US 2012O315.630A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0315630 A1 Gong et al. (43) Pub. Date: Dec. 13, 2012
(54) METHODS FOR DIAGNOSINGIRRITABLE (60) Provisional application No. 61/256,717, filed on Oct. BOWEL, SYNDROME 30, 2009, provisional application No. 61/264,588, filed on Nov. 25, 2009, provisional application No. (75) Inventors: Hua Gong, San Diego, CA (US); 61/326,176, filed on Apr. 20, 2010. Shui Long Wang, San Diego, CA O O (US); Sharat Singh, Rancho Santa Publication Classification Fe, CA (US) (51) Int. Cl. GOIN 33/566 (2006.01) (73) Assignee: Prometheus Laboratories Inc., CI2O I/68 (2006.01) San Diego, CA (US) GOIN 33/573 (2006.01) (52) U.S. Cl...... 435/6.11: 436/501; 435/7.4; 435/7.92; (21) Appl. No.: 13/441,658 435/6.12 (57) ABSTRACT (22) Filed: Apr. 6, 2012 The invention provides novel biomarkers, kits, and methods O O of diagnosing, prognosing, and Subtyping IBS. Also provided Related U.S. Application Data are methods for aiding in the diagnosis of irritable bowel (63) Continuation of application No. PCT/US 10/54810, syndrome by detecting the serum level of novel IBS biomar filed on Oct. 29, 2010. kers identified herein. Patent Application Publication Dec. 13, 2012 Sheet 1 of 31 US 2012/0315630 A1
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METHODS FOR DIAGNOSING RRITABLE visceral pain perception, known as peripheral sensitization. BOWEL, SYNDROME This sensitization involves a reduction in the threshold and an increase in the gain of the transduction processes of primary CROSS-REFERENCES TO RELATED afferent neurons, attributable to a variety of mediators includ APPLICATIONS ing monoamines (e.g., catecholamines and indoleamines), Substance P, and a variety of cytokines and prostanoids Such 0001. This application is a continuation of PCT/US2010/ as E-type prostaglandins (see, e.g., Mayer et al., Gastroen 054810 filed Oct. 29, 2010, which application claims priority terol., 107:271-293 (1994)). Also implicated in the etiopa to U.S. Provisional Application No. 61/256,717, filed Oct. 30, thology of IBS is intestinal motor dysfunction, which leads to 2009, U.S. Provisional Application No. 61/264,588, filed abnormal handling of intraluminal contents and/or gas (see, Nov. 25, 2009, and U.S. Provisional Application No. 61/326, e.g., Kellow et al., Gastroenterol., 92: 1885-1893 (1987); 176, filed Apr. 20, 2010, the disclosures of which are herein Levitt et al., Ann. Int. Med., 124:422-424 (1996)). Psycho incorporated by reference in their entireties for all purposes. logical factors may also contribute to IBS symptoms appear The following documents are also incorporated by reference: ing in conjunction with, if not triggered by, disturbances U.S. Patent Publication Nos. 2008/0085524, filed Aug. 14, including depression and anxiety (see, e.g., Drossman et al., 2007 and published Apr. 10, 2008; 2008/0166719, filed Aug. Gastroenterol. Int., 8:47-90 (1995)). 20, 2007, published Jul. 10, 2008. 0006. The causes of IBS are not well understood. The walls of the intestines are lined with layers of muscle that BACKGROUND OF THE INVENTION contract and relax as they move food from the stomach 0002 Irritable bowel syndrome (IBS) is the most common through the intestinal tract to the rectum. Normally, these of all gastrointestinal disorders, affecting 10-20% of the gen muscles contract and relax in a coordinated rhythm. In IBS eral population and accounting for more than 50% of all patients, these contractions are typically stronger and last patients with digestive complaints. However, studies suggest longer than normal. As a result, food is forced through the that only about 10% to 50% of those afflicted with IBS actu intestines more quickly in some cases causing gas, bloating, ally seek medical attention. Patients with IBS present with and diarrhea. In other cases, the opposite occurs: food pas disparate symptoms such as, for example, abdominal pain sage slows and stools become hard and dry causing constipa predominantly related to defecation, diarrhea, constipation or tion. alternating diarrhea and constipation, abdominal distention, 0007. The precise pathophysiology of IBS remains to be gas, and excessive mucus in the stool. More than 40% of IBS elucidated. While gut dysmotility and altered visceral percep patients have symptoms so severe that they have to take time tion are considered important contributors to symptom patho off from work, curtail their social life, avoid sexual inter genesis (Quigley, Scand. J. Gastroenterol. 38(Suppl. 237): course, cancel appointments, stop traveling, take medication, 1-8 (2003); Mayer et al., Gastroenterol., 122:2032-2048 and even stay confined to their house for fear of embarrass (2002)), this condition is now generally viewed as a disorder ment. The estimated health care cost of IBS in the United of the brain-gut axis. Recently, roles for enteric infection and States is S8 billion per year (Talley et al., Gastroenterol., intestinal inflammation have also been proposed. Studies 109:1736-1741 (1995)). have documented the onset of IBS following bacteriologi 0003 IBS patients are classified into three groups accord cally confirmed gastroenteritis, while others have provided ing to their predominant bowel symptoms: constipation-pre evidence of low-grade mucosal inflammation (Spiller et al., dominant IBS (IBS-C), diarrhea-predominant IBS (IBS-D) Gut, 47:804-811 (2000); Dunlop et al., Gastroenterol. 125: and IBS with alternating symptoms of diarrhea and constipa 1651-1659 (2003); Cumberland et al., Epidemiol. Infect. tion (IBS-M). In current clinical practice, diagnosis of IBS is 130:453-460 (2003)) and immune activation (Gwee et al., based on the Rome III criteria and according to the symptoms Gut, 52:523-526 (2003); Pimentel et al., Am. J. Gastroen presented by the patients plus the exclusion of other GI dis terol.95:3503-3506 (2000)) in IBS. The enteric flora has also orders. There are no specific biological, radiographic, endo been implicated, and a recent study demonstrated the efficacy scopic or physiological biomarkers that can be used to iden of the probiotic organism Bifidobacterium in treating the tify this disorder. disorder through modulation of immune activity (O'Mahony 0004 Irritable bowel syndrome is a heterogeneous gas et al., Gastroenterol., 128:541-551 (2005)). trointestinal (GI) function disorder. There is increasing evi 0008. The hypothalamic-pituitary-adrenal axis (HPA) is dence pointing to the involvement of the immune system in its the core endocrine stress system in humans (De Wied et al., pathogenesis. GI infection may be a triggering factor for Front. Neuroendocrinol., 14:251-302 (1993)) and provides causing the onset of IBS symptoms. On the other hand, IBS is an important link between the brain and the gut immune often described as a “brain-gut disorder. Alterations in GI system. Activation of the axis takes place in response to both motility and secretion mediated by dysregulation of the 5-HT physical and psychological stressors (Dinan, Br. J. Psychia signaling pathway may underlie the irregularities in bowel try, 164:365-371 (1994)), both of which have been implicated habits. Activation of mast cells in proximity to colonic nerves in the pathophysiology of IBS (Cumberland et al., Epidemiol. correlated with the abnormal pain experienced by patients Infect. 130:453-460 (2003)). Patients with IBS have been with IBS. Mast cells are well known to be capable of produc reported as having an increased rate of sexual and physical ing and releasing a variety of inflammatory mediators upon abuse in childhood together with higher rates of stressful life activation. However, it is not clear how these different path events in adulthood (Gaynes et al., Baillieres Clin. Gastroen ways communicate with each other and whether their inter terol. 13:437-452 (1999)). Such psychosocial trauma or poor actions behave in the same manner in IBS patients as it is in cognitive coping strategy profoundly affects symptom sever healthy subjects. ity, daily functioning, and health outcome. 0005. The precise pathophysiology of IBS is not well 0009. Although the etiology of IBS is not fully character understood. Nevertheless, there is a heightened sensitivity to ized, the medical community has developed a consensus defi US 2012/03 15630 A1 Dec. 13, 2012
nition and criteria, known as the Rome II criteria, to aid in the table bowel syndrome (IBS) in a subject, the method com diagnosis of IBS based upon patient history. The Rome II prising: (a) contacting a first blood or serum sample taken criteria requires three months of continuous or recurrent from the subject at a first time with an IBS marker binding abdominal pain or discomfort over a one-year period that is moiety under conditions suitable to transform the IBS marker relieved by defecation and/or associated with a change in present in the sample into a complex comprising the IBS stool frequency or consistency as well as two or more of the marker and the IBS marker binding moiety; (b) determining following: altered stool frequency, altered stool form, altered the level of the complex, thereby determining the level of the stool passage, passage of mucus, or bloating and abdominal IBS marker present in the first sample; (c) contacting a second distention. The absence of any structural or biochemical dis blood or serum sample taken from the Subjectata second time orders that could be causing the symptoms is also a necessary with an IBS marker binding moiety under conditions suitable condition. As a result, the Rome II criteria can be used only to transform the IBS marker present in the sample into a when there is a substantial patient history and is reliable only complex comprising the IBS marker and the IBS marker when there is no abnormal intestinal anatomy or metabolic binding moiety; (d) determining the level of the complex, process that would otherwise explain the symptoms. Simi thereby determining the level of the IBS marker present in the larly, the Rome III criteria recently developed by the medical second sample; and (e) comparing the level of the IBS marker community can be used only when there is presentation of a present in the first sample to the level of the IBS marker specific set of symptoms, a detailed patient history, and a present in the second sample, wherein a higher level of the physical examination. IBS marker in the second sample relative to the first sample is 0010. It is well documented that diagnosing a patient as indicative of the progression of IBS in the subject and a lower having IBS can be challenging due to the similarity in Symp level of the IBS marker in the second sample relative to the toms between IBS and other diseases or disorders. In fact, first sample is indicative of the regression of IBS in the sub because the symptoms of IBS are similar or identical to the ject. symptoms of so many other intestinal illnesses, it can take 0014. In one aspect, the present invention provides a com years before a correct diagnosis is made. For example, puter-readable medium comprising code for controlling one patients who have inflammatory bowel disease (IBD), but or more processors to classify whether a serum or blood who exhibit mild signs and symptoms Such as bloating, diar sample from an subject is associated with irritable bowel rhea, constipation, and abdominal pain, may be difficult to syndrome (IBS), the code comprising instructions to apply a distinguish from patients with IBS. As a result, the similarity statistical process to a data set comprising a diagnostic marker in symptoms between IBS and IBD renders rapid and accu profile to produce a statistically derived decision classifying rate diagnosis difficult. The difficulty in differentially diag the sample as an IBS sample or non-IBS sample based upon nosing IBS and IBD hampers early and effective treatment of the diagnostic marker profile, wherein the diagnostic marker these diseases. Unfortunately, rapid and accurate diagnostic profile indicates the level of at least IBS diagnostic marker methods for definitively distinguishing IBS from other intes provided herein. tinal diseases or disorders presenting with similar symptoms 0015. In another aspect, the present invention provides a are currently not available. The present invention satisfies this system for classifying whether a serum or blood sample from need and provides related advantages as well. a subject is associated with irritable bowel syndrome (IBS), the system comprising: (a) a data acquisition module config BRIEF SUMMARY OF THE INVENTION ured to produce a data set comprising a diagnostic marker 0011. The present invention provides methods, assays, profile, wherein the diagnostic marker profile indicates the systems, and code for accurately classifying whether a presence or level of at least one IBS diagnostic marker pro sample from an individual is associated with irritable bowel vided herein; (b) a data processing module configured to syndrome (IBS). As a non-limiting example, the present process the data set by applying a statistical process to the invention is useful for classifying a sample from an individual data set to produce a statistically derived decision classifying as an IBS sample using a statistical algorithm and/or empiri the sample as an IBS sample or non-IBS sample based upon cal data. Similarly, the present invention also provides meth the diagnostic marker profile; and (c) a display module con ods, assays, systems, and code for aiding in the diagnosis of figured to display the statistically derived decision. irritable bowel syndrome (IBS) in a subject. The present 0016. In one aspect, the present invention provides a invention is also useful for ruling out one or more diseases or method for classifying whethera sample from an individual is disorders that present with IBS-like symptoms and ruling in associated with IBS, the method comprising: (a) determining IBS using a combination of Statistical algorithms and/or a diagnostic marker profile by detecting the presence or level empirical data. Thus, the present invention provides an accu of at least one diagnostic marker in the sample; and (b) clas rate diagnostic prediction of IBS and prognostic information Sifying the sample as an IBS sample or non-IBS sample using useful for guiding treatment decisions. an algorithm based upon the diagnostic marker profile. 0012. In one aspect, the present invention provides a 0017. In certain embodiments of the diagnostic methods method for aiding in the diagnosis of irritable bowel syn provided herein, the IBS marker is selected from the group drome (IBS) in a subject, the method comprising: (a) contact consisting of a cytokine, growth factor, anti-neutrophil anti ing a blood or serum sample from the subject with an IBS body, anti-Saccharomyces cerevisiae antibody (ASCA), anti marker binding moiety under conditions suitable to transform microbial antibody, mast cell marker, stress marker, gas the IBS marker present in the sample into a complex com trointestinal hormone, serotonin metabolite, serotonin prising the IBS marker and the IBS marker binding moiety; pathway marker, carbohydrate deficient transferrin (CDT), and (b) determining the level of the complex, thereby deter lactoferrin, anti-tissue transglutaminase (tTG) antibody, mining the level of the IBS marker present in the sample. lipocalin, matrix metalloproteinase (MMP), tissue inhibitor 0013. In one aspect, the present invention provides a of metalloproteinase (TIMP), alpha-globulin, actin-severing method for monitoring the progression or regression of irri protein, S100 protein, fibrinopeptide, calcitonin gene-related US 2012/03 15630 A1 Dec. 13, 2012 peptide (CGRP), tachykinin, ghrelin, neurotensin, corticotro pin-releasing hormone, serine protease, prostaglandin, hista TABLE 1-continued mine, and combinations thereof. Exemplary diagnostic markers Suitable 0018. In preferred embodiments of the diagnostic methods for use in IBS classification. provided herein, the IBS marker is selected from the group consisting of carbohydrate deficient transferrin, urocortin, Family Biomarker corticotrophin-releasing hormone-binding protein, cortisol, Anti-tissue transglutaminase (tTG) antibody adrenocorticotropic hormone, Substance P. nerve growth fac Carbohydrate Deficient Transferrin (CDT) tor, neurokinin A, neurokinin B. vasoactive intestinal peptide, Stress Markers Urocortin (Ucn) glucagon-like peptide 2, motilin, pituitary adenylate cyclase Corticotropin-releasing hormone-binding protein (CRFBP) activating peptide, serotonin, tryptophan, serotonin O-sul Cortisol fate, 5-hydroxyindoleacetic acid, 5-HT glucuronide, Adrenocorticotropic hormone (ACTH, tyrosine, phenylalanine, UDP-glucuronosyltransferase 1-6. corticotrophin) serotonin reuptake transporter, tryptophan hydroxylase 1, Mast Cell Tryptase Markers Histamine monoamine oxidase A, monoamine oxidase B, and hydrox Prostaglandin E2 (PGE2) ytryptamine (serotonin) receptor 3A, and combinations Gastrointestinal Calcitonin gene-related peptide (CGRP) thereof. hormones Substance P Nerve Growth Factor (NGF) 0019. In preferred embodiments of the various methods Neurokinin A and assays of the present invention, the presence or level of 1. Neurokinin B 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Vasoactive Intestinal Peptide (VIP) Glucagon-Like Peptide 2 (GLP-2) 21, 22, 23, 24, 25, or more of the biomarkers shown in Table Motilin 1 is detected to generate a diagnostic marker profile that is Pituitary Adenylate Cyclase-Activating Peptide (PACAP) useful for predicting IBS. In certain instances, the biomarkers Serotonin Serotonin described herein are analyzed using an immunoassay Such as Metabolites Tryptophan an enzyme-linked immunosorbent assay (ELISA) or an Serotonin O-sulfate (5-Hydroxytryptamine O-sulfate; 5-HT-O-sulfate) immunohistochemical assay. 5-Hydroxyindoleacetic acid (5-HIAA) 5-HT glucuronide (5-HT-GA) TABLE 1. Serum Tyrosine Metabolites Phenylalanine Exemplary diagnostic markers Suitable Serotonin UDP-glucuronosyltransferase 1-6 (UGT1A6) for use in IBS classification. Pathway serotonin reuptake transporter (SERT) Markers Tryptophan hydroxylase 1 (TPH1) Family Biomarker Monoamine oxidase A (MAO-A) Monoamine oxidase B (MAO-B) Cytokine CXCL8 IL-8 Hydroxytryptamine (serotonin) receptor 3A IL-1B (5-HT3A, 5-HT3R) TNF-related weak inducer of apoptosis (TWEAK) Leptin Osteoprotegerin (OPG) BRIEF DESCRIPTION OF THE DRAWINGS CXCL1 GRO1 GROC CXCL4, PF-4 0020 FIG. 1 illustrates a dose response curve in one embodiment of the present invention. INFB2/IL-6 IL-12 0021 FIG. 2 illustrates a dose response curve in one CSIFIL-10 embodiment of the present invention. Tumor necrosis factor-alpha (TNF-C.) 0022 FIG. 3 illustrates a standard curve in one embodi Growth Factor Epidermal growth factor (EGF) ment of the present invention. Rabbit anti-human tryptase Vascular endothelial growth factor (VEGF) Pigment epithelium-derived factor (PEDF) was coated onto an immunoassay plate and blocked with Brain-derived neurotrophic factor (BDNF) assay buffer (5% BSA in PBS). Different concentrations of Schwaninoma-derived growth factor (SDGF). human tryptase (Standard curve) or human serum samples amphiregulin Anti-neutrophil Anti-neutrophil cytoplasmic antibody (ANCA) (diluted in assay buffer 1:5 and 1:10) were added to the wells antibody Perinuclear anti-neutrophil cytoplasmic antibody and incubated for 2 hours at RT. The plate was washed and (pANCA) alkaline phosphatase (AP) labeled McAb against tryptase Anti-I2 antibody (G3) were added and incubated for 2 hours at RT. The plate ASCA ASCA-IgA ASCA-IgG was washed and AP substrate (CSPD) was added to each well. Antimicrobial Anti-outer membrane protein C (OmpC) antibody Aluminescence plate reader was used to detect the lumines antibody Anti-Cbir-1 flagellin antibody cent light. Serum tryptase concentration was calculated using Lipocalin Neutrophilgelatinase-associated lipocalin (NGAL) the standard curve. (Tryptase detection range 0.019-5000 MMP MMP-9 ng/ml. EC50=65 ng/ml: Recovery 81.5% with 20 ng/ml of TIMP TIMP-1 Alpha-globulin Alpha-2-macroglobulin (C2-MG) tryptase spiked in normal pooled serum). See Example 1. Haptoglobin precursor alpha-2 (HpC2) 0023 FIG. 4 illustrates a bar graph with tryptase concen Orosomucoid trations in serum of GI control, IBS-C and IBS-D. Actin-severing Gelsolin protein 0024 FIG. 5 illustrates a bar graph with tryptase log value S100 protein Calgranulin AS100A8/MRP-8 distribution. Fibrinopeptide Fibrinopeptide A (FIBA) 0025 FIG. 6 illustrates a density analysis for tryptase data. Others Lactoferrin 0026 FIG. 7 illustrates a bar graph with tryptase concen trations in serum of GI control, IBS-C, IBS-D and IBS-A. US 2012/03 15630 A1 Dec. 13, 2012
