Compositions and methods for detecting gastrointestinal diseases

Abstract

Compositions and methods for detecting gastrointestinal diseases are provided. The present invention relates to compositions and methods for detecting and treating gastrointestinal diseases. The present invention relates to the use of an agent for determining intestinal alkaline phosphatase (iAP) activity and the amount of iAP in a child for the preparation of a kit for use in a method for determining the prognosis of inflammatory bowel disease (IBD) in said child.

Description

[0001] This application is a divisional application with the same invention name as the parent case. The Chinese application number of the parent case is 202080027193.3, the international application number is PCT / US2020 / 016646, and the application date is February 4, 2020.

[0002] This application claims priority to U.S. Patent Application No. 16 / 267,120, filed on February 4, 2019, the entire contents of each of which are incorporated herein by reference.

[0003] All patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. The disclosures of these publications are incorporated herein by reference in their entirety in order to more fully describe the prior art known to those skilled in the art as of the date of the present invention described and claimed herein.

[0004] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights whatsoever.

[0005] Government interests

[0006] This invention was made with government support under Grant No. R01GM097350 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0007] The present invention relates to compositions and methods for detecting and treating gastrointestinal diseases. Background Art

[0008] Gastrointestinal diseases refer to disorders involving the gastrointestinal tract. For example, necrotizing enterocolitis (NEC) is an acquired gastrointestinal disease that is common in premature infants. In NEC, bacteria invade the intestinal wall, causing localized infection and inflammation. NEC is characterized by high mortality and long-term morbidity, including short bowel syndrome, recurrent infections, nutritional deficiencies, and neurodevelopmental delays. Despite an overall net decrease in mortality among premature infants, deaths associated with NEC have increased. NEC is often difficult to diagnose and manage due to initial nonspecific symptoms and rapid deterioration. Clinicians currently rely on radiographic evidence to make a diagnosis in the late stages of the disease. Summary of the invention

[0009] The present invention provides a method for determining the prognosis of necrotizing enterocolitis (NEC) in a patient. In an embodiment, the method includes fitting a Markov model using a two-state transition matrix and a propensity value measured between multiple subjects, wherein the two-state transition matrix includes a first state and a second state, wherein the first state includes a non-necrotizing enterocolitis state, wherein the second state includes a necrotizing enterocolitis state (NEC), wherein the propensity value is a function of the intestinal alkaline phosphatase (iAP) activity value and the amount of iAP found in a subject in the multiple subjects; using the patient's propensity value and the fitted Markov model to estimate the probability of transitioning from the first state to the second state, wherein the fitted model indicates that an increase in the patient's propensity level significantly increases the probability of transitioning from the first state to the second state; treating the patient when the propensity value is greater than or equal to a threshold of about 0.5.

[0010] In embodiments, the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus a first ratio, wherein the first ratio comprises an iAP activity value for a subject from the plurality of subjects divided by a maximum iAP activity value observed in the plurality of subjects, and wherein the second value comprises a second ratio, wherein the second ratio comprises an iAP amount from an immunoassay value for a subject from the plurality of subjects divided by a maximum iAP amount from an immunoassay value observed in the sample.

[0011] Non-limiting examples of immunoassays include Western blot analysis, ELISA or immunoprecipitation. For example, an immunoassay can include a Western blot analysis using a chemiluminescent reporter gene. In another example, an immunoassay can include a Western blot analysis using a fluorescent reporter gene. The response of the fluorescent reporter can be more linear.

[0012] In embodiments, the immunoassay readout can be considered a 'mini ELISA' because the abundance of the patient sample iAP is quantified against a 2-point curve. For example, the signal in the patient sample can be compared by ratioing the difference between the signal in the patient sample and human small intestine lysate (a positive control with the highest level of iAP or 100%) and bovine iAP (a negative control because the anti-human iAP antibody does not detect bovine iAP or 0%). The maximum WB value in Example 9 is for human small intestine lysate.

[0013] In embodiments, the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus an iAP activity value for a subject in the plurality of subjects, and wherein the second value comprises the iAP amount from an immunoassay value for the subject in the plurality of subjects.

[0014] In embodiments, the plurality of subjects comprises patients.

[0015] In embodiments, treatment comprises withholding food, administering antibiotics, or a combination thereof.

[0016] In embodiments, the sample is human small intestine lysate.

[0017] In embodiments, the immunoassay comprises Western blot, ELISA, or immunoprecipitation.

[0018] The present invention provides methods for identifying a subject with a gastrointestinal (GI) disease. One aspect of the present invention relates to methods for diagnosing a subject with a gastrointestinal disease. Another aspect of the present invention relates to methods for identifying a subject at risk for a gastrointestinal disease. Embodiments as described herein can further identify early stages of gastrointestinal disease and late stages of gastrointestinal disease. Certain embodiments can distinguish between early stage and late stage gastrointestinal disease. For example, embodiments as described herein can diagnose late inflammatory states, such as those determined by radiological findings of intestinal gas (portal vein or biliary gas). As another example, embodiments can identify early stages of a disease before it is physiologically apparent that there is rampant inflammation of the intestine.

[0019] In embodiments, the method includes incubating a biological sample from a subject with an agent that binds intestinal alkaline phosphatase (iAP), detecting the agent that binds iAP in the sample, and detecting and / or measuring the amount or activity of iAP in the sample, whether or not bound to the agent. In embodiments, the agent that binds iAP is at least one GI disease biomarker, which includes AP enzyme activity of iAP, iAP protein level, iAP dimerization / dissociation, post-translationally modified iAP, total protein, such as total fecal protein, or a combination thereof. In some embodiments, GI disease biomarkers can be used to diagnose subjects with gastrointestinal diseases, and can also indicate subjects with gastrointestinal (GI) diseases and / or subjects at risk of developing GI diseases. In some embodiments, subjects with gastrointestinal (GI) diseases may include early and late stages of the disease.

[0020] In an embodiment, the iAP is not bound to an iAP binding reagent. For example, a substrate can be provided to an enzyme in a sample and changes in the substrate monitored. In an embodiment, the iAP is not bound for an activity assay. For example, a substrate can be provided to an enzyme in a sample and changes in the enzyme can be monitored. Without being bound by theory, this can also be the same for an AP bound to an enzyme, where the substrate changes measured in an immunoassay are for the protein bound by the antibody system, as well as for any free AP in the sample.

[0021] The present invention further provides a method for diagnosing a GI disease, such as necrotizing enterocolitis, in a subject, comprising incubating a biological sample from the subject with an agent that binds intestinal alkaline phosphatase (iAP), detecting the agent that binds iAP in the sample, and detecting and / or measuring the amount or activity of the agent that binds iAP in the sample. In an embodiment, the agent that binds iAP is at least one GI disease biomarker, which includes iAP enzyme 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 indicates a subject with a gastrointestinal (GI) disease. The agent that binds iAP of the present invention can be an iAP that binds 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 include small molecule activators or inhibitors of catalytic reactions, metal ions (tungsten is a transition state effector of alkaline phosphatase), agents that cause allosteric release of products, labile chemical moieties that serve as chemical, enzymatic or photolytic triggers, or substrates that bind to labeled forms of iAP.

[0022] Embodiments can further include diagnosing the subject as having a GI disease. For example, 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 / 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. if the iAP activity is less 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, 110 mU / mg, 120 mU / mg, 130 mU / mg, 140 mU / mg, 150 mU / mg, 160 mU / mg, 170 mU / mg, 180 mU / mg, 190 mU / mg, 200 mU / mg, 210 mU / mg, 220 mU / mg, 230 mU / mg, 240 mU / mg, 250 mU / mg, 260 mU / mg, 270 mU / mg, 280 mU / mg, 290 mU / mg, 300 mU / mg, 310 mU / mg, 320 mU / mg, 330 mU / mg, 340 mU / mg, 350 mU / mg, 360 mU / mg, 370 mU / mg, 380 mU / mg, 390 mU / mg, 400 mU / mg, 410 mU / mg, 420 mU / mg, 430 0mU / mg、100mU / mg、200mU / mg、300mU / mg、400mU / mg、500mU / mg、600mU / mg、700mU / mg、800mU / mg、900mU / mg、1000mU / mg、1050mU / mg、1100mU / mg、1 If or combinations thereof. The iAP activity is less than about 5 U / mg, 10 U / mg, 50 U / mg, 100 U / mg, 200 U / mg, 300 U / mg, 400 U / mg, 500 U / mg, 600 U / mg, 700 U / mg, 800 U / mg, 900 U / mg, 1000 U / mg; or if the level of iAP protein is 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%, or a combination thereof of that of a control sample.Without being bound by theory, the total protein concentration in a sample as described herein may be that of a stool sample where the fresh weight for the buffer is set to 1 g / mL. Otherwise, the skilled artisan will know that the sample may be diluted or concentrated to alter the protein concentration.

[0023] In embodiments, the subject may be diagnosed with a GI disease if the total protein concentration in the sample is greater than about 1.8 mg / ml, if the iAP activity is less than about 979 mU / mg, if the level of iAP protein is greater than 10.7% of a control sample, or a combination thereof. In some embodiments, the subject may be diagnosed with a GI disease if the total protein concentration in the sample is greater than about 1.6 mg / ml, if the iAP activity is less than about 1256 mU / mg, if the level of iAP protein is greater than 4.8% of a control sample, or a combination thereof.

[0024] The present invention provides a method for 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 may include an intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to a profile obtained from a control sample, and treating the subject. In embodiments, the control sample may include two or more control samples.

[0025] 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 may include an intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to a profile obtained from a control sample, and treating the subject. In embodiments, the control sample may include two or more control samples.

[0026] The present invention further provides a method of improving 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, wherein the GI disease biomarker may include an intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to a profile obtained from a control sample, and treating the subject. In embodiments, the control sample may include two or more control samples.

[0027] In an embodiment, treating a subject diagnosed with a GI disease comprises administering an effective amount of an antibiotic, a probiotic, an intravenous fluid, or a combination thereof; stopping oral feeding; administering an iAP replacement composition; an anti-inflammatory drug; a therapist; a catalytically active small molecule activator and / or effector; parenteral (or intravenous) nutrition, or a combination thereof.

[0028] Non-limiting examples of therapeutic agents that can be used according to the present invention include Toll-like receptor (TLR) inhibitors (Neal et al. Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors. PloS One 12, e65779) and disruption 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).

[0029] Non-limiting examples of small molecule effectors with catalytic activity include 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. Novel inhibitors of alkaline phosphatases press vascular smooth muscle cell calcification. Journal of Bone and Mineral Research 22, 1700-1710; al-Rashida and Iqbal. Inhibition of alkalinephosphatase: an emerging new drug target. Minireviews in Medicinal Chemistry15, 41-51.

[0030] The non-limiting examples of such antibiotics include vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), gentamicin, Flagyl (generic metronidazole), meropenem, metronidazole, cefotaxime, clindamycin or any combination thereof. In some embodiments, an antifungal agent may be further administered. In other embodiments, the antifungal agent may be fluconazole, terconazole, voriconazole, posaconazole, pentamidine, itraconazole and ketoconazole.

[0031] Non-limiting examples of probiotic organisms include those of the genera Lactobacillus, Lactococcus, Bifidobacterium, Pediococcus, Saccharomyces boulardii, and related bacteria and yeasts.

[0032] Non-limiting examples of such intravenous fluids include saline (e.g., 0.9% NaCl in water or 0.45% saline in water), lactated Ringer's solution (0.9% NaCl with electrolytes and buffer), D 5 W (5% glucose aqueous solution), D 5 NS (5% glucose in 0.9% saline), D 5 1 / 2NS (5% glucose in 0.45% saline), D 5 LR (5% dextrose in lactated Ringer's) or Normosol-R. In embodiments, the intravenous fluid solution may be isotonic. In other embodiments, the intravenous fluid solution may be hypotonic.

[0033] Non-limiting examples of parenteral (or intravenous) nutrition include intravenous dextrose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or combinations thereof.

[0034] Anti-inflammatory agents can be selected from a variety of steroidal, non-steroidal and salicylate water-soluble and water-insoluble drugs and acid addition salts or metal salts thereof. Organic and inorganic salts can be used, provided that the anti-inflammatory agent maintains its medicinal value. Anti-inflammatory agents can be selected from a wide range of therapeutic agents and mixtures of therapeutic agents, which can be administered in the form of sustained release or extended action. Non-limiting examples of anti-inflammatory agents include ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenac sodium, fenoproben calcium, ketoprofen, mefenamic acid, naproxen, ketorolac tromethamine, diclofenac and evening primrose oil (containing about 72% linoleic acid and about 9% gamma linoleic acid). Non-limiting examples of salicylate anti-inflammatory agents include acetylsalicylic acid, mesalazine, disalicylate, diflunisal, salicylsalicylic acid and choline magnesium trisalicylate. Non-limiting examples of steroidal anti-inflammatory agents include flunisolide, triamcinolone, triamcinolone acetonide, beclomethasone dipropionate, betamethasone dipropionate, hydrocortisone, cortisone, dexamethasone, prednisone, methylprednisolone, and prednisolone.

[0035] In embodiments, gastrointestinal diseases may include colitis, inflammatory bowel disease (IBD), or a combination thereof. In embodiments, colitis may include necrotizing enterocolitis (NEC), adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.

[0036] In embodiments, the sample may include a biological sample obtained from a subject. For example, the biological sample may be a biological fluid, a biological solid, or a biological semisolid. In embodiments, the sample may include feces, meconium, vomitus, peripheral blood, serum, plasma, or urine.

[0037] In embodiments, GI disease biomarkers may include iAP enzyme activity, AP enzyme activity, iAP protein level, iAP dimerization / dissociation, post-translationally modified iAP, total protein, such as total fecal protein, or a combination thereof. In embodiments, post-translational modifications may include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, fatty acylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopantetheinylation, prenylation, propionylation, ribosylation, succinylation, sulfation, SUMOylation, or ubiquitination.

[0038] In embodiments, GI disease biomarkers may include NEC biomarkers.

[0039] In embodiments, detection may include immunoassays, colorimetric assays, fluorescent assays, or a combination of both. In embodiments, immunoassays may include Western blot assays, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, single molecule immunoassays in femtoliter chamber arrays, digital enzyme assays in single and multiple formats, or combinations thereof. In embodiments, detection includes contacting the sample with an anti-iAP antibody. In embodiments, the anti-iAP antibody is a polyclonal or monoclonal antibody. In embodiments, detection may include kinetic assays, endpoint assays, Bradford assays, bicinchoninic acid (BCA) assays, Lowry assays, pyrogallol red protein dye binding assays, Coomassie blue dye binding assays, or combinations thereof.

[0040] In other embodiments, detection may include techniques known to those skilled in the art, such as mass spectrometry (MS), RNA sequencing, and immunostaining of patient samples. For example, RNA sequencing of intestinal alkaline phosphatase or other alkaline phosphatases is a rapid method for detecting molecules such as iAP (Knight et al. Non-invasive analysis of intestinal development in preterm and terminfants using RNA-sequencing. 2014. Scientific Reports 4, 5453).

[0041] In embodiments, the assay can detect phosphatase activity. Non-limiting examples of such assays include fluorescent, chemiluminescent or colorimetric detection methods, assays that detect ATP hydrolysis and / or ATP hydrolysis products, or combinations thereof.

[0042] In embodiments, detection may include a kinetic assay comprising the use of 4-methylumbelliferyl phosphate, CPD Star (2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[3.3.1.1 3.7 ]decane])-4-yl]-1-phenyl phosphate disodium), AttosPho (2'-[2-benzothiazolyl]-6'-hydroxybenzothiazole phosphate [BBTP]), or any other fluorescent or colorimetric signal, an assay that detects ATP hydrolysis and / or ATP hydrolysis products (e.g., malachite green, NADH-coupled or other proprietary variants), or a combination thereof.

[0043] In embodiments, alkaline phosphatase activity, such as intestinal alkaline phosphatase activity, can be directly detected and / or measured by mixing a chromogenic substrate and / or a fluorogenic substrate for alkaline phosphatase, such as an iAP, with a biological sample for a period of time. For example, 4-methylumbelliferyl phosphate (MUP) is a fluorogenic substrate for alkaline phosphatase, and alkaline phosphatase-mediated hydrolysis of its phosphate substituent produces blue fluorescent 4-methylumbelliferyl (excitation / emission ~386 / 448 nm). In embodiments, MUP can be directly mixed with a biological sample, such as feces, thereby allowing direct detection of the presence of alkaline phosphatase or measurement of its activity.

[0044] Non-limiting examples of alkaline phosphatase (AP) substrates include 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-indolyl phosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / INT substrate, tan precipitate). , Biomeda), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT substrate, blue / purple), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / NBT / INT, brown precipitate, DAKO, Fast Red (red), Magenta Phosphorus (magenta), Naphthol AS-Bi-phosphate (NABP) / Fast 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), VECTOR TM Black (black), VECTOR TM Blue (blue), VECTOR TMRed (red), Vega Red (raspberry red), fluorescein diacetate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl casein, 4-methylumbelliferyl-α-L-arabinopyranoside, 4-methylumbelliferyl-β-D-fucopyranoside, 4-methylumbelliferyl-α-L-fucopyranoside, 4-methylumbelliferyl-β-L-fucopyranoside, 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-galactopyranoside ,4-methylumbelliferyl-β-D-galactopyranoside, 4-methylumbelliferyl-α-D-glucopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl-β-D-glucuronide, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl phosphate, bis(4-methylumbelliferyl)phosphate, 4-methylumbelliferyl pyrophosphate diester, 4-methylumbelliferyl-β-D-xylopyranoside.

[0045] Non-limiting examples of suitable chromogenic substrates for use in the present invention include o-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-galactopyranoside, o-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-glucuronide, p-nitrophenyl phosphate, o-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-D-xylopyranoside, p-nitrophenyl-β-D-xylopyranoside, and phenolphthalein-β-D-glucuronide.

[0046] In embodiments, the methods as described herein further comprise diagnosing the subject as having a gastrointestinal disease if the protein level of iAP in the sample is at least two standard deviations higher than the mean protein level of the control sample. In embodiments, the methods as described herein further comprise diagnosing the subject as having 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 higher than the mean protein level of the control sample. In embodiments, the control sample may comprise two or more control samples. Embodiments may 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 feces / mL sterile water or buffer. For example, an embodiment may comprise 200 mg fresh weight feces / mL sterile water or buffer. In embodiments, the methods as described herein further comprise diagnosing the subject as having a gastrointestinal disease or being at risk of a gastrointestinal disease if the protein level of iAP in the sample is greater than 4.8% of the control sample. In other embodiments, the methods as described herein further comprise diagnosing the subject as having a gastrointestinal disease or being 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 methods as described herein further comprise diagnosing the subject as having a gastrointestinal disease or being 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 sample.

[0047] In embodiments, the methods as described herein further comprise diagnosing the subject as having 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 samples. In embodiments, the methods as described herein further comprise diagnosing the subject as having a gastrointestinal disease if the level of iAP enzyme activity in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations above the mean enzyme activity level of the control samples. In embodiments, the control samples may comprise two or more control samples. In embodiments, the methods as described herein further comprise if the level of iAP enzyme activity in the sample is below 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, In some embodiments, the method as described herein further comprises 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 1500mU / mg, 1000mU / mg, 1100mU / mg, 1200mU / mg, 1300mU / mg, 1400mU / mg, 5U / mg, 10U / mg, 50U / mg, 100U / mg, 300U / mg, 400U / mg, 500U / mg, 600U / mg, 700U / mg, 800U / mg, 900U / mg, 1000U / mg, such as less than 979mU / mg or less than 1256mU / mg. In embodiments, the method as described herein further comprises 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 1500mU / mg, 1000mU / mg, 500mU / mg, such as less than 1256mU / mg.

[0048] In embodiments, as described herein method further comprises if in sample, fecal protein level is higher than the average fecal protein level of control sample by at least two standard deviations, then diagnose experimenter and suffer from gastrointestinal disease.In embodiments, as described herein method further comprises if in sample, fecal protein level is higher than the average fecal protein level of control sample by at least 1,1.5,2,2.5,3,3.5,4,4.5,5 standard deviations, then diagnose experimenter and suffer from gastrointestinal disease.In embodiments, control sample can comprise two or more control samples.In embodiments, as described herein method further comprises if in sample, fecal protein level is higher than the average fecal protein level of control sample by at least 1,1.5,2,2.5,3,3.5,4,4.5,5 standard deviations, then diagnose experimenter and suffer from gastrointestinal disease.In embodiments, control sample can comprise two or more control samples.In embodiments, as described herein method further comprises if in sample, fecal protein level is higher than 1.6mg / ml, or for example, is higher than 1.8mg / ml, then diagnose experimenter and suffer from gastrointestinal disease or be in gastrointestinal disease risk. In embodiments, the methods described herein further comprise the step of: 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. , 3.0mg / ml, 3.1mg / ml, 3.2mg / ml, 3.3mg / ml, 3.4mg / ml, 3.5mg / ml, 3.6mg / ml, 3.7mg / ml, 3.8mg / ml, 3.9mg / ml, 4.0mg / ml, 4.1mg / ml, 4.2mg / ml, 4.3mg / ml, 4.4mg / ml, 4.5mg / ml, 4.6mg / ml, 4.7mg / ml, 4.8mg / ml, 4.9mg / ml, 5.0mg / ml, then the subject is diagnosed with a gastrointestinal disease or a risk of a gastrointestinal disease.

[0049] In embodiments, the methods as described herein further comprise treating the subject. In embodiments, treatment may comprise administering to a subject diagnosed with a gastrointestinal disorder an effective amount of an antibiotic, a probiotic, an intravenous fluid, a step of stopping oral feeding, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof.

[0050] In embodiments, the subject may include a mammal. In embodiments, the mammal may include a dog, a cat, a horse, a cow, a pig, or a human. In some embodiments, the biomarkers of the invention may be used to diagnose colic in horses. In embodiments, the human may include an infant. In embodiments, the infant may include a premature infant.

[0051] The present invention further provides a method for screening a subject (e.g., a subject at risk for gastrointestinal disease or a subject with asymptomatic gastrointestinal disease) for the presence of a feature, comprising obtaining a sample from the subject, measuring at least one GI disease biomarker in the sample, wherein the GI disease biomarker may comprise an intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to a profile obtained from a control or reference sample, and treating the subject. In embodiments, the control or reference sample may comprise two or more control samples. In embodiments, the sample is a stool sample.

[0052] The present invention further provides a method for identifying a subject at risk for a gastrointestinal disease or a subject with an asymptomatic gastrointestinal disease, comprising obtaining a sample from the subject, measuring at least one GI disease biomarker in the sample, wherein the GI disease biomarker may comprise an intestinal alkaline phosphatase (iAP) protein, comparing the GI disease biomarker profile to a profile obtained from a control sample, and treating the subject. In embodiments, the control sample may comprise two or more control samples.

[0053] In embodiments, gastrointestinal diseases may include colitis, inflammatory bowel disease (IBD), or a combination thereof. In embodiments, colitis may include necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.

[0054] In embodiments, the sample may include a biological sample obtained from a subject. For example, the biological sample may be a biological fluid, a biological solid, or a biological semisolid. In embodiments, the sample may include feces, meconium, vomitus, peripheral blood, serum, plasma, or urine.

[0055] In embodiments, GI disease biomarkers may also include iAP enzyme activity, total fecal protein, iAP dimerization / dissociation, post-translationally modified iAP, or a combination thereof. In embodiments, post-translational modifications may include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, fatty acylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopantetheinylation, prenylation, propionylation, ribosylation, succinylation, sulfation, SUMOylation, or ubiquitination.

[0056] In embodiments, measurement may include performing an assay to determine the total protein concentration, intestinal alkaline phosphatase activity, intestinal alkaline phosphatase protein concentration, or a combination thereof in a sample. In embodiments, measurement may include Bradford assay, bicinchoninic acid (BCA) assay, Lowry assay, pyrogallol red protein dye binding assay, Coomassie blue dye binding assay, or a combination thereof. In embodiments, measurement may include kinetic assays. In embodiments, kinetic assays may include using 4-methylumbelliferyl phosphate, nitrophenyl phosphate, or any other fluorescent or colorimetric signal, an assay to detect ATP hydrolysis, or a combination thereof. In embodiments, measurement may include immunoassays, colorimetric assays, fluorometric assays, or a combination thereof.

[0057] In embodiments, the immunoassay may comprise a Western blot assay, an enzyme-linked immunosorbent assay, an immunoprecipitation, or a combination thereof. In embodiments, the assay may comprise an anti-iAP antibody. In embodiments, the anti-iAP antibody is a monoclonal or polyclonal antibody.

[0058] In embodiments, the methods disclosed herein may further include diagnosing the subject with a gastrointestinal disease when the total protein concentration in the sample is at least two standard deviations higher than the mean value of the control sample, the concentration of intestinal alkaline phosphatase protein is at least two standard deviations higher than the mean value of the control sample, the mean value of the intestinal alkaline phosphatase activity control sample is at least two standard deviations lower, or a combination thereof. In embodiments, the methods disclosed herein may further include diagnosing the subject with a gastrointestinal disease when the total protein concentration in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations higher than the mean value 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 higher than the mean value of the control sample, the mean value of the intestinal alkaline phosphatase activity control sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations lower, or a combination thereof. In embodiments, the control sample may include two or more control samples.

[0059] In embodiments, the methods disclosed herein may further comprise treating the subject. In embodiments, treatment may comprise administering to a subject diagnosed with a gastrointestinal disorder an effective amount of an antibiotic, a probiotic, an intravenous fluid, cessation of oral feeding, an iAP replacement composition, an anti-inflammatory agent, a potential therapeutic agent, parenteral (or intravenous) nutrition, or a combination thereof.

[0060] In embodiments, if the protein concentration in the stool 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 / 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 , 3.2mg / ml, 3.3mg / ml, 3.4mg / ml, 3.5mg / ml, 3.6mg / ml, 3.7mg / ml, 3.8mg / ml, 3.9mg / ml, 4.0mg / ml, 4.1mg / ml, 4.2mg / ml, 4.3mg / ml, 4.4mg / ml, 4.5mg / ml, 4.6mg / ml, 4.7mg / ml, 4.8mg / ml, 4.9mg / ml, 5.0mg / ml, for example 1.6mg / ml or for example 1.8mg / ml, the subject can be diagnosed with or at risk of suffering from a gastrointestinal disease. In embodiments, if the iAP activity is less 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, U / mg, 1200mU / mg, 1300mU / mg, 1400mU / mg, 5U / mg, 10U / mg, 50U / mg, 100U / mg, 200U / mg, 300U / mg, 400U / mg, 500U / mg, 600U / mg, 700U / mg, 800U / mg, 900U / mg, 1000U / mg, for example 972mU / mg, or for example 1256mU / mg, the subject may be diagnosed with a gastrointestinal disease or at risk of a gastrointestinal disease. In embodiments, if the iAP protein detected by densitometry anti-iAP antibodies exceeds 4.8% of the control, the subject may be diagnosed with a gastrointestinal disease or at risk of a gastrointestinal disease. In embodiments, if the level of iAP protein is at least two standard deviations higher than the mean of the control samples, the subject may be diagnosed with a gastrointestinal disease. In embodiments, the control sample may include two or more control samples. In embodiments, the subject may include a mammal. In embodiments, the mammal may include a dog, a cat, a horse, a cow, or a human.In embodiments, the human may include an infant. In embodiments, the infant may include a premature infant.

[0061] The present invention also provides a disposable article comprising a biosensor, wherein the biosensor may comprise at least one biorecognition element, and wherein the biosensor detects iAPs in a sample.

[0062] In embodiments, the biosensor further detects iAP enzyme activity, total fecal protein, iAP dimerization / dissociation, post-translationally modified iAP, or a combination thereof. In embodiments, the post-translational modification may include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, fatty acylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopantetheinylation, prenylation, propionylation, ribosylation, succinylation, sulfation, SUMOylation, or ubiquitination.

[0063] In embodiments, the sample may include a biological sample obtained from a subject. For example, the biological sample may be a biological fluid, a biological solid, or a biological semisolid. In embodiments, the sample may include feces, meconium, vomitus, peripheral blood, serum, plasma, or urine.

[0064] In embodiments, the biosensor is an immunosensor. In embodiments, the biosensor may include a detection signal. In embodiments, the detection signal may include a colorimetric signal, a fluorescent signal, or both. In embodiments, the biorecognition element may include an anti-iAP antibody. In embodiments, the anti-iAP antibody may include a polyclonal or monoclonal antibody.

[0065] In an exemplary embodiment, the biosensor may include a lateral flow immunoassay, also referred to as an immunochromatographic assay or strip test. Lateral flow immunoassays include immunoassays adapted to operate along a single axis to accommodate test strip formats. Typical lateral flow test strips include a sample pad (an adsorbent pad to which the test sample is applied), a conjugate or reagent pad (this includes a binding agent for the target analyte conjugate, such as an antibody, which is combined with colored particles, such as colloidal gold nanoparticles or latex microspheres), a reaction membrane (usually a nitrocellulose membrane or a cellulose acetate membrane, on which an anti-target analyte binding agent such as an antibody is fixed in a line passing through the membrane to serve as a capture zone or test line. There will also be a control zone, which includes an antibody specific to the conjugate antibody) and a core or waste reservoir (another absorbent pad, which is intended to draw the sample through the reaction membrane and collect it by capillary action). The components of the strip are usually fixed on an inert backing material and can be provided in a simple dipstick form or in a plastic housing, with a sample port and a reaction window showing the capture zone and the control zone.

[0066] In embodiments, the article may include a diaper to be worn by a subject, a wipe for cleaning a subject, a dipstick, a spoon, a spatula, filter paper, or a swab.

[0067] In embodiments, the subject may include a mammal. In embodiments, the mammal may include a dog, a cat, a horse, a cow, or a human. In embodiments, the human may include an infant. In embodiments, the infant may include a premature infant.

[0068] The present invention further provides a kit for diagnosing a subject with a gastrointestinal disease. In embodiments, the kit may include disposable items as described herein. In embodiments, the gastrointestinal disease may include colitis, inflammatory bowel disease (IBD) or a combination thereof. In embodiments, colitis may include necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.

[0069] In an embodiment, the kit may comprise an iAP biorecognition element immobilized on a solid support and instructions for use thereof.

[0070] In embodiments, gastrointestinal diseases may include colitis, inflammatory bowel disease, or a combination thereof. In embodiments, colitis may include necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, radiation colitis.

[0071] In embodiments, the biorecognition element may comprise an antibody against an iAP or an oligonucleotide against an iAP, for example, directly or indirectly immobilized to a solid support. Embodiments may also comprise a fluorescent substrate or inhibitor with high binding affinity for the iAP attached to the solid support.

[0072] In embodiments, the solid support may comprise plastic, cardboard, or glass. In embodiments, the solid support may comprise a dipstick.

[0073] In embodiments, the subject may include a mammal. In embodiments, the mammal may include a dog, a cat, a horse, a cow, or a human. In embodiments, the human may include an infant. In embodiments, the infant may include a premature infant.

