900 may allow one or more samples to enter thepumping chamber 904 through the first opening 902, and exit thepumping chamber 904 through the second opening 906. Because theeffective volume of the pumping chamber 904 increases when themoveable pump head 908 is moved away from the first opening 902,the pumping mechanism 900 may draw a sample into the samplingchamber through the first opening 902 when transitioning from aclosed position depicted in the diagram 910 to an open positiondepicted in the diagram 912. In some embodiments, a one-way valvemay be incorporated into an ingestible device to prevent samplesfrom being drawn into the pumping chamber 904 through the secondopening 906 when the pumping mechanism 900 transitions between theclosed position and the open position. This may ensure that theonly sample entering the pumping chamber 904 is drawn in throughthe first opening 902.

[1070] Diagram 914 depicts the pumping mechanism 900 in a partiallyclosed position. When the pumping mechanism 900 is in the partiallyclosed position, the protrusion 908A of the moveable pump head 908is positioned adjacent to the first opening 902, or just inside thefirst opening 902. In this position, the protrusion 908A of themoveable pump head 908 effectively seals off the first opening 902,preventing any of the sample remaining in the pumping chamber 904from exiting pumping chamber 904 via the first opening 902. In thisposition, the base 908B of the moveable pump head 908 is positionedaway from the second opening 906. This may allow any sampleremaining in the pumping chamber 904 to exit the pumping chamber904 through the second opening 906. For example, if the secondopening 906 is connected to an entry port of a sampling chamber(e.g., connected via the third portion 608C of the inlet region 608to the entry port 610 of the sampling chamber 612 of the ingestibledevice 600 (FIG. 6)), this may allow the sample to flow freely fromthe pumping mechanism 900 into the sampling chamber via the entryport.

[1071] Diagram 916 depicts the pumping mechanism 900 as ittransitions between the partially closed position to the fullyclosed position. As the pumping mechanism 900 moves into the fullyclosed position, the moveable pump head 908 forces any of remainingsample contained within the pumping chamber 904 out of the pumpingchamber 904 through the second opening 906. As this happens, theprotrusion 908A of the moveable pump head 908 remains within thefirst opening 902, blocking it off and preventing the sample fromexiting the pumping chamber 904 through first opening 902. Bycomparison, the base 908B of the moveable pump head 908 does notfully cover the second opening 906, and the sample is free to exitthe pumping chamber 904 through the second opening 906. Incombination, this may result in a majority of the sample remainingin the sampling chamber being forced through the second opening 906as the pumping mechanism 900 moves from the partially closedposition depicted in diagram 914 to the fully closed positiondepicted in diagram 918.

[1072] Diagram 918 depicts the pumping mechanism 900 in the fullyclosed position, similar to diagram 910. As noted before, in thefully closed position the moveable pump head 908 is positioned toseal off the openings 902 and 906, which may prevent a sample fromentering or exiting the pumping chamber 904 from the openings 902or 904. In general, the pumping mechanism 900 may cycle between theclosed position depicted in diagrams 910 and 918 and the openposition depicted in diagram 912 any number of times in order todraw additional samples into the pumping chamber 904 through thefirst opening 902, and force the samples out of the pumping chamber904 through the second opening 906.

[1073] Although FIG. 9 depicts the protrusion 908A of the moveablepump head 908 located in the center of the moveable pump head 908,the location of the protrusion 908A may be anywhere on the moveablepump head 908. For example, the protrusion 908A of the moveablepump head 908 and the first opening 902 may be positioned on theside of the pumping chamber 904. In some embodiments, the moveablepump head 908 is split into two pieces, which may be controlled byone or more actuators. For example, the protrusion 908A and thebase 908B may be two separate pieces, each of which is moved usinga different actuator. This may allow the first opening 902 to besealed and unsealed independently from the volume of the pumpingmechanism 900 being increased or decreased.

[1074] For illustrative purposes, the diagrams 910-918 depict thebase 908B of the moveable pump head 908 being used to cover orotherwise block the second opening 906. However, in someembodiments, the moveable pump head 908 may not cover, fit within,or otherwise block the second opening 906, and it will beunderstood by one skilled in the art that the second opening 906does not need to be partially or fully blocked in order to push asample through the second opening 906. For example, the moveablepump head 908 may not include a base 908B at all. Instead, themoveable pump head 908 may be made of a flexible material thatforms a seal with the underside of the pumping chamber 904. In thiscase, the moveable pump head 908 may be moved up and down in amanner similar to a plunger in order to change the effective volumeof the pumping chamber 904. When the volume decreases, the sampleis at least partially forced out of the pumping chamber 904 throughthe second opening 906.

[1075] In general, incorporating the pumping mechanism 900 into aningestible device may not impair the function of the openings,ports, valves, membranes, sampling chambers, or other structures ofthe ingestible device, and any of the teachings or embodimentsdescribed in conjunction with the ingestible devices 100, 200, 600,or 702-706 may be combined in different embodiments of aningestible device along with the pumping mechanism 900. Forexample, the pumping mechanism 900 may replace the first valve 214in the ingestible device 200 (FIG. 2), and may be used to force thesample into the sampling chamber 212. As an alternate example, thepumping mechanism 900 may be used to force samples into thesampling chamber 304 of the osmotic valve mechanism 300 (FIG. 3).As another example, the pumping mechanism 900 may be incorporatedinto an embodiment of the ingestible device 600 (FIG. 6) where theexit port 616 is not included, and the pumping mechanism 900 may beused to force the sample into the sampling chamber 612 despite thepressure that may result from air or gas 618 being trapped withinthe sampling chamber 612.

[1076] For illustrative purposes, the examples provided by thisdisclosure focus primarily on a number of different exampleembodiments of an ingestible device, such as the ingestible devices100, 200, 600, and 702-706. However, it is understood thatvariations in the general shape and design of one or moreembodiments of the ingestible devices described in relation toFIGS. 1-9 may be made without significantly changing the functionsand operations of the device. Furthermore, it should be noted thatthe features and limitations described in any one embodiment may beapplied to any other embodiment herein, and the descriptions andexamples relating to one embodiment may be combined with any otherembodiment in a suitable manner. For example, any of the valvesdescribed in relation to FIG. 7 may be used as the valves 214 and216 described in relation to FIG. 2. As an alternate example, theabsorptive material 310 and flexible membrane 314 described inrelation to FIG. 3 may be incorporated into any of the varioussampling chambers described in various embodiments of ingestibledevices 100, 200, 600, and 702-706 in order to automatically sealthe sampling chamber. Moreover, the figures and examples providedin disclosure are intended to be only exemplary, and not limiting.Only the claims that follow are meant to set bounds as to what thepresent invention includes. It should also be noted that thesystems and/or methods described above may be applied to, or usedin accordance with, other systems and/or methods, including systemsand/or methods that may or may not be directly related toingestible devices.

[1077] FIG. 10 illustrates, in a highly schematic fashion, aningestible device 1000 having a housing 1010 that includes a firstend 1012 and a second end 1014 opposite first end 1012. Housing1010 also includes a wall 1016 that connects first end 1012 andsecond end 1014. Wall 1016 has an opening 1018 that allows fluidfrom an exterior of the ingestible device 1000 (e.g., from the GItract) and into an interior of ingestible device 1000.

[1078] FIG. 11 depicts a cross-sectional view of a portion of theinterior of ingestible device 1000. As shown in FIG. 11, theinterior of ingestible device 1000 includes a valve system 1100 anda sampling system 1200. Valve system 1100 is depicted as having aportion that is flush with the opening 1018 so that valve system1100 prevents fluid exterior to ingestible device 1000 fromentering sampling system 1200. However, as described in more detailbelow with reference to FIGS. 12-16, valve system 1100 can changeposition so that valve system 1100 allows fluid exterior toingestible device 1000 to enter sampling system 1200.

[1079] FIGS. 12 and 16 illustrate valve system 1100 in more detail.As shown in FIG. 12, valve system 1100 includes an actuationmechanism 1110, a trigger 1120, and a gate 1130. In FIGS. 12 and16, a leg 1132 of gate 1130 is flush against, and parallel with,housing wall 1016 so that gate leg 1132 covers opening 1018 toprevent fluid exterior to ingestible device 1000 (e.g., fluid inthe GI tract) from entering the interior of ingestible device 1000.A protrusion 1134 of gate 1130 engages a lip 1122 of trigger 1120.A peg 1124 of trigger 1120 engages a wax pot 1112 of actuationmechanism 1110. Referring to FIG. 16, a biasing mechanism 1140includes a compression spring 1142 that applies an upward force ongate 1130. Biasing mechanism 1140 also includes a torsion spring1144 that applies a force on trigger 1120 in the counter-clockwisedirection. In FIGS. 12 and 16, the force applied by torsion spring1144 is counter-acted by the solid wax in pot 1112, and the forceapplied by compression spring 1142 is counter-acted by lip1122.

[1080] FIG. 13A and FIG. 13B show an embodiment of the manner inwhich actuation mechanism 1110 actuates movement of trigger 1120.Similar to FIGS. 12 and 16, FIG. 13A shows a configuration in whichpeg 1124 applies a force against solid wax pot 1112 due to torsionspring 1144, and in which the solid nature of wax pot 1112 resiststhe force applied by peg 1124. A control unit 1150 is in signalcommunication with valve system 1100. During use of ingestibledevice 1000, a control unit 1150 receives a signal, indicating thatthe position of valve system 1100 should change, e.g., so thatingestible device 1000 can take a sample of a fluid in the GItract. Control unit 1150 sends a signal that causes a heatingsystem 1114 of actuation system 1100 to heat the wax in pot 1112 sothat the wax melts. As shown in FIG. 13B, the melted wax is notable to resist the force applied by peg 1124 so that, under theforce of torsion spring 1144, trigger 1120 moves in acounter-clockwise fashion.

[1081] FIGS. 14A and 14B illustrate the interaction of trigger 1120and gate 1130 before and after actuation. As shown in FIG. 14A,when wax pot 1112 is solid (corresponding to the configurationshown in FIG. 13A), protrusion 1134 engages lip 1122, whichprevents the force of compression spring 1142 from moving gate 1130upward. As shown in FIG. 14B, when the wax in pot 1112 melts (FIG.13B), trigger 1120 moves counter-clockwise, and lip 1122 disengagesfrom protrusion 1134. This allows the force of compression spring1142 to move gate 1130 upward. As seen by comparing FIG. 14A toFIG. 14B, the upward movement of gate 1130 results in an upwardmovement of an opening 1136 in gate leg 1132.

[1082] FIGS. 15A and 15B illustrate the impact of the upwardmovement of opening 1136 on the ability of ingestible device 1000to obtain a sample. As shown in FIG. 15A, when the wax in pot 1112is solid (FIGS. 13A and 14A), opening 1136 in is not aligned withopening 1018 in wall 1016 of ingestible device 1000. Instead, gateleg 1132 covers opening 1018 and blocks fluid from entering theinterior of ingestible device 1000. As shown in FIG. 15B, when thewax in pot 1112 is melted and trigger 1120 and gate 1130 have moved(FIGS. 13B and 14B), opening 1136 in gate 1130 is aligned withopening 1018 in wall 1016. In this configuration, fluid that isexterior to ingestible device 1000 (e.g., in the GI tract) canenter the interior of ingestible device 1000 via openings 1018 and1036.

[1083] While the foregoing description is made with regard to avalve system having one open position and one closed position(e.g., a two-stage valve system), the disclosure is not limited inthis sense. Rather, the concepts described above with regard to atwo stage valve system can be implemented with a valve system havemore than two stages (e.g., three stages, four stages, five stages,etc.). For example, FIGS. 17A-19C illustrate cross-sectional viewsof a three-stage valve system 1700. FIGS. 17A, 18A and 19Aillustrate different views of components of valve system 1700 inthe same position. FIGS. 17B, 18B and 19B illustrate differentviews of components of valve system 1700 in the same position.FIGS. 17C, 18C and 19C illustrate different views of components ofvalve system 1700 in the same position.

[1084] As shown in FIGS. 17A-19C, valve system 1700 includes anactuation system 1710, a trigger 1720, a gate 1730 and a biasingsystem 1740. Actuation system 1710 includes a first wax pot 1712, asecond wax pot 1714, a first heating system 1716 and a secondheating system 1718. Trigger 1720 includes a first lip 1722, asecond lip 1724, a first peg 1726 and a second peg 1728. Gate 1730includes a gate leg 1732 and a protrusion 1734. Gate leg 1732 hasan opening 1736. Biasing system 1740 includes a compression spring1742 and a torsion spring 1744. In addition, the ingestible deviceincludes a control unit 1750.

[1085] As shown in FIGS. 17A, 18A and 19A, in the first stage,protrusion 1734 engages first lip 1722, and first peg 1726 engagesfirst wax pot 1712. Compression spring 1742 applies an upward forceon gate 1730, and torsion spring 1744 applies a force on trigger1720 in the counter-clockwise direction. The force applied bytorsion spring 1744 is counter-acted by the solid wax in first pot1712, and the force applied by compression spring 1742 iscounter-acted by first lip 1722. Opening 1736 is not aligned withopening 1018.

[1086] FIGS. 17B, 18B and 19B illustrate the configuration in asecond stage, after control unit 1750 sends a signal to firstheating system 1716 to melt the wax in first pot 1712. In thesecond stage, trigger 1720 has moved counter-clockwise relative toits position in the first stage. First peg 1726 is positioned infirst pot 1712 because the melted wax cannot prevent this movement.Further counter-clockwise movement of trigger 1720 is prevented bythe engagement of second peg 1728 with the solid wax in second pot1714. With the counter-clockwise movement of trigger 1720, firstlip 1722 disengages from protrusion 1734, and gate 1730 movesupward so that opening 1736 in leg 1732 is aligned with opening1018. Further upward movement of gate 1730 is prevented by theengagement of protrusion 1734 with second lip 1724.

[1087] FIGS. 17C, 18C and 19C illustrate the configuration in athird stage, after control unit 1750 sends a signal to secondheating system 1718 to melt the wax in second pot 1714. In thethird stage, trigger 1720 has moved counter-clockwise relative toits position in the second stage. Second peg 1728 is positioned insecond pot 1714 because the melted wax cannot prevent thismovement. Further counter-clockwise rotation is prevented by theengagement of first and second pegs 1726 and 1728, respectivelywith first and second pots 1712 and 1714, respectively. Protrusion1734 is disengaged from second lip 1724, allowing the force ofcompression spring 1742 to move gate 1730 upward so that opening1736 is no longer aligned with opening 1018.

[1088] FIG. 20 illustrates another embodiment of a three stagevalve system 2000 that can be used in an ingestible device. Valvesystem 2000 that is similar to valve system 1700 except thatactuation system 2010 includes three includes wax pots 2012, 2014and 2016, respectively, that define a triangle, and trigger 2020includes three pegs 2022, 2024 and 2026, respectively, that definea corresponding triangle. Actuation system 2010 is controlled usinga control unit 2050. Actuation system 2010 also includes a firstheating system 2018 that heats the wax in pots 2012 and 2014 and sothat pegs 2022 and 2024 enters their corresponding pot, causingvalve system 2000 to move from its first stage to its second stage.Actuation system 2010 also includes a second heating system 2028that heats the wax in pot 2016 so that pegs 2026 enters pot 2016,causing valve system 2000 to move from its second stage to itsthird stage.

[1089] In the foregoing discussion, embodiments actuating systemsare described that include one or more wax pots and correspondingheating systems. But the disclosure is not limited to suchactuating systems. Generally, any actuating system can be used thatwill provide an appropriate force to resist counter-clockwisemovement of the trigger when desired and to remove that force whendesired. Examples of such actuation systems include a pot with asilicon or wax seal. A control unit may be used to rupture the sealand allow counter clock-wise movement of the trigger. Additionally,or alternatively, the actuation mechanism may use dissolvablecoating to that dissolves over time or in the presence of asubstance. As the coating dissolves, the trigger may move furtherin the counter clock-wise direction. Other actuation mechanisms mayalso apply an attractive force rather than remove a resistiveforce. For example, the actuation mechanism may include magneticpegs and slidable magnets The magnets may be located behind thepots or may slide to a position behind the pots when the valvesystem should change stages. As the magnets behind the pots slideinto range of the magnetic trigger pegs, the trigger moves in thecounterclockwise direction due to the attractive force between themagnetic peg and the magnets. The sliding mechanism to move theslidable magnets may be powered by an osmotic pump, a pressurizedchamber, or any other applicable method of movement previouslydescribed in other embodiments.

[1090] In the discussion above, embodiments of triggers aredisclosed that include one or more lips and one or more pegs.However, the disclosure is not limited to such triggers. Ingeneral, for example, any trigger design can be used that iscapable of providing the step-wise movement of the trigger. Suchtrigger designs include, for example, a releasable latch couplingor a saw toothed engagement wall. A different embodiment mayutilize a ball in socket joint to engage the trigger and gate, inwhich the "socket" is located on the trigger. It is to be notedthat such designs need not be based on counter-clockwise movementand may be, for example, designed for the controlled movement ofthe trigger in one or more of various degrees of freedom. Forexample, rather than rotate, the trigger may be configured to slidelaterally to push a peg of the trigger into a melted wax pot.

[1091] The discussion above describes embodiments of gates thatinclude a protrusion and a leg with an opening. The disclosure isnot limited to such designs. Generally, any appropriate arrangementcan be used so long as it provides the desired step-wise controlledmovement of an opening to the interior of the ingestible device.Exemplary designs include a gate that is capable of responding toor applying magnetic forces on the trigger. A saw toothed patternmay also provide a step-wise gate movement. Additionally,embodiments include a latch designed to releasably couple the gateto the trigger. A different embodiment may utilize a ball in socketjoint in which the "ball" is located on the gate. Optionally, agate can include one or regions that include one or moreappropriate sealing materials positioned to cover the opening inthe housing of the ingestible device when the gate is positioned toprevent fluid exterior to the ingestible device from entering theinterior of the device via the opening in the housing of theingestible device.

[1092] In the foregoing discussion, embodiments of biasing systemsare described that include a compression spring and a biasingspring. However, the disclosure is not limited in this sense. Ingeneral, any biasing elements can be used to provide thecounter-clockwise force to the trigger and/or to provide the upwardforce to the gate. Exemplary biasing elements include elasticbands, wherein a stretched elastic band acts similar to a stretchedcompression spring as described. Additional basing mechanisms mayinclude magnets and/or magnetic forces to induce trigger or gatemovement. For example, a magnet may be located above the gate,where, like the constant force of the stretched compression spring,the magnet also applies a constant attractive force on thegate.

[1093] As noted above in addition to a valve system, an ingestibledevice includes a sampling system. FIGS. 21A and 21B illustrate apartial cross sectional view of ingestible device 1000 withsampling system 1200 and certain components of valve system 1100.Sampling system 1200 includes a series of sponges configured toabsorb fluid from an opening, move the fluid to a location withinthe housing, and prepare the fluid for testing. Preparation fortesting may include filtering the fluid and combining the fluidwith a chemical assay. The assay may be configured to dye cells inthe filtered sample. The series of sponges includes a wickingsponge 1210, a transfer sponge 1220, a volume sponge 1230, and anassay sponge 1240.

[1094] Wicking sponge 1210 is made of an absorptive material thatabsorbs the fluid form the opening in the housing when the valve isopen i.e. when the inlet and the housing are aligned. The wickingsponge transfers the fluid from the opening to a filter. Wickingsponge 1210 includes a wicking tongue 1212 extended towards thehousing 1016. As shown in FIG. 21A, before actuation of theactuation system (FIGS. 13A, 14A, 15A), wicking tongue 1212 is notadjacent opening 1018 in wall 1016 of ingestible device 1000 sothat wicking tongue 1212 does not absorb fluid exterior toingestible device 1000. However, as shown in FIG. 21B, afteractuation of the actuation system (FIGS. 13B, 14B, 15B), wickingtongue 1212 is adjacent opening 1018 so that wicking sponge 1212 ismade of an absorptive material that absorbs fluid that passesthrough opening 1018, e.g., fluid from the GI tract. Fluid absorbedby wicking tongue 1212 can travel through wicking sponge 1210 to adistal end 1214 of wicking sponge 1210. The wicking sponge 1210 andwicking tongue 1212 may be made of a VF2 sponge, an Ahlstrom M13sponge, MF/F material, a Carwild Ivalon Polyvinyl Alcohol material,or another suitable absorptive material. Optionally, the dimensionsof the sponge material may be selected to enable all its desiredfunctions while remaining precisely packaged within the capsule. Insome embodiments, Carwild Ivalon Polyvinyl Alcohol material is cutto the dimensions 1.4 millimeters (height).times.6 millimeters(width).times.8.5 millimeters (length). In certain embodiments, oneor more of the following parameters can be considered whenselecting an appropriate material and/or its dimension: ability toload one more preservative materials; desired preservativematerial(s) to be loaded; capacity to hold one or more driedpreservatives; ability to facilitate hydration of one or more driedpreservative materials upon contact with one or more GI fluids;capacity to capture fluid (e.g., GI fluid); and swelling propertiesupon fluid uptake (generally, it is desirable to have little or noswelling upon fluid uptake). Typically, the preservative(s) is(are) selected based on the analyte of interest.

[1095] Nucleic acid preservatives can be used to prevent or reducethe rate of nucleic acid degradation or denaturation, and/orincrease the stability of nucleic acids, e.g., to maintain nucleicacid structure. In some embodiments, the nucleic acid preservativeis nuclease inhibitor (deoxyribonuclease inhibitor). In someembodiments, the nucleic acid preservative is a ribonucleaseinhibitor. Nuclease inhibitors and ribonuclease inhibitors areknown in the art, and have been described in, e.g., U.S. Pat. No.6,224,379, herein incorporated by reference in its entirety. Insome embodiments, the nucleic acid preservative mixture can includeEDTA, sodium citrate, an ammonium sulphate. In some embodiments,the RNA preservative mixture includes 2 mL of 0.5M EDTA, 1.25 ml of1 M sodium citrate, 35g of ammonium sulphate, and 46.8 mL of dH20.In some embodiments, the RNA preservative is an RNAlater.TM.stabilization solution (ThermoFisher Scientific), as described inU.S. Pat. No. 7,056,673, which is herein incorporated by referencein its entirety. In some embodiments, an RNA preservative caninclude one or more of triphenylmethane dyes (such as methyl green,crystal violet, pararosaniline, or tris-(4-aminophenyl)methane),cresyl violet, polyamines, and cobalt ions. In some embodiments, anRNA preservative can include one or more of spermine, spermidine,1,10-diamino-4,7-diazadecane, 1,11-diamino-4,8-diazaundecane,1,13-diamino-4,10-diazatridecane,1,14-diamino-4,11-diazatetradecane,1,15-diamino-4,12-diazapentadecane,1,16-diamino-4,13-diazahexadecane,1,17-diamino-4,14-diazaheptadecane,1,18-diamino-4,15-diazanonadecane, 1,19-diamino-4,16-diazaeicosane,and 1,20-diamino-4,17-diazaheneicosane.

[1096] Protein preservatives can be used to prevent or reduce therate of protein degradation or denaturation, and/or increase thestability of proteins, e.g., to maintain protein structure.Preservatives can include, by way of example, protease inhibitors,surfactants (e.g., nonionic surfactants), emulsifiers, acids,parabens, esters and protein stabilizers.

[1097] In some embodiments, the preservative can prevent or reducethe digestion or degradation of proteins by one or more proteases.In some embodiments, the preservative can be a protease inhibitor.In some embodiments, the protease inhibitor is a serine proteaseinhibitor, a metalloprotease inhibitor, an aminopeptidaseinhibitor, a cysteine peptidase inhibitor, or an aspartyl proteaseinhibitor. In some embodiments, the protease inhibitor can preventor reduce digestion by proteases such as, but not limited to,trypsin, chymotrypsin, plasmin kallikrein, thrombin, papain,cathepsin B, cathepsin L, calpain and staphopain, endoproteinaseLys-C, Kallikrein, and thrombin. In some embodiments, the proteaseinhibitor can be 4-(2-aminoethyl)benzenesulfonyl fluoridehydrochloride (AEBSF, CAS 30827-99-7), aprotinin (CAS 9087-70-1),bestatin (CAS 58970-76-6), E-64 (CAS 66701-25-5), leupeptin (CAS103476-89-7), pepstatin A (CAS 26305-03-3), orN-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK). In someembodiments, the protein biomarker preservative includes4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, CAS30827-99-7), aprotinin (CAS 9087-70-1), bestatin (CAS 58970-76-6),E-64 (CAS 66701-25-5), leupeptin (CAS 103476-89-7), pepstatin A(CAS 26305-03-3), DMSA, and bovine serum albumin, and, optionally,N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK).

[1098] In some embodiments, the preservative can be a proteinstabilizer such as, for example, Trehalose or Dextran.

[1099] A preservative as disclosed herein can be an acid. In someembodiments, the preservative can be an acid with a pKa between 3and 7. In some embodiments, the preservative can be citric acid, orsorbic acid.

[1100] In some embodiments, the preservative can be a surfactantsuch as a polysorbate. Exemplary polysorbates include, for example,polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate),polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate),polysorbate 60 (polyoxyethylene (20) sorbitan monostearate),polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate,sorbitan tristearate, and sorbitan monooleate.

[1101] In some embodiments, the preservative is a paraben,parahydroxybenzoate, or ester of parahydroxybenzoic acid(4-hydroxybenzoic acid). In some embodiments, the preservative canbe propyl paraben.

[1102] In some embodiments, the preservative can include dimethylsulfoxide (DMSA). In some embodiments, the preservative can includebovine serum albumin.

[1103] The preservative can be a mixture of two or more of aprotease inhibitor, a surfactant, an emulsifier, an acid, aparaben, and an ester. For example, a preservative as describedherein can include a mixture of two or more protease inhibitors. Insome embodiments, a preservative as described herein can include amixture of one or more protease inhibitors, and one or more acids.In some embodiments, a preservative as described herein can includea mixture of one or more protease inhibitors, one or more acids,and an ester, e.g., a paraben. In some embodiments, a preservativeas described herein can include a mixture of one or more proteaseinhibitors, one or more acids, one or more esters, and one or moresurfactants. In some embodiments, the preservative can include theHALT.TM. protease inhibitor cocktail (Thermo Fisher). In someembodiments, the preservative can include the HALT' proteaseinhibitor cocktail (Thermo Fisher) and TPCK. In some embodiments,the preservative can be bactericidal to preserve a protein, e.g., aprotein biomarker. In some embodiments, the preservative mixturethat is bactericidal includes citric acid (CAS 77-92-9), sorbicacid (CAS 110-44-1), propylparaben (CAS 94-13-3), tween 80 (CAS9005-65-6), ethanol, bovine serum albumin, and TPCK (CAS402-71-1).

[1104] In some embodiments, a preservative mixture containing oneor more protease inhibitors can be contacted with a protein in thegastrointestinal tract to stabilize the protein. In someembodiments, the protein is an immunoglobulin. In some embodiments,the protein is an IgA or IgM. In some embodiments, the protein is asecretory IgA. In an exemplary embodiment, a preservative mixturecontaining AEBSF, aprotinin, bestatin, E-64, leupeptin andpepstatin A protease inhibitors (HALT.TM., Thermo Fisher), andN-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK, Sigma Aldrich)can be used to stabilize one or more immunoglobulin proteins in thegastrointestinal tract, e.g., secretory IgA.

[1105] In some embodiments, a preservative mixture containing oneor more protease inhibitors, acids, parabens, and surfactants canbe contacted with a protein in the gastrointestinal tract tostabilize the protein. In some embodiments, the protein is not animmunoglobulin. In an exemplary embodiment, a preservative mixturecontaining AEBSF, aprotinin, bestatin, E-64, leupeptin andpepstatin A protease inhibitors (HALT.TM., Thermo Fisher),N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK, SigmaAldrich), citric acid, sorbic acid, propyl paraben, polysorbate 80(Tween 80), BSA can be used to stabilize one or morenon-immunoglobulin proteins in the gastrointestinal tract, e.g., acytokine, calprotectin, S100A12, lactoferrin, M2-pyruvate kinase,neopterin, a metalloproteinase, a myeloperoxidase,polymorphonuclear elastase, and/or alpha 1 antitrypsin eosinophilicprotein X.

[1106] In some embodiments, one or more internal controls areincluded in an ingestible device, as described herein, that is usedto collect one or more analytes. The internal control can be usedto monitor the stability and degradation of small molecules,nucleic acids, and/or proteins in the device over time. In someembodiments, the internal control can be a small molecule, anucleic acid, and/or a protein. In some embodiments, the smallmolecule internal control can be 2,4 dinitrophenol (2,4, DNP),femocene, and/or a deuterium-labeled cholesterol. In someembodiments, the nucleic acid internal control can be a DNAinternal control. In some embodiments, the nucleic acid internalcontrol can be a RNA internal control. In some embodiments, the RNAinternal control can be a G+C-rich (60%) RNA molecule withextensive secondary structure, based on a modified delta virusgenome, as described in Dingle et al., J. Clin. Microbiol.42(3):1003-1011, 2004, herein incorporated by reference in itsentirety. In some embodiments, the protein internal control can behuman serum albumin (HSA), fluorescein isothiocyanate, and/orbiotin.

[1107] In some embodiments, the preservative is a microbialpreservative. In exemplary embodiments, the preservative prevents,inhibits, or reduces the growth and/or multiplication of amicroorganism. In some embodiments, the preservative permanentlyprevents, inhibits, or reduces the growth and/or multiplication ofa microorganims. In exemplary embodiments, the preservativeprevents, inhibits, or reduces the growth and/or multiplication ofbacteria. In some embodiments, the preservative permanentlyprevents, inhibits, or reduces the growth and/or multiplication ofbacteria. In some embodiments, the preservative is one or more of abacteriostatic, bacteriocidal, and/or fixative compound.

[1108] Bacteriostatic preservatives arrest the growth ormultiplication of the bacteria. In some embodiments, thepreservative kills the bacteria, thereby preventing growth andmultiplication. Bactericidal preservatives kill bacteria. Bacteriaenter a device as described herein in the GI tract of a subject,and are contacted with a bacteriostatic preservative that arrestsbacterial growth and multiplication, or a bactericidal preservativethat kills the bacteria. As a result, the numbers of bacteria inthe device are representative of the bacterial microflora that waspresent in the GI tract at the time the bacteria first entered thedevice.

[1109] In some embodiments, the preservative can be abacteriostatic food preservative, such as, but not limited to,sorbic acid, citric acid, propyl paraben, nisin, dimethyldicarbonate, and ethylenediaminetetraacetic acid (EDTA). In someembodiments, the preservative can be sodium azide, hydroxyurea,fusidic acid, diazolidinyl urea, imidazolidinyl urea, salicylicacid, barium and nickle chloride, metallic copper, thimerosal,2-phenoxyethanol, or ProClin.TM.. In some embodiments, thepreservative can be one or more of sorbic acid, citric acid, propylparaben, nisin, dimethyl dicarbonate, ethylenediaminetetraaceticacid (EDTA), sodium azide, hydroxyurea, fusidic acid, diazolidinylurea, imidazolidinyl urea, salicylic acid, barium and nicklechloride, metallic copper, thimerosal, 2-phenoxyethanol, andProClin.TM..

[1110] In some embodiments, the preservative prevents or reducesnucleic acid degradation, in addition to preventing or inhibitingthe growth and/or multiplication of bacteria. The preservation ofnucleic acid integrity allows for the quantification of bacteriausing PCR-based DNA or RNA analysis methods, e.g., 16S ribosomalRNA PCR and sequencing. In some embodiments, the preservativeincludes EDTA.

[1111] In some embodiments, the bactericidal preservative caninclude one or more of citric acid (CAS 77-92-9), sorbic acid (CAS110-44-1), propylparaben (CAS 94-13-3), Tween 80 (CAS 9005-65-6),ethanol, bovine serum albumin, and TPCK (CAS 402-71-1). In someembodiments, the bactericidal preservative is a mixture of citricacid, sorbic acid, propyl-paraben, and Tween 80, e.g., thebactericidal preservative can include 2.5% (m/v) citric acid, 2.5%(m/v) sorbic acid, 2.5% (m/v) propyl-paraben), and 3.13% (m/v)Tween 80. In some embodiments, the bactericidal preservative is amixture of sorbic acid, Tris, EDTA, Tween 80, and NaCl, e.g., thebactericidal preservative can include 2.0% (m/v) sorbic acid, tris,EDTA, 1.0% (m/v) Tween 80, and 1.0% (m/v) NaCl. In someembodiments, the bactericidal preservative is a heavy metalbactericidal mixture. In some embodiments, the bactericidalpreservative is a mixture that includes barium chloride and nickelchloride. In some embodiments, the bactericidal preservative isthimerosal, e.g., a stabilizer that includes 0.1% thimerosal.

[1112] A cell filter 1250 is located between distal end 1214 ofwicking sponge 1210 and a first end 1222 of transfer sponge 1220.The cell filter 1250 is configured to prevent undesired cells, suchas Hela cells, from entering one or more downstream sponges insampling system 1200, particularly sponges used in testing. In someembodiments, the filter can be used to filter and/or selectivelykill eukaryotic cells. Excluding such undesired cells enhances theaccuracy of various analytical results.

[1113] Fluid that passes from wicking sponge 1210 and through cellfilter 1250 can enter transfer sponge 1220 via its first end firstend 1222. Transfer sponge 1220 is configured to move the filteredfluid from cell filter 1250 to volume sponge 1230 and/or assaysponge 1240.

[1114] To allow transfer sponge 1220 (made of an absorptivematerial) to absorb a relatively large volume of fluid, transfersponge 1220 is shaped (e.g., arc-shaped) to provide a relativelylong distance between first end 1222 of transfer sponge 1220 and asecond end 1224 of transfer sponge 1220. Second end 1224 contactsboth volume sponge 1230 and assay sponge 1240 while preventingvolume sponge 1230 and assay sponge 1240 from directly contactingeach other. A barrier 1260 is located between first end 1222 andvolume sponge 1230 to ensure that fluid absorbed in transfer sponge1220 at first end 1222 travels to second end 1224 before beingabsorbed by volume sponge 1230. Although depicted as beingarc-shaped, transfer sponge 1220 can have one or more differentconfigurations, such as, for example, an extended straight line ormultiple curves, depending, for example, on the desired volume ofsample and/or desired transfer speed. In general, the shorterand/or thinner the path of transfer sponge 1220, the quicker thetransfer speed from first end 1222 to second end 1224. The transfersponge 1220 may be made of a VF2 sponge, an Ahlstrom M13 sponge,MF/F material, or another suitable absorptive material.

[1115] Volume sponge 1230 is made of an absorptive material thatabsorbs additional fluid for testing and is in fluid communicationwith assay sponge 1240 via second end 1224 of transfer sponge 1220.Volume sponge 1230 can be particularly useful when fluorescent oroptical testing is used. In some embodiments, assay sponge 1240 andtransfer sponge 1224 may not individually contain a sufficientvolume of the sample to attain a confident test result. The volumeof volume sponge 1230, assay sponge 1240, and second end 1224 ofthe transfer sponge 1220 sum to a sufficient testing volume foroptical, and other, tests. Assay sponge 1240 contains a chemicalassay that is used to test the sample or to prepare the sample fora test. Once assay sponge 1240 is saturated, the assay chemicalsare free to flow from assay sponge 1240 and interact with sampleabsorbed by transfer sponge 1220 and volume sponge 1230. Volumesponge 1230 and the assay sponge 1240 may be made of a VF2 sponge,an Ahlstrom M13 sponge, MF/F material, or another suitableabsorptive material. Preferably, the wicking sponge, wickingtongue, transfer sponge, and assay sponge are Ahlstrom M13 sponges,and the volume sponge is a VF2 sponge.

[1116] Cell filter 1250 can be made from any appropriate materialand have any appropriate dimensions. Exemplary materials includepolycarbonate (PCTE), polyethersulfone (PES), polyester (PETE) andpolytetrafluoroethylene (PTFE). In some embodiments, the dimensionsof cell filter 1250 can be about 9.5 millimeters by about 6.5millimeters by about 0.05 millimeter.

[1117] Sampling system 1200 also includes a membrane 1270 locatedbetween assay sponge 1240 and a vent 1280 for gases to leavesampling system 1200. Membrane 1270 is configured to allow one ormore gases to leave sampling system 1200 via an opening 1280, whilemaintaining liquid in sampling system 1200.

[1118] FIG. 22 illustrates an embodiment of ingestible device 1000with a relatively detailed view of both valve system 1100 andsampling system 1200. FIG. 22 shows valve system 1100 positionedprior to actuation of actuation system 1110 (e.g., when configuredas shown in FIGS. 13A, 14A, 15A and 20A).

[1119] FIG. 23 illustrates an embodiment of an ingestible deviceincluding sampling system 1200 and three-stage valve system 1700positioned in its third stage.

[1120] FIG. 24 illustrates an embodiment of an ingestible device1000 including sampling system 1200 and valve system 2000positioned in its third stage.

[1121] FIG. 25 is a highly schematic illustration of an ingestibledevice 3000 that contains multiple different systems that cooperatefor obtaining a sample and analyzing a sample, e.g., within the GItract of a subject. Ingestible device 3000 includes a power system3100 (e.g., one or more batteries), configured to power anelectronics system 3200 (e.g., including a control system,optionally in signal communication with an external base station),and an analytic system 3500.

[1122] Exemplary analytical systems include assay systems, such as,for example, optical systems containing one or more sources ofradiation and/or one more detectors. Such systems may use, forexample, a light source that illuminates and a sample and adetector configured to detect light that is emitted by the sample(e.g., fluorescence spectroscopy), optical density (e.g., theportion of light that passes through the sample), and/or light thatis diffracted by sample (e.g., diffraction optics). An analyticalsystem may use, for example, ELISA (enzyme-linked immunosorbentassay). An analytical system may use, for example, LOCI(luminescent oxygen channeling) or LOCI (fluorescent oxygenchanneling). An analytical technique may involve incubating and/ordiluting a sample before or during the analysis/assaying of thesample. An analytical technique may involve the use ofstaining/dyeing a live cell.

[1123] Ingestible device 3000 also includes a sampling system 3400for taking in a sample from the environment exterior to ingestibledevice 3000, and a valve system 3300 that regulates the ability ofa fluid to access sampling system 3400.

[1124] FIG. 26 provides an exploded view of the ingestible device3000. FIG. 26 includes the exploded view of ingestible device 3000,showing a general configuration of the systems in FIG. 25. FIG. 26includes power system 3100 (e.g., a stack of batteries), electronicsystem 3200 (e.g., a PCB and associated wiring), valve system 3300,sampling system 3400, and analytic system 3500.

[1125] FIG. 27 illustrates a portion of an ingestible device 4000with a port 4154b in an open position to the exterior of theingestible device 4000. The ingestible device 4000 may include acylinder-shaped rotatable element 4150 that includes sampling portson the wall of the rotatable element 4150. The sampling chamber4150 is wrapped by a shell element 4140 with dividers to form aseries of dilution chambers 4151a-n between the shell element 4140and the rotatable element 4150. In operation, when the ingestibledevice 4000 determines the device itself arrives at a targetlocation within the GI tract, the rotatable element 4150 may berotated into an open position such that an aperture of the shellelement 4140 is aligned with the port 4154b on the wall of therotatable element 4150 and the port 4154b is exposed to theexterior of the ingestible device 4000 through the aperture. Inthis way, fluid from the GI tract can enter the port 4154b andoccupy the volume defined by the port 154b. In the embodiment shownin FIG. 24, the port 4154b may be a depression on the surface of arotatable element 4150 and a number of dilution chambers 4151a-nare positioned circumferentially around the axis of rotation of therotatable element 4150. As previously discussed, each of thedilution chambers 4151a-n may store a dilution fluid. In someembodiments, the depression is a cylindrical depression.Optionally, the depression may be a rectangular depression, or anyconcave depression forming a regular or irregular shape. In anotherembodiment, the port 4154b may be connected to a chamber (notshown) within the rotatable element 4150 to create an enlargedspace to store the GI fluid sample from the external environment ofthe ingestible device.

[1126] In some embodiments, the ingestible device 4000 may furtherinclude a controller and an actuator. The controller may determinethat the ingestible device 4000 is located at a target location ofthe GI tract, and then the actuator may trigger the rotation of therotatable element 4150 to align the port 4154b at the open positionto initiate the sampling. For example, the housing of ingestibledevice 4000 may have a pH-sensitive enteric coating to detect orotherwise be sensitive to a pH level of the environment external tothe ingestible device 4000, based on which the controller maydetermine whether the ingestible device has arrived at a targetlocation. For another example, the ingestible device 4000 mayinclude an optical sensing unit that transmits an illumination tothe environment and collects a reflectance, based on which, theregio-specific location of the ingestible device 4000 may beidentified based on optical characteristics of the reflectance.

[1127] FIG. 28 shows one embodiment of a portion of an ingestibledevice with a port 4154b at a first position aligned with a firstdilution chamber 4151a. In operation, the rotatable element 4150may be rotated to align the sampling port 4154b and the firstdilution chamber 4151a such that the fluid sample from the GI tractstored within the volume of the sampling port 4154b can be combinedwith dilution fluid in the first dilution chamber to form a firstdilution. The first dilution may then occupy the combined volume ofthe port 4154b and first dilution chamber 4151a. Optionally, therotatable element 4150 may be subsequently rotated to a secondposition such that the port 4154b containing a portion of the firstdilution is then moved to be aligned and in fluid communicationwith another dilution chamber, e.g., a second dilution chamber thatis next to the first dilution chamber along the rotationaldirection. In this way, the first dilution stored within the port4154b may then again be diluted with the dilution fluid storedwithin the second dilution chamber. Similarly, if the rotatableelement 4150 keeps rotating and allows the port 4154b to beserially aligned with each dilution chamber, then the original GIfluid sample may be diluted serially and each dilution chambers4151a-n may be left with a diluted GI fluid sample at a differentdilution ratio.

[1128] FIG. 29 shows an embodiment of an element 4140 forming partof a set of five dilution chambers (e.g., including 4151a-b) forsurrounding a rotatable element (e.g., 4150 in FIGS. 21-22) in aningestible device as described herein. In some embodiments, thedevice may contain a single dilution chamber. Alternatively, thedevice may contain 2, 3, 4, 5, 6, 7, 8 or greater than 8 dilutionchambers.

[1129] In some embodiments, each dilution chamber 4151a-n may befilled with a dilution fluid prior to the ingestible device 4000being administered. In another embodiment, the dilution fluid maybe stored in a separate reservoir (not shown) within the ingestibledevice 4000. At the time when the ingestible device 4000 isdetermined to be at a target location within the GI tract, apumping mechanism may pump the dilution fluid into one or moredilution chambers 4151a-b via one or more outlet (not shown) of thereservoir.