0027 FIG. 8 illustrates the dose response curve of human 0044 FIG. 25 provides structure elucidation of the 13.5 tryptase in one embodiment of the present invention. min peak by mass spectrometry. The molecular mass of the 0028 FIG. 9 illustrates the optimization of the tryptase compound present in the 13.5 min peak was determined to be ELISA. (A) Comparison of tryptase standard curve with dif 165.9 m/z (panel A), which corresponds to the molecular ferent amount of capture antibody. (B) Optimization of mass of the amino acid phenylalanine. That the identity of this detecting antibody dilutions. (C) Comparison of tryptase peak is phenylalanine was further verified by the results in standard curves in the presence of different amounts of nor panel B, which shows a prominent mass peak at m/z. 131.0 mal human serum (NHS). corresponding to the loss of H2O (18 m/z) and NH (17 m/z) 0029 FIG. 10 illustrates tryptase levels in serum from from the molecular ion (165.9 m/z); and by panel C, which healthy controls (n=139) and from subjects with IBS shows a prominent mass peak at m/Z 120.0 corresponding to (n=378). the loss of HCOOH (46 m/z) from the molecular ion. 0030 FIG. 11 illustrates the increased serum level of his 0045 FIG. 26 illustrates the dose-dependent response of tamine and PGE in IBS patients versus healthy controls. spiked in D-tyrosine to serum samples determined by HPLC 0031 FIG. 12 illustrates one embodiment of a molecular analysis. pathway derived from the IBS markers identified and dis 0046 FIG. 27 illustrates the dose-dependent response of closed herein. spiked in L-phenylalanine to serum samples determined by 0032 FIG. 13 illustrates a disease classification system HPLC analysis. (DCS) according to one embodiment of the present invention. 0047 FIG. 28 illustrates the serotonin pathway and its 0033 FIG. 14 provides representative chromatographs participation in the pathophysiology of IBS. demonstrating that the identified metabolites are present at 0048 FIG.29 illustrates the transcript levels of 5-HT path different concentrations in the serums of individuals with IBS way metabolic enzymes produced by cytokine treatment of as compared to the serums of non-IBS individuals. whole blood cells. 0034 FIG. 15 illustrates the increased serum level of the 0049 FIG.30 illustrates the normalized levels of mast cell identified metabolites in IBS patients versus healthy controls. activation markers produced by cytokine treatment of whole 0035 FIG. 16 illustrates the top variables identified in the blood cells. IBS prediction. 0050 FIG.31 illustrates the levels ofurocortin (Ucn) pro 0036 FIG. 17 provides representative chromatographs duced by cytokine treatment of whole blood cells. showing that tryptophan and 5-HT metabolite production is increased in response to TNFC. treatment of whole blood. DETAILED DESCRIPTION OF THE INVENTION 0037 FIG. 18 illustrates (A) TPH1 and (B) MAO expres sion in whole blood treated with various inflammatory cytok I. Introduction 1CS 0038 FIG. 19 illustrates Urocortin (Ucn) levels (Y-axis) in 0051 Diagnosing a patient as having irritable bowel syn whole blood treated with various inflammatory cytokines. drome (IBS) can be challenging due to the similarity in Symp 0039 FIG.20 illustrates the levels of the mast cell markers toms between IBS and other diseases or disorders. For (A) histamine, (B) PGE, and (C) tryptase in whole blood example, patients who have inflammatory bowel disease treated with various inflammatory cytokines. (IBD), but who exhibit mild signs and symptoms such as 0040 FIG.21 illustrates the interplay between leukocytes, bloating, diarrhea, constipation, and abdominal pain can be enterochromaffin cells, mast cells and platelets in the gas difficult to distinguish from patients with IBS. As a result, the trointestinal system mediated by Some of the markers pro similarity in symptoms between IBS and IBD renders rapid vided herein. and accurate diagnosis difficult and hampers early and effec 0041 FIG. 22 provides representative chromatograms tive treatment of the disease. obtained from the HPLC analysis of serum samples from IBS 0.052 Although the pathology of irritable bowel syndrome patients and healthy control (HC) subjects. (IBS) is not completely understood, many studies have led to 0042 FIG. 23 illustrates serum levels (AUC) of the 7.5 the hypothesis that IBS is a disorder caused by the dysregu min peak, the 13.5 min peak and the 17.2 min peak deter lation of the brain-gut axis (for review, see, Ohman and Sim mined by HPLC analysis of samples from IBS patients (IBS rén, Nat Rev Gastroenterol Hepatol. 2010 March; 7(3):163 C=12, IBS-D=24, IBS-M=14) and healthy control subjects 73). One observation that supports this theory is the repeated (HC–38). finding that an increased number of mast cells can be found in 0043 FIG.24 provides structure elucidation of the 7.5 min the intestinal mucosa of patients diagnosed with IBS (Gui peak by mass spectrometry. The molecular mass of the com larte, M. et al, Gut 56, 203-209 (2007); Walker, M. M. etal, pound present in the 7.5 min peak was determined to be 181.9 Pharmacol. Ther: 29,765-773 (2009); Akbar, A. etal, Gut 57, m/Z (panel A), which corresponds to the molecular mass of 923-929 (2008); Barbara, G. et al. Gastroenterology 126, the amino acid tyrosine. That the identity of this peak is 693-702 (2004); Barbara, G. et al. Gastroenterology 132, tyrosine was further verified by the results in panel B, which 26-37 (2007); Cremon, C. et al. Am. J. Gastroenterol. 104, shows a prominent mass peak at m/z, 164.9 corresponding to 392–400 (2009); and O'Sullivan, M. et al, Neurogastroen the loss of a OH group (17 m/z) from the molecularion (181.9 terol. Motil., 12, 449-457 (2000)). Similarly, some studies m/z); by panel C, which shows a prominent mass peak at m/z. have also found that levels of mediators released from these 146.9 corresponding to the loss of HO (18 m/z) and NH (17 cells, including histamine and serine proteases (e.g., m/z) from the molecular ion and panel D, which shows a tryptase), are found in the colonic mucosa of IBS patients prominent mass peak at m/z. 119 corresponding to the loss of (Buhner et al., Gastroenterology 2009 October; 137(4); Bar HCOOH (m/z 46) and a OH group (17 m/z) from the molecu bara et al., Gastroenterology, Volume: 122. Number: 4 Suppl. lar ion. 1. Page: A-276, April 2002). However, this finding remains US 2012/03 15630 A1 Dec. 13, 2012
controversial, as other studies have reported that no Such 0058. The term “irritable bowel syndrome' or “IBS” correlation exists (Guilarte et al., Gut 2007 February: 56(2): includes a group of functional bowel disorders characterized 203-9). by one or more symptoms including, but not limited to, 0053 Regardless of the existence of increased levels of abdominal pain, abdominal discomfort, change in bowel pat mast cell mediators in the colonic mucosa of IBS patients, tern, loose or more frequent bowel movements, diarrhea, and Such a correlation has a limited diagnostic value since colonic constipation, typically in the absence of any apparent struc biopsies are invasive procedures. While researchers have tural abnormality. There are at least three forms of IBS, looked for similar patterns in the blood/serum of IBS patients, depending on which symptom predominates: (1) diarrhea fluids well Suited for non-invasive diagnostics, no correlation predominant (IBS-D); (2) constipation-predominant (IBS has been reported to date (Lessof et al., Ann Allergy. 1983 C); and (3) IBS with alternating stool pattern (IBS-A). IBS August;51(2Pt.2):249-50; Guilarte, M. etal, Gut 56,203-209 can also occur in the form of a mixture of symptoms (IBS-M). (2007): Ohman and Simrén, Nat Rev Gastroenterol Hepatol. There are also various clinical subtypes of IBS, such as post 2010 March; 7(3):163–73). infectious IBS (IBS-PI). 0054 Advantageously, the present invention provides, 0059. The term “transforming the sample' includes a among other aspects, non-invasive methods and assays for the physical and/or chemical change of the sample to extract a diagnosis and classification of irritable bowel syndrome marker or to change or modify a marker as defined herein. An (IBS). In certain embodiments, these methods and assays are extraction, a manipulation, a chemical precipitation, an related to the detection of the presence or concentration level ELISA, a complexation, an immuno-extraction, a physical or of various IBS biomarkers in the blood and/or serum of sub chemical modification of the sample or marker to measure a jects Suspected of, or previously diagnosed as having IBS. In level or concentration of a marker all constitute a transforma preferred embodiments, the methods comprise the detection tion. As long as the sample or marker is not identical before of one or more IBS markers identified in Table 1, in blood and after the transformation step, the change or modification and/or serum samples from a subject. Additional markers can is a transformation. also be used. 0060. The term “sample includes any biological speci 0055. The present invention is based, in part, upon the men obtained from an individual. Suitable samples for use in Surprising discovery that the accuracy of classifying a bio the present invention include, without limitation, whole logical sample from an individual as an IBS sample can be blood, plasma, serum, Saliva, urine, Stool (i.e., feces), tears, substantially improved by detecting the presence or level of and any other bodily fluid, or a tissue sample (i.e., biopsy) certain diagnostic markers (e.g., f-tryptase, prostaglandin E, such as a small intestine or colon sample, and cellular extracts histamine, cytokines, growth factors, anti-neutrophilantibod thereof (e.g., red blood cellular extract). In a preferred ies, anti-Saccharomyces cerevisiae antibodies, antimicrobial embodiment, the sample is a blood, plasma, or serum sample. antibodies, mast cell markers, stress markers, gastrointestinal In a more preferred embodiment, the sample is a serum hormones, serotonin metabolites, serotonin pathway mark sample. The use of samples such as serum, saliva, and urine is ers, carbohydrate deficient transferrin (CDT), lactoferrin, well known in the art (see, e.g., Hashida et al., J. Clin. Lab. etc.), alone or in combination with identifying the presence or Anal., 11:267-86 (1997)). One skilled in the art will appreci severity of IBS-related symptoms based upon the individual's ate that samples such as serum samples can be diluted prior to response to one or more questions (e.g., “Are you currently the analysis of marker levels. experiencing any symptoms?”). FIG. 12 shows a non-limiting 0061. The term “biomarker” or “marker” includes any example of a molecular pathway derived from the IBS mark diagnostic marker Such as a biochemical marker, serological ers identified and disclosed herein. In some aspects, the marker, genetic marker, or other clinical or echographic char present invention uses statistical algorithms to aid in the clas acteristic that can be used to classify a sample from an indi sification of a sample as an IBS sample or non-IBS sample. In vidual as an IBS sample or to rule out one or more diseases or other aspects, the present invention uses statistical algorithms disorders associated with IBS-like symptoms in a sample for ruling out other intestinal disorders (e.g., IBD), and then from an individual. The term “biomarker” or “marker” also classifying the non-IBD sample to aid in the classification of encompasses any classification marker Such as a biochemical IBS. marker, serological marker, genetic marker, or other clinical or echographic characteristic that can be used to classify IBS II. Definitions into one of its various forms or clinical subtypes. Non-limit ing examples of diagnostic markers suitable for use in the 0056. As used herein, the following terms have the mean present invention are described below and include cytokines, ings ascribed to them unless specified otherwise. growth factors, anti-neutrophilantibodies, anti-Saccharomy 0057 The term “classifying includes “to associate' or “to ces cerevisiae antibodies, antimicrobial antibodies, mast cell categorize a sample with a disease state. In certain instances, markers, stress markers, gastrointestinal hormones, serotonin “classifying is based on Statistical evidence, empirical evi metabolites, serotonin pathway markers, carbohydrate defi dence, or both. In certain embodiments, the methods and cient transferrin (CDT), anti-tissue transglutaminase (tTG) systems of classifying use a so-called training set of samples antibodies, lipocalins, matrix metalloproteinases (MMPs), having known disease States. Once established, the training tissue inhibitor of metalloproteinases (TIMPs), alpha-globu data set serves as a basis, model, or template against which the lins, actin-severing proteins, S100 proteins, fibrinopeptides, features of an unknown sample are compared, in order to calcitonin gene-related peptide (CGRP), tachykinins, ghre classify the unknown disease state of the sample. In certain lin, neurotensin, corticotropin-releasing hormone (CRH), instances, classifying the sample is akin to diagnosing the Serine proteases (e.g., tryptase, elastase), prostaglandin (e.g., disease state of the sample. In certain other instances, classi PGE), histamine, C-reactive protein (CRP), lactoferrin, anti fying the sample is akin to differentiating the disease State of lactoferrin antibodies, calprotectin, hemoglobin, NOD2/ the sample from another disease state. CARD15, serotonin reuptake transporter (SERT), tryptophan US 2012/03 15630 A1 Dec. 13, 2012 hydroxylase-1,5-hydroxytryptamine (5-HT), lactulose, and control, IBD, and/or Celiac disease sample) or population of the like. Examples of classification markers include, without samples (e.g., greater than a median level of the same marker limitation, leptin, SERT, tryptophan hydroxylase-1,5-HT, in a comparative population of normal, GI control, IBD. antrum mucosal protein 8, keratin-8, claudin-8, Zonulin, cor and/or Celiac disease samples). In certain other instances, the ticotropin releasing hormone receptor-1 (CRHR1), corti level of a particular diagnostic marker in the individual’s cotropin releasing hormone receptor-2 (CRHR2), tryptase, sample is considered to be lowered when it is at least about histamine, prostaglandin E2 (PGE2) and the like. In some 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, embodiments, diagnostic markers can be used to classify IBS 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less than the into one of its various forms or clinical subtypes. In other level of the same marker in a comparative sample (e.g., a embodiments, classification markers can be used to classify a normal, GI control, IBD, and/or Celiac disease sample) or sample as an IBS sample or to rule out one or more diseases population of samples (e.g., less than a median level of the or disorders associated with IBS-like symptoms. One skilled same marker in a comparative population of normal, GI con in the art will know of additional diagnostic and classification trol, IBD, and/or Celiac disease samples). markers Suitable for use in the present invention. 0066. The term “individual,” “subject,” or “patient' typi 0062. In certain instances, the presence or level of at least cally refers to humans, but also to other animals including, one diagnostic marker is determined using an assay Such as a e.g., other primates, rodents, canines, felines, equines, Ovines, hybridization assay or an amplification-based assay. porcines, and the like. Examples of hybridization assays and amplification-based 0067. As used herein, the term “substantially the same assays Suitable for use in the methods of the present invention amino acid sequence' includes an amino acid sequence that is are described above. In certain other instances, the presence similar, but not identical to, the naturally-occurring amino or level of at least one diagnostic marker is determined using acid sequence. For example, an amino acid sequence that has an immunoassay or an immunohistochemical assay. Non Substantially the same amino acid sequence as a naturally limiting examples of immunoassays and immunohistochemi occurring peptide, polypeptide, or protein can have one or cal assays Suitable for use in the methods of the present more modifications such as amino acid additions, deletions, invention are described herein. or Substitutions relative to the amino acid sequence of the 0063 As used herein, the term “binding moiety” refers to naturally-occurring peptide, polypeptide, or protein, pro any class of molecules capable of specifically recognizing a vided that the modified sequence retains substantially at least biomarker of interest. Non-limiting examples of binding moi one biological activity of the naturally-occurring peptide, eties that are well suited for use in the methods provided polypeptide, or protein such as immunoreactivity. Compari herein include proteins, such as monoclonal or polyclonal son for Substantial similarity between amino acid sequences antibodies and functional fragments thereof (e.g., minibod is usually performed with sequences between about 6 and 100 ies, diabodies, triabodies, single chain Fv, F(ab)', and the residues, preferably between about 10 and 100 residues, and like), combinatorially derived proteins from phage display or more preferably between about 25 and 35 residues. A particu ribosome display, peptides, nucleic acids, such as aptamers, larly useful modification of a peptide, polypeptide, or protein and other molecules that are capable of specifically recogniz of the present invention, or a fragment thereof, is a modifica ing a biomarker of interest. tion that confers, for example, increased stability. Incorpora 0064. As used herein, the term “profile' includes any set of tion of one or more D-amino acids is a modification useful in data that represents the distinctive features or characteristics increasing stability of a polypeptide or polypeptide fragment. associated with a disease or disorder such as IBS or IBD. The Similarly, deletion or substitution of lysine residues can term encompasses a "diagnostic marker profile’ that analyzes increase stability by protecting the polypeptide or polypep one or more diagnostic markers in a sample, a “symptom tide fragment against degradation. profile’ that identifies one or more IBS-related clinical fac 0068. The term “monitoring the progression or regression tors (i.e., symptoms) an individual is experiencing or has of IBS includes the use of the methods, systems, and code of experienced, and combinations thereof. For example, a "diag the present invention to determine the disease state (e.g., nostic marker profile' can include a set of data that represents presence or severity of IBS) of an individual. In certain the presence or level of one or more diagnostic markers asso instances, the results of an algorithm (e.g., a learning statis ciated with IBS and/or IBD. Likewise, a “symptom profile' tical classifier system) are compared to those results obtained can include a set of data that represents the presence, severity, for the same individual at an earlier time. In some embodi frequency, and/or duration of one or more symptoms associ ments, the methods, systems, and code of the present inven ated with IBS and/or IBD. tion can be used to predict the progression of IBS, e.g., by 0065. In some embodiments, a panel for measuring one or determining a likelihood for IBS to progress either rapidly or more of the diagnostic markers and/or diagnostic profiles slowly in an individual based on an analysis of diagnostic described above can be constructed and used for classifying markers and/or the identification or IBS-related symptoms. In the sample as an IBS sample or non-IBS sample. One skilled other embodiments, the methods, systems, and code of the in the art will appreciate that the presence or level of a plu present invention can be used to predict the regression of IBS, rality of diagnostic markers can be determined simulta e.g., by determining a likelihood for IBS to regress either neously or sequentially, using, for example, an aliquot or rapidly or slowly in an individual based on an analysis of dilution of the individual’s sample. In certain instances, the diagnostic markers and/or the identification or IBS-related level of a particular diagnostic marker in the individual’s symptoms. sample is considered to be elevated when it is at least about 0069. The term “monitoring drug efficacy in an individual 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, receiving a drug useful for treating IBS includes the use of 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, the methods, systems, and code of the present invention to 600%. 700%,800%,900%, or 1000% greater than the level of determine the effectiveness of a therapeutic agent for treating the same marker in a comparative sample (e.g., a normal, GI IBS after it has been administered. In certain instances, the US 2012/03 15630 A1 Dec. 13, 2012