[0074] The present invention also provides a molecular biomarker diagnostic kit for identifying subjects who exhibit or have a tendency to develop gastrointestinal diseases. In an embodiment, the kit may include at least one of a tool for determining total fecal protein concentration, a tool for determining intestinal alkaline phosphatase (iAP) activity, and an iAP biorecognition element, which together represent a tendency to indicate the presence or development of gastrointestinal diseases in human subjects. In an embodiment, gastrointestinal diseases may include colitis, inflammatory bowel disease (IBD), or a combination thereof. In an embodiment, colitis may include necrotizing enterocolitis, adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.

[0075] In embodiments, the characteristic may include a total protein concentration that is at least two standard deviations above the mean of the control samples, an intestinal alkaline phosphatase protein concentration that is at least two standard deviations above the mean of the control samples, or an intestinal alkaline phosphatase activity that is at least two standard deviations below the mean of the control samples. In embodiments, the control samples may include two or more control samples.

[0076] In embodiments, the feature can be selected from at least two of the group consisting of a total protein concentration at least two standard deviations above the mean of the control samples, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of the control samples, and an intestinal alkaline phosphatase activity at least two standard deviations below the mean of the control samples. In embodiments, the control sample can include two or more control samples.

[0077] In embodiments, the subject may include a mammal. In embodiments, the mammal may include a dog, a cat, a horse, a cow, or a human. In embodiments, the human may include an infant. In embodiments, the infant may include a premature infant.

[0078] Aspects of the invention also relate to treating a subject with NEC by changing the feeding regimen. In one embodiment, the invention provides a method of treating a subject with NEC. In some embodiments, the method comprises measuring the amount or activity of an agent that binds to iAP in a sample obtained from a subject according to the methods described herein, wherein the agent that binds to iAP is at least one GI disease biomarker comprising iAP enzyme activity, AP enzyme activity, iAP protein level, AP protein level, iAP dimerization / dissociation, post-translationally modified iAP, total fecal protein, or a combination thereof; determining the subject's postnatal developmental age; and stopping enteral feeding for a period of time sufficient to resolve the gastrointestinal inflammatory process or signs of feeding intolerance. In some embodiments, the method further comprises administering an antibiotic or antifungal agent, alone or in combination. In some embodiments, the method further comprises administering probiotics, other biologics (e.g., stem cells or transcription factors) or therapeutic agents (e.g., TLR4 small molecules, alkaline phosphatase inhibitors or activators), antibiotics, intravenous fluids, iAP replacement compositions (e.g., provided exogenously), small molecule activators and / or catalytically active effectors, anti-inflammatory agents, parenteral (or intravenous) nutrition, alone or in combination. In some embodiments, the postnatal developmental age of a subject may be 'postmenstrual age' or 'postmenstrual developmental age'.

[0079] For example, oral feeding can be suspended for 1 day, 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.

[0080] Non-limiting examples of biological products include stem cells and transcription factors. The intestinal epithelium is in a constant dynamic flow state and is replaced every 3-6 days. This continuous renewal is necessary to maintain normal intestinal structure and function. In addition, non-limiting examples of transcription factors include those that can be expressed and used for intestinal cell differentiation, such as the Kruppel-like factor (GKLF or KLF4) family.

[0081] In embodiments, the feeding regimen may include an intermittent feeding regimen. For example, during the extended period of suspending oral feeding, there may be one or more days of feeding. For example, feeding may be suspended 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 there may be one or more days of oral feeding. For example, a doctor may suspect NEC and suspend feeding for a short period of time, such as one or two days, until laboratory tests / examinations show that the baby does not have NEC, at which time they will resume feeding again. After a few hours or days, NEC panic may occur again, and the baby may be prohibited from feeding again for a period of time. Intermittent feeding may occur once or more during the subject's hospitalization. This intermittent feeding can allow a clinician to determine a subject's tolerance to feeding. In other embodiments, intermittent feeding can be recommended for subjects at risk of gastrointestinal disease. Other objects and advantages of the present invention will become apparent from the subsequent description.

[0082] This article includes the following implementation plans:

[0083] 1. A method for determining the prognosis of necrotizing enterocolitis (NEC) in a patient, the method comprising:

[0084] a. fitting a Markov model using a two-state transition matrix and a propensity value measured between a plurality of subjects, wherein the two-state transition matrix comprises a first state and a second state, wherein the first state comprises a non-necrotizing enterocolitis state, wherein the second state comprises a necrotizing enterocolitis state (NEC), wherein the propensity value is a function of an intestinal alkaline phosphatase (iAP) activity value and an amount of iAP found in a subject in the plurality of subjects;

[0085] b. estimating a probability of transitioning from a first state to a second state using the patient's propensity value and a fitted Markov model, wherein the fitted model indicates that an increase in the patient's propensity level significantly increases the probability of transitioning from the first state to the second state;

[0086] c. treating the patient when the propensity value is greater than or equal to a threshold value of about 0.5.

[0087] 2. A method according to embodiment 1, wherein the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus a first ratio, wherein the first ratio comprises the iAP activity value of a subject from the plurality of subjects divided by the maximum iAP activity value observed in the plurality of subjects, and wherein the second value comprises a second ratio, wherein the second ratio comprises the iAP amount from the immunoassay value of the subject from the plurality of subjects divided by the maximum iAP amount from the immunoassay value observed in the sample.

[0088] 3. A method according to embodiment 1, wherein the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus the iAP activity value of a subject in the plurality of subjects, and wherein the second value comprises the iAP amount from an immunoassay value of a subject in the plurality of subjects.

[0089] 4. A method according to embodiment 1, wherein the multiple subjects include the patient.

[0090] 5. The method according to embodiment 1, wherein the treatment comprises stopping eating, administering antibiotics, or a combination thereof.

[0091] 6. A method according to embodiment 2, wherein the sample is human small intestine lysate.

[0092] 7. The method of embodiment 2 or 3, wherein the immunoassay comprises protein blot, ELISA or immunoprecipitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0094] Figure 1Longitudinal measurements of total fecal protein, iAP enzyme activity, and immunoblot detection of iAP protein in two preterm infants are shown. (Panel A) Patient 1 was born at 30 weeks of gestation and developed NEC on day 7 of life, was treated medically, and subsequently developed recurrent NEC and intestinal perforation on day 31 of life. The infant recovered after placement of an abdominal drain and an additional 10 days of bowel rest and antibiotics. Red symbols and bars represent NEC events. 7A and 7B refer to 2 separate stool samples collected on day 7 of life, one before and one after the diagnosis of NEC. (Panel B) Patient 2 was born at 25 weeks of gestation and developed abdominal distention on DOL 19, with suspected NEC (green symbols and bars). The infant responded rapidly to medical management and soon resumed enteral feedings. The patient developed definite NEC on DOL 32 (red symbols and bars), requiring assisted ventilation and aggressive medical management, but fully recovered on DOL 48. The table below each figure indicates which stool tests met criteria for NEC (+) and which did not (-). These criteria were defined by values ​​outside the 95% confidence interval of the control value. The risk of NEC was considered increased if: protein concentration exceeded 1.8 mg / ml; iAP activity was less than 979 mU / mg; or iAP protein exceeded 10.7% of the control by Western blot. Abbreviations: ip, intestinal perforation; ad, abdominal distension; MW, molecular weight ladder; and kDa, kilodaltons.

[0095] Figure 2 Increased total fecal protein concentration, decreased fecal iAP enzyme activity, and increased iAP detection on Western blots are shown at the time of NEC diagnosis. In the following panels, red circles represent individual fecal samples collected during 7 different NEC events in 6 patients; black circles represent the composite mean ± standard error of 2-17 fecal samples from 12 control patients. The red and gray columns represent the mean ± standard error of all samples collected during the NEC event and all samples from control subjects, respectively. (Panel A) Protein concentrations were higher in patient fecal samples at the time of NEC diagnosis compared to control samples (p value = 0.005). (Panel B) iAP activity was lower in patient fecal samples at the time of NEC diagnosis than in control samples (p value < 0.0001). (Panel C) The amount of iAP protein in the feces, quantified by comparison with the positive control standard, was higher in patients at the time of NEC diagnosis compared to control samples (p value = 0.002). Statistical test: Mann-Whitney.

[0096] Figure 3Fecal iAP-related measurements are shown to have high specificity and sensitivity. (Panel A) 3D scatter plot of our candidate biomarker measurements, where red diamonds represent NEC samples and black circles represent controls. (Panel B) 2D projection of the 3D scatter plot, which is specifically related to iAP activity and protein blot intensity measurements. (Panel C) Sensitivity and specificity curves for each candidate biomarker, as well as the sensitivity and specificity curves of the combined naive Bayes classifier that considers all 3 features simultaneously. The analysis covered 49 samples, including 13 NEC samples, 9 control samples derived from NEC patients, and 27 control samples derived from control patients. The Spearman correlation coefficient for the comparison of protein blot intensity and total protein content was 0.19, the Spearman correlation coefficient for total protein content and iAP activity was -0.48, and the Spearman correlation coefficient for iAP activity and protein blot intensity was -0.58.

[0097] Figure 4 Demographic information of the study subjects is shown. Means and standard deviations for gestational age and birth weight are shown. The distribution of gestational age, sex, and Bell stage is also reported.

[0098] Figure 5 Alkaline phosphatase activity was shown to be primarily due to intestinal alkaline phosphatase (iAP). L-Phe is a specific inhibitor of intestinal alkaline phosphatase activity, which inhibited activity. There was no observable difference in the degree of inhibition between NEC samples and control samples. NEC samples showed 90% inhibition with a standard deviation of + / -10. Control samples showed 91% inhibition with a standard deviation of + / -9. This represents 12 samples from 6 NEC patients and 64 samples from control patients (n=18).

[0099] Figure 6 The demographics of the patients enrolled in the study are shown.

[0100] Figure 7 Figure 3 shows lower iAP activity in stool samples from patients at the time of NEC diagnosis compared to control samples. Red circles represent iAP activity during 7 different NEC events in 6 patients (post-gestational weeks: 29-43 weeks). Each black circle represents the composite mean + / - SEM of 2-8 stool samples from each of 12 controls (post-gestational weeks 29-45 weeks). Statistical significance analysis: Mann-Whitney test, P < 0.0001.

[0101] Figure 8Shows higher total fecal protein in stool samples from patients at the time of NEC diagnosis compared to control samples. Red circles represent total fecal protein levels during 7 different NEC events in 6 patients (post-gestational weeks: 29-43 weeks). Each black circle represents the composite mean = / -SEM of 2-16 stool samples from 12 controls (post-gestational weeks: 29-45 weeks). Statistical analysis: Mann-Whitney test, P = 0.005

[0102] Fig. 9 The intensity of the iAP protein signal, quantified as a percentage of the positive control signal, is shown to be much higher in stool samples from patients at the time of NEC diagnosis compared to control samples. The red circles represent the intensity of the iAP protein signal during 7 different NEC events in 6 patients (post-gestational weeks: 29-43). Each black circle represents at least 1 stool sample from 7 controls (post-gestational weeks 29-35). Statistical analysis: Mann-Whitney test, P=0.002

[0103] Fig.10Showing decreased fecal iAP enzyme activity, increased fecal total protein, and increased iAP detection on WB at the time of NEC diagnosis. (Panel A) Illustrate longitudinal measurements of iAP activity, total fecal protein, and iAP enzyme activity in patients with NEC who subsequently developed perforation. Red dots and bars represent NEC events. Fecal iAP activity dropped dramatically corresponding to the time of diagnosis, suggesting that fecal iAP activity could serve as a diagnostic tool for NEC. Strong iAP protein detection was seen on western blot. The patient completed 14 days of treatment but developed intestinal perforation on day 31 of life (ip). After an additional 10 days of intestinal rest, anti-iAP antibodies no longer strongly detected iAP, and iAP activity trended upward. (Panel B) Decreased fecal iAP activity and increased iAP amounts were similarly observed at the time of NEC diagnosis in the second patient. Green dots represent episodes of bloody stools, corresponding to suspected NEC with intestinal distention (bd). iAP detection occurred during NEC surveillance, which did not become apparent until the diagnosis of NEC on day 32 of life. Again, note the drop in activity around NEC diagnosis (red dots and bars) and the trend toward higher activity and decreased iAP detection on WB after recovery. (Panel C) NEC is associated with low fecal iAP activity, high fecal protein, and high fecal iAP amounts (on WB). Three samples from 3 NEC patients at the time of NEC diagnosis (labeled N, red bars) were matched with 3 samples from 3 control subjects of similar gestational age and chronological age (labeled C, white bars). The differences in Group 3 were less significant and may represent subclinical disease in premature infants with feeding intolerance. (Panel D) The figure represents sequential stool sampling before diagnosis (labeled pre, white bars) and at diagnosis (labeled d, red bars) in 4 NEC patients with corresponding iAP enzyme activity, total fecal protein, and iAP detection on western blot.

[0104] Fig.11 It is shown that combining all 3 biomarkers has possible clinical utility and improvement in sensitivity and specificity. The figure represents data points from 6 NEC patients and 7 controls, including a total of 51 samples. (Panel A) shows a 3D scatter plot comparing WB data, fecal iAP activity, and fecal protein. The red diamonds represent the average total fecal protein, iAP activity, and WB percentage of fecal samples from NEC patients at diagnosis. The black circles represent the average total fecal protein, iAP activity, and WB percentage of the control points (including NEC patients in the disease-free period). (Panel B) represents a 2D scatter plot showing the relationship between fecal iAP activity and WB percentage. There is a clustering of control samples (black circles) in lower WB percentages and high activity trends. NEC samples demonstrate the opposite, with higher WB percentages and low activity. (Panel C) Schematic diagram of a naive Bayes classifier used to demonstrate the sensitivity and specificity of all three biomarkers alone and in combination to improve performance.

[0105] Fig.12 It is shown that there is a discernible trend towards lower fecal protein (panel A) and higher fecal iAP enzyme activity (panel B) in those infants who tolerate feeds well and progress quickly to full feeds without problems. The graph represents the mean total fecal protein and fecal iAP activity in feces from 10 control patients during the first month of life in relation to the duration before full enteral feeds were achieved.

[0106] Fig.13 showed that stool samples can be heterogeneous. There were two identifiable compartments of consistency in stool samples from patients with NEC. Separating the stool compartments and performing Western blot analysis on each compartment separately gave different results.

[0107] Fig.14 Relative iAP content (Panel A), iAP activity (Panel B), and protein concentration (Panel C) are shown. NEC was classified according to the criteria of Bell et al., as modified by Walsh and Kliegman. For this analysis, the term ‘suspected NEC’ is stage I, and ‘proven NEC’ is stage II and more severe; the term ‘perforated NEC’ is used to describe stage IIIB only. Information from chart review was used to diagnose stage I. Radiologic confirmation of stage II requires documentation of bowel inflation.

[0108] Fig.15 Immunohistochemical staining of intestinal tissue is shown.

[0109] Fig.16 A schematic diagram showing the many functions of the iAP.

[0110] Fig.17 showed that fecal total protein levels were higher in NEC patients than in control infants.

[0111] Fig.18 Fecal AP catalytic activity was consistently lower in the NEC population.

[0112] Fig.19 showed that in matched patient samples, AP enzyme activity was always low at the time of NEC diagnosis.

[0113] Fig. 20 showed that high levels of iAP proteins were detected that were associated with NEC.

[0114] Fig.21 Fecal iAP protein levels were shown to be increased during NEC episodes.

[0115] Fig. 22 Increased iAP protein levels and decreased iAP enzyme activity in NEC episodes were shown.

[0116] Fig.23 A schematic diagram for testing non-specific binding of secondary antibodies is shown. Without being bound by theory, enzyme assays can be performed only with secondary antibodies conjugated to AP.

[0117] Fig.24 Clinical data separating patients with NEC diagnosis matching X-ray and NEC suspicion (defined by neonatologists) from controls are shown, demonstrating that biomarkers can molecularly define NEC earlier.

[0118] Fig.25 is a bar graph showing that measurements of alkaline phosphatase can be confounded by the signal from the secondary antibody. Isolated alkaline phosphatase can catalyze the hydrolysis of MUP to form the fluorescent product MU. Secondary antibodies conjugated to AP from two different commercial manufacturers can also hydrolyze MUP to form a fluorescent product. When the alkaline phosphatase protein and the secondary antibody are in the same measurement, an increased level of catalytic activity is observed. This can be monitored by standard spectrophotometric readouts of biochemical activity and by western blotting.

[0119] Fig.26 The risks for non-NEC to NEC conversions and NEC to non-NEC conversions are shown.

[0120] Fig. 27 A schematic diagram of the conversion model is shown.

[0121] Fig.28 Bivariate analysis of the abundance and catalytic capacity of intestinal alkaline phosphatases found in feces of preterm infants is shown. Biological samples sampled at the time of illness are shown as colored circles; suspected necrotizing enterocolitis is pink, severe necrotizing enterocolitis is red, and late-onset sepsis is blue. Complementary control groups are shown as open gray circles.

[0122] Fig.29 Analysis of intestinal alkaline phosphatases found in feces of premature infants relative to radiographic clinical evidence of diagnosis is shown. The abundance of iAPs in infant fecal samples relative to human small intestinal lysate is shown in the left panel. Biological samples sampled at the time of clinical diagnosis of disease (vertical blue bars) are shown in colored circles; pre- and post-illness samples collected from infants with NEC are shown in hollow red diamonds; samples collected from non-NEC patients are shown in gray. The normalized iAP abundance and normalized iAP catalytic activity of iAPs were multiplied to produce propensity scores (NECPredict), which are shown on the right panel. The box plots show the median NECPredict at clinical diagnosis (red box on day zero on the x-axis), as well as the value two, four, and six days before radiographic diagnosis of disease. Box plots of NECPredict values ​​obtained from non-NEC infant samples are also shown. The Whisker method is adjacent data points, and the quartile method is Tukey.

[0123] Fig.30 Shown (panel A) is the relationship between NEC, sepsis, and intestinal defense mechanisms. The microbiota creates a unique ecosystem in the intestinal lumen and mucosa. A diverse array of commensal and pathogenic mucosal microbiota modulates intestinal immune function in the infant host. Activation of the innate and adaptive immune systems by the microbiota in turn modulates systemic immune responses. In sepsis, immune responses in distant organs are triggered due to extreme signaling. Host proteins iAP (green), TLR4, and IL-8 (blue) are proteins involved in early and late microbiota responses to inflammation. (panel B) Standard of care for preterm infants requires new approaches to monitor NEC disease. The gold standard for diagnosis is x-ray (blue box), which identifies only 44% of late NEC cases; other diagnostic methods (light blue box) are bedside observations but are not molecularly defined. Current management options in NEC (white box) are contrasted with potential clinical outcomes (red box) using proposed biomarkers (green).

[0124] Fig.31 The amount of iAP protein measured in fecal samples and its catalytic activity are shown. (Panel A) Immunoblot analysis shows that the relative iAP content detected in serial dilutions of human small intestine lysate has a linear relationship if the calibrator is ≤1 μg. Immunoblot bands are white on a black background. A logarithmic plot of the signal and the calibrator is superimposed; the mean and SEM of 5 replicates are shown. (Panel B) The mean ± SEM of iAP content of samples without disease (white), diagnosed with NEC (red), suspected NEC (pink), and diagnosed with sepsis (blue) are shown. The quantification of iAP in each sample was determined using human intestine as the maximum positive control within the linear range (100% iAP content) and calf iAP as a negative control (0% iAP content). Asterisks indicate significant differences between the median of disease status and control using the Mann-Whitney U test with p value <0.001. Immunoblotting efficacy was assessed by sensitivity / specificity calculations for NEC diagnosis (panel C) and sepsis (panel D) using a simple threshold-based classifier. (panel E) There was a direct correlation between fecal iAP activity and postconception age (PCA). Shown are the means and standard errors of the PCA bins. N=14-33 samples per control PCA bin. (panel F) Mean ± SEM of enzyme activity for samples with no disease (white), diagnosed with NEC (red), suspected NEC (pink), and diagnosed with sepsis (blue) are shown. Asterisks indicate p-values ​​<0.05. The sensitivity / specificity of the iAP activity biomarker was assessed for samples at the time of NEC diagnosis (panel G) and sepsis (panel H).

[0125] Fig.32Stimulus protocols that take sample size reasonableness into account are shown.

[0126] Fig.33 Results of power analyses for different effect sizes are shown.

[0127] Fig.34 iAP peptides useful as mass spectrometry quantification calibrants are shown. (Panel A) Amino acid sequence alignment of human alkaline phosphatases: intestinal alkaline phosphatase (iAP; P09923), placental alkaline phosphatase (PLAP; P05187), tissue nonspecific alkaline phosphatase (TNAP; P05186), and germ cell alkaline phosphatase (GCAP; P10696). Boxes P1-P6 have six peptides with unique mass spectrometric signatures that distinguish the four human AP proteins. (Panel B) Allele frequencies of missense, single nucleotide polymorphism catalogs in the human population. (Panel C) Sequences of six iAP peptides suitable for mass spectrometry quantification of protein abundance, and single nucleotide missense polymorphisms determined for each residue position. Peptides with the smallest deviation from zero on the x-axis and the lowest polymorphism frequency on the y-axis are the best candidates for MS reference standards.

[0128] Fig.35 Shown are associations of necrotizing enterocolitis (NEC) and late-onset sepsis with intestinal defense mechanisms. Panels A–C, Physiological and structural changes in the intestine associated with NEC, superimposed in cross-sectional views of the small intestine. Research efforts to develop biomarkers for NEC have focused on proteins involved in immune cascades and microbiota dysbiosis. Our approach focuses on host proteins involved in microbiota management. Panel D, Prospective recruitment of preterm infants with NEC and other confirmed infections. Panel E, The workflow for stool sample preparation was optimized for assay reproducibility and standardization. GI denotes gastrointestinal; IAP, intestinal alkaline phosphatase.

[0129] Fig.36 Clinical characteristics of patients with severe NEC, suspected NEC, or no NEC are shown. Abbreviations: IQR, interquartile range; NA, not applicable; NEC, necrotizing enterocolitis; NICU, neonatal intensive care unit; NPO, nil per os; PCA, postconception age. (a) Group differences were compared using appropriate methods (analysis of variance, Kruskal-Wallis, or Fisher's exact test); P < .05 indicated statistically significant differences among the 3 infant groups. (b) Parents identified as more than 1 race.

[0130] Fig.37Clinical characteristics of patients with other confirmed infections are shown. Abbreviations: GI, gastrointestinal; IQR, interquartile range; NA, not applicable; NEC, necrotizing enterocolitis; NICU, neonatal intensive care unit; NPO, fasting; PCA, postconception age. (a) Group differences were compared using appropriate methods (ANOVA, Kruskal-Wallis, or Fisher's exact test); P < .05 indicated statistically significant differences among the 3 infant groups. (b) Parents identified as more than 1 race.

[0131] Fig.38 Figure 1. Association of fecal intestinal alkaline phosphatase (IAP) content and activity with necrotizing enterocolitis (NEC) and other established infections. Panel A, Boxplots and violin plots of fecal abundance and IAP activity in samples collected during severe (n=20) and suspected NEC (n=15) are shown. Also shown are samples from patients without NEC (n=86), age-matched at the time of sample collection for NEC. Boxplot whiskers mark the 9th and 91st percentiles. Panel B, Receiver operating characteristic curves for IAP abundance (filled circles) and activity (open circles) in samples collected during severe (orange) or suspected (brown) NEC. Panel C, Boxplots and violin plots of fecal abundance and IAP activity in samples collected during sepsis (n=18), other non-gastrointestinal (GI) tract infections (n=10), and age-matched control patients (n=91) are shown. Boxplot whiskers mark the 9th and 91st percentiles. Panel D, Receiver operating characteristic curves for IAP abundance (filled circles) and activity (open circles) in samples collected during sepsis (dark blue) and other non-GI tract infections (light blue). (a) P < .001; (b) P = .005

[0132] Fig.39Control experiments are shown to demonstrate operator reproducibility, antibody reagent specificity, and biological sample specificity. Five different operators performed (Panel A) activity assay measurements and (Panel B) iAP content determinations in patient stool samples; the dashed line marks the 1:1 correspondence between replicates 1 and 2. (Panel C) Anti-human iAP antibodies used in this study were tested against human small intestinal lysate (GI), purified human placental alkaline phosphatase (PL), purified human tissue nonspecific alkaline phosphatase (TN), and calf intestinal alkaline phosphatase (cl). Densitometric quantification of total AP is presented as mean and SE: 100.0 ± 0.1% (GI); 1.0 ± 0.4% (PL); 0.9 ± 0.5% (TN); 0.3 ± 0.1% (cl); N = 5. (Panel D) Quantification by the immunoblot method used has a linear response to the amount of human intestinal alkaline phosphatase. The mean relative fluorescence units and standard errors (open triangles) of 5-7 total iAPs were: 216,692±14,533 for 7.5 μg; 233,533±20,264 for 3.75 μg; 211,176±132,267 for 1.875 μg; 142,834±13,019 for 0.938 μg; 75,727±7,637 for 0.469 μg; 44,101±1,410 for 0.234 μg; 18,234±450 for 0.117 μg; 4,918±549 for 0.059 μg; 1,164±79 for 0.029 μg; and 227±57 for 0.015 μg. (Panel E) Comparison of serum AP activity and fecal iAP activity if serum clinical test and fecal samples were collected on the sampling day. No relationship was observed between serum AP activity and fecal iAP activity measurements. N = 148; solid line is the best linear fit between fecal iAP activity and serum AP activity.

[0133] Fig.40 A sequence alignment of four human alkaline phosphatases and calf intestinal alkaline phosphatase is shown. The sequences shown are human intestinal alkaline phosphatase (iAP human; P09923 UniprotID), calf intestinal alkaline phosphatase (iAP bovine; P19111), germ cell alkaline phosphatase (GCAP human; P10696), placental-like alkaline phosphatase (PLAP human, P05187), and tissue nonspecific alkaline phosphatase (TNAP human, P05186). The signal peptide is indicated in gray at the N-terminus of the sequence. The propeptide is indicated in gray italics at the C-terminus of the sequence. Residues involved in metal binding are marked with asterisks; candidate glycosylation sites are marked with a # symbol. Secondary structural motifs are shown from a human placental alkaline phosphatase crystal (PDBID 1EW2). A color heatmap of the number of polymorphisms found in the population is overlaid on the IAP human sequence. DETAILED DESCRIPTION

[0134] Abbreviations and definitions

[0135] A detailed description of one or more preferred embodiments is provided herein. However, it should be understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but as the basis for the claims and as a representative basis for teaching those skilled in the art to apply the present invention in any appropriate manner.

[0136] Unless the context clearly dictates otherwise, the singular forms "a", "an" and "the" include plural referents. The use of the word "a" or "an" when used with the term "comprising" in the claims and / or the specification may mean "one", but is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

[0137] Unless expressly stated otherwise, any phrases "for example," "such as," "including," etc., used herein should be understood to be followed by the phrase "and not limited to." Similarly, "example," "exemplary," etc. are to be understood as non-limiting.

[0138] The term "substantially" allows deviations from the descriptive term that have no adverse effect on the intended purpose. Descriptive terms should be understood as being modified by the term "substantially" even if the word "substantially" is not explicitly mentioned.

[0139] The terms "which include" and "which contains" and "which has" and "which involves" (and similarly "includes," "including," "having," and "involving") and the like are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the conventional definition of "comprising" in U.S. patent law and is therefore interpreted as an open-ended term meaning "at least the following," and is also interpreted as not excluding other features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means that the process includes at least steps a, b, and c. Wherever the term "a" or "an" is used, it should be understood to mean "one or more" unless such an interpretation is meaningless in the context.

[0140] As used herein, the term "about" may mean approximately, roughly, around, or within the range of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20% up or down (higher or lower).

[0141] The present invention relates to compositions and methods for detecting and treating gastrointestinal diseases.

[0142] Gastrointestinal disorders

[0143] Gastrointestinal diseases are disorders involving the gastrointestinal tract. For example, necrotizing enterocolitis (NEC) is an acquired gastrointestinal disease that is common in premature infants. In NEC, bacteria invade the intestinal wall, causing localized infection and inflammation. NEC is characterized by high mortality and long-term morbidity, including short bowel syndrome, recurrent infections, nutritional deficiencies, and neurodevelopmental delays. Despite an overall net decrease in mortality among premature infants, the number of deaths associated with NEC has increased. NEC is often difficult to diagnose and manage due to initial nonspecific symptoms and rapid deterioration.

[0144] In addition to necrotizing enterocolitis in neonates and premature infants, necrotizing enterocolitis can also affect non-neonates. For example, necrotizing enterocolitis in non-neonates, such as adults, can be caused by inflammatory mediators; nutritional disorders, such as anorexia or significant weight loss; gastrointestinal dysfunction; alcohol abuse; malabsorption; drugs that block intestinal proteases; smoking; circulatory disorders, such as decreased mesenteric blood flow, intestinal ischemia, and intestinal atherosclerosis; cholelithiasis; drug administration; immune deficiencies, such as deficiencies in the secretory component of IgA or intestinal T lymphocytes, with poor antibody responses; fecal impaction or constipation; or infectious agents, such as bacterial infections, foodborne infections, and foodborne diseases.

[0145] Non-limiting examples of such drugs include those with anticholinergic properties, such as neuroleptics or phenothiazine-based neuroleptics, anesthetics, inflammatory mediators, antidepressants, iron pills, laxatives, or antacids.

[0146] Non-limiting examples of such infectious agents include bacteria such as Klebsiella, Escherichia coli, Enterobacter, Pseudomonas, Clostridium and Staphylococcus epidermidis, viruses such as coronavirus, rotavirus and enterovirus, and rare fungi such as Candida albicans. Enteropathogenic viruses are thought to infect epithelial cells, causing cell damage, necrosis and intestinal perforation.

[0147] Constipation or stool impact can have many different causes known in the art, non-limiting examples of which include antacids containing calcium or aluminum, changes in diet or activity, colon cancer, dairy products, eating disorders, neurological diseases, inactivity, dehydration, fiber depletion, overuse of laxatives, pregnancy, digestive system diseases, resisting the urge to have a bowel movement, medications, stress, or hypothyroidism.

[0148] Aspects of the present invention relate to gastrointestinal diseases. Gastrointestinal diseases refer to diseases involving the gastrointestinal tract, i.e., the esophagus, stomach, small intestine, large intestine, and rectum, as well as the digestive appendages of the liver, gallbladder, and pancreas. For example, such diseases may be caused by 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, ileus, and pseudo-obstruction or malabsorption. In addition to necrotizing enterocolitis (NEC), non-limiting examples of colitis types include adult necrotizing enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, Crohn's disease, ischemic colitis, and radiation colitis.

[0149] Intestinal alkaline phosphatase (iAP)

[0150] Using a single 100-150 mg stool sample from three healthy human donors, 234 human proteins secreted from the gastrointestinal tract were identified. Of these, a core proteome of 57 proteins shared between the three human individuals was identified. Although the presence of this core proteome was reproducible, the relative abundance of most of the shared proteins varied between the three human subjects, suggesting that the core proteome can be used to identify host-specific proteomic signatures.

