Compositions and methods for detecting gastrointestinal disorders

文档序号：245625 发布日期：2021-11-12 浏览：15次中文

阅读说明：本技术 检测胃肠道疾病的组合物和方法 (Compositions and methods for detecting gastrointestinal disorders ) 是由金宣姈于 2020-02-04 设计创作，主要内容包括：本发明涉及检测和治疗胃肠疾病的组合物和方法。(The present invention relates to compositions and methods for detecting and treating gastrointestinal disorders.)

1. A method of determining a prognosis for Necrotizing Enterocolitis (NEC) in a patient, the method comprising:

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 iAP amount found in a subject of the plurality of subjects;

b. Estimating a probability of transitioning from the first state to the second state using the trend values of the patient and a fitted markov model, wherein the fitted model indicates that an increase in the trend level of the patient significantly increases the probability of transitioning from the first state to the second state;

c. treating the patient when the predisposition value is greater than or equal to a threshold value of about 0.5.

2. The method of claim 1, wherein the predisposition 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 in the plurality divided by the maximum iAP activity value observed in the plurality, wherein the second value comprises a second ratio, wherein the second ratio comprises the iAP amount of an immunoassay value from a subject in the plurality divided by the maximum iAP amount of an immunoassay value observed in a sample.

3. A method according to claim 1, wherein the predisposition value comprises a product of a first value and a second value, wherein the first value comprises one of (1) minus the iAP activity value of the subject in the plurality of subjects, wherein the second value comprises the iAP amount from the immunoassay value of the subject in the plurality of subjects.

4. The method of claim 1, wherein the plurality of subjects comprises the patient.

5. The method of claim 1, wherein treating comprises stopping eating, administering an antibiotic, or a combination thereof.

6. The method of claim 2, wherein the sample is a human small intestine lysate.

7. The method of claim 2 or 3, wherein the immunoassay comprises western blotting, ELISA, or immunoprecipitation.

Technical Field

The present invention relates to compositions and methods for detecting and treating gastrointestinal disorders.

Background

Gastrointestinal disorders 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 local infection and inflammation. NECs are characterized by high mortality and long-term morbidity, including short bowel syndrome, recurrent infections, nutritional deficiencies, and neurodevelopmental retardation. Although the mortality rate of premature infants generally decreases net, the number of deaths associated with NEC increases. NECs are often difficult to diagnose and manage due to initial nonspecific symptoms and rapid deterioration. Clinicians currently rely on radiological evidence to make a diagnosis in the advanced stages of the disease.

Disclosure of Invention

The present invention provides a method of determining the prognosis of Necrotizing Enterocolitis (NEC) in a patient. In an embodiment, the method comprises 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 iAP amount found in a subject of the plurality of subjects; estimating a probability of transitioning from the first state to the second state using the trend values of the patient and a fitted markov model, wherein the fitted model indicates that an increase in the trend level of the patient significantly increases the probability of transitioning from the first state to the second state; treating the patient when the predisposition value is greater than or equal to a threshold value of about 0.5.

In embodiments, the predisposition 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 in the plurality divided by the maximum iAP activity value observed in the plurality, wherein the second value comprises a second ratio, wherein the second ratio comprises the iAP amount of an immunoassay value from a subject in the plurality divided by the maximum iAP amount from an immunoassay value observed in the sample.

Non-limiting examples of immunoassays include western blot analysis, ELISA, or immunoprecipitation. For example, an immunoassay may include western blot analysis using a chemiluminescent reporter gene. In another example, the immunoassay may comprise western blot analysis using a fluorescent reporter gene. The response of the fluorescent report may be more linear.

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

In embodiments, the predisposition 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, wherein the second value comprises the iAP amount of the immunoassay value from the subject in the plurality.

In embodiments, the plurality of subjects comprises patients.

In embodiments, the treatment comprises stopping eating, administering an antibiotic, or a combination thereof.

In embodiments, the sample is a human small intestine lysate.

In embodiments, the immunoassay comprises western blotting, ELISA, or immunoprecipitation.

The present invention provides methods of identifying a subject having a Gastrointestinal (GI) disease. One aspect of the invention relates to a method for diagnosing a subject with a gastrointestinal disease. Another aspect of the invention relates to a method for identifying a subject at risk for gastrointestinal disease. Embodiments as described herein may 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 may diagnose an advanced inflammatory state, such as one determined by radiologic findings of intestinal gas (portal vein or biliary tract gas). As another example, embodiments can identify early stages of disease before rampant inflammation of the intestinal tract is physiologically evident.

In embodiments, the method comprises incubating a biological sample from the subject with an agent that binds Intestinal Alkaline Phosphatase (iAP), detecting the agent that binds the iAP in the sample, and detecting and/or measuring the amount or activity of the 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 including AP enzyme activity of iAP, iAP protein levels, iAP dimerization/dissociation, post-translationally modified iAP, total protein, e.g., total fecal protein, or a combination thereof. In some embodiments, a GI disease biomarker may be used to diagnose a subject with a gastrointestinal disease, and may also indicate a subject with a Gastrointestinal (GI) disease and/or a subject at risk of developing a GI disease. In some embodiments, a subject with Gastrointestinal (GI) disease may include early and late stages of the disease.

In embodiments, the iAP is not bound to an iAP binding agent. For example, a substrate may be provided for an enzyme in a sample and a change in the substrate monitored. In one embodiment, the iAP is not bound for activity assays. For example, a substrate may be provided for an enzyme in a sample and changes in the enzyme may be monitored. Without being bound by theory, this may also be the same for AP bound to an enzyme, where the substrate change measured in the immunoassay is to the protein bound by the antibody system, and also to any free AP in the sample.

The invention further provides a method of 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 the iAP in the sample, and detecting and/or measuring the amount or activity of the agent that binds the iAP in the sample. In embodiments, the iAP-binding agent is at least one GI disease biomarker comprising iAP enzyme activity, iAP protein levels, iAP dimerization/dissociation, post-translationally modified iAP, total protein, e.g., total fecal protein, or a combination thereof, and wherein the GI disease biomarker is indicative of a subject having a Gastrointestinal (GI) disease. The iAP-binding reagents of the invention can be iAP bound to its cognate substrate, antibodies that recognize and bind iAP, short peptide sequences directed against and bind iAP, and the like, non-limiting examples of which include small molecule activators or inhibitors that catalyze reactions, metal ions (tungsten is a transition state effector of alkaline phosphatase), reagents that cause allosteric release of products, labile chemical moieties that act as chemical, enzymatic, or photolytic triggers, or matrices that bind iAP in labeled form.

Embodiments may further comprise diagnosing the subject as having a GI disorder. For example, a subject may be diagnosed with a GI disorder: if the total protein concentration in the sample is greater than about 1.0mg/ml, 1.1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml, 2.1mg/ml, 2.2mg/ml, 2.3mg/ml, 2.4mg/ml, 2.5mg/ml, 2.6mg/ml, 2.7mg/ml, 2.8mg/ml, 2.9mg/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, 4mg/ml, 4.4mg/ml, 4mg/ml, 3.5mg/ml, 4mg/ml, 4.6mg/ml, 4mg/ml, 3.7mg/ml, 3.9mg/ml, 4mg/ml, 3.4mg/ml, 3mg/ml, 3.4mg/ml, 3mg/ml, 3.0mg/ml, 3mg/ml, 3.4mg/ml, 3mg/ml, 3.4mg/ml, 3mg/ml, 3.4mg/ml, 3mg/ml, 3.0mg/ml, 3.4mg/ml, 3.0mg/ml, 3mg/ml, 3.4mg/ml, 3mg/ml, 3.4mg/ml, 3, 4.5mg/ml, 4.6mg/ml, 4.7mg/ml, 4.8mg/ml, 4.9mg/ml or 5.0 mg/ml; if the iAP activity is less than about 10mU/mg, 20mU/mg, 30mU/mg, 40mU/mg, 50mU/mg, 60mU/mg, 70mU/mg, 80mU/mg, 90mU/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, 1150mU/mg, 1200mU/mg, 1250mU/mg, 1300mU/mg, 1350mU/mg 100mU/mg, 1450mU/mg, 1500mU/mg, 1600mU/mg, 1700mU/mg, 1800mU/mg, 1900 mU/mg; if the iAP activity is less than about 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; 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% of the control sample, or a combination thereof. Without being bound by theory, the total protein concentration in a sample as described herein may be that of a fecal sample, with the fresh weight for the buffer set at 1 g/mL. Otherwise, the skilled person knows that the sample can be diluted or concentrated to change the protein concentration.

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

The present invention provides a method of diagnosing a Gastrointestinal (GI) disease in a subject, comprising obtaining a sample from the subject, detecting the presence of at least one GI disease biomarker in the sample, wherein the GI disease biomarker 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.

The invention further provides a method of preventing progression of 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 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.

The invention further provides a method of ameliorating a symptom 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 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.

In embodiments, 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 intake of food; administering an iAP replacement composition; anti-inflammatory agents; a therapist; catalytically active small molecule activators and/or effectors; parenteral (or intravenous) nutrition or a combination thereof.

Non-limiting examples of therapeutic agents that may be used in accordance with the present invention include Toll-like receptor (TLR) inhibitors (New et al discovery and validation of a new class of small molecule Toll-like receptor 4(TLR4) inhibitors. plos One 12, e65779) and interruption of eNOS-NO-nitrite signaling (Yazji et al endo-TLR 4 activation assays within the National Academy of Science USA 110, 9451-9456).

Non-limiting examples of small molecule effectors having catalytic activity include levamisole, theophylline, triazole-based compounds, sulfonamide derivatives, phosphatase derivatives, metals and amino acids (Borgers M. the biochemical application of new potential inhibitors of alkaline phosphatase. journal of biochemistry & cytology 21, 812. 824; Klemer et al. the inhibition of alkaline phosphatase by fibrous. journal of Biological Chemistry 180, 281. 288; Bobkova et al. modules of alkaline phosphate. Metal Biol. 1053, 135. 144; Nariswa et al. No. 1 linkage of alkaline sludge of Biological 34. journal of chemical sludge of alkaline sludge of Biological 23. and 11. Mineral of Biological cement of 11. calcium of alkaline sludge of Biological cement 23. 35. and 11. calcium of alkaline sludge of Biological cement of 11. journal of Biological 23. 11. and 51. Mineral of chemical sludge of Biological cement and 11. calcium of alkaline sludge of Biological cement of 11. calcium carbonate of calcium carbonate of calcium carbonate of calcium carbonate of calcium carbonate of calcium of.

Non-limiting examples of such antibiotics include vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), gentamicin, Flagyl (metronidazole mimetic), 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.

Non-limiting examples of probiotic organisms include those of the genera lactobacillus, lactococcus, bifidobacterium, pediococcus, saccharomyces boulardii, and related bacteria and yeasts.

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 buffers), D₅W (5% aqueous glucose solution), D₅NS (5% glucose in 0.9% saline), D₅1/2NS (5% glucose in 0.45% saline), D₅LR (5% glucose in ringer's lactate) or Normosol-R. In embodiments, the intravenous fluid solution may be isotonic. In other embodiments, the intravenous fluid solution may be hypotonic.

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.

The anti-inflammatory agent may be selected from a wide variety of steroidal, non-steroidal, and salicylate water-soluble and water-insoluble drugs, and acid addition or metal salts thereof. Organic and inorganic salts may be used provided that the anti-inflammatory agent retains its medicinal value. The anti-inflammatory agent may be selected from a wide range of therapeutic agents and mixtures of therapeutic agents, which may be administered in sustained release or prolonged action forms. Non-limiting examples of anti-inflammatory agents include ibuprofen, naproxen, sulindac, diflunisal, piroxicam, indomethacin, etodolac, meclofenamate sodium, fenoprofen, 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, mesalamine, salsalate, diflunisal, salicylsalicylic acid, and choline magnesium trisalicylate. Non-limiting examples of steroidal anti-inflammatory agents include flunisolide, triamcinolone acetonide, beclomethasone dipropionate, betamethasone dipropionate, hydrocortisone, cortisone, dexamethasone, prednisone, methylprednisolone, and prednisolone.

In embodiments, the gastrointestinal disease may comprise 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.

In embodiments, the sample may comprise a biological sample obtained from a subject. For example, the biological sample may be a biological fluid, a biological solid, or a biological semi-solid. In embodiments, the sample may comprise stool, meconium, vomit, peripheral blood, serum, plasma, or urine.

In embodiments, the GI disease biomarker may comprise iAP enzyme activity, AP enzyme activity, iAP protein levels, iAP dimerization/dissociation, post-translationally modified iAP, total protein, e.g., total fecal protein, or a combination thereof. In embodiments, the post-translational modification may include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glycosylphosphatidylinositol (phosphorylation), glycosylation, hydroxylation, iodination, ISG, lipoylation, malonation, methylation, myristoylation, palmitoylation, phosphorylation, phospho-phosphorylation, prenylation, propionylation, ribosylation, succinylation, sulfation, sumoylation, or ubiquitination.

In embodiments, the GI disease biomarker may comprise a NEC biomarker.

In embodiments, the detection may comprise an immunoassay, a colorimetric assay, a fluorescent assay, or a combination of both. In embodiments, the immunoassay may comprise a western blot assay, an enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, a single molecule immunoassay in a femto-cell array, single and multiplex formats of digital enzyme assays, or combinations thereof. In embodiments, detecting comprises contacting the sample with an anti-iAP antibody. In embodiments, the anti-iAP antibody is a polyclonal or monoclonal antibody. In embodiments, the detection can comprise a kinetic assay, an endpoint assay, a bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, a pyrogallol hemoglobin dye binding assay, a coomassie blue dye binding assay, or a combination thereof.

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 of detecting molecules such as iAP (Knight et al. non-innovative analysis of intestinal degradation in preterm and term factors using RNA-sequencing.2014.scientific Reports 4,5453).

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 hydrolysate, or combinations thereof.

In embodiments, the detection may comprise a kinetic assay comprising the use of 4-methylumbelliferone phosphate, CPD Star (2-chloro-5- (4-methoxyspiro [1, 2-dioxetane-3, 2' - (5-chlorotricyclo [ 3.3.1.1)^3.7]Decane]) -4-yl]Disodium-1-phenylphosphate), AttosPho (2' - [ 2-benzothiazolyl)]-6' -hydroxybenzothiazole phosphate [ BBTP]) Or any other fluorescent or colorimetric signal, detecting ATP hydrolysis and/or ATP hydrolysis products (e.g., malachite)Green, NADH-coupled, or other proprietary variants), or a combination thereof.

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

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 tetrazole/iodo nitrotetrazole (BCIP/INT substrate, tawny precipitate, Biomeda), 5-bromo, 4-chloro, 3-indolyl phosphate/nitro blue tetrazole (BCIP/NBT substrate, blue/violet), 5-bromo, 4-chloro, 3-indolyl phosphate/nitro blue tetrazole/iodo nitrotetrazole (BCIP/NBT/INT, brown precipitate, DAKO, fast red (red), magenta phosphorus (magenta), naphthol AS-bisphosphate (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^TMBlack (black), VECTOR^TMBlue (blue), VECTOR^TMRed (Red), Vega Red (raspberry Red), fluorescein diacetate, 4-methylumbelliferyl acetate, 4-methylumbelliferyl casein, 4-methylumbelliferyl-alpha-L-arabinopyranoside, 4-methylumbelliferyl-beta-D-fucopyranoside, 4-methylumbelliferyl-alpha-L-fucopyranoside, 4-methylumbelliferyl-beta-L-fucopyranoside, 4-methylumbelliferyl-alpha-D-galactopyranoside, 4-methylumbelliferyl-beta-D-hemi-pyranoside Lactoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-beta-D-glucuronide, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl phosphate, bis (4-methylumbelliferyl) phosphate, 4-methylumbelliferyl pyrophosphate, 4-methylumbelliferyl-beta-D-xylopyranoside.

Non-limiting examples of suitable chromogenic substrates for use in the present invention include o-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-galactopyranoside, o-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-alpha-D-glucopyranoside, p-nitrophenyl-beta-D-glucuronide, p-nitrophenylphosphate, o-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-alpha-D-xylopyranoside, p-nitrophenyl-beta-D-xylopyranoside and phenolphthalein-beta-D-glucuronide.

In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the protein level of iAP in the sample is at least two standard deviations higher than the average protein level of the control sample. In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the protein level of iAP in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations higher than the average protein level of the control sample. In embodiments, the control sample may comprise two or more control samples. Embodiments may include 10mg, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 80mg, 90mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, or 300mg fresh weight feces per mL sterile water or buffer. For example, embodiments may comprise 200mg fresh weight feces per mL sterile water or buffer. In embodiments, the method as described herein further comprises diagnosing the subject as having or at risk of having 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 described herein further comprise diagnosing the subject as having or at risk of having a gastrointestinal disease if the protein level of iAP in the sample is greater than 107% of the control sample. In embodiments, the methods described herein further comprise diagnosing the subject as having or at risk of having 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.

In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the level of iAP enzyme activity in the sample is at least two standard deviations lower than the average iAP enzyme activity of the control sample. In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the iAP enzyme activity level in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations higher than the average enzyme activity level of the control sample. In embodiments, the control sample may comprise two or more control samples. In embodiments, the methods as described herein further comprise determining if the level of iAP enzyme activity in the sample is less than about 10mU/mg, 20mU/mg, 30mU/mg, 40mU/mg, 50mU/mg, 60mU/mg, 70mU/mg, 80mU/mg, 90mU/mg, 100mU/mg, 200mU/mg, 300mU/mg, 400mU/mg, 500mU/mg, 600mU/mg, 700mU/mg, 800mU/mg, 900mU/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, or a combination thereof, 800U/mg, 900U/mg, 1000U/mg, for example less than 979mU/mg or less than 1256mU/mg, then the subject is diagnosed with or at risk of gastrointestinal disease. In embodiments, the method as described herein further comprises diagnosing the subject as having or at risk of having gastrointestinal disease if the level of iAP enzyme activity in the sample is less than 1500, 1000, 500, e.g., less than 1256 mU/mg.

In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the fecal protein level in the sample is at least two standard deviations higher than the average fecal protein level of the control sample. In embodiments, the method as described herein further comprises diagnosing the subject with a gastrointestinal disease if the fecal protein level in the sample is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations higher than the average fecal protein level of the control sample. In embodiments, the control sample may comprise two or more control samples. In embodiments, the method as described herein further comprises diagnosing the subject as having or at risk of having a gastrointestinal disease if the fecal protein level in the sample is above 1.6mg/ml, or for example above 1.8 mg/ml. In embodiments, the method as described herein further comprises if the fecal protein level in the sample exceeds 1.0mg/ml, 1.1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml, 2.1mg/ml, 2.2mg/ml, 2.3mg/ml, 2.4mg/ml, 2.5mg/ml, 2.6mg/ml, 2.7mg/ml, 2.8mg/ml, 2.9mg/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, 3.4mg/ml, 4mg/ml, 1.6mg/ml, 3.7mg/ml, 3.8mg/ml, 3.9mg/ml, 4mg/ml, 1.6mg/ml, 4mg/ml, 1mg/ml, 1.6mg/ml, 1mg/ml, 2mg/ml, 1mg/ml, 1.7mg/ml, 2mg/ml, 2.6mg/ml, 2mg/ml, 2.6mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 1mg/ml, 2mg/ml, 1mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 1mg/ml, 2mg/ml, 1mg/ml, 2mg/ml, 2.4mg/ml, 1mg/ml, 2.7mg/ml, 2mg/ml, 2.4mg/ml, 1mg/ml, 2mg/ml, 1mg/ml, 2mg/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 diagnosing that the subject has or is at risk of having a gastrointestinal disease.

In embodiments, the method as described herein further comprises treating the subject. In embodiments, the treatment may comprise administering 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 to a subject diagnosed with a gastrointestinal disorder.

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

The invention further provides a method for screening for the presence of a characteristic in a subject (e.g., a subject at risk for gastrointestinal disease or a subject with 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 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.

The invention further provides a method for identifying a subject at risk of having a gastrointestinal disease or a subject having 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.

In embodiments, the gastrointestinal disease may comprise 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.

In embodiments, the GI disease biomarker may further comprise 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, glycosylphosphatidylinositol (phosphorylation), glycosylation, hydroxylation, iodination, ISG, lipoylation, malonation, methylation, myristoylation, palmitoylation, phosphorylation, phospho-phosphorylation, prenylation, propionylation, ribosylation, succinylation, sulfation, sumoylation, or ubiquitination.

In embodiments, measuring may comprise performing an assay to determine the total protein concentration, intestinal alkaline phosphatase activity, intestinal alkaline phosphatase protein concentration, or a combination thereof in the sample. In embodiments, the measurement may comprise a bradford assay, a bicinchoninic acid (BCA) assay, a Lowry assay, a pyrogallol hemoglobin dye binding assay, a coomassie blue dye binding assay, or a combination thereof. In embodiments, the measuring may comprise a kinetic assay. In embodiments, kinetic assays may include the use of 4-methyl umbelliferyl phosphate, nitrophenyl phosphate, or any other fluorescent or colorimetric signal, assays that detect ATP hydrolysis, or combinations thereof. In embodiments, the measurement may comprise an immunoassay, a colorimetric assay, a fluorescent assay, or a combination of both.

In embodiments, the immunoassay may comprise a western blot assay, an enzyme-linked immunosorbent assay, 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.

In embodiments, the methods disclosed herein can further comprise diagnosing the subject as having a gastrointestinal disease when the total protein concentration in the sample is at least two standard deviations above the mean of the control sample, the concentration of intestinal alkaline phosphatase protein is at least two standard deviations above the mean of the control sample, the average of the intestinal alkaline phosphatase activity control sample is at least two standard deviations below, or a combination thereof. In embodiments, the methods disclosed herein can further comprise diagnosing the subject as having 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 greater than the average of the control samples, 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 greater than the average of the control samples, the average of the intestinal alkaline phosphatase activity control samples is at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 standard deviations less, or a combination thereof. In embodiments, the control sample may comprise two or more control samples.

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

In embodiments, if the protein concentration in the fecal sample is greater than about 1.0mg/ml, 1.1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml, 2.1mg/ml, 2.2mg/ml, 2.3mg/ml, 2.4mg/ml, 2.5mg/ml, 2.6mg/ml, 2.7mg/ml, 2.8mg/ml, 2.9mg/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, 4mg/ml, 4.4mg/ml, 3.5mg/ml, 3.6mg/ml, 3.7mg/ml, 3.8mg/ml, 3.9mg/ml, 4mg/ml, 4.4mg/ml, 4mg/ml, 2.4mg/ml, 2.6mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 2.7mg/ml, 3.7mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 2.4mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 2.7mg/ml, 2mg/ml, 2.7mg/ml, 2, 4.4mg/ml, 4.5mg/ml, 4.6mg/ml, 4.7mg/ml, 4.8mg/ml, 4.9mg/ml, 5.0mg/ml, such as 1.6mg/ml or such as 1.8mg/ml, the subject may be diagnosed with or at risk of developing a gastrointestinal disease. In embodiments, if the iAP activity is less than about 10mU/mg, 20mU/mg, 30mU/mg, 40mU/mg, 50mU/mg, 60mU/mg, 70mU/mg, 80mU/mg, 90mU/mg, 100mU/mg, 200mU/mg, 300mU/mg, 400mU/mg, 500mU/mg, 600mU/mg, 700mU/mg, 800mU/mg, 900mU/mg, 1000mU/mg, 1100mU/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, 100U/mg, 200U/mg, 300U/mg, 400U/mg, 500U/mg, 600U/mg, 700U/mg, 800U/mg, 900U/mg, 100U/mg, and/mg, 1000U/mg, such as 972mU/mg, or such as 1256mU/mg, the subject may be diagnosed with or at risk of having gastrointestinal disease. In embodiments, a subject can be diagnosed with or at risk of having gastrointestinal disease if the iAP protein is detected by densitometric anti-iAP antibodies by more than 4.8% of the control. In embodiments, a subject can be diagnosed with a gastrointestinal disease if the level of iAP protein is at least two standard deviations above the mean of the control samples. In embodiments, the control sample may comprise two or more control samples. In embodiments, the subject may comprise a mammal. In embodiments, the mammal may comprise a dog, cat, horse, cow, or human. In embodiments, the human may comprise an infant. In embodiments, the infant may comprise a premature infant.

The present invention also provides a disposable article comprising a biosensor, wherein the biosensor may comprise at least one biological recognition element, and wherein the biosensor detects iAP in a sample.

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, glycosylphosphatidylinositol (phosphorylation), glycosylation, hydroxylation, iodination, ISG, lipoylation, malonation, methylation, myristoylation, palmitoylation, phosphorylation, phospho-phosphorylation, prenylation, propionylation, ribosylation, succinylation, sulfation, sumoylation, or ubiquitination.

In embodiments, the biosensor is an immunosensor. In embodiments, the biosensor may comprise a detection signal. In embodiments, the detection signal may comprise a colorimetric signal, a fluorescent signal, or both. In embodiments, the biological recognition element can comprise an anti-iAP antibody. In embodiments, the anti-iAP antibody can comprise a polyclonal or monoclonal antibody.

In exemplary embodiments, the biosensor may comprise a lateral flow immunoassay, also known as an immunochromatographic assay or a strip test. Lateral flow immunoassays include immunoassays adapted to operate along a single axis to accommodate the test strip format. A typical lateral flow test strip includes a sample pad (an absorbent pad to which the test sample is applied), a conjugate or reagent pad (this contains a binding agent for the analyte of interest, e.g. an antibody, which binds to coloured particles, e.g. colloidal gold nanoparticles or latex microspheres), a reactive membrane (typically a nitrocellulose or cellulose acetate membrane, on which an anti-analyte binding agent, e.g. an antibody, is immobilised in a line across the membrane to act as a capture zone or test line. The assembly of strips, typically secured to an inert backing material, may be provided in the form of a simple dipstick or within a plastic housing, the sample port and reaction window of which display the capture and control zones.

In embodiments, the article may comprise a diaper worn by the subject, a wipe for cleaning the subject, a dipstick, a spoon, a small scoop, filter paper, or a swab.

In embodiments, the subject may comprise a mammal. In embodiments, the mammal may comprise a dog, cat, horse, cow, or human. In embodiments, the human may comprise an infant. In embodiments, the infant may comprise a premature infant.

The invention further provides kits for diagnosing a subject with a gastrointestinal disease. In embodiments, the kit may comprise a disposable article as described herein. In embodiments, the gastrointestinal disease may comprise 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.

In embodiments, the kit can comprise an iAP biorecognition element immobilized to a solid support and instructions for its use.

In embodiments, the gastrointestinal disease may comprise 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.

In embodiments, the biological recognition element can comprise an antibody to iAP or an oligonucleotide to iAP, e.g., immobilized directly or indirectly to a solid support. Embodiments may also comprise fluorogenic substrates or inhibitors with high binding affinity for iAP attached to a solid support.

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

The invention also provides a diagnostic kit of molecular biomarkers for identifying a subject exhibiting or having a predisposition to develop gastrointestinal disease. In embodiments, the kit may comprise at least one of means for determining total fecal protein concentration, means for determining Intestinal Alkaline Phosphatase (iAP) activity, and an iAP biorecognition element, wherein together represent a predisposition indicating the presence or development of gastrointestinal disease in a human subject. In embodiments, the gastrointestinal disease may comprise 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.

In embodiments, the characteristic may comprise a total protein concentration at least two standard deviations above the mean of the control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of the control sample, or an intestinal alkaline phosphatase activity at least two standard deviations below the mean of the control sample. In embodiments, the control sample may comprise two or more control samples.

In embodiments, the characteristic may be selected from at least two of the group comprising a total protein concentration at least two standard deviations above the mean of the control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of the control sample, and an intestinal alkaline phosphatase activity at least two standard deviations below the mean of the control sample. In embodiments, the control sample may comprise two or more control samples.