results of an algorithm (e.g., a learning statistical classifier monoamine oxidase A, monoamine oxidase B. hydrox system) are compared to those results obtained for the same ytryptamine (serotonin) receptor 3A, and a combination individual before initiation of use of the therapeutic agent or thereof. at an earlier time in therapy. As used herein, a drug useful for 0074. In certain embodiments, methods are provided for treating IBS is any compound or drug used to improve the aiding in the diagnosis of irritable bowel syndrome in a Sub health of the individual and includes, without limitation, IBS drugs such as serotonergic agents, antidepressants, chloride ject by determining the level of at least one biomarker channel activators, chloride channel blockers, guanylate selected from carbohydrate deficient transferrin, urocortin, cyclase agonists, antibiotics, opioids, neurokinin antagonists, corticotrophin-releasing hormone-binding protein, cortisol, antispasmodic or anticholinergic agents, belladonna alka adrenocorticotropic hormone, Substance P. nerve growth fac loids, barbiturates, glucagon-like peptide-1 (GLP-1) analogs, tor, neurokinin A, neurokinin B. vasoactive intestinal peptide, corticotropin releasing factor (CRF) antagonists, probiotics, glucagon-like peptide 2, motilin, pituitary adenylate cyclase free bases thereof, pharmaceutically acceptable salts thereof, activating peptide, serotonin, tryptophan, serotonin O-sul derivatives thereof, analogs thereof, and combinations fate, 5-hydroxyindoleacetic acid, 5-HT glucuronide, thereof. tyrosine, phenylalanine, UDP-glucuronosyltransferase 1-6. 0070. The term “therapeutically effective amount or dose” serotonin reuptake transporter, tryptophan hydroxylase 1, includes a dose of a drug that is capable of achieving a monoamine oxidase A, monoamine oxidase B. hydrox therapeutic effect in a subject in need thereof. For example, a ytryptamine (serotonin) receptor 3A, and a combination therapeutically effective amount of a drug useful for treating thereof in conjunction with at least one IBS biomarker IBS can be the amount that is capable of preventing or reliev selected from the group consisting of Brain-Derived Neuro ing one or more symptoms associated with IBS. The exact tropic Factor (BDNF), Neutrophil Gelatinase-Associated amount can be ascertainable by one skilled in the art using Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis known techniques (see, e.g., Lieberman, Pharmaceutical (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), Dosage Forms, Vols. 1-3 (1992); Lloyd, The Art, Science and Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein Technology of Pharmaceutical Compounding (1999); Pickar, ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody Dosage Calculations (1999); and Remington. The Science (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human and Practice of Pharmacy, 20th Edition, Gennaro, Ed., Lip Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human pincott, Williams & Wilkins (2003)). Tissue Transglutaminase IgA (tTG). B-tryptase, histamine, prostaglandin E. (PGE), and a combination thereof. III. Description of the Embodiments 0075. In yet other embodiments, methods are provided for aiding in the diagnosis of irritable bowel syndrome in a Sub 0071. The present invention provides methods, systems, ject by determining the level of at least one biomarker and code for aiding in the diagnosis of irritable bowel syn selected from carbohydrate deficient transferrin, urocortin, drome in a Subject. Similarly, the present invention provides corticotrophin-releasing hormone-binding protein, cortisol, methods, systems, and code for accurately classifying adrenocorticotropic hormone, Substance P. nerve growth fac whether a sample from an individual is associated with irri tor, neurokinin A, neurokinin B. vasoactive intestinal peptide, table bowel syndrome (IBS). In some embodiments, the glucagon-like peptide 2, motilin, pituitary adenylate cyclase present invention relies on the use of a statistical algorithm activating peptide, serotonin, tryptophan, serotonin O-sul (e.g., a learning statistical classifier system) and/or empirical fate, 5-hydroxyindoleacetic acid, 5-HT glucuronide, data (e.g., the presence or level of an IBS marker). The present tyrosine, phenylalanine, UDP-glucuronosyltransferase 1-6. invention is also useful for ruling out one or more diseases or serotonin reuptake transporter, tryptophan hydroxylase 1, disorders that present with IBS-like symptoms and ruling in monoamine oxidase A, monoamine oxidase B. hydrox IBS using a combination of Statistical algorithms and/or ytryptamine (serotonin) receptor 3A, and a combination empirical data. Accordingly, the present invention provides thereof in conjunction with at least one biomarker selected an accurate diagnostic prediction of IBS and prognostic infor from the group consisting of a cytokine (e.g., IL-8, IL-B, mation useful for guiding treatment decisions. TWEAK, leptin, OPG, MIP-3|B, GROC, CXCL4/PF-4, and/ 0072 A. Methods for Aiding in the Diagnosis of Irritable or CXCL7/NAP-2), growth factor (e.g., EGF, VEGF, PEDF, Bowel Syndrome (IBS) BDNF, and/or SDGF), anti-neutrophilantibody (e.g., ANCA, 0073. In one aspect, the present invention provides meth pANCA, cANCA, NSNA, and/or SAPPA), ASCA (e.g., ods for aiding in the diagnosis of irritable bowel syndrome in ASCA-IgA, ASCA-IgG, and/or ASCA-IgM), antimicrobial a subject by determining the level of 1,2,3,4,5,6,7,8,9, 10. antibody (e.g., anti-Ompo antibody, anti-flagellin antibody, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or and/or anti-I2 antibody), lactoferrin, anti-tTG antibody, more of the biomarkers shown in Table 1. In preferred lipocalin (e.g., NGAL, NGAL/MMP-9 complex), MMP embodiments, the methods provided herein rely on the detec (e.g., MMP-9), TIMP (e.g., TIMP-1), alpha-globulin (e.g., tion of at least one biomarker selected from the group con alpha-2-macroglobulin, haptoglobin, and/or orosomucoid), sisting of carbohydrate deficient transferrin, urocortin, corti actin-severing protein (e.g., gelsolin), S100 protein (e.g., cal cotrophin-releasing hormone-binding protein, cortisol, granulin), fibrinopeptide (e.g., FIBA), CGRP, tachykinin adrenocorticotropic hormone, Substance P. nerve growth fac (e.g., Substance P), ghrelin, neurotensin, corticotropin-re tor, neurokinin A, neurokinin B. vasoactive intestinal peptide, leasing hormone, and combinations thereof. In yet other glucagon-like peptide 2, motilin, pituitary adenylate cyclase embodiments, the presence or level of other diagnostic mark activating peptide, serotonin, tryptophan, serotonin O-sul erS Such as, for example, anti-lactoferrin antibody, L-selectin/ fate, 5-hydroxyindoleacetic acid, 5-HT glucuronide, CD62L, elastase, C-reactive protein (CRP), calprotectin, tyrosine, phenylalanine, UDP-glucuronosyltransferase 1-6. anti-U1-70 kDa autoantibody, Zona occludens 1 (ZO-1), serotonin reuptake transporter, tryptophan hydroxylase 1, vasoactive intestinal peptide (VIP), serum amyloid A, gastrin, US 2012/03 15630 A1 Dec. 13, 2012
B-tryptase, histamine, prostaglandin E2 (PGE2), and a com sample into a complex comprising the IBS marker and the bination thereof may also be detected. IBS marker binding moiety; and (b) determining the level of 0076. In another aspect, the present invention provides a the complex, thereby determining the level of the IBS marker method for classifying whethera sample from an individual is present in the sample, wherein the IBS marker is selected associated with IBS, the method comprising: (a) determining from the group consisting of carbohydrate deficient transfer a diagnostic marker profile by detecting the presence or level rin, urocortin, corticotrophin-releasing hormone-binding of at least one diagnostic marker in the sample; (b) classifying protein, cortisol, adrenocorticotropic hormone, Substance P. the sample as an IBD Sample or non-IBD Sample using a first nerve growth factor, neurokinin A, neurokinin B. vasoactive statistical algorithm based upon the diagnostic marker pro intestinal peptide, glucagon-like peptide 2, motilin, pituitary file; and if the sample is classified as a non-IBD Sample, (c) adenylate cyclase-activating peptide, serotonin, tryptophan, classifying the non-IBD sample as an IBS sample or non-IBS serotonin O-sulfate, 5-hydroxyindoleacetic acid, 5-HT glu sample using a second statistical algorithm based upon the curonide, tyrosine, phenylalanine, UDP-glucuronosyltrans same diagnostic marker profile as determined in step (a) or a ferase 1-6, serotonin reuptake transporter, tryptophan different diagnostic marker profile. hydroxylase 1, monoamine oxidase A, monoamine oxidase 0077. The diagnostic markers used for ruling out IBD can B, and hydroxytryptamine (serotonin) receptor 3A. In one be the same as the diagnostic markers used for ruling in IBS. embodiment, the method further comprises: (c) comparing Alternatively, the diagnostic markers used for ruling out IBD the level of the IBS marker present in the sample to a control can be different than the diagnostic markers used for ruling in level, wherein a difference in the level of the IBS marker IBS. present in the sample relative to the control level is indicative 0078. In some embodiments, the method of first ruling out of an increased likelihood of the subject having IBS. IBD (i.e., classifying the sample as an IBD Sample or non I0083. In a specific embodiment, the present invention pro IBD sample) and then ruling in IBS (i.e., classifying the vides an assay to aid in the diagnosis of IBS, the assay com non-IBD sample as an IBS sample or non-IBS sample) com prising: (a) contacting a sample having an IBS marker con prises determining a diagnostic marker profile optionally in tained therein under conditions suitable to transform the IBS combination with a symptom profile, wherein the symptom marker into a complex comprising the IBS marker and a profile is determined by identifying the presence or severity capture anti-IBS marker antibody; (b) contacting the complex of at least one symptom in the individual; classifying the with an enzyme labeled indicator antibody to transform the sample as an IBD Sample or non-IBD Sample using a first complex into a labeled complex; (c) contacting the labeled statistical algorithm based upon the diagnostic marker profile complex with a substrate for the enzyme; and (d) detecting the and the symptom profile; and if the sample is classified as a presence or level of the IBS marker in the sample, wherein the non-IBD sample, classifying the non-IBD sample as an IBS IBS marker is selected from the group consisting of carbohy sample or non-IBS Sample using a second statistical algo drate deficient transferrin, urocortin, corticotrophin-releasing rithm based upon the same profiles as determined in step (a) hormone-binding protein, cortisol, adrenocorticotropic hor or different profiles. One skilled in the art will appreciate that mone, Substance P. nerve growth factor, neurokinin A, neu the diagnostic marker profile and the symptom profile can be rokinin B. vasoactive intestinal peptide, glucagon-like pep determined simultaneously or sequentially in any order. tide 2, motilin, pituitary adenylate cyclase-activating peptide, 0079. In additional embodiments, the methods of the serotonin, tryptophan, serotonin O-sulfate, 5-hydroxyin present invention further comprise ruling out intestinal doleacetic acid, 5-HT glucuronide, tyrosine, phenylalanine, inflammation. Non-limiting examples of intestinal inflamma UDP-glucuronosyltransferase 1-6, serotonin reuptake trans tion include acute inflammation, diverticulitis, ileal pouch porter, tryptophan hydroxylase 1, monoamine oxidase A, anal anastomosis, microscopic colitis, infectious diarrhea, monoamine oxidase B, and hydroxytryptamine (serotonin) and combinations thereof. In some instances, the intestinal receptor 3A. inflammation is ruled out based upon the presence or level of 0084. In certain embodiments, a control level is the level C-reactive protein (CRP), lactoferrin, calprotectin, or combi of the IBS marker present in a blood or serum sample from a nations thereof. healthy subject or an average level of the IBS marker present 0080. As described herein, the methods of the present in blood or serum samples from a cohort of healthy subjects. invention can further comprise sending the IBS classification In other embodiments, a control level is the level of the IBS results to a clinician, e.g., a gastroenterologist or a general marker present in a blood or serum sample from a non-IBS practitioner. The methods can also provide a diagnosis in the subject oran average level of the IBS marker present in blood form of a probability that the individual has IBS. For example, or serum samples from a cohort of non-IBS subjects. In the individual can have about a 0%, 5%, 10%, 15%, 20%, another embodiment, a control level is the level of the IBS 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, marker present in a blood or serum sample from a diseased 75%, 80%, 85%, 90%, 95%, or greater probability of having subject oran average level of the IBS marker present in blood IBS. In some instances, the methods of the present invention or serum samples from a cohort of diseased Subjects. Non further provide a prognosis of IBS in the individual. For limiting examples of diseased subjects that are useful for example, the prognosis can be Surgery, development of a determining a control level from include subjects with IBS, category or clinical subtype of IBS, development of one or Subjects with a non-IBS gastrointestinal disease, Subjects more symptoms, or recovery from the disease. with inflammatory bowel disease (IBD), subjects with ulcer 0081 1. IBS Markers ative colitis (UC), subjects with Crohn's disease (CD), sub 0082 In one aspect, a method for aiding in the diagnosis of jects with celiac disease, Subjects with gastroesophageal irritable bowel syndrome (IBS) in a subject is provided, the reflux disease (GERD), subjects with cancer, subjects with a method comprising: (a) contacting a blood or serum sample cancer of the gastrointestinal tract, Subjects with a cancer of from the subject with an IBS marker binding moiety under the stomach, Subjects with a cancer of the Small or large conditions suitable to transform the IBS marker present in the bowel, and the like. US 2012/03 15630 A1 Dec. 13, 2012
0085. In cases where the control level is a level of the IBS I0089. The sample used for detecting or determining the marker in a blood or serum sample of a healthy Subject or presence or level of at least one diagnostic marker is typically healthy subjects, an increased level of the IBS marker present whole blood, plasma, serum, saliva, urine, stool (i.e., feces), in a sample from a subject, relative to the control level, is tears, and any other bodily fluid, or a tissue sample (i.e., indicative of an increased likelihood of the subject having biopsy) Such as a small intestine or colon sample. Preferably, IBS. Conversely, where the control is a healthy subject or the sample is serum, whole blood, plasma, stool, urine, or a subjects, a similar or reduced level of the IBS marker present tissue biopsy. In certain instances, the methods of the present in a sample from a subject, relative to the control level, is invention further comprise obtaining the sample from the indicative of an increased likelihood of the subject not having individual prior to detecting or determining the presence or IBS. level of at least one diagnostic marker in the sample. In a 0086. In cases where the control level is a level of the IBS preferred embodiment, the additional biomarker may be marker in a blood or serum sample of a subject or Subjects detected from a blood or serum sample. In other embodi with IBS, a similar or increased level of the IBS marker ments, the additional biomarker may be detected from a stool present in a sample from a Subject, relative to the control sample or a biopsy from the bowel of the subject. level, is indicative of an increased likelihood of the subject having IBS. Conversely, where the control is a subject or 0090. In one embodiment, the method comprises detect subjects with IBS, a reduced level of the IBS marker present ing or determining the level of the IBS marker from a blood or in a sample from a subject, relative to the control level, is serum sample and Brain-Derived Neurotropic Factor indicative of an increased likelihood of the subject not having (BDNF) from a biological sample. In a preferred embodi IBS. ment, Brain-Derived Neurotropic Factor (BDNF) is detected in a blood or serum sample from the Subject. In another 0087. In certain aspects of the invention, methods are pro preferred embodiment, Brain-Derived Neurotropic Factor vided for aiding in the diagnosis of irritable bowel syndrome (BDNF) is detected in a stool sample or a biopsy from the (IBS) in a subject comprising the detection of an IBS marker bowel of the subject. In other embodiments, at least one in a blood or serum sample and at least one additional blood additional biomarker is also detected. or serum biomarker selected from B-tryptase, histamine and prostaglandin E. (PGE). In one embodiment, the method 0091. In one embodiment, the method comprises detect comprises detecting or determining the level of the IBS ing or determining the level of the IBS marker from a blood or marker and B-tryptase from a blood or serum sample from a serum sample and Neutrophil Gelatinase-Associated Lipoca subject. In another embodiment, the method comprises lin (NGAL) from a biological sample. In a preferred embodi detecting or determining the level of the IBS marker and ment, Neutrophil Gelatinase-Associated Lipocalin (NGAL) PGE, from a blood or serum sample from a subject. In a third is detected in a blood or serum sample from the subject. In embodiment, the method comprises detecting or determining another preferred embodiment, Neutrophil Gelatinase-Asso the level of the IBS marker and histamine from a blood or ciated Lipocalin (NGAL) is detected in a stool sample or a serum sample from a Subject. In yet another embodiment, the biopsy from the bowel of the subject. In other embodiments, method comprises detecting or determining the level of the at least one additional biomarker is also detected. IBS marker, B-tryptase, histamine, and PGE, from a blood or 0092. In one embodiment, the method comprises detect serum sample from a subject. Preferably, the IBS marker, ing or determining the level of the IBS marker from a blood or f3-tryptase, histamine, and/or PGE are detected from the serum sample and TNF-related Weak Inducer of Apoptosis same blood or serum sample, although in certain instances the (TWEAK) from a biological sample. In a preferred embodi biomarkers may be detected in different blood or serum ment, TNF-related Weak Inducer of Apoptosis (TWEAK) is samples taken from the same individual, for example, at the detected in a blood or serum sample from the subject. In same time or at different times. In certain embodiments, the another preferred embodiment, TNF-related Weak Inducer of biomarkers may be detected in separate assays performed Apoptosis (TWEAK) is detected in a stool sample or a biopsy with different aliquots of a blood or serum sample from a from the bowel of the subject. In other embodiments, at least subject. In other embodiments, the biomarkers may be one additional biomarker is also detected. detected in a single multiplex detection assay, for example, in 0093. In one embodiment, the method comprises detect a LumineXOR, XMAP(R) assay. ing or determining the level of the IBS marker from a blood or 0088. In yet other aspects of the invention, methods are serum sample and Growth-Related Oncogene Alpha (GRO provided for aiding in the diagnosis of irritable bowel syn C.) from a biological sample. In a preferred embodiment, drome (IBS) in a subject comprising the detection of the IBS Growth-Related Oncogene Alpha (GRO-O.) is detected in a marker in a blood or serum sample and at least one additional blood or serum sample from the subject. In another preferred biomarker selected from Brain-Derived Neurotropic Factor embodiment, Growth-Related Oncogene Alpha (GRO-O.) is (BDNF), Neutrophil Gelatinase-Associated Lipocalin detected in a stool sample or a biopsy from the bowel of the (NGAL). TNF-related Weak Inducer of Apoptosis Subject. In other embodiments, at least one additional biom (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), arker is also detected. Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein 0094. In one embodiment, the method comprises detect ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody ing or determining the level of the IBS marker from a blood or (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human serum sample and Interleukin-1 Beta (IL-B) from a biological Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human sample. In a preferred embodiment, Interleukin-1 Beta (IL-B) Tissue Transglutaminase IgA (tTG). B-tryptase, histamine, is detected in a blood or serum sample from the subject. In PGE, and a combination thereof. In certain embodiments, at another preferred embodiment, Interleukin-1 Beta (IL-f) is least 2 of the additional biomarkers may be detected. In other detected in a stool sample or a biopsy from the bowel of the embodiments, at least 3, 4, 5, 6,7,8,9, or 10 of the additional Subject. In other embodiments, at least one additional biom biomarkers may be detected. arker is also detected. US 2012/03 15630 A1 Dec. 13, 2012
0095. In one embodiment, the method comprises detect 0101. In certain embodiments, the presence or level of at ing or determining the level of the IBS marker from a blood or least one diagnostic marker is determined using an assay Such serum sample and Tissue Inhibitor of Metalloproteinase-1 as a hybridization assay or an amplification-based assay. (TIMP-1) from a biological sample. In a preferred embodi Examples of hybridization assays suitable for use in the meth ment, Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is ods of the present invention include, but are not limited to, detected in a blood or serum sample from the subject. In Northern blotting, dot blotting, RNase protection, and a com another preferred embodiment, Tissue Inhibitor of Metallo bination thereof. A non-limiting example of an amplification proteinase-1 (TIMP-1) is detected in a stool sample or a based assay suitable for use in the methods of the present biopsy from the bowel of the subject. In other embodiments, invention includes a reverse transcriptase-polymerase chain at least one additional biomarker is also detected. reaction (RT-PCR). 