[0151] Intestinal alkaline phosphatase (iAP) is expressed in small intestinal enterocytes, co-secreted into the intestinal lumen and systemic circulation, and plays an integral role in maintaining intestinal barrier function by detoxifying bacterial lipopolysaccharide and maintaining microbial homeostasis. As the major alkaline phosphatase in feces, iAP has been identified as one of 57 proteins in the core human fecal proteome.

[0152] Aspects of the invention relate to methods for diagnosing a gastrointestinal disease in a subject. For example, the method comprises the following steps: obtaining a sample from a subject; detecting the presence of at least one GI disease biomarker in the sample, wherein the GI disease biomarker comprises an intestinal alkaline phosphatase (iAP) protein; comparing the GI disease biomarker profile with a profile obtained from a control sample; and treating the subject. Embodiments may also relate to preventing the progression of a gastrointestinal disease in a subject in need thereof, and improving symptoms associated with a gastrointestinal disease in a subject in need thereof. In embodiments, a control sample may comprise two or more control samples.

[0153] As used herein, "altered compared to a control sample or subject" is understood to mean that the level of the analyte to be detected or the diagnostic or therapeutic indicator (e.g., a marker, such as iAP) is at a level that is statistically different from that of a sample from a normal, untreated or abnormal state control sample. The determination of statistical significance is within the capabilities of those skilled in the art, for example, the number of standard deviations constituting the mean of a positive or negative result and the statistical analysis to reach these intervals.

[0154] If the subject is diagnosed with a gastrointestinal disease, embodiments of the present invention include treating the subject. For example, treating the subject may include administering to the subject an effective amount of an antibiotic, a probiotic, an intravenous fluid, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof. Another treatment method may be to withhold food from the subject. Non-limiting examples of iAP replacement compositions include gene or protein replacement compositions.

[0155] The term "administering" or "administering" can refer to the introduction of a substance, such as an iAP protein or an antibiotic and / or antifungal agent, into a subject. Generally, any route of administration can be used, including, for example, intracoronary, intramyocardial, intravenous, intraarterial, or any combination thereof. For example, the iAP can be administered to a subject prior to, concurrently with, or after diagnosis of a GI disease, such as NEC.

[0156] Protein therapy can be accomplished by any method that effectively introduces an iAP protein or a fragment thereof into a subject to restore or enhance iAP activity. An effective amount of an iAP protein (e.g., an amount sufficient to reduce or eliminate symptoms associated with a gastrointestinal disease) can be administered alone or in combination with an agent that promotes protein administration or activity. An "effective amount" can be determined by one skilled in the art based on, for example, the type and severity of the symptoms being treated, the subject's weight and / or age, the subject's past medical history, and the selected route of administration of the agent.

[0157] In embodiments, the iAP protein can be bound to a lipid, such as a detergent or other amphiphilic micelle, membrane vesicle, liposome, virosome or microsome. Lipid compositions that are naturally fused or can be designed to be fused (e.g., by incorporating the fusion protein into a lipid) are particularly preferred. Fusion proteins can be obtained from viruses, such as parainfluenza virus 1-3, respiratory syncytial virus (RSV), influenza A, Sendai virus, and togavirus fusion proteins. Non-viral fusion proteins include normal cell proteins that mediate cell-cell fusion. Other non-viral fusion proteins include sperm protein PH-30, which is an integral membrane protein located on the surface of sperm cells and is believed to mediate fusion between sperm and egg. Other non-viral fusion proteins include chimeric PH-30 proteins, such as PH-30 and hemagglutinin from influenza virus and binding components of PH-30 and disintegrins (e.g., bitistatin, barbourin, kistrin, and echistatin). Additionally, lipid membranes can be fused using conventional chemical fusogens such as polyethylene glycol (PEG).

[0158] In an embodiment, a subject can be treated by administering an effective amount of an iAP protein, optionally in a pharmaceutically acceptable carrier or diluent. An effective amount of an iAP protein can be an amount sufficient to alleviate the symptoms of a gastrointestinal disease. The iAP can be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, parenterally, orally, submucosally, by inhalation (e.g., aerosolized pharmaceutical compositions), or other appropriate routes of administration within an effective dose range. If a specific mode of administration is desired, the iAP can be encapsulated in a material that protects it from enzymatic degradation. In addition, prior to administration, it may be useful to administer an agent that clears bacterial infection.

[0159] Alternatively, a preparation of a gene encoding an iAP or a fragment thereof may be incorporated into a suitable vector to deliver the gene to the subject's cells. In embodiments, iAP gene therapy may be temporary and require repeated delivery to the subject. In other embodiments, gene therapy may cure gastrointestinal diseases. For example, if the genetic material encoding an iAP is integrated into a subject's stem cells, all subsequent generations of such cells may produce authentic iAPs from the integrated sequence and correct the defect. Non-limiting examples of methods and vectors that can be used to perform iAP gene therapy include retroviruses, adeno-associated viruses, naked DNA, DNA-lipid complexes, receptor-mediated entry, or adenoviruses.

[0160] Non-limiting modes of administration for treatment include intravenous (IV); intramucosal; intramuscular; subcutaneous, and non-invasive modes of administration such as oral, intranasal, buccal, intrapulmonary, intrabronchial, and transdermal.

[0161] Aspects of the invention also relate to methods for screening for the presence of features in subjects at risk for gastrointestinal disease or subjects with asymptomatic gastrointestinal disease. For example, the steps of the method include obtaining a sample from a subject; detecting at least one GI disease biomarker in the sample, wherein the GI disease biomarker includes an intestinal alkaline phosphatase (iAP) protein; comparing the GI disease biomarker profile with a profile obtained from a control sample; and treating the subject. Similarly, aspects may also relate to methods for identifying subjects at risk for gastrointestinal disease or subjects with asymptomatic gastrointestinal disease. In embodiments, the control sample may include two or more control samples.

[0162] Aspects of the invention include measuring the total protein concentration in a sample, the intestinal alkaline phosphatase protein concentration in a sample, the intestinal alkaline phosphatase activity in a sample, or a combination thereof. The samples used in such methods and the assays for collecting such measurements are described herein. For example, if the 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 / 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, 4.1 mg / ml, 4.2 mg / ml, 4.3 mg / ml, 4.4 mg / ml, 4.5 mg / ml, 4.6 mg / ml, 4.7 mg / ml, 4.8 mg / ml, 4.9 mg / ml, 5.1 mg / ml, 5. 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, 4.3 mg / ml, 4.4 mg / ml, 4.5 mg / ml, 4.6 mg / ml, 4.7 mg / ml, 4.8 mg / ml, 4.9 mg / ml, and 5.0 mg / ml, the subject can be diagnosed with a GI disease. As another example, if the iAP activity is less 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, g, 1000 mU / mg, 1100 mU / mg, 1200 mU / mg, 1300 mU / mg, 1400 mU / mg, 5 U / mg, 10 U / mg, 50 U / mg, 100 U / mg, 200 U / mg, 300 U / mg, 400 U / mg, 500 U / mg, 600 U / mg, 700 U / mg, 800 U / mg, 900 U / mg, 1000 U / mg, the subject can be diagnosed with a GI disease. For example, if the protein concentration in the stool sample is greater than about 1.6 mg / ml, or greater than about 1.8 mg / ml; if the iAP activity is less than about 979 mU / m, or less than about 1256 mU / mg; or if the level of iAP protein is at least two standard deviations higher than the mean of the control samples, the subject can be diagnosed with a gastrointestinal disease.As another example, if the iAP protein level is greater than about 0.05%, 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 the control sample, the subject can be diagnosed with a GI disease. For example, if the iAP protein detected by an anti-iAP antibody as determined by densitometry exceeds 10.7 of the control, or exceeds 4.8% of the control as determined by densitometry, the subject can be diagnosed with a gastrointestinal disease. In other embodiments, if two thresholds are met, or if all three thresholds are met, the subject can be diagnosed with a gastrointestinal disease. In embodiments, the control sample can include two or more control samples.

[0163] The term "threshold value," e.g., a threshold value indicative of NEC, refers to a value for a biomarker, e.g., iAP protein level, iAP catalytic activity, or total fecal protein level, derived from a plurality of biological samples (e.g., donor stool samples), above which the threshold value is associated with an increased likelihood of having and / or developing a gastrointestinal disease, such as NEC.

[0164] Embodiments of the invention include diagnosing a subject with a gastrointestinal disease if the protein level of iAP in the sample, the level of iAP enzymatic activity in the sample, or the level of fecal protein is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 10 standard deviations higher than the average level of the control sample. In other embodiments, the subject may be diagnosed with a gastrointestinal disease if two thresholds are met, or if all three thresholds are met. In embodiments, the control sample may include two or more control samples.

[0165] Embodiments of the present invention include machine learning techniques or applications to determine appropriate clinical thresholds. For example, such techniques include techniques known in the art, including Naive Bayesian classifiers (NBC), linear discriminant analysis (LDA) or support vector machines (SVM), and support vector machine options. It will be readily appreciated by those skilled in the art that such thresholds can vary according to the sample size analyzed and the statistical analysis employed.

[0166] Aspects of the present invention also include identifying and / or diagnosing the early stages of gastrointestinal diseases and the late stages of gastrointestinal diseases. Certain embodiments can distinguish between early and late stage gastrointestinal diseases. For example, embodiments as described herein can diagnose late inflammatory states, such as inflammatory states determined by radiological findings of intestinal gas (portal vein or biliary gas). As another example, embodiments can identify the early stages of the disease before physiologically obvious rampant inflammation of the intestine. Currently, doctors suspect gastrointestinal diseases from a series of signs, such as abdominal distension, abdominal tenderness, decreased bowel sounds, blood in the stool, increased apnea, unstable body temperature, bile secretions, and feeding intolerance. The clinical symptoms of suspected diseases are intestinal loop expansion and radiologically displayed intestinal wall thickening. The laboratory test results of suspected diseases are thrombocytopenia, decreased or increased white blood cell counts, increased band counts, and metabolic acidosis. The embodiment can match the identification of radiological findings of intestinal gas (portal vein or biliary gas) in the late stage of inflammation. The method can also identify the early stages of the disease before physiologically obvious rampant intestinal inflammation.

[0167] sample

[0168] Aspects of the present invention include measuring or detecting biomarkers of gastrointestinal diseases in biological samples. The biomarkers of the present invention can be measured in different types of biological samples. Non-limiting examples of biological samples that can be used in the methods of the present invention include feces, plasma, umbilical cord blood, neonatal blood, cerebrospinal fluid, tears, vomitus, saliva, urine, feces and meconium. If necessary, the sample can be prepared to enhance the detectability of the biomarker. For example, the sample from the subject can be fractionated. Any method for enriching the biomarker polypeptide of interest can be used. Sample preparation, such as a pre-fractionation scheme, is optional and may or may not be necessary to improve the detectability of the biomarker depending on the detection method used. For example, if an antibody that specifically binds to the biomarker is used to detect the presence of the biomarker in the sample, sample preparation may be unnecessary. Sample preparation can involve the fractionation of the sample and the collection of the fractions containing the biomarker. Pre-fractionation methods include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis, mass spectrometry and liquid chromatography.

[0169] The methods described herein may involve obtaining a biological sample from a subject, such as an infant. As used herein, the phrase "obtaining a biological sample" refers to any process of obtaining a biological sample directly or indirectly from a subject. For example, a biological sample (e.g., at a nursing facility, such as a doctor's office, hospital, laboratory facility) can be obtained by obtaining a tissue or fluid sample (e.g., blood draw, bone marrow sample, spinal tap) from a subject. Alternatively, a biological sample can be obtained by receiving a biological sample (e.g., at a laboratory facility) from one or more people who directly obtain a sample from a subject. The biological sample can be, for example, feces such as stool, tissue (e.g., blood), cells (e.g., hematopoietic cells, such as hematopoietic stem cells, leukocytes or reticulocytes, stem cells or plasma cells), vesicles, biomolecule aggregates or platelets from a subject.

[0170] Detection and antibodies

[0171] Aspects of the invention include biomarkers for GI disease. For example, aspects include biomarkers for necrotizing enterocolitis. For example, biomarkers for GI disease include iAP enzyme activity, iAP protein level, iAP dimerization / dissociation, post-translationally modified iAP, total fecal protein, or a combination thereof.

[0172] Aspects of the invention include assays that measure iAP enzyme activity. Aspects of the invention include assays that measure iAP protein levels. Aspects of the invention include assays that measure iAP dimerization / dissociation. Aspects of the invention include assays that measure post-translationally modified iAPs. Aspects of the invention include assays that measure total fecal protein.

[0173] Non-limiting examples of post-translational modifications include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glypiation, glycosylation, hydroxylation, iodination, ISGylation, fatty acylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phosphopantetheinylation, prenylation, propionylation, ribosylation, succinylation, sulfation, SUMOylation, or ubiquitination.

[0174] iAP is a homodimer; each protomer binds four divalent (Zn 2+ and Mg 2+ ) ions, which are essential for maintaining the structural integrity and catalytic activity of the enzyme. iAP is one of four different alkaline phosphatases found in human tissues with physiological functions. Although high concentrations of iAP are found in intraluminal vesicles secreted by enterocytes on the brush border of microvilli, small amounts of iAP are released into the blood and intestinal lumen, the latter of which travels in the intestine.

[0175] Embodiments of the present invention include measuring or detecting such biomarkers using assays known in the art. Non-limiting examples of assays include immunoassays, colorimetric assays, fluorescence assays, or combinations thereof. Non-limiting examples of immunoassays include Western blot assays, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, or combinations thereof. For example, a biological sample collected from a subject can be incubated with a biomarker-specific antibody, such as an anti-iAP antibody or a fragment thereof, and the binding of the antibody to the biomarker in the sample is detected or measured.

[0176] In an embodiment, the antibody or fragment thereof may be specific for iAP (anti-iAP). The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody or fragment thereof may be linked to a molecule capable of recognition, visualization or localization using known methods. Suitable detectable labels include radioisotope labels, enzyme labels, non-radioisotope labels, fluorescent labels, toxin labels, affinity labels, and chemiluminescent labels.

[0177] Examples of assays that can be used in the methods of the invention, although not intended to be limiting, include Bradford assays, bicinchoninic acid (BCA) assays, Lowry assays, pyrogallol red protein dye binding assays, Coomassie blue dye binding assays, endpoint assays, kinetic assays, such as kinetic assays using fluorescent substrates such as 4-methylumbelliferyl phosphate, chemiluminescent substrates such as CSPD and CDP-Star, DynaLight substrates with RapidGlow enhancers, or colorimetric 4-nitrophenyl phosphate, assays for detecting phosphatase reactions, assays for detecting ATP hydrolysis, or combinations thereof. In embodiments, assays can be provided in a porous format, such as 6-, 12-, 24-, 48- or 96-well plates. In embodiments, assays can be provided in standard cuvettes, such as 1 ml cuvettes.

[0178] Total protein, such as total fecal protein, can be measured by assays known to those skilled in the art (see, for example, Cardinal Health catalogue, Dublin, Ohio, 2013, pages 7, 27, and 85, which are incorporated herein by reference in their entirety, see Roche total protein / TP2, Cobas c502 TPUC3, or Abbott total protein kit). For example, the Pyrogallol Red Molybdate dye binding method provides a total protein quantitative colorimetric method with higher linearity, which uses microliter volumes of biological samples in manual or automated systems. As described herein, pyrogallol red can be provided in a kit form, the kit comprising reagents, controls, and reagent standards, such as 25 mg / dL, 50 mg / dL, 100 mg / dL, and 200 mg / dL.

[0179] The enzyme used in the embodiments of the present invention, for example, for detecting protein levels or enzyme activity, can be, for example, alkaline phosphatase, horseradish peroxidase, β-galactosidase and / or glucose oxidase; the substrate can be an alkaline phosphatase, horseradish peroxidase, β-galactosidase or glucose oxidase substrate, respectively (see Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition (2010), Invitrogen, which is incorporated herein by reference in its entirety).

[0180] In embodiments, an enzyme, such as alkaline phosphatase or horseradish peroxidase, can be linked to a secondary antibody. Without being bound by theory, measurements of alkaline phosphatase can be confounded by signals from the secondary antibody. Isolated alkaline phosphatase can catalyze the hydrolysis of MUP to form a fluorescent product, MU. Secondary antibodies bound to AP from two different commercial manufacturers, for example, can also hydrolyze MUP to form a fluorescent product. When alkaline phosphatase protein and a secondary antibody are in the same measurement, an increased level of catalytic activity is observed. This activity can be monitored by standard spectrophotometric readings of biochemical activity and Western blots.

[0181] 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-indolyl phosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / INT substrate, tan precipitate, B iomeda), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT substrate, blue / purple), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / NBT / INT, brown precipitate, DAKO, Fast Red (red), Magenta Phosphorus (magenta), Naphthol AS-Bi-phosphate (NABP) / Fast 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), VECTOR TM Black (black), VECTOR TM Blue (blue), VECTOR TM Red (red), Vega Red (raspberry red).

[0182] Horseradish peroxidase (HRP, sometimes abbreviated as PO) substrates include, but are not limited to, 2,2'Azino-di-3-ethylbenzothiazoline sulfonate (ABTS, green, water soluble), aminoethylcarbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC, red). α-naphtholpyranose (red), 4-chloro-1-naphthol (4C1N, blue, blue-black), 3,3'-diaminobenzidine tetrahydrochloride (DAB, brown), o-benzidine (green), o-phenylenediamine (OPD, brown, water soluble), TACS Blue (blue), TACS Red (red), 3,3',5,5'tetramethylbenzidine (TMB, green or green / blue), TRUE BLUE TM (blue), VECTOR TM VIP(purple), VECTOR TM SG (smoky blue-grey) and Zymed Blue HRP substrate (bright blue).

[0183] Glucose oxidase (GO) substrates include, but are not limited to, nitro blue tetrazolium (NBT, purple precipitate), tetranitro blue tetrazolium (TNBT, black precipitate), 2-(4-iodophenyl)-5-(4-nitrobenzene)-3-phenyltetrazolium chloride (INT, red or orange precipitate), tetrazolium blue (blue), nitro tetrazolium violet (purple) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, purple). All tetrazolium substrates require glucose as a co-substrate. Glucose is oxidized and tetrazolium salts are reduced to form insoluble formazan, which forms a colored precipitate.

[0184] β-galactosidase substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal, blue precipitate).

[0185] Other examples of alkaline and acid phosphatase substrates include 9H-(1,3-dichloro-9,9-dimethylacridin-2-on-7-yl) phosphate, diammonium salt (DDAO phosphate), 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), fluorescein diphosphate, tetraammonium salt (FDP), 4-methylumbelliferyl phosphate, free acid (MUP) and 4-methylumbelliferyl phosphate, dicyclohexylammonium salt, trihydrate (MUP DCA salt).

[0186] Alkaline phosphatase activity, such as intestinal alkaline phosphatase activity, can be detected and / or measured using chromogenic and / or fluorescent substrates for alkaline phosphatase. For example, 4-methylumbelliferyl phosphate (MUP) is a fluorescent substrate for alkaline phosphatase, and alkaline phosphatase-mediated hydrolysis of its phosphate substituent produces blue fluorescent 4-methylumbelliferyl (excitation / emission ˜386 / 448 nm). In embodiments, alkaline phosphatase substrates can be directly mixed with biological samples such as feces, thereby allowing direct detection of the presence of alkaline phosphatase or measurement of its activity.

[0187] 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-indolyl phosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / INT substrate, tan precipitate, B iomeda), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT substrate, blue / purple), 5-bromo, 4-chloro, 3-indolyl phosphate / nitro blue tetrazolium / iodonitrotetrazolium (BCIP / NBT / INT, brown precipitate, DAKO, Fast Red (red), Magenta Phosphorus (magenta), Naphthol AS-Bi-phosphate (NABP) / Fast 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), VECTOR TM Black (black), VECTOR TM Blue (blue), VECTOR TM Red (red), Vega Red (raspberry red).

[0188] Other substrates known in the art, including those described herein, can be used in embodiments of the present invention (see Molecular Probes Handbook-A Guide to Fluorescent Probes and Labeling Technologies, 11th Edition (2010), Invitrogen, which is incorporated herein by reference in its entirety). In addition, various fluorophores known in the art can be covalently linked to substrates such as MUPs as desired.

[0189] The enzyme reaction can provide a highly specific, rapid and sensitive assay for detecting specific proteins in a sample, such as iAP in feces. Examples of suitable fluorescent substrates that can be used in the present invention include fluorescein diacetate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl casein, 4-methylumbelliferyl-α-L-arabinopyranoside, 4-methylumbelliferyl-β-D-fucopyranoside, 4-methylumbelliferyl-α-L-fucopyranoside, 4-methylumbelliferyl-β-L-fucopyranoside, 4-methylumbelliferyl-α-D-galactopyranoside, 4-methylumbelliferyl-β-D-fucopyranoside, 4-methylumbelliferyl-α-L ... 4-Methylumbelliferyl-β-D-galactopyranoside, 4-methylumbelliferyl-α-D-glucopyranoside, 4-methylumbelliferyl-β-D-glucopyranoside, 4-methylumbelliferyl-β-D-glucuronide, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl phosphate, bis(4-methylumbelliferyl)phosphate, 4-methylumbelliferyl pyrophosphate diester, 4-methylumbelliferyl β-D-xylopyranoside.

[0190] Non-limiting examples of suitable chromogenic substrates for use in the present invention include o-nitrophenyl-β-D-galactopyranoside, p-nitrophenyl-β-D-galactopyranoside, o-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-D-glucuronide, p-nitrophenyl phosphate, o-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-D-xylopyranoside, p-nitrophenyl-β-D-xylopyranoside, and phenolphthalein-β-D-glucuronide.

[0191] Subjects

[0192] As described herein, embodiments of the present invention include measuring or detecting gastrointestinal biomarkers in a subject. The term "subject" or "patient" may refer to any organism to which aspects of the present invention may be applied, for example, for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects to which the compounds of the present disclosure may be applied are mammals, particularly primates, particularly humans. For veterinary applications, a wide variety of subjects will be suitable, such as livestock such as cattle, sheep, goats, dairy cows, pigs, etc.; poultry, such as chickens, ducks, geese, turkeys, etc.; and domesticated animals, particularly pets, such as dogs and cats. For diagnostic or research applications, a variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and pigs such as inbred pigs, etc. The term "living subject" refers to the above-mentioned subject or another living organism. The term "living subject" refers to the entire subject or organism, not just a portion (e.g., liver or other organ) removed from a living subject.

[0193] In the embodiments herein, the subject includes a mammal, such as a human or a vertebrate. Examples of this type include, but are not limited to, dogs, cats, horses, cattle, pigs, sheep, goats, chickens, primates, such as monkeys, fish (aquaculture species), such as salmon, rats and mice. People include premature infants, infants, children, teenagers, adults or the elderly.

[0194] Although aspects of the invention described herein relate to human gastrointestinal diseases, aspects of the invention are also applicable to other non-human vertebrates. Aspects of the invention are applicable to veterinary use, such as for livestock. In general, various aspects will vary depending on the type of use and mode of administration and the specific requirements of the individual subject.

[0195] In embodiments, the subject may be in an antibiotic regimen. The term "antibiotic regimen" refers to the treatment or prevention of a disease such as infection, or a method for achieving a desired change, such as reducing or preventing infection, wherein the treatment includes administering antibiotics to the subject so that it effectively treats the disease or produces physiological changes. The antibiotic regimen may include changes known to those skilled in the art, such as antibiotic selection (e.g., including correct drug selection, route of administration and dosing regimen), administration time and duration. Non-limiting examples of such antibiotics include vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), gentamicin, Flagyl (generic metronidazole), meropenem, metronidazole, cefotaxime, clindamycin or any combination thereof. In some embodiments, an antifungal agent may be further administered. In other embodiments, the antifungal agent may be fluconazole, terconazole, voriconazole, posaconazole, pentamidine, itraconazole, ketoconazole. In embodiments, the method disclosed herein may further include treating the subject. In embodiments, treatment may include administering to a subject diagnosed with a gastrointestinal disorder an effective amount of antibiotics, probiotics, intravenous fluids, cessation of oral feeding, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof.

[0196] In one embodiment, for example, for a subject with NEC, antibiotics can be given to the subject for a sufficient time, for example, 10-14 days, wherein infant antibiotics are given. For other embodiments, for example, for a subject with sepsis, 7 days of antibiotics can be administered to the patient. For example, antibiotic administration and / or prescription can be used for broad spectrum coverage, for example, for (i) gram-positive bacteria, (ii) gram-negative bacteria and (iii) anaerobic bacteria. Non-limiting examples of such schemes include vancomycin (gram-positive, including MRSA), ceftazidime (third generation cephalosporin-gram-negative, some gram-positive and pseudomonas), metronidazole (anaerobic coverage), oxacillin (gram-positive). Non-limiting examples of general antibiotic schemes include ampicillin+gentamicin for vertically acquired infections that may be from mothers, and vancomycin+cetazidime for possible hospital-acquired infections. Based on responses from 46 neonatologists at an April 2017 NEC workshop, the commonly used antibiotics / antifungals for NEC treatment were gentamicin (32%), vancomycin (28%), ampicillin (25%), Zosyn (a combination of piperacillin and tazobactam; 15%), Flagyl (generic metronidazole; 19%), clindamycin (6%), meropenem (4%), fluconazole (antifungal, 7%), and others (1%).

[0197] In some embodiments, probiotics can also be administered to the subject. As used herein, probiotics refer to single or mixed cultures of live microorganisms that can help reestablish normal flora in the gastrointestinal tract. Probiotics can enhance immune responses, trigger the production of enzymes that degrade toxins and / or block colonic attachment sites. See, see McFarland, J. Medic. Microbiol. 2005, 54: 101-111. Non-limiting examples of probiotic organisms include those of the genera Bifidobacterium, Lactobacillus, Lactococcus, and Pediococcus, Saccharomyces boulardii, and related bacteria and yeasts.

[0198] In some embodiments, intravenous fluids or intravenous therapy may be administered to the subject. Intravenous therapy may refer to the infusion of a liquid substance directly into the vein of the subject. Non-limiting examples of such fluids include saline (e.g., 0.9% NaCl in water or 0.45% saline in water), lactated Ringer's solution (0.9% NaCl with electrolytes and buffer), D 5 W (5% glucose aqueous solution), D 5 NS (5% glucose in 0.9% saline), D 5 1 / 2NS (5% glucose in 0.45% saline), D 5LR (5% dextrose in lactated Ringer's) or Normosol-R. In embodiments, the solution may be isotonic. In other embodiments, the solution may be hypotonic.

[0199] In some embodiments, parenteral (or intravenous) nutrition may be administered to the subject. Non-limiting examples of parenteral (or intravenous) nutrition include intravenous glucose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or combinations thereof.

[0200] In embodiments, feeding, such as oral feeding, can be stopped for the subject until feeding tolerance can be demonstrated. For example, when the premature infant can safely ingest and digest the prescribed enteral (by mouth) feeding without complications associated with gastrointestinal dysfunction or infection, feeding tolerance can be demonstrated. Clinical evidence of feeding tolerance in very low birth weight premature infants can include the number of days required to reach a complete feeding amount (reporting range is 100-160 mL per kg per day), the number of episodes of feeding intolerance, the number of days of stopping feeding due to feeding intolerance symptoms, the time to restore birth weight, calf growth, weight gain, occipital head circumference and length.

[0201] In the embodiment, feeding refers to the intake of infant formula, such as EleCare (Abbott Nutrition), Neosure (Similac), 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).

[0202] In an embodiment, iAP replacement therapy may refer to protein replacement therapy. The term "protein replacement" may refer to the introduction of non-natural, purified proteins such as iAPs into individuals lacking such proteins. The administered protein may be obtained from a natural source or by recombinant expression. The term also refers to the introduction of purified proteins into individuals who otherwise need or benefit from the administration of purified proteins, for example, those suffering from protein deficiency. The introduced protein may be a purified recombinant protein produced in vitro, or from isolated tissues or body fluids, such as placenta or animal milk, or a protein purified from a plant. For example, bifidobacterium, Klebsiella, and Escherichia coli alkaline phosphatases have also been detected in human feces of premature infants (Swittink et al., 2017. Metaproteomics reveals functional differences in intestinal microbiota development of preterm infants. Molecular & Cellular Proteomics. DOI: 10.1074 / mcp. RA117.000102 (in press)), and therefore may be a source of iAP proteins for protein replacement therapy. Thus, in embodiments, increased AP activity may be a result of bacterial flora and not solely from human iAPs.

[0203] Disposable items

[0204] Aspects of the invention include disposables for detecting or measuring biomarkers of gastrointestinal disease. The disposable may include a biosensor and may optionally include other components known in the art. In embodiments, the biosensor may include at least one biological recognition element.

[0205] In embodiments, the biosensor can detect or measure iAP in a sample. In other embodiments, the biosensor can detect or measure iAP enzyme activity, total fecal protein, iAP dimerization / dissociation, post-translationally modified iAP, or a combination thereof. Non-limiting examples of post-translation modifications and samples are described herein.

[0206] In an embodiment, the biosensor can be an immunosensor and can further include a detection signal. Non-limiting examples of detection signals include radioactive signals, colorimetric signals, fluorescent signals, chemiluminescent signals, or combinations thereof. For example, the biosensor can produce a new color or a change in spectral absorption. In an embodiment, the biosensor of the present invention includes a biorecognition element or a molecular recognition element, which provides highly specific binding or detection selectivity for a specific analyte such as iAP. The biorecognition element or system can be a biological source material, such as an enzyme or enzyme sequence; an antibody or a fragment thereof; a membrane receptor protein; DNA; an organelle, a natural or synthetic cell membrane; a complete or partially viable or inactive bacterial, plant or animal cell; or a piece of plant or mammalian tissue, and is generally used to interact specifically with a target biological analyte. The biorecognition element is responsible for selectively identifying the analyte and providing a physical and chemical signal as a basis for the output signal. The physical and chemical signals generated by one or more biorecognition elements can be visually communicated to the wearer or caregiver (i.e., by a color change visible to the human eye). Other embodiments can generate light signals, which may require other instruments to enhance the signal. These include fluorescence, bioluminescence, total internal reflection resonance, surface plasmon resonance, Raman methods, and other laser-based methods.

[0207] Alternatively, the signal may be processed by an associated transducer, which may, for example, generate an electrical signal (e.g., current, potential, inductance, or impedance) that may be displayed (e.g., on a readout such as an LED or LCD display) or trigger an audible or tactile (e.g., vibration) signal or a triggerable actuator, as described herein. The signal may be qualitative (e.g., indicating the presence of a target biological analyte) or quantitative (i.e., a measurement of the amount or concentration of a target biological analyte). In such embodiments, the transducer may optionally generate a light, heat, or acoustic signal.

[0208] In any case, the signal may also be persistent (i.e., stable and readable over a period of time, typically at least of the same order of magnitude as the useful life of the article) or transient (i.e., recording a real-time measurement). In addition, the signal may be transmitted to a remote indicator location (e.g., via wires or a transmitter, such as an infrared or radio frequency transmitter), including other locations within or on the article or remote device. In addition, the biosensor 60 or any component thereof may be adapted to detect and / or signal only concentrations of the target biological analyte above a predetermined threshold level (e.g., in the case where the target biological analyte is typically present in bodily waste or when the concentration of the analyte is below a known "dangerous" level).