Aspects of the invention also relate to treating a subject having NEC by altering the feeding regimen. In one embodiment, the present invention provides a method of treating a subject having NEC. In some embodiments, the method comprises measuring the amount or activity of an iAP-binding agent in a sample obtained from the subject according to the methods described herein, wherein the iAP-binding agent 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 postpartum development age of the subject; and stopping enteral feeding for a period of time sufficient to address the gastrointestinal inflammatory process or the signs of food 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, alone or in combination thereof, a probiotic, other biologic (e.g., stem cells or transcription factors), or therapeutic agent (e.g., TLR4 small molecule, alkaline phosphatase inhibitor or activator), antibiotic, intravenous fluid, iAP replacement composition (e.g., exogenously provided), small molecule activator and/or catalytically active effector, anti-inflammatory agent, parenteral (or intravenous) nutrition. In some embodiments, the post-partum developmental age of the subject may be 'post-menstrual age' or 'post-menstrual developmental age'.

For example, oral feeding may 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.

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

In embodiments, the feeding regimen may comprise an intermittent feeding regimen. For example, during an extended period of suspended oral feeding, there may be one or more days of feeding. For example, food intake 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 one or more days of oral food intake may occur. For example, a physician may suspect NEC and pause feeding for a short period of time, for example one or two days, until laboratory testing/inspection indicates that the infant does not have NEC, at which time they will resume feeding again. After hours or days, NEC panic may reappear and the infant is prohibited from eating again for a period of time. Intermittent feeding may occur one or more times during the hospitalization of the subject. Such intermittent feeding may allow a clinician to determine the subject's tolerance to feeding. In other embodiments, intermittent feeding may be recommended for subjects at risk of gastrointestinal disease. Other objects and advantages of the present invention will become apparent from the ensuing description.

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Fig. 1 shows longitudinal measurements of immunoblot detection of total fecal protein, iAP enzyme activity and iAP protein in two preterm infants. (A) Patient 1 born at 30 weeks gestation, developed NEC at postnatal day 7, received drug therapy, followed by recurrent NEC and intestinal perforation at postnatal day 31. The infant healed after placing the peritoneal drainage tube and intestinal rest and antibiotics for an additional 10 days. Red symbols and bars represent NEC events. 7A and 7B refer to 2 individual stool samples collected on day 7 of life, one before and one after NEC diagnosis. (B) Patient 2 was born 25 weeks gestation with abdominal distension at DOL 19, with NEC suspected (green symbols and bars). Infants respond quickly to medical management and quickly resume enteral feeding. Patients presented with a clear NEC (red symbols and bars) on DOL 32, requiring assisted ventilation and active medical management, but fully recovered at DOL 48. The table below each figure illustrates which stool tests meet NEC criteria (+) and which do not. These criteria are defined by values outside the 95% confidence interval of the control values. NEC risk is considered increased if the following occurs: the protein concentration is more than 1.8 mg/ml; the iAP activity is less than 979 mU/mg; or detection of iAP protein by western blot over 10.7% of control. Abbreviations: ip, intestinal perforation; ad, abdominal distension; MW, molecular weight step; and kDa, kilodaltons.

Figure 2 shows the increase in total fecal protein concentration, decrease in fecal iAP enzyme activity and increase in iAP detection on western blot at NEC diagnosis. In the following panels, red circles represent individual stool samples collected during 7 different NEC events for 6 patients; the black circles represent the composite mean ± sem of 2-17 fecal samples from 12 control patients. Red and gray bars represent the mean ± standard error of all samples collected in NEC events and all samples from control subjects, respectively. (A) The protein concentration in the patient stool samples at NEC diagnosis was higher (p-value 0.005) compared to the control samples. (B) iAP activity in patient stool samples at NEC diagnosis was lower than control samples (p-value < 0.0001). (C) The amount of iAP protein in the stool quantified by comparison with the positive control standard was higher in patients at NEC diagnosis compared to the control samples (p-value ═ 0.002). And (4) statistical test: Mann-Whitney.

Fig. 3 shows that stool iAP-related measurements are highly specific and sensitive. (A) 3-dimensional scatter plots of our candidate biomarker measurements, where red diamonds represent NEC samples and black circles represent controls. (B) 2-dimensional projection of 3-dimensional scatter plots, which correlate exclusively with iAP activity and western blot intensity measurements. (C) Sensitivity and specificity curves for each candidate biomarker, and for a combined naive bayes classifier considering all 3 features simultaneously. The analysis covered 49 samples, 13 NEC samples, 9 NEC patient-derived control samples and 27 control patient-derived control samples. The Spearman correlation coefficient for western blot intensity versus 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 western blot intensity was-0.58.

Figure 4 shows the demographic information of the study subjects. Mean and standard deviation of gestational age and birth weight are shown. The distribution of gestational age, gender and bell stages is also reported.

Figure 5 shows that alkaline phosphatase activity is primarily due to Intestinal Alkaline Phosphatase (iAP). L-Phe is a specific inhibitor of intestinal alkaline phosphatase activity, which inhibits activity. There was no observable difference in the degree of inhibition between NEC and control samples. NEC samples showed 90% inhibition with a standard deviation of +/-10. The control sample 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).

Figure 6 shows the demographics of patient enrollment in the study.

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

Figure 8 shows that total fecal protein is higher in fecal samples from patients at NEC diagnosis compared to control samples. The red circles represent the total fecal protein level during 7 different NEC events in 6 patients (number of post-pregnancy weeks: 29-43 weeks). Each black circle represents the composite mean/-SEM of 2-16 stool samples from 12 controls (number of post-pregnancy weeks: 29-45 weeks). Statistical analysis: Mann-Whitney test, P ═ 0.005

Figure 9 shows that the intensity of the iAP protein signal quantified as a percentage of the positive control signal is much higher in the stool sample from the patient at NEC diagnosis compared to the control sample. The red circles represent the intensity of the iAP protein signal during 7 different NEC events in 6 patients (number of weeks post-pregnancy: 29-43). Each black circle represents at least 1 fecal sample from 7 controls (29-35 weeks post-pregnancy). Statistical analysis: Mann-Whitney test, P ═ 0.002

Figure 10 shows decreased fecal iAP enzymatic activity, increased fecal total protein and increased iAP detection of WB at NEC diagnosis. (A) Longitudinal measurements of iAP activity, total fecal protein and iAP enzyme activity of NEC patients who subsequently underwent perforation are demonstrated. Red dots and bars represent NEC events. The dramatic decline in fecal iAP activity corresponding to the time of diagnosis indicates that fecal iAP activity can be used as a diagnostic tool for NEC. Strong iAP protein detection occurred on western blots. The patient completed treatment for 14 days, but had an intestinal perforation (ip) on day 31 of life. After an additional 10 days of intestinal rest, the anti-iAP antibodies no longer strongly detect iAP and iAP activity is on an increasing trend. (B) A decrease in stool iAP activity and an increase in iAP volume was also observed at NEC diagnosis in the second patient. The green dots represent bloody stool episodes, corresponding to suspected NEC with intestinal distension (bd). The iAP test occurred during NEC monitoring, which did not become apparent until NEC diagnosis on life day 32. Again, the activity around NEC diagnosis (red dots and bars) was reduced and tended to be higher after recovery and decreased iAP detection on WB. (C) NEC was associated with low fecal iAP activity, high fecal protein and high fecal iAP amounts (on WB). Three samples from 3 NEC patients at NEC diagnosis (labeled N, red bars) were matched with 3 samples from 3 control subjects with similar gestational age and actual age (labeled C, white bars). The differences in group 3 were less significant and likely represent subclinical disease in preterm infants with food intolerance (D), and the plots represent sequential stool sampling in 4 NEC patients with corresponding iAP enzyme activity, total stool protein and iAP detection on western blot, pre-diagnosis (labeled pre, white bar) and at diagnosis (labeled D, red bar).

Figure 11 shows that combining all 3 biomarkers has possible clinical utility and improvement in sensitivity and specificity. The plots represent data points from 6 NEC patients and 7 controls, for a total of 51 samples. (A) A 3D scatter plot comparing WB data, fecal iAP activity and fecal protein is shown. The red diamonds represent the mean total fecal protein, iAP activity and WB percentage of fecal samples from NEC patients at diagnosis. Black circles represent mean total fecal protein, iAP activity and WB percentage of control points (including non-disease NEC patients). (B) A 2D scatter plot is represented showing the relationship between fecal iAP activity and WB percentage. There was clustering of the control samples (black circles) in the lower WB percentage and high activity trend. NEC samples demonstrated the opposite, i.e. higher WB percentage and low activity. (C) A schematic diagram illustrating a naive bayes classifier for demonstrating the sensitivity and specificity of all three biomarkers, alone and in combination, to improve performance.

Figure 12 shows that in those infants who were well tolerated and progressed rapidly to full feeding without problems, there was an identifiable trend towards lower fecal protein (panel a) and higher fecal iAP enzyme activity (panel B). The graph shows that the average total fecal protein and fecal iAP activity in the stools from 10 control patients during the first month of life correlates with the duration before complete enteral feeding is achieved.

Fig. 13 shows that the stool sample may be heterogeneous. There are two identifiable consistency compartments in the stool samples from NEC patients. The faecal compartments were separated and each compartment was subjected to western blot analysis with different results.

Fig. 14 shows relative iAP content, iAP activity and protein concentration. NEC was classified according to Bell et al and modified by Walsh and Kliegman. For this analysis, the term 'suspected NEC' is phase I, 'NEC' has been demonstrated to be phase II and more severe; the term 'perforated NEC' is used only to describe phase IIIB. Information from chart review was used to diagnose stage I. Phase II radiology requires a record of intestinal aeration.

FIG. 15 shows immunohistochemical staining of intestinal tissue.

Fig. 16 shows a schematic diagram of many functions of the iAP.

Figure 17 shows that NEC patients have higher total fecal protein content than control infants.

Figure 18 shows that fecal AP catalytic activity was consistently lower in NEC population.

Figure 19 shows that in matched patient samples, AP enzyme activity was always lower at NEC diagnosis.

Figure 20 shows that high levels of iAP protein associated with NEC were detected.

Figure 21 shows increased fecal iAP protein levels in the onset of NEC.

Figure 22 shows increased iAP protein levels and decreased iAP enzyme activity in the NEC episode.

FIG. 23 shows a schematic for testing non-specific binding of secondary antibodies. Without being bound by theory, the enzyme assay may be performed with only a secondary antibody conjugated to AP.

Figure 24 shows clinical data separating NEC diagnosis from controls matching X-ray and NEC suspicion (defined by neonatologists) with controls demonstrating that biomarkers can define NEC molecularly earlier.

Figure 25 is a bar graph showing that alkaline phosphatase measurements may be confounded by signals from secondary antibodies. The isolated alkaline phosphatase may catalyze the hydrolysis of MUP to form the fluorescent product MU. Secondary antibodies from two different commercial manufacturers that bind AP can also hydrolyze MUP to form fluorescent products. When alkaline phosphatase protein and secondary antibody were in the same measurement, an increased level of catalytic activity was observed. This can be monitored by standard spectrophotometric readings of biochemical activity and western blots.

Fig. 26 shows non-NEC to NEC transitions and the risk of NEC to non NEC transitions.

Fig. 27 shows a schematic diagram of a conversion model.

Figure 28 shows a two-variable analysis of the abundance and catalytic ability of intestinal alkaline phosphatase found in feces of preterm infants. Biological samples taken at the time of disease are shown as colored circles; suspected necrotizing enterocolitis is pink, severe necrotizing enterocolitis is red, and delayed septicemia is blue. The complementary control group is shown as a hollow gray circle.

Figure 29 shows analysis of the intestinal alkaline phosphatase found in feces of premature infants relative to diagnostic radiographic clinical evidence. The abundance of iAP in infant fecal samples relative to human small intestine lysate is shown in the left panel. Biological samples (vertical blue bars) sampled at the time of clinical determination of disease are shown as colored circles; pre-and post-disease samples collected from infants with NEC are shown as open red diamonds; samples collected from non-NEC patients are shown in gray. The normalized iAP abundance of iAP and normalized iAP catalytic activity were multiplied to generate trend scores (neccpredict), which are shown on the right panel. The boxplot shows the median value of neccpredict at clinical diagnosis (red box on day zero x axis), and the values two, four and six days before radiologic determination of disease. Boxplots of necprep values obtained from non-NEC infant samples are also shown. The Whisker method is the adjacent data point and the quartile method is Tukey.

Fig. 30 shows the relationship between (a) NEC, sepsis, and gut defense mechanisms. The microbiota creates a unique ecosystem in the intestinal lumen and mucosa. Various commensal and pathogenic mucosal microbiota regulate the intestinal immune function of the infant host. Activation of the innate and adaptive immune system by microbiota in turn modulates the systemic immune response. In sepsis, the immune response of the distant organs is triggered due to extreme signals. Host proteins iAP (green), TLR4 and IL-8 (blue) are proteins involved in the early and late microbiota response to inflammation. (B) The standard of care for premature infants requires new approaches to monitor NEC disease. The gold standard for diagnosis is x-ray (blue box), recognizing only 44% of advanced NEC cases; other diagnostic methods (light blue boxes) are bedside observable states, but are not molecular definitions. Current management options in NEC (white boxes) are contrasted with potential clinical outcomes (red boxes) using proposed biomarkers (green).

Fig. 31 shows the amount of iAP protein and its catalytic activity measured in fecal samples. (A) Immunoblot analysis showed that if the calibrator was ≦ 1 μ g, the relative iAP content detected in serial dilutions of human small intestine lysate had a linear relationship. Immunoblot bands were white against a black background. Superimposing a log plot of the signal and the calibrator; mean and SEM of 5 replicates are shown. (B) Mean ± SEM of iAP content for disease-free (white), NEC diagnosed (red), NEC suspected (pink) and sepsis diagnosed (blue) samples are shown. Quantitation of iAP in each sample was determined using the human intestinal tract as the maximum positive control (100% iAP content) and calf iAP as the negative control (0% iAP content) in the linear range. Asterisks indicate significant differences between p-values <0.001 and median disease status from control using the Mann-Whitney U test. Immunoblot efficacy was assessed by sensitivity/specificity calculations for NEC diagnosis (C) and sepsis (D) using a simple threshold-based classifier. (E) There is a direct correlation between fecal iAP activity and post-conception age (PCA). Mean and standard error binned by PCA are shown. Each control PCA chamber N-14-33 samples. (F) Mean ± SEM of enzyme activity for disease-free (white), NEC diagnosed (red), NEC suspected (pink) and sepsis diagnosed (blue) samples are shown. Asterisks indicate p-value < 0.05. Samples were assessed for sensitivity/specificity of iAP activity biomarkers at NEC diagnosis (G) and sepsis (H).

Figure 32 shows a stimulation protocol that takes into account sample size rationality.

Figure 33 shows the results of efficacy analysis for different effect sizes.

Fig. 34 shows iAP peptides that can be used as mass spectrometric quantitation calibrators. (A) Amino acid sequence alignment of human alkaline phosphatase: intestinal alkaline phosphatase (iAP; P09923), placenta-like alkaline phosphatase (PLAP; P05187), tissue non-specific alkaline phosphatase (TNAP; P05186) and Germ Cell Alkaline Phosphatase (GCAP); p10696). Boxes P1-P6 have 6 peptides with unique mass spectral characteristics that distinguish the four human AP proteins. (B) Allele frequencies of missense, single nucleotide polymorphism catalogs in the human population. (C) Sequences of six iAP peptides suitable for mass spectrometric quantification of protein abundance, and single nucleotide missense polymorphisms determined for each residue position. The peptide with the smallest deviation from zero on the x-axis and the lowest polymorphic frequency on the y-axis is the best candidate for the MS reference standard.

Figure 35 shows the association of Necrotizing Enterocolitis (NEC) and late sepsis with gut defense mechanisms. a-C, the intestinal physiological and structural changes associated with NEC, are superimposed in the cross-sectional view of the small intestine. Research efforts to develop NEC biomarkers have focused on proteins in the immune cascade and dysregulation of microbiota. Our approach focuses on host proteins involved in microbiota management. D, prospective recruitment of preterm infants with NEC and other established infections. E, the workflow of stool sample preparation was optimized for assay reproducibility and standardization. GI means gastrointestinal tract; IAP, intestinal alkaline phosphatase.

Figure 36 shows clinical features of patients with severe NEC, suspected NEC, or no NEC. Abbreviations: IQR, four-bit spacing; NA, not applicable; NEC, necrotizing enterocolitis; NICU, neonatal intensive care unit; NPO, fasting (nil per os); PCA, age after conception. (a) Differences between groups were compared using appropriate methods (ANOVA, Kruskal-Wallis or Fisher's exact test), and P <.05 indicated statistically significant differences between the 3 infant populations. (b) By parents, is identified as more than 1 ethnicity.

FIG. 37 shows the clinical features of other patients diagnosed with infection. Abbreviations: GI, gastrointestinal; IQR, four-bit spacing; NA, not applicable; NEC, necrotizing enterocolitis; NICU, neonatal intensive care unit; NPO, fasted; PCA, age after conception. (a) Differences between groups were compared using appropriate methods (ANOVA, Kruskal-Wallis or Fisher's exact test), and P <.05 indicated statistically significant differences between the 3 infant populations. (b) By parents, is identified as more than 1 ethnicity.

Figure 38 shows the correlation of fecal Intestinal Alkaline Phosphatase (IAP) content and activity with Necrotizing Enterocolitis (NEC) and other confirmed infections. A, boxplot and violin plots showing stool abundance and IAP activity for samples collected at severe (n-20) and suspected NEC (n-15). Samples from patients without NEC (n-86) are also shown, age-matched at the time samples were collected for NEC. The box plot must mark the 9 th and 91 th percentiles. B, recipient operating profile, IAP abundance (filled circles) and activity (open circles) in samples collected during severe (orange) or suspected (brown) NEC. C, box and violin plots showing fecal abundance and IAP activity of samples collected during sepsis (n ═ 18), other non-Gastrointestinal (GI) tract infections (n ═ 10), and age-matched control patients (n ═ 91). The box plot must mark the 9 th and 91 th percentiles. Receiver IAP abundance (filled circles) and activity (open circles) in samples collected during sepsis (dark blue) and other non-GI tract infections (light blue) receiver IAP abundance (filled circles) and activity (open circles) receiver operating profiles. (a) P <. 001; (b) 005 ═ P

FIG. 39 shows that the control experiments demonstrate operator reproducibility, antibody reagent specificity, and biological sample specificity. Five different operators performed (a) activity assay measurements and (B) iAP content assays in patient stool samples; the dashed line marks the 1:1 correspondence between repeat 1 and repeat 2. (C) The anti-human iAP antibodies used in this study were tested against human small intestine lysate (GI), purified human placental alkaline Phosphatase (PL), purified human tissue non-specific alkaline phosphatase (TN), and bovine intestinal alkaline phosphatase (cI). Densitometric quantification of total AP is listed 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 is 5. (D) Quantitation of the immunoblots used had a linear response to the amount of human intestinal alkaline phosphatase. The average relative fluorescence units and standard error (open triangles) for 5-7 total iAP were: 216,692 ± 14,533 for 7.5 μ g; 233,533 ± 20,264 for 3.75 μ g; 211,176 ± 132,267 for 1.875 μ g; 142,834 ± 13,019 for 0.938 μ g; 75,727 + -7,637 for 0.469 μ g; 44,101 + -1,410 for 0.234 μ g; 18,234 + -450 for 0.117 μ g; for 0.059 μ g, 4,918 ± 549; 1,164 ± 79 for 0.029 μ g; and 227 ± 57 for 0.015 μ g. (E) Serum AP activity and fecal iAP activity were compared if serum clinical trials and fecal samples were collected on the day of sampling. No relationship between serum AP activity and fecal iAP activity measurements was observed. N-148; the solid line is the best linear fit between fecal iAP activity and serum AP activity.

FIG. 40 shows the sequence alignment of 4 human alkaline phosphatases and calf intestinal alkaline phosphatase. The sequences shown are human intestinal alkaline phosphatase (iAP human; P09923UniprotID), calf intestinal alkaline phosphatase (iAP bovine; P19111), germ cell alkaline phosphatase (GCAP human; P10696), placenta-like alkaline phosphatase (PLAP human, P05187) and tissue non-specific alkaline phosphatase (TNAP human, P05186). The signal peptide is shown in grey at the N-terminus of the sequence. The propeptide is shown in grey italics at the C-terminus of the sequence. Residues involved in metal binding are marked with an asterisk; candidate glycosylation sites bear the # symbol. Secondary structural motifs were shown from human placental alkaline phosphatase crystals (PDB ID 1EW 2). A color heatmap of the number of polymorphisms found in the population is overlaid on the IAP human sequences.

Detailed Description

Abbreviations and Definitions

A detailed description of one or more preferred embodiments is provided herein. However, it should be understood that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed manner.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction 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".

Where any phrase "for example," such as, "" including, "or the like is used herein, it is to be understood that it is followed by the phrase" and is not limited thereto, unless expressly stated otherwise. Similarly, "examples," "exemplary," etc. are to be construed as non-limiting.

The term "substantially" allows for variations in the descriptors that do not adversely affect the intended purpose. Descriptive terms are to be understood as being modified by the term "substantially", even if the term "substantially" is not explicitly mentioned.

The terms "including" and "comprising" and "having" and "relating to" (and similarly "including", "comprising", "having" and "relating to") and the like are used interchangeably and have the same meaning. In particular, the definition of each term is consistent with the conventional definition of "comprising" in U.S. patent law, and thus is to be construed as an open-ended term meaning "at least the following," and is also to be construed as not excluding other features, limitations, aspects, and the like. 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 terms "a" or "an" are used, it should be understood that "one or more" unless such an interpretation is meaningless in context.

As used herein, the term "about" may refer to approximately, roughly, around, or within the range of … …. When the term "about" is used in conjunction with a range of values, it modifies the range by extending the boundaries above and below the stated values. In general, the term "about" is used herein to modify numerical values above and below the stated values by a variance of 20% up or down (higher or lower).

The present invention relates to compositions and methods for detecting and treating gastrointestinal disorders.

Gastrointestinal diseases

In addition to necrotizing enterocolitis found in newborns and premature infants, necrotizing enterocolitis also affects non-newborns. For example, necrotizing enterocolitis in non-newborns, such as adults, can be caused by: inflammatory mediators; nutritional disorders such as anorexia or significant weight loss; gastrointestinal dysfunction; alcoholism; poor absorption; drugs that block intestinal proteases; smoking; circulatory disorders such as decreased mesenteric blood flow, intestinal ischemia, intestinal atherosclerosis; cholelithiasis; administration of a drug; immunodeficiency, such as a defect in the IgA secretory component or intestinal T lymphocytes, with poor antibody response; fecal impaction or constipation; or infectious agents, such as bacterial infections, food-borne infections and food-borne diseases.

Non-limiting examples of such drugs include those with anticholinergic properties, such as neuroleptic or phenothiazine-based neuroleptic, anesthetic, inflammatory mediator, antidepressant, iron pill, laxative or antacid.

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, leading to cell destruction, necrosis, and intestinal perforation.

Constipation or fecal effects may 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 consumption, overuse of laxatives, pregnancy, digestive diseases, impulse to resist defecation, drugs, stress, or hypothyroidism.

Aspects of the invention relate to gastrointestinal disorders. Gastrointestinal disorders refer to disorders involving the gastrointestinal tract, i.e., the esophagus, stomach, small intestine, large intestine, and rectum, as well as the liver, gallbladder, and pancreas, which are digestive, accessory organs. 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 types of colitis include Adult Necrotizing Enterocolitis (ANEC), pseudomembranous enterocolitis, infectious colitis, ulcerative colitis, crohn's disease, ischemic colitis, radiation colitis.

Intestinal Alkaline Phosphatase (iAP)

Using a single 100-150mg fecal sample from three healthy human donors, 234 human proteins secreted from the gastrointestinal tract were identified. Among these, the core proteome of 57 proteins common among the three human individuals was determined. Although the presence of this core proteome can be repeated, the relative abundance of most shared proteins varies among the three human subjects, suggesting that the core proteome can be used to identify host-specific proteomic features.

Intestinal Alkaline Phosphatase (iAP) is expressed in small intestine enterocytes, is secreted into the intestinal lumen and systemic circulation together, and plays an essential role in maintaining intestinal barrier function by detoxifying bacterial lipopolysaccharides and maintaining microbial homeostasis. As the main alkaline phosphatase in feces, iAP has been identified as one of the 57 proteins in the core human fecal proteome.

Aspects of the invention relate to methods for diagnosing gastrointestinal disease in a subject. For example, the method comprises the steps of: 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 to a profile obtained from a control sample; and treating the subject. Embodiments may also relate to preventing the progression of gastrointestinal disease in a subject in need thereof, and ameliorating symptoms associated with gastrointestinal disease in a subject in need thereof. In embodiments, the control sample may comprise two or more control samples.

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

If the subject is diagnosed with a gastrointestinal disorder, embodiments of the invention include treating the subject. For example, treating the subject may comprise 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 may be to not allow the subject to eat. Non-limiting examples of iAP replacement compositions include gene or protein replacement compositions.

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

Protein therapy can be accomplished by any method effective to introduce an iAP protein or 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 gastrointestinal disease) can be administered alone or in combination with an agent that facilitates protein administration or activity. An "effective amount" can be determined by one of skill in the art based on factors such as the type and severity of the condition being treated, the weight and/or age of the subject, the subject's past medical history, and the selected route of administration of the agent.

In embodiments, the iAP protein may be associated with a lipid, such as a detergent or other amphipathic micelle, membrane vesicle, liposome, virosome, or microsome. Lipid compositions that are naturally fused or that can be designed to be fused (e.g., by incorporating the fusion protein into a lipid) are particularly preferred. The 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 cellular proteins that mediate cell-cell fusion. Other non-viral fusion proteins include sperm protein PH-30, an integral membrane protein located on the surface of sperm cells and 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 the binding and depolymerizing elements of PH-30 (e.g., bitistatin, barbourin, viper toxin (kistin), and snake venom saw-scale viper (echistatin)). In addition, lipid membranes can be fused using conventional chemical fusion agents such as polyethylene glycol (PEG).

In embodiments, a subject may 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 symptoms of gastrointestinal disease. The iAP can be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, parenterally, orally, submucosally, by inhalation (e.g., nebulized pharmaceutical compositions), or other suitable route of administration within an effective dosage range. If a particular mode of administration is desired, the iAP can be encapsulated in a material that protects it from enzymatic degradation. In addition, it may be useful to administer an agent that clears the bacterial infection prior to administration.

Alternatively, preparations of the gene encoding iAP or fragments thereof can be incorporated into a suitable vector for delivering the gene into the cells of a subject. In embodiments, the iAP gene therapy can be transient and require repeated delivery to the subject. In other embodiments, gene therapy can cure gastrointestinal diseases. For example, if the genetic material encoding the iAP is integrated into a subject's stem cells, all subsequent generations of such cells can produce the true iAP 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.

Non-limiting modes of administration of treatment include Intravenous (IV); in the mucosa; in muscle; subcutaneous, and non-invasive modes of administration, such as oral, intranasal, buccal, intrapulmonary, intrabronchial, and transdermal.

Aspects of the invention also relate to methods for screening for the presence of a characteristic in a subject at risk of having a gastrointestinal disease or a subject having an 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 comprises an Intestinal Alkaline Phosphatase (iAP) protein; comparing the GI disease biomarker profile to a profile obtained from a control sample; and treating the subject. Similarly, aspects may also relate to methods for identifying a subject at risk for gastrointestinal disease or a subject with asymptomatic gastrointestinal disease. In embodiments, the control sample may comprise two or more control samples.

