WO2020163381A1 - Compositions et procédés pour détecter une maladie gastro-intestinale - Google Patents

Compositions et procédés pour détecter une maladie gastro-intestinale Download PDF

Info

Publication number
WO2020163381A1
WO2020163381A1 PCT/US2020/016646 US2020016646W WO2020163381A1 WO 2020163381 A1 WO2020163381 A1 WO 2020163381A1 US 2020016646 W US2020016646 W US 2020016646W WO 2020163381 A1 WO2020163381 A1 WO 2020163381A1
Authority
WO
WIPO (PCT)
Prior art keywords
iap
nec
activity
protein
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/016646
Other languages
English (en)
Inventor
Sunyoung Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Louisiana State University
Original Assignee
Louisiana State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/267,120 external-priority patent/US11493515B2/en
Application filed by Louisiana State University filed Critical Louisiana State University
Priority to CN202510261116.9A priority Critical patent/CN120102879A/zh
Priority to CN202080027193.3A priority patent/CN113645846B/zh
Priority to US17/428,416 priority patent/US11953501B2/en
Priority to AU2020217717A priority patent/AU2020217717B2/en
Priority to CA3128826A priority patent/CA3128826A1/fr
Priority to EP20752596.5A priority patent/EP3920713A4/fr
Publication of WO2020163381A1 publication Critical patent/WO2020163381A1/fr
Anticipated expiration legal-status Critical
Priority to US18/628,482 priority patent/US20240393333A1/en
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • This invention is directed to compositions and methods to detect and tixiat gastrointestinal diseases.
  • Gastrointestinal diseases refer to diseases involving the gastrointestinal tract.
  • necrotizing enterocolitis is an acquired gastrointestinal disease often seen in pre-term infants.
  • bacteria invade the wall of the intestine, causing local infection and inflammation.
  • NEC is characterized by high mortality and long-term morbidity, including short gut syndrome, recurrent infection, nutritional deficiency and neurodeve!opmenta! delay.
  • NEC-associated deaths Despite an overall net decrease in mortality for premature infants, there has been an increase in NEC-associated deaths.
  • NEC is often difficult to diagnose and manage, due to initial nonspecific symptomatology and rapid deterioration. Clinicians currently rely on radiographic evidence to make the diagnosis in advanced stage of disease.
  • the present invention provides a method for determining the prognosis of necrotizing enterocolitis (NEC) in a patient.
  • the method comprises fitting a Markov model using a two state transition matrix and propensity values measured among a plurality of subjects, wherein the two stale transition matrix comprises a first state and a second state, wherein the first state comprises a non-necrotizing enterocolitis status, wherein the second state comprises a necrotizing enterocolitis status (NEC), wherein a propensity value is a function of intestinal Alkaline Phosphatase (i AP) activity values and the amount of iAP found in a subject of the plurality of subjects; using a propensity value of the patient and the fitted Maikov model to estimate a probability of transitioning from the first state to the second state, wherein the fitted model indicates that an increased propensity level of the patient significantly increases the probability of transitioning from the first state to the second stale; treating the patient when the propens
  • apropensity value comprises a product of a first value and a second value, wherein the first value comprises one (1) minus a first ratio, wherein the first ratio comprise an iAP activity value of a subject of the plurality of subjects divided by a maximum iAP activity value observed among the plurality of subjects, wherein the second value comprises a second ratio, wherein the second ratio comprises an amount of iAP from an immunoassay value of a subject of the plurality of subjects divided by a maximum amount of iAP from an immunoassay value observed in a sample.
  • Non-limiting examples of immunoassays comprise western blot analysis, ELISA, or immunopiecipitation.
  • the immunoassay can comprise western blot analysis utilizing a chemiluminescent reporter.
  • the immunoassay can comprise western blot analysis using a fluorescent reporter. Fluorescent reports can be more linear in response.
  • the immunoassay readings can be considered a 'mini-ELlSA,' as patient sample iAP abundance is quantitated against a 2-point curve.
  • the signal in a patient sample can be compared by ratioing it to the difference between human small intestinal lysate (positive control with the greatest level of iAP or 100%) and bovine iAP (negative control as anti-human iAP antibody does not detect cow iAP or 0%).
  • the maximum WB value in Example 9 is the human small intestinal lysate.
  • a propensity value comprises a product of a first value and a second value, wherein the first value comprises one (1 ) minus an iAP activity value of a subject of the plurality of subjects, wherein the second value comprises an amount of iAP from an immunoassay value of a subject of the plurality of subjects.
  • the plurality of subjects comprises the patient.
  • treating comprises withholding feeding, administering an antibiotic, or a combination thereof.
  • the sample is a human small Intestinal lysate.
  • the immunoassay comprises a western blot, an ELISA, or immunoprecipitation.
  • the present invention provides a method of identifying a subject afflicted with a gastrointestinal (GI) disease.
  • An aspect of the invention is directed to methods for diagnosing a subject with a gastrointestinal disease.
  • Another aspect of the invention is directed to methods for identifying a subject at risk of a gastrointestinal disease.
  • Embodiments as described herein can further identity both early stages of gastrointestinal disease and advanced stages of gastrointestinal disease. Certain embodiments can distinguish between early stage and late stage gastrointestinal disease.
  • embodiments as described herein can diagnose advanced slates of inflammation, such as that identified by radiological findings of pneumatosis intestinalis (portal vein or biliary gas).
  • embodiments can identify early stages of the disease before rampant inflammation of the gut is physiologically evident.
  • the methods comprise incubating a biological sample from a subject with an agent that binds intestinal alkaline phosphatase (iAP), detecting in the sample iAP-bound agent, and detecting and/or measuring in the sample the amount or activity of iAP in sample, whether bound to the agent or not
  • the iAP-bound agent is at least one GI disease biomarker comprising AP enzymatic activity of iAP, iAP protein level, iAP dimerization/dissociation, post-translationally modified iAP, total protein, such as total fecal protein, or a combination thereof.
  • the GI disease biomarker is useful for diagnosing a subject with a gastrointestinal disease, and can also be indicative of a subject afflicted with a gastrointestinal (GI) disease, and /or a subject at risk of developing a GI disease.
  • a subject afflicted with a gastrointestinal (GI) disease can encompass both early and advanced stages of the disease.
  • iAP is not bound by an iAP binding agent
  • a substrate can be provided for the enzyme in the sample, and the change of the substrate is monitored.
  • iAP is not bound for the activity assay.
  • substrate can be provided for the enzyme in the sample and the change in the enzyme can be monitored. Without being bound by theory, this can also be the same for AP-bound to enzyme where the measured change of substrate in immunoassays is for the protein tethered by the antibody system but also for any tree AP in the sample.
  • the present invention further provides for a method of diagnosing a GI disease in a subject, such as necrotizing enterocolitis, comprising incubating a biological sample from a subject with an agent that binds intestinal alkaline phosphatase (iAP), detecting in the sample iAP-bound agent, and detecting and/or measuring in the sample the amount or activity of iAP- bound agent
  • the iAP-bound agent is at least one GI disease biomarker comprising iAP enzymatic activity, iAP protein level, iAP dimerization/dissociation, post- translationally modified iAP, total protein, such as total fecal protein, or a combination thereof, and wherein the GI disease biomarker is indicative of a subject afflicted with a gastrointestinal (GI) disease.
  • GI gastrointestinal
  • An iAP-bound agent according to the invention can be iAP bound to its cognate substrate, an antibody that recognizes and binds to iAP, a short peptide sequence that is directed to and binds to iAP, and the like, non-limiting examples of which comprise a small-molecule activator or inhibitor of the catalytic reaction, a metal ion (tungsten is a transition state effector of alkaline phosphatases), an agent that causes allosteric release of products, a labile chemical moiety that serves as a chemical, enzymatic, or photoiytic trigger, or a matrix that binds to a tagged form of iAP.
  • a metal ion tungsten is a transition state effector of alkaline phosphatases
  • an agent that causes allosteric release of products a labile chemical moiety that serves as a chemical, enzymatic, or photoiytic trigger, or a matrix that binds to a tagged form of iAP.
  • Embodiments can further comprise diagnosing the subject as having a G1 disease.
  • a subject can be diagnosed as having a GI disease: if the total protein concentration in the sample is greater than about 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/m!, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml, 2.1 mg/mL 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3.0 mg/ml,
  • iAP activity is less than about 10 mU/mg, 20 mU/mg, 30 mU/mg, 40 mli/mg, 50 mU/mg, 60 mU/mg, 70 mll/mg, 80 mU/mg, 90 mU/mg, 100 mU/mg, 200 mU/mg, 300 mU/mg, 400 mU/m
  • a subject in embodiments, can be diagnosed as having a GI disease if the total protein concentration in the sample is greater than about 1.8 mg/ml, if the iAP activity is tower than about 979 mU/mg, if the level of iAP protein is greater than 10.7% of a control sample, or a combination thereof.
  • a subject can be diagnosed as having a GI disease if the total protein concentration in the sample is greater than about 1.6mg/ml, if the iAP activity is lower than about 1256 mU/mg, if the level of iAP protein is greater than 4.8% of a control sample, or a combination thereof
  • the present invention provides a method of diagnosing a gastrointestinal (GI) disease in a subject comprising obtaining a sample from the subject, detecting the presence of at least one GI disease biomarker in the sample, wherein the GI disease biomarker can comprise intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to that of a profile obtained from a control sample, and treating the subject
  • the control sample can comprise two or more control samples.
  • the present invention further provides a method of preventing the progression of a gastrointestinal disease in a subject in need thereof comprising obtaining a sample from the subject, detecting the presence of at least one GI disease biomarker in the sample, wherein the GI disease biomarker can comprise intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to that of a profile obtained from a control sample, and treating the subject.
  • the control sample can comprise two or more control samples.
  • the present invention further provides a method of ameliorating the symptoms associated with a gastrointestinal disease in a subject in need thereof comprising obtaining a sample from the subject, detecting the presence of at least one GI disease biomarker in the sample.
  • the GI disease bi omarker can comprise intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to that of a profile obtained from a control sample, and treating the subject
  • the control sample can comprise two or more control samples.
  • treating the subject diagnosed with a Gl disease comprises administering an effective amount of antibiotics, probiotics, intravenous fluids, or a combination thereof; withholding oral feeding; administering an iAP replacement composition; an anti-inflammatory; a therapeutic; a small molecule activator and/or effector of catalytic activity; parenteral (or intravenous) nutrition or a combination thereof.
  • Non-limiting examples of a therapeutic that can be used according to the invention comprise Toll-tike receptor (TLR) inhibitors (Neal et al. Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors. PIoS One 12, e65779) and interruption of eNOS-NO-nitrite signaling (Yazji et al. Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling. Proceeedings of the National Academy of Science USA 110, 9451 -9456).
  • TLR Toll-tike receptor
  • Non-limiting examples of a small molecule effector of catalytic activity comprise levamisole, theophylline, triazole-based compounds, sulfonamide derivatives, phosphatase derivatives, metals, and amino acids (Borgers M. The cytochemical application of new potent inhibitors of alkaline phosphatases. Journal of Histochemistry & Cytochemistry 21, 812-824; Klemperer et al. The inhibition of alkaline phosphatase by beryllium. Journal of Biological Chemistry 180, 281-288; Bobkova et al. Modulators of intestinal alkaline phosphatase. Methods Mol Biol 1053, 135-144; Narisawa et al.
  • Non-limiting examples of such antibiotics comprise Vancomycin, Ampicilim, Zosyn (combination of piperacillin and tazobactam), Gentamycin, Flagyl (metrodniazole generic), Meropenem, Metronidazole, Cefotaxime, Clindamycin, or any combination thereof.
  • an antifungal agent can further be administered.
  • the antifungal agent can be Fluconazole, Terconazole, Voriconazole, Posaconazole, Pentamidine, Itraconazole, and Ketoconazole.
  • Non-limiting examples of probiotic organisms include those in the genera Iactobacillus, Lactococcus, Bifidobacteria, Pediococcus, Saccharomyces boulardii, and related bacteria and yeast.
  • Non-limiting examples of such intravenous fluids comprise saline (such as 0.9% NaCl in water or .45% saline in water), Lactated Ringer's (0.9% NaCl with electrolytes and buffer), DsW (5% dextrose in water), DsNS (5% dextrose in 0.9% saline), Ds 1/2 NS (5% dextrose in 0.45% saline), DsLR (5% dextrose in Lactated Ringer’s), or Normosol-R.
  • the intravenous fluid solution can be isotonic. In other embodiments, the intravenous fluid solution can be hypotonic.
  • Non-limiting examples of parenteral (or intravenous) nutrition comprise intravenous dextrose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or a combination thereof.
  • the anti-inflammatory agents can be selected from a wide variety of steroidal, nonsteroidal, and salicylate water-soluble and water-insoluble drugs and their acid addition or metallic salts. Both organic and inorganic salts can be used provided the anti-inflammatory agent maintains its medicament value.
  • the anti-inflammatory agents can be selected from a wide range of therapeutic agents and mixtures of therapeutic agents that can be administered in sustained release or prolonged action form.
  • Non-limiting examples of anti-inflammatory agents comprise ibuprofen, naproxen, sulindac, diflunisal, piroxicam, mdomethacin, etodolac, meclofenamate sodium, fenoproben calcium, ketoprofen, mefenamic acid, nabumetone, ketorolac tromethamine, diclofenac, ami evening primrose oil (containing about 72% !ino!eic acid and about 9% gamma-linolenic acid).
  • Nonlimiting examples of salicylate antiinflammatory agents comprise acetylsalicylic acid, mesalamine, sa!salate, diflunisal, salicylsalicylic acid, and choline magnesium trisalicylate.
  • Nonlimiting examples of steroidal anti-inflammatory agents comprise flunisolide, triamdnoline, triamcinoline acetonide, bec!omethasone diproprionate, betamethasone diproprionate, hydrocortisone, cortisone, dexamethasone, prednisone, methyl prednisolone, and prednisolone.
  • the gastrointestinal disease can comprise colitis, inflammatory bowel disease (IBD), or a combination thereof,
  • colitis can comprise necrotizing enterocolitis (NEC), adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.
  • the sample can comprise a biological sample obtained from subject.
  • the biological sample can be a biological fluid, a biological solid, or a biological semi-solid.
  • the sample can comprise fecal matter, meconium, vomit, peripheral blood, sera, plasma, or urine.
  • the G1 disease biomarker can comprise iAP enzymatic activity, AP enzymatic activity, iAP protein level, iAP dimerization/dissociation, post-translationally modified iAP, total protein, such as total fecal protein, or a combination thereof.
  • the post-lranslational modification can comprise acetylation, acylation, alkylation, amidation, butyrylation, deamidation, ibrmylation, glypiation, glycosylation.
  • the GI disease biomarker can comprise a NEC biomarker.
  • detecting can comprise an immunoassay, a colorimetric assay, a fluorimetric assay or a combination of both.
  • the immunoassay can comprise a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunopredpitation, single molecule immunoassays in femoliter chamber arrays, digital enzyme assays in both single and multiplex forms, or a combination thereof.
  • the delecting comprise contacting the sample with an anti-iAP antibody.
  • the anti-iAP antibody is a polyclonal or monoclonal antibody.
  • detecting can comprise a kinetic assay, an endpoint assay, Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, a pyrogallol red protein dye-binding assay, a Coomassie blue dye-binding assay, or a combination thereof.
  • BCA bicinchoninic acid
  • detecting can comprise techniques known to one skilled in the art, such as mass spectrometry (MS), RNA sequencing, and immunostaining of patient samples.
  • MS mass spectrometry
  • RNA sequencing of intestinal alkaline phosphatase or other alkaline phosphatases is rapid method of detecting molecules, such as iAP (Knight et al. Non-invasive analysis of intestinal development in preterm and term infants using RNA-sequencing. 2014. Scientific Reports 4, 5453).
  • the assay can detect phosphatase activity.
  • Non-limiting examples of such assays comprise fluorescent, diemihuninescenl, or colorimetric dectection methods, an assay to detect ATP hydrolysis and/or products of ATP hydrolysis, or a combination thereof.
  • detecting can comprise a kinetic assay comprising use of 4- methyllumbeiliferyl phosphate, CPD Star (Disodium 2-chloro-5-(4-methoxyspiro[ 1,2- dioxetane-3,2'-(5-chlorotricyclo
  • CPD Star Disodium 2-chloro-5-(4-methoxyspiro[ 1,2- dioxetane-3,2'-(5-chlorotricyclo
  • alkaline phosphatase activity such as intestinal alkaline phosphatase activity
  • chromogenic substrates and/or fluorogenic substrates of alkaline phosphatase such as iAP.
  • iAP alkaline phosphatase activity
  • 4-methylumbeIliferyl phosphate (MUP) is a fluorogenic substrate for alkaline phosphatases, and alkaline phosphatase mediated hydrolysis of its phosphate substituent yields the blue-fluorescent 4-methyIumbelliferyI (excitation/emission ⁇ 386/448 nm).
  • the MUP can be directly admixed with the biological sample, such as stool, allowing for the direct dectection of the presence of alkaline phosphatase or the
  • Non-limiting examples of Alkaline phosphatase (AP) substrates comprise AP-Blue substrate (blue precipitate, Zymed catalog p. 61); AP-Orange substrate (orange, precipitate, Zymed), AP-Red substrate (red, red precipitate, Zymed), 5-bromo, 4-chioro, 3- indolypbosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indoIy!
  • BCIP/NBT substrate blue/purple
  • 5-bromo 4-chloro, 3-indolyl phospbate/nitroblue tetrazolium/i odonitrotetrazolium
  • BCIP/NBT/INT brown precipitate
  • DAKO Fast Red (Red), Magenta-phos (magenta), Naphthol AS-BI-phosphate (NABPVFast Red TR (Red), Naphthol AS-BI-phosphate (NABP)/New Fuchsin (Red), Naphthol AS-MX-phosphate (NAMP)/New Fuchsin (Red), New Fuchsin AP substrate (red), p-Nitrophenyl phosphate (PNPP, Yellow, water soluble), VECTORTM Black (black), VECTORTM Blue (blue), VECTORTM Red (red), Vega Red (raspberry red color), Fluorescein diacelate, 4- Methylumbelliferyl acetate,
  • Non-limiting examples of suitable chromogenic substrates for use within the present invention comprise o-Nitrophenyl-b-D-galactopyranoside, p-Nitrophenyl-b-D- galactopyranoside, o-Nitrophenyl-b-D-ghicopyranoside, p-Nitrophenyl-a-D-glucopyranoside, p-Nitrophenyl-b-D-ghicopyranoside, p-Nitrophenyl-b-D-glucuronide, p- Nitrophenyl phosphate, o-Nitrophenyl-b-D-xybpyranoside, p-Nitrophenyl-a-D- xylopyranoside, p-Nitropheny 1 -b-D-xylopyranoside, and Phenolphthalein-b-D-glucuronide.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the protein level of iAP in the sample is at least two standard deviations above the mean protein level of the control sample. In embodiments, the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the protein level of iAP in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean protein level of the control sample in embodiments, the control sample can comprise two or more control samples.
  • Embodiments can comprise 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or 300 mg fresh weight stool/mL sterile water or buffer.
  • embodiments can comprise 200 mg fresh weigh stool/mL sterile water or buffer.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the protein level of iAP in the sample is greater than 4.8% of the control sample.
  • the method described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the protein level of iAP in the sample is greater than 107% of the control sample. In embodiments, the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the protein level of iAP in the sample is greater than 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400% of the control sanple.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the level of iAP enzyme activity in the sample is at least two standard deviations below the mean iAP enzyme activity of the control sanple. In embodiments, the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the level of iAP enzyme activity in the sanple is at least 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean enzyme activity of the control sample. In embodiments, the control sample can comprise two or more control samples.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the level of iAP enzyme activity in the sample is lower than about 10 mU/mg, 20 mU/mg, 30 mU/mg, 40 mU/mg, 50 mU/mg, 60 mU/mg, 70 mU/mg, 80 mU/mg, 90 mU/mg, 100 mU/mg, 200 mU/mg, 300 mU/mg, 400 mU/mg, 500 mU/mg, 60GmU/mg, 700 mU/mg, 800 mU/mg, 900 mU/mg, 1000 mli/mg, 1100 mU/mg, 1200 mU/mg, 1300 mU/mg, 1400 mU/mg, 5 U/mg, 10 U/mg, 50 U/mg, 5 U
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the level of iAP enzyme activity in the sample is less than 1500 mU/mg, 1000 mU/mg, 500 mU/mg, for example less than 1256 mU/mg.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the fecal protein level in the sample is at least two standard deviations above the mean fecal protan level of the control sample. In embodiments, the method as described herein further comprise diagnosing the subject with a gastrointestinal disease if the fecal protein level in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean fecal protein level of the control sample. In embodiments, the control sample can comprise two or more control samples.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the fecal protein level in the sample exceeds 1.6 mg/ml, or for example exceeds 1.8 mg/ml.