[1130] In some embodiments, the shell element 4140 may have valvesor pumps (not shown) between the dilution chambers 4151a-n. Forexample, the diluted fluid from a first dilution chamber may bepumped into a second dilution chamber via a valve between the twochambers.

[1131] Devices of the type depicted in FIGS. 27-29 optionally caninclude a sampling system as disclosed herein.

[1132] In certain embodiments, an ingestible device includes amicroscopic evaluation system. In some embodiments, bacterial cellsin a sample may be first labeled with fluorescent dyes (such asthose described herein), and the fluorescently-labeled cells may beimaged and counted by the microscopic evaluation using aningestible device as described herein. For example, in someembodiments, the bacterial cells in a sample may be labeled withmultiple analyte-binding reagents (e.g., multiple antibodies eachspecific for different types of analytes (e.g., bacteria ofdifferent genera, species, and/or strains)), each conjugated to adifferent dye, thereby allowing for the imaging, detection andcounting of the different types of analytes (e.g., bacteria)present in the sample. In other embodiments, thefluorescently-labeled cells are counted as they pass through anonboard flow system (e.g., microfluidic single cell channeling).Examples of flow cytometry systems include hydrodynamic focusing,small diameter capillary tube flow, and rectangular capillary tubeflow. As described herein, live bacteria cells are labeled, and theprinciples of flow cytometry are used to quantify labeled cells.Generally speaking, the photons from an incident laser beam areabsorbed by the fluorophore and raised to a higher, unstable energylevel. Within less than a nanosecond, the fluorophore re-emits thelight at a longer representative wavelength where it is passedthrough a series of dichroic filters. This reemitted light can becollected and interpreted as proportional to the number of labeledbacteria cells. In some embodiments, a sheath fluid is not used aspart of the flow system to help accommodate the volume restrictionsof the device. In some embodiments, a rectangular capillary tube isused to achieve a sufficiently large cross-sectional area andrelatively thin inspection area. The flow cytometry optical systemoperates parallel to the fluidics system and serves to observe theredirection of light passing through the cell and deliversinformation about the bacterial cells. In some embodiments, ratherthan using a conventional laser and spherical lenses to focus thelight to a point, an LED and cylindrical lenses are used to focusthe light to a line across a rectangular capillary tube. In otherembodiments, collimating lenses are used to make the light sourceparallel, while cylindrical lenses are used to refine theinspection area. An exemplary optical configuration for thisarrangement can be seen in FIG. 30. In some embodiments, opticalfilters can be added to permit the use of fluorophores. Thecharacteristic wavelength of reemitted light from the fluorophorescan be isolated and detected with the use of dichroic, bandpass,and short or long wave pass filters. Generally, multiple dichroiclenses and photomultipliers are used, however, due to spacelimitations, only a single side-scatter detector and forwardscatter detector may be used in certain embodiments.

[1133] One of the design challenges of integrating flow cytometryinto the device is to provide a pumping mechanism. Without movingfluid, individual bacteria cells cannot be identified and accountedfor by flow cytometry within a fixed volume of fluid. In someembodiments, a gear motor is to move fluid through the device. Forexample, a micromotor including a planetary gearhead (e.g., with a25:1 reduction) can provide the desired amount of torque to createfluid flow. In another embodiment, a series of piezoelectricresistors embedded in the surface of a microfabricated plate isused to create flow. In yet another embodiment, a micropump thatincludes a pair of one-way valves and uses a magnetic pump membraneactuated by an external magnetic field is used to create flow.

[1134] In some embodiments, the system architecture includes anopening and sealing mechanism combined with a rotary wiper whichcreates a pressure driven flow via a gear motor. The gear motor canbe used for other functions in the device. As shown in FIG. 31, thecomponents of the optics and flow chamber systems fit within thedevice. In some embodiments, the sample fluid is absorbed via aflexible membrane at the top of the capsule. In some embodiments,the gear motor has 270.degree. of permissible travel which servesto open and fill the fluid chamber. During closure, the motorcloses the ingress port while simultaneously pushing the fluidthrough the rectangular capillary tube where the optical system islocated. The threaded component allows the flexible membrane toclose and seal the ingress channel without changing the wiperheight. In some embodiments, the volume of the sample chamber is 25.mu.L, 50 .mu.L, 75 .mu.L or more. In some embodiments, two or moresamples are taken from the GI tract to procure a sufficient samplesize. Referring to FIG. 31, an LED on the left side of thecapillary tube and the low-light photodetector on the right forcapturing forward and side scatter are shown. Once the fluid passesthrough the capillary tube, it exits the capsule via a one-wayvalve. In certain embodiments, the flow system allows for thedetection of cell size and internal cell complexity, in addition tocell quantitation.

[1135] The foregoing discussion is not exhaustive with respect tovarious ingestible device designs, either with respect to samplingcomponentry or absorbent (sponge) design.

[1136] As an example, while ingestible devices have been describedthat include one or more optical systems incorporated into theingestible device, in some embodiments, an ingestible device doesnot include an optical system. Optionally, such ingestible devicesmay also not include any other analytical componentry. Inembodiments of an ingestible device, which do not include anoptical system and/or other analytical componentry, there may bemore room inside the ingestible device to store one or moresamples.

[1137] FIG. 32 shows a partial view of an exemplary embodiment ofan ingestible device 5010 in which a portion of the enclosure ofingestible device 5010 has been removed. Ingestible device 5010 maybe used for collecting substances. Ingestible device 5010 maygenerally be in the shape of a capsule, like a conventional pill.Accordingly, the shape of ingestible device 5010 provides foreasier ingestion and is also familiar to healthcare practitionersand patients.

[1138] The structure of ingestible device 5010 includes firstportion 5012 and second portion 5014. First portion 5012 includescontrol electronics, a power supply, and a communication system.Second portion 5014 is generally configured to interact with the GItract, such as, for example but not limited to, sample collection,substance delivery and environmental monitoring. Second portion5014 includes a storage sub-unit 5016 with one or more chambers5018 and a chamber enclosure 5020 that encloses or overlays astorage sub-unit 5016. Each chamber 5018 has a correspondingchamber opening 5022. Chamber enclosure 5020 has an access port5024. In this example embodiment, ingestible device 5010 includesthree chambers 5018, but there can be other embodiments that haveone, two or more than three chambers.

[1139] FIGS. 33A-33C illustrate operation of ingestible device5010. Generally, chamber enclosure 5020 operates as a "closed-loop"revolver mechanism. Chamber enclosure 5020 rotates, in a controlledmanner, to align the access port 5024 with each of chamber openings5022 for collecting, at targeted locations, samples of the contentsin the GI into corresponding chambers 5018 (shown in FIG. 32),and/or for delivering substances stored in chambers 5018 (shown inFIG. 32) to targeted locations within the body.

[1140] Generally, during collection of samples, the rotation ofchamber enclosure 5020 may be described as a "closed-loop" revolvermechanism because each chamber opening 5022 is exposed only onceduring the passage of ingestible device 5010 within the body inorder to avoid cross-contamination of the collected samples. Inother words, in some embodiments, chamber enclosure 5020 ideallyrotates only once when collecting samples during each usage ofingestible device 5010 so that access port 5024 aligns with each ofchamber openings 5022 serially and only once. That is, duringcollection of samples, access port 2224 does not bypass any chamberopening 5022 and also does not return to a previous chamber opening5022 during its rotation.

[1141] In some embodiments, chamber enclosure 5020 can rotate in abidirectional motion before completing one revolution and/orperform multiple revolutions during one usage of the ingestibledevice 5010 so that at least one chamber opening 5022 is exposedmultiple times. A chamber opening 5022 may need to be exposedmultiple times if its corresponding chamber stores solids orsemi-solid reagents, sensors or cleaning agents for cleaning the GItract.

[1142] As illustrated in FIG. 33A, shown therein generally isingestible device 5010 in an open position 5010a in which accessport 5024 on chamber enclosure 5020 is aligned with a chamberopening 5022. In this configuration, ingestible device 5010 maycollect substances through chamber opening 5022. In other words,the contents of the GI tract may be forced into exposed chamber5018 (shown in FIG. 32) through muscular contractions (e.g.,peristalsis).

[1143] Thereafter, chamber enclosure 5020 may rotate to sealchamber opening 5022. FIG. 33B shows ingestible device 5010 with apartially open/partially closed position 5010b in which access port5024 has been rotated such that chamber enclosure 5020 partiallyseals chamber opening 5022.

[1144] FIG. 33C shows ingestible device 5010 in a closed position5010c, in which the chamber enclosure 5020 has been rotated adistance such that access port 5024 completely seals chamberopening 5022. If chamber enclosure 5020 has not rotated onerevolution, chamber enclosure 5020 may continue to rotate in thesame direction in order to align access port 5024 with anotherchamber opening 5022 depending if ingestible device 5010 has beenconfigured to perform another operation (i.e. sampling ordistribution).

[1145] In another example embodiment, chamber enclosure 5020 may bestationary and storage sub-unit 5016 (shown in FIG. 32) may insteadrotate to align its one or more chamber openings 5022 with accessport 5024. Rotating storage sub-unit 5016 instead of chamberenclosure 5020 may provide greater control over the rotation motionand a more constant motion since storage sub-unit 5016 would not besubjected to a varying viscosity arising from the contents in theGI tract. This arrangement, however, may limit a volume of at leastone of chambers 5018.

[1146] In some embodiments, chamber enclosure 5020 or storagesub-unit 5016 may rotate in a predetermined sequence ofbidirectional rotational motions. As described above, when storagesub-unit 5016 is configured to rotate instead of chamber enclosure5020, the volume of at least one of chambers 5018 can be limited.In order to avoid having to limit the volume of the chambers 5018,non-recess areas that may be used to separate different chambers5018 in storage sub-unit 5016 may be minimized in volume orremoved. Ingestible device 5010 can rotate in a first direction foraligning access port 5024 with one of the two adjacent chambers.Ingestible device 5010 can be configured to rotate in a seconddirection that is opposite to the first direction in order to avoidcross contamination between samples collected into or substancesreleased from those two adjacent chambers.

[1147] Ingestible device 5010 may be used for collecting usablesamples from the contents of the GI tract (e.g., 100 .mu.L sizedsamples) and maintaining each sample in isolation from one anotheruntil the samples are extracted.

[1148] In some embodiments, ingestible device 5010 may also beconfigured to conduct in-vivo measurements. Ingestible device 5010is introduced into the body with some of chambers 5018 being emptyand some of chambers 5018 carrying at least one reagents. At apredefined location in the body, ingestible device 5010 isconfigured to collect a sample from the GI tract and to store thesample into a chamber carrying at least one reagent. Aftercollection, in-vivo analysis may be conducted based on how thecollected sample interacts with the reagent inside chamber 5018.For example, ingestible device 5010 may use a biochemistry assay,such as an enzyme-linked immunosorbent assay (ELISA), forperforming in-situ experiments on collected samples. Alternatively,peripherals can be included into chambers 5018 for changing thedynamics of several in-vivo analysis and measurements. Theperipherals may include a light source, a receiver, a transducer, aheater, and the like. In general, the in-vivo experiments varyaccording to the type of information that is being sought.

[1149] FIG. 34 illustrates an exploded view of the components ofingestible device 5010 in one example embodiment. First portion5012 of ingestible device 5010 includes an end closure 5030, andelectronic components embedded on a main printed circuit board(PCB) 5032 including a communication subsystem having communicationperipherals 5034 and a transceiver 5036, a main microcontroller(i.e. processor) 5038, a power supply 5040 and other peripheralcomponents, including a magnetic switch 5039, described in furtherdetail below. Second portion 5014 of ingestible device 5010generally includes a motor 5042 with a shaft 5042s protrusing frommotor 5042, storage sub-unit 5016, a secondary PCB 5044, anencoding magnet arrangement 5046m and the chamber enclosure 5020.Generally, by placing main PCB 5032 and secondary PCB 5044 indistinct regions inside ingestible device 5010, they may beprevented from experiencing the same electrical or physicalhazards. Motor 5042 is inserted into a motor compartment 5054 thatis located in the center of storage sub-unit 5016. PCB 5044 isannular and includes one or more peripheral electronic components(e.g., a capacitor and a resistor, which can be used as a pull-upresistor), and a sensor 5064. Storage sub-unit 5016 furtherincludes chambers 5018, with chamber openings 5022, for storing oneor more collected samples and/or for storing one or moredispensable substances. Access holes 5056 are also located onstorage sub-unit 5016 orineted towards the first portion 5030.

[1150] End enclosure 5030 provides a hollow space defined by aninner wall that is cylindrical with a domed end portion. Endenclosure 5030 also includes engagement members for aligning andreleasably engaging with storage sub-unit 5016 to releasably lockend enclosure 5030 in place during operation. In particular,engagement members releasably engage complementary structures 5052in storage sub-unit 5016. When end enclosure 5030 locks withstorage sub-unit 5016, end enclosure 5030 overlaps with a rear ofstorage sub-unit 5016 and creates a seal. In some embodiments, theoverlap between end enclosure 5030 and storage sub-unit 5016 mayspan a width of 3 millimeters.

[1151] Some or all of the sponges of the above-described samplingsystems may contain one or more preservatives (see discussionabove). Typically, the assay sponge and/or the volume sponge and/orthe transfer sponge contain one or more preservatives. Typically,the preservative(s) are selected based on the analyte of interest,e.g., an analyte (such as a nucleic acid or protein biomarker) fora GI disorder.

[1152] In some embodiments, an ingestible is configured to deliveryone or more substances (e.g., one more therapeutic substances).FIGS. 35-55 provide illustrative and non-limiting examples of suchingestible devices. It is to be understood that one more featuresfrom such an ingestible device can be combined with one or morefeatures of an ingestible device configured to take one moresamples, such as, for example, described above with regarding toFIGS. 1-34.

[1153] FIG. 35 provides an example mock-up diagram illustratingaspects of a structure of an ingestible device 1600 for deliveringa dispensable substance, according to some embodiments describedherein. In some embodiments, the ingestible device 1600 maygenerally be in the shape of a capsule, a pill or any swallowableform that may be orally consumed by an individual. In this way, theingestible device 1600 may be ingested by a patient and may beprescribed by healthcare practitioners and patients.

[1154] The ingestible device 1600 includes a housing 1601 that maytake a shape similar to a capsule, a pill, and/or the like, whichmay include two ends 1602a-b. The housing 1601 may be designed towithstand the chemical and mechanical environment of the GI tract(e.g., effects of muscle contractile forces and concentratedhydrochloric acid in the stomach). A broad range of materials thatmay be used for the housing 1601. Examples of these materialsinclude, but are not limited to, thermoplastics, fluoropolymers,elastomers, stainless steel and glass complying with ISO 10993 andUSP Class VI specifications for biocompatibility; and any othersuitable materials and combinations thereof.

[1155] In some embodiment, the wall of the housing 1601 may have athickness of 0.5 mm-1 mm, which is sufficient to sustain aninternal explosion (e.g., caused by hydrogen ignition or overpressure inside the housing).

[1156] The housing 1601 may or may not have a pH-sensitive entericcoating to detect or otherwise be sensitive to a pH level of theenvironment external to the ingestible device. As discussedelsewhere in the application in more detail, the ingestible device1600 may additionally or alternatively include one more sensors,e.g., temperature sensor, pH sensor, impedance sensor, opticalsensor.

[1157] The housing 1601 may be formed by coupling two enclosureportions together. The ingestible device 1600 may include anelectronic component within the housing 1600. The electroniccomponent may be placed proximally to an end 1602b of the housing,and includes a printed circuit board (PCB), a battery, an opticalsensing unit, and/or the like.

[1158] The ingestible device 1600 further includes a gas generatingcell 1603 that is configured to generate gas and thus cause aninternal pressure within the housing 1601. In some embodiments, thegas generating cell may include or be connected to a separatechannel or valve of the ingestible device such that gas may berelease through the channel or valve to create a motion to alterthe position of the ingestible device within the GI tract. Such gasrelease can also be used to position the ingestible device relativeto the intestinal lining. In another embodiment, gas may bereleased through the separate channel or valve to alter the surfaceorientation of the intestinal tissue prior to delivery of thedispensable substance.

[1159] A traveling plunger 1604 may be placed on top of the gasgenerating cell 1603 within the housing 1601. The traveling plunger1604 is a membrane that separates the gas generating cell 1603 anda storage reservoir that stores the dispensable substance 1605. Insome embodiments, the traveling plunger 1604 may be a movablepiston. In some embodiments, the traveling plunger 1604 may insteadbe a flexible membrane such as but not limited to a diaphragm. Insome embodiments, the traveling plunger 1604, which may have theform of a flexible diaphragm, may be placed along an axialdirection of the housing 1601, instead of being placed on top ofthe gas generating cell 1603. The traveling plunger or the membrane1604 may move (when the membrane 1604 is a piston) or deform (whenthe membrane 1604 is a diaphragm) towards a direction of the end1602a of the housing, when the gas generating cell 1603 generatesgas to create an internal pressure that pushes the membrane 1604.In this way, the membrane or traveling plunger 1604 may push thedispensable substance 1605 out of the housing via a dispensingoutlet 1607.

[1160] The housing 1601 may include a storage reservoir storing oneor more dispensable substances 1605 adjacent to the travelingplunger 1604. The dispensable substance 1605 may take the form of apowder, a compressed powder, a fluid, a semi-liquid gel, or anyother dispensable or deliverable form. The delivery of thedispensable substance 1605 may take a form such as but not limitedto bolus, semi-bolus, continuous, systemic, burst delivery, and/orthe like.

[1161] In some embodiments, the storage reservoir may includemultiple chambers, and each chamber stores a different dispensablesubstance. For example, the different dispensable substances can bereleased at the same time via the dispensing outlet 1607.Alternatively, the multiple chambers may take a form of differentlayers within the storage reservoir such that the differentdispensable substance from each chamber is delivered sequentiallyin an order. In one example, each of the multiple chambers iscontrolled by a separate traveling plunger, which may be propelledby gas generation. The electronic component may control the gasgenerating cell 1603 to generate gas to propel a specific travelingplunger, e.g., via a separate gas generation chamber, etc., todelivery the respective substance. In some embodiments, the contentof the multiple chambers may be mixed or combined prior torelease.

[1162] The ingestible device 1600 may include a dispensing outlet1607 at one end 1602a of the housing 1601 to direct the dispensablesubstance 1605 out of the housing. The dispensing outlet 1607 mayinclude an exit valve, a slit or a hole, a jet injection nozzlewith a syringe, and/or the like. When the traveling plunger 1604moves towards the end 1602a of the housing 1601, an internalpressure within the storage reservoir may increase and push thedispensing outlet to be open to let the dispensable substance 1605be released out of the housing 1601.

[1163] In an embodiment, a pressure relief device 1606 may beplaced within the housing 1601, e.g., at the end 1602a of thehousing 1601.

[1164] In some embodiments, the housing 1601 may include smallholes (e.g., with a diameter smaller than 2 mm), e.g., on the sideof the housing 1601, or at the end 1602a to facilitate loading thedispensable substance into the storage reservoir.

[1165] In some embodiments, a feedback control circuit (e.g., afeedback resistor, etc.) may be added to send feedback from the gasgenerating cell 1603 to the electronic component such that when theinternal pressure reaches a threshold level, the electroniccomponent may control the gas generating cell 1603 to turn off gasgeneration, or to activate other safety mechanism (e.g.,feedback-controlled release valve, etc.). For example, an internalpressure sensor may be used to measure the internal pressure withinthe ingestible device and generate feedback to the feedback controlcircuit.

[1166] FIG. 36 provides an example diagram illustrating aspects ofa mechanism for a gas generating cell 1603 configured to generate agas to dispense a substance, according to some embodimentsdescribed herein. As shown in FIG. 36, the gas generating cell 1603generates a gas 1611 which can propel the dispensable substance1605 out of the dispensing outlet 1607. A variable resistor 1608may be connected to a circuit with the gas generating cell 1603such that the variable resistor 1608 may be used to control anintensity and/or an amount of gas 1611 (e.g., hydrogen) generatedby the cell 1603. Specifically, the gas generating cell 1603 may bea battery form factor cell that is capable of generating hydrogenwhen a resistor is applied. In this way, as the gas generating cell1603 only needs the use of a resistor only without any active powerrequirements, the gas generating cell 1603 may be integrated intoan ingestible device such as a capsule with limited energy/poweravailable. For example, the gas generating cell 1603 may becompatible with a capsule at a size of 26 mm.times.13 mm orsmaller.

[1167] In some embodiments, based on the elution rate of gas fromthe cell, and an internal volume of the ingestible device, it maytake time to generate sufficient gas 1611 to deliver the substance1605, and the time may be 30 seconds or longer. For example, thetime to generate a volume of hydrogen equivalent to 500 .mu.L offluid would be approximately 5 minutes. A longer period of time maybe needed based upon non-ideal conditions within the ingestibledevice, such as friction, etc. Thus, given that the production ofgas (e.g., hydrogen) may take time, gas generation may need tostart prior to the ingestible device arriving at the site ofdelivery to build pressure up within the device. The ingestibledevice may then need to know when it is approaching the site ofdelivery. For example, the device may start producing gas on an"entry transition," which is determined by temperature, so as toproduce enough gas to be close to the pressure high enough todeliver the dispensable substance. The ingestible device may thenonly start producing gas again when it arrives at the site ofdelivery, which will cause the internal pressure within theingestible device to reach a level required by the dispensingoutlet to release the dispensable substance. Also, forregio-specific delivery, the ingestible device may estimate thetime it takes to build up enough pressure to deliver thedispensable substance before the ingestible device arrives at aspecific location, to activate gas generation.

[1168] FIGS. 37-39 illustrate an example of an ingestible devicefor localized delivery of a dispensable substance. The ingestibledevice 1600 includes a piston or drive element 1634 to push forsubstance delivery, in accordance with particular implementationsdescribed herein. The ingestible device 1600 may have one or morebatteries 1639 placed at one end 1602a of a housing 1601 to providepower for the ingestible device 1600. A printed circuit board (PCB)1632 may be placed adjacent to a battery or other power source1639, and a gas generating cell 1603 may be mounted on or above thePCB 1632. The gas generating cell 1603 may be sealed from thebottom chamber (e.g., space including 1639 and 1632) of theingestible device 1600. A movable piston 1634 may be placedadjacent to the gas generating cell 1603. In this way, gasgeneration from the gas generating cell 1603 may propel a piston1634 to move towards another end 1602b of the housing 1601 suchthat the dispensable substance in a reservoir compartment 1635 canbe pushed out of the housing through a dispensing outlet 1607,e.g., the movement is shown at 1636, with the piston 1634 at aposition after dispensing the substance. The dispensing outlet 1607may include a plug. The reservoir compartment 1635 can store thedispensable substance, or alternatively the reservoir compartmentcan house a storage reservoir 1661 which includes the dispensablesubstance. The reservoir compartment 1635 or storage reservoir 1661may have a volume of approximately 600 .mu.L or even moredispensable substance, which may be dispensed in a single bolus, orgradually over a period of time.

[1169] FIGS. 40-42 provide example structural diagrams illustratingaspects of anchoring mechanisms of an ingestible device to anchorthe ingestible device to the intestine for dispensable substancedelivery. As shown in FIG. 40, the ingestible device 101100 can beanchored within the intestine by extending hooks 101203a-d from theingestible device 101100 after it has entered the region ofinterest. At 101201, as the ingestible device 101100 travels alongthe GI tract, the hooks 101203a-d are contained within theingestible device. At 101202, when the ingestible device 101100determines it has arrived at a location within the GI tract, thehooks 101203a-d can be actuated to extend outside of the ingestibledevice 101100 to catch in the intestinal wall and hold theingestible device 101100 in the respective location. The hooks101203a-d can be oriented to catch the intestinal wall regardlessof the instant orientation of the ingestible device 101100. Thehooks 101203a-d can also retract, dissolve, or detach from theintestinal wall after the dispensable substance has been deliveredat the anchored location.

[1170] As shown in FIG. 41, the hooks 101203a-d could also extendradially from the ingestible device, and pierce into the intestinalwall to hold the ingestible device 101100 in place. As shown inFIG. 42, if the extending hooks (e.g., 101203a-b) are hollow, thehooks can be used to both anchor the ingestible device and injectthe dispensable substance into the intestinal wall.

[1171] FIG. 43 illustrates an ingestible device 4500 including apre-pressurized actuator chamber 4503 and a sliding piston 4504,according to some embodiments described herein.

[1172] Ingestible device 4500 includes a device housing 4501. Thedevice housing 4501 is composed of a cap portion 4502a and a baseportion 4502b in the illustrated embodiments. Ingestible device4500 also includes a pre-pressurized actuator chamber 4503 that ispressurized to a target pressure, for example during manufacture orvia air fill port 4506 prior to ingestion. The capsule incorporatesan active release mechanism that activates as the capsule reachesthe target location. As the release mechanism activates, slidingpiston 4504 will rapidly move to the left, pushing a high pressurejet of dispensable substance through the nozzle.

[1173] Depending on the material used to form the walls of thedevice housing 4501, the material could diffuse the compressed gasin the pre-pressurized actuator chamber 4503 over time, decreasingthe internal pressure. To ensure that pressure is maintained in theingestible device 4500 over a period between fabrication andpatient use, packaging could be pressurized to equal the internalpressure of the pill in certain embodiments; therefore, preventingthe permeation of compressed gas from the ingestible device 4500.Assuming the gas expansion within the capsule occurs very fast andan adiabatic polytropic process takes place, gas laws are used tocorrelate the initial and final pressure of the gas with its volumechange ratio.

[1174] FIG. 44A illustrates a burst disc 4608 with an in linenozzle 4509. FIG. 44B illustrates a partial sectional view of aburst disc holder 4610, according to some embodiments describedherein. A burst disc 4608 may enable the release of a dispensablesubstance, (for example from reservoir 4505) by purposefullyfracturing at a targeted pressure allowing the dispensablesubstance to exit a nozzle 4509 to a target location within the GItract. A burst disc 4608 can be used as the sole occlusioncomponent in certain embodiments and can be used to provideisolation between upstream contamination and the dispensablesubstance payload in embodiments including another occlusioncomponent. The burst disc 4608 can be held in place via clampedouter rings 4611 of disc holder 4610 as demonstrated in FIG.44B.

[1175] FIG. 45 illustrates an ingestible device 4900 including amagnetic occlusion component 4908b, a burst disc 4608, and apre-pressurized actuator chamber 4903, according to someembodiments described herein. FIG. 46 illustrates an ingestibledevice 5000 including a magnetic occlusion component, apre-pressurized actuator chamber 4903 and a bioabsorbable plug5008, according to some embodiments described herein. A magneticstack, which upon peristaltic or osmotic pressure applicationreleases pneumatic pressure, allowing for the delivery of a jet ofdispensable substance through a conduit 4509. Osmotic pressure maybe used to reconfigure the occlusion component that includesmagnets 4908a and 4908b. The enteric coating 4908c dissolves whenexposed to luminal fluid, exposing the membrane 4908d and osmogen4908e. The membrane 4908d and osmogen 4908e facilitate the movementof liquid to create osmotic pressure on the magnet 4908a. As theosmotic pressure builds up, magnet 4908a will be pushed up inproximity to magnet 4908b. Magnet 4908b will be pulled downproviding a flow through path for a gas from pressurized chamber4905 to interact with the reservoir 4905 via connecting conduit4911. The advantage of this system is that the mechanism may becompletely sealed from the exterior of the capsule, allowing forpressure to only project into the chamber 4905. Note that anenteric coating/membrane stack 4908c, 4908d could be replaced by amethod of leveraging peristalsis for pushing magnet 4908a. FIG. 45is implemented with a burst disc 4608 as the sealing/releasemechanism once the chamber 4905 is exposed to the pressurizedchamber 4903. FIG. 46 is implemented with a bioabsorbable plug 5008(e.g. enteric coating) that is dissolved and expelled once thereservoir 4905 is exposed to the pressurized actuator chamber4903.

[1176] FIG. 47 illustrates an ingestible device 5100 includingenteric sliding occlusion component 5102, a pre-pressurizedactuator chamber 4903 and a sliding component 5108, according tosome embodiments described herein. An osmotic drive 4908, includingan enteric coating 5102 and semipermeable membrane 5104, isconfigured to move a sliding component 5108. The sliding component5108, once pushed by the osmotic drive 4908, will allow aflow-through port 4911 to connect the pressurized actuator chamber4903 to the reservoir 4905, providing dispensable substancedelivery through the nozzle 5108.

[1177] FIG. 48 illustrates an ingestible device 5200 includingdissolvable pin occlusion component, a chamber 5202, apre-pressurized chamber 5204 and a sliding piston 5206, accordingto some embodiments described herein. In another embodiment, anenteric coating 5208b is dissolved, exposing a structural pin 5208a(such as a glucose spike or hydrogel) that dissolves in thepresence of intestinal luminal fluid. With this design, as long asthe pin 5208a is in place, the force exerted on the piston 5206 andthe chamber 5202 is not large enough for the burst disk 4608 torupture. The enteric coating 5208b and pin 5208a will dissolve asthe capsule 5200 is ingested and as a result, the pressure force onthe piston 5206 will increase. The full force of thepre-pressurized chamber 5204 translated onto the chamber 5202 viathe piston 5206 is large enough to rupture the burst disk 4608. Therupture of the burst disk 4608 results in a pressurized jet ofliquid being delivered from the chamber 5202 through the nozzle4509.

[1178] FIG. 49 illustrates an ingestible device 5300 including waxplug 5308a with wire lead activators 5308b, according to someembodiments described herein. In this method, the dispensing siteis identified based on collected reflected light. The reflectanceof light in green and red spectrums (with iterations to thismethodology and algorithm actively being pursued) are measured andan algorithm is used to correlate the measured reflectance with thelocation in the Gastrointestinal (GI) tract. This method provides anon-pH based system to determine the anatomical locations of thecapsule during fasted transit. As the capsule 5300 reaches thetarget location, a signal is generated which will be used toactivate an alternative release mechanism.

[1179] FIG. 50 illustrates an ingestible 5500 device including aspring actuator 5503 and a sliding piston 5504, according to someembodiments described herein. Ingestible device 5500 uses thepotential energy stored in a spring 5503 when compressed as thedriving or actuating mechanism for jet delivery of the dispensablesubstance. The occlusion component or release mechanism consists ofbioabsorbable plug 5508a separated from the reservoir 5505 by aprotectant layer 5508b. In this embodiment, the inner volume of thecapsule 5500 is divided into two sections separated by a slidingpiston 5504. The left section (e.g., reservoir 5505) is filled withdispensable substance and a spring 5503 is mounted in the rightsection. The piston 5504 can freely move to the right or leftdepending on the net force exerted on the piston 5504. An O-ring5511 is used to provide the sealing desired between the twosections, with alternative sealing means possible. Compressedspring 5503 applies a force on the piston 5504 and the piston 5504transfers this force to the liquid dispensable substance in form ofpressure. The same pressure will be transferred to the plug 5508asealing the nozzle 5513. However, this pressure acts on a smallarea (area of the plug 5508a). Therefore, the large force exertedby the spring 5503 translates into a small force on the sealingplug 5508a. As the capsule 5500 is digested, it moves through GItract and the bioabsorbable sealing plug 5508a will startdissolving. After certain amount of time, the plug will weaken orfully dissolve in GI fluid. As soon as the plug 5508a weakens tothe design threshold, the pressure inside the reservoir 5503 drops,the spring 5503 will expand delivering dispensable substance (e.g.,in the form of a high-pressure jet of fluid) through theopening.

[1180] FIG. 51 illustrates an ingestible device 5600 including aspring actuated slidable housing portion 5602b, according to someembodiments described herein. Ingestible device 5600 consists of apressurized actuator 5603 chamber, a reservoir 5605 separated fromthe pressure actuator chamber 5603 by a deformable body 5604 suchas bellows and a spring/enteric coating release mechanism Thespring 5608a is mounted on the polycarbonate cap 5602a from one endand to a sliding cap 5602b on the other end. The stainless steeltop slider 5602b can slide to the left and right opening andclosing the nozzle 5611. An enteric ring 5608b is used to keep thetop slider closed. An O-ring and a bioabsorbable plug 5609 are usedto provide the desired sealing. An adhesive seal 5612 is located onthe housing, on the opposite end of the capsule 5600 from thespring 5608a. Compressed gas applies a force on the bellows 5604and the bellows 5604 transfer this force to the liquid dispensablesubstance in form of pressure. The same pressure will betransferred to the slider 5602b in form of a radial force. However,this pressure acts on a small area (area of the exit orifice 5607).Therefore, the transverse load on the slider 5602b is relativelysmall. When the capsule 5600 is assembled, the spring 5608a iscompressed (slider 5602b in closed mode), and the enteric coating5608b keeps the slider 5602b in position. As the capsule 5600 isdigested, it moves through GI tract. The enteric coating 5608b willdissolve when the capsule 5600 passes through the intestinal fluid.With the dissolution of the enteric coating 5608b, the spring 5608awill push the slider 5602b back away from the capsule 5600 (openmode). As a result, the exit orifice 5607 becomes concentric withthe nozzle 5611 and the jet of fluid will be released.

[1181] FIG. 52 illustrates an ingestible device 5700 with anotherspring actuated slidable housing portion 5712, according to someembodiments described herein. Ingestible device 5700 uses acompressed spring (spring 5703) as the drive mechanism and acompressed spring 5708a (spring with sliding top cap 5712 as therelease mechanism. A piston 5704 separates the reservoir 5705 fromthe spring chamber and an enteric coating 5708b is used to initiatethe release mechanism. An O-ring 5710 is used to provide sealingbetween the piston 5704 and cylinder. Compressed spring 5703applies a force on the piston 5704 and the piston 5704 transfersthis force to the liquid dispensable substance in the form ofpressure. The same pressure will be transferred to the top capslider 5712 in form of a radial force. However, this pressure actson a small area (area of the exit orifice 5714) resulting in asmall transverse force on the top slider 5712. When the capsule5700 is assembled, spring 5703 is left in compressed mode (slider5712 in closed position). As the capsule 5700 is digested, it movesthrough GI tract. The enteric coating 5708b will dissolve when thecapsule 5700 passes through the intestinal fluid. With thedissolution of the enteric coating 5708b, the spring 5708a willpush the slider 5712 back away from the capsule 5700 (open mode).As a result, the exit orifice 5714 becomes concentric with thenozzle 5716 and the jet of fluid will be released.

[1182] FIG. 53 illustrates an ingestible device 5800 including amelt away occlusion component 5808a and a pressurized chamber 5803,according to some embodiments described herein. Ingestible device5800 consists of two chambers, one chamber is filled withdispensable substance and the other chamber is filled withpressurized gas. A wax valve 5808a actuated by localization board5822 is used as the occlusion component. A large section of thepressure chamber 5803 is occupied by the release mechanism and thebatteries 5821. Wax valve wires 5808b are connected to the waxvalve 5808a and will melt the wax using an electric current. Thetiming of this operation is controlled by the localization board5822. In this embodiment, a fully controlled release mechanism isused. As the capsule 5800 reaches target area, the localization kitwill activate and direct a predetermined electric current towardthe wax valve 5808a. A heating element will receive this currentand will melt or weaken the wax valve 5808a. With weakening orremoval of the wax from the nozzle 5810, gas pressure from thepressurized chamber 5803 will push the bellows 5804 resulting in apressurized jet of liquid dispensable substance exiting the nozzle5810, thus delivering the dispensable substance.

[1183] FIG. 54 illustrates an ingestible device 5900 including adissolvable pin occlusion component 5908 and a spring actuatedsliding piston 5914, according to some embodiments describedherein. One of the main challenges of designing an effectivecapsule is the sealing between the two chambers inside the capsulesince there is a significant pressure difference between the twochambers, the dispensable substance tends to move from thedispensable substance chamber into the pressure or spring chamber.Certain embodiments address this by reducing the pressuredifference between the two chambers during the shelf life andbefore jet delivery. For example, ingestible device 5900 includes acompressed spring 5903 is retained using a dissolvable pin 5908.Additionally, an O-ring 5912 is used to provide sealing between thepiston 5914 and housing. With this design, as long as the pin 5908is in place, there is no force exerted on the piston 5904 and theliquid in chamber 5906. The force exerted by the spring 5903 willresult in shear stress on the pin 5908. The pin 5908 will dissolveas the capsule 5900 is ingested and as a result, the spring forcewill translate into a pressurized jet of liquid. An enteric coatingon the ends of the pin 5908 could further enhance the specificityof the triggering location. During the shelf life and beforeingestion of the capsule 5900, there is not a significant amount ofpressure acting on the dispensable substance and consequently,sealing challenges are easier to address. With a 200-psi designpressure, the pin would be expected to hold approximately 20 lbf,and would involve design consideration to the shear strength of thedissolvable pin. As the capsule 5900 passes through the GI tract,the pin 5908 will start dissolving. As the pin 5908 dissolves,there is no support for the piston 5904 to keep the piston 5904 inplace. The force of the spring 5903 will result in a significantpressure in the fluid. At a certain point the pin 5908 will failand the piston 5904 will move to the left releasing a high-pressurejet of fluid through the nozzle 5910.

[1184] FIG. 55 illustrates an ingestible device 6000 includingshuttle slider occlusion component 6012 and a pressurized chamber6010, according to some embodiments described herein. Ingestibledevice 6000 includes two chambers separated by a wall 6002 made ofpolycarbonate. The right chamber is an adhesive seal 6028 and apressurized chamber 6010, pressurized with gas, and a bellows 6006is installed in the left chamber. There are no openings connectingthe two chambers 6006, 6010. An osmotic release mechanism is usedto connect the two chambers 6006, 6010 through a sliding valve6012. Osmogen 6014 is contained within a small container below thesliding valve 6012. Osmogen 6014 is separated from the GI fluid bya water permeable membrane 6016 covered with enteric coating 6018.On the top of the osmogen 6014, a shuttle slider 6012 is mounted.The slider 6012 has an opening 6020 in the middle. The slidershuttle 6012 is sandwiched between two slabs of polycarbonate witha pressure through port 6022. When the slider shuttle 6012 is inclosed form, the holes on the polycarbonate slabs are notconcentric with the hole on the slider shuttle 6012. When theslider shuttle 6012 is in open mode, the holes of the slider andpolycarbonate slabs surrounding it all will be concentric lettinggas and pressure exchange between the two chambers 6006, 6010.

[1185] In certain embodiments, an ingestible device is configuredto determine its location (e.g., within the GI tract of a subject).FIGS. 56-70 provide illustrative and non-limiting examples of suchingestible devices and associated methods. It is to be understoodthat one more features from such embodiments can be combined withone or more features of an ingestible device configured to take onemore samples, such as, for example, described above with regardingto FIGS. 1-34, and/or with one or more features of an ingestibledevice configured to deliver one or more substances (e.g., one ormore therapeutic substances), such as, for example, described abovewith respect to FIGS. 35-55.

[1186] In some embodiments, the location of the ingestible devicewithin the GI tract of the subject can be determined to an accuracyof at least 85%, e.g., at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, 100%. In such embodiments, the portion ofthe portion of the GI tract of the subject can include, forexample, the esophagus, the stomach, duodenum, the jejunum, and/orthe terminal ileum, cecum and colon.

[1187] In certain embodiments, the location of the ingestibledevice within the esophagus of the subject can be determined to anaccuracy of at least 85%, e.g., at least 90%, at least 95%, atleast 97%, at least 98%, at least 99%, 100%.

[1188] In some embodiments, the location of the ingestible devicewithin the stomach of the subject can be determined to an accuracyof at least 85%, e.g., at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, 100%.

[1189] In certain embodiments, the location of the ingestibledevice within the duodenum of the subject can be determined to anaccuracy of at least 85%, e.g., at least 90%, at least 95%, atleast 97%, at least 98%, at least 99%, 100%.

[1190] In some embodiments, the location of the ingestible devicewithin the jejunum of the subject can be determined to an accuracyof at least 85%, e.g., at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, 100%.

[1191] In certain embodiments, the location of the ingestibledevice within the terminal ileum, cecum and colon of the subjectcan be determined to an accuracy of at least 85%, e.g., at least90%, at least 95%, at least 97%, at least 98%, at least 99%,100%.

[1192] In some embodiments, the location of the ingestible devicewithin the cecum of the subject can be determined to an accuracy ofat least 85%, e.g., at least 90%, at least 95%, at least 97%, atleast 98%, at least 99%, 100%.

[1193] As used herein, the term "reflectance" refers to a valuederived from light emitted by the device, reflected back to thedevice, and received by a detector in or on the device. Forexample, in some embodiments this refers to light emitted by thedevice, wherein a portion of the light is reflected by a surfaceexternal to the device, and the light is received by a detectorlocated in or on the device.

[1194] As used herein, the term "illumination" refers to anyelectromagnetic emission. In some embodiments, an illumination maybe within the range of Infrared Light (IR), the visible spectrumand ultraviolet light (UV), and an illumination may have a majorityof its power centered at a particular wavelength in the range of100 nm to 1000 nm. In some embodiments, it may be advantageous touse an illumination with a majority of its power limited to one ofthe infrared (750 nm-1000 nm), red (600 nm-750 nm), green (495nm-600 nm), blue (400 nm-495 nm), or ultraviolet (100 nm-400 nm)spectrums. In some embodiments a plurality of illuminations withdifferent wavelengths may be used. For illustrative purposes, theembodiments described herein may refer to the use of green or bluespectrums of light. However, it is understood that theseembodiments may use any suitable light having a wavelength that issubstantially or approximately within the green or blue spectradefined above, and the localization systems and methods describedherein may use any suitable spectra of light.

[1195] Referring now to FIG. 56, shown therein is a view of anexample embodiment of an ingestible device 65100, which may be usedto identify a location within a gastrointestinal (GI) tract. It isto be understood that certain details regarding the design ofingestible device 65100 are not shown in FIG. 56 and the followingfigures, and that, in general, various aspect of ingestible devicesdescribed elsewhere herein can be implemented in ingestible device65100 and the ingestible devices shown in the followingfigures.

[1196] In some embodiments, ingestible device 65100 may beconfigured to autonomously determine whether it is located in thestomach, a particular portion of the small intestine such as aduodenum, jejunum, or ileum, or the large intestine by utilizingsensors operating with different wavelengths of light.Additionally, ingestible device 65100 may be configured toautonomously determine whether it is located within certainportions of the small intestine or large intestine, such as theduodenum, the jejunum, the cecum, or the colon.

[1197] Ingestible device 65100 may have a housing 65102 shapedsimilar to a pill or capsule. The housing 65102 of ingestibledevice 65100 may have a first end portion 65104, and a second endportion 65106. The first end portion 65104 may include a first wallportion 65108, and second end portion 65106 may include a secondwall portion 65110. In some embodiments, first end portion 65104and second end portion 65106 of ingestible device 65100 may bemanufactured separately, and may be affixed together by aconnecting portion 65112.