Aspects of the invention include measuring the total protein concentration in the sample, the intestinal alkaline phosphatase activity in the sample, or a combination thereof. Samples used in such methods and assays for collecting such measurements are described herein. For example, if the protein concentration in the sample is greater than about 1.0mg/ml, 1.1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml, 1.9mg/ml, 2.0mg/ml, 2.1mg/ml, 2.2mg/ml, 2.3mg/ml, 2.4mg/ml, 2.5mg/ml, 2.6mg/ml, 2.7mg/ml, 2.8mg/ml, 2.9mg/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, 4mg/ml, 4.4mg/ml, 4mg/ml, 3.5mg/ml, 4mg/ml, 4.6mg/ml, 4mg/ml, 3.7mg/ml, 3.8mg/ml, 3.9mg/ml, 4mg/ml, 3.4mg/ml, 4mg/ml, 3.4mg/ml, 4mg/ml, 3.4mg/ml, 4mg/ml, 3.0mg/ml, 3.4mg/ml, 3.0mg/ml, 3mg/ml, 3.0mg/ml, 3.4mg/ml, 3mg/ml, 3.0mg/ml, 3.4mg/ml, 4mg/ml, 3.4mg/ml, 3.0mg/ml, 3.4mg/ml, 3mg/ml, 3.4mg/, 4.5mg/ml, 4.6mg/ml, 4.7mg/ml, 4.8mg/ml, 4.9mg/ml, 5.0mg/ml, the subject may be diagnosed with GI disease. As another example, if the iAP activity is less than about 10mU/mg, 20mU/mg, 30mU/mg, 40mU/mg, 50mU/mg, 60mU/mg, 70mU/mg, 80mU/mg, 90mU/mg, 100mU/mg, 200mU/mg, 300mU/mg, 400mU/mg, 500mU/mg, 600mU/mg, 700mU/mg, 800mU/mg, 900mU/mg, 1000mU/mg, 1100mU/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, 100U/mg, 200U/mg, 300U/mg, 400U/mg, 500U/mg, 600U/mg, 700U/mg, 800U/mg, 900U/mg, etc, 1000U/mg, the subject may be diagnosed with GI disease. For example, if the protein concentration in the fecal sample is greater than about 1.6mg/ml, or greater than about 1.8 mg/ml; if the iAP activity is less than about 979mU/m, or less than about 1256 mU/mg; or if the level of iAP protein is at least two standard deviations above the mean of the control samples, the subject can be diagnosed with gastrointestinal disease. As another example, a subject may be diagnosed with a GI disease 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 a control sample. For example, a subject can be diagnosed with a gastrointestinal disease if the iAP protein detected by the anti-iAP antibody by densitometry exceeds 10.7% of the control, or 4.8% of the control by densitometry. In other embodiments, a subject may be diagnosed with gastrointestinal disease if two thresholds are met, or if all three thresholds are met. In embodiments, the control sample may comprise two or more control samples.

The term "threshold", e.g. indicating a threshold for 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 fecal samples), above which threshold a likelihood of having and/or developing a gastrointestinal disease, e.g. NEC, is associated.

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

Embodiments of the invention include machine learning techniques or applications to determine appropriate clinical thresholds. For example, such techniques include those known in the art, including Naive Bayes Classifier (NBC), Linear Discriminant Analysis (LDA), or Support Vector Machine (SVM), as well as support vector machine options. One skilled in the art will readily appreciate that such a threshold may vary depending on the sample size being analyzed and the statistical analysis employed.

Aspects of the invention also include identifying and/or diagnosing 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 may diagnose an advanced inflammatory state, such as one determined by radiologic findings of intestinal gas (portal vein or biliary tract gas). As another example, embodiments can identify early stages of disease before rampant inflammation of the intestinal tract is physiologically evident. Physicians currently suspect gastrointestinal disorders from a range of signs, such as bloating, abdominal tenderness, decreased bowel sounds, hematochezia, increased apneas, unstable body temperatures, bile secretions, and eating intolerance. The clinical symptoms of suspected disease are distension of the intestinal loop and radioactively manifested thickening of the intestinal wall. Laboratory examination of suspected diseases results in thrombocytopenia, decreased or increased white blood cell counts, increased band counts and metabolic acidosis. Embodiments can match the identification of radiological findings of intestinal gas (portal vein or biliary tract gas) at the late stage of inflammation. The method can also identify early stages of disease before physiologically significant intestinal inflammation is rampant.

Sample (I)

Aspects of the invention include measuring or detecting biomarkers of gastrointestinal disease in a biological sample. The biomarkers of the invention can be measured in different types of biological samples. Non-limiting examples of biological samples that may be used in the methods of the invention include stool, plasma, cord blood, newborn blood, cerebrospinal fluid, tears, vomit, saliva, urine, stool, and meconium. If desired, the sample can be prepared to enhance the detectability of the biomarkers. For example, a sample from a subject may be fractionated. Any method of enriching for the biomarker polypeptide of interest may 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 biomarkers depending on the detection method used. For example, if an antibody that specifically binds to a biomarker is used to detect the presence of the biomarker in a sample, sample preparation may not be necessary. Sample preparation may involve fractionation of the sample and collection of the fraction determined to contain the biomarker. Methods of prefractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis, mass spectrometry, and liquid chromatography.

The methods described herein 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 from a subject, either directly or indirectly. For example, a biological sample may be obtained (e.g., at a care facility, such as a doctor's office, hospital, laboratory facility) by obtaining a tissue or fluid sample from a subject (e.g., blood draw, bone marrow sample, spinal fluid draw). Alternatively, the biological sample may be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who obtained the sample directly from the subject. The biological sample may be, for example, stool from a subject, such as stool, tissue (e.g., blood), cells (e.g., hematopoietic cells, e.g., hematopoietic stem cells, white blood cells, or reticulocytes, stem cells, or plasma cells), vesicles, biomolecular aggregates, or platelets.

Detection and antibodies

Aspects of the invention include biomarkers of GI disease. For example, aspects include biomarkers of necrotizing enterocolitis. For example, biomarkers of GI disease include iAP enzyme activity, iAP protein levels, iAP dimerization/dissociation, post-translationally modified iAP, total fecal protein, or combinations thereof.

Aspects of the invention include assays for measuring iAP enzyme activity. Aspects of the invention include assays that measure the levels of iAP protein. Aspects of the invention include assays to measure iAP dimerization/dissociation. Aspects of the invention include assays for measuring post-translationally modified iAP. Aspects of the invention include assays for measuring total fecal protein.

Non-limiting examples of post-translational modifications include acetylation, acylation, alkylation, amidation, butyrylation, deamidation, formylation, glycosylphosphatidylinositol (phosphorylation), glycosylation, hydroxylation, iodination, ISG, lipoylation, malonylation, methylation, myristoylation, palmitoylation, phosphorylation, phospho-phosphorylation, prenylation, propionylation, ribosylation, succinylation, sulfation, SUMO-acylation, or ubiquitination.

iAP is a homodimer; 4 bivalent (Zn) bonds per protomer²⁺And Mg²⁺) Ions, which are critical for maintaining the structural integrity and catalytic activity of the enzyme. iAP is one of four different alkaline phosphatases found in human tissue that are associated with physiological functions. Although high concentrations of iAP are found in the luminal vesicles secreted by the intestinal cells at the brush border of the microvilli, a small amount of iAP is released into the blood and the intestinal lumen, which travels in the intestinal tract.

Embodiments of the invention include the use of assays known in the art to measure or detect such biomarkers. Non-limiting examples of assays include immunoassays, colorimetric assays, fluorescent assays, or combinations thereof. Non-limiting examples of immunoassays include western blot assays, enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, 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 fragment thereof, and binding of the antibody to the biomarker in the sample is detected or measured.

In embodiments, the antibody or fragment thereof may be specific for iAP (anti-iAP). The antibody may be a polyclonal antibody or a monoclonal antibody. The antibodies or fragments thereof can be linked to molecules 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.

Examples of assays that can be used in the methods of the invention, while not intended to be limiting, include bradford assays, bicinchoninic acid (BCA) assays, Lowry assays, pyrogallol dye binding assays, coomassie blue dye binding assays, endpoint assays, kinetic assays, e.g., kinetic assays using a fluorescent substrate such as 4-methylumbelliferyl phosphate, a chemiluminescent substrate such as CSPD and CDP-Star, a DynaLight substrate with a RapidGlow enhancer, or a colorimetric 4-nitrophenyl phosphate, assays that detect phosphatase reactions, assays that detect ATP hydrolysis, or combinations thereof. In embodiments, the assay may be provided in a multi-well format, such as a 6-, 12-, 24-, 48-, or 96-well plate. In embodiments, the assay may be provided in a standard cuvette, such as a 1ml cuvette.

Total protein, e.g., total fecal protein, can be measured by assays known to those of skill in the art (see, e.g., Cardinal Health catalog, Dublin, Ohio, pages 7, 27, and 85 of 2013, which is incorporated herein by reference in its entirety, see Roche total protein/TP 2, Cobas c502 TPUC3, or Abbott total protein kit). For example, the pyromaloll Red Molybdate dye binding method provides a total protein quantitative colorimetric method with higher linearity using microliter volumes of biological samples in a manual or automated system. As described herein, pyrogallol red can be provided in a kit that includes reagents, controls, and reagent standards, e.g., 25mg/dL, 50mg/dL, 100mg/dL, and 200 mg/dL.

The enzyme used in embodiments herein, e.g., for detecting protein levels or enzyme activity, may be, e.g., alkaline phosphatase, horseradish peroxidase, beta-galactosidase and/or glucose oxidase; the substrate may be an alkaline phosphatase, horseradish peroxidase, beta-galactosidase or glucose oxidase substrate, respectively (see Molecular Probes Handbook-A Guide to Fluorescent Probes and laboratory Technologies,11th Edition (2010), Invitrogen, which is incorporated herein by reference in its entirety).

In embodiments, an enzyme, such as alkaline phosphatase or horseradish peroxidase, can be linked to the secondary antibody. Without being bound by theory, the measurement of alkaline phosphatase may be confounded by the signal from the secondary antibody. The isolated alkaline phosphatase may catalyze the hydrolysis of MUP to form the fluorescent product MU. Secondary antibodies from two different commercial manufacturers that bind AP, for example, can also hydrolyze MUP to form a fluorescent product. When alkaline phosphatase protein and secondary antibody were in the same measurement, an increased level of catalytic activity was observed. This activity can be monitored by standard spectrophotometric readings of biochemical activity and western blots.

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 tetrazole/iodo nitrotetrazole (BCIP/INT substrate, tawny precipitate, Biomeda), 5-bromo, 4-chloro, 3-indolyl phosphate/nitro blue tetrazole (BCIP/NBT substrate, blue/violet), 5-bromo, 4-chloro, 3-indolyl phosphate/nitro blue tetrazole/iodo nitrotetrazole (BCIP/NBT/INT, brown precipitate, DAKO, fast red (red), magenta phosphorus (magenta), naphthol AS-bisphosphate (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 ^TMBlack (black), VECTOR^TMBlue (blue), VECTOR^TMRed (Red), Vega Red (raspberry Red).

Horseradish peroxidase (HRP, sometimes abbreviated as PO) substrates include, but are not limited to, 2' Azino-di-3-ethylbenzothiazoline sulfonate (ABTS, green, water soluble), aminoethylcarbazole, 3-amino, 9-ethylcarbazole AEC (3A9EC, red). α -Naphtholpyran (Red), 4-chloro-1-naphthol (4C1N, Blue, bluish Black), 3' -diaminobenzidine Tetrahydrochloride (DAB, Brown), ortho-benzidine (Green), ortho-phenylenediamine (OPD, Brown, Water soluble), TACS Blue (Blue), TACS Red (Red), 3',5,5' tetramethylbenzidine (TMB, Green or Green/Blue), TRUE BLUE^TM(blue), VECTOR^TMVIP (purple), VECTOR^TMSG (smoky Blue grey) and Zymed Blue HRP substrate (bright Blue).

Glucose Oxidase (GO) substrates include, but are not limited to, nitroblue tetrazolium (NBT, purple precipitate), tetranitroblue tetrazolium (TNBT, black precipitate), 2- (4-iodophenyl) -5- (4-nitrophenyl) -3-phenyltetrazolium chloride (INT, red or orange precipitate), tetrazolium blue (blue), nitrotetrazolium violet (purple), and 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT, purple). All tetrazole substrates require glucose as a co-substrate. Glucose is oxidized and the tetrazolium salt is reduced to form insoluble formazan, which forms a colored precipitate.

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

Other examples of alkaline and acid phosphatase substrates include 9H- (1, 3-dichloro-9, 9-dimethylacridin-2-one-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).

Alkaline phosphatase activity, e.g., intestinal alkaline phosphatase activity, can be detected and/or measured using a chromogenic substrate and/or a fluorogenic substrate for alkaline phosphatase. For example, 4-methylumbelliferone phosphate (MUP) is a fluorogenic substrate for alkaline phosphatase, and alkaline phosphatase-mediated hydrolysis of its phosphate substituent produces blue fluorescent 4-methylumbelliferone (-386/448 nm excitation/emission). In embodiments, the alkaline phosphatase substrate may be mixed directly with the biological sample, e.g., stool, thereby allowing direct detection of the presence of alkaline phosphatase or a measurement of its activity.

Alkaline Phosphatase (AP) substrates include, but are not limited to, AP-blue substrate (blue precipitate, Zymed catalog p.61); AP-orange substrate (orange, precipitate, Zymed), AP-red substrate (red, red precipitate, Zymed), 5-bromo, 4-chloro, 3-indolyl phosphate (BCIP substrate, turquoise precipitate), 5-bromo, 4-chloro, 3-indolyl phosphate/nitrobluetetrazolium/iodonitrotetrazolium (BCIP/INT substrate, tawny precipitate, Biomeda), 5-bromo, 4-chloro, 3-indolyl phosphate/nitrobluetetrazolium (BCIP/NBT substrate, blue/violet ) 5-bromo, 4-chloro, 3-indolyl phosphate/nitro blue tetrazolium/iodo nitro tetrazolium (BCIP/NBT/INT, brown precipitate, DAKO, fast Red (Red), magenta phosphorous (magenta), Naphthol AS-bisphosphate (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^TMBlack (black), VECTOR^TMBlue (blue), VECTOR^TMRed (Red), Vega Red (raspberry Red).

Other substrates known in the art, including those described herein, can be used in embodiments of the invention (see Molecular Probes Handbook-A Guide to Fluorescent Probes and laboratory Technologies, 11 th edition (2010), Invitrogen, which is incorporated herein by reference in its entirety). In addition, various fluorophores known in the art can be covalently attached to a substrate, such as MUP, as desired.

The enzymatic reaction can provide a highly specific, rapid and sensitive assay for detecting specific proteins in a sample (e.g., iAP in stool). Examples of suitable fluorescent substrates that can be used in the present invention include fluorescein diacetate, 4-methyl umbelliferyl acetate, 4-methyl umbelliferyl casein, 4-methyl umbelliferyl-alpha-L-arabinopyranoside, 4-methyl umbelliferyl-beta-D-fucopyranoside, 4-methyl umbelliferyl-alpha-L-fucopyranoside, 4-methyl umbelliferyl-beta-L-fucopyranoside, 4-methyl umbelliferyl-alpha-D-galactopyranoside, 4-methyl umbelliferyl-beta-D-galactopyranoside, 4-methyl umbelliferyl-alpha-D-glucopyranoside, 4-methyl umbelliferyl-beta-D-glucopyranoside, 4-methyl umbelliferyl-alpha-D-glucopyranoside, 4-beta-D-glucopyranoside, and, 4-methylumbelliferyl-beta-D-glucuronide, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl oleate, 4-methylumbelliferyl phosphate, bis (4-methylumbelliferyl) phosphate, 4-methylumbelliferyl pyrophosphate diester, 4-methylumbelliferyl beta-D-xylopyranoside.

Test subject

As described herein, embodiments of the invention include measuring or detecting a gastrointestinal biomarker in a subject. The term "subject" or "patient" may refer to any organism to which aspects of the invention may be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects to which the compounds of the present disclosure can be administered are mammals, particularly primates, and particularly humans. For veterinary applications, a wide variety of subjects will be suitable, for example livestock such as cattle, sheep, goats, cows, pigs, etc.; poultry, such as chickens, ducks, geese, turkeys, and the like; 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, among others. The term "living subject" refers to the subject or another living organism. The term "living subject" refers to an entire subject or organism, not just a portion (e.g., liver or other organ) excised from a living subject.

In embodiments herein, the subject comprises a mammal, such as a human or a vertebrate. Examples of such include, but are not limited to, dogs, cats, horses, cows, pigs, sheep, goats, chickens, primates, such as monkeys, fish (aquaculture species), such as salmon, rats and mice. Humans include premature infants, children, adolescents, adults or the elderly.

Although aspects of the invention described herein relate to human gastrointestinal diseases, aspects of the invention are applicable to other non-human vertebrates as well. Aspects of the invention are suitable for veterinary use, for example for livestock. In general, aspects will vary according to the type of use and mode of administration, as well as the specific requirements of the individual subject.

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 an infection, or a method of achieving a desired change, such as a reduction or prevention of an infection, wherein the treatment comprises administering an antibiotic to a subject such that it is effective to treat the disease or produce a physiological change. The antibiotic regimen may include variations known to those skilled in the art such as antibiotic selection (e.g., including proper drug selection, route of administration, and dosing regimen), time of administration, and duration. Non-limiting examples of such antibiotics include vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), gentamicin, Flagyl (metronidazole mimetic), 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 methods disclosed herein may further comprise treating the subject. In embodiments, the treatment may comprise administering an effective amount of an antibiotic, a probiotic, an intravenous fluid, cessation of oral feeding, an iAP replacement composition, parenteral (or intravenous) nutrition, or a combination thereof to a subject diagnosed with a gastrointestinal disorder.

In one embodiment, e.g., a subject with NEC, the antibiotic may be administered to the subject for a sufficient time, e.g., 10-14 days, in which the antibiotic is administered to the infant. For other embodiments, e.g., a subject with sepsis, the patient may be administered an antibiotic for 7 days. For example, antibiotic administration and/or prescription can be used for broad spectrum coverage, e.g., for (i) gram-positive bacteria, (ii) gram-negative bacteria, and (iii) anaerobic bacteria. Non-limiting examples of such regimens include vancomycin (gram positive, including MRSA), ceftazidime (third generation cephalosporin-gram negative, some gram positive and pseudomonas), metronidazole (anaerobic bacterial coverage), oxacillin (gram positive). Non-limiting examples of general antibiotic regimens include ampicillin + gentamicin for a vertically acquired infection, possibly from the mother, and vancomycin + cetrimide for a hospital acquired infection, possibly. From the replies of 46 new pediatricians at the NEC seminar of 4 months in 2017, the commonly used antibiotics/antifungals for NEC treatment were gentamicin (32%), vancomycin (28%), ampicillin (25%), Zosyn (combination of piperacillin and tazobactam; 15%), flag (metronidazole mimetic; 19%), clindamycin (6%), meropenem (4%), fluconazole (antifungal, 7%) and others (1%).

In some embodiments, the probiotic may also be administered to the subject. As used herein, probiotic refers to a single or mixed culture of living microorganisms that can help to reestablish the normal flora in the gastrointestinal tract. Probiotics may enhance the immune response, trigger the production of enzymes that degrade toxins and/or block colonic attachment sites. See, McFarland, J.Medic.Microbiol.2005, 54: 101-. Non-limiting examples of probiotic organisms include those of the genera bifidobacterium, lactobacillus, lactococcus and pediococcus, saccharomyces boulardii and related bacteria and yeasts.

In some embodiments, the subject may be administered an intravenous fluid or intravenous therapy. Intravenous therapy may refer to the infusion of a liquid substance directly into a vein of a subject. Non-limiting examples of such fluids include saline (e.g., 0.9% NaCl in water or 0.45% saline in water), ringer's lactate (0.9% NaCl with electrolytes and buffers), D₅W (5% aqueous glucose solution), D₅NS (5% glucose in 0.9% saline), D₅1/2NS (5% glucose in 0.45% saline), D₅LR (5% glucose in ringer's lactate) or Normosol-R. In embodiments, the solution may be isotonic. In other embodiments, the solution may be hypotonic.

In some embodiments, parenteral (or intravenous) nutrition may be administered to a 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.

In embodiments, the subject may be stopped from eating, e.g., orally, until eating tolerance can be demonstrated. For example, food tolerance can be demonstrated when a preterm infant is able to safely ingest and digest prescribed enteral (via the oral cavity) food without complications associated with gastrointestinal dysfunction or infection. Clinical evidence for food tolerance in very low birth weight preterm infants can include the number of days required to reach full food intake (reported in the range of 100 mL per kg), the number of food intolerance episodes, the number of days to stop eating due to food intolerance symptoms, the time to recover birth weight, calf growth, weight gain, pillow circumference, and length.

In embodiments, feeding refers to the intake of infant formula, such as EleCare (Abbott Nutrition), neocure (Similac), EnfaCare (Enfamil), Pregestimil (Enfamil), Similac Special Care, or SSC (Similac), Gentlease (Enfamil). Eating may also refer to ingestion of a supplement, such as, for example, micro-fat (Nestle Health Science).

In embodiments, iAP replacement therapy may refer to protein replacement therapy. The term "protein substitution" may refer to the introduction of a non-native, purified protein, such as iAP, into an individual lacking such a protein. The protein administered may be obtained from a natural source or by recombinant expression. The term also refers to introducing the purified protein into an individual who otherwise requires or benefits from administration of the purified protein, e.g., has a protein deficiency. The introduced protein may be a purified recombinant protein produced in vitro, or a protein purified from an isolated tissue or body fluid, such as placenta or animal milk, or from a plant. For example, Bifidobacterium, Klebsiella and Escherichia coli alkaline phosphatase (Swittink et al, 2017.Metaproteomics novel functional variants of prevention of molecular and Cellular proteomics. DOI:10.1074/mcp. RA117.000102 (in printing)) are also detected in human stools of preterm infants and can therefore be a source of iAP proteins for protein replacement therapy. Thus, in embodiments, the increased AP activity may be the result of a bacterial flora, and not just from human iAP.

Disposable article

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

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-translational modifications and samples are described herein.

In embodiments, the biosensor may be an immunosensor, and may 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 may produce a new change in color or spectral absorption. In embodiments, the biosensors of the invention include a biological recognition element or a molecular recognition element that provides a high degree of specific binding or detection selectivity for a particular analyte, such as iAP. The biological recognition element or system may be a material of biological origin, such as an enzyme or enzyme sequence; an antibody or fragment thereof; a membrane receptor protein; DNA; organelles, natural or synthetic cell membranes; whole or partial viable or non-viable bacterial, plant or animal cells; or a piece of plant or mammalian tissue, and is typically used to specifically interact with a target biological analyte. The biological recognition element is responsible for selectively recognizing the analyte and providing the physicochemical signal on which the output signal is based. The physicochemical signal generated by the one or more biological recognition elements may be communicated visually to the wearer or caregiver (i.e., by a color change visible to the human eye). Other embodiments may generate optical signals, which may require other instrumentation to enhance the signal. These include fluorescence, bioluminescence, total internal reflection resonance, surface plasmon resonance, raman methods, and other laser-based methods.

Alternatively, the signal may be processed by an associated transducer, e.g., the transducer may 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 may trigger an actuator, as described herein. The signal may be qualitative (e.g., indicative of the presence of the target biological analyte) or quantitative (i.e., a measure of the amount or concentration of the target biological analyte). In such embodiments, the transducer may optionally generate an optical, thermal or acoustic signal.

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). Further, the signal may be transmitted to a remote indicator site (e.g., by a wire or transmitter, such as an infrared or radio frequency transmitter), which includes other locations within or on the article or remote device. Furthermore, the biosensor 60, or any component thereof, may be adapted to only detect and/or signal concentrations of the target biological analyte that are above a predetermined threshold level (e.g., where the target biological analyte is typically present in bodily waste or when the concentration of the analyte is below a known "dangerous" level).

In one embodiment, the disposable article may be a diaper to be worn by a subject. Non-limiting examples of additional disposable items include wipes, dipsticks, spoons, scoops, filter paper, or swabs for cleaning a subject.

In aspects of the invention, a disposable article as described herein may be a component of a kit useful for diagnosing a subject as having a gastrointestinal disorder. Additional components of the kits of the invention may include a biological recognition element, a support structure, and instructions for use thereof. For example, an iAP biorecognition element, such as an antibody described herein, can be immobilized to a solid support structure.

Non-limiting examples of the composition of the solid support structure include plastic, cardboard, glass, perspex, tin, paper, or combinations thereof. The solid support may also include a dipstick, a spoon, a small spatula, filter paper or swab.

Aspects of the invention also relate to diagnostic kits for identifying molecular biomarkers for a subject exhibiting or having a predisposition to develop gastrointestinal disease. In embodiments, the kit comprises at least one of means for determining total fecal protein concentration, means for determining Intestinal Alkaline Phosphatase (iAP) activity, and an iAP biorecognition element, wherein together represent a predisposition indicating the presence or development of gastrointestinal disease in a human subject. In embodiments, the characteristic comprises a total protein concentration at least two standard deviations above the mean of the control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of the control sample, or an intestinal alkaline phosphatase activity at least two standard deviations below the mean of the control sample. In yet other embodiments, the characteristic may be selected from at least two of the group comprising a total protein concentration at least two standard deviations above the mean of the control sample, an intestinal alkaline phosphatase protein concentration at least two standard deviations above the mean of the control sample, and an intestinal alkaline phosphatase activity at least two standard deviations below the mean of the control sample. In embodiments, the control sample may comprise two or more control samples.

In one embodiment, the kit comprises (a) a container comprising the components and a support structure as described herein, and optionally (b) an informational material. The informational material may be descriptive, instructive, marketable, or other material related to the use of the methods and/or reagents described herein for diagnostic purposes. In one embodiment, the kit further comprises a therapeutic agent, such as an antibiotic, probiotic, or iAP replacement composition.

The form of the information material of the kit is not limited. In one embodiment, the informational material may include information regarding the production of the kit components, such as molecular weight, concentration, expiration date, batch or production site information, and the like. In one embodiment, the informational material relates to a method of using the kit of parts (e.g., diagnosing a subject with a GI disorder). The information may be provided in a variety of formats, including printed text, computer readable material, video or audio recordings, or information providing a link or address to substantive material.

The kit may include other ingredients such as solvents or buffers, stabilizers or preservatives. Optionally, the kit may comprise a therapeutic agent, such as an iAP replacement composition or an antibiotic, which may be provided in any form, such as a liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the reagents are provided as liquid solutions, the liquid solutions are preferably aqueous solutions. When the reagents are provided in dry form, reconstitution is typically by addition of a suitable solvent. Solvents, such as sterile water or buffers, may optionally be provided in the kit.

Examples

The following examples are provided to facilitate a more complete understanding of the invention. The following examples illustrate exemplary ways of making and practicing the invention. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be utilized to achieve similar results.

Example 1

Described herein are methods for diagnosing acquired gastrointestinal emergencies common to premature infants. This disease (necrotizing enterocolitis or NEC) occurs in 12% of premature infants; 30% of NEC patients are non-viable. In the united states, a total of approximately 5000 infants have NEC annually. Due to non-specific symptoms, the 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 indicated 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 by western blot alone is closely related to NEC diagnosis and can be used in ELISA format.

Current clinical diagnostic methods rely on imaging: x-ray, CT, and ultrasound. The success rate of radiography diagnosis is only 48% at most. The embodiments as described herein have a true positive rate of 93% and a true negative rate of 95% for disease diagnosis. Embodiments as described herein may have the potential to risk assess and monitor disease.

The method is relatively fast and inexpensive compared to proteomics efforts and mass spectrometry. In addition, other patent applications use serum or urine; serum is invasive and requires fluid extraction from very fragile patients, whereas urinalysis cannot directly read gastrointestinal discomfort.

Example 2

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

Summary of the invention

The target is as follows: necrotizing Enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants, with high mortality and morbidity. Diagnosis and management can be difficult due to non-specific symptoms, inconsistent radiological findings, and rapid exacerbations. This investigation was done to test whether fecal Intestinal Alkaline Phosphatase (iAP) is a specific biomarker for NEC.

Research and design: in a prospective, longitudinal, case-control study, serial stool samples of 6 NEC patients and 12 control infants were collected for measurement of total stool protein, iAP activity and detection of iAP protein by western blot. In classifier-based analysis, data is evaluated by longitudinal assessment, inter-group comparison, and sensitivity/specificity assessment of individual patients.

As a result: there was no significant difference in gestational age or birth weight between groups 2. In 2 patients at longitudinal follow-up, fecal protein increased, iAP activity decreased, and iAP protein was detected by western blot after NEC development. NEC patients were diagnosed with higher mean fecal protein content (p ═ 0.005), lower iAP activity (p <0.0001) and higher intensity of the specific iAP protein band on western blot (p ═ 0.002) compared to controls. The 3-feature naive bayes classifier distinguished NEC from control samples with 93% sensitivity and 95% specificity.

And (4) conclusion: despite the limited number of subjects and samples, the results of the study indicate that specific changes in fecal protein, iAP activity and iAP western blot intensity occur during NEC. Preliminary sensitivity and specificity studies indicate that three-component biomarkers have potential as a non-invasive diagnostic and monitoring tool for NEC.