  • the method as described herein further comprise diagnosing the subject with a gastrointestinal disease or at risk of a gastrointestinal disease if the fecal protein level in the sample exceeds 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3.0 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3,4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, 4.0 mg
  • the method as described herein further comprise treating the subject.
  • treating can comprise administering to the subject diagnosed with a gastrointestinal disease an effective amount of antibiotics, probiotics, intravenous fluids, steps to withhold oral feeding, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof.
  • the subject can comprise a mammal.
  • the mammal can comprise a dog, cat, horse, cow, pig, or human.
  • the biomarkers of the invention can be useful for diagnosing colic in horses.
  • the human can comprise an infant.
  • the infant can comprise a preterm infant
  • the present invention further provides a method for screening the presence of a signature in a subject, such as a subject at risk of developing a gastrointestinal disease or a subject with a iron-symptomatic gastrointestinal disease, comprising obtaining a sample from the subject, measuring at least one GI disease biomarker in the sample, wherein the GI disease biomarker can comprise intestinal alkaline phosphatase (iAP) protein, comparing the Gl disease biomarker profile to that of a profile obtained from a control or reference sample, and treating the subject
  • the control or reference sample can comprise two or more control samples.
  • the sample is a fecal sample.
  • the present invention further provides a method for identifying a subject at risk for a gastrointestinal disease or a subject with a non-symptomatic gastrointestinal disease comprising obtaining a sample from the subject, measuring at least one GI disease biomarker in the sample, wherein the GI disease biomarker can comprise intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to that of a profile obtained from a control sample, and treating the subject
  • the control sample can comprise two or more control samples.
  • the gastrointestinal disease can comprise colitis, inflammatory bowel disease (IBD), or a combination thereof.
  • colitis can comprise necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.
  • the sample can comprise a biological sample obtained from subject.
  • the biological sample can be a biological fluid, a biological solid, or a biological semi-solid.
  • the sample can comprise fecal matter, meconium, vomit, peripheral blood, sera, plasma, or urine.
  • the GI disease biomarker further can comprise iAP enzymatic activity, total fecal protein, iAP dimerization/dissociation, post-translationally modified iAP, or a combination thereof.
  • the post-translational modification can comprise acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, lipoylation, matonylation, methy!ation, myristoylation, palmitoylation, phosphorylation, phosphopanletheinylation, prenylation, propionylation, ribosylation succinylation, sulfation, SUMOylation, or ubiquitination.
  • measuring can comprise performing an assay to determine total protein concentration, intestinal alkaline phosphatase enzyme activity, intestinal alkaline phosphatase protein concentration, or a combination thereof in the sample.
  • measuring can comprise a Bradford assay , a bicinchonimc acid (BC A) assay, a Lowry assay, a pyrogallol red protein dye-binding assay, a Coomassie blue dye-binding assay, or a combination thereof.
  • measuring can comprise a kinetic assay.
  • the kinetic assay can comprise use of 4-methyllumbelliferyI phosphate, nitrophenyl phosphate, or any other fluoromelric or colorimetric signal, an assay to detect ATP hydrolysis, or a combination thereof.
  • measuring can comprise an immunoassay, a colorimetric assay, fluorimetric assay or a combination of both.
  • the immunoassay can comprise a western blot assay, an enzyme- linked immunosorbent assay, immnnoprectpitation or a combination thereof.
  • the assay can comprise an anti-iAP antibody.
  • the anti-iAP antibody is a monoclonal or polyclonal antibody.
  • methods as disclosed herein can further comprise diagnosing the subject with a gastrointestinal disease when total protein concentration in the sample is at least two standard deviations above the mean of the control sample, the concentration of intestinal alkaline phosphatase protein is at least two standard deviations above the mean of the control sample, intestinal alkaline phosphatase activity is at least two standard deviations below the mean of the control sample, or a combination thereof.
  • methods as disclosed herein can further comprise diagnosing the subject with a gastrointestinal disease when total protein concentration in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean of the control sample, the concentration of intestinal alkaline phosphatase protein is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean of the control sample, intestinal alkaline phosphatase activity is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations below the mean of the control sample, or a combination thereof.
  • the control sample can comprise two or more control samples.
  • methods as disclosed herein can further comprise treating the subject.
  • treating can comprise administering to the subject diagnosed with a gastrointestinal disease an effective amount of antibiotics, probiotics, intravenous fluids, withholding oral feeding, an iAP replacement composition, an anti-inflammatory, a potential therapeutic, parenteral (or intravenous) nutrition, or a combination thereof.
  • the subject can be diagnosed with a gastrointestinal disease or at risk of a gastrointestinal disease if the protein concentration in fecal sample is greater than about 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ral, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/mi, 2.0 mg/ml, 2.1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml ⁇ .5 mg/ml, 2.6 mg/mi, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3.0 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3,4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/mi, 3.8 mg/mi, 3.9 mg/ml, 4.0 mg/m
  • the subject can be diagnosed with a gastrointestinal disease or at risk of a gastrointestinal disease if the iAP activity is lower than about 10 mU/mg, 20 mU/mg, 30 mU/mg, 40 mU/mg, 50 mU/mg, 60 mU/mg, 70 mU/mg, 80 mU/mg, 90 mU/mg, 100 mU/mg, 200 mU/mg, 300 mU/mg, 400 mU/mg, 500 mU/mg, 600 mU/mg, 700 mU/mg, 800 mU/mg, 900 mU/mg, 1000 mU/mg, 1100 mU/mg, 1200 mU/mg, 1300 mU/mg, 1400 mU/rag, 5 U/mg 10U/mg, 50 U/mg, 100 U/mg, 200 U/mg, 300 U/mg,
  • the subject can be diagnosed with a gastrointestinal disease or at risk of a gastrointestinal disease if the iAP protein detection by anti-iAP antibody exceeds 4.8% of control via densitometry. In embodiments, the subject can be diagnosed with a gastrointestinal disease if the level of iAP protein is at least two standard deviations above the mean of the control sample. In embodiments, the control sample can comprise two or more control samples. In embodiments, the subject can comprise a mammal. In embodiments, the mammal can comprise a dog, cat, horse, cow, or human. In embodiments, the human can comprise an infant. In embodiments, the infant can comprise a preterm infant,
  • the present invention further provides for a disposable article, the disposable article comprising a biosensor, wherein the biosensor can comprise at least one bio-recognition element, and wherein the biosensor detects iAP in a sample.
  • the biosensor further detects iAP enzymatic activity, total fecal protein, iAP dimerization/dissociation, post-translalionally modified iAP, or a combination thereof.
  • the post-translational modification can comprise acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosyiation, hydroxylation, iodination, ISGylation, Hpoylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopantetheinylation, prenylation, propionylation, ribosylation suecinylation, sulfation, SUMOylation, or ubiquitination.
  • the sample can comprise a biological sample obtained from subject.
  • the biological sample can be a biological fluid, a biological solid, or a biological semi-solid.
  • the sample can comprise fecal matter, meconium, vomit, peripheral blood, sera, plasma, or urine.
  • the biosensor is an immunosensor.
  • the biosensor can comprise a detection signal.
  • the detection signal can comprise a colorimetric signal, a fluorescent signal, or both.
  • the bio-recognition element can comprise an anti-iAP antibody.
  • the anti-iAP antibody can comprise a polyclonal or monoclonal antibody.
  • the biosensor can comprise lateral flow immunoassays, also known as immunochromatography assay or a strip test
  • Lateral flow immunoassays comprise immunoassays adapted to operate along a single axis to suit the test strip format.
  • a typical lateral flow test strip comprises a sample pad (an adsorbent pad onto which the test sample is applied), a conjugate or reagent pad (this contains binding agents, such as antibodies, specific to the target analyte conjugate to colored particles, such as colloidal gold nanoparticles or latex microspheres), reaction membrane (typically a nitrocellulose or- cellulose acetate membrane onto which anti-target analyte binding agents, such as antibodies, are immobilized in a line that crosses the membrane to act as a capture zone or test line.
  • a control zone Will also be present, containing antibodies specific for the conjugate antibodies), and a wick or waste reservoir (a further absorbent pad designed to draw the sample across the reaction membrane by capillary action and collect it).
  • the components of the strip are usually fixed to an inert backing material and may be presented in a simple dipstick format or within a plastic casing with a sample port and reaction window showing the capture and control zones.
  • the article can comprise a diaper to be worn by a subject, wipe for cleaning a subject, dipstick, spoon, scoopula, filter paper or swab.
  • the subject can comprise a mammal.
  • the mammal can comprise a dog, cat, horse, cow, or human.
  • the human can comprise an infant.
  • the infant can comprise a preterm infant.
  • the present invention further provides for a kit for diagnosing a subject with a gastrointestinal disease.
  • the kit can comprise a disposable «tide as described herein.
  • the gastrointestinal disease can comprise colitis, inflammatory bowel disease (IBD), or a combination thereof.
  • colitis can comprise necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.
  • the kit can comprise an iAP bio-recognition element immobilized to a solid support and instructions for use of same,
  • the gastrointestinal disease can comprise colitis, inflammatory bowel disease, or a combination thereof.
  • colitis can comprise necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.
  • the bio-recognition element can comprise an antibody directed to iAP or an oligonucleotide directed to iAP, for example, that is affixed directly or indirectly to a solid support.
  • Embodiments can also comprise a fluorescent substrate or inhibitor with high binding affinity to iAP attached to the solid support.
  • the solid support can comprise plastic, cardboard, or glass. In embodiments, the solid support can comprise a dip stick.
  • the subject can comprise a mammal.
  • the mammal can comprise a dog, cat, horse, cow, or human.
  • the human can comprise an infant
  • the infant can comprise a preterm infant.
  • the present invention further provides a diagnostic kit of molecular biomarkers for identifying a subject exhibiting or having a predisposition to develop a gastrointestinal disease.
  • the kit can comprise at least one of a means for determining total fecal protein concentration, a means for determining intestinal alkaline phosphatase (iAP) activity, and an iAP bio-recognition element, wherein together represent a molecular signature that is indicative of the presence of or a predisposition to development of a gastrointestinal disease in a human subject.
  • the gastrointestinal disease can comprise colitis, inflammatory bowel disease (IBD), or a combination thereof.
  • colitis can comprise necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.
  • the signature can comprise total protein concentration at least two standard deviations above the mean of a control sample, intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, or intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample.
  • the control sample can comprise two or more control samples.
  • the signature can be selected from at least two of the group comprising total protein concentration at least two standard deviations above the mean of a control sample, intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, and intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample.
  • the control sample can comprise two or more control samples.
  • the subject can comprise a mammal.
  • the mammal can comprise a dog, cat, horse, cow, or human.
  • the human can comprise an infant, in embodiments, the infant can comprise a preterm infant.
  • the invention provides for a method for mealing an NEC-afflicted subject.
  • the method comprises measuring, in a sample obtained from the subject according to the methods described herein, the amount or activity of iAP-bound agent, wherein the iAP-bound agent is at least one GI disease biomarker comprising iAP en/ymatic activity, AP enzymatic activity, IAP protein level, AP protein level, iAP dimerization/dissociation, post-translationally modified iAP, total fecal protein, or a combination thereof; determining the post-partum developmental age of the subject; and withholding enteral feeding for a period of time sufficient to resolve gastrointestinal inflammatory processes or signs of feeding intolerance
  • the method further comprises administering an antibiotic or an antifungal, either alone or in combination.
  • the method further comprises administering a probiotic, other biologic (such as stem cells or transcription factors), or therapeutic (such as TLR4 small molecules, alkaline phosphatase inhibitors or activators), antibiotics, intravenous fluids, an iAP replacement composition (such as that provide exogenously), a small molecule activator and/or effector of catalytic activity, an anti-inflammatory, parenteral (or intravenous) nutrition, either alone or in a combination thereof.
  • a probiotic such as stem cells or transcription factors
  • therapeutic such as TLR4 small molecules, alkaline phosphatase inhibitors or activators
  • antibiotics such as that provide exogenously
  • an iAP replacement composition such as that provide exogenously
  • a small molecule activator and/or effector of catalytic activity an anti-inflammatory, parenteral (or intravenous) nutrition, either alone or in a combination thereof.
  • the post-partum developmental age of a subject can be‘post-menstrual age’ or a‘post-menstrual developmental
  • oral feeding can be withheld for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days.
  • Non-limiting examples of biologies comprise stem cells and transcription factors.
  • the small intestine epithelium is in a constant dynamic state of flux and replaces itself every 3-6 days. This continuous renewal is necessary for maintenance of normal gut structure and function.
  • transcription factors include those that can be expressed and used for enterocyte differentiation, such as the Kruppel-like factor (GKLF or KLF4) family.
  • the feeding regiment can comprise an intermittent feeding regiment.
  • feeding can be withheld for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days, during which time there may be one or more days of oral feeding.
  • a doctor may suspect NEC and withhold feeding for a short period of time, such as one day or two days, until lab tests/examinations suggest that the baby does not have NEC, at which time they would resume feeding again. Several hours or days later, there may be another NEC scare and food would be withheld from the baby again for a period of time.
  • Intermittent feed can occur one or more times throughout a subjects stay in the hospital. Such intermittent feeding may allow the clinician to determine the tolerability to feeding of the subject In other embodiments, intermittent feeding may be recommended for a subject at risk of developing a gastrointestinal disorder.
  • FIG. 1 shows the longitudinal measurements of total fecal protein, iAP enzymatic activity, and immunoblol detection of iAP protein is depicted for two pre-term infants.
  • Patient 1 was bom at 30 weeks of gestation, developed NEC at 7 days of life, was treated medically and subsequently had recurrent NEC and an intestinal perforation on day of life 31. Infant recovered after placement of an intraperitoneal drain and 10 additional days of bowel rest and antibiotics. Red symbols and columns represent NEC episodes.
  • 7A and 7B refer to 2 separate stool samples collected on day of life 7, one prior to and one following the diagnosis of NEC.
  • FIG. 2 shows increased concentration of total fecal protein, decreased fecal iAP enzymatic activity, and increased iAP detection on weste blot at the time of NEC diagnosis, in the following panels, red circles represent individual stool samples collected during 7 distinct NEC events from 6 patients; black circles represent a composite average ⁇ standard error of 2-17 stool samples from 12 control patients. Red and grey columns represent the mean ⁇ standard error of all samples collected in NEC events and of all samples from control subjects, respectively.
  • (A) Protein concentrations were higher in stool samples from patients at the tin» ofNEC diagnosis compared with control samples (p-vahte 0.005).
  • FIG.3 shows fecal iAP-related measurements have high specificity and sensitivity.
  • A 3 -dimensional scalterplot of measurements for our biomarker candidates, where red diamonds represent NEC samples and black circles represent controls.
  • B A 2 -dimensional projection of the 3 -dimensional scatterplot that relates exclusively to iAP activity and western blot intensity measurements.
  • C Sensitivity and specificity curves for each biomarker candidate individually, as well as for a combined Naive Bayes Classifier that considers all 3 features simultaneously. Analysis covers 49 samples with 13 NEC samples, 9 NEC patient- derived control samples, and 27 control patient-derived control samples. The Spearman correlation coefficient was 0.19 for comparison of western blot intensity and total protein content, -0.48 for total protein content and iAP activity, and -0.58 for iAP activity and western blot intensity.
  • FIG. 4 shows demographic information of the study subjects. Averages and standard deviations are shown for gestational age and birth weight. Distribution of gestational age, gender, and Bell stage is also reported.
  • FIG. 6 shows demographics of patient enrollment in study.
  • FIG. 7 shows iAP activity was lower in stool samples from patients at time of NEC diagnosis compared to control samples.
  • the red circles represent iAP activity during 7 distinct NEC events of 6 patients (post conceptual age: 29-43 weeks).
  • Each black circle represents a composite average +/- SEM of 2-8 stool samples from each of 12 controls (post-conceptual ages 29-45 weeks).
  • Statistical significance analysis Mann-Whitney Test, P ⁇ 0.0001.
  • FIG. S shows total fecal protein was higher in stool samples from patients at the time of NEC diagnosis compared to control samples.
  • the red circles represent total fecal protein levels during 7 distinct NEC events of 6 patients (post conceptual age: 29-43 weeks).
  • FIG. 9 shows the intensity of iAP protein signed quantified to a percentage of positive control signal was much higher in stool samples from patients at the time of NEC diagnosis compared to control samples.
  • the red circles represent the intensity of iAP protein signal during 7 distinct NEC events of 6 patients (post conceptual age: 29-43) Each black circle represents at least 1 stool sample from 7 controls (post conceptual age 29-35 weeks).
  • Statistical analysis Mann-Whitney Test, P-0.002
  • FIG. 10 shows there are decreased fecal iAP enzymatic activity, increased total fiscal protein and increased iAP detection on WB at the time of NEC diagnosis.
  • A Illustrates longitudinal measurements of iAP activity, total fecal protein and iAP enzymatic activity in a NEC patient who subsequently developed perforation. The red points and columns represent NEC episodes. There is a precipitous decrease in fecal iAP activity corresponding to time of diagnosis demonstrating fecal iAP activity as a diagnostic tool for NEC. There is emergence of intense iAP protein detection on western blot. The patient completes 14 days of treatment, but had an intestinal perforation (ip) on day of life 31.
  • ip intestinal perforation
  • NEC was associated wife low fecal iAP activity, high fecal protein and high fecal iAP amounts (on WB).
  • FIG. 11 shows dial combining all 3 biomarkers has possible clinical utility aid improvement in sensitivity and specificity.
  • (A) illustrates a 3D scatterplot graph comparing WB data, fecal iAP activity and fecal protein
  • the red diamonds represent averaged total fecal protein, iAP activity and WB percentage of fecal samples from NEC patients at the time of diagnosis.
  • the black circles represent averaged total fecal protein, iAP activity and WB percentage of control points (inclusive of NEC patients in disease-free period)
  • (B) Represents a 2D scatterplot graph that shows the relationship between fecal iAP activity and WB percentage. There is clustering of control samples (black circles) in lower WB percentage and tendency towards high activity. The opposite is demonstrated with NEC samples whereby there is higher WB percentage and low activity.
  • C Schematic illustrating Naive Bayes Classifier used to demonstrate sensitivity and specificity of all three biomarkers individually and in combination to improve performance.
  • FIG. 12 shows there was an identifiable trend towatds lower fecal protein (Panel A) and higher fecal iAP enzymatic activity (Panel B) in those infants who tolerated feeds well and advanced rapidly to full feeds without issue.
  • Graph represents average total fecal protein and fecal iAP activity in stool from 10 control patients during the first month of life related to the duration of time until achievement of full enteral feeds.
  • FIG. 13 shows stool samples can be heterogeneous. There were two identifiable consistency compartments in a stool sample from a NEC patient. The stool compartments were separated and western blots were run on each compartment separately with differing results.
  • FIG. 14 shows relative iAP content, iAP activity, and protein concentration.
  • NEC was classified according to the criteria of Bell et al. and modified by Walsh and Kliegman. For this analysis, the term 'suspected NEC * is stage I and the‘proven NEC’ is stage II and more severe stages; the term‘perforated NEC * was used only to describe stage IIIB. Information from chart review was used to diagnose stage I. Radiographic determination of stage II required a record of pneumotosis intestinalis.
  • FIG. 15 shows immunohistochemical staining of intestinal tissue.
  • FIG. 16 shows schematic showing the many functions of iAP.