[1198] In some embodiments, ingestible device 65100 may include anoptically transparent window 65114. Optically transparent window65114 may be transparent to various types of illumination in thevisible spectrum, infrared spectrum, or ultraviolet light spectrum,and ingestible device 65100 may have various sensors andilluminators located within the housing 65102, and behind thetransparent window 65114. This may allow ingestible device 65100 tobe configured to transmit illumination at different wavelengthsthrough transparent window 65114 to an environment external tohousing 65102 of ingestible device 65100, and to detect areflectance from a portion of the illumination that is reflectedback through transparent window 65114 from the environment externalto housing 65102. Ingestible device 65100 may then use the detectedlevel of reflectance in order to determine a location of ingestibledevice 65100 within a GI tract. In some embodiments, opticallytransparent window 65114 may be of any shape and size, and may wraparound the circumference of ingestible device 65100. In this case,ingestible device 65100 may have multiple sets of sensors andilluminators positioned at different locations azimuthally behindwindow 65114.

[1199] In some embodiments, ingestible device 65100 may optionallyinclude an opening 65116 in the second wall portion 65110. In someembodiments, the second wall portion 65110 may be configured torotate around the longitudinal axis of ingestible device 65100(e.g., via a suitable motor or other actuator housed withiningestible device 65100). This may allow ingestible device 65100 toobtain a fluid sample from the GI tract, or release a substanceinto the GI tract, through opening 65116.

[1200] FIG. 57 shows an exploded view of ingestible device 65100.In some embodiments, ingestible device 65100 may optionally includea rotation assembly 65118. Optional rotation assembly 65118 mayinclude a motor 65118-1 driven by a microcontroller (e.g., amicrocontroller coupled to printed circuit board 65120), a rotationposition sensing ring 65118-2, and a storage sub-unit 65118-3configured to fit snugly within the second end portion 65104. Insome embodiments, rotation assembly 65118 may cause second endportion 65104, and opening 65116, to rotate relative to the storagesub-unit 65118-3. In some embodiments, there may be cavities on theside of storage sub-unit 65118-3 that function as storage chambers.When the opening 65116 is aligned with a cavity on the side of thestorage sub-unit 65118-3, the cavity on the side of the storagesub-unit 65118-3 may be exposed to the environment external to thehousing 65102 of ingestible device 65100. In some embodiments, thestorage sub-unit 65118-3 may be loaded with a medicament or othersubstance prior to the ingestible device 65100 being administeredto a subject. In this case, the medicament or other substance maybe released from the ingestible device 65100 by aligning opening65116 with the cavity within storage sub-unit 65118-3. In someembodiments, the storage sub-unit 65118-3 may be configured to holda fluid sample obtained from the GI tract. For example, ingestibledevice 65100 may be configured to align opening 65116 with thecavity within storage sub-unit 65118-3, thus allowing a fluidsample from the GI tract to enter the cavity within storagesub-unit 65118-3. Afterwards, ingestible device 65100 may beconfigured to seal the fluid sample within storage sub-unit 65118-3by further rotating the second end portion 65106 relative tostorage sub-unit 65118-3. In some embodiments, storage sub-unit118-3 may also contain a hydrophilic sponge, which may enableingestible device 65100 to better draw certain types of fluidsamples into ingestible device 65100. In some embodiments,ingestible device 65100 may be configured to either obtain a samplefrom within the GI tract, or to release a substance into the GItract, in response to determining that ingestible device 65100 hasreached a predetermined location within the GI tract. For example,ingestible device 65100 may be configured to obtain a fluid samplefrom the GI tract in response to determining that the ingestibledevice has entered the jejunum portion of the small intestine(e.g., as determined by process 65900 discussed elsewhere herein).It is understood that any suitable method of obtaining samples orreleasing substances may be incorporated into some of theembodiments of the ingestible devices disclosed herein, and thatthe systems and methods for determining a location of an ingestibledevice may be incorporated into any suitable type of ingestibledevice.

[1201] Ingestible device 65100 may include a printed circuit board(PCB) 65120, and a battery 65128 configured to power PCB 65120. PCB65120 may include a programmable microcontroller, and control andmemory circuitry for holding and executing firmware or software forcoordinating the operation of ingestible device 65100, and thevarious components of ingestible device 65100. For example, PCB65120 may include memory circuitry for storing data, such as datasets of measurements collected by sensing sub-unit 65126, orinstructions to be executed by control circuitry to implement alocalization process, such as, for example, one or more of theprocesses, discussed herein, including those discussed below inconnection with one or more of the associated flow charts. PCB65120 may include a detector 65122 and an illuminator 65124, whichtogether form sensing sub-unit 65126. In some embodiments, controlcircuitry within PCB 65120 may include processing units,communication circuitry, or any other suitable type of circuitryfor operating ingestible device 65100. For illustrative purposes,only a single detector 65122 and a single illuminator 65124 forminga single sensing sub-unit 65126 are shown. However, it isunderstood that in some embodiments there may be multiple sensingsub-units, each with a separate illuminator and detector, withiningestible device 65100. For example, there may be several sensingsub-units spaced azimuthally around the circumference of the PCB65120, which may enable ingestible device 65100 to transmitillumination and detect reflectances or ambient light in alldirections around the circumference of the device. In someembodiments, sensing sub-unit 65126 may be configured to generatean illumination using illuminator 65124, which is directed throughthe window 65114 in a radial direction away from ingestible device65100. This illumination may reflect off of the environmentexternal to ingestible device 65100, and the reflected light comingback into ingestible device 65100 through window 65114 may bedetected as a reflectance by detector 65122.

[1202] In some embodiments, window 65114 may be of any suitableshape and size. For example, window 65114 may extend around a fullcircumference of ingestible device 65100. In some embodiments theremay be a plurality of sensing sub-units (e.g., similar to sensingsub-unit 65126) located at different positions behind the window.For example, three sensing sub-units may be positioned behind thewindow at the same longitudinal location, but spaced 120 degreesapart azimuthally. This may enable ingestible device 65100 totransmit illuminations in all directions radially around ingestibledevice 65100, and to measure each of the correspondingreflectances.

[1203] In some embodiments, illuminator 65124 may be capable ofproducing illumination at a variety of different wavelengths in theultraviolet, infrared, or visible spectrum. For example,illuminator 65124 may be implemented by using Red-Green-BlueLight-Emitting diode packages (RGB-LED). These types of RGB-LEDpackages are able to transmit red, blue, or green illumination, orcombinations of red, blue, or green illumination. Similarly,detector 65122 may be configured to sense reflected light of thesame wavelengths as the illumination produced by illuminator 65124.For example, if illuminator 65124 is configured to produce red,blue, or green illumination, detector 65122 may be configured todetect different reflectances produced by red, blue, or greenillumination (e.g., through the use of an appropriately configuredphotodiode). These detected reflectances may be stored byingestible device 65100 (e.g., within memory circuitry of PCB 65120(FIG. 57)), and may then be used by ingestible device 65100 indetermining a location of ingestible device 65100 within the GItract (e.g., through the use of one or more processes describedherein).

[1204] It is understood that ingestible device 65100 is intended tobe illustrative, and not limiting. It will be understood thatmodifications to the general shape and structure of the variousdevices and mechanisms described in relation to FIG. 56 and FIG. 57may be made without significantly changing the functions andoperations of the devices and mechanisms. For example, ingestibledevice 65100 may have a housing formed from a single piece ofmolded plastic, rather than being divided into a first end portion65104 and a second end portion 65106. As an alternate example, thelocation of window 65114 within ingestible device 65100 may bemoved to some other location, such as the center of ingestibledevice 65100, or to one of the ends of ingestible device 65100.Moreover, the systems and methods discussed in relation to FIGS.56-70 may be implemented on any suitable type of ingestible device,provided that the ingestible device is capable of detectingreflectances or levels of illumination in some capacity. Forexample, in some embodiments ingestible device 65100 may bemodified to replace detector 65122 with an image sensor, and theingestible device may be configured to measure relative levels ofred, blue, or green light by decomposing a recorded image into itsindividual spectral components. It should be noted that thefeatures and limitations described in any one embodiment may beapplied to any other embodiment herein, and the descriptions andexamples relating to one embodiment may be combined with any otherembodiment in a suitable manner.

[1205] FIG. 58 is a diagram of an ingestible device during anexample transit through a gastrointestinal (GI) tract, inaccordance with some embodiments of the disclosure. The ngestibledevice may include any portion of any other ingestible devicediscussed in this disclosure, and may be any suitable type ofingestible device with localization capabilities. For example, theingestible device may be without an optional opening for samplingor optional rotation assembly for sampling. In some embodiments,the ingestible device may be ingested by a subject, and as theingestible device traverses the GI tract, the ingestible devicedetermines its location within the GI tract. For example, themovement of the ingestible device and the amount of light detectedby the ingestible device (e.g., via a detector as describedelsewhere herein) may vary substantially depending on the locationof the ingestible device within the GI tract, and the ingestibledevice may be configured to use this information to determine alocation of the ingestible device within the GI tract. Forinstance, the ingestible device may detect ambient light from thesurrounding environment, or reflectances based on illuminationgenerated by the ingestible device (e.g., generated by anilluminator as described elsewhere herein), and use thisinformation to determine a location of the ingestible devicethrough processes, such as described herein. The current locationof the ingestible device, and the time that the ingestible devicedetected each transition between the various portions of the GItract, may then be stored by the ingestible device (e.g., in memorycircuitry of a PCB as described elsewhere herein), and may be usedfor any suitable purpose.

[1206] Shortly after the ingestible device is ingested, theingestible device will traverse the esophagus 65302, which mayconnect the subject's mouth to a stomach 65306. In someembodiments, the ingestible device may be configured to determinethat it has entered the esophagus portion GI tract by measuring theamount and type of light (e.g., via a detector as describedelsewhere herein) in the environment surrounding the the ingestibledevice. For instance, the ingestible device may detect higherlevels of light in the visible spectrum (e.g., via a detector asdescribed elsewhere herein) while outside the subject's body, ascompared to the levels of light detected while within the GI tract.In some embodiments, the ingestible device may have previouslystored data (e.g., on memory circuitry of a PCB as describedelsewhere herein) indicating a typical level of light detected whenoutside of the body, and the the ingestible device may beconfigured to determine that entry to the body has occurred when adetected level of light (e.g., detected via a detector as describedelsewhere herein) has been reduced beyond a threshold level (e.g.,at least a 20-30% reduction) for a sufficient period of time (e.g.,5.0 seconds).

[1207] In some embodiments, the ingestible device may be configuredto detect a transition from esophagus 65302 to stomach 65306 bypassing through sphincter 65304. In some embodiments, ingestibledevice 65300 may be configured to determine whether it has enteredstomach 65306 based at least in part on a plurality of parameters,such as but not limited to the use of light or temperaturemeasurements (e.g., via a detector as described elsewhere herein orvia a thermometer within the ingestible device), pH measurements(e.g., via a pH meter within the ingestible device), timemeasurements (e.g., as detected through the use of clock circuitryincluded within a PCB as described elsewhere herein), or any othersuitable information. For instance, the ingestible device may beconfigured to determine that the ingestible device has enteredstomach 65306 after detecting that a measured temperature of theingestible device exceeds 31 degrees Celsius. Additionally, oralternately, the ingestible device may be configured toautomatically determine it has entered stomach 65306 after oneminute (or another pre-set time duration parameter, 80 seconds, 90seconds, etc.) has elapsed from the time that the ingestible devicewas ingested, or one minute (or another pre-set time durationparameter, 80 seconds, 90 seconds, etc.) from the time that theingestible device detected that it has entered the GI tract.

[1208] Stomach 65306 is a relatively large, open, and cavernousorgan, and therefore the ingestible device may have a relativelylarge range of motion. By comparison, the motion of the ingestibledevice is relatively restricted within the tube-like structure ofthe duodenum 65310, the jejunum 65314, and the ileum (not shown),all of which collectively form the small intestine. Additionally,the interior of stomach 65306 has distinct optical properties fromduodenum 65310 and jejunum 65314, which may enable the ingestibledevice to detect a transition from stomach 65306 to duodenum 65310through the appropriate use of measured reflectances (e.g., throughthe use of reflectances measured by a detector as describedelsewhere herein), as used in conjunction with a process65600).

[1209] In some embodiments, the ingestible device may be configuredto detect a pyloric transition from stomach 65306 to duodenum 65310through the pylorus 65308. For instance, in some embodiments, theingestible device may be configured to periodically generateillumination in the green and blue wavelengths (e.g., via anilluminator as described elsewhere herein), and measure theresulting reflectances (e.g., via a detector as described elsewhereherein). The ingestible device may be configured to then use aratio of the detected green reflectance to the detected bluereflectance to determine whether the ingestible device is locatedwithin the stomach 65306, or duodenum 65310 (e.g., via process65600). In turn, this may enable the ingestible device to detect apyloric transition from stomach 65306 to duodenum 65310, an exampleof which is discussed in relation to FIG. 61.

[1210] Similarly, in some embodiments, the ingestible device may beconfigured to detect a reverse pyloric transition from duodenum65310 to stomach 65306. The ingestible device will typicallytransition naturally from stomach 65306 to duodenum 65310, andonward to jejunum 65314 and the remainder of the GI tract. However,similar to other ingested substances, the ingestible device mayoccasionally transition from duodenum 65310 back to stomach 65306as a result of motion of the subject, or due to the naturalbehavior of the organs with the GI tract. To accommodate thispossibility, the ingestible device may be configured to continue toperiodically generate illumination in the green and bluewavelengths (e.g., via an illuminator as described elsewhereherein), and measure the resulting reflectances (e.g., via adetector as described elsewhere herein) to detect whether or notthe ingestible device has returned to stomach 65306. An exemplarydetection process is described in additional detail in relation toFIG. 61.

[1211] After entering duodenum 65310, the ingestible device may beconfigured to detect a transition to the jejunum 65314 through theduodenojejunal flexure 65312. For example, the ingestible devicemay be configured to use reflectances to detect peristaltic waveswithin the jejunum 65314, caused by the contraction of the smoothmuscle tissue lining the walls of the jejunum 65314. In particular,the ingestible device may be configured to begin periodicallytransmitting illumination (and measuring the resulting reflectances(e.g., via a detector and an illuminator of a sensing sub-unit asdescribed elsewhere herein) at a sufficiently high frequency inorder to detect muscle contractions within the jejunum 65314. Theingestible device may then determine that it has entered thejejunum 65314 in response to having detected either a first musclecontraction, or a predetermined number of muscle contractions(e.g., after having detected three muscle contractions insequence). The interaction of the ingestible device with the wallsof jejunum 65314 is also discussed in relation to FIG. 59, and anexample of this detection process is described in additional detailin relation to FIG. 64.

[1212] FIG. 59 is a diagram of an ingestible device during anexample transit through a jejunum, in accordance with someembodiments of the disclosure. Diagrams 65410, 65420, 65430, and65440 depict ingestible device 65400 as it traverses through ajejunum (e.g., jejunum 65314), and how ingestible device 65400interacts with peristaltic waves formed by walls 65406A and 65406B(collectively, walls 65406) of the jejunum. In someimplementations, ingestible device 65400 may include any portion ofany other ingestible device discussed in this disclosure, and maybe any suitable type of ingestible device with localizationcapabilities.

[1213] Diagram 65410 depicts ingestible device 65400 within thejejunum, when the walls 65406 of the jejunum are relaxed. In someembodiments, the confined tube-like structure of the jejunumnaturally causes ingestible device 65400 to be orientedlongitudinally along the length of the jejunum, with window 65404facing walls 65406. In this orientation, ingestible device 65400may use sensing sub-unit 65402 to generate illumination (e.g., viaan illuminator as described elsewhere herein) oriented towardswalls 65406, and to detect the resulting reflectances (e.g., via adetector as described elsewhere herein) from the portion of theillumination reflected off of walls 65406 and back through window65404. In some embodiments, ingestible device 65400 may beconfigured to use sensing sub-unit 65402 to generate illuminationand measure the resulting reflectance with sufficient frequency todetect peristaltic waves within the jejunum. For instance, in ahealthy human subject, peristaltic waves may occur at a rate ofapproximately 0.05 Hz to 0.33 Hz. Therefore, the ingestible device65400 may be configured to generate illumination and measure theresulting reflectance at least once every 2.5 seconds (i.e.,potentially minimum rate to detect a 0.2 Hz signal), and preferablyat a higher rate, such as once every 0.5 seconds, which may improvethe overall reliability of the detection process due to more datapoints being available. It is understood that the ingestible device65400 need not gather measurements at precise intervals, and insome embodiments the ingestible device 65400 may be adapted toanalyze data gathered at more irregular intervals, provided thatthere are still a sufficient number of appropriately spaced datapoints to detect 0.05 Hz to 0.33 Hz signals.

[1214] Diagram 65420 depicts ingestible device 65400 within thejejunum, when the walls 65406 of the jejunum begin to contract andform a peristaltic wave. Diagram 65420 depicts contracting portion65408A of wall 65406A and contracting portion 65408B of wall 65406B(collectively, contracting portion 65408 of wall 65406) that form aperistaltic wave within the jejunum. The peristaltic wave proceedsalong the length of the jejunum as different portions of wall 65406contract and relax, causing it to appear as if contracting portions65408 of wall 65406 proceed along the length of the jejunum (i.e.,as depicted by contracting portions 65408 proceeding from left toright in diagrams 65410-65430). While in this position, ingestibledevice 65400 may detect a similar level of reflectance (e.g.,through the use of an illuminator and a detector of a sensingsub-unit as described elsewhere herein) as detected when there isno peristaltic wave occurring (e.g., as detected when ingestibledevice 65400 is in the position indicated in diagram 65410).

[1215] Diagram 65430 depicts ingestible device 65400 within thejejunum, when the walls 65406 of the jejunum continue to contract,squeezing around ingestible device 65400. As the peristaltic waveproceeds along the length of the jejunum, contracting portions65408 of wall 65406 may squeeze tightly around ingestible device65400, bringing the inner surface of wall 65406 into contact withwindow 65404. While in this position, ingestible device 65400 maydetect a change in a reflectance detected as a result ofillumination produced by sensing sub-unit 65402. The absolute valueof the change in the measured reflectance may depend on severalfactors, such as the optical properties of the window 65404, thespectral components of the illumination, and the optical propertiesof the walls 65406. However, ingestible device 65400 may beconfigured to store a data set with the reflectance values overtime, and search for periodic changes in the data set consistentwith the frequency of the peristaltic waves (e.g., by analyzing thedata set in the frequency domain, and searching for peaks between0.05 Hz to 0.33 Hz). This may enable ingestible device 65400 todetect muscle contractions due to peristaltic waves withoutforeknowledge of the exact changes in reflectance signal amplitudethat may occur as a result of detecting the muscle contractions ofthe peristaltic wave. An example procedure for detecting musclecontractions is discussed further in relation to FIG. 64, and anexample of a reflectance data set gathered while ingestible device65400 is located within the jejunum is discussed in relation toFIG. 65.

[1216] Diagram 65440 depicts ingestible device 65400 within thejejunum, when the peristaltic wave has moved past ingestible device65400. Diagram 65440 depicts contracting portions 65408 that formthe peristaltic wave within the jejunum having moved past the endof ingestible device 65400. The peristaltic wave proceeds along thelength of the jejunum as different portions of wall 65406 contractand relax, causing it to appear as if contracting portions 65408 ofwall 65406 proceed along the length of the jejunum (i.e., asdepicted by contracting portions 65408 proceeding from left toright in diagrams 65410-65430). While in this position, ingestibledevice 65400 may detect a similar level of reflectance (e.g.,through the use of an illuminator and a detector of a sensingsub-unit as described elsewhere herein) as detected when there isno peristaltic wave occurring (e.g., as detected when ingestibledevice 65400 is in the position indicated in diagram 65410, ordiagram 65420).

[1217] Depending on the species of the subject, peristaltic wavesmay occur with relatively predictable regularity. After theperistaltic wave has passed over ingestible device 65400 (e.g., asdepicted in diagram 65440), the walls 65406 of the jejunum mayrelax again (e.g., as depicted in diagram 65410), until the nextperistaltic wave begins to form. In some embodiments, ingestibledevice 65400 may be configured to continue to gather reflectancevalue data while it is within the GI tract, and may store a dataset with the reflectance values over time. This may allowingestible device 65400 to detect each of the muscle contractionsas the peristaltic wave passes over ingestible device 65400 (e.g.,as depicted in diagram 65430), and may enable ingestible device65400 to both count the number of muscle contractions that occur,and to determine that a current location of the ingestible device65400 is within the jejunum. For example, ingestible device 65400may be configured to monitor for possible muscle contractions whileis inside either the stomach or the duodenum, and may determinethat ingestible device 65400 has moved to the jejunum in responseto detecting a muscle contraction consistent with a peristalticwave.

[1218] FIG. 60 is a flowchart illustrating some aspects of alocalization process used by the ingestible device. In general, theprocess described in FIG. 60 can be used with any ingestible devicedisclosed herein. Furthermore, the features of FIG. 60 may becombined with any other systems, methods or processes described inthis application. For example, portions of the process in FIG. 60may be integrated into or combined with the pyloric transitiondetection procedure described by FIG. 61, or the jejunum detectionprocess described by FIG. 64.

[1219] At 65502, the ingestible device gathers measurements (e.g.,through a detector as described elsewhere herein) of ambient light.For example, the ingestible device may be configured toperiodically measure (e.g., through a detector as describedelsewhere herein) the level of ambient light in the environmentsurrounding the ingestible device. In some embodiments, the type ofambient light being measured may depend on the configuration of thedetector within the ingestible device. For example, if the detectoris configured to measure red, green, and blue wavelengths of light,the ingestible device may be configured to measure the ambientamount of red, green, and blue light from the surroundingenvironment. In some embodiments, the amount of ambient lightmeasured by the ingestible device will be larger in the areaexternal to the body (e.g., a well-lit room where the ingestibledevice is being administered to a subject) and in the oral cavityof the subject, as compared to the ambient level of light measuredby the ingestible device when inside of an esophagus, stomach, orother portion of the GI tract (e.g., esophagus, stomach, duodenum,or jejunum).

[1220] At 65504, the ingestible device determines (e.g., viacontrol circuitry within a PCB as described elsewhere herein)whether the ingestible device has detected entry into the GI tract.For example, the ingestible device may be configured to determinewhen the most recent measurement of ambient light (e.g., themeasurement gathered at 65502) indicates that the ingestible devicehas entered the GI tract. For instance, the first time that theingestible device gatherers a measurement of ambient light at65502, the ingestible device may store that measurement (e.g., viastorage circuitry within a PCB) as a typical level of ambient lightexternal to the body. The ingestible device may be configured tothen compare the most recent measurement of ambient light to thetypical level of ambient light external to the body (e.g., viacontrol circuitry within a PCB as described elsewhere herein), anddetermine that the ingestible device has entered the GI tract whenthe most recent measurement of ambient light is substantiallysmaller than the typical level of ambient light external to thebody. For example, the ingestible device may be configured todetect that it has entered the GI tract in response to determiningthat the most recent measurement of ambient light is less than orequal to 20% of the typical level of ambient light external to thebody. If the ingestible device determines that it has detectedentry into the GI tract (e.g., that the ingestible device hasentered at least the esophagus), process 65500 proceeds to 65506.Alternately, if the ingestible device determines that it has notdetected entry into the GI tract (e.g., as a result of the mostrecent measurement being similar to the typical level of ambientlight external to the body), process 65500 proceeds back to 65502where the ingestible device gathers further measurements. Forinstance, the ingestible device may be configured to wait apredetermined amount of time (e.g., five seconds, ten seconds,etc.), and then gather another measurement of the level of ambientlight from the environment surrounding the ingestible device.

[1221] At 65506, the ingestible device waits for a transition fromthe esophagus to the stomach (e.g., from the esophagus to thestomach). For example, the ingestible device may be configured todetermine that it has entered the stomach (e.g., the stomach) afterwaiting a predetermined period of time after having entered the GItract. For instance, a typical esophageal transit time in a humanpatient may be on the order of 15-30 seconds. In this case, afterhaving detected that the ingestible device has entered the GI tractat 65504 (i.e., after detecting that the ingestible device hasreached at least the esophagus), the ingestible device may beconfigured to wait one minute, or a similar amount of time longerthan the typical esophageal transmit time (e.g., ninety-seconds),before automatically determining that the ingestible device hasentered at least the stomach (e.g., the stomach).

[1222] In some embodiments, the ingestible device may alsodetermine whether it has entered the stomach based on measurementsof pH or temperature. For example, the ingestible device may beconfigured to determine that it has entered the stomach if atemperature of ingestible device has increased to at least 31degrees Celsius (i.e., consistent with the temperature inside thestomach), or if a measured pH of the environment surrounding theingestible device is sufficiently acidic (i.e., consistent with theacidic nature of gastric juices that may be found inside thestomach).

[1223] At 65508, the ingestible device (stores data indicating theingestible device has entered the stomach (e.g., the stomach). Forexample, after having waited a sufficient amount of time at 65506,the ingestible device may store data (e.g., within storagecircuitry of a PCB 65120 as described elsewhere herein) indicativeof the ingestible device having entered at least the stomach. Oncethe ingestible device reaches at least the stomach, process 65500proceeds to 65510 where the ingestible device may be configured togather data to detect entry into the duodenum (e.g., theduodenum).

[1224] In some embodiments, process 65500 may also simultaneouslyproceed from 65508 to 65520, where the ingestible device may beconfigured to gather data in order to detect muscle contractionsand detect entry into the jejunum (e.g., the jejunum). In someembodiments, the ingestible device may be configured tosimultaneously monitor for entry into the duodenum at 65516-65518,as well as detect for entry into the jejunum at 65520-65524. Thismay allow the ingestible device to determine when it has enteredthe jejunum (e.g., as a result of detecting muscle contractions),even when it fails to first detect entry into the duodenum (e.g.,as a result of very quick transit times of the ingestible devicethrough the duodenum).

[1225] At 65510, the ingestible device gathers measurements ofgreen and blue reflectance levels (e.g., through the use of anilluminator and a detector of a sensing sub-unit as describedelsewhere herein) while in the stomach. For example, the ingestibledevice may be configured to periodically gather measurements ofgreen and blue reflectance levels while in the stomach. Forinstance, the ingestible device may be configured to transmit agreen illumination and a blue illumination (e.g., via anilluminator as described elsewhere herein) every five to fifteenseconds, and measure the resulting reflectance (e.g., via adetector as described elsewhere herein). Every time that theingestible device gathers a new set of measurements, themeasurements may be added to a stored data set (e.g., stored withinmemory circuitry of a PCB as described elsewhere herein). Theingestible device may then use this data set to determine whetheror not the ingestible device is still within a stomach or aduodenum.

[1226] In some embodiments, the ingestible device may be configuredto detect a first reflectance based on generating an illuminationof a first wavelength in approximately the green spectrum of light(between 495-600 nm), and detecting a second reflectance based ongenerating an illumination of the second wavelength inapproximately the blue spectrum of light (between 400-495 nm). Insome embodiments, the ingestible device may ensure that theillumination in the green spectrum and the illumination in the bluespectrum have wavelengths separated by at least 50 nm. This mayenable the ingestible device to sufficiently distinguish betweenthe two wavelengths when detecting the reflectances (e.g., via adetector as described elsewhere herein). It is understood that theseparation of 50 nm is intended to be illustrative, and notlimiting, and depending on the accuracy of the detectors within theingestible device, smaller separations may be possible to beused.

[1227] At 65512, the ingestible device determines (e.g., usingcontrol circuitry within a PCB as described elsewhere herein)whether the ingestible device has detected a transition from thestomach to a duodenum based on a ratio of green and blue (G/B)reflectance levels. For example, the ingestible device may obtain(e.g., from memory circuitry of a PCB as described elsewhereherein) a data set containing historical data for the respectiveratio of the green reflectance to the blue reflectance as measuredat a respective time. Generally speaking, a duodenum of a humansubject reflects a higher ratio of green light to blue light, ascompared to the ratio of green light to blue light that isreflected by a stomach. Based on this, the ingestible device may beconfigured to take a first set of ratios from the data set,representing the result of recent measurements, and compare them toa second set of ratios from the data set, representing the resultsof past measurements. When the the ingestible device determinesthat the mean value of the first set of ratios is substantiallylarger than the mean value of the second set of ratios (i.e., thatthe ratio of reflected green light to reflected blue light hasincreased), the ingestible device may determine that it has enteredthe duodenum (from the stomach. If the ingestible device detects atransition from the stomach to a duodenum (process 65500 proceedsto 65514, where the ingestible device stores data indicating thatthe ingestible device has entered the duodenum. Alternatively, ifthe ingestible device determines that the ingestible device has nottransitioned from the stomach to the duodenum, process 65500proceeds back to 65510 to gather more measurements of green andblue reflectance levels while still in the stomach. An exampleprocedure for using measurements of green and blue reflectances tomonitor for transitions between the stomach and the duodenum isdiscussed in greater detail in relation to FIG. 61.

[1228] In some embodiments, the first time that detects atransition from the stomach to the duodenum, the ingestible devicemay be configured to take a mean of the second set of data, (e.g.,the set of data previously recorded while in the stomach) and storethis as a typical ratio of green light to blue light detectedwithin the stomach (e.g., the stomach) (e.g., within memorycircuitry of a PCB 65120 (FIG. 57) as described elsewhere herein).This stored information may later be used by the ingestible deviceto determine when the ingestible device re-enters the stomach fromthe duodenum as a result of a reverse pyloric transition.

[1229] At 65514, the ingestible device stores data indicating thatthe ingestible device has entered the duodenum. For example, theingestible device may store a flag within local memory (e.g.,memory circuitry of a PCB as described elsewhere herein) indicatingthat the the ingestible device is currently in the duodenum. Insome embodiments, the ingestible device may also store a timestampindicating the time when the ingestible device entered theduodenum. Once the ingestible device reaches the duodenum, process65500 proceeds to 65520 where the ingestible device may beconfigured to gather data in order to detect muscle contractionsand detect entry into the jejunum. Process 65500 also proceeds from65514 to 65516, where the ingestible device may be configured togather data additional data in order to detect re-entry into thestomach from the duodenum.

[1230] At 65516, the ingestible device gathers measurements (e.g.,via a sensing sub-unit as described elsewhere herein) of green andblue reflectance levels while in the duodenum. For example, theingestible device may be configured to periodically gathermeasurements (e.g., via a sensing sub-unit as described elsewhereherein) of green and blue reflectance levels while in the duodenum,similar to the measurements made at 65510 while in the stomach. Forinstance, the ingestible device may be configured to transmit agreen illumination and a blue illumination (e.g., via anilluminator as described elsewhere herein) every five to fifteenseconds, and measure the resulting reflectance (e.g., via adetector as described elsewhere herein). Every time that theingestible device gathers a new set of measurements, themeasurements may be added to a stored data set (e.g., stored withinmemory circuitry of a PCB). The the ingestible device may then usethis data set to determine whether or the ingestible device isstill within the duodenum, or if the ingestible device hastransitioned back into the stomach).

[1231] At 65518, the ingestible device determines a transition fromthe duodenum to the stomach based on a ratio of the measured greenreflectance levels to the measured blue reflectance levels. In someembodiments, the ingestible device may compare the ratio of themeasured green reflectance levels to the measured blue reflectancelevels recently gathered by the ingestible device (e.g.,measurements gathered at 65516), and determine whether or not theratio of the measured green reflectance levels to the measured bluereflectance levels is similar to the average ratio of the measuredgreen reflectance levels to the measured blue reflectance levelsseen in the stomach. For instance, the ingestible device mayretrieve data (e.g., from memory circuitry of a PCB (FIG. 57))indicative of the average ratio of the measured green reflectancelevels to the measured blue reflectance levels seen in the stomach,and determine that the ingestible device has transitioned back tothe stomach if the recently measured ratio of the measured greenreflectance levels to the measured blue reflectance levels issufficiently similar to the average level in the stomach (e.g.,within 20% of the average ratio of the measured green reflectancelevels to the measured blue reflectance levels seen in the stomach,or within any other suitable threshold level). If the ingestibledevice detects a transition from the duodenum to the stomach,process 65500 proceeds to 65508 to store data indicating theingestible device has entered the stomach, and continues to monitorfor further transitions. Alternatively, if the ingestible devicedoes not detect a transition from the duodenum to the stomach,process 65500 proceeds to 65516 to gather additional measurementsof green and blue reflectance levels while in the duodenum, whichmay be used to continuously monitor for possible transitions backinto the stomach. An example procedure for using measurements ofgreen and blue reflectances to monitor for transitions between thestomach and the duodenum is discussed in greater detail in relationto FIG. 61.

[1232] At 65520, the ingestible device gathers periodicmeasurements of the reflectance levels (e.g., via a sensingsub-unit) while in the duodenum. In some embodiments, theingestible device may gather similar periodic measurements while inthe stomach as well. In some embodiments, these periodicmeasurements may enable the ingestible device to detect musclecontractions (e.g., muscle contractions due to a peristaltic waveas discussed in relation to FIG. 59), which may be indicative ofentry into a jejunum. The ingestible device may be configured togather periodic measurements using any suitable wavelength ofillumination (e.g., by generating illumination using an illuminatorand detecting the resulting reflectance using a detector), orcombinations of wavelengths of illumination. For example, in someembodiments, the ingestible device may be configured to generatered, green, and blue illumination, store separate data setsindicative of red, green, and blue illumination, and analyze eachof the data sets separately to search for frequency components inthe recorded data indicative of detected muscle contractions. Insome embodiments, the measurements gathered by the ingestibledevice at 65520 may be sufficiently fast as to detect peristalticwaves in a subject. For instance, in a healthy human subject,peristaltic waves may occur at a rate of approximately 0.05 Hz to0.33 Hz. Therefore, the ingestible device may be configured togenerate illumination and measure the resulting reflectance atleast once every 2.5 seconds (i.e., potentially minimum rate todetect a 0.2 Hz signal), and preferably at a higher rate, such asonce every 0.5 seconds or faster, and store values indicative ofthe resulting reflectances in a data set (e.g., within memorycircuitry of a PCB). After gathering additional data (e.g., aftergathering one new data point, or a predetermined number of new datapoints), process 65500 proceeds to 65522, where the ingestibledevice determines whether or not a muscle contraction has beendetected.

[1233] At 65522, the ingestible device determines (e.g., viacontrol circuitry within a PCB) whether the ingestible devicedetects a muscle contraction based on the measurements ofreflectance levels (e.g., as gathered by a sensing sub-unit). Forexample, the ingestible device may obtain a fixed amount of datastored as a result of measurements made at 65520 (e.g., retrievethe past minute of data from memory circuitry within a PCB. Theingestible device may then convert the obtained data into thefrequency domain, and search for peaks in a frequency range thatwould be consistent with peristaltic waves. For example, in ahealthy human subject, peristaltic waves may occur at a rate ofapproximately 0.05 Hz to 0.33 Hz, and the ingestible device may beconfigured to search for peaks in the frequency domainrepresentation of the data between 0.05 Hz to 0.33 Hz above athreshold value. If the ingestible device detects a contractionbased on the reflectance levels (e.g., based on detecting peaks inthe frequency domain representation of the data between 0.05 Hz to0.33 Hz), process 65500 proceeds to 65524 to store data indicatingthat the device has entered the jejunum. Alternatively, if theingestible device does not detect a muscle contraction, process65500 proceeds to 65520 to gather periodic measurements of thereflectance levels while in the duodenum. In some embodiments, theingestible device may store data (e.g., within memory circuitry ofa PCB) indicating that a muscle contraction was detected, andprocess 65500 will not proceed from 65522 to 65524 until asufficient number of muscle contractions have been detected.

[1234] At 65524, the ingestible device stores data (e.g., withinmemory circuitry of a PCB) indicating that the device has enteredthe jejunum). For example, in response to detecting that musclecontraction has occurred at 65522, the ingestible device maydetermine that it has entered the jejunum 65314, and is no longerinside of the duodenum or the stomach. In some embodiments, theingestible device may continue to measure muscle contractions whilein the jejunum, and may store data indicative of the frequency,number, or strength of the muscle contractions over time (e.g.,within memory circuitry of a PCB). In some embodiments, theingestible device may also be configured to monitor for one or moretransitions. Such transitions can include a transition from thejejunum to the ileum, an ileoceacal transition from the ileum tothe cecum, a transition from the cecum to the colon, or detect exitfrom the body (e.g., by measuring reflectances, temperature, orlevels of ambient light).

[1235] In some embodiments, the ingestible device may alsodetermine that it has entered the jejunum after a pre-determinedamount of time has passed after having detected entry into theduodenum. For example, barring a reverse pyloric transition fromthe duodenum back to the stomach, the typical transit time for aningestible device to reach the jejunum from the duodenum in ahealthy human subject is less than three minutes. In someembodiments, the ingestible device may therefore be configured toautomatically determine that it has entered the jejunum afterspending at least three minutes within the duodenum. Thisdetermination may be made separately from the determination madebased on measured muscle contractions (e.g., the determination madeat 65522), and in some embodiments, the ingestible device maydetermine that it has entered the jejunum in response to eitherdetecting muscle contractions, or after three minutes has elapsedfrom having entered the duodenum (e.g., as determined by storingdata at 65514 indicative of the time that ingestible device enteredthe duodenum).

[1236] For illustrative purposes, 65512-65518 of process 65500describe the ingestible device measuring green reflectances andblue reflectances, calculating a ratio of the two reflectances, andusing this information to determine when the ingestible device hastransitioned between the duodenum and stomach. However, in someembodiments, other wavelengths of light may be used other thangreen and blue, provided that the wavelengths of light chosen havedifferent reflective properties within the stomach and the duodenum(e.g., as a result of different reflection coefficients of thestomach tissue and the tissue of the duodenum).

[1237] It will be understood that the steps and descriptions of theflowcharts of this disclosure, including FIG. 60, are merelyillustrative. Any of the steps and descriptions of the flowcharts,including FIG. 60, may be modified, omitted, rearranged, andperformed in alternate orders or in parallel, two or more of thesteps may be combined, or any additional steps may be added,without departing from the scope of the present disclosure. Forexample, the ingestible device may calculate the mean and thestandard deviation of multiple data sets in parallel in order tospeed up the overall computation time. As another example, theingestible device may gather data periodic measurements and detectpossible muscle contractions (e.g., at 65520-65522) whilesimultaneously gathering green and blue reflectance levels todetermine transitions to and from the stomach and duodenum (e.g.,at 65510-65518). Furthermore, it should be noted that the steps anddescriptions of FIG. 60 may be combined with any other system,device, or method described in this application, includingprocesses 65600 and 65900, and any of the ingestible devices orsystems discussed in this application could be used to perform oneor more of the steps in FIG. 60.

[1238] FIG. 61 is a flowchart illustrating some aspects of aprocess for detecting transitions from a stomach to a duodenum andfrom a duodenum back to a stomach, which may be used whendetermining a location of an ingestible device as it transitsthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure. In some embodiments, process 65600may begin when an ingestible device first detects that it hasentered the stomach, and will continue as long as the ingestibledevice determines that it is within the stomach or the duodenum. Insome embodiments, process 65600 may only be terminated when aningestible device determines that it has entered the jejunum, orotherwise progressed past the duodenum and the stomach. Theduodenum detection process 65600 described in FIG. 61 may beapplied to any device discussed in this application, and any of theingestible devices may be used to perform one or more parts of theprocess described in FIG. 61. Furthermore, the features of FIG. 61may be combined with any other systems, methods or processesdescribed in this application. For example, portions of the processdescribed by the process in FIG. 61 may be integrated into process65500 discussed in relation to FIG. 60.

[1239] At 65602, the ingestible device retrieves a data set (e.g.,from memory circuitry within a PCB) with ratios of the measuredgreen reflectance levels to the measured blue reflectance levelsover time. For example, the ingestible device may retrieve a dataset from a PCB containing recently recorded ratios of the measuredgreen reflectance levels to the measured blue reflectance levels(e.g., as recorded at 65510 or 65516 of process 65500). In someembodiments, the retrieved data set may include the ratios of themeasured green reflectance levels to the measured blue reflectancelevels over time. Example plots of data sets of ratios of themeasured green reflectance levels to the measured blue reflectancelevels are discussed further in relation to FIG. 62 and FIG.63.

[1240] At 65604, the ingestible device includes a new measurement(e.g., as made with a sensing sub-unit) of a ratio of the measuredgreen reflectance level to the measured blue reflectance level inthe data set. For example, the ingestible device may be configuredto occasionally record new data by transmitting green and blueillumination (e.g., via an illuminator), detecting the amount ofreflectance received due to the green and blue illumination (e.g.,via a detector), and storing data indicative of the amount of thereceived reflectance (e.g., in memory circuitry of a PCB). Theingestible device may be configured to record new data every fiveto fifteen seconds, or at any other convenient interval of time.For illustrative purposes, the ingestible device is described asstoring and retrieving the ratio of the measured green reflectancelevels to the measured blue reflectance levels (e.g., if the amountof detected green reflectance was identical to the amount ofdetected blue reflectance at a given time, the ratio of the greenand blue reflectances would be "1.0" at that given time); however,it is understood that the green reflectance data and the bluereflectance data may be stored separately within the memory of theingestible device (e.g., stored as two separate data sets withinmemory circuitry of a PCB).

[1241] At 65606, the ingestible device retrieves a first subset ofrecent data by applying a first sliding window filter to the dataset. For example, the ingestible device may use a sliding windowfilter to obtain a predetermined amount of the most recent datawithin the data set, which may include any new values of the ratioof the measured green reflectance level to the measured bluereflectance level obtained at 65604. For instance, the ingestibledevice may be configured to select between ten and forty datapoints from the data set, or the ingestible device may beconfigured to select a predetermined range of data values betweenfifteen seconds of data and five minutes of data. In someembodiments, other ranges of data may be selected, depending on howfrequently measurements are recorded, and the particularapplication at hand. For instance, any suitable amount of data maybe selected in the sliding window, provided that it is sufficientto detect statistically significant differences between the dataselected in a second sliding window (e.g., the second subset ofdata selected at 65614).

[1242] In some embodiments, the ingestible device may also beconfigured to remove outliers from the data set, or to smooth outunwanted noise in the data set. For example, the ingestible devicemay select the first subset of data, or any other subset of data,by first obtaining a raw set of values by applying a window filterto the data set (e.g., selecting a particular range of data to beincluded). The ingestible device may then be configured to identifyoutliers in the raw set of values; for instance, by identifyingdata points that are over three standard deviations away from themean value of the raw set of values, or any other suitablethreshold. The ingestible device may then determine the subset ofdata by removing outliers from the raw set of values. This mayenable the ingestible device to avoid spurious information whendetermining whether or not it is located within the stomach or theduodenum.