Introduction to the design reside in

Necrotizing Enterocolitis (NEC) is a serious inflammatory disease of the gastrointestinal tract, affecting annually>5000 very low birth weight (less than or equal to 1500g) infants.^1,2It is characterized by high mortality (up to 30%) and long-term morbidity, including short bowel syndrome, recurrent infections, nutritional deficiencies, and neurodevelopmental delay.^3,4Although the mortality rate of premature infants generally decreases net, the number of deaths associated with NEC increases.⁵The disease is often difficult to diagnose and manage due to initial nonspecific symptoms and rapid deterioration. Clinicians rely on radiological evidence such as intestinal gas to make a diagnosis, but sensitivity to this finding is reported to be as low as 44%.⁶Although many NEC biomarkers are under investigation,⁷none of them are currently widely used in clinical practice.

Without being bound by theory, Intestinal Alkaline Phosphatase (iAP) measured in feces provides diagnostic value as a marker of intestinal pathology. The protein is expressed in small intestine enterocyte and secreted into intestinal cavity and systemic circulation⁸And plays an indispensable role in maintaining the intestinal barrier function by detoxifying bacterial lipopolysaccharides and maintaining the microbial homeostasis.^9,10As the main alkaline phosphatase in feces, there is a group of enzymes, ^3,4iAP has been identified as one of the 57 proteins in the core human fecal proteome.¹¹Because of its protective role, it has been studied in animal models as a potential treatment for NEC.^12-15However, most studies have not evaluated iAP as a diagnostic tool, and only a few have examined iAP in humans. In this investigation, we examined faecesiAP serves as a potential biomarker for non-invasively monitoring the development of NEC in newborns. To our knowledge, this study was the first study to investigate the level of iAP in human preterm feces to determine its relationship to NEC.

Method

Study design and participants. This prospective, longitudinal case control study was approved by the institutional review board of the louisiana state university medical college. It is carried out according to the moral guidelines of the world medical association (declaration of helsinki). 18 preterm infants with gestational age of 23-37 weeks were admitted to the new orleand children hospital and charlo hospital after obtaining written informed consent from parents. Demographic data for 6 NEC patients and 12 control infants are shown in table 1. All patient samples were de-labeled prior to analysis. Patient records were evaluated retrospectively to determine clinical relevance. No patients in this study were known to have chromosomal or congenital abnormalities that would not allow enteral feeding.

Sample collection/preparation: fecal samples were continuously collected from the diapers of the study subjects after spontaneous bowel movements. Feces are stored briefly in hospital specimen refrigerators until transported to the laboratory in refrigerated storage. In the initial processing step, approximately 200mg of feces was measured, and then sterile molecular grade water (Sigma Aldrich) was added to reach the desired concentration of 200 mg/ml. The mixture was vortexed vigorously for 30-60 seconds, or until well-mixed slurry appeared. The mixture was then centrifuged at 22,000Xg for 30 minutes at 4 ℃. The supernatant was collected and stored at-20 ℃ until assayed.

Protein concentration: the concentration of total protein in the fecal supernatant was determined by the bradford assay (coomassie protein assay reagent, Thermo-Scientific) using bovine serum albumin as a standard.

Denaturing gel electrophoresis and western blotting: the supernatant of the fecal sample was mixed with 6 Xgel loading buffer (375mM Tris pH 6.8, 50% (w/v) glycerol, 600mM dithiothreitol, 420mM sodium dodecyl sulfate) and boiled for 5 minutes. Each lane of denatured 4-12% Bis-Tris gels (Novex, Life Technologies) was loaded with a total of 10. mu.g of total protein. The positive control was small intestine 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 the proteins in each lane and the proteins in the second were transferred to PVDF membranes for immunoblot detection of intestinal alkaline phosphatase. Membranes were serially blocked in 5% (w/v) skim milk in 50mM Tris-HCl pH 7.5, 150mM NaCl and 0.1% Tween, incubated with anti-human iAP rabbit primary polyclonal antibody (Abcam, ab7322 or ab198101), washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Abcam, ab6721) at room temperature. Chemiluminescent signal was initiated using Pierce ECL western 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 digital western blot, the 60kDa band corresponding to iAP was manually identified. Equivalent area was quantified for each lane of each western blot. Negative controls were subtracted from each patient sample and the difference was calculated as a percentage of the positive control standard.

Feces iAP catalytic activity: alkaline phosphatase activity was measured using 4-methylumbelliferyl phosphate (MUP) as fluorogenic substrate (Abcam, ab83371) in the presence and absence of the iAP inhibitor L-phenylalanine. Relative Fluorescence Units (RFU) at 360/440nm were measured using a Spectra Max M2e spectrophotometer (Molecular Devices, Sunnyvale, Calif.). A ninety-six hole black optical backplane was 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 × V), where B is nmol of product; v is the sample volume added to the well; t is the reaction time; u is the amount of enzyme that causes hydrolysis of 1. mu. mol MUP per minute at pH 10.0 and 25 ℃. Stock solutions of 100mM L-phenylalanine (98% purity; Sigma Aldrich) were freshly prepared in molecular-grade water each day of use. The final assay concentration of 10mM Phe was used to assess inhibition of iAP-specific activity.

Statistical and computational analysis of iAP biomarker classification: total fecal protein, iAP activity, was tested between NEC and control groups using a non-parametric Mann-Whitney U assayAnd the mean difference in 60kDaiAP band intensity on western blot; p value<0.05 was considered significant. Potential biomarker efficacy was assessed by sensitivity (true positive rate) and specificity (true negative rate) calculations. Of the 49 unique stool samples analyzed, 13 were obtained from NEC patients at the time of clinical diagnosis. Thirty-six samples were labeled as controls, with 27 from control subjects and 9 from NEC patients during the healthy interval. For each variable of interest, specificity and sensitivity were initially obtained using a simple threshold-based classifier. Subsequently, using scimit-spare packet in Python, ¹⁶Multivariate classifier performance is performed by training a Naive Bayes Classifier (NBC). NBC assumes that each feature is statistically independent; however, it may work well on the multi-feature classification problem, even when the assumption of statistically independent features is not valid.¹⁷For each classifier, we calculated the standard error of our sensitivity and specificity estimates by performing five rounds of hierarchical jack knife resampling, where each round of resampling excludes 20% of the data. We used a 5-fold hierarchical cross-validation scheme in which NBC was trained on 80% of the data for each fold and the resulting classifier was tested for sensitivity and specificity to the remaining 20% of the data.

Results

Longitudinal study: to explore whether these 3 fecal parameters correlate with NEC, two preterm infants were observed over time and their fecal samples were repeatedly monitored.

Patient 1 (fig. 1A) was diagnosed with NEC at day 7 of life (DOL). After 14 days of drug treatment, including bowel rest and antibiotic treatment, clinical symptoms and intestinal gas were resolved. Enteral feeding was resumed with varying degrees of success until the infant had recurrent NEC and subsequent intestinal perforation at DOL 31. Three stool analyses were performed: soluble protein concentration, catalytic activity of iAP and immunoblot detection of iAP. Two stool samples were obtained on DOL 7: bloody stools before NEC diagnosis (7A, fig. 1A) and later on the day (7B, fig. 1A). Data from five other fecal samples (DOL 13, 20, 29, 32, and 42) are also provided.

Longitudinal monitoring of patient 1 indicated that 3 candidate biomarkers were of diagnostic value (fig. 1A). The protein concentration of the DOL 7A fecal sample was 1.85mg/mL, the catalytic activity was 2218U/g, and no 60kDa signal was detected on the Western blot. The DOL 7B fecal sample collected after several hours had a protein concentration of 2.1mg/mL, a catalytic activity of 250U/g, and a clear immunoassay for iAP was performed. Comparison of two stool samples from the same patient before and after NEC onset indicated that a sharp decrease in iAP activity and an increase in iAP protein were characteristic of NEC.

These biomarkers also showed monitoring value. Following initial NEC diagnosis of DOL 7, immunodetection of low iAP enzyme activity and high iAP protein levels persisted in fecal samples collected during drug treatment, significant "recovery" and re-enteral feeding. The infant then re-develops NEC on day 31 of birth with intestinal perforation. In combination, increased fecal protein, low iAP enzyme activity and high iAP protein levels in western blots indicate perforation. Over 10 days of treatment, including peritoneal drainage, bowel rest and antibacterial treatment, the measured values of stool collected at DOL 42 were close to those before diagnosis of NEC.

Longitudinal monitoring also indicates the prognostic potential of the three candidate biomarkers. Patient 2 (fig. 1B) was diagnosed with suspected NEC at DOL 19, and maintained "NEC observation" (NEC monitoring, bowel rest, antibiotics) for several days before resuming enteral feeding. Although no definitive diagnosis of NEC was made until DOL 32, two-thirds of the biomarkers in the fecal samples of DOL 13 and 19 were within the positive range of NEC association, likely predictive NEC.

Cross-sectional studies: in a survey of fecal material collected from 6 NEC and 12 control infants, there were significant differences between 3 in vitro measurements between the fecal samples of active NEC patients and the fecal samples of the control group (fig. 2). Mean ± SEM protein concentrations in the patient fecal samples at NEC diagnosis were 2.62 ± 0.33mg/mL, with a significant difference in the 0.98 ± 0.25mg/mL levels found in the fecal samples of age-matched control patients post-conception (p ═ 0.005, fig. 2A). Furthermore, NEC patients had more than ten times lower mean fecal iAP enzyme activity at diagnosis compared to age-matched controls post-conception (162 ± 30 mn/mg vs.1826 ± 376 mn/mg, NEC vs. control, p <0.0001, fig. 2B, respectively).

Finally, samples from 2 patient populations were significantly different in the relative amount of a particular iAP protein (p ═ 0.002), as determined by densitometric analysis of immunoblots probed with anti-human iAP antibodies, and expressed as a percentage of standard positive controls. We found that iAP protein in stool samples of NEC patients was nearly 30-fold higher than in samples of healthy preterm subjects (215.0 ± 47.6% vs.7.2 ± 2.3%, NEC vs. control, fig. 2C, respectively). In summary, stool samples from NEC infants had increased total protein, decreased iAP enzyme activity, and increased iAP protein at diagnosis compared to healthy controls.

Study of sensitivity specificity: in the 3-dimensional scatter plot (fig. 3A), biomarkers from NEC samples (red circles) aggregated independently of controls (black circles), indicating that these biomarkers can achieve high sensitivity and specificity. Despite some overlap, the potential to distinguish NEC from control patient samples was still evident even after total fecal protein levels were removed as variables (fig. 3B). We evaluated sensitivity and specificity directly using the univariate threshold classifier and the Naive Bayes Classifier (NBC), which classified the samples based on the integration of all 3 tests (fig. 3C). Clearly, there is a trade-off between sensitivity and specificity. The 3-signature NBC biomarker performed best if maximum sensitivity or true positive rate was the primary target, with 100% sensitivity and 92% specificity. However, if the goal is to maximize both sensitivity and specificity, 3-feature NBC shows performance of 93% sensitivity and 95% specificity. When a threshold of 300mU/mg iAP activity was used, the level of iAP activity considered alone also performed almost equally well in this case, with sensitivity and specificity averaging 92%. However, it may not be surprising that fecal total protein levels alone are not reliable biomarkers for NEC. At 92% sensitivity, the specificity dropped to 67% using a protein threshold of 1.35 mg/mL. Thus, fecal iAP activity levels and 60kDa western blot intensity levels are expected to be candidates for NEC biomarkers alone. However, total fecal protein activity levels may only be useful when considered as part of a multi-characteristic diagnostic assessment.

Discussion of the related Art

The diagnosis and management of NEC is complicated by our current inability to accurately identify disease before irreversible intestinal injury occurs. Disease progression cannot be accurately predicted in most patients based solely on clinical parameters.¹⁸Despite the fact that the presence of radiography, NEC diagnosis and staging cornerstone,¹⁹this disease pathology measurement provides a qualitative rather than quantitative endpoint, which is rapid and accessible in the intensive care unit. There is well documented variability in the interpretation of observable radiological signs that determine disease severity.^6,20,21Disturbingly, only 44% of pathologically confirmed NECs report a marked radiological finding, namely intestinal gas accumulation²²。

To better understand NEC, for example to distinguish normal and pathobiological processes or to monitor response to clinical intervention, quantitative markers measured on a ratio or interval scale are still needed. The clinical definition of NEC can be significantly improved, moving from relying only on clinical impression and imaging results to an extended diagnostic palette that includes reliable molecular biomarkers. Identification of molecular NEC biomarkers suitable for use in clinical practice has the potential to reduce neonatal mortality, morbidity and associated healthcare costs. Furthermore, characterization of these parameters may provide insight into cellular integrity, protein expression, and changes in gastrointestinal metabolism. The discovery of NEC candidate biomarkers obtained from serum, urine, feces and buccal swabs is the focus of current research. ^23-25

Our study demonstrated a correlation between 3 extra-individual stool parameters and patient pathology, which is consistent with the results of animal studies. The increased total fecal protein concentration measured in NEC patients may be associated with mucosal detachment and disease-related inflammatory products such as serum amyloid a, anaphylatoxins, C-reactive proteins, platelet activating factor, calprotectin, and alpha-1 antitrypsin.^26-31In these inflammation-based biomarkersOf these, the latter three were measured directly in neonatal stool samples.^27,28,32-34However, biomarkers associated with inflammation, while generally associated with gastrointestinal pathology, are not specific for NEC diagnosis.

In our study, increased iAP detected by western blot correlates negatively with lower intestinal AP enzyme activity in stool samples from NEC patients at diagnosis. Our findings are consistent with other reports in the literature. First, biopsy intestinal tissue from patients with inflammatory bowel disease showed lower AP activity based on enzyme histochemical analysis.³⁵Second, serum iAP shows an increase in patients who will continue to develop NEC; however, AP levels were only monitored by gel electrophoresis and no positive detection of intestinal alkaline phosphatase was specified. ³⁶Third, rat ileal terminal tissue samples showed reduced protein content, activity and alkaline phosphatase specific immunofluorescence in animal models inducing NEC.^14,37Finally, a decrease in mucosal AP activity was also reported in animal models following ischemia reperfusion.³⁸Increased shedding of mucosal proteins, including inactivated iAP, may explain our findings.

We have several criteria to evaluate the transformation prospects of our NEC biomarkers. First, the molecular characteristics should be a direct readout of gastrointestinal disease and easy to detect. In this study, we evaluated three candidates: total fecal protein, specific iAP activity and western blot band intensity of iAP. Monitoring total protein levels in feces is pathophysiologically justified because high protein levels in feces are closely associated with poor integrity of immature neonatal intestinal mucosa, which may be exacerbated by inflammation. The attractiveness of iAP as a biomarker is its tissue-specific expression in the small intestine and its secretion into the mucus layer and intestinal lumen.^39,40It is also responsible for most of the AP enzymatic activity in feces^41,42And have been used as a measure of small intestine toxic injury in animal models. ^43,44All three of our stool biomarker candidates showed significant mean differences between NEC patients and control subjects at diagnosis(FIG. 2).

Second, the basic 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 disposable items for ease of packaging and shipping. The measurement of the protein concentration takes less than 30 minutes. In current practice, iAP enzymatic detection and western blotting can be accomplished within one or two hours, respectively.

We assessed the third criterion for NEC biomarkers as potential for superiority over radiological diagnosis. For reference, the 7-parameter analysis report of clinical diagnostic criteria developed by WHO integrated management of childhood disease programs showed that 85% sensitivity and 75% specificity were high.⁴⁵The sensitivity of the detection of intestinal gas accumulation was only 44%.²²In contrast, performance data for our diagnostic methods, including specific iAP activity, immunoblot detection and three-parameter NBC, are promising because both sensitivity and specificity are greater than 90%. We also note that in general, the marker performance of positive diagnostic readings (e.g. increased immunoblot detection and protein concentration) is more robust than negative diagnostic readings: the latter mode is more susceptible to false positive (and false negative) diagnosis. Although future studies involving a larger patient population may alter our performance data, we conclude that this stool sample analysis has potential clinical utility for improving NEC prognosis diagnosis and follow-up monitoring.

The challenges of using our fecal biomarker analysis as a diagnostic tool for NEC are the heterogeneous composition of some fecal samples, the intermittent and variable patterns of defecation in some newborns, and the lack of immediate, on-demand test results. However, our 3-feature fecal biomarker analysis has the advantage of requiring less time, requiring no special training or expertise, and is inexpensive compared to proteomics or mass spectrometry techniques. Adapting this test to continuous, non-invasive monitoring of premature infants in neonatal intensive care units would provide objective measures to assess mucosal integrity, help assess risks associated with feeding regimes, and help us understand NEC.

In future studies, our naive bayes classifier approach can be extended to simultaneously analyze iAP enzyme activity, western blot signals, and other candidate NEC biomarkers, such as fecal calprotectin and platelet activating factor.⁷These fecal biomarkers can be added to our classification scheme without the need for additional neonatal patient blood or urine samples. The performance of the classifier for distinguishing NEC from septic patients by protein biomarkers in urine samples was analyzed and indeed, the efficiency of distinguishing NEC from septic patients was reduced compared to distinguishing NEC from normal patients. ⁴⁶If our proposed biomarker protocol is able to maintain high sensitivity and specificity for a larger patient population with more complex control groups, machine learning analysis of iAP measurements in combination with other biomarker candidates may lead to significant advances in NEC diagnosis and management.

References cited in the examples

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Example 3

Introduction to the design reside in

Necrotizing Enterocolitis (NEC) is an extremely severe inflammatory disease of the gastrointestinal tract, affecting mainly premature infants. It occurs in up to 10% of very low birth weight infants (less than 1500g at birth), and is characterized by a high mortality rate (up to 30%) and significant long-term morbidity, including infant short bowel syndrome, recurrent infections, extra-intestinal nutrition-related cholestasis, nutritional deficiencies, and neurodevelopmental retardation (1). Despite advances in the field of neonatology, NEC is responsible for an increased number of deaths in very premature infants (2). The exact cause of the disease is not well understood, which makes diagnosis and management a challenge. The course of the disease usually involves initial nonspecific symptoms and rapid clinical deterioration. While many biomarkers are currently being investigated as potential aids for diagnosing NEC, none is widely used to determine the true integrity of the attacked gut (3).

Intestinal Alkaline Phosphatase (iAP) has become an enzyme in the study of gastrointestinal disorders. Produced and secreted by intestinal cells in the proximal small intestine, and iAP activity was found throughout both 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 gut (7). It dephosphorylates ATP and has been shown to influence microbial homeostasis by this interaction (8).

With such a wide range of functions involving intestinal homeostasis, one may expect alterations in iAP in NEC. In rats, Biesterveld et al demonstrated a decrease in endogenous iAP catalytic activity during induction of NEC, followed by an increase 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 stool iAP could be used as a parameter for toxic injury to the small intestine (10). Thomas and Henton later studied the use of fecal iAP as a potential marker of intestinal injury, but found to be very different (11). They suggest the use of a longitudinal approach to determine clinical effectiveness (11). Supplementation with iAP has been shown to reduce 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 towards high iAP levels in infants that later developed NEC (15). These observations suggest the hypothesis that iAP may be a useful biomarker for NEC.

Our research focus was on determining whether stool iAP could be used as a diagnostic tool for NEC. Stool iAP measurements are less invasive than serum measurements of preterm newborns that have received multiple serological examinations. To date, no study has been published investigating human neonatal stool iAP and its relationship to NEC. We hypothesized that stool iAP can be used as an objective and specific biomarker for diagnosing NEC and monitoring the course of NEC after disease determination.

Development of the method

In preparing this study, i sought and obtained approval for institutional committee review. I designed a written informed consent and recruited a total of 20 infants. I sought for the help of NICU nurses to help with the collection and proposed a tagging system that could protect patient privacy. Stool samples were prospectively collected and charts were reviewed retrospectively to determine clinical relevance. There is little literature on stool iAP measurements. I have found a reference describing how to mix rat fecal material with water and then centrifuge to obtain supernatant 16, and therefore I continue to use a similar method to treat human fecal samples. We determined that 200mg of stool measured was sufficient to easily collect the supernatant and quantify the protein. Stool consistency varies greatly making wet weight an unreliable parameter. Total fecal protein content (determined by bradford assay) was used to normalize iAP measurements.

To confirm the presence of IAPs in the feces, we chose Western Blot (WB) as the initial assay. Surprisingly, in our healthy controls, positive detection of proteins in feces is extremely difficult. In fact, more than 10 samples were analyzed that produced negative results with no band signal or signals located far below the expected position. Initially, we believe that our negative results are due to processing and/or storage failures. However, even the measurements performed on freshly obtained fecal samples on the same day, there was no evidence of production of anti-iAP antibody recognition proteins on western blots. Only after analysis of stool samples from our first NEC patients, we found evidence of full-length protein on western blots. When we recruited a second NEC patient, we obtained a positive result when we mapped iAP on WB again.

The determination of the presence of the iAP protein on WB in at least some experiments led us to be confident to continue our study. We then performed experiments to determine the ideal processing and storage technique. We placed some samples in a refrigerator at 4 ℃ for several days, and after 5 days in the refrigerator, the proteins were not significantly degraded. Western blot showed persistent positive detection of iAP for anti-iAP. Enzyme activity measurements showed minimal change per day. We also frozen our supernatant at-20 ℃, but determined that short term storage in hospital refrigerators is acceptable.

We believe that 200mg feces are required to produce reliable results. In one case, although we collected only 10mg of stool at NEC diagnosis, we were still able to demonstrate a strong band of stool iAP on western blot, suggesting that the test is useful even if a small amount of stool is available.

We chose a fluorometric assay to measure activity because it was more sensitive than a colorimetric assay (detection sensitivity of approximately 1 μ U) (17). We have surprisingly found that there is evidence of alkaline phosphatase activity even in samples where no signal was produced on WB. Since our fluorescence activity assay is not specific for intestinal alkaline phosphatase, other alkaline phosphatases in stool may lead to differences between WB and activity assays. Our next set of studies aimed at measuring the actual activity of iAP by performing sample assays in the presence and absence of L-phenylalanine (L-Phe), a specific inhibitor of iAP (4, 18). L-Phe blocked 90% + -10% (SD) AP activity in NEC patient samples and 91% + -9% (SD) AP activity in control samples, indicating that iAP is a major contributor to alkaline phosphatase activity in the stool samples studied (FIG. 5). This finding is consistent with previous reports (3,4), confirming that iAP is the most common AP in stool.

Method

Study design and participants-this is a prospective case-control study. 20 preterm infants With Gestational Age (WGA) of 23-37 weeks were admitted to new orleans children hospital and charlo hospital after obtaining parental consent. 6 infants had NEC as defined by Bell stage (19). Infants with known chromosomal or congenital abnormalities that failed to feed were excluded. Stool samples from 2 subjects were excluded from the statistical analysis due to the different treatment modalities. The demographic data for the remaining 18 subjects (6 NEC patients and 12 controls) are shown in table 1. Fecal management — fecal samples were collected continuously from the diapers of the study subjects after spontaneous bowel movements. Feces are stored briefly in a specimen refrigerator in a hospital. The samples were transported in a refrigerator to a laboratory for preliminary processing. Approximately 200mg of feces, where possible, was measured and molecular water was added to achieve the desired concentration of 200 mg/ml. The mixture was vortexed vigorously for 30 seconds to 1 minute, or until a well-mixed slurry appeared. The mixture was then centrifuged at 22,000Xg for 30 minutes at 4 ℃. The supernatant was collected and stored at-20 ℃ until assayed.

Determination of protein concentration-total protein concentration in stool supernatant was determined by the bradford assay (coomassie protein assay reagent, Thermo-Scientific) using bovine serum albumin as a standard.

Denaturing gel electrophoresis and Western blotting-the supernatant of a stool sample was mixed with gel loading buffer (375mM Tris pH 6.8, 50% (w/v) glycerol, 600mM dithiothreitol, 420mM sodium dodecyl sulfate) and then boiled for 5 minutes. A total of 10. mu.g of total protein was loaded per lane of a pre-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 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 full-length human iAPab7322/ab198101(Abcam) primary antibody and horseradish peroxidase conjugated goat anti-rabbit secondary antibody ab6721 (Abcam). We used Pierce ECL Western blot substrate (Thermo-scientific) as the chemiluminescent peroxidase substrate. Developer (AFP Imaging; Mount Kisco, NY) was used to produce films after WB and imager (Biorad Gel-Doc XR; Hercules, Calif.) was used to scan Western blots and gels. Densitometry was performed to analyze digitized images of western blots. We manually identified a 60kDa band on the western blot, which corresponds to intestinal alkaline phosphatase. We then calculated the area of each 60kDa band relative to the background and then expressed this value as a percentage of the positive control. Positive controls were either hepatocellular carcinoma whole cell lysate or small intestine tissue lysate (Abcam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control.

Fecal iAP activity-alkaline phosphatase activity was determined using 4-methylumbelliferyl phosphate as fluorogenic substrate ab83371 (Abcam). Substrate background control and background control were performed to improve accuracy. Relative Fluorescence Units (RFU) at 360/440nm wavelength were measured using a Spectra Max M2e spectrophotometer (Molecular Devices, Sunnyvale, Calif.). A ninety-six hole black optical backplane was used. Reaction wells for samples, standards and negative background controls were prepared for each test run and total alkaline phosphatase activity was determined using the following method:

ALP Activity (mU/ml) ═ B/T)/Vx dilution factor

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 hydrolysis of 1. mu. mol product per minute at pH 10.0 and 25 ℃ (glycine buffer). 100mM L-phenylalanine stock solutions (purity > 98%; Sigma Aldrich) were freshly prepared in molecular-grade water and assayed daily. L-Phe at a final concentration of 10mM was added to each well to inhibit iAP activity.

Calculation and statistical analysis of iAP biomarker classification-due to consistency of fecal management and similar gestational age, we included 18 infants in the analysis of fecal protein and fecal iAP datasets. Mean differences in total fecal protein, iAP activity and iAP protein band intensity on WB between NEC and control were tested using a non-parametric Mann-Whitney U test (GraphPad Instat v.3; La Jolla, Calif.). Linear regression analysis was used to determine the correlation between the days before complete feeding and total fecal protein and between the days before complete feeding and iAP activity (GraphPad Prism v 7; La Jolla, Calif.). P values less than 0.05 were considered significant. Igor Pro (Lake Oswego, OR) was used to generate FIG. 10.

A separate data subset was used for 51 stool samples (from 6 NEC patients and 7 controls) for which we could measure specific iAP activity, WB and stool protein. We analyzed the distribution of these measurements for western blot band intensity and total fecal protein level, 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 the classifier, while specificity is the FPR (false positive rate) of the 1-classifier. For each of the three variables we are interested in, we first investigated the specificity and sensitivity obtained using a simple threshold-based classifier. For each of these classifiers, we calculated the standard error of our sensitivity and specificity estimates by performing five rounds of jack knife resampling, with 20% of the data excluded from the sensitivity and specificity estimates for each round of resampling. Our data was stratified by category label during the resampling process, so 6-7 control samples and 3-4 NEC samples (20% of the total number of each category) per round of resampling were excluded from the analysis.

After studying the univariate classifier, we explored the utility of the multivariate classifier by training the Naive Bayes Classifier (NBC) using the scinit-leann package in Python (20). The naive bayes classifier assumes that each feature used in the classification is statistically independent (21). This naive hypothesis was incorrect for our three characteristics (iAP activity level, total protein and WB intensity). However, previous work by machine learning communities showed that NBC may perform well on the multi-feature classification problem even if the assumption of statistically independent features is not true (21). To avoid overfitting of the multi-feature classifier, we used a 5-fold hierarchical cross-validation scheme in which NBC was trained on 80% of the data for each fold, and then the resulting classifier data was tested on the remaining 20% to estimate sensitivity and specificity.

Results

Stool from NEC patients at diagnosis had decreased iAP activity (figure 7), increased total stool protein (figure 8), and increased iAP protein detection (figure 9) compared to multiple samples from controls of approximately matched gestational age and actual age. Stool iAP activity was lower when NEC was diagnosed compared to the mean control. There was statistical significance between the groups when the Mann-Whitney test was applied. The mean value for fecal iAP activity was 184mU/mg with a Standard Error of Measurement (SEM) of 34, while the control group was 1932mU/mg with an SEM of 433(P < 0.0001). This is shown in figure 2.