  • FIG. 17 shows fecal total protein content was highs: in NEC patients than in control infants;
  • FIG.18 shows fecal AP catalytic activity was consistently lows in NEC population.
  • FIG. 19 shows AP enzyme activity al time of NEC diagnosis was always lower in matched patient samples.
  • FIG.20 shows high levels of iAP protein are detected in association with
  • FIG. 21 shbwS increased Seal iAP protein levels in NEC episodes.
  • FIG.22 shows increased iAP protein levels and decreased iAP enzyme activity in NEC episodes.
  • FIG.23 shows a schematic for testing for non-specific binding of secondary antibody.
  • the enzyme assay can be conducted with just a secondary antibody conjugated with AP.
  • FIG. 24 shows clinical data that separates NEC diagnosis that matches X-ray and NEC suspicion (defined by neonatologists) and controls, demonstrating that tire biomarker(s) can mo!ecular!y define NEC earlier.
  • FIG.25 is a bar graph showing (hat measurement of alkaline phosphatase can be confounded by signal from secondary antibodies.
  • Isolated alkaline phosphatase can catalylically hydrolyze MUP to form the fluorescent product MU.
  • Secondary antibodies, conjugated to AP, from two different commercial manufacturers can also hydrolyze MUP to form fluorescent product.
  • both alkaline phosphatase protein and the secondary antibody are in the same measurement, there is an increased level of catalytic activity observed. This can be monitored by both standard spectrophotometric readings of biochemical activity and by Western blot.
  • FIG.26 shows the risk of non-NEC to NEC transition and NEC to non-NEC transition.
  • FIG.27 shows a schematic of a Transition Model.
  • FIG. 28 shows bivariate analysis of abundance and catalytic ability of intestinal alkaline phosphatase found in preterm infant stool. Biospecimens sampled at the time of disease are shown in colored circles; suspected necrotizing enterocolitis is in pink, severe necrotizing enterocolitis is in red, late-onset sepsis is in blue. Complementary control groups are shown in open grey circles
  • FIG.29 shows analyses of intestinal alkaline phosphatase, found in preterm infant stool, relative to radiographic clinical evidence of diagnosis. Abundance of iAP in infant stool samples, relatively to human small intestinal lysate, is shown in the left panel Biospecimens sampled at the time of clinical determination of disease (vertical blue bar) are shown in colored circles; samples collected prim ⁇ and post-disease, from infants who did have NEC, are shown in open red diamonds; samples collected from non-NEC patients are shown in grey. Normalized iAP abundance and normalized iAP catalytic activity of iAP were multiplied to yield a propensity score (NECPredict) and these scores are drown on title right panel.
  • NECPredict propensity score
  • FIG.30 shows (A) relationship between NEC, sepsis and gut defense mechanisms. Microhiota create distinct ecosystems in the gut lumen and mucosa. Various mucosal microbiota, both commensal and pathogenic, regulate intestinal immune function of the infant host. Activation of innate & adaptive immune systems by the raicrobiota in turn regulates systemic immune responses. In sepsis, immune responses in distant organ are triggered due to extreme signaling. Host proteins iAP (green), TLR4 and iL-8 (blue) are proteins involved in microbiota responses at early and later stages of inflammation. (B) Preemie standard of care requires new methods to monitor NEC disease.
  • Gold standard for diagnosis is x-ray (blue box) and identifies only 44% of advanced NEC cases; other diagnostic methods (light blue boxes) are bedside observable states, but not molecular definitions
  • Current management choices in NEC white boxes are contrasted with potential clinical outcomes (red boxes) from use of proposed biomarkers (green).
  • FIG. 31 shows amount of iAP protein and its catalytic activity were measured in stool samples.
  • A Immunoblot-analysis showed that relative iAP content detected in a serial dilution of human small intestinal lysate had a linear relationship, if calibrator is ⁇ 1 pg. Immunobiot bands are while against a black background. Log-log plot of signal versus calibrator is superimposed; averages and SEM of 5 replicates are shown.
  • B Average ⁇ SEM of iAP content for samples free of disease (white), diagnosed with NEC (red), suspected of having NEC (pink) and diagnosed with sepsis (blue) are shown.
  • FIG.32 shows stimulation scenarios considered for sample size justification.
  • FIG. 33 shows power analysis results for different effect sizes.
  • FIG. 34 shows iAP peptides that can be used as calibrators for mass spectral quantification.
  • A Amino acid sequence alignment of human alkaline phosphatases:
  • iAP intestinal alkaline phosphatase
  • FLAP placental-like alkaline phosphatase
  • TNAP tissue nonspecific alkaline phosphatase
  • GCAP germ-cell alkaline phosphatase
  • Boxes P1-P6 feature 6 peptides which have unique mass spectral signatures that discriminate between the four human AP proteins.
  • B Allelic frequency of missense, single nucleotide polymorphism catalogues in the human population.
  • C Sequences of the six iAP peptides amenable for mass spectral quantification of protein abundance mid single nucleotide missense polymorphisms identified for each residue position. Peptide with smallest deviation from zero on the x-axis and lowest polymorphic frequency on y-axis is best candidate for MS reference standards.
  • FIG.35 shows association of Necrotizing Enterocolitis (NEC) and Lale-Onset
  • A-C Physiological and structural changes in the gut, associated with NEC, are overlaid in the cross-sectional view of the small intestine.
  • Research efforts to develop an NEC biomarker has focused on proteins in immunity cascades and in dysbiosis of the microbiome. Our approach focused on host proteins involved in microbiota management.
  • D Prospective enrollment of premature infants with NEC and other confirmed infections.
  • E Workflow of stool sample preparation was optimized for assay reproducibility and standardization.
  • GI indicates gastrointestinal; IAP, intestinal alkaline phosphatase.
  • FIG. 36 shows clinical characteristics of patients with severe NEC, suspected
  • NEC no NEC.
  • IQR interquartile range
  • NA not applicable
  • NEC necrotizing enterocolitis
  • NICU neonatal intensive care unit
  • NPO nil per os
  • PCA postconceptual age.
  • FIG.37 shows clinical characteristics of patients with other confirmed infections.
  • Gl gastrointestinal
  • IQR interquartile range
  • NA not applicable
  • NEC necrotizing enterocolitis
  • NICU neonatal intensive care unit
  • NPO nil per os
  • PCA postconceptual age.
  • FIG. 38 shows association of Fecal intestinal Alkaline Phosphatase (1AP) content and activity with necrotizing enterocolitis (NEC) and other confirmed infections.
  • B Receiver operating characteristic curves for IAP abundance (filled circles) and activity (open circles) in samples collected during severe (orange) or suspected (brown) NEC.
  • FIG. 39 shows control experiments demonstrated operator reproducibility, antibody reagent specificity, and biospecimen specifity.
  • Five different operators performed (A) activity assay measurements and (B) iAP content determinations in the patient stool samples; dotted line marks the 1 : 1 correspondence between replicate 1 and replicate 2.
  • Q The anti-human iAP antibody used in this study was tested against human small intestine lysate (Gl), purified human placental alkaline phosphatase (PL), purified human tissue nonspecific alkaline phosphatase (TN), and bovine intestinal alkaline phosphatase (cl).
  • FIG.40 shows sequence alignment of 4 human Alkaline Phosphatases and
  • Calf Intestinal Alkaline Phosphatases Sequences were shown are human intestinal alkaline phosphatase (iAP human; P09923 Uniprot ID), calf intestinal alkaline phosphatase (iAP bovine; P19111), germ cell alkaline phosphatase (GCAP human; P10696), placenta- Like alkaline phosphatase (FLAP human, P05187), and tissue-nonspecific alkaline phosphatase (TNAP human, P05186). Signal peptide is in grey at the N-terminus of the sequences.
  • Propeptide is in grey italics at the C-terminus of the sequences. Residues involved in metal binding are annotated with an asterisk; candidate glycosylation sites have a # symbol.
  • the term“about” can refer to approximately, roughly, around, or in the region of. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
  • the present invention is directed to compositions and methods to detect and treat gastrointestinal diseases.
  • Gastrointestinal diseases refer to diseases involving the gastrointestinal tracts.
  • necrotizing enterocolitis is an acquired gastrointestinal disease often seen in pre-term infants.
  • bacteria invade the wall of the intestine, causing local infection and inflammation.
  • NEC is characterized by high mortality and long-term morbidity, including short gut syndrome, recurrent infection, nutritional deficiency and neurodevelopmental delay.
  • NEC-associated deaths Despite an overall net decrease in mortality for premature infants, there has been an increase in NEC-associated deaths. NEC is often difficult to diagnose and manage, due to initial nonspecific symptomatology and rapid deterioration.
  • necrotizing enterocolitis can also affect non-neonates.
  • necrotizing enterocolitis in non-neonates can result from inflammatory mediators; nutritional disorders, such as anorexia or significant weight loss; gastrointestinal dysfunction; alcoholism; malabsorption; agents that block intestinal proteases; smoking; circulatory disturbances, such as reduced mesenteric blood flow, bowel ischemia, atherosclerosis of the bowel arteries; cholelithiasis; administration of drags; immunological deficiencies, such as of the IgA secretory component or intestinal T-lymphocytes coupled with poor antibody response; fecal impaction or constipation; or infectious agents, such as bacterial infections, food borne infections and food borne illnesses.
  • Nan-limiting examples of such drugs include those with anticholinergic properties, such as neuroleptics or phenothiazine-based neuroleptics, narcotics, inflammatory mediators, antidepressants, iron pills, laxatives, or antacids.
  • anticholinergic properties such as neuroleptics or phenothiazine-based neuroleptics, narcotics, inflammatory mediators, antidepressants, iron pills, laxatives, or antacids.
  • Non-limiting examples of such infectious agents comprise bacteria like Klebsiella, E. coli, Enlerobacter, Pseudomonas, Clostridia and Staphylococcus epidermidis, viruses like Corona virus. Rota virus and Emero vims and rarely, fungi like Candida albicans. Enteropathogenic viruses are believed to infect epithelial cells resulting in cell destruction, necrosis and intestinal perforation.
  • Constipation or fecal impact can have many different causes known to art, nonlimiting examples of which include antacid medicines containing calcium or aluminum, changes in diet or activities, colon cancer, dairy products, eating disorders, neurological conditions, inactivity, dehydration, consuming fiber, overuse of laxatives, pregnancy, digestive disorders, resisting the urge to have a bowel movement, medications, stress, or hypothyroidism.
  • Gastrointestinal diseases refer to diseases involving the gastrointestinal tract, namely the esophagus, stomach, small intestine, large intestine and rectum, and the accessory organs of digestion, the liver, gallbladder, ami pancreas.
  • diseases can result from infectious, autoimmune, and physiological states.
  • Non-limiting examples of gastrointestinal diseases include colitis, inflammatory bowel disease (IBD), gastritis, gastroenteritis, pyloric stenosis, gastric cancer, infectious diarrhea, fecal impaction, constipation, intestinal obstruction and pseudo- obstruction, or malabsorption.
  • IBD inflammatory bowel disease
  • gastritis gastritis
  • gastroenteritis gastroenteritis
  • pyloric stenosis gastric cancer
  • infectious diarrhea fecal impaction
  • constipation constipation
  • intestinal obstruction and pseudo- obstruction or malabsorption.
  • necrotizing enterocolitis In addition to necrotizing enterocolitis (NEC), non-limiting examples of types of colitis comprise adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.
  • ANEC adult necrotizing enterocolitis
  • pseudomembranous enterocolitis infectious colitis
  • ulcerative colitis Crohn's disease
  • ischemic colitis ischemic colitis
  • radiation colitis radiation colitis.
  • Intestinal alkaline phosphatase is expressed in small intestinal emerocytes, co-secreted into the intestinal lumen and systemic circulation and plays an integral role in maintaining got barrier function by detoxifying bacterial lipopolysaccharides and maintaining microbial homeostasis.
  • iAP Intestinal alkaline phosphatase
  • aspects of the invention pertain to methods for diagnosing a gastrointestinal disease in a subject.
  • the method comprises the steps of obtaining a sample from the subject; detecting the presence of at least one GI disease biomatker in the sample, wherein the GI disease biomarker comprises intestinal alkaline phosphatase (iAP) protein; comparing the GI disease biomarker profile to that of a profile obtained from a control sample; and treating the subject.
  • iAP intestinal alkaline phosphatase
  • Embodiments can also be directed towards preventing the progression of a gastrointestinal disease in a subject in need thereof, and ameliorating the symptoms associated with a gastrointestinal disease in a subject in need thereof.
  • the control sample can comprise a two or more control samples.
  • “changed as compared to a control” sample or subject is understood as having a level of the analyte or diagnostic or therapeutic indicator (e.g., marker such as iAP) to be detected at a level that is statistically different than a sample from a normal, untreated, or abnormal state control sample. Determination of statistical significance is within the ability of those skilled in the art, e.g., the number of standard deviations from the mean that constitute a positive or negative result and the statistical analyses to arrive at these intervals.
  • analyte or diagnostic or therapeutic indicator e.g., marker such as iAP
  • embodiments of the invention comprise treating the subject
  • treating the subject can conoprise administering to the subject an effective amount of antibiotics, probiotics, intravenous fluids, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof.
  • An additional therapeutic approach can be withholding food from the subject.
  • Non-limiting examples of an iAP replacement composition comprise gene or protein replacement compositions.
  • administering can refer to introducing a substance, such as iAP protein or an antibiotic and/or an antifungal, into a subject.
  • a substance such as iAP protein or an antibiotic and/or an antifungal
  • any route of administration can be utilized including, for example, intracoronarilly, intramyocardially , intravenously, intraarterially, or any combination thereof.
  • the IAP can be administered to the subject prior to, concurrent with, or subsequent to diagnosis of a GI disease such as NEC.
  • Protein therapy can be accomplished by any method that effectively introduces iAP protein or a fragment thereof into the subject to restore or enhance iAP activity.
  • An effective amount of an iAP 1 protein (far example an amount sufficient to reduce or eliminate the symptoms associated with gastrointestinal diseases) can be administered alone or in association with an agent that facilitates the administration or activity of the protein.
  • the "effective amount" can be determined by one of skill in the art based on such factors as the type and severity of symptoms being treated, the weight and/or age of the subject, the previous medical history of the subject, and the selected route for administration of the agent.
  • iAP protein can be associated with lipids, such as detergents or other amphipalhic molecule micelles, membrane vesicles, liposomes, virosotnes, or microsomes.
  • lipids such as detergents or other amphipalhic molecule micelles, membrane vesicles, liposomes, virosotnes, or microsomes.
  • Lipid compositions that are naturally fusogenic or can be engineered to become fusogenic (e.g. by incorporating a fusion protein into the lipid) are especially preferred.
  • Fusion proteins can be obtained from viruses such as parainfluenza viruses 1-3, respiratory syncytial virus (RSV), influenza A, Sendai virus, and togavirus fusion protein.
  • Non viral fusion proteins include normal cellular proteins that mediate cell-cell fusion.
  • nonviral fusion proteins include the sperm protein PH-30 which is an integral membrane protein located on the surface of sperm cells that is believed to mediate fusion between the sperm and the egg.
  • Still other nonviral fusion proteins include chimeric PH-30 proteins such as PH-30 and the binding component of hemaghitinin from influenza virus and PH-30 and a disintegrin (e.g. bitistatin, barbourin, kistrin, and echistatin).
  • lipid membranes can be fused using traditional chemical fiisogens such as polyethylene glycol (PEG).
  • a subject can be treated by administration of an effective amount of iAP protein, optionally in a pharmaceutically acceptable carrier or diluent
  • An effective amount of iAP protein can be an amount sufficient to alleviate the symptoms of a gastrointestinal disease.
  • iAP can be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, parenterally, orally, submucosaily, by inhalation (for example of an aerosolized pharmaceutical composition), or other appropriate route of administration in an effective dosage range. If necessitated by a particular mode of administration, iAP can be encapsulated within a material that protects it from enzymatic degradation. In addition, prior to administration, it can be useful to administer agents to clear bacterial infection.
  • a preparation of the gene encoding iAP or a fragment thereof can be incorporated into a suitable vector for delivering the gene into a subject's cells.
  • the iAP gene therapy can be transient and require repeated delivery to the subject in other embodiments, gene therapy can offer a cure for the gastrointestinal disease.
  • gene therapy can offer a cure for the gastrointestinal disease.
  • genetic material encoding iAP is incorporated into stem cells of a subject, all subsequent generations of such cells can make authentic iAP from the integrated sequences and would correct the defect.
  • approaches and vectors that can be useful for performing iAP gene therapy include retroviruses, adeno-associated viruses, naked DNA, DNA-lipid complexes, receptor mediated entry, or adenovirus.
  • Non-limiting modes of administration of treatment comprise intravenous (IV); intramucosal; intramuscular; subcutaneously, and nan-invasive modes of administration, such as oral, intranasal, buccal, intrapulmonary, intrabronchial, and transdermai.
  • aspects of the invention further pertain to methods for screening for the presence of a signature in a subject at risk of developing a gastrointestinal disease or a subject with a non-symptomatic gastrointestinal disease.
  • steps of the method comprise obtaining a sample from the subject; measuring at least one GI disease biomarker in the sample, wherein the Gl disease biomarker comprises intestinal alkaline phosphatase (iAP) protein; comparing the Gl disease biomarker profile to that of a profile obtained from a control sample; and treating the subject
  • aspects can also be directed towards methods for identifying a subject at risk for a gastrointestinal disease or a subject with a non-symptomatic gastrointestinal disease.
  • the control sample can comprise two or more control samples.
  • aspects of the invention comprise measuring total protein concentration in a sample, intestinal alkaline phosphatase protein concentration in a sample, intestinal alkaline phosphatase enzyme activity in a sample, or a combination thereof. Samples used in such methods, and assays used to collect such measurements are described herein.
  • a subject can be diagnosed as having a Gl disease if the protein concentration in the sample is greater than about i.O mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/mi, 2,1 mg/ml, 2.2 mg/ml, 2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9 mg/ml, 3.0 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3.4 mg/ml, 3.5 mg/ml, 3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, 4.0 mg/ml, 4.1 mg/ml, 4.2 mg/ml
  • a subject can be diagnosed as having a Gl disease if the iAP activity is lower than about 10 mU/mg, 20 mU/mg, 30 mU/mg, 40 mU/mg, 50 mU/mg, 60 mU/mg, 70 mU/mg, 80 mU/mg, 9Q mU/mg, lOO mU/mg, 200 mU/mg, 300 mU/mg, 400 mU/mg.
  • a subject can be diagnosed with a gastrointestinal disease if the protein concentration in fecal sample is greater than about 1.6 mg/ml, or greater than about 1.8 mg/ml; if the iAP activity is lower than about 979 mU/m, or lower than about 1256 mU/mg; or if the level of iAP protein is at least two standard deviations above the mean of the control sample.
  • a subject can be diagnosed as having a GI disease if the level of iAP protein is greater than about .05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275% of a control sample.
  • the subject can be diagnosed with a gastrointestinal disease if the iAP protein detection by anti -iAP antibody exceeds 10.7 of control via densitometry, or in excess of 4.8% of control via densitometry.
  • a subject can be diagnosed with a gastrointestinal disease if two of the thresholds are met, or if all three thresholds are met.
  • the control sample can comprise a two or more control samples.
  • threshold for example a threshold indicative of NEC, refers to a value derived from a plurality of biological samples, such as donor stool samples, for a biomarker, such as iAP protein levels, iAP catalytic activity, or total fecal protein levels, above which threshold is associated with an increased likelihood of having and/or developing a gastrointestinal disease such as NEC.
  • Embodiments of the invention comprise diagnosing the subject with a gastrointestinal disease if the protein level of iAP in the sample, the level of iAP enzyme activity in the sample, or the fecal protein level is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 10 standard deviations above the mean level of the control sample.
  • a subject can be diagnosed with a gastrointestinal disease if two of the thresholds are met, or if all three thresholds are met.
  • the control sample can comprise two or more control samples.
  • Embodiments of the invention comprise machine learning techniques or applications to determine appropriate clinical thresholds.
  • such techniques comprise those known to the field, including Naive Bayes classifiers (NBC), linear discriminant analysis (LDA), or support vector machines (SVM). and support vector machine options.
  • NBC Naive Bayes classifiers
  • LDA linear discriminant analysis
  • SVM support vector machines
  • One of skill in the art readily understands that such a threshold value can vary depending cm the sample size analyzed and the statistical analyses employed.
  • aspects of the invention further comprise identifying and/or diagnosing both early stages of gastrointestinal disease and advanced stages of gastrointestinal disease.
  • Certain embodiments can distinguish between early stage and late stage gastrointestinal disease.