[1243] At 65608, the ingestible device determines whether the mostrecently detected location was the duodenum. In some embodiments,the ingestible device may store a data flag (e.g., within memorycircuitry of a PCB 65120 (FIG. 57)) indicating the most recentportion of the GI tract that the ingestible device detected itselfto be within. For instance, every time the ingestible devicedetects entry to the stomach (e.g., detects entry into stomach65306 as a result of the decision made at 65610), a flag is storedin memory indicating the ingestible device is in the stomach (e.g.,as part of storing data at 65612). If the ingestible devicesubsequently detects entry into the duodenum (e.g., detects entryinto the duodenum 65310 as a result of a decision made at 65624),another different flag is stored in memory indicating that theingestible device is in the duodenum (e.g., as part of storing dataat 65624). In this case, the ingestible device may retrieve themost recently stored flag at 65608, and determine whether or notthe flag indicates that the ingestible device was most recentlywithin the duodenum. If the ingestible device detects that it wasmost recently in the duodenum, process 65600 proceeds to 65610where the ingestible device compares the recent measurements of theratios of the measured green reflectance levels to the measuredblue reflectance levels (e.g., measurements that include the recentmeasurement made at 65606) to the typical ratios measured withinthe stomach, and uses this information to determine whether areverse pyloric transition from the duodenum back to the stomachhas occurred. Alternately, if the ingestible device detects that itwas not most recently in the duodenum (e.g., because it was in thestomach instead), process 65600 proceeds to 65614 where theingestible device compares the recent measurements of the ratios ofthe measured green reflectance levels to the measured bluereflectance levels (e.g., measurements that include the recentmeasurement made at 65606) to past measurements, and uses thisinformation to determine whether a pyloric transition from thestomach to the duodenum has occurred.

[1244] Process 65600 proceeds from 65608 to 65610 when theingestible device determined that it was most recently in theduodenum. At 65610, the ingestible device determines (e.g., viacontrol circuitry within a PCB) whether the current G/B signal issimilar to a recorded average G/B signal in the stomach. Forexample, the ingestible device may be configured to have previouslystored data (e.g., within memory circuitry of a PCB 65120 (FIG.57)) indicative of the average ratio of the measured greenreflectance levels to the measured blue reflectance levels measuredin the stomach. The ingestible device may then retrieve this storeddata indicative of the average ratio of the measured greenreflectance levels to the measured blue reflectance levels in thestomach, and compare this against the recent measurements in orderto determine whether or not the ingestible device has returned backto the stomach from the duodenum. For instance, the ingestibledevice may determine if the mean value of the first subset ofrecent data (i.e., the average value of the recently measuredratios of the measured green reflectance levels to the measuredblue reflectance levels) is less than the average ratio of themeasured green reflectance levels to the measured blue reflectancelevels within the stomach, or less that the average ratio measuredwithin the stomach plus a predetermined number times the standarddeviation of the ratios measured within the stomach. For instance,if the average ratio of the measured green reflectance levels tothe measured blue reflectance levels in the stomach was "1," with astandard deviation of "0.2," ingestible device may determinewhether or not the mean value of the first subset of data is lessthan "1.0+k*0.2," where "k" is a number between zero and five. Itis understood that, in some embodiments, the ingestible device maybe configured to use a different threshold level to determinewhether or not the mean value of the first subset of recent data issufficiently similar to the average ratio of the measured greenreflectance levels to the measured blue reflectance levels withinthe stomach. In response to determining that the recent ratio ofthe measured green reflectance levels to the measured bluereflectance levels is similar to the average ratio of measuredgreen and blue reflectance levels seen in the stomach, process65600 proceeds to 65612 where the ingestible device stores dataindicating that it has re-entered the stomach from the duodenum.Alternately, in response to determining that the recent ratio ofmeasured green and blue reflectance levels is sufficientlydifferent from the average ratio of measured green and bluereflectance levels seen in the stomach, the ingestible deviceproceeds directly to 65604, and continues to obtain new data on anongoing basis.

[1245] At 65612, the ingestible device stores data indicating areverse pyloric transition from the duodenum to the stomach wasdetected. For example, the ingestible device may store a data flag(e.g., within memory circuitry of a PCB) indicating that theingestible device most recently detected itself to be within thestomach portion of the GI tract. In some embodiments, theingestible device may also store data (e.g., within memorycircuitry of a PCB) indicating a time that the ingestible devicedetected the reverse pyloric transition from the duodenum to thestomach. This information may be used by the ingestible device at65608, and as a result process 65600 may proceed from 65608 to65614, rather than proceeding from 65618 to 65610. After theingestible device stores the data indicating a reverse pylorictransition from the duodenum to the stomach was detected, process65600 proceeds to 65604 where the ingestible device continues togather additional measurements, and continues to monitor forfurther transitions between the stomach and the duodenum.

[1246] Process 65600 proceeds from 65608 to 65614 when theingestible device determined that it was not most recently in theduodenum (e.g., as a result of having most recently been in thestomach instead). At 65614, the ingestible device retrieves asecond subset of previous data by applying a second sliding windowfilter to the data set. For example, the ingestible device may usea sliding window filter to obtain a predetermined amount of olderdata from a past time range, which may be separated from recenttime range used to select the first subset of data gathered at65606 by a predetermined period of time. In some embodiments, anysuitable amount of data may be selected by the first and secondwindow filters, and the first and second window filters may beseparated by any appropriate predetermined amount of time. Forexample, in some embodiments, the first window filter and thesecond window filter may each be configured to select apredetermined range of data values from the data set, thepredetermined range being between fifteen seconds of data and fiveminutes of data. In some embodiments, the recent measurements andthe past measurements may then be separated by a predeterminedperiod of time that is between one to five times the predeterminedrange of data values. For instance, the ingestible device mayselect the first subset of data and the second subset of data toeach be one minute of data selected from the dataset (i.e.,selected to have a predetermined range of one minute), and thefirst subset of data and the second subset of data are selectedfrom recorded measurements that are at least two minutes apart(i.e., the predetermined period of time is two minutes, which istwice the range used to select the subsets of data using the windowfilters). As another example, the ingestible device may select thefirst subset of data and the second subset of data to each be fiveminutes of data selected from the dataset (i.e., selected to have apredetermined range of five minutes), and the first subset of dataand the second subset of data are selected from recordedmeasurements that are at least 10 minutes apart (i.e., thepredetermined period of time is two minutes, which is twice therange used to select the subsets of data using the windowfilters).

[1247] In some embodiments, if the ingestible device recentlytransitioned to the stomach from the duodenum (e.g., as determinedby checking for recent data stored within the ingestible device at65612), the ingestible device may select the second subset of dataat 65614 from a time frame when the ingestible device is known tobe within the stomach. In some embodiments, the ingestible devicemay alternately select a previously recorded average and standarddeviation for ratios of green reflectances and blue reflectanceswithin the stomach (e.g., an average and standard deviation typicalof data recorded within the stomach, as previously recorded withinmemory circuitry of a PCB at 65620) in place of the second subsetof data. In this case, the ingestible device may simply use thepreviously recorded average and previously recorded standarddeviation when making a determination at 65616, rather thanexpending resources to calculate the mean and standard deviation ofthe second subset.

[1248] At 65616, the ingestible device determines whether thedifference between the mean of the second subset and the mean ofthe first subset is greater than a predetermined multiple of thestandard deviation of the first subset. For example, the ingestibledevice may compute a difference between a mean of the first subsetof recent data and a mean of a second subset of past data, anddetermine whether this difference is greater than three times thestandard deviation of the second subset of past data. In someembodiments, it is understood that any convenient threshold levelmay be used other than three times the standard deviation, such asany value between one and five times the standard deviation. Also,in some embodiments, the ingestible device may instead set thethreshold level based on the standard deviation of the secondsubset instead of the first subset. In response to determining thatthe difference between the mean of the first subset and the mean ofthe second subset is greater than a predetermined multiple of thestandard deviation of the second subset, process 65600 proceeds to65618. Otherwise, process 65600 proceeds back to 65604, where theingestible device 65604 continues to gather new data to be used inmonitoring for transitions between the stomach and theduodenum.

[1249] At 65618, the ingestible device determines (e.g., viacontrol circuitry within a PCB) whether the determination made at65616 is the first time that the difference between the mean of thefirst subset of recent data and the mean of the second subset ofpast data is calculated to be greater than the standard deviationof the second subset. If the ingestible device determines that thisis the first time that the difference between the mean of the firstsubset and the mean of the second subset is calculated to begreater than the standard deviation of the second subset, process65600 proceeds to 65620 to store the mean of the second subset ofpast data as an average G/B signal in the stomach. Alternatively,if the ingestible device determines that the immediately precedingdetermination made at 65616 is not the first time that thedifference between the mean of the first subset of recent data andthe mean of the second subset of past data is calculated to begreater than the standard deviation of the second subset, process65600 proceeds directly to 65622.

[1250] At 65620, the ingestible device stores the mean of thesecond subset as an average G/B signal in the stomach. For example,the ingestible device may be configured to store the mean of thesecond subset of past data (e.g., store within memory circuitry ofa PCB 65120) as the average ratio of the measured green reflectancelevels to the measured blue reflectance levels measured in thestomach. In some embodiments, the ingestible device may also storethe standard deviation of the second subset of past data as atypical standard deviation of the ratios of the measured greenreflectance levels to the measured blue reflectance levels detectedwithin the stomach. This stored information may be used by theingestible device later on (e.g., at 65610) to compare againstfuture data, which may enable the ingestible device to detectreverse pyloric transitions from the duodenum back to the stomach,and may generally be used in place of other experimental datagathered from the stomach (e.g., in place of the second subset ofdata at 65616). After storing the mean of the second subset as anaverage G/B signal in the stomach, process 65600 proceeds to65622.

[1251] At 65622, the ingestible device determines whether adifference of the mean of the first subset of recent data to themean of the second subset of past data is greater than apredetermined threshold, "M". In some embodiments, thepredetermined threshold, "M," will be sufficiently large to ensurethat the mean of the first subset is substantially larger than themean of the second subset, and may enable the ingestible device toensure that it detected an actual transition to the duodenum. Thismay be particularly advantageous when the determination made at65616 is potentially unreliable due to the standard deviation ofthe second subset of past data being abnormally small. For example,a typical value of the predetermined threshold "M," may be on theorder of 0.1 to 0.5. If the ingestible device determines that thedifference of the mean of the first subset of recent data to thesecond subset of past data is greater than a predeterminedthreshold, process 65600 proceeds to 65624 to store data indicatingthat a pyloric transition from the stomach to the duodenum wasdetected. Alternatively, if the ingestible device determines thatthe ratio of the mean of the first subset to the second subset isless than or equal to the predetermined threshold, "M" (i.e.,determines that a transition to the duodenum has not occurred),process 65600 proceeds directly to 65604 where the ingestibledevice continues to make new measurements and monitor for possibletransitions between the stomach and the duodenum.

[1252] In some embodiments, instead of using a difference of themean of the first subset of recent data to the mean of the secondsubset of past data, the ingestible device determines whether theratio of the mean of the first subset of recent data to the mean ofthe second subset of past data is greater than a predeterminedthreshold, "M". In some embodiments, the predetermined threshold,"M," will be sufficiently large to ensure that the mean of thefirst subset is substantially larger than the mean of the secondsubset, and may enable the ingestible device to ensure that itdetected an actual transition to the duodenum. This may beparticularly advantageous when the determination made at 65616 ispotentially unreliable due to the standard deviation of the secondsubset of past data being abnormally small. For example, a typicalvalue of the predetermined threshold "M," may be on the order of1.2 to 2.0. It is understood any convenient type of threshold orcalculation may be used to determine whether or not the firstsubset of data and the second subset of data are both statisticallydistinct from one another, and also substantially different fromone another in terms of overall average value.

[1253] At 65624, the ingestible device stores data indicating apyloric transition from the stomach to the duodenum was detected.For example, the ingestible device may store a data flag (e.g.,within memory circuitry of a PCB) indicating that the ingestibledevice most recently detected itself to be within the duodenumportion of the GI tract. In some embodiments, the ingestible devicemay also store data (e.g., within memory circuitry of a PCB)indicating a time that the ingestible device detected the pylorictransition from the stomach to the duodenum. This information maybe used by the ingestible device at 65608, and as a result process65600 may proceed from 65608 to 65610, rather than proceeding from65618 to 65614. After the ingestible device stores the dataindicating a pyloric transition from the stomach to the duodenumwas detected, process 65600 proceeds to 65604 where the ingestibledevice continues to gather additional measurements, and continuesto monitor for further transitions between the stomach and theduodenum.

[1254] It will be understood that the steps and descriptions of theflowcharts of this disclosure, including FIG. 61, are merelyillustrative. Any of the steps and descriptions of the flowcharts,including FIG. 61, may be modified, omitted, rearranged, andperformed in alternate orders or in parallel, two or more of thesteps may be combined, or any additional steps may be added,without departing from the scope of the present disclosure. Forexample, the ingestible device may calculate the mean and thestandard deviation of multiple data sets in parallel in order tospeed up the overall computation time. Furthermore, it should benoted that the steps and descriptions of FIG. 61 may be combinedwith any other system, device, or method described in thisapplication, and any of the ingestible devices or systems discussedin this application could be used to perform one or more of thesteps in FIG. 61. For example, portions of process 65600 may beincorporated into 65508-65516 of process 65500, and may be part ofa more general process for determining a location of the ingestibledevice. As another example, the ratio of detected blue and greenlight (e.g., as measured and added to the data set at 65604) maycontinue even outside of the stomach or duodenum, and similarinformation may be recorded by the ingestible device throughout itstransit in the GI tract. Example plots of data sets of ratios ofmeasured green and blue reflectance levels, which may be gatheredthroughout the GI tract, are discussed further in relation to FIG.62 and FIG. 62 below.

[1255] FIG. 62 is a plot illustrating data collected during anexample operation of an ingestible device, which may be used whendetermining a location of an ingestible device as it transitsthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure.

[1256] Although FIG. 62 may be described in connection with theingestible device for illustrative purposes, this is not intendedto be limiting, and plot 65700 and data set 65702 may be typical ofdata gathered by any device discussed in this application. Plot65700 depicts the ratios of the measured green reflectance levelsto the measured blue reflectance levels over time. For example, theingestible device may have computed the value for each point in thedata set 65702 by transmitting green and blue illumination at agiven time (e.g., via a illuminator), measuring the resulting greenand blue reflectances (e.g., via a detector), calculating the ratioof the resulting reflectances, and storing the ratio in the dataset along with a timestamp indicating the time that thereflectances were gathered.

[1257] At 65704, shortly after the ingestible device beginsoperation, the ingestible device determines that it has reached atleast the stomach (e.g., as a result of making a determinationsimilar to the determination discussed in relation to 65506 inprocess 65500). The ingestible device continues to gatheradditional measurements of green and blue reflectance levels, andat 65706 the ingestible device determines that a pyloric transitionhas occurred from the stomach to the duodenum (e.g., as a result ofmaking a determination similar to the determinations discussed inrelation to 65616-65624 of process 65600). Notably, the values indata set 65702 around 65706 jump up precipitously, which isindicative of the higher ratios of measured green reflectancelevels to measured blue reflectance levels typical of theduodenum.

[1258] The remainder of the data set 65702 depicts the ratios ofthe measured green reflectance levels to the measured bluereflectance levels throughout the remainder of the GI tract. At65708, the ingestible device has reached the jejunum (e.g., asdetermined through measurements of muscle contractions, asdiscussed in relation to FIG. 64), and by 65710, the ingestibledevice has reached the cecum. It is understood that, in someembodiments, the overall character and appearance of data set 65702changes within the small intestine (i.e., the duodenum, jejunum,and ileum) versus the cecum. Within the jejunum and ileum, theremay typically be a wide variation in the ratios of the measuredgreen reflectance levels to the measured blue reflectance levels,resulting in relatively noisy data with a high standard deviation.By comparison, within the cecum the ingestible device may measure arelatively stable ratio of the measured green reflectance levels tothe measured blue reflectance levels. In some embodiments, theingestible device may be configured to determine transitions fromthe small intestine to the cecum based on these differences. Forexample, the ingestible device may compare recent windows of datato past windows of data, and detect a transition to the cecum inresponse to determining that the standard deviation of the ratiosin the recent window of data is substantially less than thestandard deviation of the ratios in the past window of data.

[1259] FIG. 63 is another plot illustrating data collected duringan example operation of an ingestible device, which may be usedwhen determining a location of an ingestible device as it transitsthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure. Similar to FIG. 62, FIG. 63 may bedescribed in connection with the ingestible device for illustrativepurposes. However, this is not intended to be limiting, and plot65800 and data set 65802 may be typical of data gathered by anydevice discussed in this application.

[1260] At 65804, shortly after the ingestible device beginsoperation, the ingestible device determines that it has reached atleast the stomach (e.g., as a result of making a determinationsimilar to the determination discussed in relation to 65506 inprocess 65500). The ingestible device continues to gatheradditional measurements of green and blue reflectance levels (e.g.,via a sensing sub-unit), and at 65806 the ingestible devicedetermines that a pyloric transition has occurred from the stomachto the duodenum (e.g., as a result of making a determinationsimilar to the determinations discussed in relation to 65616-65624of process 65600). Notably, the values in data set 65802 around65806 jump up precipitously, which is indicative of the higherratios of measured green reflectance levels to measured bluereflectance levels typical of the duodenum, before falling shortlythereafter. As a result of the reduced values in data set 65802,the ingestible device determines that a reverse pyloric transitionhas occurred from the duodenum back to the stomach at 65808 (e.g.,as a result of making a determination similar to the determinationsdiscussed in relation to 65610-65612 of process 65600). At 65810,as a result of the values in data set 65802 increasing again, theingestible device determines that another pyloric transition hasoccurred from the stomach to the duodenum, and shortly thereafterthe ingestible device proceeds onwards to the jejunum, ileum, andcecum.

[1261] The remainder of the data set 65802 depicts the ratios ofthe measured green reflectance levels to the measured bluereflectance levels throughout the remainder of the GI tract.Notably, at 65812, ingestible device reaches the transition pointbetween the ileum and the cecum. As discussed above in relation toFIG. 62, the transition to the cecum is marked by a reducedstandard deviation in the ratios of measured green reflectances andmeasured blue reflectances over time, and the ingestible device maybe configured to detect a transition to the cecum based ondetermining that the standard deviation of a recent set ofmeasurements is substantially smaller than the standard deviationof past measurements taken from the jejunum or ileum.

[1262] FIG. 64 is a flowchart of illustrative steps for detecting atransition from a duodenum to a jejunum, which may be used whendetermining a location of an ingestible device as it transitsthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure. Although FIG. 64 may be described inconnection with the ingestible device for illustrative purposes,this is not intended to be limiting, and either portions or theentirety of process 65900 described in FIG. 64 may be applied toany device discussed in this application, and any of theseingestible devices may be used to perform one or more parts of theprocess described in FIG. 64. Furthermore, the features of FIG. 64may be combined with any other systems, methods or processesdescribed in this application. For example, portions of the processdescribed by the process in FIG. 64 may be integrated into thelocalization process 65500 (e.g., as part of 65520-65524). In someembodiments, the ingestible device may perform process 65900 whilein the duodenum, or in response to detecting entry to the duodenum.In other embodiments, the ingestible device may perform process65900 while in the stomach, or in response to detecting entry intothe GI tract. It is also understood that process 65900 may beperformed in parallel with any other process described in thisdisclosure (e.g., process 65600), which may enable the ingestibledevice to detect entry into various portions of the GI tract,without necessarily detecting entry into a preceding portion of theGI tract.

[1263] For illustrative purposes, FIG. 64 may be discussed in termsof the ingestible device generating and making determinations basedon a single set of reflectance levels generated at a singlewavelength by a single sensing sub-unit (e.g., sensing sub-unit65126). However, it is understood that the ingestible device maygenerate multiple wavelengths of illumination from multipledifferent sensing sub-units positioned around the circumference ofingestible device (e.g., multiple sensing sub-units positioned atdifferent locations behind window 65114 of the ingestible device,and each of the resulting reflectances may be stored as a separatedata set. Moreover, each of these sets of reflectance levels may beused to detect muscle contractions by running multiple versions ofprocess 65900, each one of which processes data for a different setof reflectances corresponding to data sets obtained frommeasurements of different wavelengths or measurements made bydifferent sensing sub-units.

[1264] At 65902, the ingestible device retrieves a set ofreflectance levels. For example, the ingestible device may retrievea data set of previously recorded reflectance levels from memory(e.g., from memory circuitry of a PCB). Each of the reflectancelevels may correspond to reflectances previously detected by theingestible device (e.g., via a detector) from illuminationgenerated by the ingestible device (e.g., via an illuminator), andmay represent a value indicative of an amount of light detected ina given reflectance. However, it is understood that any suitablefrequency of light may be used, such as light in the infrared,visible, or ultraviolet spectrums. In some embodiments, thereflectance levels may correspond to reflectances previouslydetected by the ingestible device at periodic intervals.

[1265] At 65904, the ingestible device includes new measurements ofreflectance levels in the data set. For example, the ingestibledevice may be configured to detect a new reflectance (e.g.,transmit illumination and detect the resulting reflectance using asensing sub-unit) at regular intervals, or with sufficient speed asto detect peristaltic waves. For example, the ingestible device maybe configured to generate illumination and measure the resultingreflectance once every three seconds (i.e., potentially minimumrate to detect a 0.17 Hz signal), and preferably at a higher rate,as fast at 0.1 second or even faster. It is understood that theperiodic interval between measurements may be adapted as neededbased on the species of the subject, and the expected frequency ofthe peristaltic waves to be measured. Every time the ingestibledevice makes a new reflectance level measurement at 65904, the newdata is included to the data set (e.g., a data set stored withinmemory circuitry of a PCB).

[1266] At 65906, the ingestible device obtains a first subset ofrecent data by applying a sliding window filter to the data set.For example, the ingestible device may retrieve a one-minute worthof data from the data set. If the data set includes values forreflectances measured every second, this would be approximately 60data points worth of data. Any suitable type of window size may beused, provided that the size of the window is sufficiently large todetect peristaltic waves (e.g., fluctuations on the order of 0.05Hz to 0.33 Hz for healthy human subjects). In some embodiments, theingestible device may also clean the data, for example, by removingoutliers from the first subset of data obtained through the use ofthe sliding window filter.

[1267] At 65908, the ingestible device obtains a second subset ofrecent data by interpolating the first subset of recent data. Forexample, the ingestible device may interpolate the first subset ofdata in order to generate a second subset of data with a sufficientnumber of data points (e.g., data points spaced every 0.5 secondsor greater). In some embodiments, this may enable the ingestibledevice to also replace any outlier data points that may have beenremoved as part of applying the window filter at 65906.

[1268] At 65910, the ingestible device calculates a normalizedfrequency spectrum from the second subset of data. For example, theingestible device may be configured to perform a fast Fouriertransform to convert the second subset of data from a time domainrepresentation into a frequency domain representation. It isunderstood that depending on the application being used, and thenature of the subset of data, any number of suitable procedures(e.g., Fourier transform procedures) may be used to determine afrequency spectrum for the second subset of data. For example, thesampling frequency and size of the second subset of data may beknown in advance, and the ingestible device may be configured tohave pre-stored values of a normalized discreet Fourier transform(DFT) matrix, or the rows of the DFT matrix corresponding to the0.05 Hz to 0.33 Hz frequency components of interest, within memory(e.g., memory circuitry of a PCB). In this case, the ingestibledevice may use matrix multiplication between the DFT matrix and thedata set to generate an appropriate frequency spectrum. An exampledata set and corresponding frequency spectrum that may be obtainedby the ingestible device is discussed in greater detail in relationto FIG. 65.

[1269] At 65912, the ingestible device determines whether at leasta portion of the normalized frequency spectrum is between 00.05 Hzto 0.33 Hz above a threshold value of 0.5 Hz. Peristaltic waves ina healthy human subject occur at a rate between 0.05 Hz to 0.33 Hz,and an ingestible device experiencing peristaltic waves (e.g., aningestible device detecting contractions in the walls of thejejunum) may detect sinusoidal variations in the amplitude ofdetected reflectances levels that follow a similar 0.05 Hz to 0.33Hz frequency. If the ingestible device determines that a portion ofthe normalized frequency spectrum between 0.05 Hz to 0.33 Hz isabove a threshold value of 0.5 Hz, this measurement may beconsistent with peristaltic waves in a healthy human subject, andprocess 65900 proceeds to 65914 where the ingestible device storesdata indicating a muscle contraction was detected. Alternatively,if the ingestible device determines that no portion of thenormalized frequency spectrum between 0.05 Hz to 0.33 Hz above athreshold value of 0.5, process 65900 proceeds directly to 65904 tomake new measurements and to continue to monitor for new musclecontractions. It is understood that a threshold value other than0.5 may be used, and that the exact threshold may depend on thesampling frequency and type of frequency spectrum used by theingestible device.

[1270] At 65914, the ingestible device stores data indicating amuscle contraction was detected. For example, the ingestible devicemay store data in memory (e.g., memory circuitry of a PCB)indicating that a muscle contraction was detected, and indicatingthe time that the muscle contraction was detected. In someembodiments, the ingestible device may also monitor the totalnumber of muscle contractions detected, or the number of musclecontractions detected in a given time frame. In some embodiments,detecting a particular number of muscle contractions may beconsistent with the ingestible device being within the jejunum) ofa healthy human subject. After detecting a muscle contraction,process 65900 proceeds to 65916.

[1271] At 65916, the ingestible device determines whether a totalnumber of muscle contractions exceeds a predetermined thresholdnumber. For example, the ingestible device may retrieve the totalnumber of muscle contractions detected from memory (e.g., frommemory circuitry of a PCB), and compare the total number to athreshold value. In some embodiments, the threshold value may beone, or any number larger than one. The larger the threshold value,the more muscle contractions need to be detected before theingestible device stores data indicating that it has entered thejejunum. In practice, setting the threshold value as three orhigher may prevent the ingestible device from detecting falsepositives (e.g., due to natural movement of the GI tract organs, ordue to movement of the subject). If the total number ofcontractions exceeds the predetermined threshold number, process65900 proceeds to 65918 to store data indicating detection of atransition from the duodenum to the jejunum. Alternatively, if thetotal number of contractions does not exceed a predeterminedthreshold number, process 65900 proceeds to 65904 to include newmeasurements of reflectance levels in the data set. An example plotof the muscle contractions detected over time is discussed ingreater detail in relation to FIG. 66.

[1272] At 65918, the ingestible device stores data indicatingdetection of a transition from the duodenum to the jejunum. Forexample, the ingestible device may store data in memory (e.g., frommemory circuitry of a PCB) indicating that the jejunum has beenreached. In some embodiments, if the ingestible device isconfigured to perform all or part of process 65900 while in thestomach, the ingestible device may store data at 65918 indicatingdetection of a transition from the stomach directly to the jejunum(e.g., as a result of transitioning too quickly through theduodenum for the pyloric transition to be detected using process65600).

[1273] In some embodiments, the ingestible device may be configuredto obtain a fluid sample from the environment external to a housingof the ingestible device in response to identifying a change in thelocation of the ingestible device. For example, the ingestibledevice may be configured to obtain a fluid sample from theenvironment external to the housing of the ingestible device (e.g.,through the use of optional opening 65116 and optional rotatingassembly 65118) in response to determining that the ingestibledevice is located within the jejunum. In some embodiments, theingestible device may also be equipped with appropriate diagnosticsto detect certain medical conditions based on the retrieved fluidsample, such as small intestinal bacterial overgrowth (SIBO).

[1274] In some embodiments, the ingestible device may be configuredto deliver a dispensable substance that is pre-stored within theingestible device from the ingestible device into the GI tract inresponse to identifying the change in the location of theingestible device. For example, the ingestible device may have adispensable substance pre-stored within the ingestible device(e.g., within a storage chamber or cavity on an optional storagesub-unitGI and the ingestible device may be configured to dispensethe substance into the gastrointestinal tract (e.g., through theuse of an optional opening and an optional rotating assembly) whenthe ingestible device detects that the ingestible device is locatedwithin the jejunum. In some embodiments, this may enable theingestible device to deliver substances (e.g., therapeutics andmedicaments) at targeted locations within the GI tract.

[1275] In some embodiments, the ingestible device may be configuredto perform an action based on the total number of detected musclecontractions. For example, the ingestible device may be configuredto retrieve data indicative of the total number of musclecontractions (e.g., from memory circuitry of a PCB), and comparethat to an expected number of muscle contractions in a healthyindividual. In response, the ingestible device may either dispensea substance into the GI tract (e.g., through the use of an optionalopening and an optional rotating assembly), or may obtain a fluidsample from the environment external to the housing of theingestible device (e.g., through the use of an optional opening andan optional rotating assembly). For instance, the ingestible devicemay be configured to obtain a sample in response to determiningthat a number of detected muscle contractions is abnormal, anddiffers greatly from the expected number. As another example, theingestible device may be configured to deliver a substance into theGI tract (such as a medicament), in response to determining thatthe detected muscle contractions are consistent with a functioningGI tract in a healthy individual.

[1276] It will be understood that the steps and descriptions of theflowcharts of this disclosure are merely illustrative. Any of thesteps and descriptions of the flowcharts may be modified, omitted,rearranged, and/or performed in alternate orders or in parallel,two or more of the steps may be combined, or any additional stepsmay be added, without departing from the scope of the presentdisclosure. For example, the ingestible device may calculate themean and the standard deviation of multiple data sets in parallel(e.g., multiple data sets, each one corresponding to a differentwavelength of reflectance or different sensing sub-unit used todetect the reflectance) in order to speed up the overallcomputation time. Furthermore, it should be noted that the stepsand descriptions of FIG. 64 may be combined with any other system,device, or method described in this application, and any of theingestible devices or systems discussed in this application couldbe used to perform one or more of the steps in FIG. 64.

[1277] FIG. 65 is a plot illustrating data collected during anexample operation of an ingestible device, which may be used whendetecting a transition from a duodenum to a jejunum, in accordancewith some embodiments of the disclosure. Diagram 651000 depicts atime domain plot 651002 of a data set of reflectance levelsmeasured by an ingestible device (e.g., the second subset of datadiscussed in relation to 65908). In some embodiments, theingestible device may be configured to gather data points atsemi-regular intervals approximately 0.5 seconds apart. Bycomparison, diagram 651050 depicts a frequency domain plot 651004of the same data set of reflectance levels measured by aningestible device (e.g., as a result of the ingestible devicecalculating a frequency spectrum at 65910). In some embodiments,the ingestible device may be configured to calculate the frequencyspectrum through any convenient means.

[1278] In diagram 651050, the range of frequencies 651006 between0.05 Hz to 0.33 Hz may be the range of frequencies that theingestible device searches in order to detect muscle contractions.As shown in diagram 651050, there is a strong peak in the frequencydomain plot 651004 around 0.14 Hz, which is consistent with thefrequency of peristaltic motion in a healthy human individual. Inthis case, the ingestible device analyzing frequency domain plot651004 may be configured to determine that the data is consistentwith a detected muscle contraction (e.g., using a process similarto 65912 of process 65900), and may store data (e.g., in memorycircuitry of a PCB) indicating that a muscle contraction has beendetected. Because the muscle contraction was detected from theone-minute window of data ending at 118 minutes, the ingestibledevice may also store data indicating that the muscle contractionwas detected at the 118-minute mark (i.e., which may indicate thatthe ingestible device was turned on and ingested by the subject 118minutes ago).

[1279] FIG. 66 is a plot illustrating muscle contractions detectedby an ingestible device over time, which may be used whendetermining a location of an ingestible device as it transitsthrough a gastrointestinal (GI) tract, in accordance with someembodiments of the disclosure. In some embodiments, the ingestibledevice may be configured to detect muscle contractions, and storedata indicative of when each muscle contraction is detected (e.g.,as part of 65914 of process 65900). Plot 651100 depicts thedetected muscle contractions 651106 over time, with each musclecontraction being represented by a vertical line reaching from "0"to "1" on the y-axis.

[1280] At 651102, around the 10-minute mark, the ingestible devicefirst enters the duodenum (e.g., as determined by the ingestibledevice performing process 65600). Shortly thereafter, at 651108,the ingestible device begins to detect several muscle contractions651106 in quick succession, which may be indicative of the strongperistaltic waves that form in the jejunum. Later, around 651110,the ingestible device continues to detect intermittent musclecontractions, which may be consistent with the ingestible devicewithin the ileum. Finally, at 651104, the ingestible devicetransitions out of the small intestine, and into the cecum.Notably, the ingestible device detects more frequent musclecontractions in the jejunum portion of the small intestine ascompared to the ileum portion of the small intestine, and theingestible device does not measure any muscle contractions afterhaving exited the small intestine. In some embodiments, theingestible device may incorporate this information into alocalization process. For example, the ingestible device may beconfigured to detect a transition from a jejunum to an ileum inresponse to determining that a frequency of detected musclecontractions (e.g., the number of muscle contractions measured in agiven 10-minute window) has fallen below a threshold number. Asanother example, the ingestible device may be configured to detecta transition from an ileum to a cecum in response to determiningthat no muscle contractions have been detected for a thresholdperiod of time. It is understood that these examples are intendedto be illustrative, and not limiting, and that measurements ofmuscle contractions may be combined with any of the otherprocesses, systems, or methods discussed in this disclosure.

[1281] FIG. 66 is a flowchart 651200 for certain embodiments fordetermining a transition of the device from the jejunum to theileum. It is to be noted that, in general, the jejunum is redderand more vascular than the ileum. Moreover, generally, incomparison to the ileum, the jejunum has a thicker intestine wallwith more mesentery fat. These differences between the jejunum andthe ileum are expected to result in differences in opticalresponses in the jejunum relative to the ileum. Optionally, one ormore optical signals may be used to investigate the differences inoptical responses. For example, the process can include monitoringa change in optical response in reflected red light, blue light,green light, ratio of red light to green light, ratio of red lightto blue light, and/or ratio of green light to blue light. In someembodiments, reflected red light is detected in the process.

[1282] Flowchart 651200 represents a single sliding window process.In step 651210, the jejunum reference signal is determined based onoptical reflection. Typically, this signal is as the average signal(e.g., reflected red light) over a period of time since the devicewas determined to enter the jejunum. The period of time can be, forexample, from five minutes to 40 minutes (e.g., from 10 minutes to30 minutes, from 15 minutes to 25 minutes). In step 651220, thedetected signal (e.g., reflected red light) just after the periodof time used in step 651210 is normalized to the reference signaldetermined in step 651210. In step 651230, the signal (e.g.,reflected red light) is detected. In step 651240, the mean signaldetected based on the single sliding window is compared to a signalthreshold. The signal threshold in step 651240 is generally afraction of the reference signal of the jejunum reference signaldetermined in step 651210. For example, the signal threshold can befrom 60% to 90% (e.g., from 70% to 80%) of the jejunum referencesignal. If the mean signal exceeds the signal threshold, then theprocess determines that the device has entered the ileum at step651250. If the mean signal does not exceed the signal threshold,then the process returns to step 651230.

[1283] FIG. 68 is a flowchart 651200 for certain embodiments fordetermining a transition of the device from the jejunum to theileum using a two sliding window process. In step 651310, thejejunum reference signal is determined based on optical reflection.Typically, this signal is as the average signal (e.g., reflectedred light) over a period of time since the device was determined toenter the jejunum. The period of time can be, for example, fromfive minutes to 40 minutes (e.g., from 10 minutes to 30 minutes,from 15 minutes to 25 minutes). In step 651320, the detected signal(e.g., reflected red light) just after the period of time used instep 651310 is normalized to the reference signal determined instep 651310. In step 651330, the signal (e.g., reflected red light)is detected. In step 651340, the mean difference in the signaldetected based on the two sliding windows is compared to a signalthreshold. The signal threshold in step 651340 is based on whetherthe mean difference in the detected signal exceeds a multiple(e.g., from 1.5 times to five times, from two times to four times)of the detected signal of the first window. If signal threshold isexceeded, then the process determines that the device has enteredthe ileum at step 651350. If the signal threshold is not exceeded,then the process returns to step 651330.

[1284] FIG. 69 is a flowchart 651400 for a process for certainembodiments for determining a transition of the device from theileum to the cecum. In general, the process involves detectingchanges in the reflected optical signal (e.g., red light, bluelight, green light, ratio of red light to green light, ratio of redlight to blue light, and/or ratio of green light to blue light). Insome embodiments, the process includes detecting changes in theratio of reflected red light to reflected green light, and alsodetecting changes in the ratio of reflected green light toreflected blue light. Generally, in the process 651400, the slidingwindow analysis (first and second windows) discussed with respectto process 65600 is continued.

[1285] Step 651410 includes setting a first threshold in a detectedsignal, e.g., ratio of detected red light to detected green light,and setting a second threshold for the coefficient of variation fora detected signal, e.g., the coefficient of variation for the ratioof detected green light to detected blue light. The first thresholdcan be set to a fraction (e.g., from 0.5 to 0.9, from 0.6 to 0.8)of the average signal (e.g., ratio of detected red light todetected green light) in the first window, or a fraction (e.g.,from 0.4 to 0.8, from 0.5 to 0.7) of the mean difference betweenthe detected signal (e.g., ratio of detected red light to detectedgreen light) in the two windows. The second threshold can be set to0.1 (e.g., 0.05, 0.02).

[1286] Step 651420 includes detecting the signals in the first andsecond windows that are to be used for comparing to the first andsecond thresholds.

[1287] Step 651430 includes comparing the detected signals to thefirst and second thresholds. If the corresponding value is notbelow the first threshold or the corresponding value is not belowthe second threshold, then it is determined that the device has notleft the ileum and entered the cecum, and the process returns tostep 651420. If the corresponding value is below the firstthreshold and the corresponding value is below the secondthreshold, then it is determined that the device has left the ileumand entered the cecum, and the proceeds to step 651440.

[1288] Step 651450 includes determining whether it is the firsttime that that the device was determined to leave the ileum andenter the cecum. If it is the first time that the device wasdetermined to leave the ileum and enter the cecum, then the processproceeds to step 651460. If it is not the first time that thedevice has left the ileum and entered the cecum, then the processproceeds to step 651470.

[1289] Step 651460 includes setting a reference signal. In thisstep the optical signal (e.g., ratio of detected red light todetected green light) as a reference signal.

[1290] Step 651470 includes determining whether the device may haveleft the cecum and returned to the ileum. The device is determinedto have left the cecum and returned to the ileum if thecorresponding detected signal (e.g., ratio of detected red light todetected green light) is statistically comparable to the referencesignal (determined in step 651460) and the coefficient of variationfor the corresponding detected signal (e.g., ratio of detectedgreen light to detected blue light) exceeds the second threshold.If it is determined that the device may have left the cecum andreturned to the ileum, the process proceeds to step 651480.

[1291] Step 651480 includes continuing to detect the relevantoptical signals for a period of time (e.g., at least one minute,from five minutes to 15 minutes).

[1292] Step 651490 includes determining whether the signalsdetermined in step 651480 indicate (using the methodology discussedin step 651470) that the device re-entered the ileum. If thesignals indicate that the device re-entered the ileum, the processproceeds to step 651420. If the signals indicate that the device isin the cecum, the process proceeds to step 651492.

[1293] Step 651492 includes continuing to monitor the relevantoptical signals for a period of time (e.g., at least 30 minutes, atleast one hour, at least two hours).

[1294] Step 651494 includes determining whether the signalsdetermined in step 651492 indicate (using the methodology discussedin step 651470) that the device re-entered the ileum. If thesignals indicate that the device re-entered the ileum, the processproceeds to step 651420. If the signals indicate that the device isin the cecum, the process proceeds to step 651496.

[1295] At step 651496, the process determines that the device is inthe cecum.

[1296] FIG. 70 is a flowchart 651500 for a process for certainembodiments for determining a transition of the device from thececum to the colon. In general, the process involves detectingchanges in the reflected optical signal (e.g., red light, bluelight, green light, ratio of red light to green light, ratio of redlight to blue light, and/or ratio of green light to blue light). Insome embodiments, the process includes detecting changes in theratio of reflected red light to reflected green light, and alsodetecting changes in the ratio of reflected blue light. Generally,in the process 651500, the sliding window analysis (first andsecond windows) discussed with respect to process 651400 iscontinued.

[1297] In step 651510, optical signals (e.g., the ratio ofreflected red signal to reflected green signal, and reflected bluesignal) are collected for a period of time (e.g., at least oneminute, at least five minutes, at least 10 minutes) while thedevice is in the cecum (e.g., during step 651480). The averagevalues for the recorded optical signals (e.g., the ratio ofreflected red signal to reflected green signal, and reflected bluesignal) establish the cecum reference signals.

[1298] In step 651520, the optical signals are detected after ithas been determined that the device entered the cecum (e.g., atstep 651440). The optical signals are normalized to the cecumreference signals.

[1299] Step 651530 involves determining whether the device hasentered the colon. This includes determining whether any of threedifferent criteria are satisfied. The first criterion is satisfiedif the mean difference in the ratio of a detected optical signal(e.g., ratio of detected red signal to the detected green) is amultiple greater than one (e.g., 2.times., 3.times., 4.times.) thestandard deviation of the corresponding signal (e.g., ratio ofdetected red signal to the detected green) in the second window.The second criterion is satisfied if the mean of a detected opticalsignal (e.g., a ratio of detected red light to detected greenlight) exceeds a given value (e.g., exceeds one). The thirdcriterion is satisfied if the coefficient of variation of anoptical signal (e.g., detected blue light) in the first windowexceeds a given value (e.g., exceeds 0.2). If any of the threecriteria are satisfied, then the process proceeds to step 651540.Otherwise, none of the three criteria are satisfied, the processreturns to step 651520.

[1300] For illustrative purposes the disclosure focuses primarilyon a number of different example embodiments of an ingestibledevice, and example embodiments of methods for determining alocation of an ingestible device within a GI tract. However, thepossible ingestible devices that may be constructed are not limitedto these embodiments, and variations in the shape and design may bemade without significantly changing the functions and operations ofthe device. Similarly, the possible procedures for determining alocation of the ingestible device within the GI tract are notlimited to the specific procedures and embodiments discussed (e.g.,process 65500, process 65600, process 65900, process 651200,process 651300, process 651400 and process 651500). Also, theapplications of the ingestible devices described herein are notlimited merely to gathering data, sampling and testing portions ofthe GI tract, or delivering medicament. For example, in someembodiments the ingestible device may be adapted to include anumber of chemical, electrical, or optical diagnostics fordiagnosing a number of diseases. Similarly, a number of differentsensors for measuring bodily phenomenon or other physiologicalqualities may be included on the ingestible device. For example,the ingestible device may be adapted to measure elevated levels ofcertain chemical compounds or impurities in the GI tract, or thecombination of localization, sampling, and appropriate diagnosticand assay techniques incorporated into a sampling chamber may beparticularly well suited to determine the presence of smallintestinal bacterial overgrowth (SIBO).