0096. In one embodiment, the method comprises detect 0102. In certain other embodiments, the presence or level ing or determining the level of the IBS marker from a blood or of at least one diagnostic marker is determined using an serum sample and Anti-Saccharomyces cerevisiae Antibody immunoassay or an immunohistochemical assay. A non-lim (ASCA-IgA) from a biological sample. In a preferred iting example of an immunoassay Suitable for use in the embodiment, Anti-Saccharomyces cerevisiae Antibody methods of the present invention includes an enzyme-linked (ASCA-IgA) is detected in a blood or serum sample from the immunosorbent assay (ELISA). Examples of immunohis Subject. In another preferred embodiment, Anti-Saccharomy tochemical assays suitable for use in the methods of the ces cerevisiae Antibody (ASCA-IgA) is detected in a stool present invention include, but are not limited to, immunof sample or a biopsy from the bowel of the subject. In other luorescence assays Such as direct fluorescentantibody assays, embodiments, at least one additional biomarker is also indirect fluorescent antibody (IFA) assays, anticomplement detected. immunofluorescence assays, and avidin-biotin immunofluo 0097. In one embodiment, the method comprises detect rescence assays. Other types of immunohistochemical assays ing or determining the level of the IBS marker from a blood or include immunoperoxidase assays. serum sample and Anti-CBir-1 Antibody (CBirl) from a bio 0103) In a preferred embodiment, the presence or level of logical sample. In a preferred embodiment, Anti-CBir-1Anti the IBS marker is determined using a sandwich enzyme body (CBirl) is detected in a blood or serum sample from the linked immunosorbent assay (ELISA). Any suitable antibody subject. In another preferred embodiment, Anti-CBir-1 Anti pair may be used for the capture and detecting antibodies in a body (CBirl) is detected in a stool sample or a biopsy from sandwich ELISA. One of skill in the art will know and appre the bowel of the subject. In other embodiments, at least one ciate how to select an appropriate antibody pair for the assay. additional biomarker is also detected. Generally, two antibodies are selected that bind to the target 0098. In one embodiment, the method comprises detect of interest, e.g., the IBS marker, at different epitopes such that ing or determining the level of the IBS marker from a blood or the binding of the first (capture) antibody does not interfere serum sample and Anti-Human Neutrophil Cytoplasmic with the second (detecting) antibody. In certain embodi Antibody (ANCA) from a biological sample. In a preferred ments, the detecting antibody will be conjugated to an embodiment, Anti-Human Neutrophil Cytoplasmic Antibody enzyme, for example, alkaline phophatase, to aid in the detec (ANCA) is detected in a blood or serum sample from the tion of the complex. In other embodiments, a secondary anti subject. In another preferred embodiment, Anti-Human Neu body conjugated to an enzyme (e.g., alkaline phophatase), trophil Cytoplasmic Antibody (ANCA) is detected in a stool which binds to the detecting antibody, may be used in the sample or a biopsy from the bowel of the subject. In other assay. Generally, the complex will then be detected by the use embodiments, at least one additional biomarker is also of aluminescent substrate, for example, Ultra LITETM (NAG detected. Research Laboratories); SensoLyte.R. (AnaSpec); SuperSig 0099. In one embodiment, the method comprises detect nal ELISA Femto Maximum Sensitivity Substrate (Thermo ing or determining the level of the IBS marker from a blood or Scientific); SuperSignal ELISA Pico Chemiluminescent serum sample and Anti-Human Tissue Transglutaminase IgA Substrate (Thermo Scientific); CPSD (disodium 3-(4-meth (tTG) from a biological sample. In a preferred embodiment, oxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7 Anti-Human Tissue Transglutaminase IgA (tTG) is detected decan-4-yl)phenyl phosphate; Tropix. Inc). In a preferred in a blood or serum sample from the Subject. In another embodiment, the B-tryptase sandwich ELISA comprises the preferred embodiment, Anti-Human Tissue Transglutami use of an alkaline phosphatase conjugated anti-tryptase anti nase IgA (tTG) is detected in a stool sample or a biopsy from body as the detecting antibody and a CPSD containing lumi the bowel of the subject. In other embodiments, at least one nescent substrate to enhance the assay sensitivity. The CPSD additional biomarker is also detected. Substrate can be found in chemiluminescent detection sys 0100. In one embodiment, the method comprises detect tems, such as the ELISA-LightTM System (Applied Biosys ing or determining the level of the IBS marker from a blood or tems). serum sample and all of Brain-Derived Neurotropic Factor 0104. In some embodiments, the methods provided herein (BDNF), Neutrophil Gelatinase-Associated Lipocalin further include the step of determining a symptom profile for (NGAL). TNF-related Weak Inducer of Apoptosis the subject, wherein the symptom profile is determined by (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), identifying the presence or severity of at least one symptom in Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein the subject. In a preferred embodiment, the method com ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody prises: (d) determining a symptom profile for the Subject, (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human wherein the symptom profile is determined by identifying the Neutrophil Cytoplasmic Antibody (ANCA), and Anti-Hu presence or severity of at least one symptom in the Subject; man Tissue Transglutaminase IgA (tTG) from a blood or and (e) diagnosing the Subject as having IBS or not having serum sample, a stool sample, or a biopsy from the bowel of IBS using an algorithm based upon the level of an IBS biom the subject. arker and the system profile. In a preferred embodiment, the US 2012/03 15630 A1 Dec. 13, 2012 method for aiding in the diagnosis of irritable bowel syn transform the complex into a labeled complex; (c) contacting drome (IBS) in a subject comprises determining a diagnostic the labeled complex with a substrate for the enzyme; and (d) marker profile, for example for the IBS marker or a combi detecting the presence or level of the IBS marker in the nation thereofas described herein, optionally in combination sample. with a symptom profile, wherein the symptom profile is deter mined by identifying the presence or severity of at least one 0111. In some instances, the assay to aid in the differen symptom in the individual; and classifying the sample as an tiation of IBS-D and IBS-A from IBS-C further comprises IBS sample or non-IBS sample using an algorithm based detecting the presence or level of B-tryptase, a prostaglandin upon the diagnostic marker profile and the symptom profile. (e.g., PGE), and/or histamine in the sample. 0105. The symptom profile is typically determined by 0112. In yet other embodiments, methods provided herein identifying the presence or severity of at least one symptom further include diagnosing a subject not having IBS as having selected from the group consisting of chest pain, chest dis IBD, as not having IBD, as having celiac disease, as not comfort, heartburn, uncomfortable fullness after having a having celiac disease, as being a healthy Subject, or as not regular-sized meal, inability to finish a regular-sized meal, having a gastrointestinal disease. In a related embodiment, abdominal pain, abdominal discomfort, constipation, diar methods are provided herein that further classify a non-IBS rhea, bloating, abdominal distension, negative thoughts or sample as an IBD Sample, a non-IBD sample, a healthy feelings associated with having pain or discomfort, and com sample, a non-gastrointestinal disease sample, a celiac binations thereof. sample, and the like. 0106. In preferred embodiments, the presence or severity 0113. In one embodiment, the methods provided herein of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms comprise the use of an algorithm based upon the level of an described herein is identified to generate a symptom profile IBS biomarker. In certain embodiments, the algorithm is fur that is useful for predicting IBS. In certain instances, a ques ther based upon the system profile. In a preferred embodi tionnaire or other form of written, verbal, or telephone survey is used to produce the symptom profile. The questionnaire or ment, the algorithm comprises a statistical algorithm, for Survey typically comprises a standardized set of questions example a learning statistical classifier system. In a more and answers for the purpose of gathering information from preferred embodiment, the algorithm comprises a combina respondents regarding their current and/or recent IBS-related tion of at least two learning statistical classifier systems. In a symptoms. most preferred embodiment, the combination of at least two 0107. In some embodiments, the symptom profile is pro learning statistical classifier systems comprises a random duced by compiling and/or analyzing all or a Subset of the forest classifier and a neural network classifier. answers to the questions set forth in the questionnaire or 0114 2. Stress Markers Survey. In other embodiments, the symptom profile is pro 0.115. In one embodiment, the present invention provides a duced based upon the individual’s response to the following method for aiding in the diagnosis of irritable bowel syn question: "Are you currently experiencing any symptoms? drome (IBS) in a subject by detecting the level or concentra The symptom profile generated in accordance with either of tion of a stress marker in a biological sample from the Subject these embodiments can be used in combination with a diag (e.g., a blood or serum sample). The level or concentration of nostic marker profile in the algorithmic-based methods the stress marker may be determined by various methods described herein to improve the accuracy of predicting IBS. known in the art, including without limitation, immunoas 0108. In some embodiments, the methods provided herein says, for example, enzyme immunoassays (EIA) Such as further include providing a probability that a subject has IBS. enzyme multiplied immunoassay technique (EMIT), In certain embodiments, the method may comprise providing enzyme-linked immunosorbent assay (ELISA), antigen cap a probability that the is subject highly unlikely, unlikely, ture ELISA, sandwich ELISA, IgM antibody capture ELISA likely, or highly likely has IBS. In related embodiments, (MAC ELISA), and microparticle enzyme immunoassay methods are provided that further include providing a prob (MEIA); capillary electrophoresis immunoassays (CEIA); ability that a sample classified as an IBS sample or a non-IBS radioimmunoassays (RIA); immunoradiometric assays sample is from a subject with IBS. In certain embodiments, (IRMA); fluorescence polarization immunoassays (FPIA); the method may comprise providing a probability that the is and chemiluminescence assays (CL); marker purification, for sample is from a subject that highly unlikely, unlikely, likely, example, by high pressure liquid chromatography (HPLC), or highly likely has IBS. alone or in combination with mass spectrometry (e.g., 0109. In other embodiments, the methods provided herein MALDI/MS, MALDI-TOF/MS, SELDI-TOF/MS, tandem further include classifying a diagnosis of IBS as IBS-consti MS, etc.); and other well known methods including, without pation (IBS-C), IBS-diarrhea (IBS-D), IBS-mixed (IBS-M), limitation, Bradford assays, Coomassie blue staining, silver IBS-alternating (IBS-A), or post-infectious IBS (IBS-PI). In staining, assays for radiolabeled protein, and mass spectrom related embodiments, methods are provided herein to further etry. classify a sample as an IBS-constipation (IBS-C), IBS-diar 0116. In a specific embodiment, a method is provided for rhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating (IBS-A), aiding in the diagnosis of irritable bowel syndrome (IBS) in a or post-infectious IBS (IBS-PI) sample. Subject, said method comprising: contacting a blood or serum 0110. In one embodiment, the present invention provides sample from the Subject with a stress marker binding moiety an assay to aid in the differentiation of IBS-D and IBS-A from under conditions Suitable to transform the stress marker IBS-C, the assay comprising: (a) contacting a sample having present in the sample into a complex comprising the stress an IBS marker contained therein under conditions suitable to marker and the stress marker binding moiety; and determin transform the IBS marker into a complex comprising the IBS ing the level of said complex, thereby determining the level or marker and a capture anti-IBS marker antibody; (b) contact concentration of the stress marker present in the sample. In a ing the complex with an enzyme labeled indicator antibody to preferred embodiment, the stress marker is selected from US 2012/03 15630 A1 Dec. 13, 2012
urocortin (Ucn), corticotrophin-releasing hormone-binding I0122. In another specific embodiment, a method is pro protein (CRFBP), cortisol, adrenocorticotropic hormone, and vided for aiding in the diagnosis of irritable bowel syndrome a combination thereof. (IBS) in a Subject, said method comprising: contacting a 0117. In one embodiment, the method provided above, blood or serum sample from the subject with a corticotrophin further comprises a step of comparing the level or concentra releasing hormone-binding protein binding moiety under tion of the stress marker present in the sample to a control conditions Suitable to transform corticotrophin-releasing hor level or concentration, wherein a difference in the level or mone-binding protein present in the sample into a complex concentration of the stress marker present in the sample rela comprising corticotrophin-releasing hormone-binding pro tive to the control level or concentration is indicative of an tein and the corticotrophin-releasing hormone-binding pro increased likelihood of said subject having IBS. tein binding moiety; and determining the level of said com 0118. In one embodiment, wherein the control level or plex, thereby determining the level or concentration of concentration is the level or concentration of the stress marker corticotrophin-releasing hormone-binding protein present in present in a blood or serum sample from a healthy Subject, an increased level or concentration of the stress marker present the sample. In one embodiment, the method further com in the sample relative to the control level or concentration is prises detecting the level or concentration of at least one indicative of an increased likelihood of said subject having additional IBS marker selected from Brain-Derived Neuro IBS, while the same or a reduced level or concentration of the tropic Factor (BDNF), Neutrophil Gelatinase-Associated stress marker present in the sample relative to the control level Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis or concentration is indicative of an increased likelihood of (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), said subject not having IBS. Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein 0119. In another embodiment, wherein the control level or ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody concentration is the level or concentration of the stress marker (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human present in a blood or serum sample from a subject with IBS, Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human the same or an increased level or concentration of the stress Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, marker present in the sample relative to the control level or and prostaglandin E. (PGE). concentration is indicative of an increased likelihood of said I0123. In another specific embodiment, a method is pro subject having IBS, while a reduced level or concentration of vided for aiding in the diagnosis of irritable bowel syndrome the stress marker present in the sample relative to the control (IBS) in a Subject, said method comprising: contacting a level or concentration is indicative of an increased likelihood blood or serum sample from the subject with a cortisol bind of said subject not having IBS. ing moiety under conditions suitable to transform cortisol 0120 In another related embodiment, methods are pro present in the sample into a complex comprising cortisoland vided for aiding in the diagnosis of irritable bowel syndrome the cortisol binding moiety; and determining the level of said (IBS) in a subject by detecting the level or concentration of a complex, thereby determining the level or concentration of stress marker and at least one additional IBS marker selected cortisol present in the sample. In one embodiment, the from Brain-Derived Neurotropic Factor (BDNF), Neutrophil method further comprises detecting the level or concentration Gelatinase-Associated Lipocalin (NGAL). TNF-related of at least one additional IBS marker selected from Brain Weak Inducer of Apoptosis (TWEAK), Growth-Related Derived Neurotropic Factor (BDNF), Neutrophil Gelatinase Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B), Tissue Associated Lipocalin (NGAL), TNF-related Weak Inducer of Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Saccharo Apoptosis (TWEAK), Growth-Related Oncogene Alpha myces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Anti (GRO-O.), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of Met body (CBirl), Anti-Human Neutrophil Cytoplasmic Anti alloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae body (ANCA), Anti-Human Tissue Transglutaminase IgA Antibody (ASCA-IgA), Anti-CBir-1Antibody (CBirl), Anti (tTG), histamine, f-tryptase, prostaglandin E. (PGE), and a Human Neutrophil Cytoplasmic Antibody (ANCA), Anti combination thereof. Human Tissue Transglutaminase IgA (tTG), histamine, 0121. In a specific embodiment, a method is provided for B-tryptase, and prostaglandin E2 (PGE). aiding in the diagnosis of irritable bowel syndrome (IBS) in a 0.124. In yet another specific embodiment, a method is Subject, said method comprising: contacting a blood or serum provided for aiding in the diagnosis of irritable bowel syn sample from the Subject with a urocortin binding moiety drome (IBS) in a Subject, said method comprising: contacting underconditions Suitable to transform urocortin present in the a blood or serum sample from the Subject with an adrenocor sample into a complex comprising urocortin and the urocortin ticotropic hormone binding moiety under conditions Suitable binding moiety; and determining the level of said complex, to transform adrenocorticotropic hormone present in the thereby determining the level or concentration of urocortin sample into a complex comprising adrenocorticotropic hor present in the sample. In one embodiment, the method further mone and the adrenocorticotropic hormone binding moiety; comprises detecting the level or concentration of at least one and determining the level of said complex, thereby determin additional IBS marker selected from Brain-Derived Neuro ing the level or concentration of adrenocorticotropic hormone tropic Factor (BDNF), Neutrophil Gelatinase-Associated present in the sample. In one embodiment, the method further Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis comprises detecting the level or concentration of at least one (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), additional IBS marker selected from Brain-Derived Neuro Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein tropic Factor (BDNF), Neutrophil Gelatinase-Associated ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody and prostaglandin E. (PGE). (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human US 2012/03 15630 A1 Dec. 13, 2012
Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human tinal hormone present in a blood or serum sample from a Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, subject with IBS, the same or an increased level or concen and prostaglandin E. (PGE). tration of the gastrointestinal hormone present in the sample 0125 3. Gastrointestinal Hormones relative to the control level or concentration is indicative of an 0126. In one embodiment, the present invention provides a increased likelihood of said subject having IBS, while a method for aiding in the diagnosis of irritable bowel syn reduced level or concentration of the gastrointestinal hor drome (IBS) in a subject by detecting the level or concentra mone present in the sample relative to the control level or tion of a gastrointestinal hormone in a biological sample from concentration is indicative of an increased likelihood of said the Subject (e.g., a blood or serum sample). The level or subject not having IBS. concentration of the gastrointestinal hormone may be deter I0131. In another related embodiment, methods are pro mined by various methods known in the art, including without vided for aiding in the diagnosis of irritable bowel syndrome limitation, immunoassays, for example, enzyme immunoas (IBS) in a subject by detecting the level or concentration of a says (EIA) Such as enzyme multiplied immunoassay tech gastrointestinal hormone and at least one additional IBS nique (EMIT), enzyme-linked immunosorbent assay marker selected from Brain-Derived Neurotropic Factor (ELISA), antigen capture ELISA, sandwich ELISA, IgM (BDNF), Neutrophil Gelatinase-Associated Lipocalin antibody capture ELISA (MAC ELISA), and microparticle (NGAL). TNF-related Weak Inducer of Apoptosis enzyme immunoassay (MEIA); capillary electrophoresis (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), immunoassays (CEIA); radioimmunoassays (RIA); immu Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein noradiometric assays (IRMA); fluorescence polarization ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody immunoassays (FPIA); and chemiluminescence assays (CL): (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human marker purification, for example, by high pressure liquid Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human chromatography (HPLC), alone or in combination with mass Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI prostaglandin E2 (PGE2), and a combination thereof. TOF/MS, tandem MS, etc.); and other well known methods 0.132. In a specific embodiment, a method is provided for including, without limitation, Bradford assays, Coomassie aiding in the diagnosis of irritable bowel syndrome (IBS) in a blue staining, silver staining, assays for radiolabeled protein, Subject, said method comprising: contacting a blood or serum and mass spectrometry. sample from the subject with a substance P binding moiety 0127. In a specific embodiment, a method is provided for under conditions suitable to transform substance P present in aiding in the diagnosis of irritable bowel syndrome (IBS) in a the sample into a complex comprising substance P and the Subject, said method comprising: contacting a blood or serum substance Pbinding moiety; and determining the level of said sample from the Subject with a gastrointestinal hormone complex, thereby determining the level or concentration of binding moiety under conditions Suitable to transform the Substance P present in the sample. In one embodiment, the gastrointestinal hormone present in the sample into a com method further comprises detecting the level or concentration plex comprising the gastrointestinal hormone and the gas of at least one additional IBS marker selected from Brain trointestinal hormone binding moiety; and determining the Derived Neurotropic Factor (BDNF), Neutrophil Gelatinase level of said complex, thereby determining the level or con Associated Lipocalin (NGAL), TNF-related Weak Inducer of centration of the gastrointestinal hormone present in the Apoptosis (TWEAK), Growth-Related Oncogene Alpha sample. In a preferred embodiment, the gastrointestinal hor (GRO-O.), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of Met mone is selected from substance P. nerve growth factor alloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae (NGF), neurokinin A, neurokinin B. vasoactive intestinal Antibody (ASCA-IgA), Anti-CBir-1Antibody (CBirl), Anti peptide (VIP), glucagon-like peptide 2 (GLP-2), motilin, Human Neutrophil Cytoplasmic Antibody (ANCA), Anti pituitary adenylate cyclase-activating peptide (PACAP), and Human Tissue Transglutaminase IgA (tTG), histamine, a combination thereof. f-tryptase, and prostaglandin E. (PGE). 0128. In one embodiment, the method provided above, I0133. In another specific embodiment, a method is pro further comprises a step of comparing the level or concentra vided for aiding in the diagnosis of irritable bowel syndrome tion of the gastrointestinal hormone present in the sample to (IBS) in a Subject, said method comprising: contacting a a control level or concentration, wherein a difference in the blood or serum sample from the subject with a nerve growth level or concentration of the gastrointestinal hormone present factor binding moiety under conditions Suitable to transform in the sample relative to the control level or concentration is nerve growth factor present in the sample into a complex indicative of an increased likelihood of said subject having comprising nerve growth factor and the nerve growth factor IBS. binding moiety; and determining the level of said complex, 0129. In one embodiment, wherein the control level or thereby determining the level or concentration of nerve concentration is the level or concentration of the gastrointes growth factor present in the sample. In one embodiment, the tinal hormone present in a blood or serum sample from a method further comprises detecting the level or concentration healthy Subject, an increased level or concentration of the of at least one additional IBS marker selected from Brain gastrointestinal hormone present in the sample relative to the Derived Neurotropic Factor (BDNF), Neutrophil Gelatinase control level or concentration is indicative of an increased Associated Lipocalin (NGAL), TNF-related Weak Inducer of likelihood of said subject having IBS, while the same or a Apoptosis (TWEAK), Growth-Related Oncogene Alpha reduced level or concentration of the gastrointestinal hor (GRO-O.), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of Met mone present in the sample relative to the control level or alloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae concentration is indicative of an increased likelihood of said Antibody (ASCA-IgA), Anti-CBir-1Antibody (CBirl), Anti subject not having IBS. Human Neutrophil Cytoplasmic Antibody (ANCA), Anti 0130. In another embodiment, wherein the control level or Human Tissue Transglutaminase IgA (tTG), histamine, concentration is the level or concentration of the gastrointes B-tryptase, and prostaglandin E2 (PGE). US 2012/03 15630 A1 Dec. 13, 2012