[0209] In one embodiment, the disposable article can be a diaper to be worn by the subject. Non-limiting examples of additional disposable articles include wipes, dipsticks, spoons, spatulas, filter papers, or swabs for cleaning the subject.

[0210] In aspects of the invention, a disposable article as described herein can be a component of a kit that can be used to diagnose a subject with a gastrointestinal disease. Additional components of the kit of the invention can include a biorecognition element, a support structure, and instructions for use thereof. For example, an iAP biorecognition element, such as an antibody as described herein, can be fixed to a solid support structure.

[0211] Non-limiting examples of compositions of the solid support structure include plastic, cardboard, glass, plexiglass, tin, paper, or combinations thereof. The solid support may also include a dipstick, a spoon, a spatula, filter paper, or a swab.

[0212] Aspects of the present invention also relate to diagnostic kits for molecular biomarkers for identifying subjects who exhibit or have a tendency to develop gastrointestinal diseases. In embodiments, the kit comprises at least one of a tool for determining total fecal protein concentration, a tool for determining intestinal alkaline phosphatase (iAP) activity, and an iAP biorecognition element, which together represent a tendency to indicate the presence or development of gastrointestinal diseases in human subjects. In embodiments, features include a total protein concentration that is at least two standard deviations higher than the mean of a control sample, an intestinal alkaline phosphatase protein concentration that is at least two standard deviations higher than the mean of a control sample, or an intestinal alkaline phosphatase activity that is at least two standard deviations lower than the mean of a control sample. In yet other embodiments, features may be selected from at least two of the group comprising a total protein concentration that is at least two standard deviations higher than the mean of a control sample, an intestinal alkaline phosphatase protein concentration that is at least two standard deviations higher than the mean of a control sample, and an intestinal alkaline phosphatase activity that is at least two standard deviations lower than the mean of a control sample. In embodiments, a control sample may include two or more control samples.

[0213] In one embodiment, the kit includes (a) a container containing components and a support structure as described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material related to the use of the methods and / or reagents described herein for diagnostic purposes. In one embodiment, the kit also includes a therapeutic agent, such as an antibiotic, a probiotic, or an iAP replacement composition.

[0214] The form of the informational material of the kit is not limited. In one embodiment, the informational material can include information about the production of the kit components, such as molecular weight, concentration, expiration date, batch or production site information, etc. In one embodiment, the informational material relates to methods of using the kit components (e.g., diagnosing a subject with a GI disease). The information can be provided in a variety of formats, including printed text, computer readable material, video recording or audio recording, or information providing links or addresses to substantive materials.

[0215] The kit may include other ingredients, such as solvents or buffers, stabilizers or preservatives. Optionally, the kit may include a therapeutic agent, such as an iAP replacement composition or an antibiotic, which may be provided in any form, such as a liquid, dry or lyophilized form, preferably substantially pure and / or sterile. When the agent is provided as a liquid solution, the liquid solution is preferably an aqueous solution. When the agent is provided in dry form, reconstitution is typically by adding a suitable solvent. A solvent, such as sterile water or a buffer, may optionally be provided in the kit.

[0216] Example

[0217] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are intended for illustrative purposes only, because alternative methods may be utilized to obtain similar results.

[0218] Example 1

[0219] This article describes methods for diagnosing a common acquired gastrointestinal emergency in premature infants. This disease (necrotizing enterocolitis or NEC) occurs in 12% of premature infants; 30% of NEC patients do not survive. In the United States, a total of approximately 5,000 infants are diagnosed with NEC each year. Due to nonspecific symptoms, this medical condition is delayed and poorly diagnosed. Biomarkers for reliable diagnosis are needed. Using infant stool samples, three biomarker measurements were performed; classifier analysis of a total of three biomarkers showed that NEC can be diagnosed by high total protein concentration, low intestinal alkaline (iAP) phosphatase activity, and high levels of intestinal alkaline phosphatase protein. Detection of intestinal alkaline phosphatase protein alone by Western blot is closely associated with NEC diagnosis and can be used in ELISA format.

[0220] Current clinical diagnostic methods rely on imaging: X-ray, CT and ultrasound. The diagnostic success rate of radiography is only 48% at best. The embodiments described herein have a true positive rate of 93% and a true negative rate of 95% for disease diagnosis. The embodiments described herein may have the potential for risk assessment and monitoring of disease.

[0221] Compared to proteomics work and mass spectrometry, the method is relatively fast and cheap. In addition, other patent applications use serum or urine; serum is invasive and requires extracting fluid from very fragile patients, while urine analysis does not provide a direct readout of gastrointestinal distress.

[0222] Example 2

[0223] Abbreviations: AP, alkaline phosphatase; DOL, days of life; iAP, intestinal alkaline phosphatase; MUP, 4-methylumbelliferyl phosphate; NBC, naive Bayes classifier; NEC, necrotizing enterocolitis; WB, Western blot

[0224] summary

[0225] Objective: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is associated with significant mortality and morbidity. Diagnosis and management can be difficult due to nonspecific symptoms, inconsistent radiological findings, and rapid deterioration. This investigation was performed to test whether fecal intestinal alkaline phosphatase (iAP) is a specific biomarker for NEC.

[0226] Study design: In a prospective, longitudinal, case-control study, serial stool samples were collected from six NEC patients and 12 control infants for measurement of total fecal protein, iAP activity, and detection of iAP protein by Western blot. Data were evaluated with longitudinal assessment of individual patients, between-group comparisons, and sensitivity / specificity assessments in classifier-based analyses.

[0227] Results: There were no significant differences in gestational age or birth weight between the 2 groups. In the 2 patients followed longitudinally, fecal protein increased, iAP activity decreased, and iAP protein was detected on Western blot after the development of NEC. NEC patients had higher mean fecal protein at diagnosis (p = 0.005), lower iAP activity (p < 0.0001), and higher specific iAP protein band intensity on Western blot (p = 0.002) compared with controls. The 3-feature naive Bayes classifier distinguished NEC from control samples with 93% sensitivity and 95% specificity.

[0228] Conclusions: Despite the limited number of subjects and samples, our results suggest that specific changes in fecal protein, iAP activity, and iAP western blot intensity occur during NEC. Preliminary sensitivity and specificity studies suggest that the three-component biomarker has the potential to serve as a noninvasive diagnostic and monitoring tool for NEC.

[0229] introduction

[0230] Necrotizing enterocolitis (NEC) is a severe 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 bowel syndrome, recurrent infections, nutritional deficiencies, and neurodevelopmental delays. 3,4 Despite an overall net decrease in premature mortality, deaths related to NEC have increased.5 The disease is often difficult to diagnose and manage due to initial nonspecific symptoms and rapid deterioration. Clinicians rely on radiographic evidence such as bowel gas to make the diagnosis, but the sensitivity of this finding has been reported to be as low as 44%. 6 Although many NEC biomarkers are under investigation, 7 None is currently widely used in clinical practice.

[0231] Without being bound by theory, intestinal alkaline phosphatase (iAP) measured in feces provides diagnostic value as a marker of intestinal pathology. This protein is expressed in small intestinal enterocytes and co-secreted into the intestinal lumen and systemic circulation. 8 It also plays an indispensable role in maintaining intestinal barrier function by detoxifying bacterial lipopolysaccharide and maintaining microbial homeostasis. 9,10 As the main alkaline phosphatase in feces, 3,4 iAP has been identified as one of 57 proteins in the core human fecal proteome. 11 Due to its protective effects, it has been investigated as a potential treatment for NEC in animal models. 12-15 However, most studies have not evaluated iAP as a diagnostic tool, and only a few studies have examined iAP in humans. In this investigation, we examined fecal iAP as a potential biomarker for noninvasive monitoring of the development of NEC in neonates. To our knowledge, this study is the first to investigate fecal iAP levels in human preterm infants to determine its association with NEC.

[0232] method

[0233] Study design and participants. This prospective, longitudinal case-control study was approved by the Institutional Review Board of the Louisiana State University School of Medicine. It was conducted in accordance with the ethical guidelines of the World Medical Association (Declaration of Helsinki). Eighteen preterm infants with a gestational age of 23–37 weeks were enrolled at Children's Hospital New Orleans and Touro Hospital after written informed consent was obtained from their parents. Demographic data of the six NEC patients and 12 control infants are shown in Table 1 . All patient samples were de-identified before analysis. Patient records were reviewed retrospectively to determine clinical relevance. No patient in this study had known chromosomal abnormalities or congenital anomalies that precluded enteral feeding.

[0234] Sample Collection / Preparation: Stool samples were collected serially from the diapers of study subjects following spontaneous bowel movements. Stool was stored briefly in a hospital specimen refrigerator until transported to the laboratory in a refrigerated container. In the initial processing step, approximately 200 mg of stool was measured and sterile molecular grade water (Sigma Aldrich) was added to achieve the desired concentration of 200 mg / ml. The mixture was vortexed vigorously for 30-60 seconds, or until a well-mixed slurry appeared. The mixture was then centrifuged at 22,000xg for 30 minutes at 4°C. The supernatant was collected and stored at -20°C until assayed.

[0235] Protein concentration: The concentration of total protein in fecal supernatants was determined by Bradford assay (Coomassie Plus protein assay reagent, Thermo-Scientific) using bovine serum albumin as a standard.

[0236] Denaturing gel electrophoresis and Western blotting: The supernatant of the fecal sample was mixed with 6X gel loading buffer (375mMTris pH 6.8, 50% (w / v) glycerol, 600mM dithiothreitol, 420mM sodium dodecyl sulfate) and boiled for 5 minutes. A total of 10 μg of total protein was loaded per lane of a denatured 4-12% Bis-Tris gel (Novex, Life Technologies). The positive control was a small intestinal tissue lysate (Abcam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control. Duplicate gels were run: one was Coomassie stained to show all proteins in each lane, and the proteins in the second were transferred to a PVDF membrane for immunoblotting detection of intestinal alkaline phosphatase. The membrane was blocked in 5% (w / v) skim milk in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% Tween, incubated with rabbit primary polyclonal antibodies against human iAP (Abcam, ab7322 or ab198101), washed, and incubated with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Abcam, ab6721) at room temperature. Chemiluminescent signals were initiated using Pierce ECL protein blotting substrate (ThermoScientific) and captured on developed photographic film (AFP Imaging). Western blot densitometry was performed on scanned films (Biorad GelDoc XR) using Image J. In the digitized protein blot, the 60 kDa band corresponding to iAP was manually identified. The equivalent area was quantified for each lane of each protein blot. Negative controls were subtracted from each patient sample, and the difference was calculated as a percentage of the positive control standard.

[0237] Fecal iAP catalytic activity: Alkaline phosphatase activity was measured using 4-methylumbelliferyl phosphate (MUP) as a fluorogenic substrate (Abcam, ab83371) in the presence and absence of the iAP inhibitor L-phenylalanine. Relative fluorescence units (RFU) at 360 / 440 nm were measured using a SpectraMax M2e spectrophotometer (Molecular Devices, Sunnyvale, CA). Ninety-six-well black optical bottom plates were used. Standards and negative controls were prepared for each plate run. Total AP activity was determined as: AP activity (mU / mL) = (B x dilution factor) / (T x V), where B is nmol of product; V is the sample volume added to the well; T is the reaction time; and U is the amount of enzyme that results in the hydrolysis of 1 μmol of MUP per minute at pH 10.0 and 25°C. A 100 mM L-phenylalanine stock solution (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.

[0238] Statistical and computational analysis for iAP biomarker classification: Mean differences in total fecal protein, iAP activity, and 60kDaiAP band intensity on western blot between NEC and control groups were tested using the nonparametric Mann-Whitney U test; p-values ​​< 0.05 were considered significant. Potential biomarker efficacy was evaluated by sensitivity (true positive rate) and specificity (true negative rate) calculations. Of the 49 unique fecal samples analyzed, 13 were obtained from NEC patients at the time of clinical diagnosis. Thirty-six samples were labeled as controls, of which 27 were from control subjects and 9 were from NEC patients during a healthy interval. For each variable of interest, specificity and sensitivity were initially obtained using a simple threshold-based classifier. Subsequently, the scikit-learn package in Python was used to 16 Multivariate classifier performance is performed by training a Naive Bayes classifier (NBC). NBC assumes that 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 For each classifier, we calculated the standard errors of our sensitivity and specificity estimates by performing five rounds of stratified jackknife resampling, where each round of resampling excluded 20% of the data. We used a 5-fold stratified cross-validation scheme, where, for each fold, NBC was trained on 80% of the data, and the sensitivity and specificity of the resulting classifier were tested on the remaining 20% ​​of the data.

[0239] result

[0240] Longitudinal study: To explore whether these 3 stool parameters were associated with NEC, two premature infants were observed over time and their stool samples were repeatedly monitored.

[0241] Patient 1 (small Figure 1 A) was diagnosed with NEC on day 7 of life (DOL). After 14 days of medical therapy, including bowel rest and antibiotics, clinical signs and bowel gas resolved. Enteral feedings were reinitiated with varying degrees of success until the infant developed recurrent NEC and subsequent intestinal perforation on DOL 31. Three stool analyses were performed: soluble protein concentration, catalytic activity of iAPs, and immunoblot detection of iAPs. Two stool samples were obtained on DOL 7: before the diagnosis of NEC (7A, Figure 1 A) and bloody stools later that day (7B, small Figure 1 A). Data from five additional stool samples (DOL 13, 20, 29, 32, and 42) are also presented.

[0242] Longitudinal monitoring of patient 1 showed that 3 candidate biomarkers had diagnostic value (small Figure 1 A). The DOL 7A stool sample had a protein concentration of 1.85 mg / mL, a catalytic activity of 2218 U / g, and no 60 kDa signal was detected in the western blot. The DOL 7B stool sample collected several hours later had a protein concentration of 2.1 mg / mL, a catalytic activity of 250 U / g, and clear immunodetection of iAP. Comparison of two stool samples from the same patient before and after the onset of NEC demonstrates a dramatic decrease in iAP activity and an increase in iAP protein that are characteristic of NEC.

[0243] These biomarkers also demonstrate surveillance value. After the initial NEC diagnosis on DOL 7, low iAP enzyme activity and high iAP protein levels persisted on immunoassay in stool samples collected during medical therapy, apparent “recovery,” and reintroduction of enteral feedings. The infant subsequently developed NEC again on day 31 of life with intestinal perforation. In combination, increased fecal protein, low iAP enzyme activity, and high iAP protein levels on Western blot were predictive of perforation. After 10 days of treatment, which included peritoneal drainage, bowel rest, and antimicrobial therapy, the assay values ​​in stool collected on DOL 42 approached those before the diagnosis of NEC.

[0244] Longitudinal monitoring also demonstrated the prognostic potential of the three candidate biomarkers. Figure 1B) Diagnosed with suspected NEC on DOL 19, kept on “NEC watch” (NEC monitoring, bowel rest, antibiotics) for several days before resuming enteral feeding. Although a definitive diagnosis of NEC was not made until DOL 32, two-thirds of the biomarkers in stool samples from DOL 13 and 19 were in the positive range associated with NEC and may predict NEC.

[0245] Cross-sectional study: In an investigation of fecal material collected from 6 NEC and 12 control infants, significant differences were found in 3 in vitro measurements between fecal samples from patients with active NEC and those from controls ( Figure 2 The mean ± SEM protein concentration in stool samples from patients at the time of NEC diagnosis was 2.62 ± 0.33 mg / mL, which was significantly different from the level of 0.98 ± 0.25 mg / mL found in stool samples from age-matched control patients after conception (p = 0.005, small Figure 2 A). In addition, mean fecal iAP enzyme activity at diagnosis was more than tenfold lower in NEC patients compared with controls matched for postconception age (162±30 mU / mg vs. 1826±376 mU / mg, NEC vs. controls, respectively, p<0.0001, small Figure 2 B).

[0246] Finally, samples from the 2 patient populations differed significantly (p = 0.002) in the relative amounts of specific iAP proteins, as determined by densitometric analysis of immunoblots probed with anti-human iAP antibodies and expressed as a percentage of a standard positive control. We found that iAP protein was nearly 30-fold higher in fecal samples from NEC patients than in samples from healthy preterm subjects (215.0 ± 47.6% vs. 7.2 ± 2.3%, NEC vs. controls, respectively). Figure 2 C) In conclusion, stool samples from infants with NEC had increased total protein, decreased iAP enzyme activity, and increased iAP protein at diagnosis compared with healthy controls.

[0247] Sensitivity and specificity study: In the 3D scatter plot (small Figure 3 A), Biomarkers from NEC samples (red circles) clustered independently from controls (black circles), indicating that these biomarkers can achieve high sensitivity and specificity. Although there was some overlap, the potential to distinguish NEC from control patient samples remained evident even after total fecal protein levels were removed as a variable (small Figure 3 B). We directly assessed sensitivity and specificity using univariate threshold classifiers and naive Bayesian classifiers (NBC), which classify samples based on the integration of all 3 tests (small Figure 3C). Clearly, there is a trade-off between sensitivity and specificity. If maximum sensitivity or true positive rate is the primary goal, the 3-feature NBC biomarker performs best, with a sensitivity of 100% and a specificity of 92%. However, if the goal is to maximize both sensitivity and specificity, the 3-feature NBC shows performance of 93% sensitivity and 95% specificity. When a threshold of 300 mU / mg iAP activity is used, iAP activity levels considered alone also perform nearly as well in this setting, reaching sensitivity and specificity levels of 92%. Perhaps unsurprisingly, however, total fecal protein levels alone are not a reliable biomarker for NEC. At 92% sensitivity, specificity drops to 67% when a protein threshold of 1.35 mg / mL is used. Therefore, fecal iAP activity levels and 60 kDa protein blot intensity levels hold promise as NEC biomarker candidates alone. However, total fecal protein activity levels are only likely to be useful if considered as part of a multi-feature diagnostic assessment.

[0248] discuss

[0249] The diagnosis and management of NEC is complicated by our current inability to accurately identify the disease before irreversible intestinal damage has occurred. Clinical parameters alone cannot accurately predict disease progression in most patients. 18 Although radiography, the cornerstone of NEC diagnosis and staging, 19 Rapid and accessible in the intensive care unit, this measure of disease pathology provides a qualitative rather than quantitative endpoint. There is well-documented variability in the interpretation of observable radiographic signs that determine disease severity. 6,20,21 Disturbingly, only 44% of pathologically confirmed NEC reported the hallmark radiologic finding of pneumatosis intestinalis. 22 .

[0250] Quantitative markers measured on a ratio or interval scale are still needed to better understand NEC, such as distinguishing normal from pathological biological processes or monitoring responses to clinical interventions. The clinical definition of NEC can be significantly improved by moving from reliance solely on clinical impression and imaging findings to an expanded diagnostic palette that includes reliable molecular biomarkers. Identification of molecular NEC biomarkers suitable for adoption in clinical practice has the potential to reduce neonatal mortality, morbidity, and associated healthcare costs. In addition, characterization of these parameters can provide insight into changes in cellular integrity, protein expression, and gastrointestinal metabolism. The discovery of candidate biomarkers for NEC obtained from serum, urine, stool, and oral swabs is the focus of current research. 23-25

[0251] Our study demonstrated a correlation between the 3 in vitro fecal parameters and patient pathology, which is consistent with the results of animal studies. The increased total fecal protein concentration measured in patients with NEC may be related to mucosal shedding and disease-associated inflammatory products, such as serum amyloid A, anaphylatoxins, C-reactive protein, platelet-activating factor, calprotectin, and alpha-1 antitrypsin. 26-31 Of these inflammation-based biomarkers, the latter three are measured directly in newborn stool samples. 27,28,32-34 However, biomarkers related to inflammation, although commonly associated with gastrointestinal pathology, are not specific for NEC diagnosis.

[0252] In our study, increased iAP detected by western blot was negatively correlated with lower intestinal AP enzyme activity in stool samples of NEC patients at diagnosis. Our findings are consistent with other reports in the literature. First, biopsied intestinal tissues from patients with inflammatory bowel disease showed lower AP activity based on enzyme histochemical analysis. 35 Second, serum iAP was shown to be increased in patients who would go on to develop NEC; however, AP levels were only monitored by gel electrophoresis and did not specify positive detection of intestinal alkaline phosphatase. 36 Third, in an animal model of induced NEC, rat terminal ileum tissue samples showed decreased protein content, activity, and alkaline phosphatase-specific immunofluorescence. 14,37 Finally, decreased mucosal AP activity has also been reported in animal models following ischemia-reperfusion. 38 Increased shedding of mucosal proteins, including inactivated iAPs, could explain our findings.

[0253] We had several criteria to evaluate the translational promise of our NEC biomarkers. First, the molecular signature should be a direct readout of gastrointestinal disease and easily detectable. In this study, we evaluated three candidates: total fecal protein, specific iAP activity, and Western blot band intensity of iAPs. Monitoring total protein levels in stool has pathophysiological plausibility, as high fecal protein levels are closely associated with poor intestinal mucosal integrity in immature neonates, which may be exacerbated by inflammation. The appeal of iAPs as biomarkers lies in their tissue-specific expression in the small intestine and their secretion into the mucus layer and intestinal lumen. 39,40 It is also responsible for most of the AP enzyme activity in feces 41,42 and has been used as a measure of toxic injury to the small intestine in animal models. 43,44 All three of our fecal biomarker candidates showed significant mean differences between NEC patients and control subjects at diagnosis ( Figure 2 ).

[0254] Second, essential features of clinically useful molecular biomarkers are ease of patient sample handling and a rapid turnaround time of <3 hours. Fresh weight-volume standardization in sterile water is rapid, requires minimal reagents, and allows storage in small disposables that are convenient for packaging and shipping. Measurement of protein concentration takes less than 30 minutes. In the current implementation, iAP enzymatic detection and western blotting can be completed in one or two hours, respectively.

[0255] Our third criterion for evaluating NEC biomarkers was the potential to outperform radiological diagnosis. As a reference, a 7-parameter analysis of the clinical diagnostic criteria developed by the WHO Integrated Management of Childhood Illness program reported a high sensitivity of 85% and a specificity of 75%. 45 The sensitivity of the test for intestinal gas was only 44%. 22 In contrast, the performance data of our diagnostic methods, including specific iAP activity, immunoblot detection, and three-parameter NBC, are promising, as both sensitivity and specificity are greater than 90%. We also noted that, in general, marker performance was more robust for positive diagnostic readouts (e.g., increased immunoblot detection and protein concentration) compared with negative diagnostic readouts: the latter modality is more susceptible to false-positive (and false-negative) diagnoses. Although future studies involving larger patient populations may alter our performance data, we conclude that this stool sample analysis has the potential clinical utility to improve the diagnosis and subsequent monitoring of NEC prognosis.

[0256] Challenges to adopting our stool biomarker assay as a diagnostic tool for NEC are the heterogeneous composition of some stool samples, the intermittent and variable bowel movement patterns of some neonates, and the lack of immediate, on-demand test results. However, our 3-signature stool biomarker assay has the advantage of requiring less time, no special training or expertise, and is inexpensive compared to proteomics or mass spectrometry techniques. Adapting this test for continuous, noninvasive monitoring of premature infants in the NICU will provide an objective measure to assess mucosal integrity, help evaluate risks associated with feeding regimens, and contribute to our understanding of NEC.

[0257] In future studies, our naive Bayes classifier approach could be extended to simultaneously analyze iAP enzyme activity, western blot signals, and other candidate NEC biomarkers, such as fecal calprotectin and platelet-activating factor. 7 These fecal biomarkers can be added to our classification scheme without the need for additional blood or urine samples from neonatal patients. The classifier performance of protein biomarkers in urine samples was analyzed to distinguish NEC from sepsis patients, and indeed, the efficiency of distinguishing NEC from sepsis patients was reduced compared to that of distinguishing NEC from normal patients. 46If our proposed biomarker protocol can maintain high sensitivity and specificity for larger patient populations with more complex control groups, iAP measurement combined with machine learning analysis of other biomarker candidates may lead to major advances in NEC diagnosis and management.

[0258] References cited in this example

[0259] [1] Fitzgibbons SC, Ching Y, Yu D, Carpenter J, Kenny M, Weldon C, Lillehei C, Valim C, Horbar JD, Jaksic T. Mortality of necrotizing enterocolitis expressed by birth weight categories. JPediatr Surg. 2009; 44: 1072-6.

[0260] [2] González-Rivera R, Culverhouse RC, Hamvas A, Tarr PI, Warner BB. Theage of necrotizing enterocolitis onset: an application of Sartwell's incubation period model. J Perinat. 2011; 31:519-23.

[0261] [3] Yee WH, Soraisham AS, Shah VS, Aziz K, Yoon W, Lee SK, Canadian Neonatal Network. Incidence and timing of presentation of necrotizing enterocolitis inpreterm infants. Pediatrics. 2012; 129: e298-304.

[0262] [4] Young C, Sharma R, Handfield M, Mai V, Neu J. Biomarkers for infants at risk for necrotizing enterocolitis: clues to prevention? Pediatr Res. 2009;65:91R-7R.

[0263] [5]Patel RM,Kandefer S,Walsh MC,Bell EF,Carlo WA,Laptook AR,SanchezPJ,Shankaran S,Van Meurs KP,Ball MB,Hale EC,Newman NS,Das A,Higgins RD,StollBJ,Eunice Kennedy Shriver National Institute of Child H,Human DevelopmentNeonatal Research Network.Causes and timing of death in extremely prematureinfants from 2000 through 2011.N Engl J Med.2015;372:331-40。

[0264] [6]Tam AL,Camberos A,Applebaum H.Surgical decision making innecrotizing enterocolitis and focal intestinal perforation:predictive valueof radiologic findings.JPediatr Surg.2002;37:1688-91。

[0265] [7]Chu A,Hageman JR,Caplan MS.Necrotizing enterocolitis:predictivemarkers and preventive strategies.NeoReviews.2013;14:e113-e20。

[0266] [8]Eliakim R,Mahmood A,Alpers DH.Rat intestinal alkaline phosphatasesecretion into lumen and serum is coordinately regulated.BiochimBiophysActa.1991;1091:1-8。

[0267] [9]LallesJP.Intestinal alkaline phosphatase:multiple biological rolesin maintenance of intestinal homeostasis and modulation by diet.NutrRev.2010;68:323-32。

[0268]

[10] LallesJP.Luminal ATP:the missing link between intestinal alkalinephosphatase,the gut microbiota,and inflammation?Am J PhysiolGastrointestLiver Physiol.2014;306:G824-5。

[0269]

[11] Lichtman JS,Marcobal A,Sonnenburg JL,Elias JE.Host-centricproteomics of stool:a novel strategy focused on intestinal responses to thegut microbiota.Mol Cell Proteomics.2013;12:3310-8。

[0270]

[12] Biesterveld BE,Koehler SM,Heinzerling NP,Rentea RM,Fredrich K,Welak SR,GourlayDM.Intestinal alkaline phosphatase to treat necrotizingenterocolitis.J Surg Res.2015;196:235-40。

[0271]

[13] Riggle KM,Rentea RM,Welak SR,Pritchard KA,Jr.,Oldham KT,GourlayDM.Intestinal alkaline phosphatase prevents the systemic inflammatoryresponse associated with necrotizing enterocolitis.J Surg Res.2013;180:21-6。

[0272]

[14] Rentea RM,Liedel JL,Welak SR,Cassidy LD,Mayer AN,Pritchard KA,Jr.,Oldham KT,GourlayDM.Intestinal alkaline phosphatase administration innewborns is protective of gut barrier function in a neonatal necrotizingenterocolitis rat model.JPediatr Surg.2012;47:1135-42。

[0273]

[15] Heinzerling NP,Liedel JL,Welak SR,Fredrich K,Biesterveld BE,Pritchard KA,Jr.,GourlayDM.Intestinal alkaline phosphatase is protective tothe preterm rat pup intestine.JPediatr Surg.2014;49:954-60。

[0274]

[16] Pedregosa F,Varoquaux G,Gramfort A,Michel V,Thirion B,Grisel O,Blondel M,Prettenhofer P,Weiss R,Dubourg V.Scikit-learn:Machine learning inPython.J Machine Learning Research.2011;12:2825-30。

[0275]

[17] Rish I.An empirical study of the naive Bayes classifier.IJCAI2001workshop on empirical methods in artificial intelligence:IBM New York;2001.p.41-6。

[0276]

[18] Ji J,Ling XB,Zhao Y,Hu Z,Zheng X,Xu Z,Wen Q,Kastenberg ZJ,Li P,Abdullah F,Brandt ML,Ehrenkranz RA,Harris MC,Lee TC,Simpson BJ,Bowers C,MossRL,Sylvester KG.A data-driven algorithm integrating clinical and laboratoryfeatures for the diagnosis and prognosis of necrotizing enterocolitis.PLoSOne.2014;9:e89860。

[0277]

[19] Bell MJ,Ternberg JL,Feigin RD,Keating JP,Marshall R,Barton L,Brotherton T.Neonatal necrotizing enterocolitis.Therapeutic decisions basedupon clinical staging.Ann Surg.1978;187:1-7。

[0278]

[20] Kosloske AM,Musemeche CA,Ball WS,Jr.,Ablin DS,BhattacharyyaN.Necrotizing enterocolitis:value of radiographic findings to predictoutcome.AJR Am J Roentgenol.1988;151:771-4。

[0279]

[21] Coursey CA,Hollingsworth CL,Wriston C,Beam C,Rice H,Bisset G,3rd.Radiographic predictors of disease severity in neonates and infants withnecrotizing enterocolitis.AJR Am J Roentgenol.2009;193:1408-13。

[0280]

[22] Ballance WA,Dahms BB,Shenker N,KliegmanRM.Pathology of neonatalnecrotizing enterocolitis:a ten-year experience.J Pediatr.1990;117:S6-13。

[0281]

[23] Evennett N,Cerigioni E,Hall NJ,Pierro A,Eaton S.Smooth muscleactin as a novel serologic marker of severe intestinal damage in ratintestinal ischemia-reperfusion and human necrotisingenterocolitis.J SurgRes.2014;191:323-30。

[0282]

[24] Ng PC,Ma TP,Lam HS.The use of laboratory biomarkers forsurveillance,diagnosis and prediction of clinical outcomes in neonatal sepsisand necrotisingenterocolitis.Arch Dis Child Fetal Neonatal Ed.2015;100:F448-52。

[0283]

[25] Sylvester KG,Ling XB,Liu GY,Kastenberg ZJ,Ji J,Hu Z,Peng S,Lau K,Abdullah F,Brandt ML,Ehrenkranz RA,Harris MC,Lee TC,Simpson J,Bowers C,MossRL.A novel urine peptide biomarker-based algorithm for the prognosis ofnecrotising enterocolitis in human infants.Gut.2014;63:1284-92。

[0284]

[26] Pourcyrous M,Korones SB,Yang W,Boulden TF,Bada HS.C-reactiveprotein in the diagnosis,management,and prognosis of neonatal necrotizingenterocolitis.Pediatrics.2005;116:1064-9。

[0285]

[27] Amer MD,Hedlund E,Rochester J,Caplan MS.Platelet-activatingfactor concentration in the stool of human newborns:effects of enteralfeeding and neonatal necrotizing enterocolitis.Biol Neonate.2004;85:159-66。

[0286]

[28] Yang Q,Smith PB,Goldberg RN,CottenCM.Dynamic change of fecalcalprotectin in very low birth weight infants during the first month oflife.Neonatology.2008;94:267-71。

[0287]

[29] Tayman C,Tonbul A,Kahveci H,Uysal S,Koseoglu B,Tatli MM,DilmenU.C5a,a complement activation product,is a useful marker in predicting theseverity of necrotizing enterocolitis.Tohoku J Exp Med.2011;224:143-50。

[0288]

[30] Cetinkaya M,Ozkan H,Koksal N,Akaci O,Ozgur T.Comparison of theefficacy of serum amyloid A,C-reactive protein,and procalcitonin in thediagnosis and follow-up of necrotizing enterocolitis in prematureinfants.JPediatr Surg.2011;46:1482-9。

[0289]

[31] Ng PC,Ang IL,Chiu RW,Li K,Lam HS,Wong RP,Chui KM,Cheung HM,Ng EW,Fok TF,Sung JJ,Lo YM,Poon TC.Host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants.J ClinInvest.2010;120:2989-3000。

[0290]

[32] Rabinowitz SS,Dzakpasu P,Piecuch S,Leblanc P,Valencia G,KorneckiE.Platelet-activating factor in infants at risk for necrotizingenterocolitis.J Pediatr.2001;138:81-6。

[0291]

[33] Moussa R,Khashana A,Kamel N,ElsharqawySE.Fecal calprotectinlevels in preterm infants with and without feeding intolerance.JPediatr(RioJ).2016。

[0292]

[34] Shulman RJ,Buffone G,Wise L.Enteric protein loss in necrotizingenterocolitis as measured by fecal alpha 1-antitrypsin excretion.JPediatr.1985;107:287-9。

[0293]

[35] Tuin A,Poelstra K,de Jager-Krikken A,Bok L,Raaben W,Velders MP,Dijkstra G.Role of alkaline phosphatase in colitis in man and rats.Gut.2009;58:379-87。

[0294]

[36] Kampanatkosol R,Thomson T,Habeeb O,Glynn L,Dechristopher PJ,YongS,Jeske W,Maheshwari A,Muraskas J.The relationship between reticulatedplatelets,intestinal alkaline phosphatase,and necrotizingenterocolitis.JPediatr Surg.2014;49:273-6。

[0295]

[37] Whitehouse JS,Riggle KM,Purpi DP,Mayer AN,Pritchard KA,Jr.,OldhamKT,GourlayDM.The protective role of intestinal alkaline phosphatase innecrotizing enterocolitis.J Surg Res.2010;163:79-85。

[0296]

[38] Sisley AC,Desai TR,Hynes KL,Gewertz BL,DudejaPK.Decrease inmucosal alkaline phosphatase:a potential marker of intestinal reperfusioninjury.J Lab Clin Med.1999;133:335-41。

[0297]

[39] Goldberg RF,Austen WG,Jr.,Zhang X,Munene G,Mostafa G,Biswas S,McCormack M,Eberlin KR,Nguyen JT,Tatlidede HS,Warren HS,Narisawa S,Millan JL,HodinRA.Intestinal alkaline phosphatase is a gut mucosal defense factormaintained by enteral nutrition.Proc Natl Acad Sci U S A.2008;105:3551-6。

[0298]

[40] Shifrin DA,Jr.,McConnell RE,Nambiar R,Higginbotham JN,Coffey RJ,TyskaMJ.Enterocyte microvillus-derived vesicles detoxify bacterial productsand regulate epithelial-microbial interactions.Curr Biol.2012;22:627-31。

[0299]

[41] Horrigan FD,DanovitchSH.The origin of human fecal alkalinephosphatase.Am J Dig Dis.1974;19:603-8。

[0300]

[42] Malo MS. A high level of intestinal alkaline phosphatase is protective against type 2 diabetes mellitus irrespective of obesity. EBioMedicine. 2015;2:2016 - 23.