  • embodiments as described herein can diagnose advanced states of inflammation, such as that identified by radiological findings of pneumatosis intestinalis (portal vein or biliary gas).
  • embodiments can identify early stages of the disease before rampant inflammation of the gut is physiologically evident, Physicians currently suspect gastrointestinal disease from a range of physical signs, such as abdominal distension, abdominal tenderness, decreased bowel sounds, blood in stools, increased apnea, temperature instability, bilious aspirates, and feeding intolerance.
  • Clinical signs for suspecting disease are dilated intestinal loops and thickened bowel walls from radiology.
  • Laboratory findings for suspecting disease are decreased platelets, decreased or increased white blood cell count, increased band count, and metabolic acidosis.
  • Embodiments can match identification of radiological findings of pneumatosis intestinalis (portal vein or biliary gas) in advanced stages of inflammation. The method also identifies early stages of the disease before rampant inflammation of the gut is physiologically evident
  • Biomarkers of the invention can be measured in different types of biological samples.
  • biological samples that can be used in methods of the invention, although not intended to be limiting, include stool, plasma, cord blood, neonatal blood, cerebral spinal fluid, tears, vomit, saliva, urine, feces, and meconium.
  • a sample can be prepared to enhance detectability of the biomatkers.
  • a sample from the subject can be fractionated. Any method that enriches for a biomarker polypeptide of interest can be used.
  • Sample preparations are optional and may or may not be necessary to enhance detectability of biomatkers depending on the methods of detection used. For example, sample preparation can be unnecessary if an antibody that specifically binds a biomarker is used to detect the presence of the biomarker in a sample.
  • Sample preparation can involve fractionation of a sample and collection of fractions determined to contain the biomarkers. Methods of prefractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis, mass spectrometry, and liquid chromatography.
  • the methods described herein can involve obtaining a biological sample from the subject, such as an infant.
  • obtaining a biological sample refers to any process for directly or indirectly acquiring a biological sample from a subject.
  • a biological sample can be obtained (e.g via at a point-of-care facility, such as a physician's office, a hospital, laboratory facility) by procuring a tissue or fluid sample (e.g., blood draw, marrow sample, spinal tap) from a subject.
  • a biological sample can be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject.
  • the biological sample can be, for example, feces, such as stool, a tissue (e.g., blood), cell (e.g., hematopoietic cell such as hematopoietic stem cell, leukocyte, or reticulocyte, stem cell, CM plasma cell), vesicle, biomolecular aggregate or platelet from the subject
  • feces such as stool, a tissue (e.g., blood), cell (e.g., hematopoietic cell such as hematopoietic stem cell, leukocyte, or reticulocyte, stem cell, CM plasma cell), vesicle, biomolecular aggregate or platelet from the subject
  • aspects of the invention comprise bioraarkers of G1 disease.
  • aspects comprise biomarkers of necrotizing enterocolitis.
  • biomarkers of GI disease comprise iAP enzymatic activity, iAP protein Level, iAP dimerization/dissociation, post-translationally modified iAP, total fecal protein, or a combination thereof.
  • aspects of the invention comprise an assay that measures iAP enzymatic activity.
  • aspects of the invention comprise an assay that measures iAP protein level.
  • aspects of the invention comprise an assay that measures iAP dimerization/dissociation.
  • aspects of the invention comprise an assay that measures post-translationally modified iAP.
  • aspects of the invention comprise an assay that measures total fecal protein.
  • Non-limiting examples of post-translational modifications comprises acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formyl ation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, lipoylation, malonyl ation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopanletheinylation, prenylation, propionylation, ribosylation succinylation, sulfation, SUMOylation, or ubiquitination.
  • iAP is a homodimer; each protomer binds 4 divalent (Zh 2Y and Mg 2 *) ions, which are essential in maintaining the structural integrity and catalytic activity of the enzymes.
  • iAP is one of four different alkaline phosphatases found in human tissue that has been correlated with physiological function. Although it is found in high concentrations within luminal vesicles secreted by enterocytes on the microvilli brush border, small levels of iAP are released into the blood as well as the gut lumen, where in the latter travel throughout the intestinal tract.
  • Embodiments of the invention conoprise measuring or detecting such biomarkers using assays known to the art.
  • assays include an immunoassay, a colorimetric assay, fluorimelric assay or a combination thereof.
  • immunoassays comprise a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitalion or a combination thereof.
  • ELISA enzyme-linked immunosorbent assay
  • a biological sample collected from a subject can be incubated together with a biomarker specific antibody, such as an anti-iAP antibody or fragment thereof, and the binding of the antibody to the biomarker in the sample is detected or measured.
  • the antibody or fragment thereof can be specific for iAP (anti- iAP).
  • the antibody can be a polyclonal antibody or a monoclonal antibody.
  • the antibody or fragment thereof can be attached to a molecule that is capable of identification, visualization, or localization using known methods.
  • Suitable detectable labels include radioisotopic labels, enzyme labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, affinity labels, and chemiluminescent labels.
  • Examples of assays that can be used in methods of the invention comprise a Bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, a pyrogallo!
  • BCA bicinchoninic acid
  • red protein dye-binding assay a Coomassie blue dye-binding assay, an endpoint assay, a kinetic assay, such as a kinetic assay using a fluorometric substrate such as 4-methyilumbeliiferyI phosphate, chemiluminescent substrates such as CSPD and CDP-Star, DynaLight Substrate with RapidGlow enhancer, or colorimetric 4-nitrophenyl phosphate, an assay to detect phosphatase reactions, an assay to detect ATP hydrolysis, or a combination thereof.
  • the assays can be provided in a multi-well format, such as a 6-, 12-, 24-, 48, or 96-well plate.
  • the assays can be provided in a standard cuvette, such as a 1ml cuvette.
  • Total protein such as total fecal protein
  • Pyrogallol Red Molybdate dye binding method provides a colorimetric method fen: total protein quantitation with greater linearity, using microliter volumes of biological samples in manual or automated systems.
  • pyrogallol red can be provided in a kit comprising reagent, controls, and reagent standards, such as 25 mg/dL, 50 mg/dL, 100 mg/dL, and 200 mg/dL.
  • the enzyme employed in embodiments herein for example to detect protein levels or enzymatic activity, can be » for example, alkaline phosphatase, horseradish peroxidase, p-galactosidase and/or glucose oxidase; and the substrate can respectively be an alkaline phosphatase, horseradish peroxidase, b-galactosidase or glucose oxidase substrate (see Molecular Probes Handbook - A Guide to Fluorescent Probes and labeling Technologies , 11th Edition (2010), Invitrogen, which is incorporated by reference herein in its entirety).
  • the enzyme such as alkaline phosphatase or horseradish peroxidase
  • a secondary antibody can be attached to a secondary antibody.
  • Isolated alkaline phosphatase can catalytically hydrolyze MUP to form the fluorescent product MU.
  • Secondary antibodies, conjugated to AP, from two different commercial manufacturers, for example, can also hydrolyze MUP to form fluorescent product.
  • both alkaline phosphatase protein and the secondary antibody are in the same measurement, there is an increased level of catalytic activity observed. This activity can be monitored by both standard spectrophotomelric readings of biochemical activity and by Western blot
  • Alkaline phosphatase (AP) substrates include, but are not limited to, AP-Blue substrate (blue precipitate, Zymed catalog p. 61); AP-Orange substrate (orange, precipitate, Zymed), AP-Red substrate (red, red precipitate, Zymed), 5-bromo, 4-chloro, 3- indolyphosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indoly!
  • phosphate/nitroblue tetrazolium/i odonitrotetrazolium BCIP/NBT/INT, brown precipitate, DAKO, Fast Red (Red), Magenta-phos (magenta), Naphthol AS-BI-phosphate (NABP)/Fast Red TR (Red), Naphthol AS-BI-phosphate (NABP)/New Fuchsin (Red), Naphthol AS-MX-phosphaie (NAMPj/New Fuchsin (Red), New Fuchsin AP substrate (red), p-Nitrophenyl phosphate (PNPP, Yellow, water soluble), VECTORTM 1 Black (black), VECTORTM Blue (blue), VECTORTM Red (red), Vega Red (raspberry red color).
  • Horseradish Peroxidase (HRP, sometimes abbreviated PO) substrates include, but are not limited to, 2,2 r Azino-di-3-ethylbenz-thiazoline sulfonate (ABTS, green, water soluble), aminoethyl carbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC, red).
  • Alpha- naphthol pyronin (red), 4-chloro- 1 -naphthol (4C 1 N, blue, blue-black), 3 ,3 '-diaminobenzidine tetrahydrochloride (DAB, brown), oriho-dianisidine (green), o-phenylene diamine (OPD, brown, water soluble), TACS Blue (blue).
  • TAGS Red (red), 3, 3', 5, 5' Tetramethylbenzidine (TMB, green or green/blue)
  • TRUE BLUETM blue
  • VECTORTM VIP purple
  • VECTORTM SG silicase
  • Zymed Blue HRP substrate (vivid blue).
  • Glucose Oxidase (GO) substrates include, but are not limited to, nitroblue tetrazolium (NBT, purple precipitate), tetranitroblue tetrazolium (TNBT, black precipitate), 2- (4-iodophenyl)-5-(4-nitorphenyl)-3-pbenyltetrazolium chloride (1NT, red or orange precipitate), Tetrazolium blue (blue), Nitrotetrazolium violet (violet), and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, purple). All tetrazolium substrates require glucose as a co-substrate. The glucose gets oxidized and the tetrazolium salt gets reduced and forms an insoluble fonnazan which forms the color precipitate.
  • Beta-Galactosidase substrates include, but are not limited to, 5-bromo ⁇ 4- chloro-3-indoyl beta-D-galactopyranoside (X-gal, blue precipitate).
  • alkaline and acid phosphatase substrates comprise 9H-( 1 ,3- dich1oro-9,9 ⁇ dimeihylacridin-2-one-7-yl) phosphate, diammonium salt (DDAO phosphate), 6,8-difluoro-4-methyIumbelliferyl phosphate (DiFMUP), fluorescein diphosphate, letraammonium salt (FDP), 4-methyl umbelli fery 1 phosphate, flee acid (MUP), and 4- methy I umbelliferyl phosphate, dicyclohexylammonium salt, trihydrate (MUP DCA salt).
  • Alkaline phosphatase activity such as intestinal alkaline phosphatase activity
  • 4-methylumbelliferyl phosphate MUP
  • MUP 4-methylumbelliferyl phosphate
  • alkaline phosphatase mediated hydrolysis of its phosphate substituent yields the blue- fluorescent 4-methylumbelliferyl (excitation/emission -386/448 nm).
  • the alkaline phosphatase substrate can be directly admixed with the biological sample, such as stool, allowing for the direct declection of the presence of alkaline phosphatase or the measurement of its activity.
  • Alkaline phosphatase (AP) substrates include, but are not limited to, AP-Blue substrate (blue precipitate, Zymed catalog p. 61); AP-Orange substrate (orange, precipitate, Zymed), AP-Red substrate (red, red precipitate, Zymed), 5-bromo, 4-chloro, 3- indolyphosphate (BC1P substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate/'nilroblue tetrazolium/ iodonitrotetrazolium (BCIP/INT substrate, yellow-brown precipitate, Biomeda), 5-bromo, 4-chloro, 3-indolyphosphate/nitroblue tetrazolium (BCIP/NBT substrate, bhie/purple), 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium/lodonitrotetrazolium (BCIP/N
  • Enzyme reactions can provide a highly specific, rapid and sensitive assay for detection of specific proteins in a sample, such as iAP in stool.
  • suitable fluorogenic substrates which can be utilized within the present invention comprise
  • Fluorescein diacetale 4-Methylumbelliferyl acetate, 4-Methyhimbelliferyl casein, 4- Methylumbeiliferyl-a-L-arabinopyranoside, 4-Methylumbelliferyl-b-D-fucopyranoside, 4- Melhylumbeiliferyl-a-L-fucopyranoside, 4-Methylumbelliferyl-(3-L-fucopyranoside, 4- Methylumbellifery I -a-D-galactopynmoside, 4-Methylumbelliferyl-b-D-galactopyranoside, 4- M ethy lumbei lifery 1 -a-D-glucopyranoside, 4-Methylumbelliferyl-$-I>glucopyranoside, 4- Methyhimbellifeiyl-b-jD-glucuronide, 4-Methylumbelliferyl nonanoate, 4- Mcthylumbelliferyl oleate, 4-Methylumbellife
  • Non-limit1 ⁇ 2g examples of suitable chromogenic substrates for use within the present invention comprise o-Nitrophenyl-b-D-galactopyranoside, p-Nitrophenyl-b-D- galactopyranoside, o-N itropheny 1 -b-D-glucopyranoside, p-N itropheny 1-a-D-glncopyranoside, p-Nitrophenyl-b-D-ghicopyranoside, p-Ni tr ⁇ 3 ⁇ 4)henyl-b-D-glucuronide, p- Nitrophenyl phosphate, o-Nitrophenyl-b-D-xylopyranoside, p-Nitrophenyl-a-D- xylopyranoside, p-Nitropheny I -b-D-xylopy ranoside, and Phenolphthalein-b-D-glucuronide.
  • embodiments of the invention comprise measuring or detecting a gastrointestinal biomarker in a subject.
  • subject or“patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects to which compounds of the present disclosure can be administered will be mammals, particularly primates, especially humans.
  • a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goals, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cals.
  • a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as mixed pigs and the like.
  • rodents e.g., mice, rats, hamsters
  • rabbits e.g., primates, and swine such as mixed pigs and the like.
  • living subject refers to a subject noted above or another organism that is alive.
  • the term“living subject” refers to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject.
  • a subject comprises a mammal, such as a human or vertebrate animal.
  • a mammal such as a human or vertebrate animal. Examples of such include but are not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, primate, e.g., monkey, fish (aquaculture species), e.g. salmon, rat, and mouse.
  • a human comprises a preterm neonate, an infant, a child, an adolescent, an adult, or an elderly individual.
  • aspects of the invention as described herein relate to human gastrointestinal disorders, aspects of the invention are also applicable to other nonhuman vertebrates. Aspects of the invention are applicable for veterinary use, such as with domestic animals. In general, aspects will vary according to the type of use and mode of administration, as well as the particularized requirements of individual subjects.
  • the subject can be on an antibiotic regimen.
  • antibiotic regimen refers to the treatment or prevention of a disease, such as an infection, or method for achieving a desired change, such as the reducing or prevention of an infection, wherein said treatment comprises administering to a subject an antibiotic to effectively treat the disease or produce the physiological change.
  • An antibiotic regimen can comprise variations known to those skilled in the art, such as antibiotic choice (for example, comprises correct medication choice, route of administration and dosing schedule), timing of administration, and duration.
  • Non-limiting examples of such antibiotics comprise Vancomycin, Ampicillin, Zosyn (combination of piperacillin and tazobactam), Gentamycin, Flagyl (metrodniazole generic), Meropenem, Metronidazole, Cefotaxime, Clindamycin, or any combination thereof.
  • an antifungal agent can further be administered.
  • the antifungal agent can be Fluconazole, Terconazole, Voriconazole, Posaconazole, Pentamidine, Itraconazole, Ketoconazole.
  • methods as disclosed herein can further comprise Heating the subject.
  • treating can comprise administering to the subject diagnosed with a gastrointestinal disease an effective amount of antibiotics, probiotics, intravenous fluids, withholding oral feeding, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof
  • antibiotics can be administered to a subject for a sufficient period of time, such as 10-14 days where antibiotics are administrated to the infant
  • a sufficient period of time such as 10-14 days where antibiotics are administrated to the infant
  • 7 days of antibiotics can be administered to the patient.
  • antibiotic administration and/or prescription can be for broad spectrum coverage, such as for (i) yam-positive bacteria, (ii) gram-negative bacteria, and (iii) anaerobic bacteria.
  • Non-limiting examples of such regimens comprise Vancomycin (gram-positive including MRSA), ceftazadime (third generation cephalosporins - gram negative, some grant positive, and pseudomonas), metronidazole (anaerobic coverage), oxacillin (gram positive).
  • Non-limiting examples of general antibiotics regimes comprise ampiciilin + gentamicin for possible vertically acquired infection from mother, and vancomycin + cetazidime for possible hospital acquired infections.
  • probiotics also can be administered to the subject
  • probiotics refers to mono- or mixed cultures of live microorganisms that can help reestablish normal flora in the GI tract. Probiotics can enhance the immune response, elicit production of enzymes that degrade toxins and/or block attachment sites to the colon. See, See McFarland, J. Medic . Microbiol. 2005, 54:101-111.
  • probiotic organisms include (hose in the genera Bifidobacteria, Lactobacillus, Lactococcus, and Pediococcus, Saccharomyces boulardii, and related bacteria and yeast.
  • intravenous fluids or intravenous therapy can be administered to the subject.
  • Intravenous therapy can refer to the infusion of liquid substances directly into the vein of a subject.
  • Non-limiting examples of such fluids comprise saline (such as 0.9% NaCl in water or .45% saline m water), Lactated Ringer's (0.9% NaCl with electrolytes and buffer), DsW (5% dextrose in water), DsNS (5% dextrose in 0.9% saline), DJ 1/2 NS (5% dextrose in 0.45% saline), DsLR (5% dextrose in Lactated Ringer's), or Normosol-R.
  • the solution can be isotonic. In other embodiments, the solution can be hypotonic.
  • parenteral (or intravenous) nutrition can be administered to the subject.
  • parenteral (car intravenous) nutrition comprise intravenous dextrose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or a combination thereof.
  • feeding such as oral feeding, can be withheld from the subject until feeding tolerance can be demonstrated.
  • feeding tolerance can be demonstrated when the preterm infant is capable of safely ingesting and digesting the prescribed enteral (via mouth) feeding without complications associated with gastrointestinal dysfunction or infection.
  • Clinical evidence of feeding tolerance in very low birth weight preterm infant can include the number of days required to reach full-feeding volumes (reported ranged from 100-160 mL per kg per day), the number of episodes of feeding intolerance, the number of days feeds are withheld due to feeding intolerance symptoms, time to regain birth weight, lower leg growth, increase in weight gain, occipital-frontal head circumference, and length.
  • feeding refers to the intake of infant formula, such as EleCare (Abbott Nutrition), Neosure (Simi!ac), EnfaCare (Enfamil), Pregestimil (Enfamil), Similac Special Care or SSC (Similac), Gentlease (Enfamil).
  • Feeding can also refer to the intake of supplements, such as Microlipid (Nestle Health Science).
  • iAP replacement therapy can refer to protein replacement therapy.
  • protein replacement can refer to the introduction of a non-native, purified protein, such as iAP, into an individual having a deficiency in such protein.
  • the administered protein can be obtained from natural sources or by recombinant expression.
  • the term also refers to the introduction of a purified protein in an individual otherwise requiring or benefiting from administration of a purified protein, e.g., suffering from protein insufficiency.
  • the introduced protein can be a purified, recombinant protein produced in vitro, or protein purified from isolated tissue or fluid, such as, e.g, placenta or animal milk, or from plants.
  • Bifidobacteria, Klebsiella, and E. Coli alkaline phosphatases are also delected in human stool from preterm infants (Swittink et al. 2017. Metaproteomics reveals functional differences in intestinal microbiota development of preterm infents. Molecular & Cellular Proteomics. DOI: 10.1074/mcp.RAl 17.000102 (in press)), and can thus be sources of iAP protein for protein replacement therapy.
  • increased AP activity can be a result of bacterial flora, and not from human iAP only.
  • aspects of the invention comprise a disposable article fur- detecting or measuring biomarkers of gastrointestinal diseases.
  • the disposable article can comprise a biosensor, and can optionally comprise other components known to the art.
  • the biosensor can comprise at least one bio-recognition element [00193]
  • the biosensor can detect or measure iAP in a sample.
  • the biosensor can detect or measure iAP enzymatic activity, total fecal protein, iAP dimerization/dissociation, post-translationally modified iAP, or a combination thereof. Non-limiting examples of post-translational modifications and samples are described herein.
  • the biosensor can be an immunosensor, and can further comprise a detection signal.
  • detection signals comprise a radioactive signal, colorimetric signal, a fluorescent signal, chemiluminescent signal, or a combination thereof.
  • the biosensor can produce a new color or change in spectral absorption.
  • the biosensor of the present invention comprises a bio-recognition element, or molecular recognition element, that provides the highly specific binding or detection selectivity for a particular analyte, such as iAP.
  • the bio-recognition element can be a biologically derived material such as an enzyme or sequence of enzymes; an antibody or fragment thereof; a membrane receptor protein; DNA; an organelle, a natural or synthetic cell membrane; an intact or partial viable or nonviable bacterial, plant or animal cell; or a piece of plant or mammalian tissues, and generally functions to interact specifically with a target biological analyte.
  • the bio-recognition element is responsible for the selective recognition of the analyte and the physico-chemical signal that provides the basis for the output signal.
  • the physico-chemical signal generated by the bio-recognition element or elements can be communicated visually to the wearer or caretaker (i.e., via a color change visible to the human eye).
  • Other embodiments can produce optical signals, which can require other instrumentation to enhance the signal. These include fluorescence, bioluminescence, total internal reflectance resonance, surface plasmon resonance, Raman methods and other laser-based methods.
  • the signal can be processed via an associated transducer which, for example, can produce an electrical signal (e.g. , current, potential, inductance, or impedance) that can be displayed (e.g., on a readout such as an LED or LCD display) or which niggers an audible or tactile (e.g., vibration) signal or which can trigger an actuator, as described herein.