[1301] At least some of the elements of the various embodiments ofthe ingestible device described herein that are implemented viasoftware (e.g., software executed by control circuitry within a PCB65120) may be written in a high-level procedural language such asobject oriented programming, a scripting language or both.Accordingly, the program code may be written in C, C++ or any othersuitable programming language and may include modules or classes,as is known to those skilled in object oriented programming.Alternatively, or in addition, at least some of the elements of theembodiments of the ingestible device described herein that areimplemented via software may be written in assembly language,machine language or firmware as needed. In either case, thelanguage may be a compiled or an interpreted language.

[1302] At least some of the program code used to implement theingestible device can be stored on a storage media or on a computerreadable medium that is readable by a general or special purposeprogrammable computing device having a processor, an operatingsystem and the associated hardware and software to implement thefunctionality of at least one of the embodiments described herein.The program code, when read by the computing device, configures thecomputing device to operate in a new, specific and predefinedmanner in order to perform at least one of the methods describedherein.

[1303] Furthermore, at least some of the programs associated withthe systems, devices, and methods of the example embodimentsdescribed herein are capable of being distributed in a computerprogram product including a computer readable medium that bearscomputer usable instructions for one or more processors. The mediummay be provided in various forms, including non-transitory formssuch as, but not limited to, one or more discettes, compact discs,tapes, chips, and magnetic and electronic storage. In someembodiments, the medium may be transitory in nature such as, butnot limited to, wire-line transmissions, satellite transmissions,internet transmissions (e.g. downloads), media, digital and analogsignals, and the like. The computer useable instructions may alsobe in various formats, including compiled and non-compiledcode.

[1304] The techniques described above can be implemented usingsoftware for execution on a computer. For instance, the softwareforms procedures in one or more computer programs that execute onone or more programmed or programmable computer systems (which maybe of various architectures such as distributed, client/server, orgrid) each including at least one processor, at least one datastorage system (including volatile and non-volatile memory and/orstorage elements), at least one input device or port, and at leastone output device or port.

[1305] The software may be provided on a storage medium, such as aCD-ROM, readable by a general or special purpose programmablecomputer or delivered (encoded in a propagated signal) over acommunication medium of a network to the computer where it isexecuted. All of the functions may be performed on a specialpurpose computer, or using special-purpose hardware, such ascoprocessors. The software may be implemented in a distributedmanner in which different parts of the computation specified by thesoftware are performed by different computers. Each such computerprogram is preferably stored on or downloaded to a storage media ordevice (e.g., solid state memory or media, or magnetic or opticalmedia) readable by a general or special purpose programmablecomputer, for configuring and operating the computer when thestorage media or device is read by the computer system to performthe procedures described herein. The inventive system may also beconsidered to be implemented as a computer-readable storage medium,configured with a computer program, where the storage medium soconfigured causes a computer system to operate in a specific andpredefined manner to perform the functions described herein.

[1306] For illustrative purposes the examples given herein focusprimarily on a number of different example embodiments of aningestible device. However, the possible ingestible devices thatmay be constructed are not limited to these embodiments, andvariations in the general shape and design may be made withoutsignificantly changing the functions and operations of the device.For example, some embodiments of the ingestible device may featurea sampling chamber substantially towards the middle of the device,along with two sets of axial sensing sub-units, each located onsubstantially opposite ends of the device. In addition, theapplications of the ingestible device are not limited merely togathering data, sampling and testing portions of the GI tract, ordelivering medicament. For example, in some embodiments theingestible device may be adapted to include a number of chemical,electrical, or optical diagnostics for diagnosing a number ofdiseases. Similarly, a number of different sensors for measuringbodily phenomenon or other physiological qualities may be includedon the ingestible device. For example, the ingestible device may beadapted to measure elevated levels of certain analytes, chemicalcompounds or impurities in the GI tract, or the combination oflocalization, sampling, and appropriate diagnostic and assaytechniques incorporated into a sampling chamber may be particularlywell suited to determine the presence of small intestinal bacterialovergrowth (SIBO). It is also noted that although embodimentsdescribed herein focus on an ingestible device in the GI tract,such ingestible device described in FIGS. 1-34 may be used fordelivering substances including medicaments and therapeutics inother parts of the body, such as but not limited to the femalereproductive tract, and/or the like.

[1307] The various embodiments of systems, processes andapparatuses have been described herein by way of example only. Itis contemplated that the features and limitations described in anyone embodiment may be applied to any other embodiment herein, andflowcharts or examples relating to one embodiment may be combinedwith any other embodiment in a suitable manner, done in differentorders, or done in parallel. It should be noted, the systems and/ormethods described above may be applied to, or used in accordancewith, other systems and/or methods. Various modifications andvariations may be made to these example embodiments withoutdeparting from the spirit and scope of the embodiments, which islimited only by the appended embodiments. The appended embodimentsshould be given the broadest interpretation consistent with thedescription as a whole.

[1308] Implementations of the subject matter and the operationsdescribed in this specification can be implemented by digitalelectronic circuitry, or via computer software, firmware, orhardware, including the structures disclosed in this specificationand their structural equivalents, or in combinations of one or moreof them. Implementations of the subject matter described in thisspecification can be implemented as one or more computer programs,i.e., one or more modules of computer program instructions, encodedon computer storage medium for execution by, or to control theoperation of, data processing apparatus.

[1309] A computer storage medium can be, or be included in, acomputer-readable storage device, a computer-readable storagesubstrate, a random or serial access memory array or device, or acombination of one or more of them. Moreover, while a computerstorage medium is not a propagated signal, a computer storagemedium can be a source or destination of computer programinstructions encoded in an artificially generated propagatedsignal. The computer storage medium can also be, or be included in,one or more separate physical components or media (e.g., multipleCDs, discs, or other storage devices).

[1310] The operations described in this specification can beimplemented as operations performed by a data processing apparatuson data stored on one or more computer-readable storage devices orreceived from other sources.

[1311] The term "data processing apparatus" encompasses all kindsof apparatus, devices, and machines for processing data, includingby way of example a programmable processor, a computer, a system ona chip, or multiple ones, or combinations, of the foregoing. Theapparatus can include special purpose logic circuitry, e.g., anFPGA (field programmable gate array) or an ASIC (applicationspecific integrated circuit). The apparatus can also include, inaddition to hardware, code that creates an execution environmentfor the computer program in question, e.g., code that constitutesprocessor firmware, a protocol stack, a database management system,an operating system, a cross-platform runtime environment, avirtual machine, or a combination of one or more of them. Theapparatus and execution environment can realize various differentcomputing model infrastructures, such as web services, distributedcomputing and grid computing infrastructures.

[1312] A computer program (also known as a program, software,software application, script, or code) can be written in any formof programming language, including compiled or interpretedlanguages, declarative or procedural languages, and it can bedeployed in any form, including as a stand-alone program or as amodule, component, subroutine, object, or other unit suitable foruse in a computing environment. A computer program may, but neednot, correspond to a file in a file system. A program can be storedin a portion of a file that holds other programs or data (e.g., oneor more scripts stored in a markup language document), in a singlefile dedicated to the program in question, or in multiplecoordinated files (e.g., files that store one or more modules, subprograms, or portions of code). A computer program can be deployedto be executed on one computer or on multiple computers that arelocated at one site or distributed across multiple sites andinterconnected by a communication network.

[1313] The processes and logic flows described in thisspecification can be performed by one or more programmableprocessors executing one or more computer programs to performactions by operating on input data and generating output. Theprocesses and logic flows can also be performed by, and apparatuscan also be implemented as, special purpose logic circuitry, e.g.,a FPGA (field programmable gate array) or an ASIC (applicationspecific integrated circuit).

[1314] Processors suitable for the execution of a computer programinclude, by way of example, both general and special purposemicroprocessors, and any one or more processors of any kind ofdigital computer. Generally, a processor will receive instructionsand data from a read only memory or a random access memory or both.The essential elements of a computer are a processor for performingactions in accordance with instructions and one or more memorydevices for storing instructions and data. Generally, a computerwill also include, or be operatively coupled to receive data fromor transfer data to, or both, one or more mass storage devices forstoring data, e.g., magnetic, magneto optical discs, or opticaldiscs. However, a computer need not have such devices. Moreover, acomputer can be embedded in another device, e.g., a mobiletelephone, a personal digital assistant (PDA), a mobile audio orvideo player, a game console, a Global Positioning System (GPS)receiver, or a portable storage device (e.g., a universal serialbus (USB) flash drive), to name just a few. Devices suitable forstoring computer program instructions and data include all forms ofnon-volatile memory, media and memory devices, including by way ofexample semiconductor memory devices, e.g., EPROM, EEPROM, andflash memory devices; magnetic discs, e.g., internal hard discs orremovable discs; magneto optical discs; and CD ROM and DVD-ROMdiscs. The processor and the memory can be supplemented by, orincorporated in, special purpose logic circuitry.

[1315] To provide for interaction with a user, implementations ofthe subject matter described in this specification can beimplemented on a computer having a display device, e.g., a CRT(cathode ray tube) or LCD (liquid crystal display) monitor, fordisplaying information to the user and a keyboard and a pointingdevice, e.g., a mouse or a trackball, by which the user can provideinput to the computer. Other kinds of devices can be used toprovide for interaction with a user as well; for example, feedbackprovided to the user can be any form of sensory feedback, e.g.,visual feedback, auditory feedback, or tactile feedback; and inputfrom the user can be received in any form, including acoustic,speech, or tactile input. In addition, a computer can interact witha user by sending documents to and receiving documents from adevice that is used by the user; for example, by sending web pagesto a web browser on a user's user device in response to requestsreceived from the web browser.

[1316] Implementations of the subject matter described in thisspecification can be implemented in a computing system thatincludes a back end component, e.g., as a data server, or thatincludes a middleware component, e.g., an application server, orthat includes a front end component, e.g., a user computer having agraphical display or a Web browser through which a user caninteract with an implementation of the subject matter described inthis specification, or any combination of one or more such backend, middleware, or front end components. The components of thesystem can be interconnected by any form or medium of digital datacommunication, e.g., a communication network. Examples ofcommunication networks include a local area network ("LAN") and awide area network ("WAN"), an inter-network (e.g., the Internet),and peer-to-peer networks (e.g., ad hoc peer-to-peer networks).

[1317] The computing system can include users and servers. A userand server are generally remote from each other and typicallyinteract through a communication network. The relationship of userand server arises by virtue of computer programs running on therespective computers and having a user-server relationship to eachother. In some implementations, a server transmits data (e.g., anHTML page) to a user device (e.g., for purposes of displaying datato and receiving user input from a user interacting with the userdevice). Data generated at the user device (e.g., a result of theuser interaction) can be received from the user device at theserver.

[1318] While this specification contains many specificimplementation details, these should not be construed aslimitations on the scope of any inventions or of what may beclaimed, but rather as descriptions of features specific toparticular implementations of particular inventions. Certainfeatures that are described in this specification in the context ofseparate implementations can also be implemented in combination ina single implementation. Conversely, various features that aredescribed in the context of a single implementation can also beimplemented in multiple implementations separately or in anysuitable sub combination. Moreover, although features may bedescribed above as acting in certain combinations and eveninitially claimed as such, one or more features from a claimedcombination can in some cases be excised from the combination, andthe claimed combination may be directed to a sub combination orvariation of a sub combination.

[1319] For the purpose of this disclosure, the term "coupled" meansthe joining of two members directly or indirectly to one another.Such joining may be stationary or moveable in nature. Such joiningmay be achieved with the two members or the two members and anyadditional intermediate members being integrally formed as a singleunitary body with one another or with the two members or the twomembers and any additional intermediate members being attached toone another. Such joining may be permanent in nature or may beremovable or releasable in nature.

[1320] It should be noted that the orientation of various elementsmay differ according to other exemplary implementations, and thatsuch variations are intended to be encompassed by the presentdisclosure. It is recognized that features of the disclosedimplementations can be incorporated into other disclosedimplementations.

[1321] FIG. 71 illustrates a nonlimiting example of a system forcollecting, communicating and/or analyzing data about a subject,using an ingestible device as disclosed herein. For example, aningestible device may be configured to communicate with an externalbase station. As an example, an ingestible device can have acommunications unit that communicates with an external base stationwhich itself has a communications unit. FIG. 71 illustratesexemplary implementation of such an ingestible device. As shown inFIG. 71, a subject ingests an ingestible device as disclosedherein. Certain data about the subject (e.g., based on a collectedsample) and/or the location of the ingestible device in the GItract of the subject is collected or otherwise available andprovided to a mobile device, which then forwards the data via theinternet and a server/data store to a physician's office computer.The information collected by the ingestible device is communicatedto a receiver, such as, for example, a watch or other object wornby the subject. The information is then communicated from thereceiver to the mobile device which then forwards the data via theinternet and a server/data store to a physician's office computer.The physician is then able to analyze some or all of the data aboutthe subject to provide recommendations, such as, for example,general health recommendations, dietary health recommendationsand/or lifestyle recommendations. While FIG. 71 shows a particularapproach to collecting and transferring data about a subject, thedisclosure is not limited. As an example, one or more of thereceiver, mobile device, internet, and/or server/data store can beexcluded from the data communication channel. For example, a mobiledevice can be used as the receiver of the device data, e.g., byusing a dongle. In such embodiments, the item worn by the subjectneed not be part of the communication chain. As another example,one or more of the items in the data communication channel can bereplaced with an alternative item. For example, rather than beprovided to a physician's office computer, data may be provided toa service provider network, such as a hospital network, an HMOnetwork, or the like. In some embodiments, subject data may becollected and/or stored in one location (e.g., a server/data store)while device data may be collected and/or stored in a differentlocation (e.g., a different server/data store).

[1322] An ingestible device may include one or more environmentalsensors. Environmental sensor may be used to generate environmentaldata for the environment external to device in the GI tract of thesubject. Environmental data may be used to further characterize theGI tract of the subject either alone or in combination with thespectral data. In some embodiments, environmental data is generatedat the same location within the GI tract of the subject where asample is procured. Examples of environmental sensor include, butare not limited to a capacitance sensor, a temperature sensor, animpedance sensor, a pH level sensor, a heart rate sensor, acousticsensor, image sensor, and/or a movement sensor. In someembodiments, the ingestible device includes a plurality ofdifferent environmental sensors for generating different kinds ofenvironmental data. In some embodiments, the image sensor is avideo camera suitable for obtaining images in vivo of the tissuesforming the GI tract of the subject. In some embodiments, theenvironmental data is used to help determine one or morecharacteristics of the GI tract the subject such as for thediagnosis of a medical condition. In some embodiments, theingestible device may include a camera for generating video imagingdata of the GI tract which can be used to determine, among otherthings, the location of the device. Examples of video imagingcapsules include Medtronic's PillCam.TM., Olympus'Endocapsule.RTM., and IntroMedic's MicroCam.TM. (see Basar et al."Ingestible Wireless Capsule Technology: A Review of Developmentand Future Indication" International Journal of Antennas andPropagation (2012); 1-14). Other imaging technologies includethermal imaging cameras, and those that employ ultrasound orDoppler principles to generate different images (see Chinese patentdisclosure CN104473611: "Capsule endoscope system having ultrasonicpositioning function"). In another embodiment, the ingestibledevice described herein may be localized using a gamma scintigraphytechnique or other radio-tracker technology as employed by PhaetonResearch's Enterion.TM. capsule (See Teng, Renli, and Juan Maya."Absolute bioavailability and regional absorption of ticagrelor inhealthy volunteers." Journal of Drug Assessment 3.1 (2014): 43-50),or monitoring the magnetic field strength of permanent magnet inthe ingestible device (see T. D. Than, et al., "A review oflocalization systems for robotic endoscopic capsules," IEEE Trans.Biomed. Eng., vol. 59, no. 9, pp. 2387-2399, September 2012). Insome embodiments, the one or more environmental sensors measure pH,temperature, transit times, or combinations thereof. Examples ofdevices useful to detect pH changes include Medimetrics'IntelliCap.RTM. technology (see Becker, Dieter, et al. "Novelorally swallowable IntelliCap.RTM. device to quantify regional drugabsorption in human GI tract using diltiazem as model drug." AAPSPharmSciTech 15.6 (2014): 1490-1497) and Rani Therapeutics'Auto-Pill.TM. technology (see U.S. Pat. No. 9,149,617), herebyincorporated by reference in its entirety.

Detection Methods and Systems

[1323] Live Cell Dye

[1324] Certain systems described herein employ methods,compositions and detection systems found to accurately and reliablycorrelate fluorescence to total bacteria count (TBC) in anautonomous, ingestible device, or other similarly-sized device. Insome embodiments, the methods and devices described herein can beused for the detection of TBC in a sample from the gastrointestinaltract of the subject to determine whether the subject has or is atrisk of developing a GI disorder (e.g., SIBO). The compositionsinclude novel combinations of dyes, buffers and detergents thatallow for the selective staining of viable bacterial cells insamples that include non-bacterial cells and other components thatotherwise make detecting or quantifying live bacterial cellschallenging. In some embodiments, the systems allow for bacteria tobe quantified in near real-time and the results to be sharedtelemetrically outside of the device. Above, various types of cells(e.g., bacterial cells) are disclosed which can be detected usingthe methods described in this section.

[1325] In some embodiments, the disclosure provides a compositionincluding a dye and optionally a reagent for selective lysis ofeukaryotic cells. In some embodiments, the composition includesboth a dye and a reagent for selective lysis of eukaryotic cells.In some embodiments, the composition further comprises one or morereagents independently selected from the group consisting of: asecond reagent for selective lysis of eukaryotic cells, anelectrolyte (e.g., MgCl.sub.2), an anti-fungal reagent (e.g.,amphotericin-B), and an antibiotic. In some embodiments, thecomposition comprises water and is in the form of an aqueoussolution. In some embodiments, the composition is a solid orsemi-solid. In some embodiments, the compositions described hereare suitable for use in a kit or device for detecting orquantifying viable bacterial cells in a sample. In someembodiments, such a device is an ingestible device for detecting orquantifying viable bacterial cells in vivo (e.g., in the GI tract).In some embodiments, viable bacterial cells in a sample aredetected or quantified in the presence of one or more antibioticsto determine antibiotic resistance of the bacteria in the sample.In some embodiments, anomalous bacterial populations in a samplemay be detected or quantified, for example through the use of acomposition comprising a dye as disclosed herein, to determinewhether a subject has an infection, such as Small IntestinalBacterial Overgrowth (SIBO), or to characterize bacterialpopulations within the GI tract for diagnostic or otherpurposes.

[1326] In some embodiments, the dye suitable for use in thecomposition of the present disclosure is a dye that is capable ofbeing internalized by a viable cell, binding to or reacting with atarget component of the viable cell, and having fluorescenceproperties that are measurably altered when the dye is bound to orreacted with the target component of the viable cell. In someembodiments, the dye of the present disclosure is activelyinternalized by penetrating viable cells through a process otherthan passible diffusion across cell membranes. Such internalizationincludes, but is not limited to, internalization through cellreceptors on cell surfaces or through channels in cell membranes.In some embodiments, the target component of a viable cell to whichthe dye is bound to or reacted with is selected from the groupconsisting of: nucleic acids, actin, tubulin, enzymes,nucleotide-binding proteins, ion-transport proteins, mitochondria,cytoplasmic components, and membrane components. In someembodiments, the dye suitable for use herein is a fluorogenic dyethat is capable of being internalized and metabolized by a viablecell, and wherein the dye fluoresces when metabolized by the viablecell. In some embodiments, the dye is a chemiluminescent dye thatis capable of being internalized and metabolized by a viable cell,and wherein the dye becomes chemiluminescent when metabolized bythe viable cell.

[1327] In some embodiments, the composition includes a dye thatfluoresces when bound to nucleic acids. Examples of such dyesinclude, but are not limited to, acridine orange (U.S. Pat. No.4,190,328); calcein-AM (U.S. Pat. No. 5,314,805); DAPI; Hoechst33342; Hoechst 33258; PicoGreen.TM.; SYTO.RTM. 16; SYBR.RTM. GreenI; Texas Red.RTM.; Redmond Red.TM.; Bodipy.RTM. Dyes; OregonGreen.TM.; ethidium bromide; and propidium iodide.

[1328] In some embodiments, the composition includes a lipophilicdye that fluoresces when metabolized by a cell. In someembodiments, the dye fluoresces when reduced by a cell or a cellcomponent. Examples of dyes that fluoresce when reduced include,but are not limited to, resazurin; C.sup.12-resazurin;7-hydroxy-9H-(1,3 dichloro-9,9-dimethylacridin-2-ol)N-oxide;6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine; and tetrazoliumsalts. In some embodiment, the dye fluoresces when oxidized by acell or a cell component. Examples of such dyes include, but arenot limited to, dihydrocalcein AM; dihydrorhodamine 123;dihydroethidium; 2,3,4,5,6-pentafluorotetramethyldihydrorosamine;and 3'-(p-aminophenyl) fluorescein.

[1329] In some embodiments, the composition includes a dye thatbecomes chemiluminescent when oxidized by a cell or a cellcomponent, such as luminol.

[1330] In some embodiments, the composition includes a dye thatfluoresces when de-acetylated and/or oxidized by a cell or a cellcomponent. Examples of such dyes include, but are not limited to,dihydrorhodamines; dihydrofluoresceins; 2',7'-dichlorodihydrofluorescein diacetate; 5-(and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; and chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester.

[1331] In some embodiments, the composition includes a dye thatfluoresces when reacted with a peptidase. Examples of such dyesinclude, but are not limited to, (CBZ-Ala-Ala-Ala-Ala)2-R110elastase 2; (CBZ-Ala-Ala-Asp)2-R110 granzyme B; and7-amino-4-methylcoumarin,N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide.

[1332] In some embodiments, the composition of this disclosureincludes a dye selected from the group consisting of resazurin,fluorescein diacetate fluorescein diacetate (FDA), Calcein AM, andSYTO.RTM. 9. In some embodiments, the dye is FDA or SYTO.RTM. 9. Insome embodiments, the methods described herein may make use of morethan one dye (e.g., two, three, four, five, six, seven, eight,nine, ten, or more dyes). The use of multiple dyes allows for thedetection of multiple analytes (e.g., multiplexing), for example,when each dye is detectable at a different wavelength. Moregenerally, multiple dyes operating with different fluorescentwavelenths can be used as appropriate.

[1333] SYTO.RTM. 9, when used alone, labels nucleic acids ofbacteria cells. The excitation/emission wavelengths for SYTO.RTM. 9is 480/500 nm, with the background remaining non-fluorescent. See,e.g., J. Appl. Bacteriol. 72, 410 (1992); Lett. Appl. Microbiol.13, 58 (1991); Curr. Microbiol. 4, 321 (1980); J. Microbiol.Methods 13, 87 (1991); and Microbiol. Rev. 51, 365 (1987); and J.Med. Microbiol. 39, 147 (1993).

[1334] FDA is a non-polar, non-fluorescent compound that can crossthe membranes of mammalian and bacterial cells. The acetylesterases (present only within viable cells) hydrolyze the FDA intothe fluorescent compound fluorescein. Fluorescein is a fluorescentpolar compound that is retained within these cells. Living cellscan be visualized in a photospectrometer when assayed with anexcitation wavelength of 494 nm and an emission wavelength of 518nm. See, e.g., Brunius, G. (1980). Technical aspects of the use of3', 6' Diacetyl fluorescein for vital fluorescent staining ofbacteria. Current Microbiol. 4: 321-323; Jones, K. H. and Senft, J.A. (1985). An improved method to determine cellviability bysimultaneous staining with fluorescein diacetate--propidium iodide.J. Histochem. Cytochem. 33: 77-79; Ross, R. D., Joneckis, C. C.,Ordonez, J. V., Sisk, A. M., Wu, R. K., Hamburger, A. W., and Nora,R. E. (1989). Estimation of cell survival by flow cytometricquantification offluorescein diacetate/propidium iodide viable cellnumber. Cancer Research. 49: 3776-3782.

[1335] Calcein-AM, which is an acetoxylmethyl ester of calcein, ishighly lipophilic and cell permeable. Calcein-AM in itself is notfluorescent, but the calcein generated by esterase in a viable cellemits a green fluorescence with an excitation wavelength of 490 nmand an emission of 515 nm. Therefore, Calcein-AM can only stainviable cells. See, e.g., Kimura, K., et al., Neurosci. Lett., 208,53 (1998); Shimokawa, I., et al., J. Geronto., 51a, b49 (1998);Yoshida, S., et al., Clin. Nephrol., 49, 273 (1998); and Tominaga,H., et al., Anal. Commun., 36, 47 (1999).

[1336] Resazurin (also known as Alamar Blue) is a blue compoundthat can be reduced to pink resorufin which is fluorescent. Thisdye is mainly used in viability assays for mammalian cells.C.sup.12-resazurin has better cell permeability than resazurin.When lipohilic C.sup.12-resazurin crosses the cell membranes, it issubsequently reduced by living cells to make a red fluorescentresorufin. The adsorption/emission of C.sup.12-resazurin is 563/587nm. See, e.g., Appl Environ Microbiol 56, 3785 (1990); J Dairy Res57, 239 (1990); J Neurosci Methods 70, 195 (1996); J ImmunolMethods 210, 25 (1997); J Immunol Methods 213, 157 (1998);Antimicrob Agents Chemother 41, 1004 (1997).

[1337] In some embodiments, the composition of this disclosureoptionally further includes a reagent for selective lysis ofeukaryotic cells. In some embodiments, the composition includes adye as described herein and a reagent for selective lysis ofeukaryotic cells. In some embodiments, the reagent for selectivelysis of eukaryotic cells is a detergent, such as a non-ionic or anionic detergent. Examples of the reagent for selective lysis ofeukaryotic cells include, but are not limited to, alkylglycosides,Brij 35 (C12E23 Polyoxyethyleneglycol dodecyl ether), Brij 58(C16E20 Polyoxyethyleneglycol dodecyl ether), Genapol, glucanidssuch as MEGA-8, -9, -10, octylglucoside, Pluronic F127, TritonX-100.TM. (C.sub.14H.sub.22O(C.sub.2H.sub.4O).sub.n), Triton X-114(C.sub.24H.sub.42O.sub.6), Tween 20 (Polysorbate 20) and Tween 80(Polysorbate 80), Nonidet P40, deoxycholate, reduced TritonX-100.TM. and/or Igepal CA 630. In some embodiments, thecomposition of this disclosure includes a dye as described hereinand deoxycholate (e.g., sodium deoxycholate) as a reagent forselective lysis of eukaryotic cells. In some embodiments, thecomposition of this disclosure includes deoxycholate at aconcentration selected from 0.0001% to 1 wt %. In some embodiments,the composition of this disclosure includes deoxycholate at aconcentration of 0.005 wt %. In some embodiments, the compositionmay include more than one reagent for selective lysis of eukaryoticcells.

[1338] In some embodiments, the composition may include twodifferent reagents for selective lysis of eukaryotic cells. In someinstances, when more than one selective lysis reagents are used,more effective and/or complete selective lysis of eukaryotic cellsin a sample may be achieved. For example, the composition mayinclude deoxycholate (e.g., sodium deoxycholate) and TritonX-100.TM. as two different reagents for selective lysis ofeukaryotic cells. In some embodiments, the composition includesdeoxycholate (e.g., sodium deoxycholate) at a concentrationselected from 0.0001% to 1 wt % (e.g., 0.005 wt %) and TritonX-100.TM. at a concentration selected from 0.1 to 0.05 wt %.

[1339] In some embodiments, after a sample (e.g., a biologicalsample) is treated or contacted with a composition including a dyeand one or more reagents for selective lysis of eukaryotic cells asdescribed herein, the eukaryotic cells (e.g., animal cells) in thesample are selectively lysed whereby a substantial percentage(e.g., more than 20%, 40%, 60%, 80%, 90% or even more that 95%) ofthe bacterial cells in the same sample remains intact or alive.

[1340] In some embodiments, the composition does not include areagent for selective lysis of eukaryotic cells, and such acomposition is useful for detecting or quantifying viable bacterialcells in a sample (e.g., an environmental sample such as a watersample) that does not contain any eukaryotic cells.

[1341] In some embodiments, the composition of this disclosurefurther includes an electrolyte, such as a divalent electrolyte(e.g., MgCl.sub.2). In some embodiments, the composition includesMgCl.sub.2 at a concentration selected from 0.1 mM to 100 mM (e.g.,a concentration selected from 0.5 mM to 50 mM).

[1342] In some embodiments, the composition of this disclosurefurther includes water and is in a form of an aqueous solution. Insome embodiments, the composition has a pH selected from 5-8 (e.g.,a pH selected from 6-7.8, such as pH being 6.0). In someembodiments, the composition is a solid or a semi-solid.

[1343] In some embodiments, the composition further includes ananti-fungal agent. Suitable anti-fungal agents for use hereininclude, but are not limited to, fungicidal and fungistatic agentsincluding terbinafine, itraconazole, micronazole nitrate,thiapendazole, tolnaftate, clotrimazole and griseofulvin. In someembodiments, the anti-fungal agent is a polyene anti-fungal agent,such as amphotericin-B, nystatin, and pimaricin.

[1344] In some embodiments, the composition does not contain anyanti-fungal agent. In some embodiments, the composition containsbroad spectrum antibiotics but not any anti-fungal agent. Suchcompositions that do not contain anti-fungal agents but containbroad spectrum antibiotics may be useful in detecting orquantifying fungi (e.g., yeast) in a sample.

[1345] In some embodiments, the composition does not contain anyanti-fungal agent or any antibiotics. Such compositions that do notselectively lyse mammalian cells may be useful in detecting orquantifying mammalian cells (e.g., cells from the GI tract) in asample since many dyes have a higher affinity for mammalian ascompared to bacteria or fungi cells. In some embodiments, thecomposition contains broad spectrum antibiotics and one or moreanti-fungal agents. Such compositions that contain anti-fungalagents and broad spectrum antibiotics may be useful in detecting orquantifying mammalian cells (e.g., cells from the GI tract) in asample. The detection or quantification of mammalian cells may beuseful for determining cell turnover in a subject. High cellturnover is sometimes associated with a GI injury (e.g., lesion),the presence of a tumor(s), or radiation-induced colitis orradiation enteropathy.

[1346] In some embodiments, the composition further includes anantibiotic agent as described herein. Such a composition may beuseful in detecting or quantifying antibiotic-resistant strains ofbacteria in a sample.

[1347] In some embodiments, the composition of this disclosureincludes Triton X-100, deoxycholate, resazurin, and MgCl.sub.2. Insome embodiments, the composition includes Triton X100.TM.,deoxycholate, resazurin, amphotericin-B and MgCl.sub.2. In someembodiments, the composition includes 0.1 wt % or 0.05 wt % TritonX-100.TM.; 0.005 wt % deoxycholate; 10 mM resazurin; 2.5 mg/Lamphotericin-B and 50 mM MgCl.sub.2. In some embodiments, thecomposition has a pH of 6.0.

[1348] In some embodiments, the compositions of this disclosure aresuitable for use in a kit or device, e.g., for detecting orquantifying viable bacterial cells in a sample. In someembodiments, such a device is an ingestible device for detecting orquantifying viable bacterial cells in vivo (e.g., in the GItract).

[1349] In one aspect, this disclosure provides a method fordetecting the presence of viable bacterial cells in a sample,including: (a) contacting the sample with a composition asdescribed herein; and (b) measuring total fluorescence or rate ofchange of fluorescence as a function of time of the sample, therebydetecting viable bacterial cells in the sample.

[1350] In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured over multiple time points for an extended period of timein step (b), thereby detecting viable bacterial cells in thesample. For instance, in some embodiments, the total fluorescenceor the rate of change of fluorescence as a function of time of thesample is measured continuously for a period of 0-1800 minutes,0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes,0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the sample is measuredcontinuously for a period of 0-330 minutes. In some embodiments,the method further includes correlating the total fluorescence orthe rate of change of fluorescence as a function of time determinedin step (b) to the number of viable bacterial cells in the sample.In some embodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver.

[1351] In some embodiments, a control may be employed in the methodas described herein. Such a control may be a positive control,e.g., a composition as described herein further including a knownnumber of viable bacterial cells. In some embodiments, the controlmay be a negative control, e.g., a composition as described hereinthat has not been contacted with any viable bacterial cells. Insome embodiments, this disclosure provides a method for detectingthe presence of viable bacterial cells in a sample, including: (a)contacting the sample with a composition as described herein; (b)measuring total fluorescence or rate of change of fluorescence as afunction of time of the sample; and (c) comparing the totalfluorescence measured in step (b) to the total fluorescenceproduced by a control as described herein, or comparing the rate ofchange of fluorescence as a function of time measured in step (b)to the rate of change of fluorescence as a function of timeproduced by a control as described herein, thereby detecting viablebacterial cells.

[1352] In some embodiments of the method, the control may be (1) acomposition identical to the one used in step (a) but has not beencontacted with any viable bacterial cells; or (2) a compositionidentical to the one used in step (a) further including a knownnumber of viable bacterial cells (e.g., a composition identical tothe one used in step (a) further including 10.sup.2, 10.sup.3,10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 CFU/mL of bacterialcells). In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured over multiple time points for an extended period of timein step (b), thereby detecting viable bacterial cells in thesample. For instance, in some embodiments, the total fluorescenceor the rate of change of fluorescence as a function of time of thesample is measured continuously for a period of 0-1800 minutes,0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes,0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the sample is measuredcontinuously for a period of 0-330 minutes. In some embodiments,the method further includes correlating the comparative totalfluorescence determined in step (c) to the number of viablebacterial cells in the sample. In some embodiments, the rate ofchange of fluorescence as a function of time of the sample measuredover multiple time points is determined and compared to the rate ofchange of fluorescence as a function of time of a control measuredover the same time points to determine the number of viablebacterial cells in the sample. In some embodiments, the method doesnot require ex vivo plating or culturing. In some embodiments, themethod does not require aspiration. In some embodiments, the methodis performed in vivo (e.g., in an ingestible device in vivo). Insome embodiments, the method includes communicating the results ofthe onboard assay(s) to an ex vivo receiver.

[1353] In some embodiments, methods as described herein are highlysensitive in detecting or quantifying viable bacterial cells invarious samples. In some embodiments, the lowest detection orquantification limit of the present methods is 10.sup.2 CFU/mL. Insome embodiments, the highest detection or quantification limit ofthe present methods is 10.sup.7 CFU/mL, 10.sup.8 CFU/mL, 10.sup.9CFU/mL, 10.sup.10 CFU/mL or more. In some embodiments, the methodsallow detection or quantification of 10.sup.2 to 10.sup.7 CFU/mLbacterial cells in various samples. In some embodiments, methods ofthis disclosure may be used to distinguish samples bases on thequantity of viable bacterial cells contained therein. For instance,the methods may be used to distinguish among samples including10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7CFU/mL of bacterial cells.

[1354] In one aspect, this disclosure provides a kit including acomposition as described herein and instructions, e.g., fordetecting or quantifying viable bacterial cells in a sample. Inanother aspect, this disclosure provides a device (e.g., aningestible device) including a composition as described herein,e.g., for detecting or quantifying viable bacterial cells in asample. The detection of live cells is the gold standard of viableplate counting and represents one of the advantages of exemplarycompositions and methods described herein.

[1355] In one aspect, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) obtaining a sample from the GI tract of the subject;(b) contacting the sample with a composition as described herein;(c) measuring total fluorescence or rate of change of fluorescenceas a function of time of the sample; and (d) correlating the totalfluorescence or the rate of change of fluorescence as a function oftime measured in step (c) to the number of viable bacterial cellsin the sample, wherein the number of the viable bacterial cellsdetermined in step (d) greater than about 10.sup.5 CFU/mL indicatesa need for treatment, e.g., with an antibiotic agent as describedherein.

[1356] In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured over multiple time points for an extended period of timein step (c). For instance, in some embodiments, the totalfluorescence or the rate of change of fluorescence as a function oftime of the sample is measured continuously for a period of 0-1800minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220minutes In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured continuously for a period of 0-330 minutes. In someembodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver.

[1357] In some embodiments, a control may be used in the method ofassessing the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the GI tract. Such a control maybe a positive control, e.g., a composition as described hereinfurther including a known number of viable bacterial cells. In someembodiments, the control may be a negative control, e.g., acomposition as described herein that has not been contacted withany viable bacterial cells. In some embodiments, this disclosureprovides a method of assessing or monitoring the need to treat asubject suffering from or at risk of overgrowth of bacterial cellsin the GI tract, including: (a) obtaining a sample from the GItract of the subject; (b) contacting the sample with a compositionas described herein; (c) measuring total fluorescence of thesample; (d) comparing the total fluorescence measured in step (c)to the total fluorescence produced by a control as describedherein; and (e) correlating the comparative fluorescence determinedin step (d) to the number of viable bacterial cells in the sample,wherein the number of the viable bacterial cells determined in step(e) greater than about 10.sup.5 CFU/mL indicates a need fortreatment, e.g., with an antibiotic agent as described herein.

[1358] In some embodiments, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) obtaining a sample from the GI tract of the subject;(b) contacting the sample with a composition as described herein;(c) measuring rate of change of fluorescence as a function of timeof the sample; (d) comparing the rate of change of fluorescence asa function of time measured in step (c) to the rate of change offluorescence as a function of time produced by a control asdescribed herein; and (e) correlating the comparative rate ofchange of fluorescence as a function of time determined in step (d)to the number of viable bacterial cells in the sample. The numberof the viable bacterial cells determined in step (e) greater thanabout 10.sup.5 CFU/mL indicates a need for treatment, e.g., with anantibiotic agent as described herein.

[1359] In some embodiments of the method, the control may be (1) acomposition identical to the one used in step (b) but has not beencontacted with any viable bacterial cells; or (2) a compositionidentical to the one used in step (b) further including a knownnumber of viable bacterial cells (e.g., a composition identical tothe one used in step (b) further including 10.sup.2, 10.sup.3,10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 CFU/mL of bacterialcells). In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured over multiple time points for an extended period of timein step (c), thereby detecting viable bacterial cells in thesample. For instance, in some embodiments, the total fluorescenceor the rate of change of fluorescence as a function of time of thesample is measured continuously for a period of 0-1800 minutes,0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes,0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the sample is measuredcontinuously for a period of 0-330 minutes. In some embodiments,the rate of change of fluorescence as a function of time of thesample measured over multiple time points is determined andcompared to the rate of change of fluorescence as a function oftime of a control measured over the same time points to determinethe number of viable bacterial cells in the sample. In someembodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver. In some embodiments, the method may befurther used to monitor the subject after the treatment (e.g., withan antibiotic). In some embodiments, the method may be used toassess the efficacy of the treatment. For example, efficacioustreatment may be indicated by the decrease of the number of viablebacterial cells in a sample from the GI tract of the subjectpost-treatment. Efficacy of the treatment may be evaluated by therate of decrease of the number of viable bacterial cells in asample from the GI tract of the subject post-treatment. In someembodiments, the method may be used to detect infection withantibiotic-resistant strains of bacteria in a subject. Forinstance, such infection may be indicated where the number ofviable bacterial cells in a sample from the GI tract of the subjectdoes not substantially decrease after antibiotic treatment.

[1360] In one aspect, the present disclosure provides a member madeof an absorptive material (e.g., an absorptive sponge) havingabsorbed therein a composition (e.g., a composition as describedherein) including a dye and a reagent for selective lysis ofeukaryotic cells. In some embodiments, the absorptive sponge is ahydrophilic sponge. In some embodiments, the absorptive sponge isselected from the group consisting of: fibers of cotton, rayon,glass, polyester, polyethylene, polyurethane, nitrocellulose, andthe like. In some embodiments, the absorptive sponge is polyesteror polyethylene. In some embodiments, the absorptive sponge isselected from the group consisting of: Ahlstrom Grade 6613H, Porex1/16'' Fine Sheet 4897, Porex 1/8'' Fine Sheet 4898, Porex 45880.024'' Conjugate release pad, Porex PSU-567, and Filter Papers. Insome embodiments, the absorptive sponge is Ahlstrom Grade 6613H(Lot 150191) or Porex PSU-567.

[1361] The present disclosure further provides a method forpreparing an absorptive sponge as described herein, including thestep of injecting into the absorptive sponge an aqueous solutionincluding a composition of the present disclosure. In someembodiments, the method including a step of drying the absorptivesponge having absorbed therein the aqueous solution at atemperature in the range of 0-100.degree. C., 0-50.degree. C.,0-40.degree. C., 0-30.degree. C., 0-20.degree. C., 0-10.degree. C.,or 0-4.degree. C.), for a time period sufficient to reduce thetotal water content to below 50%, 40%, 30%, 20%, 15%, 10%, 7%, 5%,3%, 1%, 0.7%, 0.5%, 0.3%, or 0.1% by weight.

[1362] In some embodiments, the absorptive sponge of thisdisclosure are suitable for use in a kit or device, e.g., fordetecting or quantifying viable bacterial cells in a sample. Insome embodiments, such a device is an ingestible device fordetecting or quantifying viable bacterial cells in vivo (e.g., inthe GI tract).

[1363] In one aspect, this disclosure provides a method fordetecting the presence of viable bacterial cells in a sample,including: (a) fully or partially saturating (e.g., at 50% or halfsaturation) an absorptive sponge as described herein, or anabsorptive sponge prepared according to a method as describedherein, with the sample; and (b) measuring total fluorescence orrate of change of fluorescence as a function of time of the fullyor partially saturated sponge (e.g., 50% or half-saturated)prepared in step (a), thereby detecting viable bacterial cells inthe sample.

[1364] In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sponge ismeasured over multiple time points for an extended period of timein step (b), thereby detecting viable bacterial cells in thesample. For instance, in some embodiments, the total fluorescenceor the rate of change of fluorescence as a function of time of thesample is measured continuously for a period of 0-1000 minutes,0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600 minutes, 0-500minutes, 0-400 minutes, 0-350 minutes, or 0-330 minutes. In someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the sample is measuredcontinuously for a period of 0-330 minutes. In some embodiments,the method further includes correlating the total fluorescence orthe rate of change of fluorescence as a function of time determinedin step (b) to the number of viable bacterial cells in the sample.In some embodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver.

[1365] In some embodiments, a control may be employed in the methodas described herein. Such a control may be a positive control,e.g., an absorptive sponge as described herein further including aknown number of viable bacterial cells. In some embodiments, thecontrol may be a negative control, e.g., an absorptive sponge asdescribed herein that has not been contacted with any viablebacterial cells.

[1366] In some embodiments, this disclosure provides a method fordetecting the presence of viable bacterial cells in a sample,including: (a) fully or partially saturating (e.g., at 50% or halfsaturation) an absorptive sponge as described herein, or anabsorptive sponge prepared according to a method as describedherein, with the sample; (b) measuring total fluorescence or rateof change of fluorescence as a function of time of the fully orpartially saturated sponge (e.g., at 50% or half saturation)prepared in step (a); and (c) comparing the total fluorescencemeasured in step (b) to the total fluorescence produced by acontrol as described herein, or comparing the rate of change offluorescence as a function of time measured in step (b) to the rateof change of fluorescence as a function of time produced by acontrol as described herein, thereby detecting viable bacterialcells.