[0301]

[43] Lehmann FG, Hufnagel H, Lorenz - Meyer H. Fecal intestinal alkaline phosphatase: a parameter for toxic damage of the small intestinal mucosa. Digestion. 1981;21:156 - 62.

[0302]

[44] Thomas DW, Henton DH. The use of fecal alkaline phosphatase as an indicator of intestinal damage. Digestion. 1985;31:82 - 8.

[0303]

[45] Group YICSS. Clinical signs that predict severe illness in children under age 2 months: a multi - centre study. The Lancet. 2008;371:135 - 42.

[0304]

[46] Sylvester KG, Ling XB, Liu GY, Kastenberg ZJ, Ji J, Hu Z, Wu S, Peng S, Abdullah F, Brandt ML, Ehrenkranz RA, Harris MC, Lee TC, Simpson BJ, Bowers C, Moss RL. Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants. J Pediatr. 2014;164:607 - 12e1 - 7.

[0305] Example 3

[0306] introduction

[0307] Necrotizing enterocolitis (NEC) is an extremely severe inflammatory disease of the gastrointestinal tract that primarily affects premature infants. It occurs in up to 10% of extremely low birth weight infants (≤1500 g at birth) and is characterized by high mortality (up to 30%) and significant long-term morbidity, including short bowel syndrome, recurrent infections, parenteral nutrition-related cholestasis, nutritional deficiencies, and neurodevelopmental delays (1). Despite advances in the field of neonatology, NEC is responsible for an increasing number of deaths in extremely premature infants (2). The exact cause of the disease remains poorly understood, making diagnosis and management a challenge. The course of the disease typically includes initial nonspecific symptoms followed by rapid clinical deterioration. Although many biomarkers are currently being investigated as potential aids in diagnosing NEC, none are widely used to determine the true integrity of the affected intestine (3).

[0308] Intestinal alkaline phosphatase (iAP) has emerged as an enzyme of interest in the study of gastrointestinal diseases. Produced and secreted by enterocytes in the proximal small intestine, iAP activity is found throughout the small and large intestine (4). It is the major alkaline phosphatase (AP) detected in feces (5,6). It has multiple functions, such as cleaving lipopolysaccharide (LPS) produced by Gram-negative bacteria and interfering with the activation of Toll-like receptors in the intestine (7). It dephosphorylates ATP and has been shown to influence microbial homeostasis through this interaction (8).

[0309] Given such a broad range of functions involved in intestinal homeostasis, one might expect iAP to be altered in NEC. In rats, Biesterveld et al. demonstrated that endogenous iAP catalytic activity was reduced during induction of NEC and subsequently increased during recovery from injury (9). Lehmann and Lorenz Meyer observed an increase in fecal iAP following toxic injury to the small intestine in rats, followed by a significant decrease in fecal iAP (10). They suggested that fecal iAP could be used as a parameter for toxic injury to the small intestine (10). Thomas and Henton later investigated the use of fecal iAP as a potential marker for intestinal injury but found considerable variability (11). They recommended a longitudinal approach to determine clinical validity (11). iAP supplementation has been shown to attenuate some of the systemic inflammatory responses associated with NEC (9,12,14). A recent study proposed serum iAP as a potential biomarker and found a trend toward higher iAP levels in infants who later developed NEC (15). These observations support the hypothesis that iAP may be a useful biomarker for NEC.

[0310] The focus of our study was to determine whether fecal iAP could be used as a diagnostic tool for NEC. Fecal iAP measurement is less invasive than serum measurements in preterm neonates who have undergone multiple serological tests. To date, no studies have been published investigating fecal iAP in human neonates and its association with NEC. We hypothesized that fecal iAP could be used as an objective and specific biomarker for diagnosing NEC and monitoring the course of NEC once the disease has been established.

[0311] Development of the method

[0312] In preparation for this study, I sought and received approval from an institutional board review. I designed an informed consent form and enrolled a total of 20 infants. I enlisted the help of NICU nurses to assist with collection and came up with a labeling system that would protect patient privacy. Fecal samples were collected prospectively, and charts were reviewed retrospectively to determine clinical relevance. There is little literature on fecal iAP measurement. I found a reference that described how rat fecal matter could be mixed with water and then centrifuged to obtain a supernatant16, so I proceeded with a similar approach for human fecal samples. We determined that 200 mg of measured feces was sufficient to easily collect the supernatant and quantify protein. The consistency of feces varies greatly, making wet weight an unreliable parameter. Fecal total protein content (determined by the Bradford assay) was used to standardize iAP measurements.

[0313] To confirm the presence of IAPs in feces, we chose western blotting (WB) as an initial assay. Surprisingly, positive detection of the protein in feces was extremely difficult in our healthy controls. In fact, more than 10 samples that yielded negative results were analyzed with either no band signal or a signal located far below the expected position. Initially, we believed that our negative results were due to handling and / or storage failures. However, even measurements performed on freshly obtained fecal samples that day produced no evidence of protein recognition by the anti-iAP antibodies on western blots. Only after analyzing a fecal sample from our first NEC patient did we find evidence of the full-length protein on western blot. When we recruited a second NEC patient, we again obtained positive results when localizing iAPs on WB.

[0314] Confirming the presence of iAP protein on WB in at least some experiments gave us the confidence to proceed with our studies. We then performed experiments to determine ideal handling and storage techniques. We placed some samples in a 4°C refrigerator for several days, and after 5 days in the refrigerator, there was no apparent degradation of the protein. Western blots showed consistent positive detection of iAP by the anti-iAP. Enzyme activity measurements showed minimal day-to-day variation. We also froze our supernatants at -20°C, but determined that short-term storage in a hospital refrigerator was acceptable.

[0315] We believe that 200 mg of stool is required to produce reliable results. In one case, although we collected only 10 mg of stool at the time of NEC diagnosis, we were still able to demonstrate a strong band for fecal iAP on western blot, indicating that the test is useful even when small amounts of stool are available.

[0316] We chose a fluorometric assay to measure activity because it is more sensitive than colorimetric methods (detection sensitivity is approximately 1 μU)(17). We were surprised to find that there was evidence of alkaline phosphatase activity even in samples that produced no signal on WB. Because our fluorometric activity assay is not specific for intestinal alkaline phosphatase, other alkaline phosphatases in the stool may have contributed to the discrepancy between the WB and activity assays. Our next set of studies was designed to measure the actual activity of iAPs by assaying samples in the presence and absence of L-phenylalanine (L-Phe), a specific inhibitor of iAPs(4,18). L-Phe blocked 90% ± 10% (SD) of AP activity in samples from NEC patients and 91% ± 9% (SD) in samples from controls, indicating that iAPs were the major contributor to alkaline phosphatase activity in the stool samples studied( Figure 5 This finding is consistent with previous reports (3,4) confirming that iAP is the most common AP in stool.

[0317] method

[0318] Study Design and Participants— This was a prospective case-control study. After obtaining parental informed consent, 20 preterm infants with a gestational age (WGA) of 23–37 weeks were enrolled at Children's Hospital New Orleans and Touro Hospital. Six infants had NEC as defined by Bell staging (19). Infants with known chromosomal abnormalities or congenital anomalies who were unable to feed were excluded. Stool samples from two subjects were excluded from statistical analysis because of differences in processing. Demographic data for the remaining 18 subjects (6 NEC patients and 12 controls) are shown in Table 1 . Processing of Stool— Stool samples were collected serially from the diapers of study subjects after spontaneous bowel movements. Stool was stored briefly in a specimen refrigerator at the hospital. Samples were transported in a refrigerator to the laboratory for initial processing. Approximately 200 mg of stool was measured, when possible, and molecular grade water was added to achieve the desired concentration of 200 mg / ml. The mixture was vortexed vigorously for 30 s to 1 min or until a well-mixed slurry appeared. The mixture was then centrifuged at 22,000 x g for 30 min at 4° C. The supernatant was collected and stored at −20° C. until assayed.

[0319] Determination of protein concentration—Total protein concentration in fecal supernatants was determined by Bradford assay (Coomassie Plus protein assay reagent, Thermo-Scientific) using bovine serum albumin as a standard.

[0320] Denaturing gel electrophoresis and Western blotting - The supernatant of the fecal sample was mixed with gel loading buffer (375mMTris pH 6.8, 50% (w / v) glycerol, 600mM dithiothreitol, 420mM sodium dodecyl sulfate) and then boiled for 5 minutes. A total of 10 micrograms of total protein was loaded in each lane of a precast denatured 4-12% Bis-Tris gel (Novex, Life Technologies). Duplicate gels were run. One gel was stained with Coomassie and the other gel was electroblotted onto a polyvinylidene fluoride membrane (PVDF) and blocked with Tris buffered saline and Tween 20 (50mM Tris HCl, 150mM NaCl, Tween 20) in 5% skim milk powder. PVDF was incubated with primary antibodies of full-length human iAPab7322 / ab198101 (Abcam) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody ab6721 (Abcam). We used Pierce ECL protein blotting substrate (Thermo-scientific) as a chemiluminescent peroxidase substrate. Developer (AFP Imaging; Mount Kisco, NY) was used to produce film after WB, and imager (Biorad Gel-Doc XR; Hercules, CA) was used to scan protein blots and gels. Densitometry was performed to analyze digitized images of protein blots. We manually identified the 60kDa band on the protein blot, which corresponds to intestinal alkaline phosphatase. We then calculated the area of ​​each 60kDa band relative to the background, and then expressed the value as a percentage of the positive control. The positive control was a whole cell lysate of hepatocellular carcinoma or a small intestinal tissue lysate (Abcam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control.

[0321] Fecal iAP Activity – Alkaline phosphatase activity was determined using 4-methylumbelliferyl phosphate as a fluorogenic substrate ab83371 (Abcam). Substrate background control and background control were performed to improve accuracy. Relative fluorescence units (RFU) 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. Sample, standard, and negative background control wells were prepared for each assay and total alkaline phosphatase activity was determined using the following method:

[0322] ALP activity (mU / ml) = (B / T) / V x dilution factor

[0323] Where B is nmol of 4-methylumbelliferone (4-MU), V is the sample volume added to the well, and T is the reaction time. U is the amount of enzyme that results in the hydrolysis of 1 μmol of product per minute at pH 10.0 and 25°C (glycine buffer). A 100 mM L-phenylalanine stock solution (purity >98%; Sigma Aldrich) was freshly prepared in molecular grade water and assayed daily. L-Phe was added to each well at a final concentration of 10 mM to inhibit iAP activity.

[0324] Computational and statistical analysis of iAP biomarker categories – We included 18 infants in the analysis of fecal protein and fecal iAP datasets due to consistency in stool processing and similar gestational age. Differences in mean values ​​for total fecal protein, iAP activity, and iAP protein band intensity on WB between NEC and control groups were tested using the nonparametric Mann-Whitney U test (GraphPad Instat v.3; La Jolla, CA). Linear regression analysis was used to determine the correlation between days before full feeding and total fecal protein and between days before full feeding and iAP activity (GraphPad Prism v7; La Jolla, CA). P values ​​less than 0.05 were considered significant. Igor Pro (Lake Oswego, OR) was used to generate Fig.10 .

[0325] Separate data subsets were used for 51 stool samples (from 6 NEC patients and 7 controls) for which we could measure specific iAP activity, WB, and fecal protein. For western blot band intensity and total fecal protein levels, we analyzed the distribution of these measurements and investigated the sensitivity and specificity of these measurements when used as prognostic and diagnostic biomarkers. Sensitivity is equivalent to the true positive rate of a classifier, while specificity is 1–the FPR (false positive rate) of the classifier. For each of our three variables of interest, we first investigated the specificity and sensitivity obtained using a simple threshold-based classifier. For each of these classifiers, we calculated the standard errors of our sensitivity and specificity estimates by performing five rounds of jackknife resampling, in which 20% of the data were excluded from the sensitivity and specificity estimates at each resampling round. Our data were stratified by class label during the resampling process, so that 6–7 control samples and 3–4 NEC samples (20% of the total for each class) were excluded from the analysis at each resampling round.

[0326] After investigating univariate classifiers, we explored the utility of multivariate classifiers by training a naive Bayes classifier (NBC) using the scikit-learn package in Python (20). The naive Bayes classifier assumes that each feature used in the classification is statistically independent (21). For our three features (iAP activity level, total protein, and WB intensity), this naive assumption is not correct. However, previous work in the machine learning community has shown that NBC can perform well on multi-feature classification problems even when the assumption of statistically independent features does not hold (21). To avoid overfitting of the multi-feature classifier, 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 then tested on the remaining 20% ​​of the data to estimate sensitivity and specificity.

[0327] result

[0328] Compared with multiple samples from controls approximately matched for gestational and chronological age, stool from patients with NEC at diagnosis had reduced iAP activity ( Figure 7 ), increased total fecal protein ( Figure 8 ) and increased iAP protein detection ( Fig. 9 ). Fecal iAP activity was lower at the time of diagnosis of NEC compared to mean controls. When the Mann-Whitney test was applied, there was statistical significance between the groups. The mean value of fecal iAP activity was 184 mU / mg with a standard error of measurement (SEM) of 34, while the control group had a value of 1932 mU / mg with a SEM of 433 (P<0.0001). Figure 2 shown.

[0329] We found that NEC patients had significantly higher fecal protein levels at diagnosis than matched controls of postconception age. We averaged protein levels in individual non-NEC patients between 29-43 PCA and compared them to patient fecal samples at NEC diagnosis, for a total of 6 patients with 7 events ( Figure 3 ). Nonparametric tests (Mann-Whitney) were used to compare non-normally distributed data, and statistically significant differences were found between the groups (P = 0.005). In the NEC patient sample, the mean at diagnosis was 2.62, SEM 0.33, while the mean in the average control was 0.98, SEM 0.25.

[0330] WB quantification was performed as a percentage of positive control using 7 NEC events from 6 patients and 7 control patients. The mean WB percentage in NEC patient samples was 193% compared to 6% in controls (P=0.0022). The standard error of measurement was 45 for NEC and 1.9 for controls. Figure 4 ).

[0331] The antibodies we selected for western blot analysis did not readily detect iAPs in feces, except in the case of NEC. Longitudinal observations of 2 NEC patients are shown in Fig.10 Small Figure A and Fig.10 Panel B. Panel A highlights a premature infant with a prolonged course of NEC and subsequent perforation. It shows the acute drop in iAP activity and appearance of iAP protein on WB at the time of initial NEC diagnosis, continued low iAP activity and evidence of iAP protein on WB during initial treatment until the subsequent perforation. Ten days after Penrose drain placement and bowel rest, there was no longer evidence of high iAP protein on WB, but fecal iAP activity increased. Panel B highlights a different infant with multiple NEC surveillance events (one event represented by a green dot) prior to NEC. The suspected NEC event was associated with evidence of iAP protein on WB, but normal iAP activity. This resolved prior to the acute drop in iAP activity and appearance of high iAP protein on WB at the time of NEC. Following medical management and recovery from NEC, iAP activity increased and there was no longer iAP protein on WB. Fig.10 Panel C shows 3 groups of samples, each consisting of stool from NEC patients at diagnosis compared to stool from closely matched controls. Groups 1 and 2 clearly show increased total fecal protein, decreased iAP activity, and evidence of iAP protein on WB. In Group 3, there were no significant differences in iAP activity and total fecal protein, but Western blot clearly distinguished NEC from control samples. Fig.10 Panel D shows stool from 4 different NEC patients before and at diagnosis. Using each patient as his or her own control, the diagnosis of NEC was associated with decreased iAP activity, increased total fecal protein, and demonstration of iAP protein on WB.

[0332] Fig.11 Panel A is a 3D scatter plot showing fecal iAP activity, fecal protein, and WB data points for NEC samples and controls. NEC samples are marked in red. Fig.11 Panel B depicts a 2D scatter plot showing iAP activity and WB percentage. There was high activity and low WB percentage in the control. Despite the small sample size and number of patients, combining all 3 biochemical analyses improved the observed sensitivity and specificity. Fig.11Panel C demonstrates the utility of examining multiple features simultaneously by depicting the tradeoffs between sensitivity and specificity for multiple thresholds and multiple feature selections. For a detection threshold of 10% of the positive control band intensity, western blot intensity considered alone performed best at 100% sensitivity and 70% specificity. However, if 100% sensitivity is not required and the goal is to maximize both sensitivity and specificity, then the 3-feature naive Bayes classifier performs best, achieving 95% sensitivity and 93% specificity. When a threshold of 300 mU / mg iAP activity is used, iAP activity level considered alone also performs nearly as well in this case, with a sensitivity level of 95% and a specificity of 91%. When western blot intensity level is considered alone at 95% sensitivity, the specificity level using a threshold of 30% of the positive control band intensity is 88%. However, perhaps unsurprisingly, total fecal protein level alone is not specific for NEC. To achieve 95% sensitivity for total fecal protein level, the specificity must be reduced to 44% using an interpolated threshold of 1.02 mg / mL. Therefore, fecal iAP activity levels and 60 kDa protein blot intensity levels are expected to serve as NEC biomarker candidates alone.

[0333] There was a clear trend toward higher fecal protein in the control group with feeding intolerance, as defined by the number of days required to reach target enteral feeding volume ( Fig.12 ). An opposite trend was seen in iAP activity, although the association was less significant.

[0334] Fecal heterogeneity may interfere with test reliability. In a given heterogeneous stool, the mucus-containing fraction ( Fig.13 A) is similar to other NEC samples, while the more solid portion of the stool ( Fig.13 B) Similar to the control in terms of iAP protein and iAP activity on WB.

[0335] discuss

[0336] In our study, we observed changes in total fecal protein, iAP protein measured by WB, and iAP activity in stool at the time of diagnosis in patients with NEC. High protein levels in the stool of patients with NEC at the time of diagnosis could reflect the loss of mucosal integrity in an already immature intestine and the inflammatory products associated with the disease. Shulman et al. showed a similar trend, in which fecal α1 antitrypsin was significantly increased at the time of NEC diagnosis compared with controls (22).

[0337] 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 diseases such as NEC. It is tightly adherent to the membrane but also sheds into the lumen (4). Shifrin et al. recently demonstrated that iAP is distributed into the mucus layer and intestinal lumen via shedding of microvillous vesicles (23). During inflammatory injury and intestinal necrosis, disruption of the mucosal barrier and cell death as well as shedding of the mucosal lining result in increased release of mucosal proteins such as iAP into the feces. The rarity of iAP signals on Western blots but high iAP activity in the feces of control subjects remains unclear. Perhaps the normal shedding process alters the free, luminal iAP structure in a way that does not allow recognition by our specific antibody. The immunogen for this antibody is full-length native human iAP from small intestinal tissue, and it may be more sensitive to the membrane-bound full-length protein. Alternatively, there are 2 known isoenzymes of iAP, namely fetal and adult forms that undergo developmental changes (24,25). NEC inflammation may be associated with the production of the isozyme recognized by our antibody, whereas the iAP produced under normal conditions is another unrecognized isozyme. In any case, the difference in WB results is highly suggestive of differences in the conformation of fecal IAPs between healthy and diseased states.

[0338] The abrupt decrease in overall fecal iAP activity that we found in stool samples from NEC patients at the time of NEC diagnosis was also found in rat pups in which NEC was induced (13, 26). Whitehouse et al. demonstrated reduced tissue iAP protein and activity using intestinal histology and terminal ileal tissue sampling (26). The cellular loss and putative luminal shedding that have been used to explain the reduced iAP protein and activity at the tissue level also help explain the increased iAP protein and reduced activity in the feces of NEC patients. Interestingly, adult patients with inflammatory bowel disease have been found to exhibit reduced AP activity in biopsied intestinal tissue (27).

[0339] The mechanism of low iAP activity in NEC does not appear to be due to an initial defect, as we observed a rapid decrease in activity from normal levels in some NEC patients. Loss of iAP enzyme activity could reflect damage to the enzyme catalytic site. In animal models, Sisley et al. demonstrated decreased mucosal AP activity after ischemia-reperfusion and suggested that metal-binding sites may be more susceptible to oxidative damage (28).

[0340] The decrease in fecal iAP activity is accompanied by a corresponding sudden appearance of fecal iAP protein on WB, sometimes occurring hours before diagnosis. This observation suggests that the use of activity assays and Western blots to detect iAP may provide diagnostic value for the initial event. In situations where NEC can be easily identified by traditional methods (bloody stools, intestinal gas, etc.), this additional biomarker provides little benefit. However, in patients with subclinical disease or those lacking definitive imaging evidence, the use of fecal iAP is most helpful in confirming the diagnosis. Although considered a hallmark of NEC, Ballance et al. demonstrated that in only 48% of the NEC patient population, pathology confirmed the presence of intestinal gas (29). Fecal iAP may also be used during recovery from NEC to gauge intestinal integrity and guide feeding strategies in our most vulnerable patient populations and determine the length of time required for recovery. Without being bound by theory, the needs of some children may vary from the 7-14 days of treatment considered standard management.

[0341] These biomarkers may be useful even in patients without NEC. We observed a trend toward higher total fecal protein and lower iAP activity in control infants with feed intolerance and delayed achievement of full enteral feedings. Control infants who tolerated feeds well showed low total fecal protein and very high iAP activity. We do not yet have information on the presence or absence of iAP protein by WB as it is associated with feed intolerance. If this is confirmed in larger studies, it would provide more motivation to explore the potential benefit of iAP supplementation in these settings. More studies are needed to determine normal values ​​for each parameter for all gestational and chronological ages and to identify dietary and other factors that may influence them.

[0342] No method is without limitations. A potential confounding factor for fecal iAP detection is the heterogeneity of stool. We collected a stool sample at the time of NEC diagnosis that had two different consistencies, mucus and normal-appearing stool. We separated these fractions and the normal-appearing stool had similar results to controls, whereas the mucus-containing fraction had the expected low iAP activity and high iAP signal on western blot ( Fig.13 ). Due to the wide variability in stool preparation, we did not include these data points in our analysis. This was the only sample with this issue and the frequency of occurrence is unknown. Another challenge we faced was the infrequent and sporadic bowel movement patterns associated with preterm birth, which does not allow for accurate standardized collection times across subjects. In the clinical arena, reliance on stool samples alone may result in delayed diagnosis due to poor bowel movements. No biomarker can replace a good physical examination and clinical expertise. The best use of stool iAP as a biomarker may be as an aid in establishing a diagnosis of NEC, monitoring disease progression, and monitoring or surveillance of high-risk populations.

[0343] In our examination of 3 potential NEC biomarkers related to intestinal alkaline phosphatase, we have demonstrated that stool sample analysis has potential clinical utility to improve the diagnosis of necrotizing enterocolitis. We have shown that, when considered independently, both specific iAP activity levels and Western blot band intensities can be used to identify NEC patient stool samples with high sensitivity and specificity. We also showed that multiple features can be combined using a naive Bayes classifier to achieve better sensitivity and specificity levels simultaneously. Furthermore, in future work, our naive Bayes classifier approach could be extended to simultaneously analyze iAP activity, Western blot band intensity, and a variety of other candidate NEC biomarkers, which are beyond the scope of the current study.

[0344] in conclusion

[0345] Fecal iAP protein and total fecal protein on WB were increased, but fecal iAP activity was decreased in patients with NEC at diagnosis. Measurement of iAP protein on WB, iAP activity, and fecal protein amount are individually useful biomarkers, but the sensitivity and specificity of diagnosis may be improved by combining the 3 parameters. More studies are needed to determine the sensitivity and specificity of each assay individually and in combination.

[0346] References cited in this example

[0347] 1Neu, J. & Walker, WANecrotizing enterocolitis. NEngl J Med 364, 255-264, doi:10.1056 / NEJMra1005408 (2011).

[0348] 2Patel,RMet al.Causes and timing of death in extremely premature infants from 2000 through 2011.N Engl J Med 372,331-340,doi:10.1056 / NEJMoa1403489(2015).

[0349] 3Ng, PC, Chan, KY & Poon, TCBiomarkers for prediction and diagnosis of necrotizing enterocolitis. ClinPerinatol 40, 149-159, doi: 10.1016 / j.clp.2012.12.005 (2013).

[0350] 4Goldberg,R.F.et al.Intestinal alkaline phosphatase is a gut mucosaldefense factor maintained by enteral nutrition.Proc Natl Acad Sci U S A 105,3551-3556,doi:10.1073 / pnas.0712140105(2008)。

[0351] 5Horrigan,F.D.&Danovitch,S.H.The origin of human fecal alkalinephosphatase.Am J Dig Dis 19,603-608(1974)。

[0352] 6 Malo,M.S.A High Level of Intestinal Alkaline Phosphatase IsProtective Against Type 2Diabetes Mellitus Irrespective ofObesity.EBioMedicine2,2016-2023,doi:10.1016 / j.ebiom.2015.11.027(2015)。

[0353] 7 Fawley,J.&Gourlay,D.M.Intestinal alkaline phosphatase:a summary ofits role in clinical disease.J Surg Res 202,225-234,doi:10.1016 / j.jss.2015.12.008(2016)。

[0354] 8 Malo,M.S.et al.Intestinal alkaline phosphatase promotes gutbacterial growth by reducing the concentration of luminal nucleotidetriphosphates.Am J PhysiolGastrointest Liver Physiol306,G826-838,doi:10.1152 / ajpgi.00357.2013(2014)。

[0355] 9 Biesterveld,B.E.et al.Intestinal alkaline phosphatase to treatnecrotizing enterocolitis.J Surg Res 196,235-240,doi:10.1016 / j.jss.2015.02.030(2015)。

[0356] 10 Lehmann,F.G.,Hufnagel,H.&Lorenz-Meyer,H.Fecal intestinal alkalinephosphatase:a parameter for toxic damage of the small intestinalmucosa.Digestion21,156-162(1981)。

[0357] 11 Thomas,D.W.&Henton,D.H.The use of fecal alkaline phosphatase as anindicator of intestinal damage.Digestion31,82-88(1985)。

[0358] 12 Riggle,K.M.et al.Intestinal alkaline phosphatase prevents thesystemic inflammatory response associated with necrotizing enterocolitis.JSurg Res 180,21-26,doi:10.1016 / j.jss.2012.10.042(2013)。

[0359] 13 Rentea,R.M.et al.Intestinal alkaline phosphatase administration innewborns is protective of gut barrier function in a neonatal necrotizingenterocolitis rat model.JPediatr Surg 47,1135-1142,doi:10.1016 / j.jpedsurg.2012.03.018(2012)。

[0360] 14 Heinzerling,N.P.et al.Intestinal alkaline phosphatase isprotective to the preterm rat pup intestine.JPediatr Surg 49,954-960;discussion 960,doi:10.1016 / j.jpedsurg.2014.01.031(2014)。

[0361] 15 Kampanatkosol,R.et al.The relationship between reticulatedplatelets,intestinal alkaline phosphatase,and necrotizingenterocolitis.JPediatr Surg 49,273-276,doi:10.1016 / j.jpedsurg.2013.11.037(2014)。

[0362] 16 Malo,M.S.et al.Intestinal alkaline phosphatase preserves thenormal homeostasis of gut microbiota.Gut59,1476-1484,doi:10.1136 / gut.2010.211706(2010)。

[0363] 17 Porstmann,B.,Porstmann,T.,Nugel,E.&Evers,U.Which of the commonlyused marker enzymes gives the best results in colorimetric and fluorimetricenzyme immunoassays:horseradish peroxidase,alkaline phosphatase or beta-galactosidase?J Immunol Methods 79,27-37(1985)。

[0364] 18 McLachlan,R.,Coakley,J.,Murton,L.&Campbell,N.Plasma intestinalalkaline phosphatase isoenzymes in neonates with bowel necrosis.J ClinPathol46,654-659(1993)。

[0365] 19 Uauy,R.D.et al.Necrotizing enterocolitis in very low birth weightinfants:biodemographic and clinical correlates.National Institute of ChildHealth and Human Development Neonatal Research Network.JPediatr119,630-638(1991)。

[0366] 20 Pedregosa,F.V.G.,Gramfort A,et al..Scikit-learn:Machine learningin{P}ython.J Mach Learn Res 12,2825-2830(2011)。

[0367] 21 Rish,I.An empirical study of the Naive Bayes Classifier IJCAI2001Work Empir Methods ArtifIntell22230,41-46(2001)。

[0368] 22 Shulman,R.J.,Buffone,G.&Wise,L.Enteric protein loss in necrotizingenterocolitis as measured by fecal alpha 1-antitrypsin excretion.JPediatr107,287-289(1985)。

[0369] 23 Shifrin,D.A.,Jr.et al.Enterocyte microvillus-derived vesiclesdetoxify bacterial products and regulate epithelial-microbialinteractions.Curr Biol 22,627-631,doi:10.1016 / j.cub.2012.02.022(2012)。

[0370] 24 Mulivor,R.A.,Hannig,V.L.&Harris,H.Developmental change in humanintestinal alkaline phosphatase.Proc Natl Acad Sci U S A 75,3909-3912(1978)。

[0371] 25 Vockley,J.,Meyer,L.J.&Harris,H.Differentiation of human adult andfetal intestinal alkaline phosphatases with monoclonal antibodies.Am J HumGenet 36,987-1000(1984)。

[0372] 26 Whitehouse,J.S.et al.The protective role of intestinal alkalinephosphatase in necrotizing enterocolitis.J Surg Res 163,79-85,doi:10.1016 / j.jss.2010.04.048(2010)。

[0373] 27 Tuin,A.et al.Role of alkaline phosphatase in colitis in man andrats.Gut58,379-387,doi:10.1136 / gut.2007.128868(2009)。

[0374] 28 Sisley, AC, Desai, TR, Hynes, KL, Gewertz, BL & Dudeja, PKDecreasein mucosal alkaline phosphatase: a potential marker of intestinal reperfusioninjury. J Lab Clin Med 133, 335-341 (1999).