  • the signal can be qualitative (e.g., indicating the presence of the target biological analyte) or quantitative (le., a measurement of the amount or concentration of the target biological analyte).
  • the transducer can optionally produce an optical, thermal or acoustic signal,
  • the signal can also be durable (i.edeem stable and readable over a length of time typically at least of the same magnitude as the usage life of the article) or transient (i.edeem registering a real-time measurement). Additionally, the signal can be transmitted to a remote indicator site (e.g., via a wire, or transmitter, such as an infrared or if transmitter) including other locations within or on the article or remote devices.
  • a remote indicator site e.g., via a wire, or transmitter, such as an infrared or if transmitter
  • the biosensor 60 can be adapted to detect and/or signal only concentrations of the target biological analyte above a predefined threshold level (e.g., in cases wherein the target biological analyte is normally present in the bodily waste or when the concentration of the analyte is below a known“danger’ * level).
  • a predefined threshold level e.g., in cases wherein the target biological analyte is normally present in the bodily waste or when the concentration of the analyte is below a known“danger’ * level.
  • the disposable article can be a diaper to be worn by a subject.
  • Non-limiting examples of additional disposable articles include wipe for cleaning a subject, dipstick, spoon, scoopula, filter paper, or swab.
  • kits of the invention can comprise a bio-recognition element, a support structure, and instructions for use thereof.
  • an iAP bio-recognition element such as an antibody as described herein, can be immobilized to a solid support structure.
  • Non-limiting examples of the composition of the solid support structure comprise plastic, cardboard, glass, plexiglass, tin, paper, or a combination thereof
  • the solid support can also comprise a dip slick, spoon, scoopula, filter paper or swab.
  • kits of molecular biomarkers for identifying a subject exhibiting or having a predisposition to develop a gastrointestinal disease.
  • the kit comprises at least one of a means for determining total fecal protein concentration, a means for determining intestinal alkaline phosphatase (iAP) activity, and an iAP bio-recognition element, wherein together represent a molecular signature that is indicative of the presence of or a predisposition to development of a gastrointestinal disease in a human subject
  • the signature comprises total protein concentration at least two standard deviations above the mean of a control sample, intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, or intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample.
  • the signature is selected from at least two of the group comprising total protein concentration at least two standard deviations above the mean of a control sample, intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of a control sample, and intestinal alkaline phosphatase activity at least two standard deviations below the mean of a control sample
  • the control sample can comprise two or more control samples.
  • the kit includes (a) a container that contains components and support structures as described herein, and optionally (b) informational material.
  • the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for diagnostic purposes.
  • (be kit includes also includes a therapeutics, such as antibiotics, probiotics, or an IAP replacement composition.
  • the informational material of the kits is not limited in its form.
  • the informational material can include information about production of the components of the kit, such as molecular weight, concentration, date of expiration, batch or production site information, and so forth.
  • the informational material relates to methods of using the components of the kit, (e.g., to diagnose a subject with a GI disorder).
  • the information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.
  • the kit can include other ingredients, such as solvents or buffers, a stabilizer, or a preservative.
  • the kit can comprise therapeutic agents, such as iAP replacement compositions or antibiotics, that can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile.
  • therapeutic agents such as iAP replacement compositions or antibiotics
  • the liquid solution preferably is an aqueous solution.
  • the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent.
  • the solvent e.g., sterile water or buffer, can optionally be provided in the kit.
  • This disease necrotizing enterocolitis or NEC
  • This disease occurs in 12% of pre-term infants; 30% of NEC patients do not survive.
  • Biomarkers for reliable diagnosis are required. Using infant stool samples, three biomarker measurements are performed; classifier analysis of the three biomarkers together showed that NEC can be diagnosed with high total protein concentration, low intestinal alkaline (iAP) phosphatase activity, and high levels of intestinal alkaline phosphatase protein.
  • iAP low intestinal alkaline
  • the detection of intestinal alkaline phosphatase protein by western blot alone is strongly correlated with NEC diagnosis and can be used in ELISA format.
  • Embodiments as described herein have a 93% true positive rate and 95% true negative rate for disease diagnosis. Embodiments as described herein can have the potential for risk assessment and surveillance of the disease.
  • the method is relatively fast and inexpensive in comparison to proteomic efforts and mass spectrometry.
  • other patent applications use serum or urine; serum is invasive and requires extraction of fluids from very fragile patients, whereas urine analysis does not provide a direct readout of the gastrointestinal distress.
  • AP alkaline phosphatase
  • DOL day of life
  • iAP intestinal alkaline phosphatase
  • MUP 4-methy!umbelliferyl phosphate
  • NBC Naive Bayes classifier
  • NEC necrotizing enterocolitis
  • WB wester blot
  • NEC Necrotizing enterocolitis
  • Study design In a prospective, longitudinal, case control study, serial stool samples were collected from 6 NEC patients and 12 control infants for the measurement of total fecal protein, iAP activity and iAP protein detection by western blot. Data were evaluated by longitudinal assessment of individual patients, intergroup comparison and sensitivity/specificity evaluation m a classifier-based analysis.
  • Necrotizing enterocolitis is a serious inflammatory disease of the gastrointestinal tract that affects >5000 very low birth weight ( ⁇ 1500 g) infants each year. 1 ⁇ 2 It is characterized by high mortality (up to 30%) and long-term morbidity, including short gut syndrome, recurrent infection, nutritional deficiency and neurodevelopmental delay. 3,4 Despite an overall net decrease in mortality for premature infants, there has been an increase in NEC- associated deaths/ The -disease is often difficult to diagnose and manage, due to initial nonspecific symptomatology and rapid deterioration.
  • intestinal alkaline phosphatase measured in stool, offers diagnostic value as a marker for intestinal pathology. This protein is expressed in small intestinal enterocytes, co-secreted into the intestinal lumen and systemic circulation 8 and plays an integral role in maintaining gut barrier function by detoxifying bacterial lipopolysaccharides and maintaining microbial homeostasis.
  • Sample Collection/Preparation Stool samples were collected serially from diapers of study subjects after spontaneous stooling. Stool was stored briefly in hospital specimen refrigerators, until transport to the lab in cooler boxes in the initial processing step, about 200 mg of stool was measured, and sterile, molecular grade water (Sigma Aldrich) was added to make a desired concentration of 200 mg/ml. The mixture was vigorously vortexed for 30-60 s, or until a well-mixed shiny was evident. The mixture was then centrifuged at 22,000*$: for 30 min at 4 °C. The supernatant was collected and was stored at -20 °C until assays were performed. [00220] Protein concentration: The concentration of total protein in the stool supernatant was determined by Bradford assay (Coomassie Plus Protein Assay Reagent, Thermo- Scientific), using bovine serum albumin as the standard.
  • Denaturing gel electrophoresis and western blot Supernatants of stool samples were mixed with 6X gel loading buffer (375 mM Tris pH 6.8, 50% (w/v) glycerol, 600 mM dithiothreitol, 420 mM sodium dodeeyl sulfate) and boiled for 5 mins. A total of 10 pg of total protein was loaded per lane of a denaturing 4-12% Bis-Tris gel (Novex, Life Technologies). The positive control was small intestinal tissue lysate (Abeam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control.
  • 6X gel loading buffer 375 mM Tris pH 6.8, 50% (w/v) glycerol, 600 mM dithiothreitol, 420 mM sodium dodeeyl sulfate
  • Duplicate gels were run: one was Coomassie-stained to visualize all proteins in each lane and proteins in the second were transferred onto a PVDF membrane for immunoblotting detection of intestinal alkaline phosphatase.
  • the membrane was serially blocked in 5% (w/v) nonfat dry milk in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% Tween, incubated with primary rabbit polyclonal antibodies against human iAP (Abeam, at>7322 or abl98I01), washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Abeam, ab6721 ) at room temperature.
  • Chemiluminescent signal was initiated using Pierce ECL western blotting substrate (ThermoScientific) and captured cm developed photographic film (AFP Imaging). Western blot densitometry was performed on scanned films (Biorad GeiDoc XR) using Image J. In the digitized western blots, the 60 kDa band, which corresponded to iAP, was manually identified. Equivalent areas were quantitated for each lane of each western blot The negative control was subtracted from each patient sample and the difference was calculated as a percentage of the positive control standard.
  • Fecal iAP catalytic activity Alkaline phosphatase activity was measured with use of 4-methylumbelliferyl phosphate (MUP) as a fluorescent substrate (Abeam, ab83371 ) in the presence and absence of L-phenylalanine, an inhibitor of iAP.
  • MUP 4-methylumbelliferyl phosphate
  • REUs Relative fluorescence units at 360/440 nm were measured using a Spectra Max M2e spectrophotometer (Molecular Devices, Sunnyvale, CA). Ninety-six- well black optical bottom plates were used. Standards and negative controls were prepared for each plate nm.
  • AP activity (mU/rnL) (B x dilution factor)/(T x V), in which B is nmol of product; V is yohune of sample added to the well; T is reaction time; and U is the amount of enzyme causing hydrolysis of 1 praol of MUP pea * minute at pH 10.0 and 25°C.
  • a 100 mM stock of L- phenylalanine (purity >98%; Sigma Aldrich) was freshly prepared in molecular grade water each day of use. A final assay concentration of 10 mM Phe was used to assess inhibition of iAP-specific activity.
  • NBC Naive Bayes Classifiers
  • An NBC assumes each feature is statistically independent; however, it can perform well on multi-feature classification problems, even when the assumption of statistically independent features does not hold. 17
  • standard error for each classifier, we computed standard error for our sensitivity and specificity estimations by performing five rounds of stratified jackknife resampling, in which 20% of the data was excluded ibr each round of resampling.
  • We used a 5-fold stratified cross-validation scheme where, for each fold, the NBC was trained on 80% of the data, and the resulting classifier was tested for sensitivity and specificity on the remaining 20% of the data.
  • Fig. 1A was diagnosed with NEC on day of life (DOL) 7. After 14 days of medical treatment, including bowel rest and antibiotics, clinical symptoms and pneumatosis inlestlnalis resolved. Enteral feeding was restarted with variable success until the baby experienced recurrent NEC and subsequent intestinal perforation on DOL 31. Three fecal analyses were performed: soluble protein concentration, catalytic activity of iAP, and immunoblot detection of iAP. Two stool samples were obtained on DOL 7: before NEC diagnosis (7A, Fig. 1 A) and the bloody stool later in the day (7B, Fig, 1 A). Data from fi ve other stool samples (DOL 13, 20, 29, 32, and 42) also are presented,
  • Fig. 1A Longitudinal monitoring of patient 1 showed that the 3 candidate biomarkers had diagnostic value (Fig. 1A).
  • the DOL 7 A stool sample had a protein concentration of 1.85 mg/mL, catalytic activity of 2218 U/g, and no detectable signal at 60 kDa in the western blot.
  • the DOL 7B stool sample collected several hours later, had a protein concentration of 2.1 mg/mL, catalytic activity of 250 U/g, and dear immunodetection of iAP.
  • NEC patients had a more than ten-fold lower mean fecal iAP enzymatic activity at the time of diagnosis compared with controls matched for post-conceptual age (162 ⁇ 30 mU/mg vs. 1826 ⁇ 376 mU/mg, NEC vs. control, respectively, /K0.Q001 , Fig, 2B).
  • iAP protein was found nearly a 30-fold higher in stool samples from NEC patients compared with samples from healthy premature subjects (215.0 * 47.6% vs. 7.2 ⁇ 2.3%, NEC vs. control, respectively, Fig. 2C).
  • stool samples from infants with NEC have increased total protein, decreased iAP enzyme activity, and increased iAP protein at the time of diagnosis compared with healthy controls.
  • the 3-feature NBC biomarker performs best with 100% sensitivity and 92% specificity. However, if the goal is to simultaneously maximize both sensitivity and specificity, then the 3-feature NBC showed performance of 93% sensitivity and 95% specificity.
  • the iAP activity level considered alone also performs almost as well in this case by achieving both sensitivity and specificity levels of 92%, when using a threshold of 300 mU/mg iAP activity. Perhaps unsurprisingly, though, total fecal protein level alone is not as robust a biomarker for NEC. At 92% sensitivity, specificity falls to 67% using a protein threshold of 1.35 mg/mL.
  • fecal iAP activity level and 60 kDa western blot intensity levels hold promise individually as NEC biomarker candidates.
  • total fecal protein activity level is only likely to have utility if considered as part of a multi-feature diagnostic evaluation.
  • Quantitative markers measured as a ratio or on an interval scale, are stilt needed to achieve a better understanding of NEC, e.g., to distinguish between normal and pathological biological processes or monitor a response to clinical interventions.
  • the clinical definition of NEC could be significantly improved with a shift from sole dependence on clinical impression and imaging findings to an expanded diagnostic palette including reliable molecular biomarkers.
  • Identification of molecular NEC biomarkers amenable to adoption in clinical practice has the potential to reduce neonatal deaths, morbidity, and associated healthcare costs.
  • characterization of such parameters can provide insight into cellular integrity, protein expression, and changes in gastrointestinal metabolism. Obtained from serum, urine, feces, and buccal swabs, the discovery of candidate biomarkers for NEC is a focus of current research. 23 25
  • marker performance is more robust for a positive diagnostic readout, such as increased immunoblot detection and protein concentration, than for a negative diagnostic readout: the latter mode is more susceptible to false positive (and false negative) diagnoses.
  • Neonatal Network Incidence and timing of presentation of necrotizing enterocolitis in preterm infants. Pediatrics. 2012;129:e298-304.
  • Gourlay DM Intestinal alkaline phosphatase prevents the systemic inflammatory response associated with necrotizing enterocolitis. J Surg Res. 2013;180:21-6,
  • Ng PC, Ma TP, Lam HS The use of laboratory biomarkers for surveillance, diagnosis and prediction of clinical outcomes in neonatal sepsis and necrotising enterocolitis. Arch Dis Child Fetiit Neonatal Ed. 2015;100:F448-52.
  • HM high-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants. J Clin Invest 2010;120:2989-3000.
  • Tyska MJ Enterocyte microvillus-derived vesicles detoxify bacterial products and regulate epithelial-microbial interactions. Cunr Biol. 2012;22:627-31.
  • Necrotizing enterocolitis is an extremely serious inflammatory disease of the gastrointestinal tract that primarily affects premature infants. It occurs in up to 10% of very low birth weight infants ( ⁇ 1500g at birth) and is characterized by high mortality (up to 30%) and significant long term morbidity, including infantile short gut syndrome, recurrent infection, parenteral nutrition related cholestasis, nutritional deficiency and neurodevelopmenta] delay (1). Despite advances in the field of neonatology, NEC is responsible for increasing deaths in very premature infants (2). The exact cause of the disease is still not well understood, making diagnosis and management a challenge. The course of the disease often involves initial nonspecific symptomatology and rapid clinical deterioration.
  • Intestinal alkaline phosphatase has become an enzyme of interest in the study of gastrointestinal disease. Produced and secreted by enterocytes in the proximal small intestine, iAP activity is found throughout the small and large intestine (4). It is the primary alkaline phosphatase (AP) detected in stool (5,6). It has a variety of functions such as cleaving of lipopolysaccharide (LPS) produced by gram-negative bacteria and interfering with activation of Toll-like receptors in the gut (7). It dephosphorylates ATP and has been shown to affect microbial homeostasis through this interaction (8).
  • LPS lipopolysaccharide
  • the samples were transported in cooler boxes to the lab for initial processing. About 200 mg of stool was measured out, when possible, and molecular grade water was added to make a desired concentration of 200 mg/ml. The mixture was vigorously vortexed for 30 secs to 1 min or until a well-mixed slurry was evident. The mixture was then centrifuged at 22,000xg for 30 min at 4 °C. The supernatant was collected and was stored at -20 °C until assays were performed.
  • PVDF polyvinylidene difluoride membrane
  • a developer (AFP Imaging; Mount Kisco, NY) was used to produce films after WB and an imager (Biorad Gel-Doc XR; Hercules, CA) was used to scan western blots and gels. Densitometry was done to analyze digitized images of western blots. We manually identified bands at 60 kDa on the western blots, which corresponded to intestinal alkaline phosphatase. We then calculated the area relative to background for each 60 kDa band and then expressed this value as a percentage of the positive control. Positive controls were hepatocellular carcinoma whole cell lysate or small intestine tissue lysate (Abeam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control.
  • Fecal iAP Activity - Alkaline phosphatase activity was ascertained with the use of 4-methylumbelliferyl phosphate as a fluorescent substrate ab83371 (Abeam). Substrate background controls and background controls were done to improve accuracy. Relative fluorescence units (RFUs) at 360/440 nm wavelengths were measured using a Spectra Max M2e spectrophotometer (Molecular Devices, Sunnyvale, CA). Ninety-six well black, optical bottom plates were used. Reaction wells for samples, standards and negative background controls were prepared each time the assay was performed and total alkaline phosphatase activity was determined using:
  • B is nmol of 4-methyhimbeiloferone (4-MU)
  • V is volume of sample added to the well
  • T is reaction time.
  • U is the amount of enzyme causing hydrolysis of 1 pmol of product per minute at pH 10.0 and 25°C (glycine buffer).
  • NEC patients had significantly higher amounts of fecal protein at the time of diagnosis compared to matched controls for post conceptual age.
  • Nonparametric tests Mann- Whitney
  • Panel B highlights a different infant with multiple NEC surveillance events (one event is represented by the green point) prior to NEC.
  • the suspected NEC episode was associated with evidence of iAP protein on WB, but normal iAP activity. This resolved before the precipitous drop in the iAP activity and emergence of high iAP protein on WB at the lime of NEC.
  • Figure 10 Panel C shows 3 groups of samples, each consisting of stool from a NEC patient at the lime of diagnosis compared with that from a closely matched control. Groups 1 and 2 clearly show increased total fecal protein, decreased iAP activity, and evidence of iAP protein on WB.
  • FIG. 1 is a 3D scaUerplot illustrating NEC samples mid controls with fecal iAP activity, fecal protein and WB data points. NEC samples are labeled in red. Panel B depicts a 2D scatterplot showing iAP activity and WB percentage.
  • FIG. 6 panel C demonstrates the utility of examining multiple features simultaneously by depicting the tradeoff between sensitivity and specificity for multiple threshold values and multiple feature selections.
  • Western blot intensity considered alone performs best with 100% sensitivity at 70% specificity for a detection threshold of 10% positive control band intensity.
  • 100% sensitivity is not required and the goal is to simultaneously maximize both sensitivity and specificity, then the 3 feature Naive Bayes Classifier performs best by reaching a sensitivity of 95% and a specificity of 93%.
  • the iAP activity level considered alone performs almost as well in this case by achieving a sensitivity level of 95% and a specificity of 91% when using a threshold of 300 mU/mg iAP activity.
  • the specificity level is 88% using a 30% positive control band intensity threshold.
  • total fecal protein level alone is not specific for NEC.
  • the specificity must drop to 44% using an interpolated threshold of 1.02 mg/mL. Consequently, fecal iAP activity level and 60 kDa western blot intensity levels hold promise individually as NEC biomarker candidates.
  • Intestinal alkaline phosphatase is expressed primarily in the apical enterocytes of the small intestine, making it an ideal, relatively specific candidate biomarker for localizing gastrointestinal disorders such as NEC. It adheres closely to the membrane hit is also shed into the lumen (4).
  • iAP was recently demonstrated by Shifrin et al. to be distributed into the mucus layer and gut lumen via microvillar vesicle shedding (23). During inflammatory insult and bowel necrosis, disruption of the mucosal barrier and cell death, as well as shedding of the mucosal lining, would lead to an increased release of mucosal proteins such as iAP into the feces.
  • the rarity of iAP signal on western blot, but high iAP activity in the stock of control subjects is still not well understood. Perhaps the normal shedding process changes free luminal iAP structure in a way that does not allow for recognition by our particular antibody.
  • the immunogen for the antibody is foil length native human iAP from small intestinal tissue and it can be more sensitive to the membrane bound foil length protein.
  • the difference in WB findings is highly suggestive of a conformational difference in fecal IAP between the healthy and diseased state.