[1367] In some embodiments of the method, the control may be (1) anabsorptive sponge identical to the one used in step (a) that hasnot been contacted with any viable bacterial cells, or (2) anabsorptive sponge identical to the one used in step (a) and isfully or partially saturated with a solution including a knownnumber of viable bacterial cells (e.g., an absorptive spongeidentical to the one used in step (a) further including 10.sup.2,10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 CFU/mL ofbacterial cells). In some embodiments, the total fluorescence orthe rate of change of fluorescence as a function of time of thesponge is measured over multiple time points for an extended periodof time in step (b), thereby detecting viable bacterial cells inthe sample. For instance, in some embodiments, the totalfluorescence or the rate of change of fluorescence as a function oftime of the sample is measured continuously for a period of 0-1800minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220minutes. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured continuously for a period of 0-330 minutes. In someembodiments, the method further includes correlating thecomparative total fluorescence determined in step (c) to the numberof viable bacterial cells in the sample. In some embodiments, therate of change of fluorescence as a function of time of the fullyor partially saturated sponge measured over multiple time points isdetermined and compared to the rate of change of fluorescence as afunction of time of a control measured over the same time points todetermine the number of viable bacterial cells in the sample. Insome embodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver.

[1368] In some embodiments, methods as described herein are highlysensitive in detecting and quantifying viable bacterial cells invarious samples. In some embodiments, the lowest detection orquantification limit of the present methods is 10.sup.2 CFU/mL. Insome embodiments, the highest detection or quantification limit ofthe present methods is 10.sup.7 CFU/mL, 10.sup.8 CFU/mL, 10.sup.9CFU/mL, 10.sup.1.degree. CFU/mL or more. In some embodiments, themethods allow detection or quantification of 10.sup.2 to 10.sup.7CFU/mL bacterial cells in various samples. In some embodiments,methods of this disclosure may be used to distinguish samples baseson the quantity of viable bacterial cells contained therein. Forinstance, the methods may be used to distinguish among samples thatcontain 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, or10.sup.7 CFU/mL of bacterial cells.

[1369] In one aspect, this disclosure provides a kit including anabsorptive sponge as described herein and instructions, e.g., fordetecting or quantifying viable cells using the absorptive sponge.In another aspect, this disclosure provides a device (e.g., aningestible device) including an absorptive sponge as describedherein, e.g., for detecting or quantifying viable bacterial cellsin a sample.

[1370] In one aspect, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) obtaining a sample from the GI tract of the subject;(b) fully or partially saturating (e.g., at 50% or half saturation)an absorptive sponge as described herein, or an absorptive spongeprepared according to a method as described herein, with thesample; (c) measuring total fluorescence or rate of change offluorescence as a function of time of the fully or partiallysaturated sponge (e.g., at 50% or half saturation) prepared in step(b); and (d) correlating the total fluorescence or the rate ofchange of fluorescence as a function of time measured in step (c)to the number of viable bacterial cells in the sample, wherein thenumber of the viable bacterial cells determined in step (d) greaterthan about 10.sup.5 CFU/mL indicates a need for treatment, e.g.,with an antibiotic agent as described herein.

[1371] In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the fully orpartially saturated sponge is measured over multiple time pointsfor an extended period of time in step (c). For instance, in someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the fully or partiallysaturated sponge is measured continuously for a period of 0-1800minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220minutes. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the fully orpartially saturated sponge is measured continuously for a period of0-330 minutes. In some embodiments, the method does not require exvivo plating or culturing. In some embodiments, the method does notrequire aspiration. In some embodiments, the method is performed invivo (e.g., in an ingestible device in vivo).

[1372] In some embodiments, a control may be used in the method ofassessing the need to treat a subject suffering from or at risk ofovergrowth of bacterial cells in the GI tract. Such a control maybe a positive control, e.g., an absorptive sponge as describedherein further including a known number of viable bacterial cells.In some embodiments, the control may be a negative control, e.g.,an absorptive sponge as described herein that has not beencontacted with any viable bacterial cells.

[1373] In some embodiments, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) obtaining a sample from the GI tract of the subject;(b) fully or partially saturating (e.g., at 50% or half saturation)an absorptive sponge as described herein, or an absorptive spongeprepared according to a method as described herein, with thesample; (c) measuring total fluorescence of the fully or partiallysaturated sponge (e.g., at 50% or half saturation) prepared in step(b); (d) comparing the total fluorescence measured in step (c) tothe total fluorescence produced by a control as described herein;and (e) correlating the comparative fluorescence determined in step(d) to the number of viable bacterial cells in the sample, whereinthe number of the viable bacterial cells determined in step (e)greater than about 10.sup.5 CFU/mL indicates a need for treatment,e.g., with an antibiotic agent as described herein.

[1374] In some embodiments, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) obtaining a sample from the GI tract of the subject;(b) fully or partially saturating (e.g., at 50% or half saturation)an absorptive sponge as described herein, or an absorptive spongeprepared according to a method as described herein, with thesample; (c) measuring rate of change of fluorescence as a functionof time of the fully or partially saturated sponge (e.g., at 50% orhalf saturation) prepared in step (b); (d) comparing the rate ofchange of fluorescence as a function of time measured in step (c)to the rate of change of fluorescence as a function of timeproduced by a control as described herein; and (e) correlating thecomparative rate of change of fluorescence as a function of timedetermined in step (d) to the number of viable bacterial cells inthe sample, wherein the number of the viable bacterial cellsdetermined in step (e) greater than about 10.sup.5 CFU/mL indicatesa need for treatment, e.g., with an antibiotic agent as describedherein.

[1375] In some embodiments of the method, the control may be (1) anabsorptive sponge identical to the one used in step (a) that hasnot been contacted with any viable bacterial cells, or (2) anabsorptive sponge identical to the one used in step (a) and isfully or partially saturated with a solution including a knownnumber of viable bacterial cells (e.g., a composition identical tothe one used in step (b) further including 10.sup.2, 10.sup.3,10.sup.4, 10.sup.5, 10.sup.6, or 10.sup.7 CFU/mL of bacterialcells). In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sponge ismeasured over multiple time points for an extended period of timein step (c), thereby detecting viable bacterial cells in thesample. For instance, in some embodiments, the total fluorescenceor the rate of change of fluorescence as a function of time of thesample is measured continuously for a period of 0-1800 minutes,0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320 minutes,0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700 minutes, 0-600minutes, 0-500 minutes, 0-400 minutes, 0-350 minutes, 0-330minutes, 0-300 minutes, 0-270 minutes, or 0-220 minutes. In someembodiments, the total fluorescence or the rate of change offluorescence as a function of time of the sample is measuredcontinuously for a period of 0-330 minutes. In some embodiments,the rate of change of fluorescence as a function of time of thefully or partially saturated sponge measured over multiple timepoints is determined and compared to the rate of change offluorescence as a function of time of a control measured over thesame time points to determine the number of viable bacterial cellsin the sample. In some embodiments, the method does not require exvivo plating or culturing. In some embodiments, the method does notrequire aspiration. In some embodiments, the method is performed invivo (e.g., in an ingestible device in vivo). In some embodiments,the method includes communicating the results of the onboardassay(s) to an ex vivo receiver. In some embodiments, the methodmay be further used to monitor the subject after the treatment(e.g., with an antibiotic). In some embodiments, the method may beused to assess the efficacy of the treatment. For example,efficacious treatment may be indicated by the decrease of thenumber of viable bacterial cells in a sample from the GI tract ofthe subject post-treatment. Efficacy of the treatment may beevaluated by the rate of decrease of the number of viable bacterialcells in a sample from the GI tract of the subject post-treatment.In some embodiments, the method may be used to detect infectionwith antibiotic-resistant strains of bacteria in a subject. Forinstance, such infection may be indicated where the number ofviable bacterial cells in a sample from the GI tract of the subjectdoes not substantially decrease after antibiotic treatment.

[1376] In some embodiments, fluorescence intensity is measured withan optical reader. The actual configuration and structure of theoptical reader may generally vary as is readily understood by thoseskilled in the art. Typically, the optical reader contains anillumination source that is capable of emitting light at a definedwavelength and a detector that is capable of registering a signal(e.g., transmitted, reflected, or fluorescence light). Opticalreaders may generally employ any known detection technique,including, for instance, luminescence (e.g., fluorescence,phosphorescence, etc.), absorbance (e.g., fluorescent ornon-fluorescent), diffraction, etc. Exemplary optical readers,illumination sources and detectors are disclosed in U.S. Pat. No.7,399,608, which is hereby incorporated by reference herein in itsentirety.

[1377] In some embodiments, the illumination source may be anydevice known in the art that is capable of providingelectromagnetic radiation, such as light in the visible ornear-visible range (e.g., infrared or ultraviolet light). Forexample, suitable illumination sources that may be used in thepresent disclosure include, but are not limited to, light emittingdiodes (LED), flashlamps, cold-cathode fluorescent lamps,electroluminescent lamps, and so forth. The illumination may bemultiplexed and/or collimated. In some embodiments, theillumination may be pulsed to reduce any background interference.In some embodiments, filters may be used to improve optics. See,e.g., Reichman, Jay, Handbook of optical filters for fluorescencemicroscopy, Chroma Technology Corporation (2000). In someembodiments, excitation source may be a LED with a band-passfilter, e.g., a filter for 500 nm+/-10 nm wavelength to selectivelyexcite a sample with 500 nm light. In some embodiments, to cut outany stray longer wavelengths from the green LED, a ThorlabsFESH0550 shortpass filter may be used for excitation (FIG. 72A). Insome embodiments, the emission from a sample is captured at a90.degree. angle with an avalanche photodiode detector with abandpass filter, e.g., a filter for 590 nm+/-20 nm wavelength,placed in front of the detector, to selectively capture lightemitted at 590 nm. In some embodiments, a Thorlabs FB580-10bandpass filter may be used as an emission filter (FIG. 72B). Across sectional view of an exemplary fluorescent assay test fixturedepicting collimating, focusing, and filtering lenses is shown inFIG. 72C. In some embodiments, a 5-50 nanosecond delay may be usedbefore emission is measured. Typical fluorophore used for timedelayed fluorescence are lanthanide metal chelates (Europium,Samarium, Terbium, etc), ruthenium complexes and others known inthe art. In some embodiments, illumination may be continuous or maycombine continuous wave (CW) and pulsed illumination where multipleillumination beams are multiplexed (e.g., a pulsed beam ismultiplexed with a CW beam), permitting signal discriminationbetween a signal induced by the CW source and a signal induced bythe pulsed source. For example, in some embodiments, LEDs (e.g.,aluminum gallium arsenide red diodes, gallium phosphide greendiodes, gallium arsenide phosphide green diodes, or indium galliumnitride violet/blue/ultraviolet (UV) diodes) are used as the pulsedillumination source. In some embodiments, the illumination sourcemay provide diffuse illumination to the dye. For example, an arrayof multiple point light sources (e.g., LEDs) may simply be employedto provide relatively diffuse illumination. In some embodiments,the illumination source is capable of providing diffuseillumination in a relatively inexpensive manner is anelectroluminescent (EL) device. An EL device is generally acapacitor structure that utilizes a luminescent material (e.g.,phosphor particles) sandwiched between electrodes, at least one ofwhich is transparent to allow light to escape. Disclosure of avoltage across the electrodes generates a changing electric fieldwithin the luminescent material that causes it to emit light.

[1378] In some embodiments, the detector may be any device known inthe art that is capable of sensing a signal. In some embodiments,the detector may be an electronic imaging detector that isconfigured for spatial discrimination. Some examples of suchelectronic imaging sensors include high speed, linearcharge-coupled devices (CCD), charge-injection devices (CID),complementary-metal-oxide-semiconductor (CMOS) devices, and soforth. Such image detectors, for instance, are generallytwo-dimensional arrays of electronic light sensors, although linearimaging detectors (e.g., linear CCD detectors) that include asingle line of detector pixels or light sensors, such as, forexample, those used for scanning images, may also be used. Eacharray includes a set of known, unique positions that may bereferred to as "addresses." Each address in an image detector isoccupied by a sensor that covers an area (e.g., an area typicallyshaped as a box or a rectangle). This area is generally referred toas a "pixel" or pixel area. A detector pixel, for instance, may bea CCD, CID, or a CMOS sensor, or any other device or sensor thatdetects or measures light. The size of detector pixels may varywidely, and may in some cases have a diameter or length as low as0.2 micrometers.

[1379] In other embodiments, the detector may be a light sensorthat lacks spatial discrimination capabilities. For instance,examples of such light sensors may include photomultiplier devices,photodiodes, such as avalanche photodiodes or silicon photodiodes,and so forth. Silicon photodiodes are sometimes advantageous inthat they are inexpensive, sensitive, capable of high-speedoperation (short risetime/high bandwidth), and easily integratedinto most other semiconductor technology and monolithic circuitry.In addition, silicon photodiodes are physically small, whichenables them to be readily incorporated into various types ofdetection systems. If silicon photodiodes are used, then thewavelength range of the emitted signal may be within their range ofsensitivity, which is 400 to 1100 nanometers. In some embodiments,a photomultiplier may be used to increase the intensity of thesignal.

[1380] In another aspect, the present disclosure providesingestible devices containing a microscopic evaluation system. Insome embodiments, bacterial cells in a sample may be first labeledwith fluorescent dyes (such as those described herein), and thefluorescently-labeled cells may be imaged and counted by themicroscopic evaluation using an ingestible device as describedherein. In other embodiments, the fluorescently-labeled cells arecounted as they pass through an onboard flow system (e.g.,microfluidic single cell channeling). Examples of flow cytometrysystems include hydrodynamic focusing, small diameter capillarytube flow, and rectangular capillary tube flow. As describedherein, live bacteria cells are labeled, and the principles of flowcytometry are used to quantify labeled cells. Generally speaking,the photons from an incident laser beam are absorbed by thefluorophore and raised to a higher, unstable energy level. Withinless than a nanosecond, the fluorophore re-emits the light at alonger representative wavelength where it is passed through aseries of dichroic filters. This reemitted light can be collectedand interpreted as proportional to the number of labeled bacteriacells. In some embodiments, a sheath fluid is not used as part ofthe flow system to help accommodate the volume restrictions of thedevice. In some embodiments, a rectangular capillary tube is usedto achieve a sufficiently large cross-sectional area and relativelythin inspection area. The flow cytometry optical system operatesparallel to the fluidics system and serves to observe theredirection of light passing through the cell and deliversinformation about the bacterial cells. In some embodiments, ratherthan using a conventional laser and spherical lenses to focus thelight to a point, an LED and cylindrical lenses are used to focusthe light to a line across a rectangular capillary tube. In otherembodiments, collimating lenses are used to make the light sourceparallel, while cylindrical lenses are used to refine theinspection area. An exemplary optical configuration for thisarrangement can be seen in FIG. 30. In some embodiments, opticalfilters can be added to permit the use of fluorophores. Thecharacteristic wavelength of reemitted light from the fluorophorescan be isolated and detected with the use of dichroic, bandpass,and short or long wave pass filters. Generally, multiple dichroiclenses and photomultipliers are used, however, due to spacelimitations, only a single side-scatter detector and forwardscatter detector may be used in certain embodiments.

[1381] One of the design challenges of integrating flow cytometryinto the device is to provide a pumping mechanism. Without movingfluid, individual bacteria cells cannot be identified and accountedfor by flow cytometry within a fixed volume of fluid. In someembodiments, a gear motor is to move fluid through the device. Forexample, a micromotor including a planetary gearhead (e.g., with a25:1 reduction) can provide the desired amount of torque to createfluid flow. In another embodiment, a series of piezoelectricresistors embedded in the surface of a microfabricated plate isused to create flow. In yet another embodiment, a micropump thatincludes a pair of one-way valves and uses a magnetic pump membraneactuated by an external magnetic field is used to create flow.

[1382] In some embodiments, the system architecture includes anopening and sealing mechanism combined with a rotary wiper whichcreates a pressure driven flow via a gear motor. The gear motor canbe used for other functions in the device. As shown in FIG. 31, thecomponents of the optics and flow chamber systems fit within thedevice. In some embodiments, the sample fluid is absorbed via aflexible membrane at the top of the capsule. In some embodiments,the gear motor has 270.degree. of permissible travel which servesto open and fill the fluid chamber. During closure, the motorcloses the ingress port while simultaneously pushing the fluidthrough the rectangular capillary tube where the optical system islocated. The threaded component allows the flexible membrane toclose and seal the ingress channel without changing the wiperheight. In some embodiments, the volume of the sample chamber is 25.mu.L, 50 .mu.L, 75 .mu.L or more. In some embodiments, two or moresamples are taken from the GI tract to procure a sufficient samplesize. Referring to FIG. 31, an LED on the left side of thecapillary tube and the two low-light detectors on the right forcapturing forward and side scatter are shown. Once the fluid passesthrough the capillary tube, it exits the capsule via a one-wayvalve. In certain embodiments, the flow system allows for thedetection of cell size and internal cell complexity, in addition tocell quantitation.

[1383] In some embodiment, the ingestible devices as describedherein may be used to analyze samples (e.g., samples from the GItract) to detect or quantify viable bacterial cells in a sample. Insome embodiments, the devices of this disclosure may be used tomeasure the concentration of viable bacteria in specific regions ofthe GI tract. Such data may be used to determine whether a subjecthas a condition in need for treatment, such as an infection, SmallIntestinal Bacterial Overgrowth (SIBO), or a SIBO-relatedcondition, or to quantify bacterial populations within the GI tract(or within specific regions of the GI tract) for other diagnosticpurposes.

[1384] An ingestible device used in a live cell dye method can beconfigured so that one or more than one samples may beanalyzed.

[1385] As an example, in some embodiments, the ingestible devicehas only one sample chamber. In such embodiments, the chamber canbe used to analyze one sample. In certain embodiments, aningestible device having a single sample chamber can be used toanalyze multiple different embodiments. The sample chamber mayinclude a sponge that is used such that the different samples areanalyzed at different points in time, such as, for example, takenat different locations within the GI tract (e.g., duodenum,jejunum, ileum) as the device passes through the GI tract. Forexample, a given sponge may be contacted multiple times and usedfor analyte detection. In some embodiments, the sponge may becontacted with non-saturating amounts of sample multiple times andused for analyte detection. Alternatively or additionally, thesample chamber may be used with multiple dyes (e.g., used in asingle reaction) that are detectable at different wavelengths.Multiple analytes can be detected, for example, using differentantibodies (e.g., detecting fluorescence at differentwavelengths)

[1386] As another example, in certain embodiments, the ingestibledevice has multiple chambers for analyzing samples. In suchembodiments, each chamber can be used to analyze different samples.Features noted in the preceding paragraph may be implemented withan ingestible device having multiple sample chambers.

[1387] In some embodiments, data may be generated after theingestible device has exited the subject, or the data may begenerated in vivo and stored on the device and recovered ex vivo.Alternatively, the data can be transmitted wirelessly from theingestible device while the device is passing through the GI tractof the subject.

[1388] In one aspect, this disclosure provides a method fordetecting the presence of viable bacterial cells in a sample,including: (a) providing an ingestible device as described herein;(b) transferring a fluid sample from the GI tract of a subject intoa sampling chamber of the ingestible device in vivo; and (c)detecting the presence of viable bacterial cells in the fluidsample (e.g., in vivo).

[1389] In some embodiments, the method for detecting the presenceof viable bacterial cells in a sample includes: (a) providing aningestible device as described herein; (b) transferring a fluidsample from the GI tract of a subject into a sampling chamber ofthe ingestible device in vivo, wherein the sampling chamber of thedevice is configured to hold an absorptive sponge as describedherein, or an absorptive sponge prepared according to the methodfor preparing an absorptive sponge as described herein; (c) fullyor partially saturating (e.g., at 50% or half saturation) theabsorptive sponge with the fluid sample; and (d) measuring totalfluorescence or rate of change of fluorescence as a function oftime of the fully or partially saturated sponge (e.g., 50% orhalf-saturated) prepared in step (c), thereby detecting viablebacterial cells in the sample (e.g., in vivo).

[1390] In some embodiments, the method described herein furtherincludes a step of calibrating the ingestible device, wherein thefluorescent properties of the absorptive sponge contained in thesampling chamber of the device are determined prior to theintroduction of the sample. In some embodiments, each ingestibledevice is calibrated by measuring the fluorescence of theabsorptive sponge held in the sampling chamber of the device andcomparing the measured florescence to a positive or negativecontrol as described herein. In some embodiments, each ingestibledevice is calibrated by measuring the fluorescence of theabsorptive sponge held in the sampling chamber of the device toprovide a baseline fluorescence. In some embodiments, the baselinefluorescence should be within a set number (900+/-450 FU). In someembodiments, a subset of ingestible devices may be treated with 0,10.sup.4, 10.sup.5, 10.sup.6 and 10.sup.7 CFU bacteria in duodenalor jejunal aspirates to generate a calibration curve. Thecalibration curve may be stored and used to quantify bacteria inall the devices in the batch. See, e.g., David Wild,Standardization and Calibration, The Immunoassay Handbook, GulfProfessional Publishing, 2005. In some embodiments, the totalfluorescence or the rate of change of fluorescence as a function oftime of the sponge is measured over multiple time points for anextended period of time in step (d), thereby detecting viablebacterial cells in the sample. For instance, in some embodiments,the total fluorescence or the rate of change of fluorescence as afunction of time of the sample is measured continuously for aperiod of 0-1800 minutes, 0-1600 minutes, 0-1500 minutes, 0-1440minutes, 0-1320 minutes, 0-1000 minutes, 0-900 minutes, 0-800minutes, 0-700 minutes, 0-600 minutes, 0-500 minutes, 0-400minutes, 0-350 minutes, 0-330 minutes, 0-300 minutes, 0-270minutes, or 0-220 minutes. In some embodiments, the totalfluorescence or the rate of change of fluorescence as a function oftime of the sample is measured continuously for a period of 0-330minutes. In some embodiments, the method further includescorrelating the total fluorescence or the rate of change offluorescence as a function of time determined in step (d) to thenumber of viable bacterial cells in the sample. In someembodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in theingestible device in vivo.

[1391] In some embodiments, a control may be employed in the methodas described herein. Such a control may be an internal control(e.g., the fluorescence coming from resorufin impurity in resazruin(900+/-450 FU) in the sponge may be used as an internal control foroptics and amount of dye in the sponge). In some embodiments, eachingestible device as described herein is individually calibratedwherein the fluorescent properties of the absorptive spongecontained in the sampling chamber of the device are determinedprior to the introduction of sample.

[1392] In some embodiments, methods as described herein are highlysensitive in detecting and quantifying viable bacterial cells invarious samples. In some embodiments, the lowest detection orquantification limit of the present methods is 10.sup.2 CFU/mL. Insome embodiments, the highest detection or quantification limit ofthe present methods is 10.sup.7 CFU/mL, 10.sup.8 CFU/mL, 10.sup.9CFU/mL, 10.sup.1.degree. CFU/mL or more. In some embodiments, themethods allow detection or quantification of 10.sup.2 to 10.sup.7CFU/mL bacterial cells in various samples. In some embodiments,methods of this disclosure may be used to distinguish samples baseson the quantity of viable bacterial cells contained therein. Forinstance, the methods may be used to distinguish among samples thatcontain 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, or10.sup.7 CFU/mL of bacterial cells.

[1393] In one aspect, this disclosure provides a method ofassessing or monitoring the need to treat a subject suffering fromor at risk of overgrowth of bacterial cells in the GI tract,including: (a) providing an ingestible device as described herein;(b) transferring a fluid sample from the GI tract of a subject intoa sampling chamber of the ingestible device in vivo; and (c)quantifying viable bacterial cells present in the fluid sample(e.g., in vivo), wherein the number of the viable bacterial cellsdetermined in step (c) greater than about 10.sup.5 CFU/mL indicatesa need for treatment, e.g., with an antibiotic agent as describedherein.

[1394] In some embodiments, the method of assessing or monitoringthe need to treat a subject suffering from or at risk of overgrowthof bacterial cells in the GI tract includes: (a) providing aningestible device as described herein; (b) transferring a fluidsample from the GI tract of a subject into a sampling chamber ofthe device in vivo, wherein the sampling chamber of the device asdescribed herein is configured to hold an absorptive sponge asdescribed herein, or an absorptive sponge prepared according to themethod for preparing an absorptive sponge as described herein; (c)fully or partially saturating (e.g., at 50% or half saturation) theabsorptive sponge in the sampling chamber with the fluid sample;(d) measuring total fluorescence of the fully or partiallysaturated sponge (e.g., at 50% or half saturation) prepared in step(c); and (e) correlating the total fluorescence measured in step(d) to the number of viable bacterial cells in the sample, whereinthe number of the viable bacterial cells determined in step (e)greater than about 10.sup.5 CFU/mL indicates a need for treatment,e.g., with an antibiotic agent as described herein.

[1395] In some embodiments, the method of assessing or monitoringthe need to treat a subject suffering from or at risk of overgrowthof bacterial cells in the GI tract includes: (a) providing aningestible device as described herein; (b) transferring a fluidsample from the GI tract of a subject into a sampling chamber ofthe device in vivo, wherein the sampling chamber of the device asdescribed herein is configured to hold an absorptive sponge asdescribed herein, or an absorptive sponge prepared according to themethod for preparing an absorptive sponge as described herein; (c)fully or partially saturating (e.g., at 50% or half saturation) theabsorptive sponge in the sampling chamber with the fluid sample;(d) measuring rate of change of fluorescence as a function of timeof the fully or partially saturated sponge (e.g., at 50% or halfsaturation) prepared in step (c); and (e) correlating the rate ofchange of fluorescence as a function of time measured in step (d)to the number of viable bacterial cells in the sample, wherein thenumber of the viable bacterial cells determined in step (e) greaterthan about 10.sup.5 CFU/mL indicates a need for treatment, e.g.,with an antibiotic agent as described herein.

[1396] In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sponge ismeasured over multiple time points for an extended period of timein step (d). For instance, in some embodiments, the totalfluorescence rate of change of fluorescence as a function of timeof the sample is measured continuously for a period of 0-1800minutes, 0-1600 minutes, 0-1500 minutes, 0-1440 minutes, 0-1320minutes, 0-1000 minutes, 0-900 minutes, 0-800 minutes, 0-700minutes, 0-600 minutes, 0-500 minutes, 0-400 minutes, 0-350minutes, 0-330 minutes, 0-300 minutes, 0-270 minutes, or 0-220minutes. In some embodiments, the total fluorescence or the rate ofchange of fluorescence as a function of time of the sample ismeasured continuously for a period of 0-330 minutes. In someembodiments, the method does not require ex vivo plating orculturing. In some embodiments, the method does not requireaspiration. In some embodiments, the method is performed in vivo(e.g., in an ingestible device in vivo). In some embodiments, themethod includes communicating the results of the onboard assay(s)to an ex vivo receiver. In some embodiments, the ingestible deviceand the method may be further used to monitor the subject after thetreatment (e.g., with an antibiotic). In some embodiments, theingestible device and the method may be used to assess the efficacyof the treatment. For example, efficacious treatment may beindicated by the decrease of the number of viable bacterial cellsin a sample from the GI tract of the subject post-treatment.Efficacy of the treatment may be evaluated by the rate of decreaseof the number of viable bacterial cells in a sample from the GItract of the subject post-treatment. In some embodiments, theingestible device and the method may be used to detect infectionwith antibiotic-resistant strains of bacteria in a subject. Forinstance, such infection may be indicated where the number ofviable bacterial cells in a sample from the GI tract of the subjectdoes not substantially decrease after antibiotic treatment.

[1397] In some embodiments, the compositions, methods and devicesdescribed herein may use a combination of (e.g., two or more)analyte-binding agents to detect, characterize and/or quantitatethe type of analyte (e.g., a microorganism and a protein, ametabolite) present in a sample. For example, in some embodiments,the compositions, methods and devices described herein may be usedto determine the types of microorganisms (e.g., bacteria,protozoans, or viruses) present in a sample. In some embodiments, afirst analyte-binding agent that binds to an analyte and comprisesa first fluorescent dye, may be used in combination with a secondanalyte-binding agent, wherein the second analyte-binding agentcomprises a second fluorescent dye that exhibits increasedfluorescence when spatially proximal to the first fluorescent dye.In some embodiments, spatial proximity between the firstfluourescent dye and the second fluorescent dye results in energytransfer from the first fluorescent dye to the second fluorescentdye. The detection of the fluorescence emitted by the secondfluorescent dye can be used, for example, to determine whether bothanalyte-binding agents are located in close proximity to each otherin the sample. Alternatively, a first analyte-binding agent thatbinds to an analyte and comprises a first fluorogenic dye may beused in combination with a second analyte-binding agent, whereinthe second analyte-binding agent comprises a second fluorescent dyethat exhibits increased fluorescence when spatially proximal to thefirst fluorogenic dye. In some embodiments, the first fluorogenicdye exhibits no fluorescence or reduce fluorescence when thefirst-analyte binding agent is not bound to the analayte. In someembodiments, the first fluorogenic dye exhibits increasedfluorescence upon binding of the first analyte-binding agent to theanalyte. In some embodiments, spatial proximity between the firstfluorogenic dye and the second fluorescent dye results in energytransfer from the first fluorogenic dye to the second fluorescentdye. The detection of the fluorescence emitted by the secondfluorescent dye can be used, for example, to determine whether bothanalyte-binding agents are located in close proximity to each otherin the sample. In some embodiments, the first and the secondanalyte-binding agents bind to the same region (e.g., epitope) ofthe analyte (e.g., a protein). For instance, in some embodiments,the first and the second analyte-binding agents comprise the sametype of analyte-binding moiety or reagent (e.g., the sameantibody). In some embodiments, the first and the secondanalyte-binding agents bind to separate regions (e.g., epitopes) ofthe analyte (e.g., a protein). In some embodiments, the first andthe second analyte-binding agents bind to the separate regions ofthe analyte (e.g., a protein) that do not spatially overlap. Insome embodiments, the first analyte-binding agent and the secondanalyte-binding agent are configured such that when bothanalyte-binding agents are bound to the analyte, their respectivedyes are in close proximity (e.g., allowing for energy transfer tooccur). In some embodiments, the first and/or second analytebinding agent(s) is an antigen-binding agent (e.g., an antibody).In some embodiments, the first and/or second analyte bindingagent(s) is an affimer. In some embodiments, the first and/orsecond analyte binding agent(s) is an antigen-binding agent is anaptamer.

[1398] In some embodiments, the compositions, methods and devicesdescribed herein make use of fluorescent oxygen channelingimmunoassay (FOCI) compositions and methods. FOCI is generallydescribed in U.S. Pat. Nos. 5,807,675; 5,616,719; and 7,635,571,the entire contents of which are expressly incorporated herein byreference. In some embodiments, a first analyte-binding agent thatis capable of binding to an analyte and comprises a photosensitizeris used in combination with a second analyte-binding agentcomprising a fluorogenic dye. In some embodiments, thephotosensitizer of the first analyte-binding agent generatessinglet oxygen in an excited state thereby causing the fluorogenicdye of the second analyte-binding agent to emit fluorescence uponreacting with the singlet oxygen. In some embodiments, the emittedfluorescence can be detected to, e.g., determine the presenceand/or absence of the analyte and/or to quantitate and/or analyzethe analyte in a sample. In some embodiments, the first and thesecond analyte-binding agents bind to the same region (e.g.,epitope) of the analyte (e.g., a protein). For example, in someembodiments, the first and the second analyte-binding agentscomprise the same type of analyte-binding moiety or reagent (e.g.,the same antibody). In some embodiments, the first and the secondanalyte-binding agents bind to separate regions (e.g., epitopes) ofthe analyte (e.g., a protein). In some embodiments, the first andthe second analyte-binding agents bind to the separate regions ofthe analyte (e.g., a protein) that do not spatially overlap. Insome embodiments, the first analyte-binding agent and the secondanalyte-binding agent are configured such that when bothanalyte-binding agents are bound to the analyte, the singlet oxygengenerated by photosensitizer of the first analyte-binding agent isin close proximity to the fluorogenic dye of the secondanalyte-binding agent. In some embodiments, the first and/or secondanalyte binding agent(s) is an antigen-binding agent (e.g., anantibody). In some embodiments, the first and/or second analytebinding agent(s) is an affimer. In some embodiments, the firstand/or second analyte binding agent(s) is an antigen-binding agentis an aptamer.

[1399] In some embodiments, the use of a combination ofanalyte-binding agents allows for the detection, analysis and/orquantitation of a multitude of analytes. Multiple combinations ofanalyte-binding agents may be used for the detection, analysisand/or quantitation of a complex mixture of analytes. For example,multiple analyte-binding agents having different dyes, and/oranalyte specificities may be used to analyze a sample. Forinstance, in order to detect different species of bacteria (ore.g., LTA vs. LPS) present in a sample, one can couple a live celldye (F1) as described herein to an antibody or to an antibioticthat is microorganism-specific (e.g., bacteria specific orbacterial species-specific). Antibodies that specifically bind to abiomolecule (e.g., a surface antigen) present in a microorganism(e.g., a bacteria) of a genus, species or strain of interest and donot cross-react with other microorganism biomolecules and/oreukaryotic biomolecules may also be used, including the exemplaryantibodies described herein. A second antibody or antibiotic havinga fluorescent dye (F2) that binds to the same microorganism andthat gets excited (via an energy transfer from F1 to F2) when inclose proximity to F1 may be employed to detect, analyze and/orquantitate the microorganism to which the antibodies and/orantibiotics bind. Two exemplary proximity assays are depicted inFIGS. 73A and 73B.

[1400] Analyte Diluting and Culturing

[1401] In some embodiments, the disclosure provides methods ofobtaining, culturing, and/or detecting cells and/or analytes invivo within the gastrointestinal (GI) tract or reproductive tractof a subject. Associated devices are also disclosed. The methodsand devices described provide a number of advantages for obtainingand/or analyzing fluid samples from a subject. In some embodiments,diluting the fluid sample increases the dynamic range of analytedetection and/or reduces background signals or interference withinthe sample. For example, interference may be caused by the presenceof non-target analytes or non-specific binding of a dye or labelwithin the sample. In some embodiments, culturing the sampleincreases the concentration of cells (e.g., a specific type ofcells) and/or analytes (e.g., a specific analyte of interest)produced by the cells thereby facilitating their detection and/orcharacterization. Above, various types of analytes are disclosedwhich may be detected and/or characterized as described herein.

[1402] In certain embodiments, the methods and devices a describedherein may be used to obtain information regarding bacterialpopulations in the GI tract of a subject. This has a number ofadvantages and is less invasive than surgical procedures such asintubation or endoscopy to obtain fluid samples from the GI tract.The use of an ingestible device as described herein also allows forfluid samples to be obtained and data to be generated on bacterialpopulations from specific regions of the GI tract.

[1403] In some embodiments, the methods and devices describedherein may be used to generate data such as by analyzing the fluidsample, dilutions thereof or cultured samples for one or more cellsand/or analytes. The data may include, but is not limited to, thetypes of bacteria present in the fluid sample or the concentrationof bacteria in specific regions of the GI tract. Such data may beused to determine whether a subject has an infection, such as SmallIntestinal Bacterial Overgrowth (SIBO), or to characterizebacterial populations within the GI tract for diagnostic or otherpurposes.

[1404] For example, in one aspect, the data may include, but is notlimited to, the concentration of bacteria in a specific region ofthe GI tract that is one or more of the duodenum, jejunum, ileum,ascending colon, transverse colon or descending colon. In oneaspect, the specific region of the GI tract is the duodenum. In oneaspect, the specific region of the GI tract is the jejunum. In oneaspect, the specific region of the GI tract is the ileum. In oneaspect, the specific region of the GI tract is the ascending colon.In one aspect, the specific region of the GI tract is thetransverse colon. In one aspect, the specific region of the GItract is the descending colon. In a related embodiment, the datamay be generated every one or more days to monitor diseaseflare-ups, or response to the therapeutic agents disclosedherein.

[1405] Data may be generated after the device has exited thesubject, or the data may be generated in vivo and stored on thedevice and recovered ex vivo. Alternatively, the data can betransmitted wirelessly from the device while the device is passingthrough the GI tract of the subject or in place within thereproductive tract of the subject.

[1406] In some embodiments, a method comprises: providing a devicecomprising one or more dilution chambers and dilution fluid;transferring all or part of a fluid sample obtained from the GItract or reproductive tract of the subject into the one or moredilution chambers in vivo; and combining the fluid sample and thedilution fluid to produce one or more diluted samples in the one ormore dilution chambers.

[1407] In certain embodiments, a method comprises: providing aningestible device comprising one or more dilution chambers;transferring all or part of a fluid sample obtained from the GItract into the one or more dilution chambers comprising sterilemedia; culturing the sample in vivo within the one or more dilutionchambers to produce one or more cultured samples; and detectingbacteria in the one or more cultured samples.

[1408] In some embodiments, a method comprises: providing a devicecomprising one or more dilution chambers; transferring all or partof a fluid sample obtained from the GI tract or reproductive tractinto the one or more dilution chambers; combining all or part ofthe fluid sample with a dilution fluid in the one or more dilutionchambers; and detecting the analyte in the one or more dilutedsamples.

[1409] In certain embodiments, a device comprises: one or moredilution chambers for diluting a fluid sample obtained from the GItract or reproductive tract; and dilution fluid for diluting thesample within the one or more dilution chambers.

[1410] In some embodiments, the device comprises: one or moredilution chambers for culturing a fluid sample obtained from the GItract; sterile media for culturing the sample within the one ormore dilution chambers; and a detection system for detectingbacteria.

[1411] In certain embodiments, a device comprises: one or moredilution chambers for culturing a fluid sample obtained from the GItract; sterile media for culturing the sample within the one ormore dilution chambers; and a detection system for detectingbacteria.

[1412] Also provided is the use of a device as described herein fordiluting one or more samples obtained from the GI tract orreproductive tract of a subject. In one embodiment, there isprovided the use of an ingestible device as described herein fordetecting cells and/or analytes in vivo within the gastrointestinal(GI) tract of a subject.

[1413] Further provided is a system comprising a device asdescribed herein and a base station. In one embodiment, the devicetransmits data to the base station, such as data indicative of theconcentration and/or types of bacteria in the GI tract of thesubject. In one embodiment, the device receives operatingparameters from the base station. Some embodiments described hereinprovide an ingestible device for obtaining one or more samples fromthe GI tract or reproductive tract of a subject and diluting and/orculturing all or part of the one or more samples. The ingestibledevice includes a cylindrical rotatable element having a port onthe wall of the cylindrical rotatable element. The ingestibledevice further includes a shell element wrapping around thecylindrical rotatable element to form a first dilution chamberbetween the cylindrical rotatable element and the shell element.The shell element has an aperture that exposes a portion of thewall of the cylindrical rotatable element to an exterior of theingestible device.

[1414] In some embodiments, the ingestible device includes one ormore dilution chambers for receiving a fluid sample from the GItract or reproductive tract of a subject or a dilution thereof. Insome embodiments, one or more dilutions of the fluid sample arecultured in one or more dilution chambers. In some embodiments, thedilution chambers each define a known volume, optionally the samevolume or different volumes. In some embodiments, the dilutionchambers define a fluid volume ranging from about 10 .mu.L to about1 mL. The dilution chambers may define a fluid volume less than orequal to about 500 .mu.L, less than or equal to about 250 .mu.L,less than or equal to about 100 .mu.L, or less than or equal toabout 50 pt. In some embodiments, the dilution chambers define afluid volume of greater than or equal to about 10 .mu.L, greaterthan or equal to about 20 .mu.L, greater than or equal to about 30.mu.L, or greater than or equal to about 50 .mu.L. In someembodiments, the dilution chambers define a fluid volume betweenabout 10 .mu.L and 500 .mu.L, between about 20 .mu.L and 250 .mu.L,between about 30 .mu.L and 100 .mu.L or about 50 pt.

[1415] In some embodiments, dilution fluid in the device iscombined with all or part of the fluid sample, or dilution thereof,to produce one or more dilutions. In some embodiments, the dilutionfluid is sterile media suitable for culturing one or more cellswithin the dilution chambers.

[1416] In some embodiments, the one or more dilution chambers maybe filled with the dilution fluid prior to a patient ingesting theingestible device. Alternatively, in another embodiment, thedilution fluid may be added into the one or more dilution chambersin vivo from a reservoir of the ingestible device. Sampling anddilution of the GI fluid sample may take place in vivo. Forexample, an actuator of the ingestible device may pump the dilutionfluid from the reservoir into a dilution chamber when it isdetermined that the ingestible device is located at a predeterminedlocation within the GI tract. In some embodiments, the dilutionchambers each contain a volume of sterile media suitable forculturing a fluid sample from the GI tract or reproductive tract.In some embodiments, the dilution chambers are at least 95%, atleast 97%, at least 98%, or at least 99% full of sterile media. Insome embodiments, the dilution chambers each contain oxygen tofacilitate aerobic bacteria growth. In another embodiment anon-dilution chamber includes oxygen and is added to one or more ofthe dilution chambers to facilitate aerobic bacteria growth.

[1417] In some embodiments, the culturing may take place in vivoimmediately after the GI fluid sample has been diluted. Oralternatively, the culturing may take place ex vivo, e.g., when theingestible device has been evacuated and recovered such that thedilution chamber containing the diluted GI fluid sample may beextracted and the culturing may be performed in a laboratory. Therecovery of the ingestible device may be performed in a similarmanner as embodiments described in U.S. Provisional Application No.62/434,188, filed on Dec. 14, 2016, which is herein expresslyincorporated by reference in its entirety.

[1418] In some embodiments, the dilution fluid includes one or moreagents for inhibiting the growth of fungus. In some embodiments,the anti-fungal agent is amphotericin B. In some embodiments, thedilution fluid contains about 2.5 mg/L of Amphotericin B.

[1419] In some embodiments, the media includes one or moreantimicrobial agents in order to determine antibioticsensitivity/resistance of bacteria within the fluid sample. Forexample, if bacteria grow in a dilution chamber containing mediawithout antibiotics but do not grow in a separate dilution chambercontaining media including antimicrobial agents, the sample may beidentified as containing bacteria sensitive to that antibiotic.Alternatively, if bacteria grow in both dilution chambers (with andwithout antibiotics), the sample can be identified as containingbacteria resistant to that antibiotic. In some embodiments, thebacteria remaining in the dilution chamber(s) are quantified using,e.g., any detection and/or quantification method described herein.In some embodiments, the presence and/or absence of a particulartype of bacteria in the dilution chamber (e.g., a bacteria of aparticular genus, species and/or strain) is detected and/orquantified using a method described herein.