[0375] 29 Ballance, WA, Dahms, BB, Shenker, N. & Kliegman, RMPathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117, S6-13 (1990).

[0376] Example 4

[0377] summary:

[0378] Results from three different biochemical tests on preterm feces were grouped by age since conception, allowing comparison of intestinal development between preterm and term infants. Measurement of relative iAP content in feces is a biomarker of intestinal infection. Measurement of iAP activity is a biomarker of intestinal maturation in preterm infants. Measurement of fecal protein concentration is associated with intestinal inflammatory responses or disease states.

[0379] Summary and Results:

[0380] Necrotizing enterocolitis (NEC) is a multifactorial disease that primarily affects premature infants and is a leading cause of late mortality and morbidity in extremely premature infants (Caplan, 2008; Christensen et al., 2010). Although the etiology of NEC remains unclear (Dominguez and Moss, 2012; Gephart et al., 2012), NEC is thought to represent a severe inflammatory disease in the intestine (Balance et al., 1990; Zhang et al., 2011). An exaggerated inflammatory response of the immature intestine to environmental insults is a hallmark of NEC (Chan et al., 2009). Specifically, increased levels of LPS / TLR4 signaling have been shown to contribute to the pathogenesis of NEC (Chan et al., 2009; Fusunyan et al., 2001; Leaphart et al., 2007; Nanthakumar et al., 2011). In animal models, inhibition of LPS / TLR4 signaling reduces intestinal inflammation and attenuates NEC pathology (Chan et al., 2009; Gribar et al., 2009).

[0381] Intestinal alkaline phosphatase (iAP) is an important component of innate intestinal immunity. This enzyme is normally anchored to the intestinal brush border and can cleave phosphate groups, thus dephosphorylating lipopolysaccharide (LPS). LPS dephosphorylation inhibits potent signaling pathways; thus, proinflammatory cytokine release and immune responses resulting from LPS activation of TLR4 (toll-like receptor 4; Lalles, 2010) are blocked by iAP. Furthermore, iAP is concentrated in specialized membrane vesicles that are released from the distal ends of enterocyte microvilli into the intestinal lumen (McConnell et al., 2009; Shilfrin et al., 2012). These released vesicles interact with bacteria and bacterial products and limit their proinflammatory potential.

[0382] Therefore, we expected that iAPs could be measured in human fecal samples; this was confirmed by the fact that iAPs are one of the core proteins in the human fecal proteome. A steady-state baseline of iAPs is detectable in enterocytes that are shed from the lumen and detected in feces. If there is a risk of bacterial-induced inflammation, fecal iAP levels would be increased due to the release of iAP-loaded membrane vesicles. Samples from non-NEC infants grouped by age after conception (grey bars, Fig.14 Panel A) shows that preterm infants have low amounts of iAP relative to a positive control from human small intestinal tissue lysate. Our data also show that infants with NEC have higher relative amounts of iAP in stool samples at the time of clinical diagnosis (120-320% of the positive control).

[0383] Second, dynamic transitions in iAP isoforms are associated with maturation of the fetal intestine (Mulivor et al., 1978; Suriura et al., 1981). Fetal isoforms of intestinal APs have low biochemical activity, whereas adult iAPs have high biochemical activity. We hypothesized that iAP activity would change with fetal development, with preterm infants having lower iAP activity than term infants. We reasoned that limited iAP biochemical activity in neonates could result in overactive LPS / TLR4 signaling. We tested this hypothesis by comparing fecal iAP activity in infants of different gestational ages. Thus, feces accurately measure iAP activity in the neonatal intestine.

[0384] Our data suggest that iAP activity is reduced in preterm infants compared with term infants. Fig.14 Panel B shows the mean iAP activity of fecal samples grouped by age after conception (grey bars), normalized to protein concentration. There was a strong positive correlation between iAP activity and age after conception. When comparing iAP activity between 24 and 41 weeks after conception, a one-way ANOVA and Tukey's multiple comparison test were performed, indicating that the samples were statistically divided into two groups: preterm (age after conception ≤ 35 weeks) and term (age after conception ≥ 36 weeks). All fecal samples from term infants (age after conception ≥ 36 weeks, n = 28) were compared with all samples from preterm infants (age after conception ≤ 35 weeks, n = 79), and the latter group was found to have significantly lower iAP activity (p < 0.0001; one-tailed t-test).

[0385] In contrast, infant stool collected on the same day of clinical NEC diagnosis (red bars) had much lower iAP activity compared to age-matched controls. Therefore, very low iAP activity is associated with NEC. Stool samples with iAP activity below 240 U / mg can be used as a biomarker to identify infants at greatest risk for NEC. The sensitivity of this univariate biomarker was 100% with a 95% CI of 66-100%. The specificity was 100% with a 95% CI of 97-100%. For this sample set, the disease prevalence was 7.8%, the positive predictive value was 100%, and the negative predictive value was 100%.

[0386] Without wishing to be bound by theory, the reduced ability of the preterm intestine to dephosphorylate pro-inflammatory LPS may increase the risk of an exaggerated inflammatory response to bacterial colonization and the development of NEC. Furthermore, based on these findings and without wishing to be bound by theory, prophylactic supplementation of preterm infants with iAPs may be further investigated as a strategy to reduce the risk of NEC. Our data also showed that the relative levels of iAPs and proteins were high in fecal samples of infants with NEC at the time of clinical diagnosis.

[0387] Example 5

[0388] Feed tolerance is demonstrated when the preterm infant is able to safely ingest and digest prescribed enteral (by mouth) feeds without complications related to gastrointestinal dysfunction or infection. Clinical evidence of feeding tolerance in very low birth weight preterm infants is most often described in the literature as the number of days required to achieve full feeds (reported range is 100-160 mL per kg per day), the number of episodes of feeding intolerance, the number of days feeds were stopped due to symptoms of feeding intolerance, the time to regain birth weight, calf growth, weight gain, occipitofrontal head circumference, and length. None of the infants studied achieved full feeds.

[0389] Formula types include, but are not limited to: EleCare (Abbott Nutrition), Neosure (Similac), EnfaCare (Enfamil), Pregestimil (Enfamil), Similac Special Care or SSC (Similac), and Gentlease (Enfamil).

[0390] Supplements may include, but are not limited to, microlipids (Nestle Health Science).

[0391] Non-limiting examples of parenteral (or intravenous) nutrition include intravenous dextrose solutions, intravenous amino acid solutions, intravenous fat emulsions, intravenous vitamin and mineral supplements, or combinations thereof.

[0392] Example 6

[0393] 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 include abdominal distention, poor gastrointestinal motility, and bloody stools. X-ray findings include intestinal gas and perforation.

[0394] The diagnosis of NEC is difficult because early presentation is nonspecific, the presence of intestinal gas is inconsistent, and clinical signs rapidly deteriorate despite aggressive management. For example, gas will be present in only 48% of cases with pathologically confirmed necrotizing enterocolitis. There are currently no biochemical measures to identify those infants at greatest risk and enable early diagnosis.

[0395] As described in this article, intestinal alkaline phosphatase (iAP) can be used as a biomarker for NEC, and the deficiency of iAP is associated with susceptibility to NEC.

[0396] iAPs are produced by apical enterocytes and secreted to the luminal brush border and catalyze the hydrolysis of phosphomonoesters. iAPs are active as homodimers and require Zn at the active site. 2+ and Mg2+ ions. Substrates of iAP include LPS and nucleotide triphosphates. iAP has multiple effects that affect intestinal barrier function and inflammation. iAP is shed in feces. iAP is a tissue-specific AP, meaning that it is produced primarily in the intestine, as demonstrated by immunohistochemical staining of intestinal tissue ( Fig.15 ).

[0397] iAPs maintain intestinal barrier function ( Fig.16 ).

[0398] This study investigated whether fecal iAP is a diagnostic tool for NEC. Serial patient stool samples were collected and processed over 4 days. A slurry of 200 mg stool / 1 ml molecular grade water was centrifuged at 14,000 rpm at 4 degrees Celsius. The supernatant was stored at -20 degrees Celsius until analysis. Biochemical analyses performed included the concentration of total protein in stool, enzymatic activity of alkaline phosphatase, and western blot for human iAP. Sixteen infants from Touro Hospital and Children's Hospital New Orleans provided samples (NEC: 5 patients (25-35 WGA); non-NEC: 11 patients (23-34 WGA)). More than 100 stool samples were processed and analyzed.

[0399] Five NEC patients at diagnosis were compared with 11 control patients, and the average total fecal protein of the control patients between the corrected gestational ages of 29-35 corresponding to the corrected gestational ages of the NEC patients was taken. The results were statistically significant, with a median total fecal protein of 2.7 mg / ml in the NEC patients and 0.7 mg / ml in the non-NEC patients. The total fecal protein content of the NEC patients was higher than that of the control infants ( Fig.17 The median (5%-95% CI) fecal protein NEC was 2.7 (1.6-3.6) for patients and 0.7 (0.2-2.1) for controls.

[0400] The determination of fecal protein content requires about 1 hour of laboratory work. A measurement of fecal protein concentration above 2 mg / ml can be used as an early indicator of the onset of NEC.

[0401] Total fecal AP is predominantly the enteric isoform. There are other alkaline phosphatases in the intestine, such as bacterial and TNAP. We quantified the proportion of enteric AP catalytic activity in feces by using L-phenylalanine, which specifically inhibits the activity of only intestinal-type alkaline phosphatase. Specifically, we obtained AP activity with and without L-Phe to determine specific iAP activity and concluded that iAP is the predominant form of AP in feces. Fecal AP catalytic activity was consistently lower (statistically significantly) in the NEC group ( Fig.18 ).

[0402] Table 1:

[0403] The median values ​​are 200 and 600. Statistically significant difference

[0404] Data Summary

[0405]

[0406] When NEC patients were matched with selected controls of similar age and gestational age, AP enzyme activity was lower ( Fig.19 ).

[0407] Measurement of low AP activity (<200 U / mg) is a potential biomarker for NEC. Alkaline phosphatase activity is uniformly reduced in NEC patients compared to matched controls. Without wishing to be bound by theory, iAP silencing may be a component of intestinal mucosal barrier dysfunction in critically ill NEC patients. Goldberg et al. Proc Natl Acad Sci 105, 3551.

[0408] Unexpectedly high levels of iAP protein were detected in association with NEC. Specific antibodies to human iAP were used in western blot analysis and surprisingly, appropriate signals were detected only in NEC samples (labeled N) and not in control samples. Each group represented a different NEC patient and age- and gestational-age-matched controls at diagnosis. At diagnosis, much higher amounts of iAP were present in the feces of NEC patients ( Fig. 20 ).

[0409] NEC episodes demonstrate increased fecal iAP protein levels. One patient with NEC was followed serially and had high iAP levels even with medical management. The patient subsequently had a perforation and subsequently underwent surgical intervention. Ten days after surgery, the feces no longer contained high levels of iAP protein. No signal was observed on day 42 ( Fig.21 ). Compared with AP activity, patients maintained low AP activity before surgical intervention. AP activity began to increase 10 days after surgery. The presence of persistently high fecal iAP levels and low activity may indicate intestinal damage leading to perforation. ( Fig. 22 ).

[0410] iAPs are developmentally regulated and their expression and activity have been shown to be reduced in premature pups in rat models. Data demonstrate that iAP activity is reduced in human NEC infants, but their expression is not reduced. Without wishing to be bound by theory, a third NEC biomarker could be a Western blot analysis or ELISA of iAP protein levels in premature infants (Rentea et al. Eur J Pediatr Surg 23, 39; Heinzerling et al. J Pediatr Surg 49, 954; Biesterveld et al. J Surg Res 196, 235).

[0411] This study provides preliminary evidence that three laboratory tests performed on stool samples can serve as biomarkers for NEC. The techniques, length of time, and equipment required vary between the three tests. Combining the three markers may increase diagnostic value compared to using a single biomarker. Subsequent studies will optimize the specificity and sensitivity of each method.

[0412] There are differences between the proximal and distal halves of the intestine in lactating rats. Structural differences include that the majority of iAPs are membrane-bound in the proximal half of the intestine; however, in the ileum, iAPs are found in the supernatant fraction of intestinal homogenates; in adults, more than 95% of iAPs are membrane-associated. Functional differences include that total alkaline phosphatase activity is higher in the ileum during lactation; as rats mature, activity decreases in the ileum and increases in the proximal intestine. Yedlin et al. J Biol Chem 256, 5620.

[0413] Test for non-specific binding of secondary antibodies ( Fig.23 ).

[0414] Methods: Fluorescence analysis. Alkaline phosphatase cleaves the phosphate group of the non-fluorescent 4-methylumbelliferyl phosphate disodium salt (MUP) substrate; resulting in an increase in the fluorescence signal after dephosphorylation; measured using a spectrophotometer.

[0415] Example 7

[0416] Antibiotics for NEC

[0417] For NEC, infants are given antibiotics for 10–14 days, but prescriptions are variable between hospital practices. Ideally, prescriptions would be for broad-spectrum coverage of (i) Gram-positive, (ii) Gram-negative, and (iii) anaerobes. Examples include vancomycin (Gram-positive, including MRSA), ceftazidime (third-generation cephalosporin—Gram-negative, some Gram-positive, and Pseudomonas), metronidazole (anaerobic coverage), oxacillin (Gram-positive).

[0418] Examples of general antibiotic regimens are: ampicillin + gentamicin for vertically acquired infections that may have come from the mother, and vancomycin + tazobactam for possible hospital-acquired infections. Commonly used antibiotics are gentamicin, vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), Flagyl (generic metronidazole), clindamycin, meropenem, fluconazole (antifungal drugs).

[0419] For sepsis, the patient will be given antibiotics for 7 days.

[0420] Feeding and nutrition plan for premature infants:

[0421] A challenging task facing neonatologists is to adequately and safely provide nutrition to premature infants. Enteral feeding (feeding by mouth) is the most challenging balance between safety and nutrition. Preterm infants often encounter signs of feed intolerance or the inability to digest enteral feeds. Intolerance to enteral feeds can be a benign condition but overlaps with necrotizing enterocolitis. In addition, there are significant adverse effects associated with fasting.

[0422] Complete parenteral nutrition (PN) solutions are provided on the first afternoon after birth. Infants receive a stock solution containing glucose (10 g / dL), amino acids (2.5 g / dL), and lipids in the first 2 hours after birth. The amino acid solution contains Aminosyn PF 10% (Hospira Inc) or TrophAmine 10% (B Braun Medical Inc). Intralipid 20% (Baxter) Liposyn III 20% and Liposyn II 20% (Hospira Inc) provide parenteral lipids. Fluids are typically provided at birth at 80 to 100 mL / kg per day and increased by 20 mL / kg per day to 140-160 mL / kg during the first week of life. The sodium and potassium acetates in the PN solution are buffers against metabolic acidosis.

[0423] PN solutions provide the majority of nutrition during the first week of life. Enteral nutrition (EN) usually provides only minimal energy by the end of the second week. The transition to full EN is usually achieved by the end of the fourth week. Infants receive breast milk from the mother when conditions allow. After tolerating 150 mL / kg of breast milk per day, infants receive supplemental human milk fortifier (Mead-Johnson). When breast milk is not available, infants receive formula prepared specifically for premature infants. The maximum caloric density of supplemental breast milk or formula is provided at 0.8 kcal / mL (80 kcal / dL; 24 kcal / oz).

[0424] Formula types: Premature Enfamil Formula (Enfamil), EleCare (Abbott Nutrition), Neosure (Similac), EnfaCare (Enfamil), Pregestimil (Enfamil), Similac Special Care or SSC (Similac), and Gentlease (Enfamil). Pregesternil and Elecare are hydrolyzed cow's milk-based formulas, often used for infants after NEC or infants with a history of feeding intolerance. Enfacare and Neosure are formulas for premature infants discharged from the hospital. Premature Enfamily Formula and Similac Special Care are hospital formulas for premature infants.

[0425] Feed tolerance is demonstrated when the preterm infant is able to safely ingest and digest prescribed enteral feeds without complications related to gastrointestinal dysfunction or infection. Clinical evidence of feed tolerance in very low birth weight preterm infants is most often described in the literature as the number of days required to achieve full orifices (reported range is 100-160 mL per kg per day), the number of episodes of feed intolerance, the number of days feeds were stopped due to symptoms of feed intolerance, the time to regain birth weight, calf growth, weight gain, and occipitofrontal head circumference and length.

[0426] Proposed prevention / treatment strategies for feeding intolerance in preterm infants include:

[0427] Table 2: Proposed prevention / treatment strategies for feeding intolerance in preterm infants.

[0428]

[0429] References cited in this example:

[0430] Herrmann and Herrman.2010.Nutrition in Clinical Practice 25,69-75

[0431] Fanaro.2013.Early Human Development 89,S13-S20

[0432] Example 8

[0433] PROP = tendency to obtain NEC = (1-activity)*WB

[0434] 1. A Markov switching model was fitted for PROP, WBC count, antibiotics (yes / no), and whether the infant had a certain amount of food (>0).

[0435] Table 3:

[0436]

[0437] Given volume = 0

[0438] Without wishing to be bound by theory, increased PROP significantly increases the risk of transitioning to NEC. Without wishing to be bound by theory, the use of antibiotics increases the likelihood of transitioning from a NEC state to a non-NEC state.

[0439] Without wishing to be bound by theory, when the data from the table is plotted, there is a symmetrical relationship between the state 1 to 2 and state 2 to 1 ratios.

[0440] For example, using a dataset without the additional testing data for 2019, PROP has a coefficient of about 11 or 12.

[0441] The resulting printout is as follows:

[0442] Maximum Likelihood Estimation

[0443] Baseline sets the covariate to its mean

[0444] Transformation strength with hazard ratios for each covariate

[0445]

[0446] Using other variables (IT ratio, platelet count, time) caused the model to not converge. Without wishing to be bound by theory, this may be due to the fact that the observations were made on days where we had missing values. For example, if it is known on Tuesday that an infant had a PROP score of .05 on Monday, and another PROP value is not obtained until Thursday, then the infant will have a PROP value of .05 on Tuesday and Wednesday.

[0447] 2. In an embodiment, some variables may be removed, for example if the model is complex and preliminary data are limited. When it comes to predicting transition to NEC status, an important variable is PROP (because the confidence interval does not contain 1). Without wishing to be bound by theory, if the model only fits this term, the table would be as follows:

[0448] Table 4:

[0449]

[0450] In another embodiment, antibiotics were left in the analysis, which appeared to be significantly associated with the switch from NEC state to non-NEC state. Without wishing to be bound by theory, in this case, the table is as follows:

[0451]

[0452] 3. Fitting of linear mixed models for PROP scores

[0453] Next, a linear mixed model was run to predict PROP scores as a function of only the current values ​​over time (i.e., we did not estimate values ​​of PROP by carrying forward). This produced 580 data points for 92 patients, of which approximately 45 (7%) contained PROP values ​​corresponding to patients diagnosed with NEC at that time. The model was only fit to the covariates NEC, antibiotics (yes / no), and oral intake > 0 (no / yes), as introducing more covariates would have reduced the full case to 78 data points. Linear and quadratic time effects were initially included, but were not constrained by theory and likelihood ratio tests indicated that this was not necessary. The results of the simplified model without time effects are as follows:

[0454]

[0455] In other embodiments, having explicit mathematical terms can allow for analysis of data from linear mixed models.

[0456] Without wishing to be bound by theory, mixed models are useful for analyzing correlated data, such as longitudinal data or information that may have multiple dependencies. A key feature of mixed models is that they allow for addressing multiple sources of variation, i.e., within-subject and between-subject variation, and interactions between combinations of discrete and continuous variables, by introducing random effects in addition to fixed effects. In one embodiment, the 't' in the information in Example 8 may refer to an abbreviation for time. In this embodiment, NEC PROP may be able to predict disease 4.93 days prior to an X-ray.

[0457] As described herein, if a patient is diagnosed with NEC on a given date, it can be predicted that they will have a significantly higher PROP score because the NEC coefficient is positive (0.0733) and the p-value is less than 0.0001. There is an interaction between NEC Prop and not eating; the Vol1 coefficient is 0.0502. This interaction is significant because it has a p-value of 0.0001. Without wishing to be bound by theory, this interaction is expected because medical staff withhold food when NEC is diagnosed. Antibiotic use has no relevant interaction with NEC propensity. This mixed model takes into account multiple observations at each time interval.

[0458] Example 9

[0459] Without wishing to be bound by theory, the PROP score may be a function of iAP activity and WB value:

[0460]

[0461] Transition Model:

[0462] Does increased PROP increase (decrease) the rates of transitions A and B?

[0463] A. PROP only

[0464] In one embodiment, the analysis carries forward the patient's PROP score to the day for which there is no data. For example, if a patient has PROP scores on days 3 and 7, their PROP scores on days 4-6 are equal to the score on day 3.

[0465] Table 5:

[0466]

[0467] Increased PROP was associated with a significantly increased risk of conversion to NEC suspicious / (+).

[0468] B. PROP and Antibiotics

[0469] The same push was made for antibiotics, which only considered whether infants were given antibiotics, not what antibiotics were used.

[0470] Table 6:

[0471]

[0472] Increased PROP was associated with an increased risk of transition to NEC. Antibiotics increased the likelihood of transition from NEC to NEC(-).

[0473] C. Full Model

[0474] Table 7:

[0475]

[0476] In the described embodiment, additional covariates cannot be considered due to convergence issues.

[0477] White blood cell counts are continued, as is whether the patient receives food.

[0478] After adjusting for white blood cell count and whether the patient received food, the results showed that PROP and antibiotics were significant predictors of conversion risk.

[0479] Data display and Fig.26 The data in is partially symmetrical.

[0480] There is no significant difference from the interval overlap inference of 1.

[0481] Linear mixed models for PROP scores

[0482] Note that we did not proceed any further; therefore, without wishing to be bound by theory, we used the 580 PROP data points observed on the 92 patients.

[0483] Model Fitting:

[0484]

[0485] PROP ij is the prop score of patient i at the jth measurement.

[0486] t ij is the time of the jth measurement for patient i (in PCA days).

[0487] Time and Time^2 are not needed in the regression model.

[0488] Table 8:

[0489] variable coefficient Test Statistics P-value Nec+ .0733 4.93 <.0001 antibiotic .0074 .086 .3876 Volume = 0 .0502 3.92 .0001

[0490] NEC was associated with a significant increase in PROP scores.

[0491] Without wishing to be bound by theory, not eating on a given day was associated with a significant increase in PROP scores.

[0492] Without wishing to be bound by theory, the assumed normality of the PROP.Aβ regression has mixed effects modeling capabilities.

[0493] Using beta regression without mixed effects, we get the same results

[0494] If desired, embodiments may use Bayesian methods to fit a beta mixture regression.

[0495] Example 10

[0496] For example, see Example 10 Fig.28 and Fig.29 We have been collecting fecal samples from premature infants and analyzing the abundance and enzymatic capacity of intestinal alkaline phosphatase (iAP). One of the original graphs I generated ( Fig.28 ) highlighted that these two biochemical characteristics separate NEC disease from non-disease. When sepsis was examined in this sample patient population, this bloodstream infection was not separated from non-bloodstream infections.

[0497] Given that both of these biochemical properties of iAPs can distinguish NEC disease, we were interested in developing a simple algebraic formula to combine the equal contributions of both parameters. We elaborated a formula in which iAP abundance is multiplied by the degree of iAP enzyme dysfunction. This product can be called the PROP score or the 'NECPredict' score.

[0498] The first term in the formula is iAP abundance. If there is an imbalance of unhelpful bacteria, iAP will be shed into the intestinal lumen and thus found in the feces. When there is a bacterial imbalance or NEC diagnosis, the normalized percentage of iAP is high relative to that found in human small intestinal lysate samples (see Heath, Maya, et al. "Association of Intestinal Alkaline Phosphatase With Necrotizing Enterocolitis Among Premature Infants." JAMAnetwork open 2.11(2019):e1914996-e1914996, which is incorporated herein by reference in its entirety). I also know that we tested the amount of iAP protein in stool samples before clinical diagnosis ( Fig.29 ).

[0499] The second term in the NECPredict formula (also known as the PROP score) is iAP dysfunction. iAPs are responsible for neutralizing signals derived from Gram-negative bacteria and triggering human innate immune responses. Humans with robust iAP function may prevent inappropriate pro-inflammatory signaling cascades in the human gut and aid in the maturation of beneficial microbiota. In infants with NEC, we found that iAPs were nonfunctional at any time during the clinical study compared to control infants. 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; this algebraic subtraction needed to be normalized to give equal weight between protein abundance and protein function. The iAP abundance and iAP function terms were multiplied to provide the propensity (NECPredict) score.

[0500] At clinical diagnosis, the median NEC Predict score is close to 1 ( Fig.29 ), significantly higher than controls. Even before clinical diagnosis, NEC Predict scores of sick infants were significantly different from those of control infants. These data suggest that there is a clear clinical-iAP biochemical relationship in NEC disease. Without wishing to be bound by theory, any NEC Predict score above 0.5 could be used as an adjunct to clinical disease intervention, such as withholding oral feedings and prescribing antibiotics in the neonatal intensive care unit.

[0501] Embodiment 11

[0502] The goal of this project was to generate data on prognostic biomarkers for the prediction of necrotizing enterocolitis (NEC), the most common and lethal gastrointestinal disease in premature infants. Such a disease-sensitive and disease-specific tool is critical to support the development of new drugs in this smallest and most vulnerable patient population. In addition, this work directly addresses a critical decision point in current clinical practice: neonatologists and patient advocacy groups in the field directly motivate us to search for windows of disease reversibility. The team first developed a diagnostic test for NEC, NECDetect. NECDetect analyzed samples from 135 premature infants at three hospitals and identified >95% true positives and >95% true negatives at the onset of the disease; importantly, it was not associated with late-onset neonatal sepsis. Without wishing to be bound by theory, the NECDetect component can be used to assess the risk of NEC before a severe onset occurs. Our prospective observational study will evaluate whether NECPredict, a calculated probability based on infant biochemical data, can predict disease 36-48 hours before clinical symptoms develop, and whether Neonatal DDx, a genetic polymorphism screen, can identify infants at birth who are predisposed to develop NEC. The enrollment goal is 150 preterm infants, with 90% statistical power. Although the small number of preterm infants limited the number of participants, this goal still exceeds the goals of most studies registered on ClinicalTrials.gov, of which 62% have fewer than 100 participants. This work is also the first study to involve the NEC Biorepository, a virtual biorepository composed of 8 different academic center hospitals. Supported by the infrastructure of the consortium, future clinical studies will be able to recruit thousands of infant patients, which will put them in the top 6% of the clinical study recruitment target. If successful, accurate, rapid and inexpensive diagnostics could enable personalized management and improved treatment of infant intestinal inflammation.

[0503] Necrotizing enterocolitis is the most common gastrointestinal disease in premature infants. With 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 within which infants can be selected for clinical trials of active management and therapeutic interventions.

[0504] Specific goals

[0505] Necrotizing enterocolitis (NEC) of prematurity is a devastating gastrointestinal disease with significant mortality and morbidity. First described 200 years ago, fundamental knowledge gaps remain about this rare disease. We do not know its cause, but it has been linked to changes in infant development, feeding, and taxa in the microbiome. Second, and more clinically pressing, no single factor or combination of known factors can explain the wide variability in the onset of NEC: we do not know who will develop the disease and when. Such insights would open new avenues of care, such as earlier and more effective management of fragile premature infants in the neonatal intensive care unit (NICU) and selection of infants for therapeutic clinical trials.

[0506] This proposal addresses the unmet need for prognostic biomarkers that predict the onset of NEC in neonates. To achieve this goal, an initial hurdle is to elucidate the molecular signatures or biomarkers that intersect dysbiosis, human epithelial function, and NEC. Without wishing to be bound by theory, aberrant biochemical communication between the host and intestinal bacteria in premature infants is a predictor of necrotizing enterocolitis. Our preliminary data highlight that biochemical assays measuring the host response to intestinal bacteria are diagnostic biomarkers for NEC. In examining biological samples from 135 very low birth weight infants from three different hospitals, the main feature of NECDetect is its improved identification of true positives at the onset of disease. Another notable feature of NECDetect is its availability; it is non-invasive, rapid, low-cost, and easily integrated into existing pathology workflows.