  • Fecal iAP could be also used during recovery from NEC to gauge the integrity of the bowel and guide feeding strategies in our most vulnerable patient population and to determine the length of lime needed for recovery'. Without being bound by theory, the needs of some children can vary from 7-14 days of treatment that is considered standard management.
  • Measurement of relative iAP content in stool is a biomarker for bowel infection.
  • Measurement of iAP activity is a biomarker for maturation of intestine in preterm infants.
  • Measurement of fecal protein concentration is correlated with an intestinal inflammation response or diseased state.
  • Necrotizing enterocolitis is a multi-factorial disease that predominately affects premature infants and is the leading cause of hue mortality and morbidity in very preterm infants (Caplan, 2008; Christensen et al, 2010). Although the etiology of NEC Is not clearly defined (Dominguez and Moss, 2012; Gephart et al., 2012), NEC is believed to represent a severe inflammatory disorder in the intestine (Balance et al., 1990; Zhang et al., 2011). Excessive inflammatory responses to environmental insults in the immature intestine are a hallmark of NEC (Chan et al., 2009).
  • LPS/TLR4 signaling has been suggested to contribute to the pathogenesis of NEC (Chan et al., 2009; Fusunyan et al., 2001; Leaphart et al, 2007; Nanthakumar et al., 2011). Inhibition of LPS/TLR4 signaling attenuates intestinal inflammation and mitigates NEC pathology in animal models (Chan et al., 2009; Gribar et al., 2009).
  • Intestinal alkaline phosphatase is a critical component of innate intestinal immunity.
  • the enzyme typically anchored to the intestinal brush bonier, cleaves phosphate groups, and as such can dephosphorylate lipopolysaccharides (LPS).
  • LPS lipopolysaccharides
  • EPS dephosphorylation inhibits a potent signaling pathway; thus, proinflammatory cytokines release and immune responses by LPS-activation ofTLR4 (toll-like receptor 4; Lalles, 2010) are blocked by iAP.
  • iAP is concentrated in specialized membrane vesicles, which are released from disial tips of enterocyte microvilli into the intestinal lumen (McConnell et at, 2009; Sangerin et al..2012). These released vesicles interact with and limit the pro- inflammatory potential of both bacteria and bacterial products.
  • iAP would be measureabie in human stool samples; this is confirmed limn iAP being one of the core proteins in the human stool proteome.
  • a steady state baseline of iAP would be detected from intestinal epithelial cells shed in the lumen and detected in stool.
  • iAP content in stool would increase from released membrane vesicles loaded with iAP, if there was risk of bacteria-induced inflammation.
  • Samples from non-NEC infants that were grouped by post-conceptual age (grey bars. Fig 14A) showed the premature infants have low amounts of iAP, relative to a positive control from human small intestine tissue lysate.
  • Our data also draw that infants who have NEC have a high relative content of iAP (l 20-320% of positive control) in their stool samples at the time of clinical diagnosis.
  • preterm infants would have lower iAP activity than full-term infants.
  • Stool thus provides an accurate measurement of iAP activity in the neonatal intestine.
  • prophylactic iAP prophylactic iAP
  • Feeding tolerance is demonstrated when the preterm infant is capable of safely ingesting and digesting the prescribed enteral (via mouth) feeding without complications associated with gastrointestinal dysfunction or infection.
  • Clinical evidence of feeding tolerance in very low birth weight preterm infant is most often described in the literature as the number of days required to reach fell-feeding volumes (reported ranged from 1CXM60 mL per kg per day), the number of episodes of feeding intolerance, the number of days feeds are withheld due to feeding intolerance symptoms, time to regain birth weight, lower leg growth, increase in weight gain, occipital-frontal head circumference, and length. None of the infants studied had reached fell-feeding volumes.
  • Types of formula include, but are not limited to: EleCare (Abbott Nutrition), Neosure (Similac), EnfaCare (Enfamil), Pregeslimil (Enfamil), Similac Special Care or SSC (Similac), and Gentlease (Enfamil).
  • Supplements can include without limitation Microlipid (Nestle Health Science).
  • Non-limiting examples of parenteral (or intravenous) nutrition comprise intravenous dextrose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or a combination thereof.
  • NEC is a devastating GI disease that primarily affects premature infants (Incidence: 4-14%; Mortality: 15-30% (up to 50%); Morbidity: up to 50% of survivors).
  • Clinical manifestations of NEC comprise abdominal distension, poor gastrointestinal motility, and bloody stools.
  • X-ray findings comprise pneumatosis intestinalis and perforation.
  • intestinal alkaline phosphatase iAP
  • iAP intestinal alkaline phosphatase
  • iAP is produced by apical enterocytes and secreted into luminal brush border and catalyzes hydrolysis of phosphomonoesters .
  • iAP is active as a homodimer and requires Zn 2 i and Mg 2* ions in the active site.
  • Substrates of iAP include LPS and nucleotide triphosphates.
  • iAP has multiple roles affecting gut barrier function and inflammation.
  • iAP is shed in stool.
  • iAP is tissue specific AP, meaning made mostly in intestine, as demonstrated by immunohistochemical staining of intestinal tissue (FIG. 15).
  • iAP maintains gut barrier function (FIG. 16).
  • Total fecal AP is predominantly intestinal isoform. There are other alkaline phosphatases in the intestine, such as bacterial and TNAP.
  • L-phenyl alanine which specifically inhibits activity of only the intestinal type alkaline phosphatase. Specifically, we obtained AP activity with and without L-Phe added to determine specific iAP activity, and concluded that iAP is the main form of AP in stool. Fecal AP catalytic activity was consistently lower (statistically significant) in NEC population (FIG. 18).
  • NEC NEC.
  • iAP silencing can be a component of gut mucosal barrier dysfunction in critically-ill NEC patients.
  • a specific antibody for human iAP was used in wester blot analyses and, surprisingly, detected appropriate signal only in the NEC samples (labeled N) and absent in control samples.
  • NEC samples labeled N
  • Each group represents different NEC patients at the time of diagnosis and the controls which were age and gestational age matched. There are much higher iAP amounts present in stool of NEC patients at the time of diagnosis (FIG. 20).
  • NEC episodes demonstrate increased fecal iAP protein levels.
  • One NEC patient serially was followed and found that the patient continued to have high iAP levels even after medical management The patient subsequently had a perforation followed by surgical intervention.
  • Stool 10 days following surgery no longer has high levels of iAP protein.
  • There is no signal on day 42 (FIG. 21).
  • Contrasted with AP activity the patient maintained lower AP activity until surgical intervention. 10 days after surgery AP activity begins to increase.
  • the presence of persistent high fecal iAP levels and tow activity could have been an indication of compromised bowel leading up to perforation. (FIG. 22).
  • iAP is deve!opmentally regulated, and its expression and activity
  • a third NEC biomarker can be western blot analysis or ELISA of iAP protein levels in preterm infants (Rentes et al Eur J Pediatr Surg 23, 39; Heinzerling et at J Pediatr Surg 49, 954; Biesterveld etal. J Surg Res 196, 235).
  • Methods fhiorimetric assays. Alkaline Phosphatase cleaves the phosphate group of the non-fiuorescent 4-Methylumbelliferyl phosphate disodium salt (MUP) substrate; Results in an increased fluorescent signal when dephosphorylated; Measured using a spectrophotometer.
  • MUP 4-Methylumbelliferyl phosphate disodium salt
  • prescription would be for broad spectrum coverage for (i) gram-positive bacteria, (ii) gram-negative bacteria, and (iii) anaerobic bacteria.
  • gram-positive bacteria gram-positive including MRSA
  • ceftazadime third generation cephalosporins - gram negative, some grant positive, and pseudomonas
  • metronidazole anaerobic coverage
  • oxacillin gram positive
  • antibiotics regimes are: ampicil!m + gentamicin for possible vertically acquired infection from mother, and vancomycin + cetazidime for possible hospital acquired infections.
  • Commonly used antibiotics are Gentamycin, Vancomycin, Ampicillin, Zosyn (combination of piperacillin and tazobactam), Flagyl (metrodniazoie generic).
  • PN parenteral nutrition
  • Infants receive stock solutions containing glucose (10 g/dL), amino acid (2.5 g/dL), and lipid s in the first 2 hours of life.
  • Amino add solutions contained AminosynPF 10% (Hospira Inc) or TrophAmine 10% (B Braun Medical Inc).
  • Intralipid 20% (Baxter) Liposyn HI 20%, and Liposyn II 20% (Hospira Inc) provided parenteral lipids.
  • Fluids typically provided 80 to 100 mL/kg per day at birth and increased by 20 mL/kg to 140- 160 mL/kg per day in the first week of life.
  • Acetate salts of sodium and potassium in PN solutions are buffers against metabolic acidosis.
  • PN solutions provided most of the nutrition in the first week of life. Enteral nutrition (EN) typically contributed only minimal energy until the end of the second week. The transition to exclusively EN was typically achieved before the end of the fourth week.
  • EN Enteral nutrition
  • infants received their mother’s breast milk. After tolerating breast milk at 150 mL/kg per day, infants received supplemental human milk fortifier (Mead-Johnson). When breast milk is not available, infants received formula specific for premature infants.
  • Premature Enfamil Formula (Enfamil), EleCare (Abbott Nutrition), Neosure (Similac), EnfaGare (Enfamil), Pregestimil (Enfamil), Similac Special Care or SSC (Similac), Gentlease (Enfamil).
  • Pregesteroil and Elecare are hydrolyzed cow- based formulas, typically used for post-NEC babies or those with history of feeding intolerance. Enftcare and Neosure are discharge preterm formula.
  • Premature Enfamily Formula and Similac Special Care are hospital premature formula.
  • Feeding tolerance is demonstrated when the preterm infant is capable of safely ingesting and digesting the prescribed enteral feeding without complications associated with gastrointestinal dysfunction or infection.
  • Clinical evidence of feeding tolerance in very low birth weight preterm infant is most often described in the literature as the number of days required to reach full-feeding volumes (reported ranged from 100- 160 tnL per kg per day), the number of episodes of feeding intolerance, the number of days feeds are withheld due to feeding intolerance symptoms, time to regain birth weight, lower leg growth, increase in weight gain, occipital-frontal head circumference, and length.
  • Proposed prevention/treatment strategies for feeding intolerance in preterm infants include:
  • Table 2 Proposed prevention/treatment strategies for feeding intolerance in preterm infants.
  • Baselines are with covariates set to their means Transition intensities with hazard ratios for each covariate
  • State 1 - State 2 0.01257 ( 0.00491, 0.03220) 18.7845 (1.302252,270.96) State 2 - State 1 1.22270 ( 0.43181. 3.46219) 0.1038 (0.006116, 1.76) State 2 - State 2 -1.22270 (-3.46219,-0.43181)
  • State 1 - State 2 0.9975 (0.9777,1.018) 0.5051 (0.1323, 1.928) State 2 - State 1 0.9726 (0.9246, 1.023) 11.9612 (3.8391,37.267) State 2 - State 2
  • PROP 525438 (5.51, 500.99) 0.2727 (0.012, 6.01)
  • antibiotics ts left in the analysts which seem to be significantly related with a switch from NEC to non-NEC status.
  • the table is as follows:
  • having the explicit mathematical terras may allow analysis of data from the linear mixed model.
  • mixed models are used for analysis of correlated data, such as longitudinal data or information that may have multiple
  • the * Y in the information in Example 8 can refer to an abbreviation for time.
  • NEC PROP may be able to predict disease 4.93 days prior to x-ray.
  • PROP Score can be a function of iAP activity mid WB value:
  • the analysis carried forth the patient's PROP score to days that did not have data. For example, if a patient had a PROP score from day 3 and 7, then their PROP score from days 4-6 are equal to that at day 3.
  • PROP 24.1 (1.85. 314) 0.14 (0.01. 2 38)
  • Antibiotics 0.39 (0.13. 1.19) 10.52 (3.59, 30.8)
  • Antibiotics increased the probability of transitioning from NEC to NEC(-).
  • PROP 18.8 (1,30.271) 0.10 (0.01, 1.7(0
  • PROPi f is the prop score of patient i at the yth measurement
  • 1 ⁇ 2 is the time (in PGA days) of the yth measurement for patient i.
  • NEC associated with a significant increase in PROP score
  • Embodiments could use Bayesian methods to fit the beta-mixed regression if desired.
  • Example 10 for example, refers to FIG. 28 and FIG. 29.
  • iAP intestinal alkaline phosphatase
  • FIG. 28 One of the original plots I generated (FIG . 28) highlights that these two biochemical properties segregate NEC disease from non-disease. When examining sepsis in this sample patient population, there is no segregation of this blood infection with non-blood infection.
  • the first term in the formula is iAP abundance. iAP will be shed into the gut lumen, mid thus found in stool, if there is an imbalance of bacteria that are not beneficial.
  • the second term in the NECPredict formula (also referred to as the PROP score) is iAP dysfunction.
  • iAP which is responsible for neutralizing signals dud originate from gram-negative bacteria and trigger the human innate immune response. Humans with robust iAP function can prevent inappropriate pro-inflammatory signal cascades in the human gut and contribute to beneficial microbiota maturation.
  • NEC non-functional, compared to control infants, at any time during the clinical study.
  • To provide a mathematical term for this dysfunction the difference between the maximum iAP activity found in our patient population and any given stool reading was determined; normalization of this algebraic subtraction was needed to give equal weight between the protein abundance and protein function.
  • the iAP abundance and iAP function terms are multipled to provide a propensity (NECPredict) score.
  • the goal offers project is to Obtain data for a prognostic biomarker that predicts necrotizing enterocolitis (NEC), the most frequent and lethal gastrointestinal disease in preterm infants.
  • NEC necrotizing enterocolitis
  • Such a tool which is sensitive and specific for the disease, is essential to support the development of new medicines in this smallest and most fragile patient population.
  • this effort directly addresses critical decision points in current clinical practice: neonalologists in the area and patient advocacy groups have directly challenged us to find fee window of disease reversibility.
  • the team first developed a diagnostic test for NEC, NECDetect.
  • NECDetect From analysis of samples from 135 preterm infants at three hospitals, NECDetect identified >95% true positives and >95% true negatives at the time of disease; importantly, it was not correlated with neonatal late-onset sepsis. Without wishing to be bound by theory, NECDetect components can be used to assess the risk of NEC, prior to its severe onset. Our prospective, observational study will evaluate if NECPredict , a calculated probability based on biochemical data from infant, forecasts disease 36-48 hours before it is clinically evident and if Neonatal DDx , a genetic polymorphism screen, identifies infants at birth wife a predisposition to develop NEC. Enrollment target is 150 preterm infants for 90% statistical power.
  • Necrotizing enterocolitis is fee most common gastrointestinal disease in preterm in&nts. Wife no diagnostics available, it is critical to better understand the pathogenesis of human-microbiome crosstalk in this disease. These studies will define the window of reversibility in which infants can be selected for proactive management and clinical trials for therapeutic interventions.
  • Necrotizing enterocolitis (NEC) in preterm infants is a devastating cause
  • AIM 1 Are IAP polymorphisms predictive of NEC predisposition? Our hypothesis is that infants diagnosed with NEC will have mutations in the gene for intestinal alkaline phosphatase, ALPI, which lower their catalytic ability to detoxify harmful Gram-negative bacteria. Methods will involve Sanger sequencing of PCR products amplified from genomic DNA of infants diagnosed with NEC and of infants without NEC. DNA will be isolated from cheek swabs or from peripheral blood cells. The significance of this effort would be the first mechanistic definition between disease severity, biochemistry, and genetic polymorphism. If achieved, this NEC predisposition screen, termed Neonatal DDx, would be indispensable in identifying the earliest possible therapeutic option and to improving long-term outcome and life quality.
  • AIM 2 Are Intestinal alkaline phosphatase (IAP) levels in stool a prognostic biomarker for NEC? This aim will determine if NEC onset can be determined at a molecular level before the most severe physical symptoms are observable at a clinical level. Without wishing to be bound by theory, increased release of iAP protein in the human gut lumen is a response to microbe-induced inflammation in NEC and measurable as a function of time. In total, over 2,000 patient samples will be longitudinally collected and analyzed for iAP protein content. In vitro results and corresponding clinical data will be used to validate associations between iAP as a biomarker and prediction of NEC diagnosis with a computational platform,
  • Mortality rate is between 30-50% [4] and it usually presents with other lethal diseases, such as sepsis. Survivors may have short gut syndrome, poor neurodevelopmental outcomes, bronchopulmonary dysplasia, and intracranial hemorrhage [5-
  • the first innovation is to evaluate non-in fiammatory proteins that precede the immune activation cascade as a biomarker for NEC.
  • intestinal alkaline phosphatase iAP; [10]
  • IAP intestinal alkaline phosphatase
  • FIG. 30, panel A an initial host regulator in microbial management
  • NEC biomarker research [13] has focused predominantly on gene products that regulate intestinal immunity, mucosal injury that permits bacterial translocation, and host inflammation (FIG. 30, panel A).
  • iAP Encoded by the human ALPI gene, iAP has a crucial role in host-microbiota interactions via restraining downstream host inflammatory responses. It is a metal loenzyme with tissue-specific expression in the small intestine that is readily detectable in the mucous layer and gut lumen [14, 15], Membrane-anchored in enterocyles, iAP only sheds into the gut lumen, and thus measurable in stool, to control bacterial colonization [15, 16] it hydrolyses phosphate from lipopolysaccharides (LPS) and thereby reduces Toll-like receptor 4 (TLR4; FIG. 30, panel A) agonist activity.
  • LPS lipopolysaccharides
  • TLR4 Toll-like receptor 4
  • TLR4 has been implicated in the pathogenesis of NEC [17-20]
  • iAP has been used as a measure of toxic damage to the small intestine in animal models [21].
  • our proposal examines iAP in human biospecimens and evaluates it over the course of the infant’s stay in the N1CU.
  • Biorepository are poised to accelerate larger-scale translational studies [29 ⁇ of NEC in the future.
  • NEC suspicion, or early NEC, also correlated with high levels of iAP in stool (pink bar, Bell stage I; FIG. 31, panel B).
  • the amount of iAP protein was statistically different from controls (pink bar, FIG. 31 , panel B).
  • NEC suspicion pink bar
  • diagnosis mean values red bar, FIG. 31 , panel B.
  • Fecal iAP levels had no measurable correlation with sepsis (blue bar, FIG. 31, panels B and D).
  • Our preliminary data show that iAP protein levels are not statistically correlated with clinical diagnosis of sepsis.
  • Clinical information This study is observational, neither invention nor deviation from standard clinical care in the NICU will be requested.
  • the following clinical data will be obtained: demographics, medical history, antibiotics/an tifungals/medications, physical examinations, complete blood counts, blood cultures, abdominal radiographs, surgical consultation notes, and feeding history.
  • the study will be conducted in compliance with institutional, local, state, and federal regulations regarding use of PHI as defined by the Health Insurance Portability and Accountability Act
  • DNA samples are collected non-invasively from cheek swabs or from residual blood draws.
  • Genomic DNA will be isolated from (i) saliva swabs from infant cheek using or (ii) either peripheral blood cells or whole blood with QIAamp DNA Blood Mini Kit.
  • ALPI variants will be identified by Sanger sequencing of PCR products amplified from genomic DNA. We regularly sequence mutations in human proteins (e.g., [46]) with Eurofins.
  • PCR will be performed with Amp!iTaq polymerase, using a GeneAmp PCR system. Primer pairs used for DNA amplification are: 5 *
  • Alkaline phosphatase enzyme activity is measured with use of 4- meihylumbelliferyl phosphate (MUP) as a fluorescent substrate in the presence said absence of 10 mM L-phenyialanine, an inhibitor of iAP [51, 52). Relative fluorescence units at 360/440 nm will be measured in samples using 96-well black optical bottom plates. Total AP activity was determined in mU/mg, in which U is the amount of enzyme hydrolysing 1 pmol of MUP/rainute at pH 10 and 25 6 C. Determination of total protein in the stool supernatant is determined using Bradford assays. Protein standards (bovine serum albumin) and patient samples will be prepared, using molecular grade water as the diluent. Standards are ran each day of data collection and must have a r2 value greater than 0.99 for acceptance.
  • MUP 4- meihylumbelliferyl phosphate
  • Statistical strategies for identification of disease-causing polymorphisms are based on nature of the disease mutation (58]. For example, statistical analyses will be conducted using a dominant model, comparing wildtype homozygous to both heterozygous and homozygous rare allele groups combined. This assumes that carrying at least one copy of the variant allele confers increased risk of disease.
  • Primary outcome measures include presence of NEC, severity of disease (Bell stage P/IP versus Bell stage 0 and NEC-related bowel perforation.
  • Hardy-Weinberg equilibrium will be determined using a chi-square test to compare the observed genotype frequencies to those expected under Hardy-Weinberg equilibrium. Ordinal logistic regression will be used to compare severity of disease.
  • Significance level will be set at /? ⁇ 0.05.
  • SPECIFIC AIM 2 Does intestinal alkaline phosphatase protein levels in stool serve as a prognostic biomarker for NEC?
  • the neonatology field is hampered by the inability to detect NEC early in (he disease process. Without wishing to be bound by theory, microbe-induced inflammation in NEC results in increasing iAP protein levels in the gut lumen and stool. If asymptomatic or only exhibiting nonspecific symptoms, infants with early NEC will have detectable amounts of iAP 36-48 hrs. prim to NEC diagnosis. Our data indicate that prognostication yforNEC is feasible.
  • NEC' a stool sample is termed‘NEC' if collected during the period from the first day of a radiological finding for NEC (Bell stage P or III) to lad, day of NEC management (antibiotic administration and no feeding by mouth). Collected sample is termed‘suspicion’ from first day of 2+ clinical signs to the last day of medical management A sample is‘control’ if obtained on a day in which no NEC diagnosis was made.