[1420] In another embodiment, the dilution fluid includes asubstrate or reagent for measuring bacterial activity or response.For example, in some embodiments, the dilution fluid includes oneor more conjugated bile acids and deconjugated bile acids or areduction in conjugated bile acids are detected in the dilutedsamples and/or cultured samples as a sign of bile salt hydrolaseactivity. In another embodiment, the substrate may be an enzyme,for example, glutamate dehydrogenase (GDH). In the case of GDH, itmay be used to detect an antigen that is produced in high amountsby C. difficile, both toxin and non-toxin producing strains. Thistest indicates if C. difficile is present but not necessarily ifthe bacteria are producing toxins.

[1421] In another embodiment, products of the bacteria are detectedor measured while the bacteria are being cultured in the media. Forexample, Clostridium difficile toxin A can be measured to detect ifbacteria are producing toxins.

[1422] In some embodiments, the methods and devices describedherein may be used to obtain, dilute, culture and/or detecteukaryotic cells from the subject. For example, epithelial cells orPBMC's from the GI tract can be diluted or cultured in theingestible device. Optionally, the eukaryotic cells may be analyzedwithin the device and/or collected once the device has exited. Insome embodiments, the dilution fluid further includes a substrateor reagent for measuring eukaryotic activity or response in vivo.For example, in some embodiments, a biomarker in the sample may bedetected, analyzed and/or quantitated. The detection of thebiomarker in the sample may be used to diagnose or monitor adisease or disorder or the treatment thereof. Exemplary biomarkersare described in detail above, including biomarkers associated withGI disorders, inflammation, and cancer. In some embodiments, thebiomarker is present on a eukaryotic cell present in the sample. Insome embodiments one or more biomarkers associated withinflammation and/or cancer are detected within the diluted samplesand/or cultured samples. In some embodiments, measuring the amountof cell growth proteins or cell-cell adhesion proteins (e.g.,beta-catenin, ErbB1, EbrB2, ErbB3, pAkt, c-Met, p53) produced maybe correlated to the presence or absence of oncogenic cells, whilemeasuring cytokines (e.g., IL-6 and TNF-alpha) may be correlatedwith inflammation.

[1423] In some embodiments, a fluid sample is transferred from theGI tract into one or more dilution chambers while the ingestibledevice is passing through the GI tract of a subject in vivo. Bycontrolling when the fluid sample is transferred into the one ormore dilution chambers, it is possible to obtain a fluid samplefrom a particular region of the GI tract. In operation, theexterior of the ingestible device will be in contact withbiological fluids in the GI tract. As the ingestible device travelsalong the GI tract, it will typically be surrounded by fluid thatis characteristic of that section of the GI tract. For example,when the ingestible device is in the stomach, the device will be incontact with stomach fluid which may include gastric acid,digestive enzymes, and partially digested food. When the ingestibledevice is in the jejunum, the device will be in contact withjejunal fluid.

[1424] In some embodiments, the device has one or more ports,valves and/or pumps that are used, either alone or in combination,for controlling the transfer of fluid from the GI tract orreproductive tract into the one or more dilution chambers. Thedevice may also contain one or more ports, valves and/or pumps forcontrolling the transfer of fluid between dilution chambers withinthe device, optionally to produce a serial dilution of the originalfluid sample from the GI tract or reproductive tract. In someembodiments, the device described herein may be used for obtaining,diluting, culturing, or detecting cells from other parts of thebody, such as but not limited to the female reproductive tract,and/or the like. Compositions and methods for sampling the GI orreproductive tract are discussed in greater detail in U.S.Provisional Application No. 62/376,688 filed Aug. 18, 2016, whichis hereby incorporated by reference herein in its entirety.

[1425] In some embodiments, the device includes a microcontrollerfor controlling the one or more ports, valves and/or pumps. In someembodiments, the microcontroller is programmed to control theports, valves and/or pumps in response to data from the one or moreenvironmental sensors. Alternatively or in addition, themicrocontroller is programmed to control the ports, valves and/orpumps in response to wireless a signal from a base station or inresponse to a signal generated by the microcontroller, such as atimer.

[1426] In some embodiments the device includes a pump having aninput conduit on the exterior of the device and an output conduitinto a first dilution chamber. In some embodiments, the pump isoperational to transfer fluid from the GI tract into the firstdilution chamber. Preferably, the pump is controllable to transfera predetermined volume of liquid from the GI tract into a dilutionchamber. In some embodiments, the device includes a pumpcontrollable to transfer a predetermined volume of liquid betweendilution chambers, optionally the same pump or a different pump asused to transfer the fluid sample into the first dilution chamber.In some embodiments, the pump is a solenoid pump. In someembodiments, the pump is controllable to transfer a fluid volumefrom about 1 .mu.L to about 50 .mu.L, about 2 .mu.L to about 20.mu.L, about 3 .mu.L to about 15 pt, or about 5 .mu.L.

[1427] In another embodiment, the device includes a port forreceiving a fluid sample from the GI tract or reproductive tract.In some embodiments, the port is exposed to the exterior of thedevice. Optionally, the device includes a cover movable to exposethe port to the exterior of the device. In operation, when the portis exposed fluid from the GI tract or reproductive tract entersinto the port through surface tension, movement of the subjectand/or peristaltic effects. Optionally, the port is coated with ahydrophilic coating to encourage fluid to flow into the port.

[1428] In some embodiments, the port is movable from an openposition such that the port is exposed to the exterior of thedevice, to a first position such that the port is in fluidcommunication with a first dilution chamber. In some embodiments,moving the port from the open position to the first positiontransfers a predetermined volume of a fluid sample from the GItract or reproductive tract into the first dilution chamber. Forexample, in some embodiments the port defines a fluid volume ofabout 1 .mu.L to about 50 .mu.L, about 2 .mu.L to about 20 .mu.L,about 3 .mu.L to about 15 .mu.L, or about 5 .mu.L.

[1429] A skilled person will appreciate that when the port is influid communication with the first dilution chamber, the port andthe first dilution chamber will define a combined volume such thatany fluid in the port and dilution chamber will mix to form adilution. In some embodiments, the mixing of fluid will be enhancedthrough the peristaltic action of the GI tract as well as movementof the subject. In some embodiments, the first incubation chambercontains dilution fluid such as sterile media and moving the portto the first position produces a first dilution including apredetermined volume of fluid sample from the GI tract and apredetermined volume of dilution fluid. For example, in someembodiments the port has a fluid volume of 5 .mu.l and the firstdilution chamber has 45 .mu.L of dilution fluid, such that thefirst dilution is a 10 fold dilution of the fluid sample.

[1430] In some embodiments, the first dilution is cultured toproduce a single cultured sample and cells and/or analytes aredetected within the cultured sample. Alternatively, in someembodiments a portion of the first dilution is transferred to oneor more additional dilution chambers to produce a serial dilutionof the fluid sample from the GI tract. In some embodiments, theserial dilution is produced by controlling the transfer of fluidbetween the dilution chambers.

[1431] For example, in some embodiments the ingestible deviceincludes a port movable to sequentially align with one or moreadditional i dilution chambers, thereby transferring a portion offirst dilution of the fluid sample to each additional dilutionchamber. Alternatively or in addition, one or more pumps and/orvalves may be used to sequentially transfer a portion of the fluidsample or dilution thereof to each dilution chamber.

[1432] In some embodiments, the device includes a port movable froma first position in fluid communication with a first dilutionchamber to a second position such that the port is in fluidcommunication with a second dilution chamber. In some embodiments,the port is movable from the second position to a third positionsuch that the port is in fluid communication with a third dilutionchamber. In some embodiments, the port is movable from the thirdposition to a fourth position such that the port is in fluidcommunication with a fourth dilution chamber. In some embodiments,the port is movable from the fourth position to a fifth positionsuch that the port is in fluid communication with a fifth dilutionchamber. Optionally, the device may contain more than five dilutionchambers for producing more than five dilutions of the originalfluid sample. In some embodiments, the dilution chambers aresuitable for culturing the dilutions in vivo within the device.

[1433] In some embodiments, the port is a depression on the surfaceof a movable element. In some embodiments, the movable element iscoupled to an actuator for moving the port relative to thepositions of the one of more dilution chambers. Optionally, theactuator is an electric motor.

[1434] In some embodiments the port is a depression on the surfaceof a rotatable element. In some embodiments, the device includes anactuator coupled to the rotatable element for rotating the port toalign with one or more dilution chambers. In some embodiments, thedilution chambers are positioned circumferentially around the axisof rotation of the rotatable element. In some embodiments, rotatingthe rotatable element sequentially moves the port from the openposition to the first position and optionally one or more of thesecond position, third position and fourth position.

[1435] FIG. 27 shows one embodiment of a portion of an ingestibledevice 4000 with a port 4154b in an open position to the exteriorof the ingestible device 400. The ingestible device 400 may includea cylinder-shaped rotatable element 4150 that includes samplingports 4154a-b on the wall of the rotatable element 4150. Thesampling chamber 4150 is wrapped by a shell element 4140 withdividers to form a series of dilution chambers 4151a-n between theshell element 4140 and the rotatable element 4150. In operation,when the ingestible device 4000 determines the device itselfarrives at a target location within the GI tract, the rotatableelement 4150 may be rotated into an open position such that anaperture of the shell element 4140 is aligned with the port 4154bon the wall of the rotatable element 4150 and the port 4154b isexposed to the exterior of the ingestible device 4000 through theaperture. In this way, fluid from the GI tract can enter the port4154b and occupy the volume defined by the port 4154b. In theembodiment shown in FIG. 27, the port 4154b may be a depression onthe surface of a rotatable element 4150 and a number of dilutionchambers 4151a-n are positioned circumferentially around the axisof rotation of the rotatable element 4150. As previously discussed,each of the dilution chambers 4151a-n may store a dilution fluid.In some embodiments, the depression is a cylindrical depression.Optionally, the depression may be a rectangular depression, or anyconcave depression forming a regular or irregular shape. In anotherembodiment, the port 4154b may be connected to a chamber (notshown) within the rotatable element 4150 to create an enlargedspace to store the GI fluid sample from the external environment ofthe ingestible device.

[1436] In some embodiments, the ingestible device 4000 may furtherinclude a controller and an actuator. The controller may determinethat the ingestible device 4000 is located at a target location ofthe GI tract, and then the actuator may trigger the rotation of therotatable element 4150 to align the port 4154b at the open positionto initiate the sampling. For example, the housing of ingestibledevice 4000 may have a pH-sensitive enteric coating to detect orotherwise be sensitive to a pH level of the environment external tothe ingestible device 4000, based on which the controller maydetermine whether the ingestible device has arrived at a targetlocation. For another example, the ingestible device 4000 mayinclude an optical sensing unit that transmits an illumination tothe environment and collects a reflectance, based on which, theregio-specific location of the ingestible device 4000 may beidentified based on optical characteristics of the reflectance.Further embodiments of localization of the ingestible device 4000may be found in PCT International Application No.PCT/US2015/052500, filed on Sep. 25, 2015, which is hereinexpressly incorporated by reference in its entirety.

[1437] FIG. 28 shows one embodiment of a portion of an ingestibledevice with a port 4154b at a first position aligned with a firstdilution chamber 4151a. In operation, the rotatable element 4150may be rotated to align the sampling port 4154b and the firstdilution chamber 4151a such that the fluid sample from the GI tractstored within the volume of the sampling port 4154b can be combinedwith dilution fluid in the first dilution chamber to form a firstdilution. The first dilution may then occupy the combined volume ofthe port 4154b and first dilution chamber 4151a. Optionally, therotatable element 4150 may be subsequently rotated to a secondposition such that the port 4154b containing a portion of the firstdilution is then moved to be aligned and in fluid communicationwith another dilution chamber, e.g., a second dilution chamber thatis next to the first dilution chamber along the rotationaldirection. In this way, the first dilution stored within the port154b may then again be diluted with the dilution fluid storedwithin the second dilution chamber. Similarly, if the rotatableelement 4150 keeps rotating and allows the port 4154b to beserially aligned with each dilution chamber, then the original GIfluid sample may be diluted serially and each dilution chambers4151a-n may be left with a diluted GI fluid sample at a differentdilution ratio.

[1438] FIG. 29 shows one embodiment of an element 4140 forming partof a set of 5 dilution chambers (e.g., including 4151a-b) forsurrounding a rotatable element (e.g., 4150 in FIGS. 28 and 29) inan ingestible device as described herein. In some embodiments, thedevice may contain a single dilution chamber. Alternatively, thedevice may contain 2, 3, 4, 5, 6, 7, 8 or greater than 8 dilutionchambers.

[1439] In some embodiments, each dilution chamber 4151a-n may befilled with a dilution fluid prior to the ingestible device 4000being administered. In another embodiment, the dilution fluid maybe stored in a separate reservoir (not shown) within the ingestibledevice 4000. At the time when the ingestible device 4000 isdetermined to be at a target location within the GI tract, apumping mechanism may pump the dilution fluid into one or moredilution chambers 4151a-b via one or more outlet (not shown) of thereservoir. The pumping mechanism and the reservoir that stores thedilution fluid, may take a form similar to the electromechanicaldelivery mechanism of an ingestible device as described in U.S.Provisional Application No. 62/385,553, filed on Sep. 9, 2016,which is herein expressly incorporated by reference in itsentirety.

[1440] In some embodiments, the shell element 4140 may have valvesor pumps (not shown) between the dilution chambers 4151a-n. Forexample, the diluted fluid from a first dilution chamber may bepumped into a second dilution chamber via a valve between the twochambers. The pump and valve mechanism may take a form similar tothe electromechanical delivery mechanism of an ingestible device asdescribed in U.S. Provisional Application No. 62/385,553, filed onSep. 9, 2016, which is herein expressly incorporated by referencein its entirety.

[1441] In some embodiments, the method and devices described hereininvolve combining a fluid sample, or dilution thereof, withdilution fluid to produce one or more dilutions of the fluidsample. For example, in some embodiments the fluid sample iscombined with dilution fluid in a first dilution chamber to producea first dilution, a portion of the first dilution is combined withdilution fluid in a second dilution chamber to produce a seconddilution, a portion of the second dilution is combined withdilution fluid in a third dilution chamber to produce a thirddilution, and optionally a portion of the third dilution iscombined with dilution fluid in a fourth dilution chamber toproduce a fourth dilution, and optionally a portion of the fourthdilution is combined with dilution fluid in a fifth dilutionchamber to produce a fifth dilution.

[1442] The relative dilution of the fluid sample will depend on therelative amount of fluid sample, or dilution thereof, and dilutionfluid that is combined in each dilution chamber. In someembodiments, the fluid sample, or dilution thereof, is combinedwith dilution fluid at a ratio between about 1:1 and about 1:1000,between about 1:1 and 1:100, between about 1:1 and about 1:20, orbetween about 1:1 and about 1:10. Optionally, the relative amountsof fluid sample, or dilution thereof, and dilution fluid that arecombined in each dilution chamber are varied such that differentdilution chambers contain different dilutions. In some embodiments,the method and devices described herein produce a series of 10-folddilutions of the fluid sample. In some embodiments, the methods anddevices described herein produce a series of dilutions of a fluidsample such that for a given bacterial concentration in the fluidsample, some of the dilutions will not be expected to contain anybacteria, and some of the dilutions will be expected to containbacteria and therefore exhibit bacterial growth when cultured.

[1443] As set out in examples below, determining the presence orabsence of bacterial growth in one or more dilutions can be used toestimate the concentration of bacteria within the original fluidsample from the GI tract. The use of a dilution series and a binarydetection system that detects the presence or absence of bacterialgrowth presents a number of advantages over more complicateddetection systems that seek to directly quantify the concentrationof bacteria within a sample. For example, binary detection systemsare robust and amenable to miniaturization and therefore suitablefor use in an ingestible device as described herein. Also, dilutingthe fluid sample increases the dynamic range while reducinginterference. Accordingly, in some embodiments the methods anddevices described herein include detecting the presence or absenceof the growth of a cell, optionally bacterial growth. In someembodiments, the methods and devices described herein includedetecting the presence or absence of bacterial growth in one ormore dilutions of the fluid sample from the GI tract.

[1444] In some embodiments, the presence or absence of bacterialgrowth in one or more dilutions is used to estimate theconcentration of bacteria in the fluid sample. For example, in someembodiments a fluid sample of about 5 .mu.L is diluted about 10000times in one of the dilution chambers and detecting the presence ofbacterial growth in the dilution chamber is indicative of abacterial concentration of 10.sup.5 or greater colony formingunits/mL (CFU/mL) in the fluid sample. 10 .mu.L of a fluid samplewith a bacterial concentration of 10.sup.4 CFU/mL would containabout 100 CFU. A 10000-fold dilution of such a 10 .mu.L fluidsample would be unlikely to contain any CFUs or bacteria(theoretically 0.01 bacteria) and therefore would not be expectedto exhibit bacterial growth when cultured.

[1445] Alternatively, in some embodiments the methods and devicesdescribed herein include detecting a level of bacterialconcentration within one or more cultured samples. For example, insome embodiments a quantifiable property of a cultured sample ismeasured in order to provide an estimate of the level of bacteriawithin the cultured samples in order to estimate the concentrationof bacteria within the original fluid sample.

[1446] In some embodiments, the devices described herein include adetection system for detecting one or more cells and/or analytes.In some embodiments the cells are bacteria (e.g., bacteria of aparticular genus, species and/or strain). Different detectionsystems known in the art for detecting bacteria may be used withthe device as described herein. In some embodiments, the detectionsystem detects the presence or absence of bacterial growth within adiluted sample. In some embodiments, the detection system detectsthe presence or absence of bacterial growth within a culturedsample. Alternatively or in addition, the detection system detectsa level of bacteria within one or more cultured samples. Forexample, in some embodiments a Coulter counter is used to detectand/or quantify bacteria in the fluid samples, dilutions thereof orcultured samples. In another embodiment, an optical detectionsystem is used to detect and/or quantify bacteria within the fluidsamples, dilutions thereof or cultured samples.

[1447] In some embodiments, the detection system detects cellsand/or analytes in a dilution or cultured sample within the one ormore dilution chambers. Alternatively, the device includes one ormore separate detection chambers and the detection system detectscells and/or analytes in the fluid sample or dilutions thereforwithin the detection chamber. In some embodiments, fluidcommunication between one or more dilution chambers and one or moredetection chambers is controlled by one or more ports, valvesand/or pumps.

[1448] In some embodiments, the detection system detects cellsand/or analytes at a plurality of time points. For example, in someembodiments the detection system detects bacteria within thesterile media prior to combining the fluid sample from the GI tractand the sterile media in order to ensure that any bacterial growthis due to the bacteria introduced into the dilution chambers fromthe GI tract. In some embodiments, the detection system detectsbacteria at a first time point and at a second time point. In someembodiments, the second time point is selected to allow for thegrowth of bacteria within the cultured fluid sample relative to thefirst time point. For example, in some embodiments the second timepoint is between about 1 hour and 6 hours, between about 1 hour and4 hours, or between about 2 hours and 4 hours after the first timepoint. In some embodiments, detecting bacteria at the first timepoint serves as a control.

[1449] In some embodiments, the detection system detects the levelof bacteria at three or more time points to determine a growthcurve for bacteria in the one or more cultured samples. Forexample, the level of bacteria may be detected within one or moreculture samples every 30 or 60 minutes after a sample is collectedfor a total of 2-12 hours; thereby producing a growth curve. Thegrowth curve may then be compared to one or more standard growthcurves. In some embodiments, the standard growth curves arerepresentative of the growth of samples with a known concentrationof bacteria. In some embodiments, the standard growth curves arerepresentative of growth curves from subjects with Small IntestinalBacterial Overgrowth (SIBO).

[1450] In some embodiments, the embodiments described herein use anoptical detection system for detecting one or more cells and/oranalytes. In some embodiments, the optical detection systemincludes a light source and a photodetector. In some embodiments,the light source and photodetector are operable to define a lightpath through a dilution chamber or detection chamber. In someembodiments, the optical detection system measures the absorbanceof light or optical density along the light path at one or morewavelengths.

[1451] In some embodiments, the optical detection system measuresthe absorbance of light at one or more wavelengths between 400 nmand 1000 nm. In some embodiments, the optical detection systemmeasures the absorbance of light at one or more wavelengths betweenabout 500 and 700 nm. In some embodiments, the optical detectionsystem measures the absorbance of light at about 600 nm.

[1452] In some embodiments, the device described herein includesone or more environmental sensors for measuring environmental dataof the GI tract or reproductive tract external to the device in thesubject. In some embodiments, the environmental data is used tohelp determine one or more characteristics of the GI tract orreproductive tract of the subject such as for the diagnosis of amedical condition. Alternatively or in addition, the environmentaldata is used to determine the location of the device within the GItract of the subject. In some embodiments, the one or moreenvironmental sensors include a capacitance sensor, a temperaturesensor, an impedance sensor, a pH level sensor and/or a lightsensor. In some embodiments, the one or more environmental sensorsmeasure pH, temperature, transit times, or combinations thereof.Examples of devices that detect pH changes include Medimetrics'IntelliCap.RTM. technology (see Becker, Dieter, et al. "Novelorally swallowable IntelliCap.RTM. device to quantify regional drugabsorption in human GI tract using diltiazem as model drug." AAPSPharmSciTech 15.6 (2014): 1490-1497) and Rani Therapeutics'Auto-Pill.TM. technology (see U.S. Pat. No. 9,149,617).

[1453] In some embodiments, data regarding the location of thedevice within the GI tract of the subject is used to determine whento obtain a fluid sample from the GI tract and transfer the fluidsample into the one or more dilution chambers. Accordingly, in someembodiments the device includes a microcontroller configured totransfer the fluid sample from the GI tract of the subject to theone or more dilution chambers based on the location of the devicewithin the GI tract.

[1454] In some embodiments, the device includes a communicationsub-unit that is configured to receive operating parameters from anexternal base station and/or transmit data to an external basestation. Also provided is a system including a device as describedherein and an external base station. In some embodiments, theoperating parameters include timing instructions for obtaining afluid sample from the GI tract and transferring the sample into oneor more dilution chambers. In some embodiments, the datatransmitted to the external base station includes data indicativeof the presence of absence of bacterial growth in the culturedsamples.

[1455] In general, it is possible for the ingestible device toobtain different samples from different predetermined regions ofthe GI tract, or for certain actions within the ingestible deviceto be triggered based on its location in the GI tract. For example,it may be possible for the ingestible device to use variouscombinations of light emitting diodes and sensors to determinewhether the device is in the stomach, small intestine, or largeintestine. This may be done by emitting light at differentwavelengths, measuring the level of light reflected at eachwavelength by the environment surrounding the ingestible device,and using this information to determine an approximate location ofthe ingestible device based on the different reflectance propertiesof the various different portions of the GI tract. Once theingestible device determines that it is in a particularpredetermined portion of the GI tract (e.g., the small intestine,or a specific part of the small intestine such as the jejunum), theingestible device may be configured to obtain a sample from thatportion of the GI tract, and store the sample in one or moresampling or incubation chambers within the ingestible device.

[1456] In another embodiment, an ingestible device may be localizedusing a gamma scintigraphy technique or other radio-trackertechnology as employed by Phaeton Research's Enterion.TM. capsule(See Teng, Renli, and Juan Maya. "Absolute bioavailability andregional absorption of ticagrelor in healthy volunteers." Journalof Drug Assessment 3.1 (2014): 43-50), or monitoring the magneticfield strength of permanent magnet in the ingestible device (see T.D. Than, et al., "A review of localization systems for roboticendoscopic capsules," IEEE Trans. Biomed. Eng., vol. 59, no. 9, pp.2387-2399, September 2012).

[1457] In still other embodiments, an ingestible device may includea camera for generating video imaging data of the GI tract whichcan be used to determine, among other things, the location of thedevice. Examples of video imaging capsules include Medtronic'sPillCam.TM. Olympus' Endocapsule.RTM., and IntroMedic'sMicroCam.TM. (see Basar et al. "Ingestible Wireless CapsuleTechnology: A Review of Development and Future Indication"International Journal of Antennas and Propagation (2012); 1-14).Other imaging technologies include thermal imaging cameras, andthose that employ ultrasound or Doppler principles to generatedifferent images (see Chinese patent application CN104473611:"Capsule endoscope system having ultrasonic positioningfunction").

[1458] LOCI

[1459] In some embodiments, the application provides an ingestibledevice for detecting an analyte in a sample, wherein the ingestibledevice includes a sampling chamber that is configured to hold acomposition including: (1) a plurality of donor particles, each ofthe plurality of donor particles including a photosensitizer andhaving coupled thereto a first analyte-binding agent (e.g., anantigen-binding agent) that binds to the analyte, wherein thephotosensitizer, in its excited state, is capable of generatingsinglet oxygen; and (2) a plurality of acceptor particles, each ofthe plurality of acceptor particles including a chemiluminescentcompound and having coupled thereto a second analyte-binding agent(e.g., an antigen-binding agent) that binds to the analyte, whereinsaid chemiluminescent compound is capable of reacting with singletoxygen to emit luminescence. In some embodiments, the first and thesecond analyte-binding agents are antigen-binding agents (e.g.,antibodies). In some embodiments, the first and the secondantigen-binding agents bind to the same epitope of the analyte(e.g., a protein). In some embodiments, the first and the secondantigen-binding agents bind to separate epitopes of the analyte(e.g., a protein) that spatially overlap. In some embodiments, thefirst and the second antigen-binding agents bind to the separateepitopes of the analyte (e.g., a protein) that do not spatiallyoverlap. In some embodiments, the first and/or second analytebinding agent(s) is an antibody. In some embodiments, the firstand/or second analyte binding agent(s) is an affimer. In someembodiments, the first and/or second analyte binding agent(s) is anantigen-binding agent is an aptamer.

[1460] In some embodiments, this application provides an ingestibledevice for detecting an analyte in a sample, wherein the ingestibledevice includes a sampling chamber that is configured to hold amember made of an absorptive material (e.g., an absorptive pad orabsorptive sponge) having absorbed therein a composition including:(1) a plurality of donor particles, each of the plurality of donorparticles including a photosensitizer and having coupled thereto afirst analyte-binding agent (e.g., an antigen-binding agent) thatbinds to the analyte, wherein the photosensitizer, in its excitedstate, is capable of generating singlet oxygen; and (2) a pluralityof acceptor particles, each of the plurality of acceptor particlesincluding a chemiluminescent compound and having coupled thereto asecond analyte-binding agent (e.g., an antigen-binding agent) thatbinds to the analyte, wherein said chemiluminescent compound iscapable of reacting with singlet oxygen to emit luminescence. Insome embodiments, the first and the second analyte-binding agentsare antigen-binding agents (e.g., antibodies). In some embodiments,the first and the second antigen-binding agents bind to the sameepitope of the analyte (e.g., a protein). In some embodiments, thefirst and the second antigen-binding agents bind to separateepitopes of the analyte (e.g., a protein) that spatially overlap.In some embodiments, the first and the second antigen-bindingagents bind to the separate epitopes of the analyte (e.g., aprotein) that do not spatially overlap.

[1461] In some embodiments, the absorptive material is anabsorptive sponge. In some embodiments, the absorptive sponge is ahydrophilic sponge. In some embodiments, the absorptive sponge isselected from the group consisting of: fibers of cotton, rayon,glass, polyester, polyethylene, polyurethane, nitrocellulose, andthe like. In some embodiments, the absorptive sponge is polyesteror polyethylene. In some embodiments, the absorptive sponge isselected from the group consisting of: Ahlstrom Grade 6613H, Porex1/16'' Fine Sheet 4897, Porex 1/8'' Fine Sheet 4898, Porex 45880.024'' Conjugate release pad, Porex PSU-567, and Filter Papers. Insome embodiments, the absorptive sponge is Ahlstrom Grade 6613H(Lot 150191) or Porex PSU-567. The present application furtherprovides a method for preparing an absorptive material as describedherein, including the step of injecting into the absorptivematerial an aqueous solution including a composition of the presentapplication. In some embodiments, the method including a step ofdrying the absorptive material having absorbed therein the aqueoussolution at a temperature in the range of 0-100.degree. C.,0-50.degree. C., 0-40.degree. C., 0-30.degree. C., 0-20.degree. C.,0-10.degree. C., or 0-4.degree. C.), for a time period sufficientto reduce the total water content to below 50%, 40%, 30%, 20%, 15%,10%, 7%, 5%, 3%, 1%, 0.7%, 0.5%, 0.3%, or 0.1% by weight.

[1462] In some embodiments, the disclosure provides a method ofmeasuring the presence, absence or amount of one or more analytesfrom one or more samples in the gastrointestinal tract. In general,in embodiments involving LOCI, the analyte is capable of beingbound by two analyte-binding agents at the same time to allow fordetection of the analyte using the methods described herein.Exemplary analytes that can be be used in embodiments involvingLOCI include, but are not limited to, proteins, peptides, andmicroorganisms (e.g., bacteria). Various examples of analytessuitable for use in embodiments involving LOCI are describedabove.

[1463] In some embodiments the one or more analytes are measuredmultiple times, for example, at different time points or atdifferent locations. In one embodiment, a single device measuresone or more analytes or more time points or locations; therebycreating a "molecular map" of a physiological region. Measurementscan be taken at any location in the gastrointestinal tract. Forexample, in one aspect, analytes from samples from one or more ofthe duodenum, jejunum, ileum, ascending colon, transverse colon ordescending colon can be measured to create a molecular map of thesmall and large intestine. In one aspect, the sample is from theduodenum. In one aspect, In one aspect, the sample is from thejejunum. In one aspect, the sample is from the ileum. In oneaspect, the sample is from the ascending colon. In one aspect, thesample is from the transverse colon. In one aspect, the sample isfrom the descending colon.

[1464] In another aspect, a series of measurements can be takenover a shorter distance of the gastrointestinal tract (e.g., theileum) to create a higher resolution molecular map. In someembodiments, previous endoscopic imaging may identify a diseasedarea for molecular mapping (e.g., biomarker mapping). For example,a gastroenterologist may use imaging (e.g., an endoscope equippedwith a camera) to identify the presence of Crohn's disease in theileum and cecum of a patient, and the methods and techniques of thepresent invention herein may be used to measureinflammation-associated analytes in this diseased area of thepatient. In a related embodiment, the inflammation-associatedanalytes, or any analyte, may be measured every one or more days tomonitor disease flare-ups, or response to therapeutics. Exemplaryinflammation-associated analytes include anti-glycan antibodies;anti-Saccharomyces cerevisiae antibodies (ASCA);anti-laminaribioside antibodies (ALCA); anti-chitobiosideantibodies (ACCA); anti-mannobioside antibodies (AMCA);anti-laminarin (anti-L) antibodies; anti-chitin (anti-C)antibodies; anti-outer membrane porin C (anti-OmpC) antibodies;anti-Cbirl flagellin antibodies; anti-I2 antibodies (see, e.g.,Mitsuyama et al. (2016) World J. Gastroenterol. 22(3): 1304-10);autoantibodies targeting the exocrine pancreas (PAB); perinuclearanti-neutrophil antibody (pANCA); calprotectin; a cytokine such asvascular endothelial growth factor (VEGF), C-reactive protein(CRP), interleukin-6 (IL-6), or tumor necrosis factor alpha(TNF-.alpha.); an adhesion molecule such as intracellular adhesionmolecule (ICAM) (e.g., ICAM-1) or vascular adhesion molecule (VCAM)(e.g., VCAM-1); or serum amyloid A (SAA).

[1465] Photosensitizers that are to be excited by light will berelatively photostable and will not react efficiently with singletoxygen. Several structural features are present in most usefulsensitizers. Most sensitizers have at least one and frequentlythree or more conjugated double or triple bonds held in a rigid,frequently aromatic structure. They will frequently contain atleast one group that accelerates intersystem crossing such as acarbonyl or imine group or a heavy atom selected from rows 3-6 ofthe periodic table, especially iodine or bromine, or they may haveextended aromatic structures. Typical sensitizers include acetone,benzophenone, 9-thioxanthone, eosin, 9,10-dibromoanthracene,methylene blue, metallo-porphyrins, such as hematoporphyrin,phthalocyanines, chlorophylls, rose bengal, buckminsterfullerene,etc., and derivatives of these compounds having substituents of 1to 50 atoms for rendering such compounds more lipophilic or morehydrophilic and/or as attaching groups for attachment. Examples ofother photosensitizers that may be utilized in the presentinvention are those that have the above properties and areenumerated in N. J. Turro, "Molecular Photochemistry," page 132, W.A. Benjamin Inc., N.Y. 1965.

[1466] In some embodiments, the photosensitizers are relativelynon-polar to assure dissolution into a lipophilic member when thephotosensitizer is incorporated in an oil droplet, liposome, latexparticle, etc.

[1467] In some embodiments, the photosensitizers suitable for useherein include other substances and compositions that can producesinglet oxygen with or without activation by an external lightsource. Thus, for example, molybdate (MoO.sub.4.sup..dbd.) saltsand chloroperoxidase and myeloperoxidase plus bromide or chlorideion (Kanofsky, J. Biol. Chem. (1983) 259 5596) have been shown tocatalyze the conversion of hydrogen peroxide to singlet oxygen andwater. Either of these compositions can, for example, be includedin particles and used in the assay method wherein hydrogen peroxideis included as an ancillary reagebly, chloroperoxidase is bound toa surface and molybdate is incorporated in the aqueous phase of aliposome. Also included within the scope of the invention asphotosensitizers are compounds that are not true sensitizers butwhich on excitation by heat, light, or chemical activation willrelease a molecule of singlet oxygen. The best known members ofthis class of compounds includes the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption oflight by these compounds releases singlet oxygen.

[1468] A chemiluminescent compound is a substance that undergoes achemical reaction with singlet oxygen to form a metastableintermediate that can decompose with the simultaneous or subsequentemission of light within the wavelength range of 250 to 1200 nm.Exemplary chemiluminescent compounds suitable for use in thepresent application include those described in U.S. Pat. Nos.6,251,581 and 7,709,273, and Patent Cooperatio Treaty (PCT)International Application Publication No. WO1999/042838. Examplerychemiluminescent compound includes the following:

TABLE-US-00003 Chemiluminescer Half-Life Emission Max Thioxene +Diphenyl anthracence: 0.6 seconds 430 nm Thioxene + Umbelliferonederivative 0.6 seconds 500 nm Thioxene + Europium chelate 0.6seconds 615 nm Thioxene + Samarium Chelate 0.6 seconds 648 nmThioxene + terbium Chelate 0.6 seconds 540 nm N-Phenyl Oxazine +Umbelliferone derivative 30 seconds 500 nm N-Phenyl Oxazine +Europium chelate 30 seconds 613 nm N-phenyl Oxazine + SamariumChelate 30 seconds 648 nm N-phenyl Oxazine + terbium Chelate 30seconds 540 nm Dioxene + Umbelliferone derivative 300 seconds 500nm Dioxene + Europium chelate 300 seconds 613 nm Dioxene + SamariumChelate 300 seconds 648 nm N-phenyl Oxazine + terbium Chelate 300seconds 540 nm

[1469] All of the above mentioned applications are herey expresslyincorporated by reference herein in their entireties. Emission willusually occur without the presence of an energy acceptor orcatalyst to cause decomposition and light emission. In someembodiments, the intermediate decomposes spontaneously withoutheating or addition of ancillary reagents following its formation.However, addition of a reagent after formation of the intermediateor the use of elevated temperature to accelerate decomposition canbe desirable for some chemiluminescent compounds. Thechemiluminescent compounds are usually electron rich compounds thatreact with singlet oxygen, frequently with formation of dioxetanesor dioxetanones. Exemplary of such compounds are enol ethers,enamines, 9-alkylidenexanthans, 9-alkylidene-N-alkylacridans, arylvinyl ethers, dioxenes, arylimidazoles and lucigenin. Otherchemiluminescent compounds give intermediates upon reaction withsinglet oxygen, which subsequently react with another reagent withlight emission. Exemplary compounds are hydrazides such as luminoland oxalate esters.

[1470] The chemiluminescent compounds of interest will generallyemit at wavelengths above 300 nanometers and usually above 400 nm.Compounds that alone or together with a fluorescent molecule emitlight at wavelengths beyond the region where serum componentsabsorb light will be of particular use in the present invention.The fluorescence of serum drops off rapidly above 500 nm andbecomes relatively unimportant above 550 nm. Therefore, when theanalyte is in serum, chemiluminescent compounds that emit lightabove 550 nm, e.g., above 600 nm may be suitable for use. In orderto avoid autosensitization of the chemiluminescent compound, insome embodiments, the chemiluminescent compounds do not absorblight used to excite the photosensitizer. In some embodiments, thesensitizer is excited with light wavelengths longer than 500 nm, itwill therefore be desirable that light absorption by thechemiluminescent compound be very low above 500 nm.

[1471] Where long wavelength emission from the chemiluminescentcompound is desired, a long wavelength emitter such as a pyrene,bound to the chemiluminescent compound can be used. Alternatively,a fluorescent molecule can be included in the medium containing thechemiluminescent compound. In some embodiments, fluorescentmolecules will be excited by the activated chemiluminescentcompound and emit at a wavelength longer than the emissionwavelength of the chemiluminescent compound, usually greater that550 nm. It is usually also desirable that the fluorescent moleculesdo not absorb at the wavelengths of light used to activate thephotosensitizer. Examples of useful dyes include rhodamine,ethidium, dansyl, Eu(fod).sub.3, Eu(TTA).sub.3,Ru(bpy).sub.3.sup.++ (wherein bpy=2,2'-dipyridyl, etc. In generalthese dyes act as acceptors in energy transfer processes and insome embodiments, have high fluorescent quantum yields and do notreact rapidly with singlet oxygen. They can be incorporated intoparticles simultaneously with the incorporation of thechemiluminescent compound into the particles.

[1472] In general, the particles are at least about 20 nm and notmore than about 20 microns, usually at least about 40 nm and lessthan about 10 microns, e.g., from about 0.10 to 2.0 micronsdiameter, normally having a volume of less than 1 picoliter.Exemplary particles (including both donor and acceptor particles)suitable for use in the present application include those describedin U.S. Pat. Nos. 6,251,581, and 7,709,273, and PCT InternationalPublication No. WO1999/042838--which are hereby expresslyincorporated by reference herein in their entireties. In someembodiments, a particle as used herein may be a bead make ofsuitable material. The particle (e.g., a bead) may be organic orinorganic, swellable or non-swellable, porous or non-porous, havingany density, but in some embodiments, of a density approximatingwater, generally from about 0.7 to about 1.5 g/ml, may besuspendible in water, and composed of material that can betransparent, partially transparent, or opaque. The particles may ormay not have a charge, and when they are charged, in someembodiments, they are negative. The particles may be solid (e.g.,polymer, metal, glass, organic and inorganic such as minerals,salts and diatoms), oil droplets (e.g., hydrocarbon, fluorocarbon,silicon fluid), or vesicles (e.g., synthetic such as phospholipidor natural such as cells and organelles). The particles may belatex particles or other particles included of organic or inorganicpolymers; lipid bilayers, e.g., liposomes, phospholipid vesicles;oil droplets; silicon particles; metal sols; cells; and dyecrystallites.

[1473] The organic particles will normally be polymers, eitheraddition or condensation polymers, which are readily dispersible inthe assay medium. The organic particles will also be adsorptive orfunctionalizable so as to bind at their surface, either directly orindirectly, an analyte-binding agent and to bind at their surfaceor incorporate within their volume a photosensitizer or achemiluminescent compound.

[1474] The particles can be derived from naturally occurringmaterials, naturally occurring materials which are syntheticallymodified and synthetic materials. Natural or synthetic assembliessuch as lipid bilayers, e.g., liposomes and non-phospholipidvesicles, are suitable for use herein. Among organic polymers ofparticular interest are polysaccharides, particularly cross-linkedpolysaccharides, such as agarose, which is available asSEPHAROSE.RTM. (Pharmacia Biotech), dextran, available asSEPHADEX.RTM. (Pharmacia Biotech) and SEPHACRYL.RTM. (PharmaciaBiotech), cellulose, starch, and the like; addition polymers, suchas polystyrene, polyacrylamide, homopolymers and copolymers ofderivatives of acrylate and methacrylate, particularly esters andamides having free hydroxyl functionalities including hydrogels,and the like. Inorganic polymers include silicones, glasses,available as Bioglas, and the like. Sols include gold, selenium,and other metals. Particles may also be dispersed water insolubledyes such as porphyrins, phthalocyanines, etc., which may also actas photosensitizers. Particles may also include diatoms, cells,viral particles, magnetosomes, cell nuclei and the like.

[1475] Where the particles are commercially available, the particlesize may be varied by breaking larger particles into smallerparticles by mechanical means, such as grinding, sonication,agitation, etc.

[1476] In some embodiments, the particles are polyfunctional or arecapable of being polyfunctionalized or are capable of being boundor coupled to or associated with an analyte-binding agent,photosensitizer, or chemiluminescent compound through specific ornon-specific covalent or non-covalent interactions. A wide varietyof functional groups are available or can be incorporated.Exemplary functional groups include carboxylic acids, aldehydes,amino groups, cyano groups, ethylene groups, hydroxyl groups,mercapto groups and the like. When covalent attachment of ananalyte-binding agent, chemiluminescent compound or photosensitizerto the particle is employed, the manner of linking is well knownand is amply illustrated in the literature. See for exampleCautrecasas, J. Biol, Chem., 245:3059 (1970). The length of alinking group may vary widely, depending upon the nature of thecompound being linked, the nature of the particle, the effect ofthe distance between the compound being linked and the particle onthe binding of analyte-binding agents and the analyte and thelike.

[1477] The photosensitizer and/or chemiluminescent compound can bechosen to dissolve in or noncovalently bind to the surface of theparticles. In some embodiments, these compounds may be hydrophobicto reduce their ability to dissociate from the particle and therebycause both compounds to associate with the same particle. Thispossibly can be further reduced by utilizing particles of only onecomposition that are associated with either the photosensitizer orchemiluminescent compound or by using two types of particles thatdiffer in composition so as to favor association of thephotosensitizer with one type of particle and association of thechemiluminescent compound with the other type of particle.

[1478] The number of photosensitizer or chemiluminescent moleculesassociated with each particle will on the average usually be atleast one and may be sufficiently high that the particle consistsentirely of photosensitizer or chemiluminescer molecules. In someembodiments, the number of molecules will be selected empiricallyto provide the highest signal to background in the assay. In somecases this will be best achieved by associating a multiplicity ofdifferent photosensitizer molecules to particles. In someembodiments, the photosensitizer or chemiluminescent compound toanalyte-binding agent ratio in the particles should be at least 1,such as at least 100 to 1 up to over 1,000 to 1.

[1479] Generally, oil droplets are fluid particles included of alipophilic compound coated and stabilized with an emulsifier thatis an amphiphilic molecule such as, for example, phospholipids,sphingomyelin, albumin and the like.