We found that NEC patients at diagnosis had significantly higher fecal protein content than the age-after-conception matched controls. We averaged the protein content of individual non-NEC patients between 29-43PCA and compared it to fecal samples of patients at NEC diagnosis, for a total of 6 patients with 7 events (fig. 3). Nonparametric tests (Mann-Whitney) were used to compare data from non-normal distributions with statistically significant differences between groups (P ═ 0.005). In NEC patient samples, the mean at diagnosis was 2.62 with an SEM of 0.33, whereas the mean in the mean control was 0.98 with an SEM of 0.25.

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% for the control group (P ═ 0.0022). The standard error of measurement for NEC was 45, while that for the control group was 1.9. (FIG. 4).

Our antibodies selected for western blot analysis did not readily detect iAP in stool, except in the case of NEC. Longitudinal observations of 2 NEC patients are shown in fig. 10A and 10B. Panel a highlights premature infants with prolonged NEC course followed by perforation. It shows a sharp decline in iAP activity at initial NEC diagnosis and appearance of iAP protein on WB, continued low iAP activity and evidence of iAP protein on WB during initial treatment until subsequent perforation. Ten days after Penrose drainage tube placement and intestinal rest, there was no evidence of high iAP protein on WB, but increased fecal iAP activity. Panel B highlights different infants with multiple NEC monitoring 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 iAP activity was normal. This was resolved before a sharp decline in iAP activity at NEC and high iAP protein on WB. Following medical management and recovery from NEC, iAP activity increased and no iAP protein was present on WB. Panel C of figure 10 shows 3 groups of samples, each group consisting of faeces at diagnosis from NEC patients compared to faeces from closely matched controls. Groups 1 and 2 clearly show evidence of increased total fecal protein, decreased iAP activity, and iAP protein on WB. In group 3, iAP activity and total fecal protein were not significantly different, but western blots clearly distinguished NEC and control samples. Fig. 5 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.

Panel a of fig. 11 is a 3D scatter plot showing fecal iAP activity, fecal protein and WB data points for NEC samples and controls. NEC samples were marked in red. Panel B depicts a 2D scatter plot showing iAP activity and WB percentage. There was high activity and low WB percentage in the control. Combining all 3 biochemical assays improves the sensitivity and specificity observed despite the small sample size and patient number. Fig. 6 panel C demonstrates the utility of examining multiple features simultaneously by describing the trade-off between sensitivity and specificity of multiple thresholds and multiple feature choices. Western blot intensities considered alone performed best at 100% sensitivity and 70% specificity for the detection threshold of 10% positive control band intensity. 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 with 95% sensitivity and 93% specificity. When a threshold of 300mU/mg iAP activity was used, the level of iAP activity considered alone also performed almost equally well in this case, with a sensitivity level of 95% and a specificity of 91%. When western blot intensity levels were considered alone with a sensitivity of 95%, the specificity level using the 30% positive control strip intensity threshold was 88%. However, perhaps not surprisingly, total fecal protein levels alone are not unique to NEC. To achieve a sensitivity of 95% for total fecal protein levels, specificity must be reduced to 44% using an interpolation threshold of 1.02 mg/mL. Thus, fecal iAP activity levels and 60kDa western blot intensity levels are expected to be candidates for NEC biomarkers alone.

Defined as the number of days required to reach the target enteral feeding, there was a clear tendency for fecal protein to increase in the control group that was intolerant to food (fig. 12). The opposite trend can be seen in iAP activity, although this association is less pronounced.

The heterogeneity of stool may interfere with the reliability of the test. In one particular heterogeneous stool, the mucus-containing fraction (fig. 13A) was similar to the other NEC samples, while the more solid fraction of the stool (fig. 13B) was similar to the control in terms of iAP protein and iAP activity on WB.

Discussion of the related Art

In our study we observed changes in stool total protein, WB measured iAP protein and iAP activity in stools in NEC patients at diagnosis. High protein levels in the faeces of NEC patients at diagnosis may reflect the loss of mucosal integrity in the already immature gut and inflammatory products associated with the disease. Shulman et al showed a similar trend, in which alpha 1 antitrypsin in feces was significantly increased at the diagnosis of NEC, compared to the control group (22).

Intestinal alkaline phosphatase is expressed primarily in apical intestinal cells of the small intestine, making it an ideal, relatively specific candidate biomarker for localizing gastrointestinal disorders (e.g., NEC). It adheres tightly to the membrane but also falls out into the cavity (4). Shifrin et al have recently demonstrated that iAP is distributed to the mucus layer and intestinal lumen by shedding of microvilli vesicles (23). During inflammatory injury and intestinal necrosis, disruption of the mucosal barrier and cell death and exfoliation of the mucosal lining result in increased release of mucosal proteins such as iAP into the stool. The rarity of iAP signal on western blot, but high iAP activity in feces of control subjects remains unclear. Perhaps the normal shedding process would alter the free luminal iAP structure in a way that would not allow for our specific antibody recognition. The immunogen for this antibody is the full-length native human iAP from small intestinal tissue, and it may be more sensitive to membrane-bound full-length proteins. Alternatively, there are 2 known iAP isozymes, fetal and adult forms that undergo developmental changes (24, 25). NEC inflammation may be associated with the production of the isozyme that we recognize by antibodies, while iAP produced under normal conditions is another unrecognized isozyme. In any case, the difference in WB results is highly suggestive of conformational differences in fecal IAPs between healthy and disease states.

The sudden decrease in global fecal iAP activity we found in the NEC diagnosis from the fecal samples of NEC patients was also found in NEC-inducing rat pups (13, 26). Whitehouse et al demonstrated a reduction in tissue iAP protein and activity by intestinal histology and ileal end tissue sampling (26). Cell loss and presumed intestinal lumen shedding at the tissue level to account for decreased iAP protein and activity also contribute to accounting for increased iAP protein and decreased activity in NEC patient stools. Interestingly, it was found that adult patients with inflammatory bowel disease showed lower AP activity in biopsied intestinal tissue (27).

The mechanism of low iAP activity at NEC does not appear to be due to initial defects, as we observed a rapid decline in activity from normal levels in some NEC patients. Loss of iAP enzymatic activity may reflect damage to the catalytic site. In animal models, Sisley et al demonstrated a decrease in mucosal AP activity following ischemia reperfusion and suggested that the metal binding site may be more susceptible to oxidative damage (28).

The decrease in fecal iAP activity is sometimes accompanied by a corresponding sudden appearance of fecal iAP protein on WB, a few hours before diagnosis. This observation indicates that detection of iAP using activity assays and western blots can provide diagnostic value for the initial event. In case NEC can be easily identified by traditional methods (bloody stools, intestinal gas accumulation, etc.), this additional biomarker offers little benefit. However, for patients with subclinical disease or patients lacking clear imaging evidence, the use of fecal iAP is most beneficial for definitive diagnosis. Although considered characteristic of NEC, balance et al showed that only 48% of pathologies in a NEC patient population confirmed the actual presence of intestinal gas accumulation (29). Stool iAP can also be used during NEC recovery to gauge the integrity of the gut and guide the feeding strategy of our most vulnerable patient population and to determine the length of time required for recovery. Without being bound by theory, the needs of some children may vary from 7-14 days of treatment, which is considered standard management.

These biomarkers can be used even in patients without NEC. We observed a trend for control infants with food intolerance and delayed achievement of complete enteral feeding to have higher total fecal protein and lower iAP activity. Control infants with good tolerance to feeding showed low total fecal protein and very high iAP activity. We have no information about the presence or absence of iAP protein through WB, as it is associated with eating intolerance. If this is confirmed by larger scale studies, more power will be provided to explore the potential benefits of supplementing iAP in these situations. More studies are needed to determine the normal values of each parameter for all gestational and actual ages, and to determine diet and other factors that may affect them.

None of the methods are without limitation. One potential confounding factor in stool iAP detection is stool heterogeneity. We collected one stool sample at NEC diagnosis with two different consistencies, mucus and normal occurring stool. We separated these fractions, with normal occurring stools with similar results as controls, while mucus-containing fractions had the expected low iAP activity and high iAP signal on western blots (fig. 13). Since stool preparations vary widely, we did not include these data points in our analysis. This is the only sample that has this problem and the frequency of occurrence is unknown. Another challenge we are faced with is the infrequent and sporadic pattern of bowel movements associated with premature birth, which does not allow for accurate standardized collection times between subjects. In the clinical field, relying solely on fecal samples may lead to delayed diagnosis due to poor defecation. No biomarkers can replace good physical examination and clinical expertise. The best use of fecal iAP as a biomarker is likely to be as an aid in establishing NEC diagnosis, monitoring disease progression, and monitoring or surveillance of high risk populations.

In our examination of 3 potential NEC biomarkers associated with intestinal alkaline phosphatase, we have demonstrated that fecal sample analysis has potential clinical utility for improving the diagnosis of necrotizing enterocolitis. We have shown that both specific iAP activity levels and western blot band intensities can be used to identify NEC patient stool samples with high sensitivity and specificity when considered independently. We also show that multiple features can be combined using a naive bayes classifier to achieve both better sensitivity and specificity levels. Furthermore, in future work, our naive bayes classifier approach can be extended to simultaneously analyze iAP activity, western blot band intensities, and a variety of other candidate NEC biomarkers, all beyond the scope of the current study.

Conclusion

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

References cited in the examples

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Example 4

Summary:

the results of three different biochemical tests performed on the feces of preterm infants were grouped by post-conception age, which allowed comparison of the intestinal development of 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 concentrations is associated with intestinal inflammatory responses or disease states.

Overview and results:

necrotizing Enterocolitis (NEC) is a multifactorial disease that mainly affects premature infants and is the leading cause of late death and morbidity in extremely premature infants (Caplan, 2008; Christensen et al, 2010). Although the etiology of NEC is not clear (Dominguez and Moss, 2012; Gephart et al, 2012), NEC is thought to represent a severe inflammatory disease in the intestinal tract (Balance et al, 1990; Zhang et al, 2011). Excessive inflammatory response of the immature intestine to environmental injury is a hallmark of NEC (Chan et al, 2009). In particular, an increase in the level of LPS/TLR4 signaling has 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). Inhibition of LPS/TLR4 signaling may reduce intestinal inflammation and reduce NEC pathology in animal models (Chan et al, 2009; Gribar et al, 2009).

Intestinal Alkaline Phosphatase (iAP) is an important component of innate intestinal immunity. This enzyme is usually immobilized at the brush border of the gut and cleaves the phosphate group and thus dephosphorylates Lipopolysaccharide (LPS). LPS dephosphorylation inhibits the effective signal pathway; thus, proinflammatory cytokine release and immune responses caused by LPS activation of TLR4 (toll-like receptor 4; Lalles, 2010) are blocked by iAP. Furthermore, the iAP is concentrated in specialized membrane vesicles that are released into the intestinal lumen from the distal end of the intestinal cell microvilli (McConnell et al, 2009; Shilfrin et al, 2012). These released vesicles interact with bacteria and bacterial products and limit their pro-inflammatory potential.

Thus, we expect that iAP can be measured in human stool samples; this was confirmed by iAP being one of the core proteins in the human fecal proteome. The steady-state baseline of iAP can be shed from the lumen and detected in intestinal epithelial cells detected in feces. If there is a risk of inflammation caused by bacteria, the iAP content in the stool will increase due to the iAP-loaded membrane vesicles released. Samples from non-NEC infants grouped by post-conception age (gray bars, fig. 14A) showed that preterm infants had low amounts of iAP relative to positive controls from human small intestine tissue lysate. Our data also show that at the time of clinical diagnosis, the relative content of iAP is higher in stool samples from infants with NEC (120-320% of the positive control).

Second, dynamic conversion of the form of the iAP isozyme is associated with maturation of the fetal gut (Mulivor et al, 1978; Suriura et al, 1981). Fetal isoforms of intestinal AP have low biochemical activity, while adult iAP has high biochemical activity. We hypothesize that iAP activity will change as the fetus develops, i.e., preterm infants have lower iAP activity than term infants. We conclude that limited biochemical activity of iAP in newborns may lead to hyperactive LPS/TLR4 signaling. We examined this hypothesis by comparing fecal iAP activity of infants of different gestational ages. Thus, stool can accurately measure iAP activity in the neonatal gut.

Our data indicate that iAP activity is reduced in preterm infants compared to full term infants. Fig. 14B shows the average iAP activity of fecal samples grouped by age after conception (grey bar), normalized to protein concentration. There is a strong positive correlation between iAP activity and age after conception. When comparing iAP activity 24-41 weeks post-conception, multiple comparison tests of one-way ANOVA and Tukey were performed, indicating that the samples were statistically divided into two groups: premature group (age after conception is less than or equal to 35 weeks) and full term group (age after conception is more than or equal to 36 weeks). Comparing all stool samples from full term infants (age ≥ 36 weeks post-conception, n ═ 28) with all samples from preterm infants (age ≤ 35 weeks post-conception, n ═ 79), the latter group was found to have significantly lower iAP activity (p < 0.0001; single tail t test).

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

Without wishing to be bound by theory, a reduced capacity of the preterm intestine to dephosphorylate pro-inflammatory LPS increases the risk of excessive inflammatory responses to bacterial colonization and NEC development. Furthermore, based on these findings and without wishing to be bound by theory, prophylactic supplementation of preterm infants with iAP may be further investigated as a strategy to reduce the risk of NEC. Our data also show that at the time of clinical diagnosis, stool samples from infants with NEC are relatively high in iAP and protein.

Example 5

Food tolerance is demonstrated when preterm infants are able to safely ingest and digest prescribed enteral (via the oral cavity) feeding without complications associated with gastrointestinal dysfunction or infection. Clinical evidence of food tolerance in preterm infants with very low birth weight is most often described in the literature as the number of days required to reach full food intake (reported in the range of 100 mL per kg), the number of food intolerance episodes, the number of days to stop eating due to the symptoms of food intolerance, the time to recover birth weight, calf growth, weight gain, pillow circumference and length. None of the infants studied reached full food intake.

Formulation types include, but are not limited to: EleCare (Abbott Nutrition), Neocure (Similac), EnfaCare (Enfamil), Pregetimi (Enfamil), Similac Special Care or SSC (Similac), and Gentlease (Enfamil).

Supplements may include, but are not limited to, micro-lipids (Nestle Health Science).

Example 6

NEC is a devastating GI disease that mainly affects premature infants (incidence: 4-14%; mortality: 15-30% (up to 50%), and morbidity: up to 50% survivors). Clinical manifestations of NEC include abdominal distension, gastrointestinal motility and bloody stools. The X-ray examination results included intestinal gas and perforation.

The diagnosis of NEC is difficult because early manifestations are non-specific, the presence of intestinal pneumatosis is inconsistent, and despite aggressive management, clinical symptoms rapidly worsen. For example, in pathologically confirmed necrotizing enterocolitis, pneumatosis occurs in only 48%. There is currently no biochemical approach to identify those infants at the greatest risk and enable early diagnosis.

As described herein, Intestinal Alkaline Phosphatase (iAP) can serve as a biomarker for NEC, and lack of iAP correlates with susceptibility to NEC.

iAP is produced by apical intestinal cells and secreted to the luminal brush border and catalyzes hydrolysis of phosphate monoesters. The iAP is active as a homodimer and requires Zn at the active site²⁺And Mg²⁺Ions. Substrates for iAP include LPS and nucleotide triphosphates. iAP has a number of effects affecting intestinal barrier function and inflammation. iAP is shed in feces. iAP is tissue-specific AP, meaning it is produced primarily in the intestine, e.g., immunoorganization of intestinal tissueAs evidenced by chemical staining (fig. 15).

iAP maintained intestinal barrier function (fig. 16).

This study investigated whether stool iAP is a diagnostic tool for NEC. Serial patient fecal samples were collected and processed over 4 days. A slurry of 200mg feces/1 ml molecular water was centrifuged at 14000rpm at 4 degrees Celsius. The supernatant was stored at-20 ℃ until analysis. The biochemical analyses performed included the concentration of total protein in the feces, the enzymatic activity of alkaline phosphatase, and western blotting of human iAP. Samples were provided from 16 infants from the New Olarch Hospital and Children Hospital (NEC: 5 patients (25-35 WGA); non-NEC: 11 patients (23-34 WGA)). More than 100 fecal samples were processed and analyzed.

5 NEC patients at diagnosis were compared to 11 control patients and total fecal protein from control patients between corrected gestational age 29-35, corresponding to corrected gestational age of NEC patients, was averaged. The results were statistically significant, with 2.7mg/ml of median total fecal protein in NEC patients and 0.7mg/ml in non NEC patients. Total fecal protein levels in NEC patients were higher than in control infants (figure 17). The median fecal protein NEC (5% -95% CI) was 2.7 (1.6-3.6); the control was 0.7 (0.2-2.1).

The determination of the faecal protein content requires about 1 hour of laboratory work. Measurements of fecal protein concentrations above 2mg/ml serve as an early indicator of the onset of NEC.

Total stool AP is predominantly an intestinal isoform. Other alkaline phosphatases, such as bacteria and TNAP, are also found in the intestinal tract. We quantified the proportion of intestinal AP catalytic activity in feces by using L-phenylalanine, which specifically inhibited only the activity of intestinal 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 low (statistically significant) in NEC population (fig. 18).

Table 1:

Median 200 and 600 statistically significant differences

Data summarization

AP enzyme activity was lower when NEC patients were matched to specific controls of similar age and gestational age (figure 19).

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

Unexpectedly high iAP protein levels were detected in association with NEC. Using antibodies specific for human iAP in western blot analysis, it was surprising that the appropriate signal was only detected in NEC samples (labeled N), but not in control samples. Each group represents different NEC patients at diagnosis and age and gestational age matched controls. At the time of diagnosis, a much higher amount of iAP was present in the stool of NEC patients (fig. 20).

NEC onset indicates increased fecal iAP protein levels. A patient with NEC was continuously followed and found to have a high iAP level even after medical management. The patient is then perforated and subsequently subjected to surgical intervention. Feces 10 days after surgery no longer contained high levels of iAP protein. No signal on day 42 (fig. 21). The patient maintained lower AP activity prior to surgical intervention compared to AP activity. AP activity began to increase 10 days after surgery. The presence of sustained high fecal iAP levels and low activity may indicate that the bowel is damaged leading to perforation. (FIG. 22).

iAP is developmentally regulated, and its expression and activity has been shown to be reduced in preterm litters in a rat model. The data demonstrate a decrease in iAP activity but not expression in human NEC infants. Without wishing to be bound by theory, the third NEC biomarker may be western blot analysis or ELISA of preterm iAP protein levels (Rentea et al eur J pediar Surg 23, 39; heinzerging et al J pediar Surg 49,954; Biesterveld et al J Surg Res 196,235).

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 between the three tests vary. Combining these three markers may increase diagnostic value compared to using a single biomarker. Subsequent studies will optimize the specificity and sensitivity of each method.

There is a difference between the proximal and distal halves of the mammalian rat intestine. Structural differences include that most iaps are membrane-bound in the proximal half of the intestine; however, in the ileum, iAP was found in the supernatant fraction of the intestinal homogenate; in adults, more than 95% of iaps are associated with membranes. Functional differences include higher ileal total alkaline phosphatase activity during lactation; as the rat matures, the activity of the ileum decreases and the activity of the proximal intestine increases. Yedlin et al J Biol Chem 256,5620.

The secondary antibodies were tested for non-specific binding (fig. 23).

The method comprises the following steps: and (4) performing fluorescence analysis. Alkaline phosphatase cleavage of the phosphate group of a non-fluorescent 4-methylumbelliferone disodium phosphate (MUP) substrate; leading to an increase in fluorescence signal after dephosphorylation; measured using a spectrophotometer.

Example 7

Antibiotics for NEC

For NEC, the infant is administered antibiotics for 10-14 days, but the prescription is variable between hospital practices. Ideally, the prescription will be for a broad spectrum covering (i) gram positive bacteria, (ii) gram negative bacteria, and (iii) anaerobic bacteria. Such as vancomycin (gram positive, including MRSA), ceftazidime (third generation cephalosporin-gram negative, some gram positive and pseudomonads), metronidazole (anaerobic bacterial overlay), oxacillin (gram positive).

Examples of general antibiotic regimens are: ampicillin + gentamicin for a vertical acquired infection, possibly from the mother, and vancomycin + cetrimidine for a hospital acquired infection, possibly. Common antibiotics are gentamicin, vancomycin, ampicillin, Zosyn (a combination of piperacillin and tazobactam), Flagyl (metronidazole mimetic), clindamycin, meropenem, fluconazole (antifungal).

For sepsis, the patient will be administered an antibiotic for 7 days.

Feeding and nutritional regimen for preterm infants:

a challenging task facing neonatal pediatricians is to provide adequate and safe nutrition to premature infants. Enteral feeding (oral feeding) is the most challenging balance between safety and nutrition. Premature infants often experience signs of food intolerance or inability to digest enteral food. Intolerance to enteral feeding may be a benign condition, but overlaps with necrotizing enterocolitis. In addition, there are significant adverse effects associated with fasting.

The first afternoon of birth is the provision of a complete Parenteral Nutrition (PN) solution. Infants received a stock solution containing glucose (10g/dL), amino acids (2.5g/dL) and lipids 2 hours prior to birth. The amino acid solution contained either AminosynPF 10% (Hospira Inc) or Trophamine 10% (B Braun Medical Inc). Intralipid 20% (Baxter) Liposon III 20% and Liposon II 20% (Hospira Inc) provide parenteral lipids. The fluid is typically provided at 80 to 100mL/kg per day at birth and increased by 20 to 140 mL/kg per day during the first week after birth. The sodium and potassium acetates of PN solution are buffers against metabolic acidosis.

The PN solution provides most of the nutrition in the first week after birth. Enteral Nutrition (EN) usually provides only minimal energy before the end of the second week. The transition to full EN is typically achieved before the end of the fourth week. Where conditions permit, the infant receives mother's breast milk. After tolerating 150mL/kg of human milk per day, the infants received a supplemental human milk fortifier (Mead-Johnson). When breast milk is absent, the infant receives formula specifically prepared for a preterm infant. The maximum caloric density of the supplemented breast milk or formula is provided at 0.8kcal/mL (80 kcal/dL; 24 kcal/oz).

The formula type is as follows: prematch Enfamil Formula (Enfamil), EleCare (Abbott Nutrition), Neosure (Similac), EnfaCare (Enfamil), Pregestimil (Enfamil), Similac Special Care or SSC (Similac), and Gentlease (Enfamil). Pregasternil and Elecare hydrolyzed cow-type formulas, commonly used in infants after NEC or infants with a history of eating intolerance. Enfacare and Neocure are formulas for discharged premature infants. Premate Enfamily Formula and Simiac Special Care are hospital preterm infant formulas.

Food tolerance is demonstrated when preterm infants are able to safely ingest and digest prescribed enteral feeding without complications associated with gastrointestinal dysfunction or infection. Clinical evidence of food tolerance in preterm infants with very low birth weight is most often described in the literature as the number of days required to reach full food intake (reported in the range of 100 mL per kg), the number of food intolerance episodes, the number of days to stop eating due to the symptoms of food intolerance, the time to recover birth weight, calf growth, weight gain, pillow circumference and length.

Prevention/treatment strategies proposed for eating intolerance in preterm infants include:

table 2: preventive/therapeutic strategies are proposed for eating intolerance in preterm infants.

References cited in this example:

hermann and Herman.2010. Nutrition in Clinical Practice 25,69-75

Fanaro.2013.Early Human Development 89,S13-S20

Example 8

Proc tendency to gain NEC (1-activity) WB

1. The markov conversion model fits the PROP, the white blood cell count, the antibiotics (yes/no) and whether the infant has a certain amount of food (> 0).

Table 3:

given volume of 0

Without being bound by theory, increased PROP significantly increases the risk of transition 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.

Without wishing to be bound by theory, there is a symmetric relationship between the state 1 to 2 and state 2 to 1 ratios when plotting data from a table.

For example, with a data set without 2019 extra test data, the coefficient for PROP is about 11 or 12.

The results are printed out as follows:

maximum likelihood estimation

Baseline sets covariates to their mean

Transition intensity with risk ratio for each covariate

The use of other variables (IT ratio, platelet count, time) leads to non-convergence of the model. Without wishing to be bound by theory, this may be due to the fact that the observation proceeded to the day we had missing values. For example, if it is known on tuesday that the PROP score for an infant on Monday is.05, and another PROP value is not obtained until Thursday, then the PROP values for that infant on Tuesday and Wednesday are.05.

2. In embodiments, certain variables may be removed, for example, if the model is complex and preliminary data is limited. When talking about a transition to the NEC state is predicted, an important variable is PROP (since the confidence interval does not contain 1). Without wishing to be bound by theory, if the model fits only this term, the table is as follows:

table 4:

in another embodiment, the antibiotic remains in the assay, which appears to be significantly associated with a switch from a NEC state to a non-NEC state. Without wishing to be bound by theory, in this case, the table is as follows:

fitting of a Linear Mixed model of PROP scores

Next, the linear mixture model is run to predict the PROP score as a function of only the currently owned values over time (i.e. we did not estimate the value of PROP by advancing). This produced 580 data points for 92 patients, of which approximately 45 (7%) contained the PROP value corresponding to the patient then diagnosed as NEC. This model only applies to the covariates NEC, antibiotics (yes/no) and food intake >0 (no/yes) as the introduction of more covariates would reduce the complete case to 78 data points. Initially including linear and quadratic time effects, but without being bound by theory, likelihood ratio tests indicate that this is not necessary. The results of the simplified model without temporal effects are as follows:

In further embodiments, having explicit mathematical terms may allow analysis of data from a linear mixture model.

Without wishing to be bound by theory, hybrid models are used to analyze relevant data, such as longitudinal data or information that may have multiple dependencies. One key feature of mixed models is that they allow to account for multiple sources of variation, i.e. intra-and inter-subject variation, as well as interactions between combinations of discrete and continuous variables, by introducing random effects in addition to fixed effects. In one embodiment,'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 X-ray.

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 no feeding; the Vol1 coefficient was 0.0502. This interaction is important because its p-value is 0.0001. Without wishing to be bound by theory, this interaction is predictable because medical personnel stop eating when NEC is diagnosed. The use of antibiotics had no associated interaction with NEC propensity. This hybrid model takes into account multiple observations over each time interval.

Example 9

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

transition model:

will increased PROP increase (decrease) the rate of transitions a and B?

A. PROP only

In one embodiment, the analysis proceeds with the PROP score of the patient to days without data. For example, if a patient has a PROP score on days 3 and 7, then his PROP score on days 4-6 equals the day 3 score.

Table 5:

increased PROP is associated with a suspected/(+) risk of transition to NEC that is significantly increased.

PROP and antibiotics

The same advances have been made for antibiotics, considering only that infants have no antibiotics, and not what antibiotics to use. For example.

Table 6:

increased PROP is associated with increased risk of transitioning to NEC. Antibiotics increase the likelihood of transition from NEC to NEC (-).

C. Full model

Table 7:

in the described embodiment, additional covariates cannot be considered due to convergence issues.

The white blood cell count continues as if the patient received food.

After adjusting the white blood cell count and whether the patient received food, the results indicate that PROP and antibiotics are important predictors of risk of conversion.

The data display is symmetrical to the data portion in fig. 26.

The interval overlap with 1 concludes no significant difference.

Linear hybrid model of PROP scores

Note that we do not proceed any further; thus, without wishing to be bound by theory, we used the 580 observed PROP data points on 92 patients

Model fitting:

PROP_ijis the prop score for patient i at the j-th measurement.

t_ijIs the time of the jth measurement (in PCA days) for patient i.

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

Table 8:

variables of	Coefficient of performance	Statistics of inspection	P value
				Nec+	.0733	4.93	<.0001
Antibiotic	.0074	.086	.3876
				Volume is 0	.0502	3.92	.0001

NEC correlates with a significant increase in PROP score.

Without wishing to be bound by theory, not eating on a given day correlates with a significant increase in PROP score.

Without wishing to be bound by theory, the assumed normality of prop.a β regression has mixed effects modeling capabilities.

The same results were obtained using beta regression without mixing effect

If desired, embodiments may fit a beta-hybrid regression using a Bayesian approach.