  • Stool is stored in a 4°C specimen NICU refrigerator, until samples are delivered to the lab. Upon receipt of each de-identtfied patient sample, stool is homogenized and a 200 mg/mL slurry is made with molecular grade water in a sterile microfuge tube. Following vortexing and centrifugation, the supernatant is collected, aliquoted, and banked at -80°C [71 ]. Safe handling (gloves, lab coats, goggles), use of absorbent bench paper, decontamination with EPA-registered hospital disinfectant and proper disposal of biohazards are followed. [00513] Determination of relative iAP protein content . Duplicate denaturing SDS-
  • PAGE gds will be run ott stool superatants to visualize all proteins in each lane and for immunoblottiog detection of iAP.
  • iBlot and iBind will be used for protein transfer and Western blot, respectively. Bands are quantified with Amersham Imager 600; relative iAP protein in stool sample is the fraction of protein found in human intestinal lysate tissue.
  • control experiments assessed the immunoblot workflow for accuracy and precision. Such experiments minimize over- or under-estimates bf true differences in protein abundance.
  • a serial dilution of a positive control (small intestinal tissue lysate) and negative control (purified bovine iAP) show our dynamic range and quantitative accuracy.
  • the 60 kDa iAP signal from patient samples is determined; this value is ratioed to the difference between the positive control and negative control measurements.
  • NECPredict will use a generalized linear mixed effects model
  • NEC diagnosis as the response
  • iAP content of the last two collected samples as tire predictors (fixed effect)
  • Dt-yandDt-2,1 are the iAP contents of the last two collected diapers e is a subject specific error term, thru incorporates the dependence on time and individual for NEC diagnosis and diaper content
  • Significant positive estimated values of a ⁇ indicate that high iAP content from the last collected diaper (about 3 days before) predicted future NEC diagnosis and ⁇ *2 > 0 indicates that high iAP from the week prior predicts future NEC diagnosis. This information indicates iAP can be established as a prognostic biomarker for NEC.
  • iAP supplementation as a preventative strategy for NEC, as enzyme replacement therapy is an low-cost, low-risk approach for rare diseases that is often successful [84].
  • enzyme replacement therapy is an low-cost, low-risk approach for rare diseases that is often successful [84].
  • these personalized biomarker approaches are not limited to children; iAP as a biomarker for infant gut inflammation has parallels to adult diseases, such as IBD.
  • Genomes Project C., et al., A map of human genome variation from population-scale sequencing : Nature, 2010. 467(7319): p. 1061-73.
  • Study Design for Aim 2 The study will be a prospective observational study with pre-term infants enrolled as they arrive. Stool samples from disposed diapers will be collected every 3-4 days until the infant reaches 37 weeks post-conceptual age or is discharged. Stool samples for patients with clinically evident NEC will be analyzed along with a random subset of stool samples from control patients.
  • Sample Size For aim 2, we will need 150 subjects to achieve 90% power. This is based on Emulation results, as existing sample size calculations are only appropriate for differences between two groups [1]. In contrast, our model requires testing the prognostic capability of a continuous biomarker.
  • FIG. 33 shows the probability of correctly declaring m > 0 across the simulations for each sample size, the probability of correctly declaring a3 ⁇ 4 > 0 and the probability of correctly declaring both ai > 0 and az > 0.
  • this triple is listed as (Pi,-,-) and far scenarios 2 and 3, this triple is listed as (Pi, Pz, Ps).
  • tissue-specific alkaline phosphatases in humans that are tissue-specific: intestinal alkaline phosphatase, placental-like alkaline phosphatase, tissue nonspecific alkaline phosphatase, and germ-cell alkaline phosphatase.
  • tissue-specific alkaline phosphatase isozymes are 86-98% identical to one another, but 52-56% identical when compared to tissue non-specific alkaline phosphatase.
  • the iAP gene, ALPI has 403 missense polymorphisms that span the entire sequence: more than 50% of the amino acids in iAP have at least one known mutation.
  • iAP intestinal alkaline phosphatase
  • Our innovation is use of two biochemical measures of intestinal alkaline phosphatase (iAP), shed in stool, as a molecular biomarker for NEC in premature infants.
  • iAP intestinal alkaline phosphatase
  • the appeal of iAP as a biomarker lies in its tissue-specific expression in the small intestine and its secretion into the gut lumen iAP would only be measurable in stool as a response to controlling bacterial colonization. Also, it is detectable in human stool samples in healthy individuals; responsible for majority of AP enzymatic activity in stool; and used as a measure of toxic damage to the small in testine in animal models.
  • iAP content in stool is expected to increase from released membrane vesicles loaded with iAP, if there was risk of bacteria-induced inflammation.
  • non-diseased patients had very low amounts of iAP protein in stool.
  • Sensitivity and specificity of fecal iAP content is >93%.
  • fecal iAP levels had no measurable correlation with other non-Gl infections or sepsis, a frequent co-morbidity that can confound diagnosis of NEC.
  • iAP is the only human alkaline phosphatase recoverable from stool proteomics mid has several candidate peptides that may be used for absolute quantitative determination of iAP abundance by mass spectrometry.
  • the combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS) provides a flexible, dynamic platform for the simultaneous identification and quantification of up to thousands of proteins in fecal samples.
  • Our initial shotgun proieomic analysis of a preterm infant stool sample showed that 635 human proteins were detected in the contents of the gut lumen, or the secreted host proteome.
  • (C) High polymorphism frequency of the IAP gene in the African American population, which may be correlated higher disease incidence, may result in spurious results for both affinity-based and MS based protein measurements. Sequence information from unrelated individuals in order to ascertain the frequency distribution of iAP polymorphisms. Only in the African-American population (n 12,487) were there iAP polymorphisms, or alleles that were greater than 1% of the population (FIG. 34, panel B). Estimated frequencies for common alleles V201, R33L, R92C, R144H, and T2071 were 4.8, 2.6, 1.9, 4.2, and 3.1%, respectively. Polymorphisms in the total population (blue circles, FIG.
  • Necrotizing enterocolitis is a common neonatal gastrointestinal (Cl) tract emergency with a high mortality rate! and long-term morbidities, including short-gut syndrome, nutritional deficiency, and neurodevelopmental detay.2,3 Suspected NEC presents with mild, nonspecific symptoms that frequently resolve with minimal intervention; no clinical test is an established criterion standard fra ⁇ suspected NEC, Radiographic evidence, such as pneumatosis inlestinalis, is used to diagnose severe or advanced disease but has a sensitivity as low as 44%, 4 has limited specificity ,5 and lacks concordance in
  • Necrotizing enterocolitis has been argued to be the antecedent of some cases oflate-onset neonatal sepsis (LOS). Neonates, particularly very low-birth-weight infants, are susceptible to sepsis owing to prolonged hospitalizations, invasive instrumentation, underdeveloped innate immunity, and altered immunological responses. The latter 2 physiological states, coupled with an immature intestinal barrier function, can give rise to NEC.18,19 From both epidemiological and clinical standpoints, sepsis can confound the use of inflammation proteins as a biomaiker for NEC. Sepsis and NEC require careful differential diagnosis, as both may be lethal if not diagnosed and treated appropriately.
  • Clinical data which included gestational age, birthweight, Apgar scores, delivery type, race/ethnicity, sex, and disposition (ie, death, discharge, or transfer to another facility), were extracted from medical records every 3 months. Of these, only race/ethnicity was defined by a parent. In-hospital data included feeding, antibiotic treatment, laboratory and radiology results, and surgical notes. Clinical findings of NEC (modified Bell stage 1-3), sepsis, mid other confirmed nou-CI tract infections were reviewed by attending physicians.
  • each enrolled patient was provided a code, which allowed for research tracking and removed any clues to the individual's identity. Every three months, patient records were evaluated to determine clinical correlatives. Clinical data were extracted from medical records into a relational clinical database. Demographic information and initial clinical data included gestational age, birth weight, Apgar scores, delivery type, race, gender, and final outcome (death, discharge, or transfer). Lastly, a second set of clinical information was obtained: antibiotic use, diet, serum AP, radiology reports, length of stay in NICU, surgery, and mortality. Human milk exposure is calculated as the mean percent of feeding from human milk as a function of the total days that the subjects were in the study.
  • NEC cases only pre-event exposures were considered for human milk. Antibiotic exposures were considered in aggregate; antibiotics were always parentally administered to subjects. Percent of days of age on antibiotics is related to the number of days that the subjects were in the study. For NEC cases, only pre-event exposures were considered for antibiotics. [00548] Disease Definitions. Different definitions of NEC have been suggested.26-29 For this study, 2 categories of NEC, derived from clinical documenlation, were used (eTable 1). Radiological signs were defining criteria for our NEC categories; abdominal signs and clinical and laboratory findings were secondary criteria. Suspected NEC was defined as concern for disease based on abnormal clinical and laboratory findings without evidence of pneumatosis intestinalis or portal venous gas on abdominal radiographic images.
  • Severe NEC was defined by radiologic evidence of pneumatosis intestinalis and/or portal venous gas. Patients diagnosed with spontaneous intestinal perforation (SIP) were excluded from the study (eTable 2). Diagnosis of neonatal LOS required the appearance of abnormal clinical findings at least 72 hours after birth and blood cultures positive for bacteria not considered a contaminanl30,31 (eTable 3). Infants with other confirmed non-GI tract infections had clinical findings with bacterial, viral, or fungal infections identified in body fluids other than blood. The summary of cohorts and diagnoses of NEC, SIP, sepsis, and non-GI tract infections are provided in eTable 4 to eTable 11.
  • NEC diagnosis was identified from review of clinical documentation.
  • the research definition of NEC did trot always align with the clinical diagnosis of fee patient.
  • NEC and suspected NEC were physician-directed clinical diagnoses in which radiologic signs were the defining criteria and abdominal signs, clinical findings, and lab findings further confirmed diagnosis (eTable 1).
  • NEC suspicion was defined as an infant wife concern for early disease based on clinical and laboratoiy abnormalities without evidence of pneumatosis intestinalis on radiography, instead, infants suspected of NEC exhibited one or more radiologic signs including mild intestinal dilation, mild intestinal ileus, thickened bowel walls, or paucity/absence of bowel gas.
  • Severe NEC Severe NEC (eTable 1) was defined by radiological evidence of pneumatosis intestinalis and/or portal venous gas or pathological findings on surgical or postmortem intestinal samples. Pneumoperitoneum, which is free intrabdominal air resulting from a perforation, was considered NEC when accompanied by evidence of pneumatosis intestinalis on radiography, as well as abdominal sigmas found in definite NEC. Additional signs included moderate to severe abdominal distention and/or abdominal tenderess and/or
  • NEC diagnosis was categorized by at least one senior clinician and two additional senior research clinicians by case review, note review, x-ray and operative findings.
  • infants diagnosed wife sepsis include only those wife laboratory and clinical findings that are confirmed after seventy-two hours of age (eTable 3).
  • Laboratory findings included blood culture or non-culture microbial testing that confirmed the presence ofbacteria in blood that was not considered a contaminants 14, SI 5
  • Clinical findings that were used to support the diagnosis of sepsis included a range of criteria from temperature instability and respiratory distress to abnormal perfusion, bleeding issues, and unexplained jaundice.
  • infants with other confirmed non-GI infections and infimts who were infection-negative were classified and documented in this study (eTable 3).
  • Other confirmed infections were those confirmed bacterial, viral or fungal infections identified in normally sterile body fluids.
  • Clinical findings were similar to those diagnosed with sepsis.
  • An infection-negative classification was comprised of infants with suspected, but not confirmed, infections and infants not suspected o f any infections for which no laboratory tests were ordered concerning an infection and were asymptomatic.
  • Those suspected of infections had laboratory findings that included, but were not limited to, leukocytosis or leukopenia, elevated immature neutrophil counts, low absolute neutrophil counts, and elevated C-reactive protein and serum alkaline phosphate.
  • Clinical findings for these infants were identical to those with confirmed sepsis diagnosis.
  • proteins most biologically relevant to host responses are unlikely to be identified, due to the expected modest representation of host-derived gut proteins in the total stool proteome,Sl8 partial proteolysis that occurs during gut transit, and a large and ill- defined microbiota proteome.
  • Protein Concentration Total protein concentration in the stool supernatant was determined by Bradford assay(ThermoFisher Scientific). Total protein was used to standardize biochemical activity measurements and protein load for quantitative IAP abundance via immunoblot analyses. Protein concentration measurement was reproducible and accurate between replicates and different operators32 (FIG. 39, eTable 12).
  • the concentration of total protein in final stool supernatant was determined by Bradford assay (Coomassie Plus Protein Assay Reagent, Thermo- Scientific) on either a Spectra Max M2e or Spectra Max i3x spectrophotometer (Molecular Devices). Protein standards (bovine serum albumin, Pierce) and patient samples were prepared, using molecular grade water (Millipore) as the diluent Five-point standard curves were generated for each day of measurement; daily r* values >0.994 were indicative of linearity of protein abundance measurements. A second measure of analytical validity was the mean error from ideal values of standards used.
  • Fecal IAP Catalytic Activity Alkaline phosphatase activity was measured with use of 4-methylumbelliferyl phosphate (Abeam) substrate in the presence and absence of L-pheny!a!anine, an inhibitor of IAP.33,34 Relative fluorescence units at 360/440 nm were measured in a multiwell format on either a Spectra Max M2e or i3x spectrophotometer (Molecular Devices). Total alkaline phosphatase catalysis and lOmM phenylalanine-inhibited alkaline phosphatase catalysis were measured in triplicate and averaged. Reported IAP activity represents the difference between these 2 averages.
  • IAP activity was reproducible between users and on different days (FIG. 39, eTable 12,).
  • Activity assays are a measure of enzymatic catalysis as a function of time and as a ratio of protein in the stool supernatant Alkaline phosphatase activity can be measured using a number of different substrates.
  • the substrate employed determines the dynamic range and sensitivity of the enzymatic reaction.
  • AP activity in (his work was measured with use of 4-methylumbelliferyl phosphate (MUP) as a fluorescent substrate (Abeam, ab83371) in the presence mid absence of L-phenylalanine, an inhibitor of iAP.
  • MUP 4-methylumbelliferyl phosphate
  • Fluorogenic substrates enable catalytic activity of AP to be measured with high sensitivity and accuracy, attributes which are ideal for basic research and biotechnology applications.
  • fluorogenic substrates typically have a detection range that is HM)X-]00()X greater than chromogenic substrates, for which product precipitates are detected after reduction of tetrazolium sails or production of colored diazo compoonds.
  • S31 The 4-MUP substrate has a lower Km than other native substrates found in human samples.
  • S32 Determination of Vmax using 4-MUP is pH-independentS33,S34 Lastly, its hydrolysis products do not lead to strong inhibition of alkaline phosphatase, S32 which would lower the measurement range and limit accuracy.
  • Relative fluorescence unite (RFUs) at 360 nm excitation/440 nm emission were measured using a Spectra Max M2e spectrophotometer or Spectra Max i3x (Molecular Devices). Ninety-six-well black optical bottom plates (TheimoScientific) were used.
  • Immunoblotting was performed using traditional or iB!ot-iBind methods (ThermoFisher Scientific).38-40 The amount of IAP in clinical samples was reported as a percent of the detected protein in an immunoblot relative to the difference in densitomeiric pixel count in a fixed area (Amersham Imager 600; GE Healthcare) that captured the IAP signal in the positive and negative controls.
  • abl 14268 purified human tissue non-specific alkaline phosphatase (abl 14267), and bovine intestinal alkaline phosphatase (Sigma, P5521).
  • Transfer of proteins in gel matrix was performed using one of two techniques: either (1) a semi-dry transfer apparatus (FisherScientific) at a constant 5V for 1 hour or (2) the iBlol 2 (ThermoFisher) dry blotting system at starting at 20V and ending at 25V for a total of 7 min.
  • Western blotting techniques were performed either using traditional methods S40-S42 or with use of an iBind system (ThermoFisher).
  • Membranes were either serially blocked in 5% (w/v) nonfat dry milk in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, tod 0.1% Tween or with the iBind solution kit (ThermoFisher) reagents. At room temperature, membranes were incubated with primary rabbit polyclonal antibodies against human iAP (Abeam, ab7322) at a 1 :13,000 dilution, washed, and incubated with horseradish peroxidase- conjugated goat anti-rabbit secondary antibodies (Abeam, ab6721) at a 1:20,000 dilution.
  • accuracy [(absolute value - measurement value)/absoiute value) x 100%.
  • the /rvalue indicates whether measurements deviate significantly from each other, it can he used to indicate whether inter- operator measurements for calibrators are either statistically similar (p-value ⁇ 0.05) or are dissimilar Q?- value >0.05).
  • dilutions of tire sample were performed to ensure the experimental measurement value fell in the middle of the linear range between the highest and lowest analyte concentration used for the standard curve.
  • FDR false discovery rate
  • a total of 136 infants were enrolled (68 [50.0%] male infants), with a median (interquartile range [IQR]) birth weight of 1050 (790-1350) g and a median (IQR) gestational age of 28.4 (26.0-30.9) weeks.
  • a total of 25 (18.4%) were classified as having severe NEC,
  • Attrition rate was 11.0% (ie, 15 infants), resulting from enrollment changes, medical changes, or inadequate biospecimen collection (FIG. 35D).
  • NEC was significantly lower compared with samples from infante who did not have NEC (FIG. 38 A). However, different levels of IAP enzyme dysfunctionwere found between patients with suspected and severe NEC. Samples at the time of severe NEC had a median (1QR) IAP activity of 183 (56-507) pmoVmin/g (95%Ci, 63-478 pmol/min/g) of stool protein.
  • median (IQR) activity for sepsis was 575 (338-1122) pmol/min/g (95%CI, 355-1073 pmol/min/g) of stool protein, for other non-GI tract infections, 319 (207- 961) pmol/min/g (95%CI, 172-1193 pmol/min/g) of stool protein, and, for the control group. 519 (180-1243) pmol/min/g (95%CI, 350-695 pmol/inin/g) of stool protein.
  • Area under the receiver operating characteristic curves showed that use of fecal ⁇ AR content or activity would randomly assign culture-confirmed bacterial sepsis and other non-Gi infection as positives or negatives for these inflammatory conditions (FIG.
  • Mean (SE) accuracy scores for IAP content were 0.52 (0.07) (95%Cl, 0.38-0.66; P - .75) at the lime of sepsis and 0.58 (0.08) (95%Cl, 0.42-0.75; P - .06) at the time of other non-Gi infection.
  • Mean (SE) accuracy scores for IAP activity were 0.52 (0.07) (95%CI, 0.39-0.67; P - .68) at the time of sepsis and 0.57 (0.08) (95%CI, 0.39-0.69; P - .66) at the lime of other non-GI infection.
  • Biomarkers such as calprotectin, are reliable indicators of intestinal inflammation in general but provide no understanding of the dominant inflammatory pathways at work in the intestinal mucosa of a patient
  • Our study required prospective inclusion of infants with NEC and concurrently tested healthy and unhealthy Controls with several inflammatory conditions in the neonatal intensive care horrin Under these real-life conditions, estimates of biomarker reliability more accurately reflected potential performance in clinical application.
  • Examination of proteins involved in organ- specific modulation of microbiota homeostasis and response distinguished NEC from other forms of inflammation.
  • lAP is the first candidate diagnostic biomarker, unique in its high positive predictive value for NEC.
  • IAP is associated with NEC and not associated with sepsis or other rtpn-GI tract infections,
  • the IAP biomatker is associated with disease severity; IAP biochemistry differentiates advanced NEC, flagged by portal venous gas or pneumatosis intestinalis, from suspected disease, for which there are no reliably observable signs by radiology. Our results also showed that this classification of NEC suspicion is supported as an explicit disease state. Our approach differed from other candidate biomarker studies. This work diverges not only by the target protein of interest but also by our use of a disease severity catalog, biospecimen choice, and molecular method of detection. We were able to segregate NEC suspicion from severe cases of NEC. There has been great effort to identify commonalities in clinical criteria to define severe NEC.
  • Nanthakumar N Meng D, Goldstein AM, et al.
  • PLoS One 201 l;6(3):el 7776. doi: 10.1371 /journal.pone.0017776
  • Buhimschi CS Buhimschi 1A
  • Abdel-Razeq S et al. Proteomic biomarkers of intra- amniotic inflammation: relationship with funisitis and early-onset sepsis in the premature neonate. Pediatr Res. 2007;61(3):318-324. doi: 10,1203/01.pdr.0000252439,48564.37
  • Burnette WN.“Western blotting” electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Nutrition Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Husbandry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)