[0507] Based on this prerequisite work to answer the ‘if’ question, this application will determine if the NECDetect component can be used as a prognostic biomarker to address the ‘who’ and ‘when’. Our approach is to conduct a prospective longitudinal study of preterm infants in two different urban NICUs with regular collection of biological samples. We will recruit 150 preterm and / or growth-restricted infants (<34 weeks gestational age; <2.5kg birth weight) from two clinical centers. This study analyzes specific biomarkers and temporal clinical correlations in patients with NEC and non-NEC. For this application, we will focus on the following objectives:

[0508] Objective 1: Can iAP polymorphisms predict NEC susceptibility? Our hypothesis is that infants diagnosed with NEC will have genetic mutations in the intestinal alkaline phosphatase ALPI that reduce their ability to detoxify harmful Gram-negative bacteria. Methods will include Sanger sequencing of PCR products amplified from genomic DNA of infants diagnosed with NEC and those without NEC. DNA will be isolated from cheek swabs or peripheral blood cells. The significance of this work will be the first mechanistic definition of the relationship between disease severity, biochemistry, and genetic polymorphisms. If achieved, this NEC susceptibility screening, called Neonatal DDx, will be essential to determine the earliest possible treatment options and improve long-term outcomes and quality of life.

[0509] Objective 2: Are intestinal alkaline phosphatase (iAP) levels in stool a prognostic biomarker for NEC? This objective will determine whether the onset of NEC can be determined at the molecular level before the most severe physical symptoms can be observed at the clinical level. Without wishing to be bound by theory, increased release of iAP protein in the human intestinal lumen is a response to microbial-induced inflammation in NEC and can be measured as a function of time. In total, more than 2,000 patient samples will be collected longitudinally and analyzed for iAP protein content. In vitro results and corresponding clinical data will be used to validate the association between iAP as a biomarker and prediction of NEC diagnosis through the computational platform NECPredict. Its significance is twofold. This work will be the first study to test a continuous time course in which patients move between clinical states during the course of the disease, rather than the traditional binary distinction between NEC events and non-NEC events. It will also identify the time window of reversibility for active rather than passive medical management.

[0510] In practical terms, these goals provide personalized predictive approaches to address human diversity, variability in infant gut development, and clinical care options. To do so, temporal granularity of patient samples and clinical information is required; such an effort is made possible by the non-invasive nature of our biospecimen acquisition. Embedded in this work is a platform for investigating operational and feasibility issues in our clinical research protocols to acquire and integrate large datasets of clinical and biochemical information between two large academic medical centers. This study optimization and data harmonization across a broad range of clinical measures will justify future multicenter studies with a larger number of NICUs coordinated through the nascent US National NEC Biobank. Importantly, this proposal will validate a much-needed biomarker that can predict and detect NEC. Such studies will be critical to improving our understanding of gastrointestinal disease in our most vulnerable infants and can be expanded to adult populations.

[0511] Research strategy

[0512] (a) Background and significance

[0513] Our goal is to identify the mechanisms that alter homeostasis between the human host and intestinal bacteria that cause gastrointestinal inflammation. Severe forms of gastrointestinal inflammation are debilitating and life-threatening, regardless of the age of onset. Its pathophysiology is poorly understood. Currently, it is thought that these complex diseases require both genetic and non-genetic factors.

[0514] For example, necrotizing enterocolitis (NEC) of prematurity remains one of the most feared and expensive neonatal illnesses [1]: we do not know who will get it, when they will get it, or if they will survive. NEC progresses rapidly from mild abdominal distention and feeding intolerance to shock, intestinal necrosis, and death. Its rapid progression and imprecise clinical presentation gave rise to the Bell staging criteria, the most commonly used classification scheme based on a broad range of bedside clinical and imaging findings [2,3]: early NEC is called Bell stage I, medical NEC is Bell stage II, and surgical NEC is Bell stage III ( Fig.30 , Panel A). However, Bell staging is not specific for NEC and does not predict disease severity. Mortality is between 30–50% [4], and it often occurs with other life-threatening conditions, such as sepsis. Survivors may develop short bowel syndrome, neurodevelopmental impairment, bronchopulmonary dysplasia, and intracranial hemorrhage [5–7].

[0515] The lack of reliable molecular biomarkers of intestinal inflammation frustrates clinicians and is a barrier to biomedical progress. Fig.30 , panel B) is the current gold standard, detecting only late, life-threatening stages of NEC (modified Bell stages II and III) and identifying only 44% of true positives [8]. Furthermore, despite its frequent and consistent use, individual radiographic signs of NEC are not easily correlated with disease severity. More importantly, biomarkers for the early, reversible stages (modified Bell stage I) are missing from the medical toolbox. Instead, a combination of phenotypic and serological information is used to guide clinical intuition.

[0516] Defining prognostic biomarkers for NEC would be of great importance to both the scientific and medical communities. It would provide insights into key mechanisms and would be essential for establishing and monitoring intestinal homeostasis in premature infants. At the clinical level, early biomarkers would mitigate surgical resection of necrotic bowel and the long-term chronic effects of the disease. In a survey of 70 physicians, we found that the minimal significant time difference for earlier identification of NEC compared to X-ray was 48 hours, a difference that would allow beneficial patient management. Timely management can reduce the need for surgery by half [9]: medical therapy typically consists of bowel rest, antibiotics, and supportive care (white box, Fig.30, Panel B). Second, prognostic biomarkers are necessary to demarcate the reversible phase of NEC. This is not trivial, as the time frame of NEC is short and there is no medical equivalent in adult GI diseases. Furthermore, this early window for disease management is necessary for drug development and enrollment in clinical trials of therapeutic agents.

[0517] (b) Innovation

[0518] Three unique aspects distinguish this proposal. The first innovation is the evaluation of non-inflammatory proteins preceding the immune activation cascade as biomarkers for NEC. Without wishing to be bound by theory, intestinal alkaline phosphatase (iAP;

[10] ), an initial host regulator of microbial management ( Fig.30 , panel A), is a biomarker of early NEC in preterm infants. The development of intestinal inflammation depends on the extent to which bacterial commensalism is accompanied by cellular signaling through innate immune mechanisms [11,12]. However, to date, NEC biomarker research

[13] has mainly focused on gene products that regulate intestinal immunity, mucosal damage that allows bacterial translocation, and host inflammation ( Fig.30 , Panel A). Unfortunately, these proteins, although associated with advanced NEC stage, are not specific biomarkers for gastrointestinal disease and, importantly, are also associated with non-gastrointestinal infections and sepsis. Therefore, they cannot serve as prognostic biomarkers and are not promising targets for therapeutic intervention.

[0519] iAP is encoded by the human ALPI gene and plays a key role in host-microbiota interactions by inhibiting downstream host inflammatory responses. It is a metalloenzyme with tissue-specific expression in the small intestine and is easily detected in the mucus layer and in the intestinal lumen [14,15]. Membrane-anchored in enterocytes, iAP flows only into the intestinal lumen and can therefore be measured in feces to control bacterial colonization [15,16]. It hydrolyzes phosphate from lipopolysaccharide (LPS), thereby reducing Toll-like receptor 4 (TLR4; Fig.30 , Panel A) Agonist activity. Notably, TLR4 has been implicated in the pathogenesis of NEC [17-20]. Therefore, iAP has been used as a measure of toxic damage to the small intestine in animal models

[21] . In contrast, our proposal examined iAP in human biospecimens and assessed it during infants’ stay in the NICU.

[0520] Our second innovative direction is to develop diagnostics for infant diseases, rather than leveraging established adult biomarkers and testing their applicability to children

[22] . Although clinical research is being conducted for all diseases, it is clear that the portfolio of trials does not meet the needs of public health or community medical practice in terms of urgency or scale. Children and adults differ in physiological capacity, pharmacokinetic profiles, and pharmacodynamics; metabolic pathways, organ function, and metabolic rates also vary greatly [23–25]. In addition, age, growth, and development are associated with disease severity in neonates, infants, and children [26,27]. Despite the clear recognition that children are not ‘small adults’, the need for pediatric-specific healthcare solutions is frustrated by invasive and harmful methods of obtaining biospecimens and the relatively small number of available participants for clinical studies and trials

[28] . In response to the former challenge, this study evaluated infant feces in discarded diapers, thereby minimizing risk to infants and being a favorable factor for study recruitment. For the latter, the PI and consortium PIs are part of the NEC Biorepository, and 8 different academic centers have agreed to share samples and clinical data from infants with NEC

[29] . If this first testing collaboration is successful, other hospital partners in the NEC Biobank are poised to accelerate larger-scale translational studies at the NEC in the future

[29] .

[0521] Third, advanced testing of clinical workflows and assay systems has enabled us to efficiently process increased numbers of infants over shorter study periods. The barriers posed by scale-up are well understood in engineering but remain relatively new in biomedical research. The preliminary data presented below demonstrate our ability to collaborate across multiple sites. This work also provides an understanding of batch effects across clinical sites, such as systematic differences in practice, documentation, and patient populations. We have invested considerable time and effort in making the analysis of biochemical and clinical data repeatable and reproducible. The data presented below had to overcome challenges in patient sample processing, biobanking, biospecimen quality, and data harmonization

[30] . As a result, we have firsthand knowledge of how to pool data from multiple sources, ensure uniform and consistent processes, and clean and apply quality control metrics to the data received and processed.

[0522] (c) Preliminary data

[0523] Our prospective study evaluated the independent association of 2 fecal biomarkers, including NECDetect ( Fig.30, Panel B) (1.1±0.5 kg; 27.6±0.8 weeks gestational age; see inclusion report). In conclusion, our data show that the presence of high amounts of iAP protein and low iAP enzyme activity in stool are biomarkers of NEC. In effect, infants with NEC are 'shooting blanks' into the intestinal lumen to control abnormal microbial development. The iAP biomarkers were not associated with sepsis or other non-gastrointestinal infections.

[0524] Infants suspected of having NEC and those with advanced NEC disease released iAP proteins into the intestinal lumen at levels that exceeded those normally found in the human small intestine.

[0525] Baseline assessment of iAP detection was performed using immunoblotting. Immunoblotting allows determination of relative protein expression in complex biological samples. Development of sensitive antibody labeling with truly quantifiable linear range and greater limits of detection through digital image analysis has allowed detection of proteins at higher resolutions than previously achievable

[31] . To illustrate our sample preparation, assay protocol, and normalization approach, a calibration curve for our positive control human intestinal lysate ( Fig.31 , Panel A) shows that the linear portion of the anti-human iAP signal detection overlaps with our working range

[32] . The positive and negative controls (calf iAP) were from a single batch and used as calibrators for our quantification; both were loaded on each gel with patient samples. Equal amounts of total protein were loaded per lane.

[0526] High iAP protein levels were associated with NEC diagnosis and suspicion of NEC, but not with sepsis. We established baseline iAP levels, which are shed in the intestinal lumen and detected in feces of unaffected infants

[33] . Control patient feces had very low amounts of iAP (≤2%; Fig.31 , Panel B). We conclude that when dysbiosis is not impending, few iAPs flow into the intestinal lumen.

[0527] A large amount of fecal iAP protein was found at the time of clinical NEC diagnosis (red bars, Bell stages II and III; Fig.31 , Panel B). If there is a risk of bacterial-induced inflammation, fecal iAP levels will increase due to the release of iAP-loaded membrane vesicles [15,16]. Our data show that iAP enters the intestinal lumen and feces at levels comparable to or higher than those found in human intestinal lysate samples, making it a molecular biomarker superior to clinical diagnosis (X-ray evidence of intestinal gas). The sensitivity and specificity of fecal iAP levels as a biomarker for NEC are greater than 95% ( Fig.31 , panel C).

[0528] NEC suspicion or early NEC was also associated with high levels of iAP in stool (pink bars, Bell stage I; Fig.31 , Panel B). In stool samples of clinically concerned advanced NEC, the amount of iAP protein was statistically different from that of controls even though no intestinal gas was detected by X-ray (pink bars, Fig.31 , Panel B). However, NEC suspicion (pink bars) and diagnostic mean (red bars, Fig.31 Fecal iAP levels were not measurably associated with sepsis (blue bars, Fig.31 , Panels B and D). Severely preterm infants (<32 weeks) not only have an inadequately formed gut but also an immature immune system. Their increased risk of sepsis [34,35] can confound the diagnosis of NEC, as it is a common comorbidity with limited diagnostic tools. Our preliminary data showed no statistical correlation between iAP protein levels and the clinical diagnosis of sepsis.

[0529] Infants with advanced NEC disease had very low enzyme activity, whereas infants with suspected NEC had intermediate iAP catalytic activity.

[0530] We evaluated iAP enzyme activity in non-NEC infants. After normalization to protein concentration, we found decreased iAP activity in feces of preterm infants compared with infants close to term (36-40 weeks postconception or PCA). A strong positive correlation was found between iAP activity and postconception age ( Fig.31 , Panel E). When comparing IAP activity in PCA from 28-40 wks, a one-way ANOVA with Tukey's multiple comparison test was performed, indicating that the samples were statistically divided into two groups: preterm (PCA ≤ 35 wks) and term (PCA ≥ 36 wks). All fecal samples from term infants (n = 28) were compared with all samples from preterm infants (n = 79), and the latter group was found to have significantly lower iAP activity (p < 0.0001; one-tailed t-test). This is consistent with the switch between fetal and 'adult' iAP isoforms at this developmental time point: dynamic shifts in IAP isoenzyme forms are associated with maturation of the fetal intestine

[36] . Fetal isoforms of iAP have low biochemical activity, whereas adult iAPs have robust catalytic rates. Therefore, iAP activity is a biomarker that is directly related to postconception age. Appropriate design and analysis of biomarker studies requires such normative data across different gestational ages.

[0531] Low iAP activity was also associated with NEC diagnosis and NEC suspicion, but not sepsis. We found that iAP activity in infant feces collected on the same day as clinical NEC diagnosis was much lower than that in age-matched controls (red bars, Fig.31, panel F), indicating that very low iAP activity is associated with NEC. Evidence suggests that a reduced ability of the premature intestine to dephosphorylate proinflammatory LPS increases the risk of an exaggerated inflammatory response to bacterial colonization and the development of NEC

[37] . Consistent with the idea that biochemical measurement of iAP is a biomarker for NEC, iAP activity (pink bars, Fig.31 , Panel F) also showed low ratios, which may indicate NEC due to the inability to control bacterial colonization. Finally, measurements of iAP activity did not correlate with sepsis ( Fig.31 , Panel F and Panel H).

[0532] (d) Methods

[0533] Specific Aim 1: Do iAP polymorphisms predict NEC susceptibility? Without wishing to be bound by theory, infants diagnosed with NEC will have single nucleotide polymorphisms (SNPs) in the intestinal alkaline phosphatase gene ALPI that reduce their catalytic capacity for lipopolysaccharide-dependent signaling detoxification of harmful Gram-negative bacteria. Our preliminary data suggest that the enzymatic capacity of the single gene product iAP is associated with NEC disease development. In patients with extreme forms of NEC (Bell stages II and III), enzyme activity is almost absent and, therefore, these infants are unable to regulate TLR4-dependent IL-8 transcription ( Fig.30 , Panel A). In early NEC (Bell stage I), iAP enzyme activity was lower than in non-NEC-like Bell stage II and III infants ( Fig.31 , panel F).

[0534] Rationale. Genetic variation in TLR signaling has been studied because this receptor has been shown to play an important role in disease. Naturally occurring single base pair changes in the genome can alter protein function and disease processes, and SNPs in the TLR2, TLR4, TLR5, IRAK1, and TIRAP genes do not appear to be associated with NEC [38–45]. Understanding the mechanisms and causal relationships will be essential to identify the earliest possible treatment options and improve long-term outcomes and quality of life. This work will lay the foundation for ALPI screening for monogenic diseases and ALPI-based treatment of NEC. This ALPI polymorphism found in NEC patients will form the basis of Neonatal DDx, a screening tool for infants at birth.

[0535] Study inclusion. Preterm infants (both non-NEC and NEC) admitted to the Neonatal Intensive Care Units (NICUs) of the LSU School of Medicine and the Washington University School of Medicine will be included in this prospective study; single IRB and IBC approvals requiring parental consent for biospecimen collection and genetic testing have been obtained. Recruitment and inclusion of low birth weight (LBW) preterm infants (<2,500 g birth weight and / or <34 wks gestational age) is a process limited by the number of infants born at our hospital. We will enroll a minimum of 120 (LBW) infants in the first year, or a minimum of 5 infants per month at each site (see Human Subjects Inclusion Reports 1 and 2). We anticipate that 25-30 will develop NEC Bell Stage II / III, and an almost equal number will develop NEC Bell Stage I.

[0536] Clinical Information. This study is observational and will not require creation or deviation from standard clinical care in the NICU. The following clinical data will be obtained: demographics, medical history, antibiotics / antifungals / medications, physical examination, complete blood count, blood cultures, abdominal radiographs, surgical consultation notes, and feeding history. This study will be conducted in accordance with institutional, local, state, and federal regulations regarding the use of PHI, as defined by the Health Insurance Portability and Accountability Act.

[0537] Specimen Collection. DNA samples are collected non-invasively from a cheek swab or residual blood draw.

[0538] Methods. Genomic DNA will be isolated from (i) saliva swabs from infant cheeks or (ii) from peripheral blood cells or whole blood using the QIAamp DNA Blood Mini Kit. For gene sequencing, ALPI variants will be identified by Sanger sequencing of PCR products amplified from genomic DNA. We routinely use Eurofins to sequence mutations in human proteins (e.g.,

[46] ). PCR will be performed using AmpliTaq polymerase using the GeneAmp PCR System. The primer pairs used for DNA amplification are: 5'GGACCTTCAGTGGTTCCAGG-3' (f) and 5'CCAAGGACCTGGTTCTGGTC-3' (r). The list of variants identified by sequencing will be the subject of filtering procedures, such as exclusion of common variants in the population, low-quality variants, and synonymous changes. Sequence data can be compared with various public databases (database of single nucleotide polymorphisms (dbSNP

[47] ); 1000GenomesProject

[48] ; and Exome Variant Server[49,50]). Comparisons will look for rare variants that occur at a frequency of <1% in controls. Initially, inherited and de novo variants in NEC patients will be classified as Neonatal DDx.

[0539] Alkaline phosphatase activity will be measured using 4-methylumbelliferyl phosphate (MUP) as a fluorogenic substrate in the presence and absence of the iAP inhibitor 10 mM L-phenylalanine [51,52]. Relative fluorescence units at 360 / 440 nm will be measured in samples using 96-well black optical bottom plates. Total AP activity is measured in mU / mg, where U is the amount of enzyme that hydrolyzes 1 μmol of MUP per minute at pH 10 and 25°C. Total protein in fecal supernatants is measured using the Bradford assay. Protein standards (bovine serum albumin) and patient samples will be prepared using molecular grade water as the diluent. Standards will be run on each day of data collection and must have an r2 value greater than 0.99 to be accepted.

[0540] Results. Without wishing to be bound by theory, infants who develop NEC will have at least one allele with a non-conserved polymorphism that causes a loss-of-function phenotype. Lack of fecal iAP activity (<240 U / mg) will confirm that the ALPI mutation causes a loss-of-function phenotype. DNA sequencing will identify SNPs in the infant's ALPI gene; their relative amino acid positions can be identified using homology models of the AP crystal structure [53-55]. Sequencing of as few as 200 unrelated patients may identify disease genes in as few as 5% of the population

[56] . Without wishing to be bound by theory, mutations will occur in the active site and / or dimerization interface of the iAP [54,57].

[0541] Statistical strategies for identifying causative polymorphisms are based on the nature of the disease mutation

[58] . For example, statistical analyses will be conducted using a dominant model, comparing wild-type homozygotes to a combined set of heterozygotes and homozygotes for the rare allele. This assumes that carrying at least one copy of the variant allele increases the risk of disease. Primary outcome measures include the presence of NEC, severity of disease (Bell stage II / III versus Bell stage I), and NEC-associated intestinal perforation.

[0542] Hardy-Weinberg equilibrium will be determined using a chi-square test to compare the observed genotype frequencies with the frequencies expected under Hardy-Weinberg equilibrium. Ordinal logistic regression will be used to compare disease severity. The significance level will be set at p < 0.05.

[0543] Alternative approaches. Statistical evaluation of sequence data may require the use of a recessive model [56,59]. If polymorphisms associated with NEC susceptibility are found, future studies will involve linkage mapping and candidate gene analysis

[60] . For autosomal dominant diseases, association with defined disease intervals is determined by linkage analysis of large pedigrees (e.g., [61,62]). De novo dominant mutations can be identified by analysis of parent-offspring triads (e.g.,

[63] ) or by intersection of heterozygous variants in unrelated probands with the same de novo autosomal dominant disease (e.g.,

[64] ).

[0544] Although modest sample sizes for less common diseases in preterm infants may limit the power to detect associations, such studies are critical as a preliminary step toward providing targeted treatments for children, uncovering important insights into factors associated with disease risk, and identifying networks involved in disease pathogenesis.

[0545] Specific Aim 2: Can intestinal alkaline phosphatase protein levels in stool serve as a prognostic biomarker for NEC? The field of neonatology has been hampered by the inability to detect NEC early in the disease process. Without wishing to be bound by theory, microbial-induced inflammation in NEC results in increased levels of iAP protein in the intestinal lumen and stool. Infants with early NEC will have detectable amounts of iAP 36–48 h before NEC diagnosis if asymptomatic or presenting only nonspecific symptoms. Our data suggest that prognostication of NEC is feasible. Although our clinical focus has been on stool collection at disease diagnosis, 5 of 25 NEC Bell II / III patients and 9 of 19 NEC Bell I infants had incidental stool samples collected before disease onset; 19 of 62 non-NEC patients had a median of 4 samples collected before 31 weeks. We will collect stool samples from each infant in a longitudinal series, measure iAP content in samples twice weekly, and model the prognostic power of the continuous biomarker NECPredict.

[0546] Statistical considerations. For this objective, the study inclusion and clinical information plan detailed in objective 1 will be followed. We require 150 subjects to achieve 90% power for objective 2. This is based on simulation results, as existing sample size calculations for prognostic biomarkers [65-67] only apply to differences between two groups

[68] . We simulated studies using four different combinations (α#, α%) and examined our probability of detecting a significant difference between these coefficients and 0. We achieved power values ​​greater than 0.8, 0.8, 0.9, and 0.95 for n=50, 100, 150, or 200. This power analysis suggests that a sample size of n>100 is required and that n=150 can provide a significant benefit compared to n=100.

[0547] Specimen collection and preparation. Continuous monitoring using novel protein biomarkers requires stool collection from study enrollment to hospital discharge. There is no identifiable risk to the patient, as noninvasive sampling from discarded diapers is painless and does not cause harm to fragile patients. Stool specimens will be collected every 3–4 days

[69] until the infant reaches 37 weeks postconception or is discharged from the hospital. The average length of stay for infants is estimated to be 49 days

[70] to 54 days (see preliminary data); for each infant, approximately 15 samples will be collected from diapers. If a stool sample was collected between the first day of radiographic discovery of NEC (Bell stage II or III) and the last day of NEC management (antibiotics administered and no oral intake), it was termed ‘NEC’. Samples collected from the first day of 2+ clinical symptoms to the last day of medical management were termed ‘suspected’. If a sample was obtained on a day when a diagnosis of NEC was not made, it was termed ‘control’.

[0548] Stool was stored in a specimen NICU refrigerator at 4°C until samples were sent to the laboratory. Upon receipt of each de-identified patient sample, stool was homogenized and slurried to 200 mg / mL in molecular-grade water in sterile microcentrifuge tubes. After vortexing and centrifugation, the supernatant was collected, aliquoted, and stored at −80°C

[71] . Safe handling (gloves, lab coat, goggles), use of absorbent tissue, decontamination with an EPA-registered hospital disinfectant, and proper disposal of biohazards were followed.

[0549] Determination of relative iAP protein content. Duplicate denaturing SDS-PAGE gels will be run on fecal supernatants to visualize all proteins in each lane and used for immunoblot detection of iAPs. iBlot and iBind will be used for protein transfer and western blotting, respectively. Bands will be quantified using Amersham Imager 600; the relative iAP protein in fecal samples is the fraction of protein found in human intestinal lysate tissue.

[0550] Arguably, the greatest source of confounding in quantitative immunoblotting is the role of protein loading and loading controls

[72] . Immunoblotting samples are often prepared based on total protein [73–75], which assumes that the average protein content per cell is constant across conditions. However, in our analyses, fecal samples were not fractionated to lyse cells: only the luminal content was assessed. Therefore, total cellular protein could not be determined, and the input had to be normalized to some estimate of protein loading. We used two loading controls as well as total protein [76–81].

[0551] exist Fig.31 In panel A, control experiments assess the accuracy and precision of the immunoblotting workflow. Such experiments minimize overestimation or underestimation of true differences in protein abundance. Serial dilutions of the positive control (small intestinal tissue lysate) and the negative control (purified bovine iAP) demonstrate our dynamic range and quantitative accuracy. The 60 kD iAP signal from the patient sample was determined; this value is proportional to the difference between the positive and negative control measurements.

[0552] Results. Without wishing to be bound by theory, we will detect iAP levels of 0.14 ± 0.10 (mean ± SE) compared to human small intestinal lysate from samples collected 7 days before NEC. Our preliminary data provide a threshold for expected immunoblot values. In stool samples at the time of NEC diagnosis, iAP levels were 1.59 ± 0.48 higher than in human small intestinal lysate. iAP levels in stool from non-NEC patients were 0.02 ± 0.01. As these protocols are established and have been successfully performed by 3 different operators, we do not anticipate technical issues.

[0553] Using NECPredict, we will model the probability of a patient developing symptoms or being diagnosed with NEC based on the amount of iAP in stool samples collected 3-5 days ago and 6-8 days before. In this test of the prognostic power of a continuous biomarker, the mean difference in relative iAP protein content between NEC and control samples will be tested by modeling the association between the probability of a NEC diagnosis and the amount of iAP on any given day in diapers 3-5 days ago and in the previous diapers (6-8 days before).

[0554] Specifically, NEPCredict uses a generalized linear mixed effects model

[82] with NEC diagnosis as the response, iAP content in the last two collected samples as predictors (fixed effects), and a patient-level error term. We model whether a patient has signs of NEC or is diagnosed with NEC as follows: logit{P[NEC t,i =1]}=α 1 D t-1,i +α 2 Dt-2,i +β+ε i , where NEC t,i = 0 if patient i was NEC negative at diaper collection time t, and 1 if patient had NEC diagnosis or NEC signs / diagnosis (analyzed separately). t-1,I and D t-2,I is the iAP content of the last two diapers collected. i is a subject-specific error term that captures the time and individual dependence of NEC diagnosis and diaper content. We used the lme4 package in R

[83] to analyze this model. The significant positive estimate α 1 showed that high iAP content in the last collected diaper (approximately 3 days ago) predicted future NEC diagnosis and α 2 >0 indicates that high iAP in the previous week predicts future NEC diagnosis. This information suggests that iAP can be used as a prognostic biomarker for NEC.

[0555] In summary, in vitro results and clinical data will confirm the association between these markers and NEC risk. Without wishing to be bound by theory, the ALPI polymorphism results in low iAP activity and Neonatal DDx can be used as a biomarker to identify infants at greatest risk for NEC. Second, the presence of iAP protein in the form of NEC Predict will have the strongest prognostic value and will identify the development of NEC before symptoms develop. These studies will be the first biochemical and physiological markers to link two NEC triggers: alterations in the microbiome and intestinal development. Another important outcome is that indiscriminate feeding withholding and broad-spectrum prophylactic antibiotics may be minimized. Thus, these studies provide the first laboratory tests to personalize therapy in the NICU. Future directions may include supplementation with iAP as a preventive strategy for NEC, as enzyme replacement therapy is a low-cost, low-risk approach for rare diseases that is often successful

[84] . Finally, these personalized biomarker approaches are not limited to children; iAP as a biomarker of intestinal inflammation in infants is similar to that in adult diseases such as IBD.

[0556] References cited in this example

[0557] 1. Hackam, D. and M. Caplan, Necrotizing enterocolitis: pathophysiology from a historical context. SeminPediatr Surg, 2018.27(1):p.11-18.

[0558] 2. Bell, M. J., Neonatal necrotizing enterocolitis. N Engl J Med, 1978. 298(5): p. 281-2.

[0559] 3. Kliegman, R. M. and M. C. Walsh, Neonatal necrotizing enterocolitis: pathogenesis, classification, and spectrum of illness. Curr Probl Pediatr, 1987. 17(4): p. 213-88.

[0560] 4. Lin, P. W., T. R. Nasr and B. J. Stoll, Necrotizing enterocolitis: recent scientific advances in pathophysiology and prevention. Semin Perinatol, 2008. 32(2): p. 70-82.

[0561] 5. Hunter, C. J., et al., Understanding the susceptibility of the premature infant to necrotizing enterocolitis (NEC). Pediatr Res, 2008. 63(2): p. 117-123.

[0562] 6. Neu, J. and W. A. Walker, Necrotizing enterocolitis. New England Journal of Medicine, 2011. 364(3): p. 255-264.

[0563] 7. Patel, J. C., et al., Neonatal necrotizing enterocolitis: The long-term perspective / discussion. Amer Surgeon, 1998. 64(6): p. 575.

[0564] 8. Tam, A. L., A. Camberos, and H. Applebaum, Surgical decision making in necrotizing enterocolitis and focal intestinal perforation: predictive value of radiologic findings. J Pediatr Surg, 2002. 37(12): p. 1688 - 91.

[0565] 9. Lin, P. W. and B. J. Stoll, Necrotising enterocolitis. Lancet, 2006. 368(9543): p. 1271 - 83.

[0566] 10. Fawley, J. and D. M. Gourlay, Intestinal alkaline phosphatase: a summary of its role in clinical disease. J Surg Res, 2016. 202(1): p. 225 - 34.

[0567] 11. Stewart, C. J., et al., Preterm gut microbiota and metabolome following discharge from intensive care. Sci Rep, 2015. 5: p. 17141.

[0568] 12. Stewart, C. J., et al., The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection. Acta Paediatr, 2012. 101(11): p. 1121 - 7.

[0569] 13. Rusconi, B., M. Good, and B. B. Warner, The microbiome and biomarkers for necrotizing enterocolitis: are we any closer to prediction? J Pediatr, 2017. 189: p. 40 - 47e2.