Example 10

For example, embodiment 10 is seen in fig. 28 and 29. We have collected stool samples from preterm infants and analyzed Intestinal Alkaline Phosphatase (iAP) abundance and enzyme capacity. One of the I generated raw figures (FIG. 28) highlights that these two biochemical properties separate NEC disease from non-disease. This blood infection was not separated from non-blood infection when sepsis was examined in this sample patient population.

Given that both biochemical properties of iAP can distinguish NEC diseases, we are interested in developing a simple algebraic formula to combine the equal contributions of both parameters. We elaborated a formula in which iAP abundance was multiplied by the degree of iAP enzyme dysfunction. This product may be referred to as the PROP score or 'necpress' score.

The first term in the formula is iAP abundance. If the bacteria are not beneficial and unbalanced, the iAP will slough into the intestinal lumen and be found in the stool. The normalized percentage of iAP is high relative to the percentage found in human small intestine lysate samples when bacterial imbalance or NEC diagnosis is present (see heat, Maya, et al, "Association of endogenous Alkaline phospholipid With crosslinking enterocolities amine precursors," JAMA network 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 prior to clinical diagnosis (fig. 29).

The second term in the necprogram formula (also called the PROP score) is iAP dysfunction. iAP, which is responsible for neutralizing signals derived from gram-negative bacteria and triggering human innate immune responses. Humans with powerful iAP functions can prevent inappropriate proinflammatory signaling cascades in the human gut and contribute to the maturation of beneficial microbiota. In infants with NEC, we found that at any time during the clinical study, iAP was not functional 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 needs to be normalized to give equal weight between protein abundance and protein function. The iAP abundance and iAP function are multiplied to provide a trend (necpress) score.

At clinical diagnosis, the median necpress score was close to 1 (fig. 29), significantly higher than the control. Even before clinical diagnosis, necpress scores of affected infants were significantly different from control infants. These data indicate that there is a clear clinical-iAP biochemical relationship for NEC disease. Without wishing to be bound by theory, any neccpredict score above 0.5 can be used as an adjunct to clinical disease intervention, such as pausing meal access through the mouth and prescribing antibiotics in neonatal intensive care units.

Example 11

The goal of this project was to obtain data that predicted prognostic biomarkers of Necrotizing Enterocolitis (NEC), the most common and most fatal gastrointestinal disease in preterm infants. This disease sensitive and specific tool is crucial to support the development of new drugs in this smallest and most vulnerable patient population. Furthermore, this work directly addresses the key decision points in current clinical practice: newborn pediatricians and patient equity groups in this field directly motivate us to find a window of disease reversibility. The team first developed a diagnostic test, necselect, for NEC. Necselect analyzed samples of 135 preterm infants from three hospitals and recognized > 95% true positives and > 95% true negatives at disease onset; importantly, it is not associated with late neonatal sepsis. Without wishing to be bound by theory, the NECDetect component can be used to assess risk of NEC before its severe onset. Our prospective observational study will evaluate whether necpress (a calculated probability based on biochemical data of infants) can predict disease 36-48 hours before clinical symptoms appear, and whether Neonatal DDx (a genetic polymorphism screening) can identify infants at birth that are predisposed to develop NEC. The objective of enrollment was 150 preterm infants with a statistical efficacy of 90%. Although the number of premature babies limited participants, this goal still exceeded the goal of most studies registered on clinical trials. gov, in which 62% of participants were less than 100. This work was also the first study involving necbiorepositor, a virtual biostore composed of 8 different academic central hospitals. With the support of this consortium infrastructure, future clinical studies will be able to recruit thousands of infant patients, which will make it the first 6% of the clinical study recruitment goals. If successful, accurate, rapid and inexpensive diagnosis may enable personalized management and improved treatment of intestinal inflammation in infants.

Necrotizing enterocolitis is the most common gastrointestinal disorder in premature infants. Since no diagnostic methods are available, it is important to better understand the pathogenesis of human-microbiome cross-talk in this disease. These studies will define a reversibility window in which infants can be selected for clinical trials for active management and therapeutic intervention.

Specific objects

Necrotizing enterocolitis premature infants (NEC) is a devastating gastrointestinal disease with high mortality and morbidity. Initially described 200 years ago, there was still a fundamental knowledge gap regarding this rare disease. We are unaware of its cause, but it is associated with changes in infant development, feeding and microbiota taxa. Secondly, it is more clinically urgent that no single factor or combination of known factors can account for the wide variability of NEC onset: we do not know who will suffer from the disease at what time. This insight will open new ways 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.

The proposal addresses an unmet need for prognostic biomarkers for the prediction of neonatal NEC pathogenesis. To achieve this goal, the initial hurdle was to elucidate molecular features or biomarkers that intersect dysbiosis, human epithelial function, and NEC. Without wishing to be bound by theory, abnormal biochemical communication between the preterm infant host and the intestinal bacteria is a predictor of necrotizing enterocolitis. Our preliminary data underscores that biochemical analysis to measure host response to intestinal bacteria is a diagnostic biomarker for NEC. A major feature of NECDetect when examining biological samples from 135 very low birth weight infants from three different hospitals is that it improves the recognition of true positives at the onset of disease. Another notable feature of necselect is its availability; it is non-invasive, fast, low cost, and easily integrated into existing pathology workflows.

Working according to this prerequisite to answer the 'if' question, the application will determine whether the NECDetect component can be used as a prognostic biomarker to solve 'who' and 'when'. Our approach was a prospective longitudinal study of preterm infants in two different urban NICUs and regular collection of biological samples. We will recruit 150 preterm and/or growth-restricted infants from two clinical centers (<34 weeks gestational age; <2.5kg birth weight). This study analyzed specific biomarkers and temporal clinical correlations for NEC and non-NEC patients. For this application, we will focus on the following goals:

target 1: is the iAP polymorphism predictive of NEC susceptibility? Our hypothesis is that the intestinal alkaline phosphatase ALPI of infants diagnosed with NEC is genetically mutated, which reduces its detoxification ability against harmful gram-negative bacteria. The method will comprise Sanger sequencing of PCR products amplified from genomic DNA of infants diagnosed with NEC and infants without NEC. DNA will be isolated from cheek swabs or peripheral blood cells. The significance of this work will be the first mechanical definition between disease severity, biochemistry and genetic polymorphisms. If implemented, this NEC susceptibility screening, called Neonatal DDx, will be essential to determine the earliest possible treatment regimen and to improve long-term outcome and quality of life.

Target 2: is Intestinal Alkaline Phosphatase (iAP) level in stool a prognostic biomarker for NEC? This objective will determine whether the onset of NEC can be determined at a molecular level before the most severe physical symptoms can be observed at a clinical level. Without wishing to be bound by theory, the increased release of iAP protein in the human intestinal lumen is responsive to microbial-induced inflammation in NEC and can be measured as a function of time. In total, 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 NEC diagnostic prediction by the computational platform necpress. Its meaning is twofold. This work would be the first study to test a continuous time course in which patients shift between clinical states during the course of the disease, rather than the traditional binary distinction between NEC and non-NEC events. It will also determine the time window of reversibility for active rather than passive medical management.

Indeed, these goals provide a personalized predictive approach to address human diversity, variability in infant gut development, and clinical care options. For this, temporal granularity of patient samples and clinical information is required; such efforts have been made only because of the non-invasive nature of our biological sample acquisition. Embedded in this work is a platform for studying operational and feasibility issues in our clinical research protocol to acquire and integrate large clinical and biochemical information datasets between two large academic medical centers. This optimization of the study across extensive clinical measures and data reconciliation will justify future multicenter studies with more NICUs, reconciled by the new national NEC biobase. Importantly, this proposal will validate an urgent need for biomarkers that can predict and detect NEC. These studies are crucial to improve our understanding of gastrointestinal diseases in the most vulnerable infants and can be extended to the adult population.

Research strategy

(a) Background and meaning

Our goal was to identify mechanisms that alter homeostasis between human hosts and intestinal bacteria that cause inflammation of the gastrointestinal tract. Regardless of age, severe forms of gastrointestinal inflammation can be debilitating and life threatening. The pathophysiology thereof is not clear. At present, it is believed that genetic and non-genetic factors are required for these complex diseases.

For example, Necrotizing Enterocolitis (NEC) in premature infants remains one of the most feared and expensive neonatal diseases [1 ]: we do not know who will get it, when they will get it, or whether they can survive. NEC rapidly progresses from mild abdominal distension and eating intolerance to shock, intestinal necrosis and death. Its rapid progression and inaccurate clinical presentation produced Bell staging criteria, the most common classification scheme based on extensive bedside clinical and imaging findings [2,3 ]: early, called Bell stage I, internal NEC, called Bell stage II, surgical NEC, Bell stage III (fig. 30, panel a). However, Bell staging is not NEC-specific and does not predict the severity of the disease. Mortality rates range from 30 to 50% [4], which commonly occur with other fatal diseases, such as sepsis. Survivors may develop short bowel syndrome, neurodevelopmental dysfunction, bronchopulmonary dysplasia and intracranial hemorrhage [5-7 ].

The lack of reliable molecular biomarkers of intestinal inflammation is frustrating to clinicians and an obstacle to biomedical advancement. Radiography (fig. 30, panel B) is the current gold standard, detecting NECs only in the late life-threatening stages (modified Bell II and III) and recognizing only 44% true positives [8 ]. Furthermore, despite frequent and consistent use of NEC, individual radiological signs of NEC are not readily correlated with disease severity. More importantly, early, reversible stage (modified Bell phase I) biomarkers are lacking in the medical toolbox. Instead, a combination of phenotypic and serological information is used to guide clinical intuition.

Defining prognostic biomarkers for NEC is of great importance to both the scientific and medical community. It will provide insight into key mechanisms and is essential for establishing and monitoring intestinal homeostasis in premature infants. At the clinical level, early biomarkers will mitigate surgical resection of necrotic bowel and the long-term chronic effects of the disease. We have conducted a survey on 70 physicians and we found that the least significant time difference to identify NEC earlier than X-ray is 48 hours, which would allow beneficial patient management. Timely management can reduce surgical requirements by half [9 ]: medication typically includes bowel rest, antibiotics, and supportive care (white box, fig. 30, panel B). Second, prognostic biomarkers are essential for the division of NEC reversible phase. This is not trivial, as NEC has a short timeframe and no medical equivalent in adult GI disease. Furthermore, this early window of disease management is essential for drug development and registration of clinical trials of therapeutic drugs.

(b) Innovation of

Three distinct aspects make this proposal distinctive. The first innovation was the evaluation of non-inflammatory proteins prior to immune activation cascade as biomarkers for NEC. Without wishing to be bound by theory, intestinal alkaline phosphatase (iAP; [10]), an initial host regulator in microbial management (FIG. 30, panel), is a biomarker for early NEC in preterm infants. The development of intestinal inflammation depends on the extent to which bacterial symbiosis is accompanied by cell signaling through innate immune mechanisms [11,12 ]. However, to date, NEC biomarker studies [13] have focused mainly on gene products that regulate gut immunity, mucosal damage that allows bacterial translocation, and host inflammation (fig. 30, panel a). Unfortunately, these proteins, although associated with the advanced NEC stage, are not specific biomarkers of gastrointestinal disease and, importantly, they are also associated with non-gastrointestinal infections and sepsis. Therefore, they cannot be used as prognostic biomarkers, nor are they promising targets for therapeutic intervention.

iAP is encoded by the human ALPI gene and plays a key role in host-microbiota interaction by inhibiting downstream host inflammatory responses. It is a metalloenzyme with tissue-specific expression in the small intestine and is readily detectable in the mucus layer and intestinal lumen [14,15 ]. Membranes are anchored in intestinal cells and iAP flows only into the intestinal lumen and can therefore be measured in feces to control bacterial colonization [15,16 ]. It hydrolyses phosphate from Lipopolysaccharide (LPS), thereby reducing Toll-like receptor 4(TLR 4; fig. 30, panel a) agonist activity. Notably, TLR4 is involved in the pathogenesis of NEC [17-20 ]. Thus, iAP has been used as a measure of enterotoxic injury in animal models [21 ]. In contrast, our proposal examines the iAP in human biological samples and evaluates them during the time the infant is left in the NICU.

Our second innovative direction was to develop diagnostic methods for infant disease, rather than using mature adult biomarkers and testing their applicability to children [22 ]. Although clinical studies were conducted for all diseases, it is clear that the combination of tests is not in compliance with the needs of public health or community medical practice in terms of urgency or scale. Differences in physiological capacity, pharmacokinetic and pharmacodynamic characteristics exist between children and adults; metabolic pathways, organ function and metabolic rates also vary widely [23-25 ]. In addition, age, growth and development are associated with disease severity in newborns, infants and children [26,27 ]. Although it is clearly recognized that children are not 'small adults', the necessity for pediatric specific healthcare solutions is frustrated due to the invasive and hazardous biological sample acquisition methods and the relatively small number of available participants for clinical studies and trials [28 ]. In response to the previous challenge, the study evaluated infant feces in diaper disposals to minimize infant risk and is a contributing factor to study recruitment. For the latter, PI and alliance PI are part of NEC bioresponsorsity, 8 different academic centre hospitals have agreed to share samples and clinical data of NEC infants [29 ]. If this first testing collaboration is successful, other hospital partners in the NEC biobank are prepared to accelerate the larger scale transformation study of NEC in the future [29 ].

Third, advanced testing of clinical workflow and assay systems enabled us to effectively treat an increased number of infants in a shorter study period. The obstacles posed by scale-up are well understood in engineering, but are still relatively new in biomedical research. The following preliminary data demonstrates our ability to collaborate in a multi-point study. This work also provides an understanding of batch effects in the clinical field, such as systematic differences in practices, documentation, patient population, and the like. We devote a great deal of time and effort to provide repeatability and reproducibility of the analysis of biochemical and clinical data. The following data must overcome challenges in patient sample handling, biological sample banking, biological sample quality, and data reconciliation [30 ]. Therefore, we have first-hand knowledge of how to aggregate data from multiple sources, ensure a uniform and consistent flow, clean up and apply quality control metrics to the accepted and processed data, and so on.

Our prospective study evaluated the independent relevance of 2 fecal biomarkers, including necselect (fig. 30, panel B), of 136 premature infants from the LSU medical school and the affiliated neonatal intensive care unit of the washington university medical school (1.1 ± 0.5 kg; 27.6 ± 0.8 weeks gestational age; see incorporated report). Taken together, our data show that the presence of large amounts of iAP protein in feces and low iAP enzyme activity are biomarkers for NEC. Indeed, NEC infants are shooting' blanks into the intestinal lumen to control abnormal microbial development. The iAP biomarkers are not associated with sepsis or other parenteral infections.

Infants suspected of having NEC and infants with advanced NEC disease release iAP protein into the intestinal lumen at levels exceeding the amounts typically found in the human small intestine.

Baseline assessment was performed for iAP detection using immunoblotting. Immunoblotting allows the determination of the relative expression of proteins in complex biological samples. Development of sensitive antibody labels with truly quantifiable linear range and larger detection limits by digital image analysis can detect proteins with higher resolution than previously achievable [31 ]. To illustrate the sample preparation, detection protocol and normalization methods in our hands, the calibration curve of our positive control human intestinal lysate (fig. 31, panel a) shows the linear part of the detection of anti-human iAP signal and our working range overlap [32 ]. Positive and negative controls (calf iAP) were from a single batch and used as our quantitative calibrator; both were loaded onto each gel with the patient sample. Equal amounts of total protein were loaded per lane.

High iAP protein levels are associated with NEC diagnosis and NEC suspicion, but not sepsis. We established a baseline iAP level, sloughed off in the intestinal lumen of non-diseased infants and detected in the feces [33 ]. The feces of control patients had very low levels of iAP (< 2%; FIG. 31, panel B) compared to human small intestine lysate and calf iAP control. We conclude that there is little iAP flow into the intestinal lumen when no dysbiosis is about to occur.

A large amount of fecal iAP protein was found at clinical NEC diagnosis (red bars, Bell II and III; FIG. 31, panel B). If there is a risk of bacterial-induced inflammation, the iAP content in feces can be increased by 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, a molecular biomarker superior to clinical diagnosis (X-ray evidence of intestinal gas accumulation). As NEC biomarkers, the sensitivity and specificity of fecal iAP content was greater than 95% (fig. 31, panel C).

NEC suspected or early NEC was also associated with high levels of iAP in stool (pink bar, Bell stage I; fig. 31, panel B). In stool samples with clinical concern for advanced NEC, the amount of iAP protein was statistically different from the control even though intestinal gas was not detectable by X-ray (pink bar, fig. 31, panel B). However, there was no difference between NEC suspicion (pink bar) and diagnostic mean (red bar, fig. 31, panel B). Stool iAP levels had no measurable correlation with sepsis (blue bar, fig. 31, panels B and D). Severely premature infants (<32 weeks) not only have an inappropriate gut formation, 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 show that iAP protein levels are not statistically relevant to clinical diagnosis of sepsis.

Infancy with advanced NEC disease has very low enzymatic activity, whereas infants suspected of NEC have moderate iAP catalytic activity.

iAP enzyme activity was assessed in non-NEC infants. After normalization to protein concentration, we found a reduction in iAP activity in the feces of preterm infants compared to near term infants (36-40 weeks post-conception or PCA). There was a strong positive correlation between iAP activity and age after conception (fig. 31, panel E). In comparing IAP activity for 28-40wksPCA, a one-way ANOVA and Tukey multiple comparison test was performed, indicating that the samples were statistically divided into two groups: preterm group (PCA ≦ 35wks) and term group (PCA ≧ 36 wks). All stool samples from full term infants (n-28) were compared to all samples from preterm infants (n-79) and the latter group was found to have significantly lower iAP activity (p < 0.0001; single tail t-test). This is consistent with the switch between the fetal and 'adult' iAP isoforms at this developmental time point: dynamic conversion of IAP isozyme forms is associated with maturation of the fetal gut [36 ]. Fetal isoforms of iAP have low biochemical activity, while adult iAP have a strong catalytic rate. Thus, iAP activity is a biomarker directly associated with the age after conception. Proper design and analysis of biomarker studies requires such normative data across different gestational ages.

Low iAP activity is also relevant for NEC diagnosis and NEC suspicion, but not sepsis. We found that the iAP activity of infant faeces collected on the same day as the clinical NEC diagnosis was much lower than the age-matched control (red bar, fig. 31, panel F), indicating that very low iAP activity was associated with NEC. Evidence suggests that a reduced capacity of the intestine of preterm infants to dephosphorylate pro-inflammatory LPS increases the risk of excessive inflammatory responses to bacterial colonization and NEC development [37 ]. Consistent with the idea that biochemical measurement of iAP is a NEC biomarker, the iAP activity of suspected NEC stool samples (pink bars, fig. 31, panel F) also showed low rates, possibly predictive of NEC due to the inability to control bacterial colonization. Finally, measurement of iAP activity had no correlation with sepsis (fig. 31, panels F and H).

(d) Method of producing a composite material

Specific object 1: is the iAP polymorphism predictive of NEC susceptibility? Without wishing to be bound by theory, infants diagnosed with NEC will have a Single Nucleotide Polymorphism (SNP) in the intestinal alkaline phosphatase gene ALPI, which reduces their catalytic ability to detoxify lipopolysaccharide-dependent signaling of harmful gram-negative bacteria. Our preliminary data indicate that the enzymatic ability of the single gene product iAP is associated with NEC disease progression. In patients with the extreme form of NEC (Bell II and III), enzyme activity was almost absent, and therefore these infants were unable to modulate TLR 4-dependent IL-8 transcription (figure 30, panel a). In early NEC (Bell I stage), iAP enzymatic activity was lower than in non-NEC-like Bell II and III infants (fig. 31, panel F).

Basic principle. Genetic variation in TLR signaling has been studied, as 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 TLR2, TLR4, TLR5, IRAK1 and TIRAP genes appear to be unrelated to NEC [38-45 ]. Understanding the mechanisms and causal relationships will be essential to determine the earliest possible treatment regimen and to improve long-term outcomes and quality of life. This work will lay the foundation for ALPI screening for monogenic diseases and ALPI-based NEC therapy. This polymorphism of ALPI found in NEC patients will underlie Neonatal DDx, a screening tool for infants at birth.

Study inclusion. In this prospective study, preterm infants (non-NEC and NEC) admitted to LSU medical school and washington university medical school Neonatal Intensive Care Unit (NICU); parents were asked to agree that single IRB and IBC approvals for biological sample collection and gene testing had been handled. Recruitment and inclusion of Low Birth Weight (LBW) preterm infants (<2,500g birth weight and/or <34wks gestational age) is a process limited by the number of infants born in our hospital. We will recruit at least 120(LBW) infants in the first year, or at least 5 infants per month (see human subjects for reports 1 and 2). We predict that 25-30 will develop NEC Bell II/III phase, and that nearly equal numbers will develop NEC Bell I phase.

Clinical information. This study is observational and does not require creation or departure from standard clinical care in NICU. The following clinical data will be obtained: demographics, medical history, antibiotics/antimycotics/drugs, physical examination, complete blood count, blood culture, abdominal radiographs, operative consultation notes, and eating history. The study will be conducted according to the rules of the institution, local, state and federal for PHI use, as defined by the Health Insurance circulation and Accountability Act.

And (6) collecting the specimen. DNA samples were collected non-invasively from cheek swabs or residual blood draws.

A method. Genomic DNA will be isolated from peripheral Blood cells or whole Blood using (i) saliva swabs of infant cheeks or (ii) the QIAamp DNA Blood Mini Kit. For gene sequencing, the ALPI variants will be identified by Sanger sequencing of PCR products amplified from genomic DNA. We used Eurofins to sequence mutations in human proteins (e.g. [46]) periodically. PCR will be performed using AmpliTaq polymerase using the GeneAmp PCR system. The primer pairs used for DNA amplification were: 5 'GGACCTTCAGTGGTTCCAGG-3' (f) and 5 'CCAAGGACCTGGTTCTGGTC-3' (r). The list of variants identified by sequencing will be the subject of a filtering procedure, e.g. to exclude common variants, low quality variants and synonymous changes in the population. The sequence data may be compared to various public databases (single nucleotide polymorphism database (dbSNP [47 ]); 1000Genomes Project [48 ]; and outer Variant Server [49,50 ]). The comparison will look for rare variations that occur with a frequency < 1% in the control. Initially, NEC patient relay variant and de novo variant will be classified as Neonatal DDx.

Alkaline phosphatase activity was measured using 4-methylumbelliferyl phosphate (MUP) as a fluorogenic substrate in the presence and absence of 10mM L-phenylalanine, an iAP inhibitor [51,52 ]. The relative fluorescence units at 360/440nm will be measured in the sample using a 96-well black optical backplane. Total AP activity was measured in mU/mg, where U is the amount of enzyme that hydrolyzes 1. mU. mol MUP per minute at pH 10 and 25 ℃. Determination of total protein in fecal supernatants was determined using the bradford assay. Protein standards (bovine serum albumin) and patient samples will be prepared using molecular-scale water as the diluent. The criteria were run every day of data collection and had to have r2 values greater than 0.99 to be acceptable.

And (6) obtaining the result. Without wishing to be bound by theory, an infant developing NEC will have at least one allele with a non-conserved polymorphism causing a loss-of-function phenotype. Lack of fecal iAP activity (<240U/mg) would confirm that the ALPI mutation caused the loss-of-function phenotype. The DNA sequence will recognize a SNP in the infant ALPI gene; their relative amino acid positions can be identified using a homology model of the AP crystal structure [53-55 ]. Only 200 unrelated patients need to be sequenced and as low as 5% of the population of disease genes can be identified [56 ]. Without wishing to be bound by theory, mutations will occur at the active site of the iAP and/or the dimerization interface [54,57 ].

Statistical strategies to identify disease-causing polymorphisms are based on the nature of the disease mutation [58 ]. For example, statistical analysis will be performed using a dominant model, comparing wild-type homozygotes to combined heterozygote and homozygote rare allele genomes. This assumes that carrying at least one copy of the variant allele increases the risk of disease. The primary outcome measures included the presence of NEC, the severity of the disease (Bell II/III and Bell I), and NEC-related intestinal perforation.

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

An alternative approach. Statistical evaluation of sequence data may require the use of implicit models [56,59 ]. If polymorphisms were found that correlated with NEC susceptibility, 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 lineages (e.g., [61,62 ]). New dominant mutations can be identified by analyzing the intersection of heterozygous variations (e.g. [64]) of three-in-one parents (e.g. [63]) or unrelated probands with the same new autosomal dominant disease.

While moderate sample size of less common diseases in premature infants may limit the ability to detect associations, such studies are crucial as a preliminary step in providing targeted therapy to children, finding important insights into factors associated with disease risk, and identifying networks associated with disease pathogenesis.

Specific object 2: is intestinal alkaline phosphatase protein levels in stool a prognostic biomarker for NEC? The field of neonatology is hampered by the inability to detect NEC early in the disease process. Without wishing to be bound by theory, the microorganism-induced inflammation in NEC results in increased levels of iAP protein in the gut lumen and feces. Early NEC infants will detect iAP levels within 36-48 hours before NEC diagnosis if asymptomatic or only non-specific symptoms are present. Our data indicate that prediction of NEC is feasible. While our clinical focus was on stool collection at disease diagnosis, stool samples were occasionally collected before onset of disease in 5 of 25 NEC Bell II/III patients and 9 of 19 NEC Bell I infants; of the 62 non-NEC patients 19 median 4 samples were collected 31 weeks ago. We will collect stool samples from each infant's longitudinal series, measure iAP content of the samples twice weekly, and model the prognostic power of the continuous biomarker necpress.

And (4) taking statistics into consideration. To this end, the study inclusion and clinical information protocol detailed in objective 1 will be followed. We required 150 subjects to achieve 90% efficacy of target 2. This is based on simulation results, since the existing sample size calculations for prognostic biomarkers [65-67] only apply to the differences between the two groups [68 ]. We performed a simulation study (α #, α%) using four different combinations and examined the probability that we detected significant differences between these coefficients and 0. For n-50, 100, 150 or 200 we achieve work values greater than 0.8, 0.9 and 0.95. This efficacy analysis indicates that a sample size of n >100 is required, and that n-150 can provide significant benefits compared to n-100.

And collecting and preparing a specimen. Continuous monitoring using the novel protein biomarkers required stool collection from study participation to discharge. There is no identifiable risk to the patient because the collection of a non-invasive sample from a discarded diaper is painless and does not cause harm to the fragile patient. Stool specimens will be collected every 3-4 days [69] until the infant reaches 37 weeks post conception or discharge. The mean hospitalization time of the infants was estimated to be 49 days [70] to 54 days (see preliminary data); for each infant, about 15 samples will be collected from the diaper. Fecal samples were collected from the first day of radiological findings of NEC (Bell II or III) to the last day of NEC management (antibiotic administration and no oral feeding) and were termed 'NEC'. From the first day of 2+ clinical symptoms to the last day of medical management, the collected sample is called 'suspect'. A sample is a 'control' if it is obtained on the day when no NEC diagnosis is made.

Feces were stored in a 4 ℃ specimen NICU refrigerator until the samples were sent to the laboratory. After each patient sample was received, the feces were homogenized and slurried with molecular water at 200mg/mL in a sterile microcentrifuge tube. After vortexing and centrifugation, the supernatant was collected, aliquoted and stored at-80 ℃ [71 ]. Follow safety procedures (gloves, lab coats, goggles), use of blotting paper, decontamination with EPA registered hospital disinfectants, and proper disposal of biohazards.

Determination of relative iAP protein content. Duplicate denaturing SDS-PAGE gels will be run on the fecal supernatant to show all proteins in each lane and for immunoblot detection of iAP. iBlot and iBind will be used for protein transfer and western blotting, respectively. Bands were quantified using Amersham Imager 600; the relative iAP protein in the fecal sample is the portion of protein found in human intestinal lysate tissue.

It can be said that the greatest source of confusion in quantitative immunoblots is the effect of protein loading and loading controls [72 ]. Immunoblot samples are usually prepared based on total protein [73-75], which assumes that the average protein content per cell is constant under different conditions. However, in our analysis, the stool sample was not fractionated to lyse the cells: only intestinal lumen content was assessed. Therefore, total cellular protein cannot be determined and the input must be normalized according to some estimate of protein loading. We used two loading controls along with total protein [76-81 ].