0134. In another specific embodiment, a method is pro 0.137 In yet another specific embodiment, a method is vided for aiding in the diagnosis of irritable bowel syndrome provided for aiding in the diagnosis of irritable bowel syn (IBS) in a Subject, said method comprising: contacting a drome (IBS) in a Subject, said method comprising: contacting blood or serum sample from the subject with a neurokinin A a blood or serum sample from the Subject with a glucagon binding moiety under conditions suitable to transform neu like peptide 2 binding moiety under conditions suitable to rokinin A present in the sample into a complex comprising transform glucagon-like peptide 2 present in the sample into neurokinin A and the neurokinin Abinding moiety; and deter a complex comprising glucagon-like peptide 2 and the glu mining the level of said complex, thereby determining the cagon-like peptide 2 binding moiety; and determining the level or concentration of neurokinin A present in the sample. level of said complex, thereby determining the level or con In one embodiment, the method further comprises detecting centration of glucagon-like peptide 2 present in the sample. In the level or concentration of at least one additional IBS one embodiment, the method further comprises detecting the marker selected from Brain-Derived Neurotropic Factor level or concentration of at least one additional IBS marker (BDNF), Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis selected from Brain-Derived Neurotropic Factor (BDNF), (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein related Weak Inducer of Apoptosis (TWEAK), Growth-Re ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic and prostaglandin E. (PGE). Antibody (ANCA), Anti-Human Tissue Transglutaminase 0135) In yet another specific embodiment, a method is IgA (tTG), histamine, B-tryptase, and prostaglandin E provided for aiding in the diagnosis of irritable bowel syn (PGE). drome (IBS) in a Subject, said method comprising: contacting 0.138. In yet another specific embodiment, a method is a blood or serum sample from the subject with a neurokinin B provided for aiding in the diagnosis of irritable bowel syn binding moiety under conditions suitable to transform neu drome (IBS) in a Subject, said method comprising: contacting rokinin B present in the sample into a complex comprising a blood or serum sample from the subject with a motilin neurokinin Band the neurokinin B binding moiety; and deter binding moiety underconditions suitable to transform motilin mining the level of said complex, thereby determining the present in the sample into a complex comprising motilin and level or concentration of neurokinin B present in the sample. the motilin binding moiety; and determining the level of said In one embodiment, the method further comprises detecting complex, thereby determining the level or concentration of the level or concentration of at least one additional IBS motilin present in the sample. In one embodiment, the method marker selected from Brain-Derived Neurotropic Factor further comprises detecting the level or concentration of at (BDNF), Neutrophil Gelatinase-Associated Lipocalin least one additional IBS marker selected from Brain-Derived (NGAL). TNF-related Weak Inducer of Apoptosis Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), and prostaglandin E. (PGE). Anti-Human Tissue Transglutaminase IgA (tTG), histamine, 0136. In yet another specific embodiment, a method is f-tryptase, and prostaglandin E. (PGE). provided for aiding in the diagnosis of irritable bowel syn 0.139. In yet another specific embodiment, a method is drome (IBS) in a Subject, said method comprising: contacting provided for aiding in the diagnosis of irritable bowel syn a blood or serum sample from the subject with a vasoactive drome (IBS) in a Subject, said method comprising: contacting intestinal peptide binding moiety under conditions suitable to a blood or serum sample from the subject with a pituitary transform vasoactive intestinal peptide present in the sample adenylate cyclase-activating peptide binding moiety under into a complex comprising vasoactive intestinal peptide and conditions Suitable to transform pituitary adenylate cyclase the vasoactive intestinal peptide binding moiety; and deter activating peptide present in the sample into a complex com mining the level of said complex, thereby determining the prising pituitary adenylate cyclase-activating peptide and the level or concentration of vasoactive intestinal peptidepresent pituitary adenylate cyclase-activating peptide binding moi in the sample. In one embodiment, the method further com ety; and determining the level of said complex, thereby deter prises detecting the level or concentration of at least one mining the level or concentration of pituitary adenylate additional IBS marker selected from Brain-Derived Neuro cyclase-activating peptide present in the sample. In one tropic Factor (BDNF), Neutrophil Gelatinase-Associated embodiment, the method further comprises detecting the Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis level or concentration of at least one additional IBS marker (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), selected from Brain-Derived Neurotropic Factor (BDNF), Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody related Weak Inducer of Apoptosis (TWEAK), Growth-Re (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 and prostaglandin E. (PGE). Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic US 2012/03 15630 A1 Dec. 13, 2012
Antibody (ANCA), Anti-Human Tissue Transglutaminase likelihood of said subject having IBS, while a reduced level or IgA (tTG), histamine, B-tryptase, and prostaglandin E concentration of the serotonin metabolite present in the (PGE). sample relative to the control level or concentration is indica 0140. 4. Serotonin Metabolites tive of an increased likelihood of said subject not having IBS. 0141. In one embodiment, the present invention provides a 0146 In another related embodiment, methods are pro method for aiding in the diagnosis of irritable bowel syn vided for aiding in the diagnosis of irritable bowel syndrome drome (IBS) in a subject by detecting the level or concentra (IBS) in a subject by detecting the level or concentration of a tion of a serotonin metabolite in a biological sample from the serotonin metabolite and at least one additional IBS marker Subject (e.g., a blood or serum sample). The level or concen selected from Brain-Derived Neurotropic Factor (BDNF), tration of the serotonin metabolite may be determined by Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF various methods known in the art, including without limita related Weak Inducer of Apoptosis (TWEAK), Growth-Re tion, immunoassays, for example, enzyme immunoassays lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). (EIA) Such as enzyme multiplied immunoassay technique Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac (EMIT), enzyme-linked immunosorbent assay (ELISA), charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 antigen capture ELISA, sandwich ELISA, IgM antibody cap Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic ture ELISA (MAC ELISA), and microparticle enzyme immu Antibody (ANCA), Anti-Human Tissue Transglutaminase noassay (MEIA); capillary electrophoresis immunoassays IgA (tTG), histamine, B-tryptase, prostaglandin E2 (PGE), (CEIA); radioimmunoassays (RIA); immunoradiometric and a combination thereof. assays (IRMA); fluorescence polarization immunoassays 0.147. In a specific embodiment, a method is provided for (FPIA); and chemiluminescence assays (CL); marker purifi aiding in the diagnosis of irritable bowel syndrome (IBS) in a cation, for example, by high pressure liquid chromatography Subject, said method comprising: contacting a blood or serum (HPLC), alone or in combination with mass spectrometry sample from the Subject with a serotonin binding moiety (e.g., MALDI/MS, MALDI-TOF/MS, SELDI-TOF/MS, tan under conditions Suitable to transform serotonin present in dem MS, etc.); and other well known methods including, the sample into a complex comprising serotonin and the sero without limitation, Bradford assays, Coomassie blue stain tonin binding moiety; and determining the level of said com ing, silver staining, assays for radiolabeled protein, and mass plex, thereby determining the level or concentration of sero spectrometry. tonin present in the sample. In one embodiment, the method 0142. In a specific embodiment, a method is provided for further comprises detecting the level or concentration of at aiding in the diagnosis of irritable bowel syndrome (IBS) in a least one additional IBS marker selected from Brain-Derived Subject, said method comprising: contacting a blood or serum Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ sample from the subject with a serotonin metabolite binding ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo moiety under conditions suitable to transform the serotonin ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO metabolite present in the sample into a complex comprising O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of the serotonin metabolite and the serotonin metabolite binding Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi moiety; and determining the level of said complex, thereby siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), determining the level or concentration of the serotonin Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), metabolite present in the sample. In a preferred embodiment, Anti-Human Tissue Transglutaminase IgA (tTG), histamine, serotonin metabolite is selected from serotonin, tryptophan, f-tryptase, and prostaglandin E. (PGE). serotonin O-sulfate, 5-hydroxyindoleacetic acid, 5-HT glu 0.148. In another specific embodiment, a method is pro curonide, and a combination thereof. vided for aiding in the diagnosis of irritable bowel syndrome 0143. In one embodiment, the method provided above, (IBS) in a Subject, said method comprising: contacting a further comprises a step of comparing the level or concentra blood or serum sample from the subject with a tryptophan tion of the serotonin metabolite present in the sample to a binding moiety under conditions suitable to transform tryp control level or concentration, wherein a difference in the tophan present in the sample into a complex comprising tryp level or concentration of the serotonin metabolite present in tophan and the tryptophan binding moiety; and determining the sample relative to the control level or concentration is the level of said complex, thereby determining the level or indicative of an increased likelihood of said subject having concentration of tryptophan present in the sample. In one IBS. embodiment, the method further comprises detecting the 0144. In one embodiment, wherein the control level or level or concentration of at least one additional IBS marker concentration is the level or concentration of the serotonin selected from Brain-Derived Neurotropic Factor (BDNF), metabolite present in a blood or serum sample from a healthy Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF Subject, an increased level or concentration of the serotonin related Weak Inducer of Apoptosis (TWEAK), Growth-Re metabolite present in the sample relative to the control level or lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). concentration is indicative of an increased likelihood of said Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac subject having IBS, while the same or a reduced level or charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 concentration of the serotonin metabolite present in the Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic sample relative to the control level or concentration is indica Antibody (ANCA), Anti-Human Tissue Transglutaminase tive of an increased likelihood of said subject not having IBS. IgA (tTG), histamine, B-tryptase, and prostaglandin E 0145. In another embodiment, wherein the control level or (PGE). concentration is the level or concentration of the serotonin 0149. In another specific embodiment, a method is pro metabolite present in a blood or serum sample from a subject vided for aiding in the diagnosis of irritable bowel syndrome with IBS, the same or an increased level or concentration of (IBS) in a Subject, said method comprising: contacting a the serotonin metabolite present in the sample relative to the blood or serum sample from the subject with a serotonin control level or concentration is indicative of an increased O-sulfate binding moiety under conditions Suitable to trans US 2012/03 15630 A1 Dec. 13, 2012 form serotonin O-sulfate present in the sample into a complex Subject (e.g., a blood or serum sample). The level or concen comprising serotonin O-sulfate and the serotonin O-sulfate tration of the serum metabolite may be determined by various binding moiety; and determining the level of said complex, methods known in the art, including without limitation, thereby determining the level or concentration of serotonin immunoassays, for example, enzyme immunoassays (EIA) O-sulfate present in the sample. In one embodiment, the Such as enzyme multiplied immunoassay technique (EMIT), method further comprises detecting the level or concentration enzyme-linked immunosorbent assay (ELISA), antigen cap of at least one additional IBS marker selected from Brain ture ELISA, sandwich ELISA, IgM antibody capture ELISA Derived Neurotropic Factor (BDNF), Neutrophil Gelatinase (MAC ELISA), and microparticle enzyme immunoassay Associated Lipocalin (NGAL), TNF-related Weak Inducer of (MEIA); capillary electrophoresis immunoassays (CEIA); Apoptosis (TWEAK), Growth-Related Oncogene Alpha radioimmunoassays (RIA); immunoradiometric assays (GRO-O.), Interleukin-1 Beta (IL-3), Tissue Inhibitor of Met (IRMA); fluorescence polarization immunoassays (FPIA); alloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae and chemiluminescence assays (CL); marker purification, for Antibody (ASCA-IgA), Anti-CBir-1Antibody (CBirl), Anti example, by high pressure liquid chromatography (HPLC), Human Neutrophil Cytoplasmic Antibody (ANCA), Anti alone or in combination with mass spectrometry (e.g., Human Tissue Transglutaminase IgA (tTG), histamine, MALDI/MS, MALDI-TOF/MS, SELDI-TOF/MS, tandem f-tryptase, and prostaglandin E. (PGE). MS, etc.); and other well known methods including, without 0150. In yet another specific embodiment, a method is limitation, Bradford assays, Coomassie blue staining, silver provided for aiding in the diagnosis of irritable bowel syn staining, assays for radiolabeled protein, and mass spectrom drome (IBS) in a Subject, said method comprising: contacting etry. a blood or serum sample from the subject with a 5-hydroxy 0154) In a specific embodiment, a method is provided for indoleacetic acid binding moiety under conditions Suitable to aiding in the diagnosis of irritable bowel syndrome (IBS) in a transform 5-hydroxyindoleacetic acid present in the sample Subject, said method comprising: contacting a blood or serum into a complex comprising 5-hydroxyindoleacetic acid and sample from the subject with a serum metabolite binding the 5-hydroxyindoleacetic acid binding moiety; and deter moiety under conditions Suitable to transform the serum mining the level of said complex, thereby determining the metabolite present in the sample into a complex comprising level or concentration of 5-hydroxyindoleacetic acid present the serum metabolite and the serum metabolite binding moi in the sample. In one embodiment, the method further com ety; and determining the level of said complex, thereby deter prises detecting the level or concentration of at least one mining the level or concentration of the serum metabolite additional IBS marker selected from Brain-Derived Neuro present in the sample. In a preferred embodiment, the serum tropic Factor (BDNF), Neutrophil Gelatinase-Associated metabolite is selected from tyrosine and/or phenylalanine. Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis 0.155. In one embodiment, the method provided above, (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), further comprises a step of comparing the level or concentra Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein tion of the serum metabolite present in the sample to a control ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody level or concentration, wherein a difference in the level or (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human concentration of the serum metabolite present in the sample Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human relative to the control level or concentration is indicative of an Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, increased likelihood of said subject having IBS. and prostaglandin E. (PGE). 0156. In one embodiment, wherein the control level or 0151. In yet another specific embodiment, a method is concentration is the level or concentration of the serum provided for aiding in the diagnosis of irritable bowel syn metabolite present in a blood or serum sample from a healthy drome (IBS) in a Subject, said method comprising: contacting Subject, an increased level or concentration of the serum a blood or serum sample from the subject with a 5-HT glu metabolite present in the sample relative to the control level or curonide binding moiety under conditions Suitable to trans concentration is indicative of an increased likelihood of said form 5-HT glucuronide present in the sample into a complex subject having IBS, while the same or a reduced level or comprising 5-HT glucuronide and the 5-HT glucuronide concentration of the serum metabolite present in the sample binding moiety; and determining the level of said complex, relative to the control level or concentration is indicative of an thereby determining the level or concentration of 5-HT glu increased likelihood of said subject not having IBS. curonide present in the sample. In one embodiment, the 0157. In another embodiment, wherein the control level or method further comprises detecting the level or concentration concentration is the level or concentration of the serum of at least one additional IBS marker selected from Brain metabolite present in a blood or serum sample from a subject Derived Neurotropic Factor (BDNF), Neutrophil Gelatinase with IBS, the same or an increased level or concentration of Associated Lipocalin (NGAL), TNF-related Weak Inducer of the serum metabolite present in the sample relative to the Apoptosis (TWEAK), Growth-Related Oncogene Alpha control level or concentration is indicative of an increased (GRO-O.), Interleukin-1 Beta (IL-3), Tissue Inhibitor of Met likelihood of said subject having IBS, while a reduced level or alloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae concentration of the serum metabolite present in the sample Antibody (ASCA-IgA), Anti-CBir-1Antibody (CBirl), Anti relative to the control level or concentration is indicative of an Human Neutrophil Cytoplasmic Antibody (ANCA), Anti increased likelihood of said subject not having IBS. Human Tissue Transglutaminase IgA (tTG), histamine, 0158. In another related embodiment, methods are pro f-tryptase, and prostaglandin E. (PGE). vided for aiding in the diagnosis of irritable bowel syndrome 0152 5. Serum Metabolites (IBS) in a subject by detecting the level or concentration of a 0153. In one embodiment, the present invention provides a serum metabolite and at least one additional IBS marker method for aiding in the diagnosis of irritable bowel syn selected from Brain-Derived Neurotropic Factor (BDNF), drome (IBS) in a subject by detecting the level or concentra Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF tion of a serum metabolite in a biological sample from the related Weak Inducer of Apoptosis (TWEAK), Growth-Re US 2012/03 15630 A1 Dec. 13, 2012
lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). immunoassays (FPIA); and chemiluminescence assays (CL): Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac marker purification, for example, by high pressure liquid charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 chromatography (HPLC), alone or in combination with mass Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI Antibody (ANCA), Anti-Human Tissue Transglutaminase TOF/MS, tandem MS, etc.); and other well known methods IgA (tTG), histamine, B-tryptase, prostaglandin E. (PGE), including, without limitation, Bradford assays, Coomassie and a combination thereof. blue staining, silver staining, assays for radiolabeled protein, 0159. In a specific embodiment, a method is provided for and mass spectrometry. aiding in the diagnosis of irritable bowel syndrome (IBS) in a 0163. In a specific embodiment, a method is provided for Subject, said method comprising: contacting a blood or serum aiding in the diagnosis of irritable bowel syndrome (IBS) in a sample from the Subject with a tyrosine binding moiety under Subject, said method comprising: contacting a blood or serum conditions suitable to transform tyrosine present in the sample from the Subject with a serotonin pathway marker sample into a complex comprising tyrosine and the tyrosine binding moiety under conditions Suitable to transform the moiety; and determining the level of said complex, thereby serotonin pathway marker present in the sample into a com determining the level or concentration of tyrosine present in plex comprising the serotonin pathway marker and the sero the sample. In one embodiment, the method further com tonin pathway marker binding moiety; and determining the prises detecting the level or concentration of at least one level of said complex, thereby determining the level or con additional IBS marker selected from Brain-Derived Neuro centration of the serotonin pathway marker present in the tropic Factor (BDNF), Neutrophil Gelatinase-Associated sample. In a preferred embodiment, the serotonin pathway Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis marker is selected from UDP-glucuronosyltransferase 1-6, (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), serotonin reuptake transporter, tryptophan hydroxylase 1, Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein monoamine oxidase A, monoamine oxidase B. hydrox ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody ytryptamine (serotonin) receptor 3A, and a combination (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human thereof. Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human 0164. In one embodiment, the method provided above, Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, further comprises a step of comparing the level or concentra and prostaglandin E. (PGE). tion of the serotonin pathway marker present in the sample to 0160. In another specific embodiment, a method is pro a control level or concentration, wherein a difference in the vided for aiding in the diagnosis of irritable bowel syndrome level or concentration of the serotonin pathway marker (IBS) in a Subject, said method comprising: contacting a present in the sample relative to the control level or concen blood or serum sample from the subject with a phenylalanine tration is indicative of an increased likelihood of said subject binding moiety under conditions suitable to phenylalanine having IBS. present in the sample into a complex comprising phenylala 0.165. In one embodiment, wherein the control level or nine and the phenylalanine binding moiety; and determining concentration is the level or concentration of the serotonin the level of said complex, thereby determining the level or pathway marker present in a blood or serum sample from a concentration of phenylalanine present in the sample. In one healthy Subject, an increased level or concentration of the embodiment, the method further comprises detecting the serotonin pathway marker present in the sample relative to the level or concentration of at least one additional IBS marker control level or concentration is indicative of an increased selected from Brain-Derived Neurotropic Factor (BDNF), likelihood of said subject having IBS, while the same or a Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF reduced level or concentration of the serotonin pathway related Weak Inducer of Apoptosis (TWEAK), Growth-Re marker present in the sample relative to the control level or lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). concentration is indicative of an increased likelihood of said Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac subject not having IBS. charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 (0166 In another embodiment, wherein the control level or Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic concentration is the level or concentration of the serotonin Antibody (ANCA), Anti-Human Tissue Transglutaminase pathway marker present in a blood or serum sample from a IgA (tTG), histamine, B-tryptase, and prostaglandin E subject with IBS, the same or an increased level or concen (PGE). tration of the serotonin pathway marker present in the sample (0161 6. Serotonin Pathway Markers relative to the control level or concentration is indicative of an 0162. In one embodiment, the present invention provides a increased likelihood of said subject having IBS, while a method for aiding in the diagnosis of irritable bowel syn reduced level or concentration of the serotonin pathway drome (IBS) in a subject by detecting the level or concentra marker present in the sample relative to the control level or tion of a serotonin pathway marker in a biological sample concentration is indicative of an increased likelihood of said from the subject (e.g., a blood or serum sample). The level or subject not having IBS. concentration of the serotonin pathway marker may be deter 0167. In another related embodiment, methods are pro mined by various methods known in the art, including without vided for aiding in the diagnosis of irritable bowel syndrome limitation, immunoassays, for example, enzyme immunoas (IBS) in a subject by detecting the level or concentration of a says (EIA) Such as enzyme multiplied immunoassay tech serotonin pathway marker and at least one additional IBS nique (EMIT), enzyme-linked immunosorbent assay marker selected from Brain-Derived Neurotropic Factor (ELISA), antigen capture ELISA, sandwich ELISA, IgM (BDNF), Neutrophil Gelatinase-Associated Lipocalin antibody capture ELISA (MAC ELISA), and microparticle (NGAL). TNF-related Weak Inducer of Apoptosis enzyme immunoassay (MEIA); capillary electrophoresis (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), immunoassays (CEIA); radioimmunoassays (RIA); immu Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein noradiometric assays (IRMA); fluorescence polarization ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody US 2012/03 15630 A1 Dec. 13, 2012
(ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human prostaglandin E. (PGE), and a combination thereof. Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human 0.168. In a specific embodiment, a method is provided for Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, aiding in the diagnosis of irritable bowel syndrome (IBS) in a and prostaglandin E. (PGE). Subject, said method comprising: contacting a blood or serum 0171 In yet another specific embodiment, a method is sample from the subject with a UDP-glucuronosyltransferase provided for aiding in the diagnosis of irritable bowel syn 1-6 binding moiety under conditions suitable to transform drome (IBS) in a Subject, said method comprising: contacting UDP-glucuronosyltransferase 1-6 present in the sample into a a blood or serum sample from the Subject with a monoamine complex comprising UDP-glucuronosyltransferase 1-6 and oxidase Abinding moiety under conditions suitable to trans the UDP-glucuronosyltransferase 1-6 binding moiety; and form monoamine oxidase A present in the sample into a determining the level of said complex, thereby determining complex comprising monoamine oxidase A and the monoam the level or concentration of UDP-glucuronosyltransferase ine oxidase A binding moiety; and determining the level of 1-6 present in the sample. In one embodiment, the method said complex, thereby determining the level or concentration further comprises detecting the level or concentration of at of monoamine oxidase A present in the sample. In one least one additional IBS marker selected from Brain-Derived embodiment, the method further comprises detecting the Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ level or concentration of at least one additional IBS marker ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo selected from Brain-Derived Neurotropic Factor (BDNF), ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of related Weak Inducer of Apoptosis (TWEAK), Growth-Re Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Anti-Human Tissue Transglutaminase IgA (tTG), histamine, Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic f-tryptase, and prostaglandin E. (PGE). Antibody (ANCA), Anti-Human Tissue Transglutaminase 0169. In another specific embodiment, a method is pro IgA (tTG), histamine, B-tryptase, and prostaglandin E vided for aiding in the diagnosis of irritable bowel syndrome (PGE). (IBS) in a subject, said method comprising: contacting a 0172. In yet another specific embodiment, a method is blood or serum sample from the subject with a serotonin provided for aiding in the diagnosis of irritable bowel syn reuptake transporter binding moiety under conditions Suit drome (IBS) in a Subject, said method comprising: contacting able to transform serotonin reuptake transporter present in the a blood or serum sample from the Subject with a monoamine sample into a complex comprising serotonin reuptake trans oxidase B binding moiety under conditions suitable to trans porter and the serotonin reuptake transporter binding moiety; form monoamine oxidase B present in the sample into a and determining the level of said complex, thereby determin complex comprising monoamine oxidase Band the monoam ing the level or concentration of serotonin reuptake trans ine oxidase B binding moiety; and determining the level of porter present in the sample. In one embodiment, the method said complex, thereby determining the level or concentration further comprises detecting the level or concentration of at of monoamine oxidase B present in the sample. In one least one additional IBS marker selected from Brain-Derived embodiment, the method further comprises detecting the Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ level or concentration of at least one additional IBS marker ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo selected from Brain-Derived Neurotropic Factor (BDNF), ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO Neutrophil Gelatinase-Associated Lipocalin (NGAL). TNF O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of related Weak Inducer of Apoptosis (TWEAK), Growth-Re Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi lated Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B). siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Anti-Human Tissue Transglutaminase IgA (tTG), histamine, Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic f-tryptase, and prostaglandin E. (PGE). Antibody (ANCA), Anti-Human Tissue Transglutaminase 0170 In another specific embodiment, a method is pro IgA (tTG), histamine, B-tryptase, and prostaglandin E vided for aiding in the diagnosis of irritable bowel syndrome (PGE). (IBS) in a Subject, said method comprising: contacting a 0173. In yet another specific embodiment, a method is blood or serum sample from the subject with a tryptophan provided for aiding in the diagnosis of irritable bowel syn hydroxylase 1 binding moiety under conditions Suitable to drome (IBS) in a Subject, said method comprising: contacting transform tryptophan hydroxylase 1 present in the sample a blood or serum sample from the subject with a hydrox into a complex comprising tryptophan hydroxylase 1 and the ytryptamine (serotonin) receptor 3A binding moiety under tryptophan hydroxylase 1 binding moiety; and determining conditions Suitable to transform hydroxytryptamine (seroto the level of said complex, thereby determining the level or nin) receptor 3A present in the sample into a complex com concentration of tryptophan hydroxylase 1 present in the prising hydroxytryptamine (serotonin) receptor 3A and the sample. In one embodiment, the method further comprises hydroxytryptamine (serotonin) receptor 3A binding moiety; detecting the level or concentration of at least one additional and determining the level of said complex, thereby determin IBS marker selected from Brain-Derived Neurotropic Factor ing the level or concentration of hydroxytryptamine (seroto (BDNF), Neutrophil Gelatinase-Associated Lipocalin nin) receptor 3A present in the sample. In one embodiment, (NGAL). TNF-related Weak Inducer of Apoptosis the method further comprises detecting the level or concen (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), tration of at least one additional IBS marker selected from US 2012/03 15630 A1 Dec. 13, 2012
Brain-Derived Neurotropic Factor (BDNF), Neutrophil 0179. In another embodiment, wherein the control level or Gelatinase-Associated Lipocalin (NGAL). TNF-related concentration is the level or concentration of carbohydrate Weak Inducer of Apoptosis (TWEAK), Growth-Related deficient transferrin present in a blood or serum sample from Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B), Tissue a subject with IBS, the same or an increased level or concen Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Saccharo tration of carbohydrate deficient transferrin present in the myces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Anti sample relative to the control level or concentration is indica body (CBirl), Anti-Human Neutrophil Cytoplasmic Anti tive of an increased likelihood of said subject having IBS, body (ANCA), Anti-Human Tissue Transglutaminase IgA while a reduced level or concentration of carbohydrate defi cient transferrin present in the sample relative to the control (tTG), histamine, B-tryptase, and prostaglandin E2 (PGE2). level or concentration is indicative of an increased likelihood (0174 7. Carbohydrate Deficient Transferrin (CDT) of said subject not having IBS. 0.175. In one embodiment, the present invention provides a 0180. In another related embodiment, methods are pro method for aiding in the diagnosis of irritable bowel syn vided for aiding in the diagnosis of irritable bowel syndrome drome (IBS) in a subject by detecting the level or concentra (IBS) in a subject by detecting the level or concentration of tion carbohydrate deficient transferrin in a biological sample carbohydrate deficient transferrin and at least one additional from the subject (e.g., a blood or serum sample). The level or IBS marker selected from Brain-Derived Neurotropic Factor concentration of carbohydrate deficient transferrin may be (BDNF), Neutrophil Gelatinase-Associated Lipocalin determined by various methods known in the art, including (NGAL). TNF-related Weak Inducer of Apoptosis without limitation, immunoassays, for example, enzyme (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), immunoassays (EIA) Such as enzyme multiplied immunoas Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein say technique (EMIT), enzyme-linked immunosorbent assay ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody (ELISA), antigen capture ELISA, sandwich ELISA, IgM (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human antibody capture ELISA (MAC ELISA), and microparticle Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human enzyme immunoassay (MEIA); capillary electrophoresis Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, immunoassays (CEIA); radioimmunoassays (RIA); immu prostaglandin E2 (PGE2), and a combination thereof. noradiometric assays (IRMA); fluorescence polarization 0181. In a specific embodiment, a method is provided for immunoassays (FPIA); and chemiluminescence assays (CL): aiding in the diagnosis of irritable bowel syndrome (IBS) in a marker purification, for example, by high pressure liquid Subject, said method comprising: contacting a blood or serum chromatography (HPLC), alone or in combination with mass sample from the subject with a carbohydrate deficient trans spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI ferrin binding moiety under conditions Suitable to transform TOF/MS, tandem MS, etc.); and other well known methods carbohydrate deficient transferrin present in the sample into a including, without limitation, Bradford assays, Coomassie complex comprising carbohydrate deficient transferrin and blue staining, silver staining, assays for radiolabeled protein, the carbohydrate deficient transferrin binding moiety; and and mass spectrometry. determining the level of said complex, thereby determining 0176). In a specific embodiment, a method is provided for the level or concentration of carbohydrate deficient transfer aiding in the diagnosis of irritable bowel syndrome (IBS) in a rin present in the sample. In one embodiment, the method Subject, said method comprising: contacting a blood or serum further comprises detecting the level or concentration of at sample from the subject with a carbohydrate deficient trans least one additional IBS marker selected from Brain-Derived ferrin binding moiety under conditions Suitable to transform Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ carbohydrate deficient transferrin present in the sample into a ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo complex comprising carbohydrate deficient transferrin and ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO the carbohydrate deficient transferrin binding moiety; and O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of determining the level of said complex, thereby determining Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi the level or concentration of carbohydrate deficient transfer siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), rin present in the sample. Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), 0177. In one embodiment, the method provided above Anti-Human Tissue Transglutaminase IgA (tTG), histamine, further comprises a step of comparing the level or concentra f-tryptase, and prostaglandin E. (PGE). tion of carbohydrate deficient transferrin present in the 0182 B. Symptom Profiles sample to a control level or concentration, wherein a differ 0183 In some embodiments, the method for aiding in the ence in the level or concentration of carbohydrate deficient diagnosis of IBS comprises determining a diagnostic marker transferrin present in the sample relative to the control level or profile optionally in combination with a symptom profile, concentration is indicative of an increased likelihood of said wherein the symptom profile is determined by identifying the subject having IBS. presence or severity of at least one symptom in the individual; 0178. In one embodiment, wherein the control level or and classifying the sample as an IBS sample or non-IBS concentration is the level or concentration of carbohydrate sample using an algorithm based upon the diagnostic marker deficient transferrin present in a blood or serum sample from profile and the symptom profile. One skilled in the art will a healthy Subject, an increased level or concentration of car appreciate that the diagnostic marker profile and the symptom bohydrate deficient transferrin present in the sample relative profile can be determined simultaneously or sequentially in to the control level or concentration is indicative of an any order. increased likelihood of said subject having IBS, while the 0.184 The symptom profile is typically determined by same or a reduced level or concentration of the stress marker identifying the presence or severity of at least one symptom present in the sample relative to the control level or concen selected from the group consisting of chest pain, chest dis tration is indicative of an increased likelihood of said subject comfort, heartburn, uncomfortable fullness after having a not having IBS. regular-sized meal, inability to finish a regular-sized meal, US 2012/03 15630 A1 Dec. 13, 2012 20 abdominal pain, abdominal discomfort, constipation, diar 95%, 96%, 97%, 98%, or 99%. As such, the classification of rhea, bloating, abdominal distension, negative thoughts or a sample as an IBS sample or non-IBS sample is useful for feelings associated with having pain or discomfort, and com aiding in the diagnosis of IBS in a Subject. binations thereof. 0190. In certain other instances, the statistical algorithm is 0185. In preferred embodiments, the presence or severity a combination of at least two learning statistical classifier of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms systems. Preferably, the combination of learning statistical described herein is identified to generate a symptom profile classifier systems comprises a RF and a NN, e.g., used in that is useful for predicting IBS. In certain instances, a ques tandem or parallel. As a non-limiting example, a RF can first tionnaire or other form of written, verbal, or telephone survey be used to generate a prediction or probability value based is used to produce the symptom profile. The questionnaire or upon the diagnostic marker profile, alone or in combination Survey typically comprises a standardized set of questions with a symptom profile, and a NN can then be used to classify and answers for the purpose of gathering information from the sample as an IBS sample or non-IBS sample based upon respondents regarding their current and/or recent IBS-related the prediction or probability value and the same or different symptoms. For instance. Example 3 provides exemplary diagnostic marker profile or combination of profiles. Advan questions that can be included in a questionnaire for identi tageously, the hybrid RF/NN learning statistical classifier fying the presence or severity of one or more IBS-related system of the present invention classifies the sample as an IBS symptoms in the individual. sample with a sensitivity, specificity, positive predictive 0186. In certain embodiments, the symptom profile is pro value, negative predictive value, and/or overall accuracy of at duced by compiling and/or analyzing all or a Subset of the least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, answers to the questions set forth in the questionnaire or 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, Survey. In certain other embodiments, the symptom profile is 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In a particularly produced based upon the individual’s response to the follow preferred embodiment, the statistical algorithm is a random ing question: “Are you currently experiencing any symp forest classifier or a combination of a random forest classifier toms?’ The symptom profile generated in accordance with and a neural network classifier. either of these embodiments can be used in combination with 0191 In some instances, the data obtained from using the a diagnostic marker profile in the algorithmic-based methods learning statistical classifier system or systems can be pro described herein to improve the accuracy of predicting IBS. cessed using a processing algorithm. Such a processing algo 0187 C. Use of Statistical Algorithms rithm can be selected, for example, from the group consisting 0188 In some embodiments, methods for aiding in the of a multilayer perceptron, backpropagation network, and diagnosis of irritable bowel syndrome (IBS) in a subject is Levenberg-Marquardt algorithm. In other instances, a com based upon the diagnostic marker profile, alone or in combi bination of such processing algorithms can be used, such as in nation with a symptom profile, in conjunction with a statisti a parallel or serial fashion. cal algorithm. In certain instances, the statistical algorithm is 0.192 In certain other embodiments, the methods of the a learning statistical classifier system. The learning statistical present invention further comprise sending the IBS classifi classifier system can be selected from the group consisting of cation results to a clinician, e.g., a gastroenterologist or a a random forest (RF), classification and regression tree general practitioner. In another embodiment, the methods of (C&RT), boosted tree, neural network (NN), support vector the present invention provide a diagnosis in the form of a machine (SVM), general chi-squared automatic interaction probability that the individual has IBS. For example, the detector model, interactive tree, multiadaptive regression individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, spline, machine learning classifier, and combinations thereof. 