Abstract

La présente invention concerne des compositions et des procédés pour détecter et traiter des maladies gastro-intestinales.
PCT/US2020/016646 2016-08-04 2020-02-04 Compositions et procédés pour détecter une maladie gastro-intestinale Ceased WO2020163381A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CN202510261116.9A CN120102879A (zh) 2019-02-04 2020-02-04 检测胃肠道疾病的组合物和方法
CN202080027193.3A CN113645846B (zh) 2019-02-04 2020-02-04 检测胃肠道疾病的组合物和方法
US17/428,416 US11953501B2 (en) 2016-08-04 2020-02-04 Compositions and methods to detect gastrointestinal disease
AU2020217717A AU2020217717B2 (en) 2019-02-04 2020-02-04 Compositions and methods to detect gastrointestinal disease
CA3128826A CA3128826A1 (fr) 2019-02-04 2020-02-04 Compositions et procedes pour detecter une maladie gastro-intestinale
EP20752596.5A EP3920713A4 (fr) 2019-02-04 2020-02-04 Compositions et procédés pour détecter une maladie gastro-intestinale
US18/628,482 US20240393333A1 (en) 2016-08-04 2024-04-05 Compositions and methods to detect gastrointestinal disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16/267,120 US11493515B2 (en) 2016-08-04 2019-02-04 Compositions and methods to detect gastrointestinal disease
US16/267,120 2019-02-04

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US16/267,120 Continuation-In-Part US11493515B2 (en) 2016-08-04 2019-02-04 Compositions and methods to detect gastrointestinal disease

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US17/428,416 A-371-Of-International US11953501B2 (en) 2016-08-04 2020-02-04 Compositions and methods to detect gastrointestinal disease
US18/628,482 Division US20240393333A1 (en) 2016-08-04 2024-04-05 Compositions and methods to detect gastrointestinal disease

Publications (1)

Publication Number Publication Date
WO2020163381A1 true WO2020163381A1 (fr) 2020-08-13

Family

ID=83366685

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/016646 Ceased WO2020163381A1 (fr) 2016-08-04 2020-02-04 Compositions et procédés pour détecter une maladie gastro-intestinale

Country Status (5)

Country Link
EP (1) EP3920713A4 (fr)
CN (2) CN120102879A (fr)
AU (1) AU2020217717B2 (fr)
CA (1) CA3128826A1 (fr)
WO (1) WO2020163381A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11741686B2 (en) 2020-12-01 2023-08-29 Raytheon Company System and method for processing facility image data
WO2023141560A3 (fr) * 2022-01-20 2023-09-21 Synthetic Biologics, Inc. Phosphatase alcaline destinée à être utilisée en oncologie
WO2025075652A1 (fr) * 2023-10-06 2025-04-10 DeLorean Artificial Intelligence, Inc. Système et méthode de gestion de soins médicaux
US12619999B2 (en) 2020-05-26 2026-05-05 DeLorean Artificial Intelligence, Inc. Predictive and interventive intelligence

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114113624B (zh) * 2020-08-28 2024-08-16 香港城市大学深圳研究院 利用免疫球蛋白关联蛋白质组开发疾病标志物的方法及装置
CN116662798B (zh) * 2023-04-23 2026-02-24 迪辅乐生物(上海)有限公司 基于肠型分型的微生物组合物、过敏预测模型的构建方法及其模型和应用
CN120673884B (zh) * 2025-08-20 2025-11-04 温州医科大学 基于ai的克林霉素磷酸酯加工水解优化方法及系统

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120253771A1 (en) * 2004-08-26 2012-10-04 Col Nananda F Syetem for Optimizing Treatment Strategies Using a Patient-Specific Rating System
US20140162370A1 (en) * 2011-06-14 2014-06-12 The Board Of Trustees Of The Leland Stanford Junior University Urine biomarkers for necrotizing enterocolitis and sepsis
US20160232324A1 (en) * 2013-09-20 2016-08-11 Georgia Tech Research Corporation Systems And Methods For Disease Progression Modeling
WO2018027179A1 (fr) * 2016-08-04 2018-02-08 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions et méthodes pour détecter une maladie gastro-intestinale

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112017014419A2 (pt) * 2015-01-09 2018-01-16 S Malo Madhu diagnose e tratamento de diabetes incipiente
CN108061556B (zh) * 2017-10-18 2021-07-09 广州傲胜机器人科技有限公司 一种基于隐马尔可夫模型的地图匹配方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120253771A1 (en) * 2004-08-26 2012-10-04 Col Nananda F Syetem for Optimizing Treatment Strategies Using a Patient-Specific Rating System
US20140162370A1 (en) * 2011-06-14 2014-06-12 The Board Of Trustees Of The Leland Stanford Junior University Urine biomarkers for necrotizing enterocolitis and sepsis
US20160232324A1 (en) * 2013-09-20 2016-08-11 Georgia Tech Research Corporation Systems And Methods For Disease Progression Modeling
WO2018027179A1 (fr) * 2016-08-04 2018-02-08 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Compositions et méthodes pour détecter une maladie gastro-intestinale

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3920713A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12619999B2 (en) 2020-05-26 2026-05-05 DeLorean Artificial Intelligence, Inc. Predictive and interventive intelligence
US11741686B2 (en) 2020-12-01 2023-08-29 Raytheon Company System and method for processing facility image data
WO2023141560A3 (fr) * 2022-01-20 2023-09-21 Synthetic Biologics, Inc. Phosphatase alcaline destinée à être utilisée en oncologie
WO2025075652A1 (fr) * 2023-10-06 2025-04-10 DeLorean Artificial Intelligence, Inc. Système et méthode de gestion de soins médicaux

Also Published As

Publication number Publication date
CN113645846B (zh) 2025-02-25
CN113645846A (zh) 2021-11-12
CN120102879A (zh) 2025-06-06
EP3920713A4 (fr) 2022-11-09
EP3920713A1 (fr) 2021-12-15
AU2020217717B2 (en) 2026-03-12
CA3128826A1 (fr) 2020-08-13
AU2020217717A1 (en) 2021-09-30

Similar Documents

Publication Publication Date Title
AU2020217717B2 (en) Compositions and methods to detect gastrointestinal disease
Mohamed et al. Predictors of mortality of severe sepsis among adult patients in the medical Intensive Care Unit
US11493515B2 (en) Compositions and methods to detect gastrointestinal disease
Chakraborty et al. Elevated serum neutrophil gelatinase-associated lipocalin is an early predictor of severity and outcome in acute pancreatitis
US8163504B2 (en) Combination of sPLA2 activity and OxPL/apoB cardiovascular risk factors for the diagnosis/prognosis of a cardiovascular disease/event
WO2011053832A1 (fr) Utilisation de la protéine ngal urinaire à des fins de diagnostic d'une septicémie chez des bébés de très petit poids de naissance
Inoue et al. Serum neutrophil gelatinase-associated lipocalin as a predictor of the development of bronchopulmonary dysplasia in preterm infants
Yamout et al. Peritoneal and plasma D‐lactate concentrations in horses with colic
Yao et al. Higher serum level of myoglobin could predict more severity and poor outcome for patients with sepsis
Cicarelli et al. Comparison of C‐reactive protein and serum amyloid A protein in septic shock patients
Jiménez et al. Primary hypolactasia diagnosis: Comparison between the gaxilose test, shortened lactose tolerance test, and clinical parameters corresponding to the C/T-13910 polymorphism
US11953501B2 (en) Compositions and methods to detect gastrointestinal disease
US9417247B2 (en) Human autoantibodies specific for PAD3 which are cross-reactive with PAD4 and their use in the diagnosis and treatment of rheumatoid arthritis and related diseases
de Andrade et al. Development and validation of a simple web-based tool for early prediction of COVID-19-associated death in kidney transplant recipients
Fiorentino et al. Improving translation from preclinical studies to clinical trials in acute kidney injury
Sharif et al. Low serum albumin concentration predicts the need for surgical intervention in neonates with necrotizing enterocolitis
US20240393333A1 (en) Compositions and methods to detect gastrointestinal disease
US12282019B2 (en) Compositions and methods to detect gastrointestinal disease
Foresta et al. Early protein profile of human embryonic secretome
EP3311164B1 (fr) Méthodes et compositions pour le diagnostic et le pronostic de l'appendicite et la différenciation des causes de la douleur abdominale
Fayed et al. Prediction of mortality and need for renal replacement therapy in patients of acute kidney injury using fibroblast growth factor 23
Lithgow et al. Resolving glycogen and related enzymes reveals correlates of Lactobacillus crispatus dominance in a cohort of young African women
JP2025078980A (ja) 間質性肺炎症急性増悪患者における肺線維化進行のリスクを予測するためのバイオマーカー
Madbouli et al. Incidence and Prognosis of Acute Kidney Injury with COVID-19 in Hospitalized Egyptian Patients
Tayman et al. A Pilot Study on the Dual Role of ADAMTS-1,-4, and-5 in Necrotizing Enterocolitis: Linking Tissue Pathology and Serum Biomarker Discovery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20752596

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3128826

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020752596

Country of ref document: EP

Effective date: 20210906

ENP Entry into the national phase

Ref document number: 2020217717

Country of ref document: AU

Date of ref document: 20200204

Kind code of ref document: A

WWD Wipo information: divisional of initial pct application

Ref document number: 818487

Country of ref document: NZ

WWG Wipo information: grant in national office

Ref document number: 202080027193.3

Country of ref document: CN

WWW Wipo information: withdrawn in national office

Ref document number: 2020752596

Country of ref document: EP