In fig. 31 panel a, control experiments evaluated the accuracy and precision of the immunoblot workflow. Such experiments minimize overestimation or underestimation of true differences in protein abundance. Serial dilutions of the positive control (small intestine tissue lysate) and the negative control (purified bovine iAP) showed our dynamic range and quantitative accuracy. Determining a 60kDaiAP signal from the patient sample; this value is proportional to the difference between the positive control and negative control measurements.

And (6) obtaining the result. Without wishing to be bound by theory, we will detect iAP levels of 0.14 ± 0.10 (mean ± SE) compared to human small intestine lysates of samples collected 7 days prior to NEC. Our preliminary data provides a threshold for expected immunoblot values. In stool samples at NEC diagnosis, iAP levels were 1.59 ± 0.48 higher than human small intestine lysate. Stool from non-NEC patients had an iAP content of 0.02 + -0.01. Since these schemes are already established and successfully implemented by 3 different operators, we expect that no technical problems will arise.

Using necbodict, we will model the probability that a patient is symptomatic or diagnosed with NEC based on the iAP content in stool samples collected 3-5 days and 6-8 days prior. In this test of prognostic power of successive biomarkers, the mean difference in relative iAP protein content between NEC and control samples will be tested by modeling the link between NEC diagnostic probability and iAP content at any given date in diapers 3-5 days ago and in diapers before (6-8 days ago).

In particular, NEPCredict will use the generalized linear mixed-effect model [82 ]]In response to NEC diagnosis, the iAP content of the last two collected samples was used as a predictor (fixed effect), as well as a patient level error term. We modeled whether the patient had evidence or diagnosis of NEC as follows: logit { P [ NEC [ ])_t,i＝1]}＝α₁D_t-1,i+α₂D_t-2,i+β+ε_iWherein NEC_t,i0-if patient i is NEC negative at the time of diaper collection t, and 1-if patient has NEC diagnosis or NEC signs/diagnosis (analysis alone). D_t-1,IAnd D_t-2,IIs the iAP content of the last two collected diapers. Epsilon_iIs a subject-specific error term that contains NEC diagnosis and the dependence of diaper content on time and individual. We used lme4 bag [83 ] in R]To analyze this model. Significant positive estimate a₁Indicating that a high iAP content of the last diaper collected (approximately 3 days ago) predicts future NEC diagnosis and alpha₂>0 indicates that a high iAP in the previous week predicts future NEC diagnosis. This information indicates that iAP can be a prognostic biomarker for NEC.

In summary, in vitro results and clinical data will determine associations with these markers and NEC risk. Without wishing to be bound by theory, the polymorphism in ALPI leads to low iAP activity and Neonatal DDx can be used as a biomarker to determine infants with the greatest risk of NEC. Second, the presence of iAP protein as necprep will have the strongest prognostic value and will identify the development of NEC before symptoms appear. These studies will be the first biochemical and physiological markers to link two NEC triggers: alteration of microbiome and gut development. Another important result is that indiscriminate inhibition of feeding and broad spectrum prophylactic antibiotics can be minimized. Thus, these studies provide the first laboratory test to personalize therapy in the NICU. Future approaches may include supplementation of iAP as a preventative strategy for NEC, since enzyme replacement therapy is a low-cost, low-risk approach for rare diseases, which is often successful [84 ]. Finally, these personalized biomarker methods are not limited to children; iAP is similar to adult diseases (such as IBD) as a biomarker of intestinal inflammation in infants.

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Statistical design and efficacy of prognostic NEC clinical studies

Statistical analysis will be performed using R (R Core Team,2018.R: A language and environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria). The significance test is a two-tailed test performed at a 5% significance level. Statistical assumptions will be tested and the model appropriately modified if necessary. Missing data will be processed by interpolation and sensitivity analysis. If the proposed statistical analysis technique is found to be unable to stand, we will use an alternative technique, possibly going back to a strategy that guarantees that a proper estimate is provided, as well as a non-parametric variability measure.

Study design of target 2: this study will be a prospective observational study in which premature infants are included at birth. Feces samples of disposable diapers will be collected every 3-4 days until the infant reaches 37 weeks post conception or discharge. Stool samples from patients with clinically significant NEC will be analyzed as well as a random subset of stool samples from control patients.

Sample size: for goal 2, we required 150 subjects to achieve 90% efficacy. This is based on simulation results, since the existing sample size calculations only apply to the differences between the two groups [1 ]. In contrast, our model requires testing the prognostic power of successive biomarkers.

We will model the probability that a patient is either physically or has been diagnosed with NEC based on the iAP content in stool samples collected 3-5 days ago and the iAP content in stool samples collected 6-8 days ago. We allow each patient to have a different level of susceptibility to NEC, which is captured in the patient's random effect term. The random effect of the patient is combined with the iAP content effect and transformed by a function (the inverse of the logit function) that limits the value between 0 and 1, enabling us to estimate the probability.

With symbolic terms, we model the probability that a patient shows signs of, or has been diagnosed with, NEC as:

logit{P[NEC_t,i＝1]}＝α₁D_t-1,i+α₂D_t-2,i+β+ε_i

wherein NEC_t,i0-if patient I is NEC negative t at the time when the diaper is collected t, and 1-if the patient has signs of NEC (Bell I stage) or NEC diagnosis (Bell II/III stage). D_t-1,iAnd D_t-2,iIs the iAP content of the last two collected diapers. Epsilon_iIs a subject-specific error term that incorporates in-individual NEC diagnosis The correlation of the probabilities. We model the above using the R program lme4 using a generalized linear mixture model. To determine how many patients we should recruit, we conducted a simulation study to determine how much we can detect significant differences in α 4 and α 8 (the magnitude of the effect of the iAP content of the last two diapers on the probability of future NEC occurrence) based on the last two collected diapers from 0, which would show a prognostic effect of iAP content on predicting NEC diagnosis or signs of NEC. This information may help the clinician predict whether the patient is at high risk for NEC.

We performed simulation studies using four different combinations (α 4, α 8) and examined the probability that we detected a significant difference between these coefficients and 0. These cases are shown in fig. 32, along with the implicit probabilities of patient NEC diagnosis/suspicion of mean iAP content for NEC and non-NEC patient groups, which are 1.59 and 0.02, respectively.

These four cases represent different effects of iAP diaper content on NEC diagnostic probability. In case 1 and case 2, only the last diaper can predict whether the patient will develop NEC the next time, since α₂0. The separation between NEC probabilities is greater for the high iAP and low iAP groups for case 1 compared to case 2. Also, cases 3 and 4 have different probability differences for the two groups. For cases 3 and 4, α ₂1, indicating that the iAP content found in diapers collected two time points (six to eight days) before was predictive of NEC status. These values were chosen in part because they resulted in 9-12% of patients in each case having NEC diagnosis, which coincided with the previously seen incidence of NEC diagnosis. With simplified assumptions, we simulated 1,000 replicates of each of the four cases, with the sample size n-50,100,150, or 200.

For each patient, we assume that diapers were collected every two weeks for a follow-up visit of 2 months, for a total of about 16 diapers per patient. We will evaluate NEC status at 3-16 days of diaper collection to see how iAP content of the last two diaper cycles predicts NEC diagnosis. For the simulation, we first plotted the NEC + index from the Bernoulli distribution with a probability of.09 that all previously tested diapers had a warp of NECAnd (6) probability testing. For each NEC patient, we generated their 16 iAP diaper content values D from a multivariate lognormal distribution of positive correlations between the mean vector-3.90, standard deviation 1.69, and each observed iAP value_1,i,…,D_16,i. To generate a multivariate lognormal sample, we generate a multivariate normal distribution using the mean vector and implicit covariance matrix described above, and then exponentiate these values. For individuals not considered NEC positive, we generated their iAP values D from the mean vector-. 68, standard deviation 1.57, and the multivariate lognormal distribution of positive correlations between each observed iAP value _1,i,…,D_16,i. These mean and standard deviation were chosen because they are the estimated maximum likelihood estimates of the log-normal distribution using the iAP values of NEC + and NEC-patients, respectively.

We then generated for the patient the probability of NEC at time t 3, …,16, α₁D_t-1,i+α₂D_t-2,i+ β, and NEC states are extracted from bernoulli random variables with this probability. FIG. 33 shows the correct declaration in simulation (correct declaration) of each sample size₁>Probability of 0, correct statement α₁>0 and alpha₂>Probability of both 0 s. For cases 1 and 2, this triplet is listed as (P)₁-, -) and for cases 2 and 3 this triplet is listed as (P)₁,P₂,P₃)。

From the above simulations we see that none of the 4 cases has a higher efficacy than.80 for n-50. For n-100 we achieved in each case a.80 or more efficacy, but only a was detected₁And alpha₂The significant difference between the two, where the probability in case 3 is.819. For n 150, our efficacy is higher than.9 for each of the four cases, and for n 200, our efficacy is higher than.95 for each of the four cases. This efficacy analysis indicates that a sample size of n ≧ 100 is desirable, and that n-150 can provide significant benefits over n-100.

References cited in the examples

Dang, Q, S.Mazumdar and P.R.Houck, Sample size and power regulation based on generated linear mixers with corrected binding schemes, comprehensive Methods Programs Biomed,2008.91(2): p.122-7.

Example 12

There are four different tissue-specific alkaline phosphatases in humans: intestinal alkaline phosphatase, placenta-like alkaline phosphatase, tissue non-specific 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 compared to tissue-non-specific alkaline phosphatase. Furthermore, 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.

Our innovation was to use two biochemical indicators of fecal Intestinal Alkaline Phosphatase (iAP) as molecular biomarkers of NEC in preterm infants. The attractiveness of iAP as a biomarker is that its tissue-specific expression in the small intestine and its secretion into the intestinal lumen can only be measured in feces as a response to control of bacterial colonization. Furthermore, it can be detected in human stool samples of healthy individuals; responsible for most of the AP enzyme activity in feces; and used as a measure of enterotoxic injury in an animal model.

(A) High iAP level detection based on immunoassay is a biomarker for necrotizing enterocolitis, but not for sepsis. Our prospective study evaluated this in human premature infants. We evaluated the independent relevance of 2 stool biomarkers, including necnect, in 136 preterm infants [ mean gestational age 28.3 weeks; 50% of women; 64% african americans, 32% whites; 4% of hispanic. A large amount of fecal iAP protein was associated with clinical NEC diagnosis; these levels are equal to or higher than those found in human small intestine enterocytes. If there is a risk of bacterial-induced inflammation, the iAP content in feces is expected to increase due to the iAP-loaded membrane vesicles released. In contrast, the iAP protein content in the stools of patients who are not diseased is very low. Thus, when no dysbiosis is imminent, little iAP flows into the intestinal lumen. Sensitivity and specificity of fecal iAP content > 95%. Unlike other candidate NEC biomarkers, fecal iAP levels have no measurable correlation with other parenteral infections or sepsis, which is a common complication that may confound the diagnosis of NEC.

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

(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 false results for affinity-based and MS-based protein measurements. Sequence information from unrelated individuals to determine the frequency distribution of iAP polymorphisms. The iAP polymorphism, or allele greater than 1% of the population, was present only in the african american population (n-12,487) (fig. 34, panel B). The estimated frequencies of the common alleles V20I, R33L, R92C, R144H, and T207I were 4.8, 2.6, 1.9, 4.2, and 3.1%, respectively. Polymorphisms in the total population (blue circles, FIG. 34, panel C) caused changes in peptide mass by changes in side chain molecular weight. Two polymorphisms (purple circles, fig. 34, panel C) common in the african american population resulted in the loss of trypsin cleavage sites, which in turn resulted in hundreds of fold changes in peptide quality. Changes in peptide mass, regardless of size, prevent MS identification and accurate quantification of proteins. These data raise concerns not only about the effectiveness of correlating SNPs with affinity-based protein measurements, but also about MS techniques, which may give false results with a minor allele frequency close to 5%.

Example 13

Necrotizing Enterocolitis (NEC) is a common Gastrointestinal (GI) emergency in newborns, with high mortality and long-term morbidity, including short bowel syndrome, nutritional deficiencies, and neurodevelopmental retardation. 2,3 suspected NEC appeared as mild nonspecific symptoms, which were often resolved with minimal intervention; no clinical trial is a established standard for suspected NEC. Radiological evidence, such as intestinal gas accumulation, is used to diagnose severe or advanced disease, but sensitivity is as low as 44%, 4 is limited in specificity, 5 and lacks consistency in interpretation. 6-8

Many efforts have been made to find molecular diagnostic biomarkers for NEC (fig. 35A). Despite the publication of 2500 more previous biomarker studies, meta-analysis failed to identify the best NEC biomarker for routine clinical use. 9-11 the design and capabilities of these studies have drawn attention: less than 30 articles per decade of analysis are considered suitable for meta-analysis. The concern for inflammation and repair proteins was problematic in these studies (fig. 35B). Advanced disease with systemic inflammatory lesions is not ideal for biomarker assessment because the reversible phase of the disease cannot be defined. 12 furthermore, positive predictions of inflammation-related proteins are of limited value, since sepsis is a complication in 35% to 60% of NEC cases. 13-17

Necrotizing enterocolitis is considered a precursor to some cases of late neonatal sepsis (LOS). Newborns, especially very low birth weight infants, are susceptible to sepsis due to prolonged hospital stays, invasive instrumentation, underdeveloped innate immunity, and altered immune responses. The latter 2 physiological states, coupled 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 NEC biomarkers. Sepsis and NEC require careful differential diagnosis, as both can be fatal if not properly diagnosed and treated.

The present study evaluated the use of Intestinal Alkaline Phosphatase (IAP) as a diagnostic biomarker for NEC. Recent findings indicate that NEC precedes and accompanies changes in gut microbiota (fig. 35C) and that it is associated with the host immune pathway leading to gut inflammation. 19,20 intestinal alkaline phosphatase detoxifies surface Lipopolysaccharide (LPS) of harmful bacteria by cleaving inorganic phosphates. LPS is a component of the cell wall of gram-negative bacteria and is a potent inducer of innate immune signals via Toll-like receptors 4. The powerful IAPs function can neutralize LPS signaling, prevent an inappropriate proinflammatory signaling cascade in the gut, and contribute to the maturation of the beneficial microbiota.

Since IAP activity precedes the initiation of the signaling cascade that triggers inflammation, we assessed IAP abundance and enzyme activity in feces as a measure of pathobiological need and the ability to maintain host-microbiota homeostasis, respectively. A multicenter, prospective diagnostic study was performed to assess the association of 2 IAP biochemical markers with disease severity. As a core protein common in the human fecal proteome, 21IAP is ideal for non-invasive detection. The level of IAP in feces is expected to increase due to the IAP-loaded membrane vesicles released if there is a risk of bacterial-induced inflammation. 22,23

Method

Design of research. During 3 years (5 months to 11 months of 2018 in 2015), preterm infants with gestational age less than 37 weeks and birth weight less than 1500 grams were enrolled at new orleand children hospital (n-29; new orleans, louisiana) and Touro infrimary hospital (n-68; new orleans, louisiana). Preterm infants born at gestational age less than 37 weeks were enrolled at the st louse hospital (n 39; st louse, missouri). Written informed consent of study participants was obtained from parents or guardians. All infants were required to be included in the study, forming a continuous sampling series.

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 (i.e., death, discharge or transfer to another institution). Wherein only race/ethnicity is defined by the parent. Hospitalization data included food intake, antibiotic treatment, laboratory and radiological results, and surgical records. The clinical findings of NEC (modified Bell stages 1-3), sepsis and other diagnosed parenteral infections were reviewed by the attending physician.

To protect confidentiality and anonymity, each recruited patient is provided with a code that allows study tracking and the removal of any clues about the identity of the individual. Every three months, patient records were evaluated to determine clinical relevance. Clinical data is extracted from medical records into relevant clinical databases. Demographic information and initial clinical data include gestational age, birth weight, Apgar score, type of labor, race, gender, and final outcome (death, discharge or transfer). Finally, a second set of clinical information is obtained: antibiotic use, diet, serum AP, radiology reports, NICU hospital stays, surgery and mortality. Human milk exposure was calculated as the average percentage of food eaten from human milk as a function of the total number of days in the study for the subject. For NEC cases, only pre-event exposure of human milk was considered. Antibiotic exposure is considered comprehensively; antibiotics are always administered to a subject by the parent. The age day percentage using antibiotics is related to the number of days the subject was in the study. For NEC cases, antibiotics only considered prior exposure.

Definition of disease. Different NEC definitions have been proposed. 26-29 in this study, class 2 NECs from clinical files were used (e Table 1). Radiological markers are the defining criteria for our NEC category; abdominal signs and clinical and laboratory examination results are secondary criteria. Suspected NEC was defined as a disease based on abnormal clinical and laboratory findings, with no evidence of intestinal or portal pneumatosis on the abdominal radiograph images. Severe NEC is defined as radiological evidence of intestinal and/or portal pneumatosis. Patients diagnosed with Spontaneous Intestinal Perforation (SIP) were excluded from the study (e table 2). LOS of neonateDiagnosis requires abnormal clinical findings at least 72 hours after birth and blood cultures positive for bacteria are not considered contaminants 30,31(e table 3). The clinical manifestations of infants with other diagnosed parenteral infections are bacterial, viral or fungal infections found in body fluids other than blood. Summary of panel and diagnosis of NEC, SIP, sepsis and parenteral infection are provided in tables 4 to 11.

Clinical findings of NEC diagnosis, NEC suspicion, sepsis and other diagnosed parenteral infections were determined by examining clinical documents. The study definition of NEC is not always consistent with clinical diagnosis of patients. For this study, NEC and suspected NEC were physician-guided clinical diagnoses, in which radiological signs were the defining criteria and abdominal signs, clinical findings, and laboratory findings further confirmed the diagnosis (e table 1). NEC suspicion (e table 1) was defined as infants with early disease fear based on clinical and laboratory abnormalities, while radiological examination showed no evidence of intestinal gas accumulation. In contrast, infants suspected of NEC exhibit one or more radiological signs, including mild bowel dilation, mild ileus, thickened intestinal walls, or rarefied/absent intestinal gas. In addition to one or more laboratory test results, one or more clinical or abdominal signs and symptoms are also required, including thrombocytopenia, leukopenia or increased, decreased absolute neutrophil count, increased number of immature neutrophils, heme-positive feces, metabolic acidosis. Clinical and abdominal signs and symptoms include bile secretion, vomiting, bloody stools, eating intolerance, increased pre-eating gastric residual amounts, apnea and/or bradycardia, temperature instability, lethargy, appearance of systemic clinical symptoms, mild to moderate abdominal distension, and discoloration of the abdominal wall.

Severe NEC (e table 1) is defined by radiological evidence of intestinal gas accumulation and/or portal vein gas or pathological consequences of surgery or post-mortem bowel samples. Pneumoperitoneum is free intra-abdominal air produced by perforation, considered NEC when accompanied by evidence of intestinal gas accumulation on the radiograph and abdominal signs found in established NEC. Other signs include moderate to severe abdominal distension and/or abdominal tenderness and/or hypoactivity/no bowel sounds and/or discoloration of the abdominal wall, abdominal cellulitis, fixed lower right abdominal mass and/or signs of peritonitis. NEC diagnosis is classified by at least one advanced clinician and two additional advanced study clinicians based on case examination, note examination, X-ray and surgical outcome.

Although clinical presentation and medical management were similar to NEC, patients with Spontaneous Intestinal Perforation (SIP) were excluded from the study and considered a completely different disease (e table 2). The main differences that distinguish NEC from SIP include the absence of intestinal gas on abdominal radiographs, earlier appearance of symptoms, focal hemorrhagic intestinal necrosis on pathological specimens (rather than the coagulative necrosis characteristic of NEC), and a generally more benign clinical course, whether pre-or post-diagnosis. Overlapping medical management of the SIP and NEC procedures includes cessation of enteral feeding, gastric decompression, intravenous antibiotics, and peritoneal drainage (if indicated). S8, S9

The diagnosis of neonatal sepsis is variable and complex by the use of biomarkers of overall low sensitivity, such as a change in the white blood cell count index, low absolute neutrophil count, high ratio of immature to total (I: T) neutrophils, and elevated serum C-reactive protein levels. Positive blood cultures of 3 days or more for S10 delayed sepsis are considered the gold standard for the diagnosis of neonatal sepsis. However, cultures are often negative, may be associated with low inoculum blood volume, which may not fully represent true bacteremia, and are exposed to prenatal antibiotics, which may inhibit bacterial growth. S11-S13

In this study, infants diagnosed with sepsis included only those with laboratory and clinical findings confirmed after seventy-two hours of age (e table 3). Laboratory test results include blood culture or non-cultured microbial tests that confirm the presence of bacteria in the blood that are not considered contaminants. S14, S15 clinical outcomes for supporting sepsis diagnosis include a range of criteria from temperature instability and respiratory distress to abnormal perfusion, bleeding problems and unexplained jaundice.

In addition, other infants diagnosed with parenteral infection and infants negative for infection were classified and documented in this study (e table 3). Other diagnosed infections are those bacterial, viral or fungal infections found in normally sterile body fluids. Clinical findings were similar to patients diagnosed with sepsis. Negative classifications of infection include infants suspected of infection but not diagnosed with infection and infants not suspected of any infection but not subjected to laboratory tests for infection and asymptomatic. Laboratory test results for suspected infected subjects include, but are not limited to, leukocytosis or leukopenia, elevated immature neutrophil counts, low absolute neutrophil counts, and elevated C-reactive protein and serum alkaline phosphate. The clinical findings of these infants were identical to those of infants diagnosed with sepsis.

Sample collection and extraction of soluble intestinal lumen contents. A simple fecal management protocol was developed for evaluating IAP processes in the intestinal lumen. After obtaining the parent's written consent, samples were collected from infant diapers every two weeks and stored in 4 ℃ specimen refrigerators at the hospital site until shipment to the laboratory. Upon receipt, fecal samples for analysis of lumen contents were prepared and 200mg/mL of slurry was prepared in a sterile microcentrifuge tube with molecular-scale water. After vortexing and centrifugation, supernatants were collected, aliquoted, and stored at-80 ℃ (fig. 35E).

Fecal samples were continuously collected from diapers discarded by the study subjects after spontaneous voiding. The initial collection of the st louis hospital is one sample per patient, but shifts to once per week per patient. Samples were prospectively collected from the new orleans children hospital and the Touro Infirmary hospital. The frequency of defecation of the recruited infants matched that reported in the literature: for the general pediatric population, the average frequency of defecation was greater than 8 times per week and did not change during the first 2 years of life. S16 according to the record of patient code and date, after the samples were collected by the caregivers, the stools were stored briefly in a refrigerator at 4 ℃ in the hospital specimen until they were transported to the laboratory by a refrigerator. Fecal samples from NEC and non-NEC patients had pH values between 6 and 7 (colorcast pH 0-14 indicator bars, Sigma), consistent with the reported median fecal pH value of 6.64. S17

Work flow of fecal material from the intestinal lumen: without being bound by theory, a particular protein should be identified byThe stimulus-dependent manner switches between insoluble and soluble fractions of the stool, e.g., the release of the luminal vesicle from the small intestine microvilli epithelium into the lumen. Thus, stool preparation is a key factor for accurate quantitative analysis. Feces are a complex matrix: not only are a variety of biological materials present (cells from the gut of the host infant, bacteria, mucins, proteolytic enzymes, etc.), but different types of biochemistry and cellular structure are not expressed in equivalent stoichiometry. For example, proteins most biologically relevant to the host response are unlikely to be identified because of the expected modest representation of host-derived gut proteins in the total fecal proteome, partial proteolysis that occurs during intestinal transit of S18, and large and undefined microbial proteomes.

In addition, previous intestinal proteomic studies S19-S21 identified or recovered much less protein than typical cell or tissue based analyses. To address these challenges, we first normalized our test protocol to the stool weight of a homogeneous patient sample, as it is the most common parameter for assessing stool characteristics. S22-S25 weighed 200mg of fresh feces, which typically contained 75% water, S17 (FIG. 35E). Sterile deionized water containing no protease and DNases water (Sigma Aldrich) was added to make a 200mg/mL (stool weight: volume) slurry, as buffer components significantly affected the results of quantitative immunoblotting and activity assay measurements. S26 then, after rapid vortexing, we applied a centrifugation protocol (fig. 35E) to separate intact free living cells, complexes associated with the cell membrane surface and other large specific substances in the pellet. The S27, S28 supernatants contained proteins secreted in the intestinal lumen, which is the focus of our analysis. Therefore, mass spectrometry confirmed that iAP was found in the supernatant and was easily detected (e table 16). We note that cell lysis conditions have a profound effect on the extracted protein and are not a variable in this study. The supernatant was aliquoted, snap frozen in liquid nitrogen and stored at-80 ℃ until use.

Protein concentration. The total protein concentration in the fecal supernatant was determined by the bradford assay (ThermoFisherScientific). Total protein is used for immunopotentiationWestern blot analysis standardized biochemical activity measurements and protein load to quantify IAP abundance. Protein concentration measurements were reproducible and accurate 32 between replicates and different operators (fig. 39, e table 12).

Biochemical measurements of secreted proteins in the intestinal lumen.Three different protein assays were performed on one supernatant (fig. 35E): determination of total protein, determination of enzyme activity monitoring alkaline phosphatase catalysis and detection of intestinal alkaline phosphatase by immunoblotting. Intestinal alkaline phosphatase is resistant to intestinal degradation by host digestive enzymes S29 and is thermotolerant. S30 quantitative measurements required standard curves for all three assays, which were evaluated repeatedly on each platform daily. Instrument and pipette calibrations were performed every six months by the external supplier.

Protein concentration.The concentration of total protein in the final fecal supernatant was determined by the bradford assay (coomassie plus protein assay reagent, Thermo-Scientific) on a Spectra Max M2e or Spectra Max i3x spectrophotometer (Molecular Devices). Protein standards (bovine serum albumin, Pierce) and patient samples will be prepared using molecular-scale water (Millipore) as a diluent. Generating a five-point standard curve for each day of measurement; r is daily ²The value ≧ 0.994 indicates the linearity of the protein abundance measurement. A second measure of the effectiveness of the analysis is the average error of the ideal value of the standard used.

Fecal IAP catalytic activity. Alkaline phosphatase activity was measured using a 4-methylumbelliferone phosphate (Abcam) substrate in the presence and absence of the IAP inhibitor L-phenylalanine. 33,34 relative fluorescence units at 360/440nm were measured in a multi-well format on a Spectra Max M2e or i3x spectrophotometer (Molecular Devices). Total alkaline phosphatase catalysis and 10mM phenylalanine inhibited alkaline phosphatase catalysis were measured in triplicate and averaged. The reported IAP activity represents the difference between these 2 means. We report IAP activity as hydrolysis of 1. mu. mol 4-methylumbelliferone phosphate per minute per gram of total protein in fecal supernatant at pH 10.0; the individual measurements are in e-table 13, e-table 14 and e-table 15. Alkaline phosphatase activity in intestinal tractReproducible between users and on different days (fig. 39, e table 12).

The activity assay is an enzyme-catalyzed measure as a function of time and as the ratio of protein in the stool supernatant. Alkaline phosphatase activity can be measured using a variety of different substrates. The substrate used determines the dynamic range and sensitivity of the enzymatic reaction. AP activity in this work was measured using 4-methylumbelliferyl phosphate (MUP) as a fluorogenic substrate (Abcam, ab83371) in the presence and absence of the iAP inhibitor L-phenylalanine. The use of MUP has technical advantages for our research. Fluorogenic substrates are capable of measuring the catalytic activity of AP with high sensitivity and accuracy, properties that are ideal for basic research and biotechnological applications. Second, the detection range of fluorescent substrates is typically 100X-1000X greater than that of chromogenic substrates, and product precipitation is detected after reduction of the tetrazolium salt or production of colored diazonium compounds. The Km of the S314-MUP substrate is lower than other natural substrates found in human samples. S32 Vmax was determined independent of pH using 4-MUP. S33, S34, finally, the hydrolysis product does not lead to a strong inhibition of alkaline phosphatase, S32 which would reduce the measurement range and limit the accuracy.