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, Preferably, the learning statistical classifier system is a tree 80%,85%, 90%.95%, or greaterprobability of having IBS. In based statistical algorithm (e.g., RF, C&RT, etc.) and/or a NN yet another embodiment, the methods of the present invention (e.g., artificial NN, etc.). Additional examples of learning further provide a prognosis of IBS in the individual. For statistical classifier systems suitable for use in the present example, the prognosis can be Surgery, development of a invention are described in U.S. patent application Ser. No. category or clinical subtype of IBS, development of one or 1 1/368,285. In certain embodiments, the methods comprise more symptoms, or recovery from the disease. classifying a sample from the Subject as an IBS sample or (0193 D. Assays and Kits non-IBS sample. 0194 Any of a variety of assays, techniques, and kits 0189 In certain instances, the statistical algorithm is a known in the art can be used to determine the presence or level single learning statistical classifier system. Preferably, the of one or more markers in a sample to classify whether the single learning statistical classifier system comprises a tree sample is associated with IBS. based statistical algorithm such as a RF or C&RT. As a non 0.195 The present invention relies, in part, on determining limiting example, a single learning statistical classifier sys the presence or level of at least one marker in a sample tem can be used to classify the sample as an IBS sample or obtained from an individual. As used herein, the term “deter non-IBS sample based upon a prediction or probability value mining the presence of at least one marker” includes deter and the presence or level of at least one diagnostic marker mining the presence of each marker of interest by using any (i.e., diagnostic marker profile), alone or in combination with quantitative or qualitative assay known to one of skill in the the presence or severity of at least one symptom (i.e., symp art. In certain instances, qualitative assays that determine the tom profile). The use of a single learning statistical classifier presence or absence of a particular trait, variable, or bio system typically classifies the sample as an IBS sample with chemical or serological Substance (e.g., protein or antibody) a sensitivity, specificity, positive predictive value, negative are Suitable for detecting each marker of interest. In certain predictive value, and/or overall accuracy of at least about other instances, quantitative assays that determine the pres 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, ence or absence of RNA, protein, antibody, or activity are 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, Suitable for detecting each marker of interest. As used herein, US 2012/03 15630 A1 Dec. 13, 2012
the term “determining the level of at least one marker” concentration, are suitable for use in the present invention. includes determining the level of each marker of interest by Nephelometry assays are commercially available from Beck using any director indirect quantitative assay known to one of man Coulter (Brea, Calif.; Kit #449430) and can be per skill in the art. In certain instances, quantitative assays that formed using a Behring Nephelometer Analyzer (Fink et al., determine, for example, the relative or absolute amount of J. Clin. Chem. Clin. Biol. Chem., 27:261-276 (1989)). RNA, protein, antibody, or activity are suitable for determin 0200 Antigen capture ELISA can be useful for determin ing the level of each marker of interest. One skilled in the art ing the presence or level of one or more markers in a sample. will appreciate that any assay useful for determining the level For example, in an antigen capture ELISA, an antibody of a marker is also useful for determining the presence or directed to a marker of interest is bound to a solid phase and absence of the marker. sample is added such that the marker is bound by the antibody. 0196. As used herein, the term “antibody' includes a After unbound proteins are removed by washing, the amount population of immunoglobulin molecules, which can be poly of bound marker can be quantitated using, e.g., a radioimmu clonal or monoclonal and of any isotype, or an immunologi noassay (see, e.g., Harlow and Lane, Antibodies: A Labora cally active fragment of an immunoglobulin molecule. Such tory Manual, Cold Spring Harbor Laboratory, New York, an immunologically active fragment contains the heavy and 1988)). Sandwich ELISA can also be suitable for use in the light chain variable regions, which make up the portion of the present invention. For example, in a two-antibody sandwich antibody molecule that specifically binds an antigen. For assay, a first antibody is bound to a solid Support, and the example, an immunologically active fragment of an immu marker of interest is allowed to bind to the first antibody. The noglobulin molecule known in the art as Fab, Fab' or F(ab') amount of the marker is quantitated by measuring the amount is included within the meaning of the term antibody. of a second antibody that binds the marker. The antibodies can 0.197 Flow cytometry can be used to determine the pres be immobilized onto a variety of Solid Supports, such as ence or level of one or more markers in a sample. Such flow magnetic or chromatographic matrix particles, the Surface of cytometric assays, including bead based immunoassays, can an assay plate (e.g., microtiter wells), pieces of a solid Sub be used to determine, e.g., antibody marker levels in the same strate material or membrane (e.g., plastic, nylon, paper), and manner as described for detecting serum antibodies to Can the like. An assay strip can be prepared by coating the anti dida albicans and HIV proteins (see, e.g., Bishop and Davis, body or a plurality of antibodies in an array on a Solid Support. J. Immunol. Methods, 210:79-87 (1997); McHugh et al., J. This strip can then be dipped into the test sample and pro Immunol. Methods, 116:213 (1989); Scillian et al., Blood, cessed quickly through washes and detection steps to gener 73:2041 (1989)). ate a measurable signal, such as a colored spot. 0198 Phage display technology for expressing a recom 0201 A radioimmunoassay using, for example, an iodine binant antigen specific for a marker can also be used to deter 125 ('I) labeled secondary antibody (Harlow and Lane, mine the presence or level of one or more markers in a sample. Supra) is also suitable for determining the presence or level of Phage particles expressing an antigen specific for, e.g., an one or more markers in a sample. A secondary antibody antibody marker can be anchored, if desired, to a multi-well labeled with a chemiluminescent marker can also be suitable plate using an antibody such as an anti-phage monoclonal for use in the present invention. A chemiluminescence assay antibody (Felici et al., “Phage-Displayed Peptides as Tools using a chemiluminescent secondary antibody is suitable for for Characterization of Human Sera’ in Abelson (Ed.), Meth sensitive, non-radioactive detection of marker levels. Such ods in Enzymol., 267, San Diego: Academic Press, Inc. secondary antibodies can be obtained commercially from (1996)). various sources, e.g., Amersham LifeSciences, Inc. (Arling 0199. A variety of immunoassay techniques, including ton Heights, Ill.). competitive and non-competitive immunoassays, can be used 0202 The immunoassays described above are particularly to determine the presence or level of one or more markers in useful for determining the presence or level of one or more a sample (see, e.g., Self and Cook, Curr: Opin. Biotechnol., markers in a sample. As a non-limiting example, an ELISA 7:60-65 (1996)). The term immunoassay encompasses tech using an IL-8-binding molecule Such as an anti-IL-8 antibody niques including, without limitation, enzyme immunoassays or an extracellular IL-8-binding protein (e.g., IL-8 receptor) (EIA) Such as enzyme multiplied immunoassay technique is useful for determining whether a sample is positive for IL-8 (EMIT), enzyme-linked immunosorbent assay (ELISA), protein or for determining IL-8 protein levels in a sample. A antigen capture ELISA, sandwich ELISA, IgM antibody cap fixed neutrophil ELISA is useful for determining whether a ture ELISA (MAC ELISA), and microparticle enzyme immu sample is positive for ANCA or for determining ANCA levels noassay (MEIA); capillary electrophoresis immunoassays in a sample. Similarly, an ELISA using yeast cell wall phos (CEIA); radioimmunoassays (RIA); immunoradiometric phopeptidomannan is useful for determining whether a assays (IRMA); fluorescence polarization immunoassays sample is positive for ASCA-IgA and/or ASCA-IgG, or for (FPIA); and chemiluminescence assays (CL). If desired, such determining ASCA-IgA and/or ASCA-IgG levels in a immunoassays can be automated. Immunoassays can also be sample. An ELISA using OmpC protein or a fragment thereof used in conjunction with laser induced fluorescence (see, e.g., is useful for determining whether a sample is positive for Schmalzing and Nashabeh, Electrophoresis, 18:2184-2 193 anti-Ompo antibodies, or for determining anti-Ompo anti (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-480 body levels in a sample. An ELISA using I2 protein or a (1997)). Liposome immunoassays, such as flow-injection fragment thereof is useful for determining whether a sample liposome immunoassays and liposome immunosensors, are is positive for anti-I2 antibodies, or for determining anti-I2 also suitable for use in the present invention (see, e.g., Rongen antibody levels in a sample. An ELISA using flagellin protein et al., J. Immunol. Methods, 204:105-133 (1997)). In addi (e.g., Cbir-1 flagellin) or a fragment thereof is useful for tion, nephelometry assays, in which the formation of protein/ determining whether a sample is positive for anti-flagellin antibody complexes results in increased light scatter that is antibodies, or for determining anti-flagellin antibody levels in converted to a peak rate signal as a function of the marker a sample. In addition, the immunoassays described above are US 2012/03 15630 A1 Dec. 13, 2012 22 particularly useful for determining the presence or level of chemiluminescence kit (New England Nuclear; Boston, other diagnostic markers in a sample. Mass.) according to the manufacturer's instructions. Autora 0203 Specific immunological binding of the antibody to diographs of the blots are analyzed using a scanning densito the marker of interest can be detected directly or indirectly. meter (Molecular Dynamics; Sunnyvale, Calif.) and normal Direct labels include fluorescent or luminescent tags, metals, ized to a positive control. Values are reported, for example, as dyes, radionuclides, and the like, attached to the antibody. An a ratio between the actual value to the positive control (den antibody labeled with iodine-125 ('I) can be used for deter sitometric index). Such methods are well known in the art as mining the levels of one or more markers in a sample. A described, for example, in Parra et al., J. Vasc. Surg., 28:669 chemiluminescence assay using a chemiluminescent anti 675 (1998). body specific for the marker is suitable for sensitive, non 0207 Alternatively, a variety of immunohistochemical radioactive detection of marker levels. An antibody labeled assay techniques can be used to determine the presence or with fluorochrome is also suitable for determining the levels level of one or more markers in a sample. The term immuno of one or more markers in a sample. Examples of fluoro histochemical assay encompasses techniques that utilize the chromes include, without limitation, DAPI, fluorescein, visual detection of fluorescent dyes or enzymes coupled (i.e., Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phyco conjugated) to antibodies that react with the marker of interest erythrin, rhodamine, Texas red, and lissamine. Secondary using fluorescent microscopy or light microscopy and antibodies linked to fluorochromes can be obtained commer includes, without limitation, direct fluorescent antibody cially, e.g., goat F(ab') anti-human IgG-FITC is available assay, indirect fluorescent antibody (IFA) assay, anticomple from Tago Immunologicals (Burlingame, Calif.). ment immunofluorescence, avidin-biotin immunofluores 0204 Indirect labels include various enzymes well-known cence, and immunoperoxidase assays. An IFA assay, for in the art, such as horseradish peroxidase (HRP), alkaline example, is useful for determining whether a sample is posi phosphatase (AP), B-galactosidase, urease, and the like. A tive for ANCA, the level of ANCA in a sample, whether a horseradish-peroxidase detection system can be used, for sample is positive for p ANCA, the level of p ANCA in a example, with the chromogenic Substrate tetramethylbenzi sample, and/or an ANCA staining pattern (e.g., cANCA, dine (TMB), which yields a soluble product in the presence of pANCA, NSNA, and/or SAPPA staining pattern). The con hydrogen peroxide that is detectable at 450 nm. An alkaline centration of ANCA in a sample can be quantitated, e.g., phosphatase detection system can be used with the chro through endpoint titration or through measuring the visual mogenic Substrate p-nitrophenyl phosphate, for example, intensity offluorescence compared to a known reference stan which yields a soluble product readily detectable at 405 nm. dard. Similarly, a B-galactosidase detection system can be used 0208 Alternatively, the presence or level of a marker of with the chromogenic Substrate o-nitrophenyl-B-D-galacto interest can be determined by detecting or quantifying the pyranoside (ONPG), which yields a soluble product detect amount of the purified marker. Purification of the marker can able at 410 nm. An urease detection system can be used with beachieved, for example, by high pressure liquid chromatog a Substrate such as urea-bromocresol purple (Sigma Immu raphy (HPLC), alone or in combination with mass spectrom nochemicals; St. Louis, Mo.). A useful secondary antibody etry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI-TOF/MS, linked to an enzyme can be obtained from a number of com tandem MS, etc.). Qualitative or quantitative detection of a mercial Sources, e.g., goat F(ab') anti-human IgG-alkaline marker of interest can also be determined by well-known phosphatase can be purchased from Jackson ImmunoRe methods including, without limitation, Bradford assays, Coo search (West Grove, Pa.). massie blue staining, silver staining, assays for radiolabeled 0205. A signal from the direct or indirect label can be protein, and mass spectrometry. analyzed, for example, using a spectrophotometer to detect 0209. The analysis of a plurality of markers may be carried color from a chromogenic Substrate; a radiation counter to out separately or simultaneously with one test sample. For detect radiation such as a gamma counterfor detection of 'I; separate or sequential assay of markers, Suitable apparatuses or a fluorometer to detect fluorescence in the presence of light include clinical laboratory analyzers such as the ElecSys of a certain wavelength. For detection of enzyme-linked anti (Roche), the AxSym (Abbott), the Access (Beckman), the bodies, a quantitative analysis of the amount of marker levels ADVIAR, the CENTAURR) (Bayer), and the NICHOLS can be made using a spectrophotometer Such as an EMAX ADVANTAGE(R) (Nichols Institute) immunoassay systems. Microplate Reader (Molecular Devices; Menlo Park, Calif.) Preferred apparatuses or protein chips perform simultaneous in accordance with the manufacturer's instructions. If assays of a plurality of markers on a single Surface. Particu desired, the assays of the present invention can be automated larly useful physical formats comprise Surfaces having a plu or performed robotically, and the signal from multiple rality of discrete, addressable locations for the detection of a samples can be detected simultaneously. plurality of different markers. Such formats include protein 0206 Quantitative western blotting can also be used to microarrays, or “protein chips' (see, e.g., Ng et al., J. Cell detect or determine the presence or level of one or more Mol. Med., 6:329-340 (2002)) and certain capillary devices markers in a sample. Western blots can be quantitated by (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments, well-known methods such as Scanning densitometry or phos each discrete Surface location may comprise antibodies to phorimaging. As a non-limiting example, protein samples are immobilize one or more markers for detection at each loca electrophoresed on 10% SDS-PAGE Laemmli gels. Primary tion. Surfaces may alternatively comprise one or more dis murine monoclonal antibodies are reacted with the blot, and crete particles (e.g., microparticles or nanoparticles) immo antibody binding can be confirmed to be linear using a pre bilized at discrete locations of a surface, where the liminary slot blot experiment. Goat anti-mouse horseradish microparticles comprise antibodies to immobilize one or peroxidase-coupled antibodies (BioRad) are used as the sec more markers for detection. ondary antibody, and signal detection performed using 0210. In addition to the above-described assays for deter chemiluminescence, for example, with the Renaissance mining the presence or level of various markers of interest, US 2012/03 15630 A1 Dec. 13, 2012 analysis of marker mRNA levels using routine techniques classifying a sample as being associated with IBS. Such a Such as Northern analysis, reverse-transcriptase polymerase panel may be constructed to determine the presence or level chain reaction (RT-PCR), or any other methods based on of 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, hybridization to a nucleic acid sequence that is complemen 20, 25, 30.35, 40, or more individual markers. The analysis of tary to a portion of the marker coding sequence (e.g., slot blot a single marker or Subsets of markers can also be carried out hybridization) are also within the scope of the present inven by one skilled in the art in various clinical settings. These tion. Applicable PCR amplification techniques are described include, but are not limited to, ambulatory, urgent care, criti in, e.g., Ausubel et al., Current Protocols in Molecular Biol cal care, intensive care, monitoring unit, inpatient, outpatient, ogy, John Wiley & Sons, Inc. New York (1999), Chapter 7 and physician office, medical clinic, and health screening set Supplement 47: Theophilus et al., “PCRMutation Detection tings. Protocols.” Humana Press, (2002); and Innis et al., PCR Pro 0214. The analysis of markers could be carried out in a tocols, San Diego, Academic Press, Inc. (1990). General variety of physical formats as well. For example, the use of nucleic acid hybridization methods are described in Ander microtiter plates or automation could be used to facilitate the son, “Nucleic Acid Hybridization.” BIOS Scientific Publish processing of large numbers of test samples. Alternatively, ers, 1999. Amplification or hybridization of a plurality of single sample formats could be developed to facilitate treat transcribed nucleic acid sequences (e.g., mRNA or cDNA) ment and diagnosis in a timely fashion. can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, “Microarrays Methods and Applica 1. EMBODIMENTS tions: Nuts & Bolts. DNA Press, 2003; and Baldi et al., 0215. In one aspect, the present invention provides an “DNA Microarrays and Gene Expression: From Experiments assay for the detection of an IBS marker in a blood or serum to Data Analysis and Modeling.” Cambridge University sample, the method comprising the steps of: (a) coating a Press, 2002. solid phase surface with a first anti-IBS marker capture anti 0211 Analysis of the genotype of a marker Such as a body; (b) contacting the solid phase surface with a blood or genetic marker can be performed using techniques known in serum sample under conditions suitable to transform the IBS the art including, without limitation, polymerase chain reac marker present in the sample into a complex comprising the tion (PCR)-based analysis, sequence analysis, and electro IBS marker and the anti-IBS marker capture antibody; (c) phoretic analysis. A non-limiting example of a PCR-based contacting the IBS marker and the anti-IBS maker complex analysis includes a Taqman R allelic discrimination assay with a second detecting antibody under conditions suitable to available from Applied Biosystems. Non-limiting examples form a ternary complex; and (d) contacting the ternary com of sequence analysis include Maxam-Gilbert sequencing, plex with a luminescent or chemiluminescent Substrate, Sanger sequencing, capillary array DNA sequencing, thermal wherein the IBS marker is selected from the group consisting cycle sequencing (Sears et al., Biotechniques, 13:626-633 of carbohydrate deficient transferrin, urocortin, corticotro (1992)). Solid-phase sequencing (Zimmerman et al., Methods phin-releasing hormone-binding protein, cortisol, adrenocor Mol. Cell. Biol., 3:39-42 (1992)), sequencing with mass spec ticotropic hormone, Substance P. nerve growth factor, neuro trometry Such as matrix-assisted laser desorption/ionization kinin A, neurokinin B. vasoactive intestinal peptide, time-of-flight mass spectrometry (MALDI-TOF/MS: Fu et glucagon-like peptide 2, motilin, pituitary adenylate cyclase al., Nature Biotech., 16:381-384 (1998)), and sequencing by activating peptide, serotonin, tryptophan, serotonin O-sul hybridization (Chee et al., Science, 274:610-614 (1996): fate, 5-hydroxyindoleacetic acid, 5-HT glucuronide, Drmanac et al., Science, 260: 1649-1652 (1993); Drmanac et tyrosine, phenylalanine, UDP-glucuronosyltransferase 1-6. al., Nature Biotech., 16:54-58 (1998)). Non-limiting serotonin reuptake transporter, tryptophan hydroxylase 1, examples of electrophoretic analysis include slab gel electro monoamine oxidase A, monoamine oxidase B, and hydrox phoresis such as agarose or polyacrylamide gel electrophore ytryptamine (serotonin) receptor 3A. sis, capillary electrophoresis, and denaturing gradient gel 0216. In one embodiment, the detecting antibody is con electrophoresis. Other methods for genotyping an individual jugated to alkaline phosphatase. In other embodiments, the at a polymorphic site in a marker include, e.g., the detecting antibody is not conjugated to an enzyme and the INVADER(R) assay from Third Wave Technologies, Inc., method further comprises the steps of (i) contacting the ter restriction fragment length polymorphism (RFLP) analysis, nary complex with a third antibody conjugated to alkaline allele-specific oligonucleotide hybridization, a heteroduplex phosphatase under conditions suitable to form a quaternary mobility assay, and single strand conformational polymor complex and (ii) contacting the quaternary complex with a phism (SSCP) analysis. luminescent or chemiluminescent Substrate. 0212. Several markers of interest may be combined into 0217. Any suitable antibody pair may be used for the one test for efficient processing of a multiple of samples. In capture and detecting antibodies in a sandwich ELISA. One addition, one skilled in the art would recognize the value of of skill in the art will know and appreciate how to select an testing multiple samples (e.g., at Successive time points, etc.) appropriate antibody pair for the assay. Generally, two anti from the same Subject. Such testing of serial samples can bodies are selected that bind to the target of interest, e.g., the allow the identification of changes in marker levels over time. IBS marker, at different epitopes such that the binding of the Increases or decreases in marker levels, as well as the absence first (capture) antibody does not interfere with the second of change in marker levels, can also provide useful informa (detecting) antibody. In certain embodiments, the detecting tion to classify IBS or to rule out diseases and disorders antibody will be conjugated to an enzyme, for example, alka associated with IBS-like symptoms. line phophatase, to aid in the detection of the complex. In 0213 A panel for measuring one or more of the markers other embodiments, a secondary antibody conjugated to an described above may be constructed to provide relevant infor enzyme (e.g., alkaline phophatase), which binds to the detect mation related to the approach of the present invention for ing antibody, may be used in the assay. US 2012/03 15630 A1 Dec. 13, 2012 24