[0570] 14. Goldberg, R.F., et al., Intestinal alkaline phosphatase is a gut mucosal defense factor maintained by enteral nutrition. Proc Natl Acad Sci U SA, 2008. 105(9): p. 3551 - 6。

[0571] 15. Shifrin, D.A., Jr., et al., Enterocyte microvillus - derived vesicles detoxify bacterial products and regulate epithelial - microbial interactions. Curr Biol, 2012. 22(7): p. 627 - 31。

[0572] 16. Shifrin, D.A., Jr. and M.J. Tyska, Ready... aim... fire into the lumen: a new role for enterocyte microvilli in gut host defense. Gut Microbes, 2012. 3(5): p. 460 - 2。

[0573] 17. Jilling, T., et al., The roles of bacteria and TLR4 in rat and murine models of necrotizing enterocolitis. J Immunol, 2006. 177(5): p. 3273 - 82。

[0574] 18. Leaphart, C.L., et al., A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair. J Immunol, 2007. 179(7): p. 4808 - 20。

[0575] 19. Gribar, S.C., et al., Reciprocal expression and signaling of TLR4 and TLR9 in the pathogenesis and treatment of necrotizing enterocolitis. J Immunol, 2009. 182(1): p. 636-46。

[0576] 20. Good, M., et al., Breast milk protects against the development of necrotizing enterocolitis through inhibition of Toll-like receptor 4 in the intestinal epithelium via activation of the epidermal growth factor receptor. Mucosal Immunol, 2015. 8(5): p. 1166-79。

[0577] 21. Thomas, D.W. and D.H. Henton, The use of fecal alkaline phosphatase as an indicator of intestinal damage. Digestion, 1985. 31(2-3): p. 82-8。

[0578] 22. Goldman, J., et al., Development of biomarkers to optimize pediatric patient management: what makes children different? Biomark Med, 2011. 5(6): p. 781-94。

[0579] 23. Smith, P.B., et al., Safety monitoring of drugs receiving pediatric marketing exclusivity. Pediatrics, 2008. 122(3): p. e628-33。

[0580] 24.Benjamin,D.K.,Jr.,et al.,Safety and transparency of pediatric drugtrials.ArchPediatrAdolesc Med,2009.163(12):p.1080-6。

[0581] 25.Williams,K.,et al.,Standard 6:age groups for pediatrictrials.Pediatrics,2012.129(Suppl 3):p.S153-60。

[0582] 26.Laventhal,N.,B.A.Tarini,and J.Lantos,Ethical issues in neonataland pediatric clinical trials.Pediatr Clin North Am,2012.59(5):p.1205-20。

[0583] 27.Joseph,P.D.,J.C.Craig,and P.H.Caldwell,Clinical trials inchildren.Br J Clin Pharmacol,2015.79(3):p.357-69。

[0584] 28.Kern,S.E.,Challenges in conducting clinical trials in children:approaches for improving performance.Expert Rev Clin Pharmacol,2009.2(6):p.609-617。

[0585] 29.Institute of Medicine.Envisioning a Transformed Clinical TrialsEnterprise in the United States:Establishing An Agenda for 2020:WorkshopSummary.2012:Washington(DC);The NationalAcademies Press。

[0586] 30. Lee, J. S., W. A. Kibbe, and R. L. Grossman, Data harmonization for a molecularly driven health system. Cell, 2018. 174(5): p. 1045 - 1048.

[0587] 31. Janes, K. A., An analysis of critical factors for quantitative immunoblotting. Sci Signal, 2015. 8(371): p. rs2.

[0588] 32. Lee, J. W., et al., Fit - for - purpose method development and validation for successful biomarker measurement. Pharm Res, 2006. 23(2): p. 312 - 28.

[0589] 33. Lichtman, J. S., et al., Host - centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota. Mol Cell Proteomics, 2013. 12(11): p. 3310 - 8.

[0590] 34. Arboleya, S., et al., Establishment and development of intestinal microbiota in preterm neonates. FEMS Microbiol Ecol, 2012. 79(3): p. 763 - 72.

[0591] 35. Arboleya, S., et al., Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics. J Pediatr, 2015. 166(3): p. 538 - 44.

[0592] 36. Mulivor, R.A., V.L. Hannig, and H. Harris, Developmental change in human intestinal alkaline phosphatase. Proc Natl Acad Sci U S A, 1978. 75(8): p. 3909-12。

[0593] 37. Milani, C., et al., The first microbial colonizers of the human gut: composition, activities, and health implications of the infant gut microbiota. Microbiol Mol Biol Rev, 2017. 81(4)。

[0594] 38. Cuna, A., L. George and V. Sampath, Genetic predisposition to necrotizing enterocolitis in premature infants: current knowledge, challenges, and future directions. Semin Fetal Neonatal Med, 2018。

[0595] 39. Sampath, V., et al., A functional ATG16L1(T300A) variant is associated with necrotizing enterocolitis in premature infants. Pediatr Res, 2017. 81(4): p. 582-588。

[0596] 40. Sampath, V., et al., Necrotizing enterocolitis is not associated with sequence variants in antioxidant response genes in premature infants. J Pediatr Gastroenterol Nutr, 2016. 62(3): p. 420-3。

[0597] 41. Franklin, A. L., et al., Are immune modulating single nucleotide polymorphisms associated with necrotizing enterocolitis?Sci Rep, 2015.5: p. 18369。

[0598] 42. Sampath, V., et al., SIGIRR genetic variants in premature infants with necrotizing enterocolitis. Pediatrics, 2015.135(6): p. e1530-4。

[0599] 43. Markel, T. A., H. Engelstad and B. B. Poindexter, Predicting disease severity of necrotizing enterocolitis: how to identify infants for future novel therapies. J Clin Neonatol, 2014.3(1): p. 1-9。

[0600] 44. Sampath, V., et al., The NFKB1(g.-24519delATTG) variant is associated with necrotizing enterocolitis (NEC) in premature infants. J Surg Res, 2011.169(1): p. e51-7。

[0601] 45. Bokodi, G., et al., Association of interferon gamma T+874A and interleukin 12p40 promoter CTCTAA / GC polymorphism with the need for respiratory support and perinatal complications in low birthweight neonates. Arch Dis Child Fetal Neonatal Ed, 2007.92(1): p. F25-9。

[0602] 46. Richard, J., et al., Allostery wiring map for kinesin energy transduction and its evolution. J Biol Chem, 2016. 291(40): p. 20932 - 20945。

[0603] 47. Sherry, S. T., et al., dbSNP: the NCBI database of genetic variation. Nucleic Acids Res, 2001. 29(1): p. 308 - 11。

[0604] 48. Genomes Project, C., et al., A map of human genome variation from population - scale sequencing. Nature, 2010. 467(7319): p. 1061 - 73。

[0605] 49. International HapMap, C., et al., Integrating common and rare genetic variation in diverse human populations. Nature, 2010. 467(7311): p. 52 - 8。

[0606] 50. Cooper, G. M. and J. Shendure, Needles in stacks of needles: finding disease - causal variants in a wealth of genomic data. Nat Rev Genet, 2011. 12(9): p. 628 - 40。

[0607] 51. Ghosh, N. K. and W. H. Fishman, On the mechanism of inhibition of intestinal alkaline phosphatase by L - phenylalanine. I. Kinetic studies. J Biol Chem, 1966. 241(11): p. 2516 - 22。

[0608] 52. Ghosh, N.K. and W.H. Fishman, L-phenylalanine inhibiton of rat intestinal alkaline phosphatase: a homosteric phenomenon. Arch Biochem Biophys, 1968. 126(2): p. 700-6。

[0609] 53. Le Du, M.H., et al., Crystal structure of alkaline phosphatase from human placenta at 1.8A resolution. Implication for a substrate specificity. J Biol Chem, 2001. 276(12): p. 9158-65。

[0610] 54. Hoylaerts, M.F., et al., Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment. Biochemistry, 2006. 45(32): p. 9756-66。

[0611] 55. Ghosh, K., et al., Crystal structure of rat intestinal alkaline phosphatase--role of crown domain in mammalian alkaline phosphatases. J Struct Biol, 2013. 184(2): p. 182-92。

[0612] 56. Zhi, D. and R. Chen, Statistical guidance for experimental design and data analysis of mutation detection in rare monogenic mendelian diseases by exome sequencing. PLoS One, 2012. 7(2): p. e31358。

[0613] 57. Fosset, M., D. Chappelet-Tordo and M. Lazdunski, Intestinal alkaline phosphatase. Physical properties and quaternary structure. Biochemistry, 1974. 13(9): p. 1783-8。

[0614] 58. Boycott, K.M., et al., Rare-disease genetics in the era of next-generation sequencing: discovery to translation. Nat Rev Genet, 2013. 14(10): p. 681-91。

[0615] 59. Walsh, T., et al., Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet, 2010. 87(1): p. 90-4。

[0616] 60. Sunyaev, S.R., Inferring causality and functional significance of human coding DNA variants. Hum Mol Genet, 2012. 21(R1): p. R10-7。

[0617] 61. Ng, S.B., et al., Exome sequencing identifies the cause of a mendelian disorder. Nat Genet, 2010. 42(1): p. 30-5。

[0618] 62. Fiskerstrand, T., et al., Familial diarrhea syndrome caused by an activating GUCY2C mutation. N Engl J Med, 2012. 366(17): p. 1586-95。

[0619] 63. Gibson, W. T., et al., Mutations in EZH2 cause Weaver syndrome. Am J Hum Genet, 2012. 90(1): p. 110 - 8。

[0620] 64. Hood, R. L., et al., Mutations in SRCAP, encoding SNF2 - related CREBBP activator protein, cause Floating - Harbor syndrome. Am J Hum Genet, 2012. 90(2): p. 308 - 13。

[0621] 65. Schmoor, C., W. Sauerbrei, and M. Schumacher, Sample size considerations for the evaluation of prognostic factors in survival analysis. Stat Med, 2000. 19(4): p. 441 - 52。

[0622] 66. Tsim, S., et al., Diagnostic and prognostic biomarkers in the rational assessment of mesothelioma (DIAPHRAGM) study: protocol of a prospective, multicentre, observational study. BMJ Open, 2016. 6(11): p. e013324。

[0623] 67. Chen, D. T., et al., Strategies for power calculations in predictive biomarker studies in survival data. Oncotarget, 2016. 7(49): p. 80373 - 80381。

[0624] 68. Dang, Q., S. Mazumdar and P. R. Houck, Sample size and power calculations based on generalized linear mixed models with correlated binary outcomes. Comput Methods Programs Biomed, 2008. 91(2): p. 122-7。

[0625] 69. Rose, C., et al., The characterization of feces and urine: a review of the literature to inform advanced treatment technology. Crit Rev Environ Sci Technol, 2015. 45(17): p. 1827-1879。

[0626] 70. Holman, R. C., et al., Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatric and perinatal epidemiology, 2006. 20(6): p. 498-506。

[0627] 71. Flores, R., et al., Collection media and delayed freezing effects on microbial composition of human stool. Microbiome, 2015. 3: p. 33。

[0628] 72. Marcus, E., Credibility and reproducibility. Cell, 2014. 159(5): p. 965-966。

[0629] 73. Olson, B. J. and J. Markwell, Assays for determination of protein concentration. Curr Protoc Protein Sci, 2007. Chapter 3: p. Unit 34。

[0630] 74. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976. 72: p. 248-54。

[0631] 75. Zor, T. and Z. Selinger, Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem, 1996. 236(2): p. 302-8。

[0632] 76. Jensen, K.J., et al., An ERK-p38 subnetwork coordinates host cell apoptosis and necrosis during coxsackievirus B3 infection. Cell Host Microbe, 2013. 13(1): p. 67-76。

[0633] 77. Kang, B.H., et al., Simultaneous profiling of 194 distinct receptor transcripts in human cells. Sci Signal, 2013. 6(287): p. rs13。

[0634] 78. Bose, A.K. and K.A. Janes, A high-throughput assay for phosphoprotein-specific phosphatase activity in cellular extracts. Mol Cell Proteomics, 2013. 12(3): p. 797-806。

[0635] 79. Eaton, S.L., et al., Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PLoS One, 2013. 8(8): p.e72457。

[0636] 80. Collins, M.A., et al., Total protein is an effective loading control for cerebrospinal fluid western blots. J Neurosci Methods, 2015. 251: p.72 - 82。

[0637] 81. Moritz, C.P., Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics, 2017. 17(20)。

[0638] 82. Breslow, N. and D. Clayton, Approximate inference in generalized linear mixed models. Journal of the American Statistical Association, 1993. 88(421): p.9 - 25。

[0639] 83. Bates, D., et al., Fitting Linear Mixed - Effects Models Using lme4. Journal of Statistical Software, 2015. 67(1): p.1 - 48。

[0640] 84. Ohashi, T., Enzyme replacement therapy for lysosomal storage diseases. Pediatr Endocrinol Rev, 2012. 10(Suppl 1): p.26 - 34。

[0641] Statistical design and power of clinical studies on the prognosis of NEC

[0642] Statistical analyses will be performed using R (R Core Team, 2018. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). Significance tests are two-tailed tests performed at the 5% significance level. Statistical assumptions will be tested and models will be modified appropriately if necessary. Missing data will be handled by imputation and sensitivity analysis. If the proposed statistical analysis technique is found to be untenable, we will use alternative techniques, possibly falling back on strategies that guarantee the provision of appropriate estimates as well as nonparametric measures of variability.

[0643] Study Design for Aim 2: This study will be a prospective observational study in which preterm infants are enrolled at birth. Fecal samples from disposable diapers will be collected every 3-4 days until the infant reaches 37 weeks post conception or is discharged from the hospital. Fecal samples from patients with clinically apparent NEC as well as a random subset of fecal samples from control patients will be analyzed.

[0644] Sample size: For Aim 2, we required 150 subjects to achieve 90% power. This was based on simulation results, as existing sample size calculations only apply to the difference between two groups [1]. In contrast, our model requires testing the prognostic power of continuous biomarkers.

[0645] We model the probability that a patient has signs or has been diagnosed with NEC based on the amount of iAP in a stool sample collected 3-5 days ago and the amount of iAP in a stool sample collected 6-8 days ago. We allow each patient to have a different level of susceptibility to NEC, which is captured in the random effect term for patient. The random effect for patient is combined with the effect for iAP amount and transformed by a function (the inverse of the logit function) that constrains the values ​​to be between 0 and 1, allowing us to estimate the probabilities.

[0646] Using the sign term, we model the probability that a patient shows signs of NEC or has been diagnosed with NEC as:

[0647] logit{P[NEC t,i =1]}=α 1 D t-1,i +α 2 D t-2,i +β+ε i

[0648] NEC t,i= 0 if patient i was NEC negative at diaper collection time t, and 1 if patient had evidence of NEC (Bell stage I) or a diagnosis of NEC (Bell stage II / III). t-1,i and D t-2,i is the iAP content of the last two diapers collected. i is a subject-specific error term that incorporates the correlation of the probability of NEC diagnosis within individuals. We modeled the above using generalized linear mixed models using the R program lme4. To determine how many patients we should recruit, we performed a simulation study to determine to what extent we could detect a significant difference (the effect size of the last two diaper iAP content on the probability of future NEC) in α4 and α8 based on the last two collected diapers starting from 0, which would show the prognostic impact of iAP content in predicting a NEC diagnosis or signs of NEC. This information could help clinicians predict whether a patient is at high risk for NEC.

[0649] We perform simulations using four different combinations (α4, α8) and examine the probability that we will detect significant differences from 0 for these coefficients. Fig.32 The implied probability of NEC diagnosis / suspicion in a patient together with the mean iAP content of the NEC and non-NEC patient groups is shown in FIG, which are 1.59 and 0.02, respectively.

[0650] These four scenarios represent different effects of iAP diaper content on the probability of NEC diagnosis. In scenarios 1 and 2, only the last diaper can predict whether the patient will have NEC in the next diaper, because α 2 =0. The separation between the NEC probabilities of the high iAP group and the low iAP group is greater in scenario 1 than in scenario 2. Similarly, scenarios 3 and 4 have different probability differences for the two groups. For scenarios 3 and 4, α 2 = 1, indicating that the amount of iAP found in diapers collected two time points earlier (six to eight days) is predictive of NEC status. These values ​​were chosen in part because they resulted in 9-12% of patients having a NEC diagnosis in each scenario, which is consistent with the incidence of NEC diagnoses seen previously. Using simplifying assumptions, we simulated 1,000 replicates of each of the four scenarios with sample sizes of n = 50, 100, 150, or 200.

[0651] For each patient, we assumed diaper collection every two weeks for 2 months of follow-up, for a total of approximately 16 diaper collections per patient. We will assess NEC status on diaper collection days 3-16 to understand how iAP content in the last two diaper cycles predicts a NEC diagnosis. To perform simulations, we first draw the NEC+ indicator from a Bernoulli distribution with probability .09, which is the empirical probability that all previously tested diapers have NEC. For each NEC patient, we generate their 16 iAP diaper content values ​​D from a multivariate lognormal distribution with a mean vector of -3.90, a standard deviation of 1.69, and a positive correlation between each observed iAP value. 1,i ,…,D 16,i To generate the multivariate lognormal samples, we generated a multivariate normal distribution using the mean vector and implicit covariance matrix described above and then exponentiated these values. For individuals who were not considered NEC positive, we generated their iAP values ​​D from a multivariate lognormal distribution with a mean vector of -.68, a standard deviation of 1.57, and a positive correlation between each observed iAP value. 1,i ,…,D 16,i These means and standard deviations were chosen because they are estimated maximum likelihood estimates of the log-normal distribution using iAP values ​​for NEC+ and NEC- patients, respectively.

[0652] We then generate the probability of NEC for the patient at time t=3,…,16 as α 1 D t-1,i +α 2 D t-2,i +β, and the NEC state is drawn from a Bernoulli random variable with this probability. Fig.33 Shows the correctly declared α in the simulation for each sample size 1 >0 probability, correct statement α 1 >0 and α 2 >0. For cases 1 and 2, this triple is listed as (P 1 ,-,-), while for cases 2 and 3, this triple is listed as (P 1 ,P 2 ,P 3 ).

[0653] From the simulations above, we see that for n = 50, none of the 4 cases had a power above .80. For n = 100, we achieved a power above .80 in each case, but only detected α 1 and α 2There is a significant difference between the two, with a probability of .819 in scenario 3. For n = 150, our power is above .9 for each of the four scenarios, and for n = 200, our power is above .95 for each of the four scenarios. This power analysis suggests that a sample size of n ≥ 100 is desired and that n = 150 can provide a significant benefit over n = 100.

[0654] References cited in this example

[0655] 1.Dang, Q., S.Mazumdar and PRHouck, Sample size and power calculations based on generalized linear mixed models with correlated binary outcomes. Comput Methods Programs Biomed, 2008.91(2):p.122-7.

[0656] Example 12

[0657] There are four different tissue-specific alkaline phosphatases in humans: intestinal alkaline phosphatase, placental alkaline phosphatase, tissue nonspecific alkaline phosphatase, and germ cell alkaline phosphatase. At the amino acid level, tissue-specific alkaline phosphatase isozymes are 86-98% identical to each other, but 52-56% identical to tissue nonspecific alkaline phosphatase. In addition, the iAP gene ALPI has 403 missense polymorphisms covering the entire sequence: more than 50% of the amino acids in iAP have at least one known mutation.

[0658] Our innovation is the use of two biochemical indices of intestinal alkaline phosphatase (iAP) excreted in the feces as molecular biomarkers of NEC in preterm infants. The appeal of iAP as a biomarker lies in its tissue-specific expression in the small intestine and its secretion into the intestinal lumen, which can only be measured in the feces as a response to control bacterial colonization. In addition, it can be detected in human fecal samples from healthy individuals; is responsible for the majority of AP enzyme activity in the feces; and is used as a measure of toxic injury to the small intestine in animal models.

[0659] (A) Immunoassay-based detection of high iAP levels is a biomarker for necrotizing enterocolitis but not for sepsis. Our prospective study evaluated this in human preterm infants. We evaluated the independent associations of 2 fecal biomarkers, including NECDetect, in 136 preterm infants [mean gestational age = 28.3 weeks; 50% female; 64% African American, 32% white; 4% Hispanic]. High levels of fecal iAP protein were associated with a clinical NEC diagnosis; these levels were equal to or higher than those found in human small intestinal enterocytes. If there is a risk of bacterial-induced inflammation, fecal iAP levels would be expected to be increased due to the release of iAP-loaded membrane vesicles. In contrast, fecal iAP protein levels in nondiseased patients are very low. Thus, when dysbiosis is not imminent, little iAP flows into the intestinal lumen. Fecal iAP levels had a sensitivity and specificity of >95%. Unlike other candidate NEC biomarkers, fecal iAP levels have no measurable correlation with other non-gastrointestinal infections or sepsis, a common comorbidity that may confound the diagnosis of NEC.

[0660] (B) iAP is the only human alkaline phosphatase that can be recovered from fecal proteomics, and several candidate peptides are available for absolute quantification of iAP abundance by mass spectrometry. The combination of liquid chromatography and tandem mass spectrometry (LC-MS / MS) provides a flexible and dynamic platform for the simultaneous identification and quantification of up to thousands of proteins in fecal samples. Our initial shotgun proteomic analysis of preterm fecal samples showed that 635 human proteins were detected in the intestinal luminal contents or secreted host proteome. This is consistent with (i) 612 proteins identified in gnobiotic mice and (ii) 234 human proteins identified in adult feces. 142 We confirmed that there are 21 unique tryptic iAP peptides ( Fig.34 ) and these peptides cover 50% of the iAP sequences. Our data are consistent with previous human proteome mapping studies but exceed their reported 32% protein coverage. Importantly, no other human alkaline phosphatases were recovered from infant feces.

[0661] (C) The high polymorphism frequency of the iAP gene in the African American population, which may be associated with a higher incidence of disease, may lead to spurious results in affinity-based and MS-based protein measurements. Sequence information from unrelated individuals was used to determine the frequency distribution of iAP polymorphisms. iAP polymorphisms that were present only in the African American population (n = 12,487) or were present in >1% of the population ( Fig.34, panel B). The estimated frequencies of the common alleles V20I, R33L, R92C, R144H, and T207I are 4.8, 2.6, 1.9, 4.2, and 3.1%, respectively. The polymorphisms in the total population (blue circles, Fig.34 , Panel C) causes changes in peptide mass through changes in side chain molecular weight. Two polymorphisms common in the African American population (purple circles, Fig.34 , panel C) results in loss of the trypsin cleavage site, which in turn results in a hundred-fold change in peptide mass. Peptide mass changes, regardless of size, hamper MS identification and accurate quantification of proteins. These data raise concerns not only about the validity of correlating SNPs to affinity-based protein measurements, but also about MS techniques that may give spurious results when minor allele frequencies approach 5%.

[0662] Example 13

[0663] Necrotizing enterocolitis (NEC) is a common neonatal gastrointestinal (GI) emergency with significant mortality and long-term morbidity, including short bowel syndrome, nutritional deficiencies, and neurodevelopmental delays. 2,3 Suspected NEC presents with mild, nonspecific symptoms that frequently resolve with minimal intervention; no clinical test is an established criterion for suspected NEC. Radiologic evidence, such as intestinal gas, is used to diagnose severe or advanced disease but has a low sensitivity of 44%, limited specificity,5 and lacks consistency in interpretation. 6-8

[0664] Many efforts have been made to discover molecular diagnostic biomarkers for NEC ( Fig.35 Panel A). Despite the publication of more than 2500 prior biomarker studies, meta-analyses have failed to identify the best NEC biomarker for routine clinical use. 9-11 The design and power of these studies have raised concerns: fewer than 30 articles per decade of analysis were considered suitable for meta-analysis. The focus on inflammatory and repair proteins in these studies is problematic ( Fig.35 Panel B). Advanced disease with systemic inflammatory damage is not ideal for biomarker assessment because a reversible phase of disease cannot be defined. 12 Furthermore, the positive predictive value of inflammation-related proteins is limited because sepsis is a comorbidity in 35% to 60% of NEC cases. 13-17

[0665] Necrotizing enterocolitis has been implicated as an antecedent in some cases of late-onset neonatal sepsis (LOS). Neonates, especially very low birth weight infants, are susceptible to sepsis due to prolonged hospital stay, invasive instrumentation, underdeveloped innate immunity, and altered immune responses. The latter 2 physiological states, combined with immature intestinal barrier function, can lead to NEC. 18,19 From an epidemiological and clinical perspective, sepsis may confound the use of inflammatory proteins as biomarkers for NEC. Sepsis and NEC require careful differential diagnosis, as both can be fatal if not properly diagnosed and treated.

[0666] This study evaluated the use of intestinal alkaline phosphatase (IAP) as a diagnostic biomarker for NEC. Recent findings suggest that NEC is preceded and accompanied by changes in the intestinal microbiota ( Fig.35 Panel C) and is associated with host immune pathways that lead to intestinal inflammation. 19,20 Intestinal alkaline phosphatases detoxify surface lipopolysaccharide (LPS) of harmful bacteria by cleaving inorganic phosphates. LPS is a component of the cell wall of Gram-negative bacteria and a potent inducer of innate immune signaling through Toll-like receptor 4. Strong IAP function can neutralize LPS signaling, prevent inappropriate proinflammatory signaling cascades in the intestine, and contribute to the maturation of beneficial microbiota.

[0667] Because IAP activity precedes the initiation of signaling cascades that trigger inflammation, we assessed the abundance and enzymatic activity of IAPs in stool as a measure of pathobiological requirements and the ability to maintain host-microbiota homeostasis, respectively. A multicenter, prospective diagnostic study was conducted to evaluate the association of 2 IAP biochemical markers with disease severity. As common core proteins in the human fecal proteome,21 IAPs are ideal for noninvasive testing. If there is a risk of bacterial-induced inflammation, the amount of IAPs in stool would be expected to be increased due to the release of IAP-loaded membrane vesicles. 22,23

[0668] method

[0669] Study Design Methods: Preterm infants born at less than 37 weeks of gestational age and birth weight less than 1500 g were recruited during a 3-year period (May 2015 to November 2018) at Children's Hospital New Orleans (n ​​= 29; New Orleans, LA) and Touro Infirmary Hospital (n = 68; New Orleans, LA). Preterm infants born at less than 37 weeks of gestational age were recruited at Saint Louis Children's Hospital (n = 39; Saint Louis, MO). Written informed consent from study participants was obtained from parents or guardians. All infants were asked to be included in the study, thus forming a consecutive sampling series.

[0670] De-identified clinical data Clinical data were extracted from medical records every 3 months, including gestational age, birth weight, Apgar score, type of delivery, race / ethnicity, sex, and disposition (ie, death, discharge, or transfer to another institution). Of these, only race / ethnicity was defined by the parents. Hospitalization data included feedings, antibiotic therapy, laboratory and radiological results, and surgical records. Clinical findings for NEC (modified Bell stages 1-3), sepsis, and other confirmed non-gastrointestinal infections were reviewed by the attending physician.

[0671] To protect confidentiality and anonymity, each recruited patient was given a code that allowed for study tracking and removed any clues to personal identity. Every three months, patient records were evaluated to determine clinical relevance. Clinical data were extracted from medical records into relevant clinical databases. Demographic information and initial clinical data included gestational age, birth weight, Apgar score, type of delivery, race, sex, and final outcome (death, discharge, or transfer). Finally, a second set of clinical information was obtained: antibiotic use, diet, serum AP, radiology reports, NICU length of stay, surgery, and mortality. Human milk exposure was calculated as the mean percentage of feeding from human milk as a function of the total number of days the subject was in the study. For NEC cases, only pre-event exposure to human milk was considered. Antibiotic exposure was considered in aggregate; antibiotics were always given to the subject by the parents. The percentage of days by age on which antibiotics were used was related to the number of days the subject was in the study. For NEC cases, only pre-event exposure to antibiotics was considered.

[0672] Disease Definition Different definitions of NEC have been proposed. 26-29 In this study, 2 categories of NEC from clinical documentation were used (eTable 1). Radiographic signs were the defining criteria for our NEC categories; abdominal signs and clinical and laboratory findings were secondary criteria. Suspected NEC was defined as disease based on abnormal clinical and laboratory findings without evidence of intestinal gas or portal venous gas on abdominal radiographic images. Severe NEC was defined as radiographic evidence of intestinal gas and / or portal venous gas. Patients diagnosed with spontaneous intestinal perforation (SIP) were excluded from the study (eTable 2). The diagnosis of neonatal LOS required the presence of abnormal clinical findings at least 72 hours after birth and blood cultures that were positive for bacteria were not considered contaminants30,31 (eTable 3). Infants with other confirmed nongastrointestinal infections had clinical findings of bacterial, viral, or fungal infection in body fluids other than blood. A summary of the groups and diagnoses of NEC, SIP, sepsis, and nongastrointestinal infections is provided in eTables 4 to eTables 11.

[0673] Clinical findings for NEC diagnosis, NEC suspicion, sepsis, and other confirmed non-gastrointestinal infections were determined by review of clinical files. Study definitions of NEC do not always agree with the patient's clinical diagnosis. For this study, NEC and suspected NEC were physician-directed clinical diagnoses, with radiographic signs as the defining criteria and abdominal signs, clinical findings, and laboratory findings further confirming the diagnosis (eTable 1). Suspected NEC (eTable 1) was defined as infants with concern for early illness based on clinical and laboratory abnormalities without radiographic evidence of intestinal gas. In contrast, infants with suspected NEC demonstrated one or more radiographic signs, including mild intestinal dilatation, mild intestinal obstruction, bowel wall thickening, or scanty / absent intestinal gas. In addition to one or more laboratory findings, one or more clinical or abdominal signs and symptoms were required, including thrombocytopenia, decreased or increased leukocytes, decreased absolute neutrophil count, increased number of immature neutrophils, heme-positive stools, and metabolic acidosis. Clinical and abdominal signs and symptoms include bilious secretions, vomiting, bloody stools, feed intolerance, increased pre-meal gastric residual volume, increased apnea and / or bradycardia, temperature instability, lethargy, the onset of systemic clinical signs, mild to moderate abdominal distension, and discoloration of the abdominal wall.

[0674] Severe NEC (e Table 1) was defined by radiographic evidence of intestinal gas and / or portal venous gas or pathological findings on surgical or postmortem intestinal samples. Pneumoperitoneum is free intraperitoneal air resulting from perforation and is considered NEC when accompanied by radiographic evidence of intestinal gas and abdominal signs found in definite NEC. Other signs included moderate to severe abdominal distention and / or abdominal tenderness and / or hypoactive / absent bowel sounds and / or abdominal wall discoloration, abdominal cellulitis, a fixed right lower quadrant abdominal mass, and / or s...

Claims

1. Use of a reagent for determining intestinal alkaline phosphatase (iAP) activity and the amount of iAP in a child in the preparation of a kit for use in a method for determining the prognosis of inflammatory bowel disease (IBD) in the child, the method comprising the following steps (a), (b), (c): (a) Fitting a Markov model using a two-state transition matrix and propensity values ​​measured across multiple subjects, wherein the two-state transition matrix comprises a first state and a second state, wherein the first state comprises a non-inflammatory bowel disease state, wherein the second state comprises an inflammatory bowel disease state, wherein the propensity value is a function of an intestinal alkaline phosphatase (iAP) activity value and an amount of iAP found in a subject of the plurality of subjects, wherein the subject is a child; (b) estimating a probability of transitioning from a first state to a second state using the child's disposition value and a fitted Markov model, wherein the fitted model indicates that an increase in the child's disposition level significantly increases the probability of transitioning from the first state to the second state; (c) recommending treatment of the child when the propensity value is greater than or equal to a threshold value of 0.

5.

2. The use according to claim 1, wherein the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus a first ratio, wherein the first ratio comprises an iAP activity value for a subject in the plurality of subjects divided by a maximum iAP activity value observed in the plurality of subjects, and wherein the second value comprises a second ratio, wherein the second ratio comprises the iAP amount from an immunoassay value for a subject in the plurality of subjects divided by a maximum iAP amount from an immunoassay value observed in a sample.

3. The use of claim 1, wherein the propensity value comprises the product of a first value and a second value, wherein the first value comprises one (1) minus the iAP activity value of the subject in the plurality of subjects, and wherein the second value comprises the iAP amount from the immunoassay value of the subject in the plurality of subjects.

4. The use according to claim 2 or 3, wherein the immunoassay comprises Western blot, ELISA or immunoprecipitation.

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