[1480] The phospholipids are based upon aliphatic carboxylic acidesters of aliphatic polyols, where at least one hydroxylic group issubstituted with a carboxylic acid ester of from about 8 to 36,more usually of from about 10 to 20 carbon atoms, which may havefrom 0 to 3, more usually from 0 to 1 site of ethylenicunsaturation and at least 1, normally only 1, hydroxyl groupsubstituted with phosphate to form a phosphate ester. The phosphategroup may be further substituted with small aliphatic compoundswhich are of di or higher functionality, generally having hydroxylor amino groups.

[1481] The oil droplets can be made in accordance with conventionalprocedures by combining the appropriate lipophilic compounds with asurfactant, anionic, cationic or nonionic, where the surfactant ispresent in from about 0.1 to 5, more usually from about 0.1 to 2weight percent of the mixture and subjecting the mixture in anaqueous medium to agitation, such as sonication or vortexing.Illustrative lipophilic compounds include hydrocarbon oils,halocarbons including fluorocarbons, alkyl phthalates, trialkylphosphates, triglycerides, etc.

[1482] An analyte-binding agent will usually be adsorbed to thesurface of the oil droplet or bonded directly or indirectly to asurface component of the oil droplet. The analyte-binding agent maybe incorporated into the liquid particles either during or afterthe preparation of the liquid particles. The analyte-binding agentwill normally be present in from about 0.5 to 100, about 1 to 90,about 5 to 80 and about 50 to 100 mole percent of the moleculespresent on the surface of the particle.

[1483] The following is a list, by way of illustration and notlimitation, of amphiphilic compounds, which may be utilized forstabilizing oil droplets: phosphatidyl ethanolamine, phosphatidylcholine, phosphatidyl serine, dimyristoylphosphatidyl choline, eggphosphatidyl choline, diapalmitoylphosphatidyl choline,phosphatidic acid, cardiolipin, lecithin, galactocerebroside,sphingomyelin, dicetylphosphate, phosphatidyl inositol,2-trihexadecylammoniumethylamine,1,3-bis(octadecylphosphate)-propanol, stearoyloxyethylenephosphate, phospholipids, dialkylphosphates, sodium dodecylsulfate, cationic detergents, anionic detergents, proteins such asalbumin, non-ionic detergents, etc.

[1484] Other compounds may also be used which have lipophilicgroups and which have been described previously. For the most part,these compounds will be alkylbenzenes, having alkyl groups of from6 to 20 carbon atoms, usually mixtures of alkyl groups, which maybe straight or branched chain, and having a carboxyl group, anhydroxylic group, a polyoxy alkylene group (alkylene of from 2 to 3carbon atoms), carboxylic group, sulfonic acid group, or aminogroup. Aliphatic fatty acids may be used which will normally be offrom about 10 to 36, more usually of from about 12 to 20 carbonatoms. Also, fatty alcohols having the carbon limits indicated forthe fatty acids, fatty amines of similar carbon limitations andvarious steroids may also find use.

[1485] The oil droplets can include a fluorocarbon oil or asilicone oil (silicon particle). Such droplets are described byGiaever in U.S. Pat. Nos. 4,634,681 and 4,619,904, each of isincorporated by reference herein in its entirety. These dropletsare formed by dispersing a fluorocarbon oil or silicone oil in anaqueous phase. The droplets are prepared by placing a small amountof the selected oil (generally, such oils are commerciallyavailable) in a container with a larger amount of the aqueousphase. The liquid system is subjected to agitation to bring aboutemulsification and then centrifuged. The homogeneous phase isremoved and the residual droplets are resuspended in an aqueousbuffered medium. The above centrifugation and decantation steps canbe repeated one or more times before the droplets are utilized.

[1486] Analyte-binding agents can be bound to the droplets in anumber of ways. As described by Giaever, the particularanalyte-binding agents, particularly a proteinoceousanalyte-binding agent, can be coated on the droplets by introducingan excess of the analyte-binding agent into the aqueous mediumprior to or after the emulsification step. Washing steps aredesirable to remove excess analyte-binding agent. Functionalizationof the oil introduces functionalities described above for linkingto analyte-binding agents. Such functionalities can also beemployed to link the droplets to a photosensitizer or achemiluminescent compound. On the other hand, the photosensitizeror chemiluminescent compound will frequently be chosen to besoluble in the oil phase of the oil droplet and will not becovalently bound. When the oil is a fluorocarbon, a fluorinatedphotosensitizer or chemiluminescent compound will often be moresoluble than the corresponding unfluorinated derivation. Other oildroplets described by Giaever also find use in the presentinvention.

[1487] In general, liposomes are microvesicles of approximatelyspherical shape and are one of the materials for use in the presentinvention. The liposomes have a diameter that is at least about 20nm and not more than about 20 microns, usually at least about 40 nmand less than about 10 microns. In some embodiments, the diameterof the liposomes will be less than about two microns so as to limitsettling or floatation.

[1488] The outer shell of a liposome consists of an amphiphilicbilayer that encloses a volume of water or an aqueous solution.Liposomes with more than one bilayer are referred to asmultilamellar vesicles. Liposomes with only one bilayer are calledunilamellar vesicles. Multilamellar vesicles are suitable for usein the present invention when using a lipophilic photosensitizer orchemiluminescent compound because of their ability to incorporatelarger quantities of these materials than unilamellar vesicles. Theamphiphilic bilayer is frequently included of phospholipids.Phospholipids employed in preparing particles utilizable in thepresent invention can be any phospholipid or phospholipid mixturefound in natural membranes including lecithin, or syntheticglyceryl phosphate diesters of saturated or unsaturated 12-carbonor 24-carbon linear fatty acids wherein the phosphate can bepresent as a monoester, or as an ester of a polar alcohol such asethanolamine, choline, inositol, serine, glycerol and the like.Suitable phospholipids include, but are not limited to,L-.alpha.-palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyloleoylphosphatidyl-glycerol (POPG),L-.alpha.-dioleoylphosphatidylglycerol,L-.alpha.(dioleoyl)-phosphatidyl ethanolamine (DOPE) andL-.alpha.(dioleoyl)-phosphatidyl.beta.-(4-(N-maleimidomethyl)-cyclohexane-1-carboxyamido)ethanol(DOPE-MCC).

[1489] The phospholipids in the bilayer may be supplemented withcholesterol and may be replaced with other amphiphilic compoundsthat have a polar head group, usually charged, and a hydrophobicportion usually included of two linear hydrocarbon chains. Examplesof such substituents include dialkylphosphate,dialkoxypropylphosphates wherein the alkyl groups have linearchains of 12-20 carbon atoms,N-(2,3-di(9-(Z)-octa-decenyloxy))-prop-1-yl-N,N,N,-trimethyl-ammoniumchloride (DOTMA), as disclosed in U.S. patent application Ser. No.811,146 filed on Dec. 19, 1985, which is hereby incorporated hereinby reference, sphingomyelin, cardiolipin, and the like.

[1490] In some embodiments, liposomes utilized in the presentinvention have a high negative charge density to stabilize thesuspension and to prevent spontaneous aggregation.

[1491] For use in the present invention the liposomes should becapable of binding to an analyte-binding agent and be capable ofhaving a photosensitizer or chemiluminescent compound associatedwith either the aqueous or the nonaqueous phase. The liposomesutilized in the present invention will usually have analyte-bindingagents bound to the outer surface of the lipid vesicle.

[1492] Liposomes may be produced by a variety of methods includinghydration and mechanical dispersion of dried phospholipid orphospholipid substitute in an aqueous solution. Liposomes preparedin this manner have a variety of dimensions, compositions andbehaviors. One method of reducing the heterogeneity andinconsistency of behavior of mechanically dispersed liposomes is bysonication. Such a method decreases the average liposome size.Alternatively, extrusion is usable as a final step during theproduction of the liposomes. U.S. Pat. No. 4,529,561 (which isincorporated by reference herein in its entirety) discloses amethod of extruding liposomes under pressure through a uniformpore-size membrane to improve size uniformity.

[1493] Preparation of liposomes containing a hydrophobic oramphiphilic photosensitizer or a chemiluminescent compounddissolved in the lipid bilayer can be carried out in a variety ofmethods, including a method described by Olsen, et al., Biochemicaet Biophysica Acta, 557(9), 1979. Briefly, a mixture of lipidscontaining the appropriate compound in an organic solvent such aschloroform is dried to a thin film on the walls of a glass vessel.The lipid film is hydrated in an appropriate buffer by shaking orvortexing. Thereafter, the lipid suspension is extruded through aseries of polycarbonate filter membranes having successivelysmaller pore sizes, for example, 2.0, 1.0, 0.8, 0.6, 0.4, and 0.2microns. Repeated filtration through any of the filters, and inparticular through the smallest filter, is desirable. The liposomescan be purified by, for example, gel filtration, such as through acolumn of SEPHACRYL.RTM. S-1000 (Pharmacia Biotech). The column canbe eluted with buffer and the liposomes collected. Storage in thecold prolongs shelf-life of the liposomes produced by this method.Alternatively the photosensitizer or chemiluminescent compound canbe added to the liquid suspension following preparation of theliposomes.

[1494] Labeling of droplets and liposomes will often involve, forexample, inclusion of thiol or maleimide or biotin groups on themolecules including the lipid bilayer. Photosensitizers,chemiluminescent molecules or analyte-binding agents may then bebound to the surface by reaction of the particles with one of thesematerials that is bound to a sulfhydryl reactive reagent, asulfhydryl group, or avidin, respectively. Sulfhydryl reactivegroups include alkylating reagents such as bromoacetamide andmaleimide.

[1495] Analyte-binding agents can be attracted to the surface ofthe liposome particles by weak hydrophobic interactions, howeversuch interactions are not generally sufficient to withstand theshear force encountered during incubation and washing. It ispossible to covalently bond analyte-binding agents to a liposomeparticle that has been functionalized, for example by use ofDOPE-MCC, as shown above, by combining said liposome with theselected analyte-binding agent functionalized with a mercaptangroup. For example, if the analyte-binding agent is an antibody, itmay be reacted with S-acetyl-mercaptosuccinic anhydride (SAMSA) andhydrolyzed to provide a sulfhydryl modified antibody.

[1496] Generally, latex signifies a particulate water suspendiblewater insoluble polymeric material usually having particledimensions of 20 nm to 20 .mu.m, e.g., 100 to 1000 nm in diameter.The latex is frequently a substituted polyethylene such as:polystyrene-butadiene, polyacrylamide polystyrene, polystyrene withamino groups, poly-acrylic acid, polymethacrylic acid,acrylonitrile-butadiene, styrene copolymers, polyvinylacetate-acrylate, polyvinyl pyrridine, vinyl-chloride acrylatecopolymers, and the like. Non-crosslinked polymers of styrene andcarboxylated styrene or styrene functionalized with other activegroups such as amino, hydroxyl, halo and the like are suitable foruse herein. In some embodiments, copolymers of substituted styreneswith dienes such as butadiene will be used.

[1497] The association of the photosensitizer or chemiluminescentcompound with latex particles utilized in the present invention mayinvolve incorporation during formation of the particles bypolymerization but will usually involve incorporation intopreformed particles, usually by noncovalent dissolution into theparticles. Usually a solution of the chemiluminescent compound orsensitizer will be employed. Solvents that may be utilized includealcohols, including ethanol, ethylene glycol and benzyl alcohol;amides such as dimethyl formamide, formamide, acetamide andtetramethyl urea and the like; sulfoxides such as dimethylsulfoxide and sulfolane; and ethers such as carbitol, ethylcarbitol, dimethoxy ethane and the like, and water. The use ofsolvents having high boiling points in which the particles areinsoluble permits the use of elevated temperatures to facilitatedissolution of the compounds into the particles and areparticularly suitable. The solvents may be used singly or incombination. In some embodiments, solvents for incorporatingphotosensitizer are those that will not quench the triplet excitedstate of the photosensitizer either because of their intrinsicproperties or because they can subsequently be removed from theparticles by virtue of their ability to be dissolved in a solventsuch as water that is insoluble in the particles. Aromatic solventsare also suitable for use herein, such as solvents that are solublein the particle. For incorporating chemiluminescent compounds inparticles a solvent should be selected that does not interfere withthe luminescence because of their intrinsic properties or abilityto be removed from the particles. In some embodiments, aromaticsolvents may be used. Typical aromatic solvents includedibutylphthalate, benzonitrile, naphthonitrile,dioctylterephthalate, dichlorobenzene, diphenylether,dimethoxybenzene, etc.

[1498] Except when the photosensitizer or chemiluminescent compoundis to be covalently bound to the particles, it may be suitable touse electronically neutral photosensitizers or chemiluminescentcompounds. In some embodiments, the liquid medium selected does notsoften the polymer beads to the point of stickiness. One techniqueincludes suspending the selected latex particles in a liquid mediumin which the photosensitizer or chemiluminescent compound has atleast limited solubility. In some embodiments, the concentrationsof the photosensitizer and chemiluminescent compound in the liquidmedia will be selected to provide particles that have the highestefficiency of singlet oxygen formation and highest quantum yield ofemission from the chemiluminescent compound in the media but lessconcentrated solutions will sometimes be used. Distortion ordissolution of the particles in the solvent can be prevented byadding a miscible cosolvent in which the particles areinsoluble.

[1499] Generally, the temperature employed during the procedurewill be chosen to maximize the singlet oxygen formation ability ofthe photosensitizer labeled particles and the quantum yield of thechemiluminescent compound particles with the proviso that theparticles should not melt or become aggregated at the selectedtemperature. Elevated temperatures are normally employed. Thetemperatures for the procedure will generally range from 20.degree.C. to 200.degree. C., more usually from 50.degree. C. to170.degree. C. It has been observed that some compounds that arenearly insoluble at room temperature are soluble in, for example,low molecular weight alcohols, such as ethanol and ethylene glycoland the like, at elevated temperatures. Carboxylated modified latexparticles have been shown to tolerate low molecular weight alcoholsat such temperatures.

[1500] An analyte-binding agent may be physically adsorbed on thesurface of the latex particle or may be covalently bonded to theparticle. In cases wherein the analyte-binding agent is only weaklybound to the surface of the latex particle, the binding may incertain cases be unable to endure particle-to-particle shear forcesencountered during incubation and washings. Therefore, it may besuitable to covalently bond analyte-binding agents to the latexparticles under conditions that will minimize adsorption. This maybe accomplished by chemically activating the surface of the latex.For example, the N-hydroxysuccinimide ester of surface carboxylgroups can be formed and the activated particles to reducenonspecific binding of assay components to the particle surface arethen contacted with a linker having amino groups that will reactwith the ester groups or directly with an analyte-binding agentthat has an amino group. The linker will usually be selected toreduce nonspecific binding of assay components to the particlesurface and will in some embodiments, provide suitablefunctionality for both attachment to the latex particle andattachment of the analyte-binding agent. Suitable materials includemaleimidated aminodextran (MAD), polylysine, aminosaccharides, andthe like. MAD can be prepared as described by Hubert, et al., Proc.Natl. Acad. Sci., 75(7), 3143, 1978.

[1501] In one method, MAD is first attached to carboxyl-containinglatex particles using a water soluble carbodiimide, for example,1-(3-dimethylaminopropyl)-3-ethyl carbodiimide. The coatedparticles are then equilibrated in reagents to prevent nonspecificbinding. Such reagents include proteins such as bovine gammaglobulin (BGG), and detergent, such as Tween.RTM. 20, (ICIAmericas, Inc.) TRITON X-100.RTM. (Rohm and Haas Company) and thelike. xAn analyte-binding agent having a sulfhydryl group, orsuitably modified to introduce a sulfhydryl group, is then added toa suspension of the particles, whereupon a covalent bond is formedbetween the analyte-binding agent and the MAD on the particles. Anyexcess unreacted analyte-binding agent can then be removed bywashing.

[1502] In general, metal sols are particles included of a heavymetal, i.e., a metal of atomic number greater than 20 such as aGroup IB metal, e.g., gold or silver or chalcogens such as seleniumor tellurium.

[1503] Metal sol particles are described, for example, by Leuveringin U.S. Pat. No. 4,313,734, the disclosure of which is incorporatedherein by reference in its entirety. Such sols include colloidalaqueous dispersion of a metal, metal compound, or polymer nucleicoated with a metal or metal compound.

[1504] The metal sols may be of metals or metal compounds, such asmetal oxides, metal hydroxides and metal salts or of polymer nucleicoated with metals or metal compounds. Examples of such metals areplatinum, gold, silver mercury, lead, palladium, and copper, and ofsuch metal compounds are silver iodide, silver bromide, copperhydrous oxide, iron oxide, iron hydroxide or hydrous oxide,aluminum hydroxide or hydrous oxide, chromium hydroxide or hydrousoxide, vanadium oxide, arsenic sulphide, manganese hydroxide, leadsulphide, mercury sulphide, barium sulphate and titanium dioxide.In general, the metals or metal compounds useful may be readilydemonstrated by means of known techniques.

[1505] In some embodiments, it may advantageous to use solsincluded of dispersed particles consisting of polymer nuclei coatedwith the above mentioned metals or metal compounds. These particleshave similar properties as the dispersed phase of pure metals ormetal compounds, but size, density and metal contact can beoptimally combined.

[1506] The metal sol particles may be prepared in a large number ofways which are in themselves known. For example, for thepreparation of a gold sol Leuvering refers to an article by G.Frens in Nature Physical Science 241, 20 (1973).

[1507] The metal sol particles can be modified to contain variousfunctional groups as described above for linking to ananalyte-binding agent or a photosensitizer or a chemiluminescentcompound. For example, polymeric bonding agents can be used to coatthe particles such as polymers containing thiol groups that bondstrongly to many heavy metals or silylating agents that can bondand form polymeric coatings as, for example, by reaction of metalparticles with trialkoxy aminoalkylsilanes as described in EPOPatent Appl. 84400952.2 by Advanced Magnetics for coating magneticparticles.

[1508] Generally, dye crystallites are microcrystals of pure ormixed solid water insoluble dyes, such as those described herein.The dye crystallites useful in the present invention have a sizerange of 20 nm to 20 .mu.m.

[1509] One method for preparing dye crystallites is described inU.S. Pat. No. 4,373,932 (Gribnau, et al.), the disclosure of whichis incorporated herein by reference in its entirety. Gribnaudescribes colloidal dye particles and aqueous dispersions of ahydrophobic dye or pigment, which may have an immunochemicallyreactive component directly or indirectly attached. The dyeparticles are prepared in general by dispersing a dye in water andthen centrifuging. A dye pellet is obtained and resuspended inwater, to which glass beads are added. This suspension is rolledfor several days at room temperature. The liquid is decanted andcentrifuged, and the dye particles are obtained after aspiration ofthe liquid.

[1510] Another method for preparing dye crystallites is by slowaddition of a solution of the dye in a water miscible solvent towater. Another method is by sonication of a suspension of the soliddye in water.

[1511] Binding of analyte-binding agents to the dye particles canbe achieved by direct or indirect adsorption or covalent chemicalattachment. The latter is governed by the presence of suitablefunctional groups in any coating material and in the dye. Forexample, functional groups can be introduced onto the surface of adye crystallite by coupling a compound containing a diazotizedaromatic amino group and the desired functional group to a phenolicor anilino group of the dye.

[1512] Where the dye has a carboxyl group, the dye crystallite canbe activated by a carbodiimide and coupled to a primary aminocomponent. Aliphatic primary amino groups and hydroxyl groups canbe activated, for example, by cyanogen bromide orhalogen-substituted di- or tri-azines, after which attachment witha primary amino component or with, for example, a componentcontaining a --SH, or --OH or group can take place. Use can also bemade of bifunctional reactive compounds. For example,glutaraldehyde can be used for the mutual coupling of primary aminocomponents of the dye and an analyte-binding agent, and, forexample, a hetero-bifunctional reagent such as N-succinimidyl3-(2-pyridyldithio) propionate can be employed for the coupling ofa primary amino component to a component containing a thiolgroup.

[1513] In some embodiments, the composition for use in theingestible devices of the present application further includes amedium having suspended therein said plurality of donor particlesand said plurality of acceptor particles. In some embodiments, themedium is an aqueous medium. In some embodiments, the aqueousmedium has a pH selected from 5-8 (e.g., a pH selected from 6-7.8,such as pH being 6.0). The standard buffer was 50 mM sodiumphosphate/0.15 M NaCl, pH 7.0. St.Av Donar beads are incubated in 1um HABA ((2-(4-HYDROXYPHENYLAZO)BENZOIC ACID) before depositingthem on the pad. The assay buffer was 0.1 M Tris'HCl/0.3 MNaCi/bovine serum albumin (1 mg/ml), pH 8.2. with 50 mM hydroxylpropyl cyclodextrin and tween-20-0.1%.

[1514] Suitable acceptor particles for use in the ingestibledevices of this application may be any type of particles asdescribed herein. In some embodiments, the acceptor particles areselected from the group consisting of latex particles, lipidbilayers, oil droplets, silica particles, and metal sols. In someembodiments, the acceptor particles are latex particles, such as,but not limited Polystyrene latex particles (175 nm) having about8.3 carboxyl groups per nm.sup.2 of surface, and/or the like.

[1515] Suitable chemiluminescent compounds for use in theingestible devices of this application include thosechemiluminescent compounds or substances as described in PCTInternational Publication No. WO1999042838 A1 (Table 1); and U.S.Pat. No. 7,709,273. In some embodiments, the chemiluminescentcompounds are selected from the group consisting of examplechemiluminescent compounds described in PCT InternationalPublication No. WO1999042838 A1 (Table 1); and U.S. Pat. No.7,709,273. The above mentioned applications are hereby expresslyincorporated by reference in their entireties.

[1516] Suitable donor particles for use in the ingestible devicesof this application may be any type of particles as describedherein. In some embodiments, the donor particles are selected fromthe group consisting of latex particles, lipid bilayers, oildroplets, silica particles, and metal sols. In some embodiments,the donor particles are latex particles, such as, but not limitedto Polystyrene latex particles (175 nm) having about 8.3 carboxylgroups per nm.sup.2 of surface. Latex particles can vary between175 nm to 800 nm. In some embodiments, the donor particles arelatex particles (e.g., any type of latex particles describedherein) that are coated with streptavidin.

[1517] Suitable photosensitizers for use in the ingestible devicesof this application include any of the photosensitizers asdescribed U.S. Pat. Nos. 6,251,581, 5,516,636, 8,907,081,6,545,012, 6,331,530, 8,247,180, 5,763,602, 5,705,622, 5,516,636,7,217,531, and U.S. Patent Publication No. 2007/0059316. In someembodiments, the photosensitizers are selected from the groupconsisting of t-Bultyl Silicon Pthalocyanine, Chlorophyll, andSilicon Napthalo cyanine.

[1518] The photosensitizer and chemiluminescent compound can beincorporated into donor and acceptor particles, respectively, byvirtue of being soluble in at least one phase of the particles, inwhich case the photosensitizer and chemiluminescent compound willbe at much higher concentration within the particle than in thebulk assay medium. When the photosensitizer and chemiluminescentcompound are covalently bound to donor and acceptor particles,respectively, the photosensitizer and chemiluminescent compound orthe particles, or components thereof, are functionalized to providea means of attaching the photosensitizer and chemiluminescentcompounds and the particles. Example ways to incorporatephotosensitier and chemiluminescent compounds in latex particlescan be found in PCT International Publication No. WO1999/042838 andU.S. Pat. No. 7,709,273. For particles that are oil droplets orliposomes the photosensitizer and chemiluminescent compound can beattached to one or more long hydrocarbon chains, each generallyhaving at least 10 to 30 carbon atoms. If the particles aredroplets of a fluorocarbon, the photosensitizer or chemiluminescentcompound incorporated into these particles may be fluorinated toenhance solubility and reduce exchange into other particles boundwith the other label, and the hydrocarbon chain used for linkingwill preferably be replaced with a fluorocarbon chain. For siliconfluid particles the photosensitizer and chemiluminescent compoundcan be bound to a polysiloxane. In order to maximize the number ofphotosensitizer or chemiluminescent compound molecules perparticle, in some embodiments, it may be desirable to minimize thecharge and polarity of the photosensitizer or chemiluminescentcompound so that it resides within the non-aqueous portion of theparticle. When the particle is a liposome and it is desired toretain the photosensitizer or chemiluminescent compound in theaqueous phase of the liposome, in some embodiments,photosensitizers or chemiluminescent compounds that are highlypolar or charged may be used.

[1519] In some embodiments, the ratio of the number of donorparticles to the number of the acceptor particles ranges from 10:1to 1:10. In some embodiments, the ratio of the number of donorparticles to the number of the acceptor particles ranges from 5:1to 1:10. In some embodiments, the ratio of the number of donorparticles to the number of the acceptor particles ranges from 5:1to 10:1.

[1520] Suitable analytes to be detected, quantified, or measured bythe ingestible devices of this application include any of theanalytes as described herein. In some embodiments, the analyte isselected from the group consisting of proteins, peptides, cellsurface receptors, receptor ligands, nucleic acids, carbohydrates,cells, microorganisms, and fragments thereof. In some embodiments,the analyte is selected from the group consisting of TNF.alpha.,lipoteichoic acid (LTA), lipopolysaccharide (LPS),lipopolysaccharide binding protein (LBP), calprotectin, cytokinesand chemokines, IL12/23, IL-6, IL-10, MADCAM, a4137 integrin, HGF,EGF, HB-EGF, TGFb, Adalimumab, Infliximab, Cimzia, Vedolizumab,Tysabri, Simponi, Remsima, bevacizumab (Avastin), and cetuximab(Erbitux).

[1521] The first analyte-binding agent coupled to or associatedwith the donor particles and the second analyte-binding agentcoupled to or associated with the acceptor particles are capable ofbinding to the same analyte. The analyte, when present, thereforeaffects the amount of the photosensitizer of the donor particlesand the chemiluminescent compound of the acceptor particles thatcan come into close proximity, wherein the short-lived singletoxygen generated by the photosensitizer can react with thechemiluminescent compound prior to its spontaneous decay and uponreaction, the chemiluminescent compound produces luminescence. Theintensity of luminescence produced is related to the amount ofanalyte in the sample. The chemiluminescent compound is capable ofactivation by singlet oxygen, and the photosensitizer catalyzes theformation of singlet oxygen usually in response to photoexcitationfollowed by energy transfer to molecular oxygen.

[1522] In some embodiments, an analyte causes molecules of thephotosensitizer and the chemiluminescent compound to be closer toeach other than their average distance in the bulk solution of theassay medium. This partitioning will depend upon the amount ofanalyte present in the sample to be analyzed. The photosensitizermolecules that do not become associated with the chemiluminescentcompound produce singlet oxygen that is unable to reach thechemiluminescent compound before undergoing decay in the aqueousmedium. However, when the photosensitizer and the chemiluminescentcompound come in close proximity with each other in response to theamount of the analyte, the singlet oxygen produced upon irradiationof the photosensitizer can activate the chemiluminescent compoundbefore undergoing decay.

[1523] In some embodiments, the first analyte-binding agent coupledto or associated with the donor particles and the secondanalyte-binding agent coupled to or associated with the acceptorparticles are independently selected from the group consisting ofantibodies, aptamers, cell surface receptors, receptor ligands,biotin, streptavidin, avidin, protein A, G, and L, and derivativesthereof. In some embodiments, the first analyte-binding agent isthe same as the second analyte-binding agent. In some embodiments,the first analyte-binding agent is different from the secondanalyte-binding agent. In some embodiments, the firstanalyte-binding agent is an antibody or a derivative thereof (e.g.,a biotinylated antibody). In some embodiments, the secondanalyte-binding agent is an antibody or a derivative thereof (e.g.,a biotinylated antibody). In some embodiments, the secondanalyte-binding agent is an antibody covalently conjugated to theacceptor particles. In some embodiments, the first analyte-bindingagent is a biotinylated antibody and the donor particles are coatedwith streptavidin. In some embodiments, the first analyte-bindingagent is a biotinylated antibody and the donor particles are coatedwith streptavidin, and the second analyte-binding agent is anantibody covalently conjugated to the acceptor particles.

[1524] In some embodiments, the first analyte-binding agent coupledto or associated with the donor particles is an antibody selectedfrom the group consisting of anti-bacterial antibodies. In someembodiments, the antibody is selected from the group consisting ofanti-Gram positive bacteria antibodies, anti-Gram positive bacteriaLTA antibodies, anti-Gram negative bacteria antibodies,anti-lipoteichoic acid antibodies, anti-E. coli antibodies,anti-lipid A antibodies, anti-TNF.alpha. antibodies, andderivatives thereof. In some embodiments, the antibody is selectedfrom the group consisting of MA1-7401 antibody, MA1-40134 antibody,ab127996 antibody, ab35654 antibody, ab35654 antibody, ab137967antibody, ab8467 antibody, and derivatives or fragmentsthereof.

[1525] In some embodiments, the second analyte-binding agentcoupled to or associated with the acceptor particles is an antibodyselected from the group consisting of anti-bacterial antibodies. Insome embodiments, the antibody is selected from the groupconsisting of anti-Gram positive bacteria antibodies, anti-Grampositive bacteria LTA antibodies, anti-Gram negative bacteriaantibodies, anti-lipoteichoic acid antibodies, anti-E. coliantibodies, anti-lipid A antibodies, anti-TNF.alpha. antibodies,and derivatives thereof. In some embodiments, the antibody isselected from the group consisting of MA1-7401 antibody, MA1-40134antibody, ab127996 antibody, ab35654 antibody, ab35654 antibody,ab137967 antibody, ab8467 antibody, and derivatives or fragmentsthereof.

[1526] In some embodiments, the donor particles include more thanone type of analyte-binding agent. In some embodiments, theacceptor particles include more than one type of analyte-bindingagent. For example, analyte-specific reagents can be used on donorand acceptor beads. For Analyte 1, parameters are set as excitationat 680 nm, emission at 615 nm (half-life 0.6 seconds). For Analyte2, parameters are set as excitation at 680 nm, emission at 615 nm(half-life 30 seconds). For Anaylte 3, parameters are set asexcitation at 680 nm, emission at 615 nm (half-life 300 seconds).After the first excitation, emission for analyte 1 is measure from:0-6 seconds; emission for analyte 2 is measured from 6-300 seconds;and emission for analyte 3 is measured from 300-600 seconds.Signals are deconvoluted as described in further detail in PCTInternational Publication No. WO1999/042838. In some embodiments,emission wavelengths may be used to evaluate multiple analytes asdiscussed herein.

[1527] In some embodiments, the composition for use in theingestible devices of the present application further includescyclodextrin with a concentration range of 25-50 mM, or 1-500mM.

[1528] In one aspect, this application provides a kit including aningestible device as described herein. In some embodiments, the kitfurther includes instructions, e.g., for detecting or quantifyingan analyte in a sample. In some embodiments, such a device is aningestible device for detecting or quantifying viable bacterialcells in vivo (e.g., in the GI tract).

[1529] Certain illustrative embodiments will now be described,including various systems and methods for obtaining samples usingingestible devices. In particular, techniques are described thatallow an ingestible device to obtain a sample from within agastrointestinal (GI) tract. These samples may include any of thefluids, solids, particulates, or other substances found within theGI tract. However, it will be understood by one of ordinary skillin the art that the systems and methods described herein may beadapted and modified as is appropriate for the applications beingaddressed, and that the systems and methods described herein may beemployed in other suitable applications, and that such otheradditions and modifications will not depart from the scope of thepresent disclosure. Generally, the ingestible devices describedherein may include actuators, sensors, valves, chambers, logicdevices, telemetry systems, microcontrollers or other devices andprocessors that may be configured using a combination of hardware,firmware, and software to carry out one or more of the methodsdescribed herein.

[1530] In some embodiments, the ingestible device further includesan illuminating source. The illuminating source is capable ofirradiating the composition held in the sampling chamber of theingestible devices with light having a wavelength with energysufficient to convert the photosensitizer to an excited state andthereby render it capable of activating molecular oxygen to singletoxygen. The excited state for the photosensitizer capable ofexciting molecular oxygen is generally a triplet state which ismore than about 20, e.g., at least 23, Kcal/mol more energetic thanthe photosensitizer ground state. In some embodiments, thecomposition is irradiated with light having a wavelength of about450 to 950 nm although shorter wavelengths can be used, forexample, 230-950 nm. The luminescence produced may be measured inany convenient manner such as photographically, visually orphotometrically to determine the amount thereof, which is relatedto the amount of analyte in the medium.

[1531] In some embodiments, the 632.6 nm emission line of ahelium-neon laser is an inexpensive light source for excitation.Photosensitizers with absorption maxima in the region of about 620to about 650 nm are compatible with the emission line of ahelium-neon laser and are, therefore, useful illuminating sourcesfor use in the present application. Example irradiating wavelengthsfor diode lasers include 680 nm, 780 nm, and/or the like. In someembodiments, the illuminating source is capable of irradiating thecomposition with light having a wavelength selected from the groupconsisting of 678 nm, 633 nm, and 780 nm.

[1532] Other means of excitation of the photosensitizer are alsocontemplated herein. In some embodiments, excitation of thephotosensitizer may be achieved by energy transfer from an excitedstate of an energy donor such as a second photosensitizer. When asecond photosensitizer is used, wavelengths of light can be usedwhich are inefficiently absorbed by the photosensitizer butefficiently absorbed by the second photosensitizer. The secondphotosensitizer may be bound to an assay component that isassociated/coupled, or becomes associated/coupled, with the firstphotosensitizer, for example, bound to a surface or incorporated inthe particle having the first photosensitizer. When a secondphotosensitizer is employed it will usually have a lowest energytriplet state at higher energy than the lowest energy triplet stateof the first photosensitizer.

[1533] In some embodiments, the ingestible device includes adetector for detecting the luminescence emitted by thechemiluminescent compound. In some embodiments, the detector is aphotodiode that is capable of detecting the luminescence emitted bythe chemiluminescent compound at a wavelength selected from 613 nmand 660 nm. Additional suitable detection wavelengths include butnot limited to 430 nm, 500 nm, 540 nm, 615 nm, 680 nm, and/or thelike.

[1534] In some embodiments, chemiluminecence intensity is measuredwith an optical reader. The actual configuration and structure ofthe optical reader may generally vary as is readily understood bythose skilled in the art. Typically, the optical reader contains anillumination source that is capable of emitting light at a definedwavelength and a detector that is capable of registering a signal(e.g., transmitted, reflected, or fluorescence light). Opticalreaders may generally employ any known detection technique,including, for instance, luminescence (e.g., fluorescence,phosphorescence, etc.), absorbance (e.g., fluorescent ornon-fluorescent), diffraction, etc. Exemplary optical readers,illumination sources and detectors are disclosed in U.S. Pat. No.7,399,608, which is hereby incorporated by reference herein in itsentirety.

[1535] In some embodiments, the illumination source may be anydevice known in the art that is capable of providingelectromagnetic radiation, such as light in the visible ornear-visible range (e.g., infrared or ultraviolet light). Forexample, suitable illumination sources that may be used in thepresent invention include, but are not limited to, light emittingdiodes (LED), flashlamps, cold-cathode fluorescent lamps,electroluminescent lamps, and so forth. The illumination may bemultiplexed and/or collimated. In some embodiments, multiplexedanalysis is enabled. For a single sample, multiple differentanalytes can be measured by detecting different wavelengths ofemitted lights. The multiplexing can be further increased by usingboth emitted light and the half-life of emitted light (e.g., 0.6seconds to 300 seconds). For example, the chemiluminescent compoundas used herein can emit light within the wavelength range of 250 to1200 nm and with emission life time of 0.6-300 seconds; and thusemitted lights of different wavelengths within the wavelength rangecan be multiplexed (e.g., up to 11, etc.). In some embodiments, theillumination may be pulsed to reduce any background interference.In some embodiments, filters may be used to improve optics. See,e.g., Reichman, Jay, Handbook of optical filters for fluorescencemicroscopy, Chroma Technology Corporation (2000). In someembodiments, excitation source may be a LED with a band-passfilter, e.g., a filter for 680 or 780 nm+/-20 nm wavelength toselectively excite a sample with 680 or 780 nm light. In someembodiments, to cut out any stray longer wavelengths from the greenLED, a Thorlabs FESH0550 shortpass filter may be used forexcitation (FIG. 72A). In some embodiments, the emission from asample is captured at a 90.degree. angle with an avalanchephotodiode detector with a bandpass filter, e.g., a filter for 430nm+/-20, 450 nm+/-20, 510 nm+/-20, 6130 nm+/-10, 648 nm+/-10 nmwavelength, placed in front of the detector, to selectively capturelight emitted at at specific nm. In some embodiments, a ThorlabsFB580-10 bandpass filter may be used as an emission filter (FIG.72B). A cross sectional view of an exemplary Chemiluminescent assaytest fixture depicting collimating, focusing, and filtering lensesis shown in FIG. 72C. In some embodiments, a 5-50 microsecond delaymay be used before emission is measured. For example, in someembodiments, LEDs (e.g., aluminum gallium arsenide red diodes,gallium phosphide green diodes, gallium arsenide phosphide greendiodes, or indium gallium nitride violet/blue/ultraviolet (UV)diodes) are used as the pulsed illumination source. In someembodiments, the illumination source may provide diffuseillumination to the dye. For example, an array of multiple pointlight sources (e.g., LEDs) may simply be employed to providerelatively diffuse illumination. In some embodiments, theillumination source is capable of providing diffuse illumination ina relatively inexpensive manner is an electroluminescent (EL)device. An EL device is generally a capacitor structure thatutilizes a luminescent material (e.g., phosphor particles)sandwiched between electrodes, at least one of which is transparentto allow light to escape. Application of a voltage across theelectrodes generates a changing electric field within theluminescent material that causes it to emit light.

[1536] In some embodiments, the detector may be any device known inthe art that is capable of sensing a signal. In some embodiments,the detector may be an electronic imaging detector that isconfigured for spatial discrimination. Some examples of suchelectronic imaging sensors include high speed, linearcharge-coupled devices (CCD), charge-injection devices (CID),complementary-metal-oxide-semiconductor (CMOS) devices, and soforth. Such image detectors, for instance, are generallytwo-dimensional arrays of electronic light sensors, although linearimaging detectors (e.g., linear CCD detectors) that include asingle line of detector pixels or light sensors, such as, forexample, those used for scanning images, may also be used. Eacharray includes a set of known, unique positions that may bereferred to as "addresses." Each address in an image detector isoccupied by a sensor that covers an area (e.g., an area typicallyshaped as a box or a rectangle). This area is generally referred toas a "pixel" or pixel area. A detector pixel, for instance, may bea CCD, CID, or a CMOS sensor, or any other device or sensor thatdetects or measures light. The size of detector pixels may varywidely, and may in some cases have a diameter or length as low as0.2 micrometers.

[1537] In other embodiments, the detector may be a light sensorthat lacks spatial discrimination capabilities. For instance,examples of such light sensors may include photomultiplier devices,photodiodes, such as avalanche photodiodes or silicon photodiodes,and so forth. Silicon photodiodes are sometimes advantageous inthat they are inexpensive, sensitive, capable of high-speedoperation (short risetime/high bandwidth), and easily integratedinto most other semiconductor technology and monolithic circuitry.In addition, silicon photodiodes are physically small, whichenables them to be readily incorporated into various types ofdetection systems. If silicon photodiodes are used, then thewavelength range of the emitted signal may be within their range ofsensitivity, which is 400 to 1100 nanometers. In some embodiments,a photomultiplier may be used to increase the intensity of thesignal.

[1538] In another aspect, the present application providesingestible devices containing a microscopic evaluation system. Insome embodiments, bacterial cells in a sample may be first labeledwith fluorescent dyes (such as those described herein), and thefluorescently-labeled cells may be imaged and counted by themicroscopic evaluation using an ingestible device as describedherein. In other embodiments, the fluorescently-labeled cells arecounted as they pass through an onboard flow system (e.g.,microfluidic single cell channeling). Examples of flow cytometrysystems include hydrodynamic focusing, small diameter capillarytube flow, and rectangular capillary tube flow. As describedherein, live bacteria cells are labeled, and the principles of flowcytometry are used to quantify labeled cells. Generally speaking,the photons from an incident laser beam are absorbed by thefluorophore and raised to a higher, unstable energy level. Withinless than a nanosecond, the fluorophore re-emits the light at alonger representative wavelength where it is passed through aseries of dichroic filters. This reemitted light can be collectedand interpreted as proportional to the number of labeled bacteriacells. In some embodiments, a sheath fluid is not used as part ofthe flow system to help accommodate the volume restrictions of thedevice. In some embodiments, a rectangular capillary tube is usedto achieve a sufficiently large cross-sectional area and relativelythin inspection area. The flow cytometry optical system operatesparallel to the fluidics system and serves to observe theredirection of light passing through the cell and deliversinformation about the bacterial cells. In some embodiments, ratherthan using a conventional laser and spherical lenses to focus thelight to a point, an LED and cylindrical lenses are used to focusthe light to a line across a rectangular capillary tube. In otherembodiments, collimating lenses are used to make the light sourceparallel, while cylindrical lenses are used to refine theinspection area. An exemplary optical configuration for thisarrangement can be seen in FIG. 30. In some embodiments, opticalfilters can be added to permit the use of fluorophores. Thecharacteristic wavelength of reemitted light from the fluorophorescan be isolated and detected with the use of dichroic, bandpass,and short or long wave pass filters. Generally, multiple dichroiclenses and photomultipliers are used, however, due to spacelimitations, only a single side-scatter detector and forwardscatter detector may be used in certain embodiments.

[1539] Where the analyte is bacteria cells, one of the designchallenges of integrating flow cytometry into the device is toprovide a pumping mechanism. Without moving fluid, individualbacteria cells cannot be identified and accounted for by flowcytometry within a fixed volume of fluid. In some embodiments, agear motor is to move fluid through the device. For example, amicromotor including a planetary gearhead (e.g., with a 25:1reduction) can provide the desired amount of torque to create fluidflow. In another embodiment, a series of piezoelectric resistorsembedded in the surface of a microfabricated plate is used tocreate flow. In yet another embodiment, a micropump that includes apair of one-way valves and uses a magnetic pump membrane actuatedby an external magnetic field is used to create flow.

[1540] In some embodiments, the system architecture includes anopening and sealing mechanism combined with a rotary wiper whichcreates a pressure driven flow via a gear motor. The gear motor canbe used for other functions in the device. As shown in FIG. 31, thecomponents of the optics and flow chamber systems fit within thedevice. In some embodiments, the sample fluid is absorbed via aflexible membrane at the top of the capsule. In some embodiments,the gear motor has 270.degree. of permissible travel which servesto open and fill the fluid chamber. During closure, the motorcloses the ingress port while simultaneously pushing the fluidthrough the rectangular capillary tube where the optical system islocated. The threaded component allows the flexible membrane toclose and seal the ingress channel without changing the wiperheight. In some embodiments, the volume of the sample chamber is 25.mu.L, 50 .mu.L, 75 .mu.L or more (e.g., within the range of 10-500.mu.ls