Relative Fluorescence Units (RFU) of 360nm excitation/440 nm emission were measured using a Spectra Max M2e spectrophotometer or Spectra Max i3x (Molecular Devices). A ninety-six hole black optical backplane (ThermoScientific) was used. Standards and negative controls were prepared for each plate run. Stock solutions of 100mM L-phenylalanine (98% purity; Sigma Aldrich) were freshly prepared in molecular-grade water each day of use. The final assay concentration of 10mM Phe was used to assess inhibition of iAP-specific activity.

Denaturing gel electrophoresis and immunoblotting. We determined IAP abundance using affinity-based methods and reported IAP abundance relative to that measured in an equivalent protein-loaded control human small intestine lysate. Prior to immunoblot detection of IAPs, duplicate pre-fabricated denaturing SDS-PAGE gels (ThermoFisher Scientific) were used to visualize proteins; each sample was run at 5. mu.g total protein. To confirm the relative protein abundance of IAP 35-37, 2 loading controls were run on each gel. Positive control is a single human batchSmall intestine lysate (Abcam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma) is our negative control. Immunoblotting was performed using either the traditional method or the iBlot-iBind method (ThermoFisher Scientific). 38-40 the amount of IAP in the clinical samples is reported as the percentage of protein detected in the immunoblots relative to the difference in density measurement pixel counts in the immobilized area (Amersham Imager 600; GE Healthcare) capturing IAP signals in the positive and negative controls. A single batch of anti-human IAP primary antibody (figure 39C) and a single batch of horseradish peroxidase-coupled secondary antibody (Abcam) that did not cross-react with other human alkaline phosphatase or negative control proteins was used for all assays. IAP levels were determined linearly with up to 1 μ g of small intestine lysate (fig. 39D).

The method compares the amount of fecal iAP, which reflects the abundance of protein in the intestinal lumen relative to iAP protein in human intestinal epithelial standards. The supernatant of the fecal sample was mixed with gel loading buffer (375mM Tris pH 6.8, 50% (w/v) glycerol, 600mM dithiothreitol, 420mM sodium dodecyl sulfate) and boiled for 5 minutes. The loading amount of each lane was prepared based on total protein. S35-S37 denaturation 4-12% iBolt Bis-Tris gels (Novex, Life Technologies) were loaded per lane for a total of 5. mu.g total protein. Duplicate gels were run: one was coomassie stained to visualize all proteins in each lane, and the second was used for immunoblotting.

Similar to ELISA measurements, in our immunoblots, more than one reference was used to quantify the relative iAP content in stool samples. This approach mitigates the risk of univariate normalization and has long been recognized in data from microarrays 38 and quantitative PCR. S39 thus, our positive control is human small intestine tissue lysate (Abcam). Purified bovine alkaline phosphatase from intestinal mucosa (Sigma Aldrich) was used as a negative control. The primary antibody used was specific for the human small intestine iAP isoform when evaluated against human small intestine lysate (Abcam; ab29276), purified human placental alkaline phosphatase (ab114268), purified human tissue non-specific alkaline phosphatase (ab114267) and bovine intestinal alkaline phosphatase (Sigma, P5521) (FIG. 39C). We positive controls from a single lot and a single negative control as all quantitative calibrators in the manuscript; both were loaded on each and every gel together with the patient sample. These two criteria are used to define the linear relationship of the anti-iAP signal of our patient samples.

Protein transfer in the gel matrix is performed using one of two techniques: (1) the semi-dry transfer device (Fisher scientific) was at constant 5V for 1 hour or (2) the iBlot2(ThermoFisher) dry track system started at 20V and ended at 25V for a total of 7 minutes. Western blotting techniques were performed using either the traditional method S40-S42 or using the iBind system (ThermoFisher). Membranes were blocked continuously in 5% (w/v) skim milk in 50mM Tris-HCl pH 7.5, 150mM NaCl and 0.1% Tween, or using the iBind solution kit (ThermoFisher) reagent. The membrane was diluted 1:13,000 with a rabbit primary anti-polyclonal antibody against human iAP (Abcam, ab7322), washed, and incubated 1:20,000 with a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Abcam, ab6721) at room temperature.

Bands were quantified on Amersham Imager 600(GE Healthcare); the CCD chip and the large-aperture FujiIon f/0.8543 mm lens can realize higher sensitivity in low-light application. The iAP protein in the positive control lane was defined manually. Equivalent areas were quantified for each lane of the immunoblot (including negative controls and patient samples). The resulting signal for each patient sample is divided by the difference between the positive and negative controls to give the final percentage of the positive control standard. To illustrate the sample preparation, detection protocol and normalization method in our hands, the calibration curve of human intestinal epithelial cell lysate (fig. 39D) (our positive control) shows an overlap between the linear part of the anti-human iAP signal detection and our working range. S43

Accuracy and reproducibility measurements.Although five different operators performed these assays, the duplicate examinations showed clear reproducibility (fig. 39A and 39B), indicating that the biological signals can be distinguished from noise in these assays.

The accuracy and reliability of each biochemical test used to calculate iAP biomarker sensitivity and specificity was evaluated (table e 16). To minimize the effects of batch effects, 44 three different operators randomly selected five independent measurements of known analyte concentrations in each biochemical analysis over an eight month period. Accuracy was assessed by comparing experimental measurements of the analyte with manufacturer-defined absolute measurements and reported as a percentage of absolute values (fig. 39A and 39B). The absolute value of each alkaline phosphatase concentration used for the activity assay was measured on a Tecan Infinite M1000 Pro (personal communications from the supplier; Abcam). The experimental measurement of the analyte was performed on SpectraMax i3x (Molecular Devices) with a photometric range of 0-0.4OD and photometric resolution of 0.001 OD. For the Bradford assay, the extinction coefficient of bovine serum albumin (BSA; 43,824M-1) and the beer's law equation were used to calculate the absolute value of each dilution of BSA for the standard curve measured on SpectraMax i3 x. The accuracy was calculated using the following formula: accuracy ═ [ (absolute-measured value)/absolute ] x 100%. The reliability, or how reproducible the analyte measurement is compared to the absolute value of the analyte, is determined by calculating the standard error and reporting the p-value. Since the p-value indicates whether the measurements deviate significantly from each other, it can be used to indicate whether the operator-measured values of the calibrator are statistically similar (p-value <0.05) or not (p-value > 0.05). For all measurements of patient samples, sample dilutions were made to ensure that experimental measurements fell in the middle of the linear range between the highest and lowest analyte concentrations for the standard curve.

And (4) mass spectrometry.MS1 scans were performed in Fusion tribrid orbitrap (Thermo Fisher Dionex, Sunnyvale, CA) on 0.5 μ g/μ L of treated fecal samples (which were subsequently reduced, alkylated, and trypsinized) with a resolution of 240,000, followed by liquid chromatography on a Dionex U3000 HPLC system (Thermo Fisher Dionex, Sunnyvale, CA). MS2 scans were performed in Orbitrap using a high energy collision dissociation (HCD) setting of 30% and a resolution of 30,000. This process was repeated for a total of three techniques. Data analysis was performed using the protein discover 2.2 using the SEQUEST HT score. The Protein FASTA database is H.sapiens version 2017-07-05. Static modifications include carbamoylmethyl (═ 57.021) on cysteine and dynamic modifications of methionine oxidationDecorations (15.9949). The parent ion tolerance was 10ppm, the fragment mass tolerance was 0.02Da, and the maximum number of missing fragmentation was set to 2. Only high scoring peptides using a False Discovery Rate (FDR) of 1% were considered.

Statistical analysis. The sample size and efficacy calculations used to plan the present study were based on preliminary data obtained from 6 NEC and 12 non-NEC fecal samples of preterm infants. From a preliminary assessment of the magnitude of the effects on IAP abundance and dysfunction, it was determined that at least 12 NEC patients were needed to demonstrate significant differences (i.e., using 5% CI, 2 side, 2 sample t-test, and 95% efficacy). Assuming a two-class outcome with an event rate of 10% (i.e., the percentage of premature infants born < 1.5kg with NEC occurring) and a natural loss rate of 10%, our target recruitment was 130 very low birth weight infants.

The association between inflammatory disease (NEC and parenteral infection), neonatal variables and the course of hospitalization was evaluated (fig. 36 and 37). When a feature or condition is considered to be predictive or concurrent with a disease pattern, the adjusted association is assessed using a logistic regression model that fits the binary disease outcome. If the results are continuous (e.g., correlation of sepsis with number of hospitalizations), then the adjusted correlation is assessed by linear regression; depending on the verification of the normality of the data, analysis of variance, t-test or Kruskal-Wallis and Wilcoxon test were used. For unadjusted comparisons or very small counts, statistical significance is given by χ²Or Fisher's exact test determination. All analyses were done using SAS version 9.4 (SAS Institute).

Each clinical pattern was considered a binary variable for age-appropriate controls. Testing the median differences in IAP activity and abundance between NEC and control groups using the Mann-Whitney U assay; in highlighting the categorical differences, 2-tailed P <0.05 was considered statistically significant. Potential biomarker efficacy was assessed by sensitivity (true positive rate) and specificity (true negative rate) calculations. For each variable of interest, specificity and sensitivity were initially obtained using a simple threshold-based classifier. Recipient operating profile analysis is used to assess the sensitivity and specificity of biomarkers to best differentiate between infant samples with or without disease. The confidence interval was determined using the Wilson-Brown method. These statistical calculations were performed using Prism version 8.1.2 (GraphPad). All numbers were generated in Igor Pro version 8.0 (Wavemetric).

Results

A total of 136 infants (68 [ 50.0% ] male) were recruited with a median birth weight (quartering distance [ IQR ]) of 1050 (790) 1350 g and a median gestational age (IQR) of 28.4(26.0-30.9) weeks. A total of 25 (18.4%) were classified as having severe NEC, 19 (14.0%) were suspected of having NEC, and 92 (66.9%) did not have NEC (i.e., control) (fig. 35D). Of the infants with severe NEC, 19 (76.0%) occurred between 26 and 35 weeks post-conception (PCA), 6 (24.0%) occurred between 36 and 40 or more weeks of PCA. For infants classified as suspected NEC, 16 cases (84.2%) occurred between 26 and 30 weeks of PCA, and 3 cases (15.8%) occurred between 31 and 35 weeks of PCA. Study participants had other forms of definitive infection in addition to NEC; 26 people (19.1%) were diagnosed with LOS, 14 people (10.3%) had a parenteral infection (fig. 35D). Equal numbers of boys and girls were recruited.

The attrition rate was 11.0% (i.e., 15 infants) due to recruitment changes, medical changes, or inadequate biological sample collection (fig. 35D). A total of 6 (4.4%) patients were excluded due to withdrawal of parental consent or death (NEC-independent lung or multi-organ failure) prior to sample collection. During a suspected or severe NEC episode, a total of 9 (6.6%) recruits were culled for SIP diagnosis, insufficient stool collection, or no stool collection. The number of remaining recruits is 121.

Demographic data and clinical history were reviewed after fecal analysis (fig. 35E). We compiled 5400 demographic and clinical course characteristics (fig. 36 and 37). Potential confounding variables for the disease were cross tabulated. Post-conception age and weight are the only pre-event clinical variables associated with NEC (fig. 36) supporting postpartum disease development as a consistent risk factor (median [ IQR ] PCA at the first NEC episode: severe NEC, 33.9[31.0-35.7] weeks; suspected NEC, 29.4[28.4-30.9] weeks; P ═ 02; median [ IQR ] weight at the first NEC episode: severe NEC, 1620[ 1110-. 18 in comparison, birth weight and gestational age were closely related to the risk of LOS (median [ IQR ] birth weight: LOS, 790[670 ] g; other parenteral infection, 830[700 ] 915 g; no other parenteral infection, 1165[912.5-1410] g; P <. 001; median [ IQR ] gestational age at birth: LOS, 25.9[25.0-29.7] weeks; other parenteral infection, 26.4[25.0-27.1] weeks; no other parenteral infection, 29.3[26.9-32.2] weeks; P <.001) (Table 2). 14

Abundance of IAP proteins and IAP enzyme activity in severe NEC, suspected NEC and NEC-free patients. At the time of clinical diagnosis, infants with NEC had high relative IAP content in their fecal samples (fig. 38A). The median (IQR) IAP content of the samples collected at severe NEC was 99.0% (51.0% -187.8%) (95% CI, 54.0% -163.0%), while the median (IQR) IAP content of the control samples was 4.8% (2.4% -9.8%) (95% CI, 3.4% -5.9%). Increased fecal IAP protein is associated not only with severe NEC, but also with suspected disease. Fecal samples collected at the time NEC was suspected to have a median (IQR) IAP content of 123.0% (31.0% -224.0%) (95% CI, 31.0% -224.0%) (fig. 38A).

Median IAP abundance in feces increases 20-fold at severe NEC and suspected NEC compared to feces collected from age-matched controls without NEC.

IAP activity was significantly reduced in samples collected during suspected and severe NEC episodes compared to samples from infants without NEC (figure 38A). However, IAP enzyme dysfunction was found to varying degrees between suspected and severe NEC patients. Samples at severe NEC had median (IQR) IAP activity of 183(56-507) μmol/min/g (95% CI,63-478 μmol/min/g) fecal protein. The median (IQR) IAP activity in the sample suspected of NEC was 355(172-608) μmol/min/g (95% CI,172-608 μmol/min/g) fecal protein, and the IAP activity in the PCA-matched control sample had a median (IQR) of 613(210-1465) μmol/min/g (95% CI,386-723 μmol/min/g) fecal protein. Thus, infants with severe NEC have only a fourth of their capacity to regulate abnormal bacterial colonization compared to infants suspected or without NEC, indicating dysfunction in host-microbe crosstalk.

Sensitivity, specificity and positive predictive value of fecal IAP measurements. The recipient working characteristic curve, which is a common tool for calculating clinical prediction rules, was used to assess the accuracy of IAP single biochemical measurements or the area under the curve (fig. 38B). The mean (SE) accuracy using IAP content as a severe NEC marker was 0.97(0.02) (95% CI, 0.93-1.00; P <.001) and the mean (SE) accuracy using IAP activity as a severe NEC marker was 0.76(0.06) (95% CI, 0.64-0.86; P <. 001). For suspected NECs, similar mean (SE) accuracy values of IAP levels of 0.97(0.02) (95% CI, 0.93-1.00; P <.001) and IAP activity of 0.62(0.07) (95% CI, 0.48-0.77; P ═ 13) were obtained.

In contrast, IAP content and activity lack accuracy in the diagnosis of sepsis and other parenteral infections (fig. 38C). IAP dropping in feces collected in the following cases was negligible: clinically defined sepsis (median [ IQR ], 6.5% [ 2.2% -23.1% ]; 95% CI, 2.2% -19.8%), other parenteral infections (median [ IQR ], 3.1% [ 0.8% -10.9% ]; 95% CI, 0.6% -15.2%) and controls (median [ IQR ], 6.2% [ 2.7% -40.0% ]; 95% CI, 4.6% -11.0%). The enzymatic capacity of IAP was not statistically different between the samples collected from these 3 cohorts (fig. 38C); the meso (IQR) activity of sepsis is 575 (338-. The area under the receiver operating characteristic curve shows that using fecal IAP content or activity randomly designates culture confirmed bacterial sepsis and other parenteral infections as positive or negative for these inflammatory conditions (fig. 38D). The mean (SE) accuracy score for IAP content was 0.52(0.07) at sepsis (95% CI, 0.38-0.66; P ═ 75) and 0.58(0.08) at other parenteral infections (95% CI, 0.42-0.75; P ═ 06). The mean (SE) accuracy score for IAP activity was 0.52(0.07) at sepsis (95% CI, 0.39-0.67; P ═ 68) and 0.57(0.08) at other parenteral infections (95% CI, 0.39-0.69; P ═ 66).

Neonatal necrotizing enterocolitis and LOS exaggerate the inflammatory response and many of the common attributes. Differential diagnosis is complicated by its overlapping manifestations, diagnostic tools with limited sensitivity, and even its evolving definition. 42,43 the current standard is abdominal radiation tablets for NEC and positive blood cultures for sepsis. However, both of these standards suffer from low sensitivity and may cause injury due to excessive radiation exposure or blood sampling. Finally, the results report problematic: the interpretation of subtle radiological findings is subjective and may vary, and culture results may take as long as 48 to 72 hours.

There have been many attempts to identify candidate markers of intestinal injury that distinguish NEC from other inflammatory conditions. The 44-48 animal NEC model indicates that the immune and microbial dysregulation associated with severe NEC is a tandem host-bacterial error due to excessive Toll-like receptor 4 signaling in response to bacterial LPS. 19,49-52 most candidate NEC biomarkers are proteins further downstream of the initial host signaling step. Elevation of platelet activating factor 3,53, meta-alpha arrestin 54, calprotectin, claudin 48, intestinal fatty acid binding protein 55 and C-reactive protein 56 in plasma is associated with the onset of NEC. In summary, the current literature indicates that diagnosis of advanced NEC is a clinical description of advanced pathological processes 29,57, suggesting that NEC biomarkers may always be confounded by sepsis.

This study challenges these theories. Biomarkers, such as calprotectin, are often a reliable indicator of intestinal inflammation, but the major inflammatory pathway that plays a role in the intestinal mucosa of patients is not understood. Our study required prospective inclusion of infants with NEC, and simultaneous testing of healthy and unhealthy controls with multiple inflammations in neonatal intensive care units. Under these realistic conditions, the estimation of biomarker reliability more accurately reflects the potential performance in clinical applications. Examination of proteins involved in organ-specific regulation of microbiota homeostasis and response distinguishes NEC from other forms of inflammation. Thus, IAP is the first candidate diagnostic biomarker, unique in its high predictive value of positivity to NEC. Importantly, IAP is associated with NEC, but not sepsis or other non-GI infections.

The use of LPS-induced proteins of proinflammatory origin as biomarkers is supported by previous studies. There are several IAP-activated models in intestinal dysbiosis: exosomes, increased intestinal permeability, and/or damage to the intestinal epithelium. It is not clear whether bacterial translocation across the gut epithelium, which can cause LOS, is a natural consequence of altered gut epithelial permeability or a consequence of a worsening of the gut barrier. Our IAP studies do not address whether gut endothelium is deteriorating in NEC or sepsis. However, the detection of such high abundance of IAP in our fecal samples during the onset of NEC suggests that there is positive regulation of lipid vesicle secretion into the intestinal lumen during active NEC disease; during LOS, no such IAP secretion was detected in the faeces. This survey does not support the view that NEC has the same pathobiological mechanisms as neonatal sepsis.

IAP biomarkers are associated with disease severity; IAP biochemistry can distinguish advanced NEC (marked by portal or intestinal pneumatosis) from suspected disease where there are no reliably observable signs by radiology. Our results also show that this classification of NEC suspicion is supported as a clear disease state. Our approach differs from other candidate biomarker studies. This work differs not only in the target protein of interest, but also in the disease severity category, biological sample selection and molecular detection methods we used. We were able to separate NEC suspicion from NEC severe cases. Great efforts have been made to determine the commonalities of clinical criteria defining severe NEC. Due to the lack of molecular diagnostic tests and defined consensus, there are few reports on NEC suspicion. The study showed that suspected and severe NEC was associated with active release of IAP in infant faeces. It also indicates that there are significant differences in IAP function for these 2 disease categories. Late NECs are associated with severe biochemical dysfunction of host IAPs, whereas NECs are suspected to only partially lose IAP enzymatic activity. In contrast, C-reactive protein and other biomarkers were not associated with Bell staging, 11, and importantly, these values did not differ significantly between suspected NEC and severe NEC.

The results discussed in this example are different from other studies evaluating IAP as a biomarker for NEC. Our study report uses not 1 but 2 measures to assess IAP biochemistry in patient samples as follows: (1) immunoblotting to quantify its relative abundance compared to the amount of IAP found in the human small intestine, and (2) enzymatic activity to determine if the protein is functional and capable of modulating microbial dysregulation. Both of these methods are necessary to differentiate between disease pathways and individual differences. Serological assay 58 of alkaline phosphatase as a biomarker for NEC reports an increased amount of IAP in blood of NEC infants compared to the control group, indicating that IAP may play a role in the pathogenesis of NEC. Serum is not an ideal source of sampling because there are 4 different alkaline phosphatases and their relative levels in serum are known to change 59 during pregnancy (fig. 39 and 40). Although the previously drawn conclusions 58 support our findings, the use of denatured protein gels alone does not provide equivalent evidence that IAPs have been identified, nor does it quantify the amount of alkaline phosphatase overall.

Taken together, the results of this study indicate that measurement of IAP dysfunction in feces is a biomarker for NEC with superior sensitivity and specificity to other candidates reported in the literature previously. While promising, fecal IAP should be considered as a biomarker for determining the diagnosis of severe NEC, monitoring disease progression and monitoring high risk infant populations. Proper design and analysis of future biomarker studies requires normative data for different PCAs to determine whether fecal IAPs can serve as diagnostic agents at the molecular level. The clinical potential of this non-invasive tool is that it can identify the infants most likely to be at risk of developing NEC, facilitate management of feeding and antibiotic regimens, and monitor response to treatment.

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e Table 1. diagnostic and suspected criteria for classifying necrotizing enterocolitis of newborn

The study classification derives from the minimum common requirement for disease severity definition. 1, 2 meet the study criteria ranking is (1) radiological signs, (2) abdominal signs, (3) clinical findings, and (4) laboratory findings; for each patient classified as NEC, one radiological sign and one criterion from the other group were identified.

e Table 2 study criteria for focal or spontaneous bowel perforation

The ranking of the study criteria met were (1) radiologic signs, (2) abdominal signs, (3) clinical findings, and (4) laboratory findings

Table 3. study criteria for defining pathogenic infections outside the gastrointestinal tract

The classification studies followed the infection definition of the U.S. centers for disease control/national health safety network (CDC/NHSN). The study criteria met are ranked as (1) laboratory findings and (2) clinical findings. Four types of infection were excluded and not reported in this study: (i) early onset sepsis (72 hours gestational age) because antibiotics are administered prophylactically to infants at birth in all three hospital centers; (ii) an accessory bloodstream infection identified as another site of infection; (iii) infections associated with the use of central lines; and (iv) laboratory detection of blastomyces, histoplasmosis, coccidiodes, paracoccus, cryptococcus, and pneumocystis, which are commonly responsible for community-related infections and rarely known to cause health-related infections.

e Table 4. summary of NEC groups at different clinical points

The number of recruited infants clinically rated as suspected or severe NEC is provided at the hospital site. The group values shown are the total number of subjects enrolled per point, grouped by Gestational Age (GA) box and the number of cases requiring surgical intervention (ascites drainage or laparotomy). Italics and percentages in parentheses relate to the number of cases by group and gestational age at birth divided by all subjects. NEC cases and suspected numbers were stratified by GA at < 27 weeks or > 27 weeks of birth. The median and accompanying quarterward values in italics and parentheses were used to identify the post-gestational age and days and gestational age at birth for the group. The number of deaths associated with necrotizing enterocolitis is provided. Abbreviations: surg inter, surgical intervention; cH, new orleans children hospital, louisiana; TI, Touro Infirmary, New Orleans, Louisiana; WU, university of bosch louisis washington.

Record and sample removal from study of recruits after transfer of infant care to national care

Two infants were discontinued from study on parental request at week 32 PCA: data after 32 weeks of PCA (disease, surgery, death, etc.) were not included. None of the infants had NEC, sepsis, confirmed non-GI site infection or sIP.

e Table 5.25 Radioactive-confirmed List of (severe) cases of necrotizing enterocolitis enrolled

Criteria defining severe NEc are shown in table el; the main criteria were confirmed by radiology evidence. Parental/ethnic self-identifying abbreviations: b-african american/black; w is white; h-hispanic. Other abbreviations: GA ═ gestational age; m ═ male; f ═ female; c, caesarean section; natural production of V ═

No sample was obtained at NEc time. All samples from infants were excluded from cross-sectional analysis if no samples were obtained during the radiologic and clinically defined NEC

Study subjects excluded due to gestational age > 37 weeks at enrollment

e Table 6. list of 19 suspected necrotizing enterocolitis cases enrolled

Criteria defining suspected NECs are shown in table el; these cases cannot be confirmed by radiologic evidence. The Bell phase recorded in the medical record by the participating oncologist is shown, which is different from our handwriting standard. Parental/ethnic self-identifying abbreviations: b-african american/black; w is white; h-hispanic. Other abbreviations: GA ═ gestational age; m ═ male; f ═ female; c ═ caesarean delivery; natural production of V ═

No sample was obtained at NEC time. All samples from infants were excluded from cross-sectional analysis if no samples were obtained during the radiology and clinical defined NEc

Multiple assessments abdominal radiology and abdominal ultrasound evidence without intestinal, portal or abdominal pneumatosis

e Table 7. list of 3 enrolled infants with Spontaneous Intestinal Perforation (SIP) and necrotizing enterocolitis

Parental/ethnic self-identifying abbreviations: b-african american/black; w is white; h-hispanic. Other abbreviations: GA-gestational age at birth; m ═ male; f ═ female; c ═ caesarean delivery; v is produced naturally; NA is not applicable

Table 8 list of 86 recruited infants neither clinically diagnosed nor suspected of necrotizing enterocolitis

Parental/ethnic self-identifying abbreviations: b-african american/black; w is white; h-hispanic. Other abbreviations: GA ═ gestational age; m ═ male; f ═ female; c ═ caesarean delivery; natural production of V ═

e table 8 (continuation) list of 86 recruited infants that were neither clinically diagnosed nor suspected of having necrotizing enterocolitis

TABLE 9 summary of sepsis and other groups of parenteral infections at different clinical sites

The number of recruited infants clinically rated as having sepsis or other parenteral infection is provided at the hospital site. The group values shown at 3, 4 are the total number of subjects enrolled for each point, grouped by Gestational Age (GA) box. Italics and percentages in parentheses relate to the number of cases by group and gestational age at birth divided by all subjects. The median and accompanying quarter-point values in italics and parentheses were used to identify post-pregnancy age, days, and birth weight. Abbreviations: GA, gestational age at birth; CH, new orleans children hospital, louisiana; TI, Touro Infirmary, New Orleans, Louisiana; WU, university of bosch louisis washington.

e Table 10. List of all 26 cases of late onset neonatal sepsis recruited

Sepsis events were closest in time to NEC onset or after study inclusion. Abbreviations: GA-gestational age at birth; m ═ male; f ═ female; b-african american/black; w is white; h-hispanic; c ═ caesarean delivery; v is produced naturally; NA is not applicable

No samples were obtained during sepsis. All samples from infants were excluded from sepsis cross-section analysis if no samples were obtained during the course of the disease

e table 11. list of all 14 cases of confirmed parenteral infections in urine, solid or trachea

Abbreviations: GA-gestational age at birth; PCA-post gestational age; m ═ male; f ═ female; c ═ caesarean delivery; v is produced naturally; t is trachea; b ═ bone; u is urine; skin (S ═ skin)

No samples were obtained during parenteral infection. If no samples were obtained during infection, all samples from the infant were excluded from the sepsis cross-section analysis

e table 12 accuracy and repeatability of in vitro measurements of intestinal lumen contents

The reference standard for total protein concentration is bovine serum albumin; the expected Absorbance (ABS) calibration standard was determined using the extinction coefficient of bovine serum albumin (43,824M-1cm-1) and beer's law. The reference standard for biochemical activity is 4-methylumbelliferyl phosphate; the kit manufacturer provides the expected relative fluorescence units. Median experimental measurements, reproducibility and accuracy for each analyte are shown. A P value of 0.05 or more indicates no significant difference between the measurements. Abbreviations: ABS ═ absorbance; RFU ═ relative fluorescence units; SE is the standard error; SI ═ small intestine; ND is not determined

e Table 13 IAP measurements on 20 stool samples at severe necrotizing enterocolitis

e Table 14 IAP measurements of 15 fecal samples at suspected necrotizing enterocolitis

e table 15 IAP measurements of 86 enrolled infants that were neither clinically diagnosed with nor suspected of necrotizing enterocolitis

e table 15 (continue) IAP measurements of 86 recruited infants that were neither clinically diagnosed with nor suspected of necrotizing enterocolitis

e table 16. proteins identified in the intestinal lumen of preterm infants (N ═ 635)

Fecal samples were analyzed by protein profiling after collection from non-NEC infants 32.57 weeks of gestational age and 1000g in weight. Proteins were ranked by sumep score. Descriptions of proteins with MS supep scores of 1400 to 96 and Uniprot ID are shown.