0218 Generally, the complex will be detected by the use 0225. In another embodiment of an assay to aid in the of a luminescent Substrate, for example, a luminescent Sub diagnosis of IBS, the assay is an enzyme-linked immunosor strate found in a kit such as Ultra LITETM (NAG Research bent assay (ELISA). Laboratories); SensoLyte R (AnaSpec); SuperSignal ELISA 0226. In another embodiment of an assay to aid in the Femto Maximum Sensitivity Substrate (Thermo Scientific); diagnosis of IBS, detecting the presence or level of the IBS SuperSignal ELISA Pico Chemiluminescent Substrate marker in the sample comprises the use of a detection device. (Thermo Scientific); or CPSD (disodium 3-(4-methox 0227. In another embodiment of an assay to aid in the yspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7 de diagnosis of IBS, the detection device comprises a lumines can-4-yl)phenyl phosphate; Tropix. Inc). cence plate reader. 0219. In a preferred embodiment, an assay for detecting 0228. In another embodiment of an assay to aid in the the presence or level of an IBS marker comprises a sandwich diagnosis of IBS, the detection device comprises a spectro ELISA that relies on the use of an alkaline phosphatase con photometer. jugated anti-IBS marker antibody as the detecting antibody 0229. In another embodiment of an assay to aid in the and a CPSD containing luminescent substrate to enhance the diagnosis of IBS, the assay further comprises detecting the assay sensitivity. The CPSD substrate can be found in chemi presence or level of B-tryptase, prostaglandin E2 (PGE), luminescent detection systems, such as the ELISA-LightTM and/or histamine in the sample. System (Applied Biosystems). 0230. In another aspect, the present invention provides an 0220. In a preferred embodiment of the assay, the detec assay to aid in the differentiation of clinical subtypes of IBS, tion limit of the IBS marker present in a blood or serum the assay comprising: (a) contacting a sample having an IBS sample is less than about 500 pg/ml. In certain embodiments, marker underconditions suitable to transform the IBS marker the detection limit of the IBS marker present in a blood or into a complex comprising the IBS marker and a capture serum sample is less than about 500 pg/ml, or less than about anti-IBS marker antibody; (b) contacting the complex with an 400 pg/ml, 300 pg/ml, 250 pg/ml, 200 pg/ml, 150 pg/ml, 100 enzyme-labeled indicator antibody to transform the complex pg/ml, 75 pg/ml, 50 pg/ml. 40 pg/ml, 30 pg/ml, 25 pg/ml, 20 into a labeled complex; (c) contacting the labeled complex pg/ml, 15 pg/ml, or less than about 10 pg/ml. In a preferred with a substrate for the enzyme; and (d) detecting the pres embodiment, the detection limit of the IBS marker present in ence or level of the IBS marker in the sample, wherein the IBS a blood or serum sample is less than about 200 pg/ml. In a marker is selected from the group consisting of carbohydrate more preferred embodiment, the detection limit of the IBS deficient transferrin, urocortin, corticotrophin-releasing hor marker present in a blood or serum sample is less than about mone-binding protein, cortisol, adrenocorticotropic hor 100 pg/ml. In a more preferred embodiment, the detection mone, Substance P. nerve growth factor, neurokinin A, neu limit of the IBS marker present in a blood or serum sample is rokinin B. vasoactive intestinal peptide, glucagon-like less than about 50 pg/ml. In a most preferred embodiment, the peptide 2, motilin, pituitary adenylate cyclase-activating pep detection limit of the IBS marker present in a blood or serum tide, serotonin, tryptophan, serotonin O-sulfate, 5-hydroxy sample is less than about 25 pg/ml. indoleacetic acid, 5-HT glucuronide, tyrosine, phenylala 0221. In another aspect, the present invention provides an nine, UDP-glucuronosyltransferase 1-6, serotonin reuptake assay to aid in the diagnosis of IBS, the assay comprising: (a) transporter, tryptophan hydroxylase 1, monoamine oxidase contacting a sample having an IBS marker under conditions A monoamine oxidase B, and hydroxytryptamine (seroto suitable to transform the IBS marker into a complex compris nin) receptor 3A. ing the IBS marker and a capture anti-IBS marker antibody; 0231. In one embodiment of an assay to aid in the differ (b) contacting the complex with an enzyme-labeled indicator entiation of clinical subtypes of IBS, the assay aids in the antibody to transform the complex into a labeled complex; (c) differentiation of IBS-D and IBS-A from IBS-C. contacting the labeled complex with a substrate for the 0232. In another embodiment of an assay to aid in the enzyme; and (d) detecting the presence or level of the IBS differentiation of clinical subtypes of IBS, the sample is marker in the sample, wherein the IBS marker is selected human serum. from the group consisting of carbohydrate deficient transfer 0233. In another embodiment of an assay to aid in the rin, urocortin, corticotrophin-releasing hormone-binding differentiation of clinical subtypes of IBS, the assay is an protein, cortisol, adrenocorticotropic hormone, Substance P. enzyme-linked immunosorbent assay (ELISA). nerve growth factor, neurokinin A, neurokinin B. vasoactive 0234. In another embodiment of an assay to aid in the intestinal peptide, glucagon-like peptide 2, motilin, pituitary differentiation of clinical subtypes of IBS, detecting the pres adenylate cyclase-activating peptide, serotonin, tryptophan, ence or level of the IBS marker in the sample comprises the serotonin O-sulfate, 5-hydroxyindoleacetic acid, 5-HT glu use of a detection device. curonide, tyrosine, phenylalanine, UDP-glucuronosyltrans 0235. In another embodiment of an assay to aid in the ferase 1-6, Serotonin reuptake transporter, tryptophan differentiation of clinical subtypes of IBS, the assay further hydroxylase 1, monoamine oxidase A, monoamine oxidase comprises detecting the presence or level off-tryptase, pros B, and hydroxytryptamine (serotonin) receptor 3A. taglandin E2 (PGE), and/or histamine in the sample. 0222. In one embodiment of an assay to aid in the diagno 0236. In certain embodiments, the assays provided herein sis of IBS, the sample is human serum. may further comprise detection of the presence or level of at 0223) In another embodiment of an assay to aid in the least one additional biomarker selected from the group con diagnosis of IBS, the sample is obtained from a subject Sus sisting of Brain-Derived Neurotropic Factor (BDNF), Neu pected of having IBS. trophil Gelatinase-Associated Lipocalin (NGAL). TNF-re 0224. In another embodiment of an assay to aid in the lated Weak Inducer of Apoptosis (TWEAK), Growth-Related diagnosis of IBS, a higher level of the IBS marker in the Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-B), Tissue sample relative to healthy controls is indicative of an Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Saccharo increased likelihood of the subject having IBS. myces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Anti US 2012/03 15630 A1 Dec. 13, 2012
body (CBirl), Anti-Human Neutrophil Cytoplasmic Anti NAP-2), growth factor (e.g., EGF, VEGF, PEDF, BDNF, and/ body (ANCA), Anti-Human Tissue Transglutaminase IgA or SDGF), anti-neutrophilantibody (e.g., ANCA, p-ANCA, (tTG). B-tryptase, prostaglandin E, histamine, and a combi cANCA, NSNA, and/or SAPPA), ASCA (e.g., ASCA-IgA, nation thereof. ASCA-IgG, and/or ASCA-IgM), antimicrobial antibody 0237. In yet other embodiments, the assays provided (e.g., anti-OmpC antibody, anti-flagellin antibody, and/or herein may further comprise detection of the presence or level anti-I2 antibody), lactoferrin, anti-tTG antibody, lipocalin of at least one additional biomarker selected from the group (e.g., NGAL, NGAL/MMP-9 complex), MMP (e.g., MMP consisting of a cytokine (e.g., IL-8, IL-1B, TWEAK, leptin, 9), TIMP (e.g., TIMP-1), alpha-globulin (e.g., alpha-2-mac OPG, MIP-3 B, GROC, CXCL4/PF-4, and/or CXCL7/NAP roglobulin, haptoglobin, and/or orosomucoid), actin-sever 2), growth factor (e.g., EGF, VEGF, PEDF, BDNF, and/or ing protein (e.g., gelsolin), S100 protein (e.g., calgranulin), SDGF), anti-neutrophil antibody (e.g., ANCA, p-ANCA, fibrinopeptide (e.g., FIBA), CGRP tachykinin (e.g., Sub cANCA, NSNA, and/or SAPPA), ASCA (e.g., ASCA-IgA, stance P), ghrelin, neurotensin, corticotropin-releasing hor ASCA-IgG, and/or ASCA-IgM), antimicrobial antibody mone, anti-lactoferrin antibody, L-selectin/CD62L, elastase, (e.g., anti-Ompo antibody, anti-flagellin antibody, and/or C-reactive protein (CRP), calprotectin, anti-U1-70 kDa anti-I2 antibody), lactoferrin, anti-tTG antibody, lipocalin autoantibody, Zona occludens 1 (ZO-1), vasoactive intestinal (e.g., NGAL, NGAL/MMP-9 complex), MMP (e.g., MMP peptide (VIP), serum amyloid A, gastrin, and combinations 9), TIMP (e.g., TIMP-1), alpha-globulin (e.g., alpha-2-mac thereof. roglobulin, haptoglobin, and/or orosomucoid), actin-sever 0242. In certain embodiments, the kits provided may con ing protein (e.g., gelsolin), S100 protein (e.g., calgranulin), tain a binding moiety for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, fibrinopeptide (e.g., FIBA), CGRP tachykinin (e.g., Sub 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, stance P), ghrelin, neurotensin, corticotropin-releasing hor 29, 30, or more of the biomarkers described herein. mone, and combinations thereof. In yet other embodiments, 0243 In a preferred embodiment, a kit is provided for the presence or level of other diagnostic markers such as, for detecting the presence or level of an IBS marker in a blood or example, anti-lactoferrin antibody, L-selectin/CD62L, serum sample. In one embodiment, the kit contains a first elastase, C-reactive protein (CRP), calprotectin, anti-U1-70 capture antibody, which is optionally attached to a solid Sur kDa autoantibody, Zona occludens 1 (ZO-1), vasoactive intes face, and a second detection antibody that binds to a different tinal peptide (VIP), serum amyloid A, gastrin, and a combi epitope on the IBS marker than the capture antibody. In cer nation thereof may also be detected. tain embodiments, the detection antibody will be conjugated 0238. In certain embodiments, the assays provided may to alkaline phosphatase. In other embodiments, the detection comprise the detection of at least 1,2,3,4,5,6,7,8,9, 10, 11, antibody will not be conjugated to an enzyme, i.e., alkaline 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, phophatase. In certain embodiments, wherein the detection 29, 30, or more of the biomarkers described herein. antibody is not enzyme-conjugated, the kit may further com 0239. In another aspect, kits are provided for aiding in the prise a third antibody specific for the detection antibody, the diagnosis of IBS in a subject, or for classifying a sample as an third antibody being conjugated to an enzyme, e.g., alkaline IBS sample or a non-IBS sample. In certain embodiments, phophatase. The kits provided herein contain a binding moiety for detect 0244. In certain embodiments, the kit will also contain a ing the presence or level of an IBS biomarker selected from luminescent or chemiluminescent Substrate, for example, a carbohydrate deficient transferrin, urocortin, corticotrophin luminescent substrate as found in Ultra LITETM (NAG releasing hormone-binding protein, cortisol, adrenocortico Research Laboratories); SensoLyte.R. (AnaSpec); SuperSig tropic hormone, Substance P. nerve growth factor, neurokinin nal ELISA Femto Maximum Sensitivity Substrate (Thermo A, neurokinin B. vasoactive intestinal peptide, glucagon-like Scientific); SuperSignal ELISA Pico Chemiluminescent peptide 2, motilin, pituitary adenylate cyclase-activating pep Substrate (Thermo Scientific); or CPSD (disodium 3-(4- tide, serotonin, tryptophan, serotonin O-sulfate, 5-hydroxy methoxyspiro 1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1. indoleacetic acid, 5-HT glucuronide, tyrosine, phenylala 13.7 decan-4-yl)phenyl phosphate; Tropix, Inc). In a pre nine, UDP-glucuronosyltransferase 1-6, serotonin reuptake ferred embodiment, the luminescent substrate is CPSD transporter, tryptophan hydroxylase 1, monoamine oxidase (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'- A monoamine oxidase B, and hydroxytryptamine (seroto chloro)tricyclo[3.3.1.13.7 decan-4-yl)phenyl phosphate. nin) receptor 3A. 0245. In certain embodiments, the kit for detecting the 0240. In other embodiments, the kit will also contain at presence or level of an IBS marker is suitable for detecting the least one binding moiety for an additional biomarker selected IBS marker present in a blood or serum sample at a concen from the group consisting of Brain-Derived Neurotropic Fac tration of less than about 500 pg/ml. In certain embodiments, tor (BDNF), Neutrophil Gelatinase-Associated Lipocalin the kit is suitable for detecting the IBS marker present in a (NGAL). TNF-related Weak Inducer of Apoptosis blood or serum sample at a concentration of less than about (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), 500 pg/ml, or less than about 400 pg/ml, 300 pg/ml, 250 Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein pg/ml, 200 pg/ml, 150 pg/ml, 100 pg/ml, 75 pg/ml, 50 pg/ml, ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody 40 pg/ml, 30 pg/ml, 25 pg/ml, 20 pg/ml, 15 pg/ml, or less than (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human about 10 pg/ml. In a preferred embodiment, the kit is suitable Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human for detecting the IBS marker present in a blood or serum Tissue Transglutaminase IgA (tTG). B-tryptase, prostaglan sample at a concentration of less than about 200 pg/ml. In a din E, histamine, and a combination thereof. more preferred embodiment, the kit is suitable for detecting 0241. In yet other embodiments, a kit will contain at least the IBS marker present in a blood or serum sample at a one binding moiety for an additional biomarker selected from concentration of less than about 100 pg/ml. In a more pre the group consisting of a cytokine (e.g., IL-8, IL-B, TWEAK, ferred embodiment, the kit is suitable for detecting the IBS leptin, OPG, MIP-3|B, GROC, CXCL4/PF-4, and/or CXCL7/ marker present in a blood or serum sample at a concentration US 2012/03 15630 A1 Dec. 13, 2012 26 of less than about 50 pg/ml. In a most preferred embodiment, cyclase agonist, asimadoline or other opioid agonist, or taln the kit is suitable for detecting the IBS marker present in a etant or other neurokinin antagonist can be administered to blood or serum sample at a concentration of less than about 25 the individual. In other instances, when the sample is classi pg/ml. fied as IBS-D and/or the individual is diagnosed with IBS-D, 0246 E. Methods for Monitoring and Assigning Therapy atherapeutically effective amount of alosetron or other 5-HT 0247. In some embodiments, the diagnosis of an indi antagonist (e.g., ramosetron, DDP-225, etc.), crofelemer or vidual as having IBS is followed by administering to the other chloride channel blocker, talnetant or other neurokinin individual atherapeutically effective amount of a drug useful antagonist (e.g., saredutant, etc.), oran antidepressant such as for treating one or more symptoms associated with IBS. Suit a tricyclic antidepressant can be administered to the indi able IBS drugs include, but are not limited to, serotonergic vidual. agents, antidepressants, chloride channel activators, chloride 0251. In yet another aspect, the present invention provides channel blockers, guanylate cyclase agonists, antibiotics, a method for monitoring the progression or regression of IBS opioid agonists, neurokinin antagonists, antispasmodic or in an individual, the method comprising: (a) determining a anticholinergic agents, belladonna alkaloids, barbiturates, diagnostic marker profile by detecting the presence or level of GLP-1 analogs, CRF antagonists, probiotics, free bases at least one diagnostic marker in the sample; and (b) deter thereof, pharmaceutically acceptable salts thereof, deriva mining the presence or severity of IBS in the individual using tives thereof, analogs thereof, and combinations thereof. an algorithm based upon the diagnostic marker profile. Other IBS drugs include bulking agents, dopamine antago 0252. In one embodiment, the method of monitoring the nists, carminatives, tranquilizers, dextofisopam, phenyloin, progression or regression of IBS comprises determining a timolol, and diltiazem. Additionally, amino acids like diagnostic marker profile optionally in combination with a glutamine and glutamic acid which regulate intestinal perme symptom profile, wherein the symptom profile is determined ability by affecting neuronal or glial cell signaling can be by identifying the presence or severity of at least one symp administered to treat patients with IBS. tom in the individual; and determining the presence or sever 0248. In other embodiments, the methods of the present ity of IBS in the individual using an algorithm based upon the invention further comprise classifying an IBS sample or a diagnostic marker profile and the symptom profile. diagnosis of IBS as an IBS-constipation (IBS-C), IBS-diar 0253) In a related aspect, the present invention provides a rhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating (IBS-A), method for monitoring drug efficacy in an individual receiv or post-infectious IBS (IBS-PI) sample or diagnosis. In cer ing a drug useful for treating IBS, the method comprising: (a) tain instances, the classification of the IBS sample or diagno determining a diagnostic marker profile by detecting the pres sis into a category, form, or clinical Subtype of IBS is based ence or level of at least one diagnostic marker in the sample: upon the presence or level of at least one, two, three, four, five, and (b) determining the effectiveness of the drug using an six, seven, eight, nine, ten, or more classification markers algorithm based upon the diagnostic marker profile. provided herein. Preferably, at least one form of IBS is dis 0254. In one embodiment, the method of monitoring IBS tinguished from at least one otherform of IBS based upon the drug efficacy comprises determining a diagnostic marker pro presence or level of one or more IBS markers provided herein. file optionally in combination with a symptom profile, In certain instances, the methods of the present invention can wherein the symptom profile is determined by identifying the be used to differentiate an IBS-C sample from an IBS-A presence or severity of at least one symptom in the individual; and/or IBS-D sample in an individual previously identified as and determining the effectiveness of the drug using an algo having IBS. In certain other instances, the methods of the rithm based upon the diagnostic marker profile and the Symp present invention can be used to classify a sample from an tom profile. individual not previously diagnosed with IBS as an IBS-A 0255. In one embodiment, the present invention provides a sample, IBS-C sample, IBS-D sample, or non-IBS sample. method for monitoring the progression or regression of irri 0249. In certain embodiments, the methods further com table bowel syndrome (IBS) in a subject, the method com prise sending the results from the classification to a clinician. prising: (a) contacting a first blood or serum sample taken In certain other embodiments, the methods further provide a from the subject at a first time with an IBS marker binding diagnosis in the form of a probability that the individual has moiety under conditions suitable to transform the IBS marker IBS-A, IBS-C, IBS-D, IBS-M, or IBS-PI. The methods of the present in the sample into a complex comprising the IBS present invention can further comprise administering to the marker and the IBS marker binding moiety; (b) determining individual atherapeutically effective amount of a drug useful the level of the complex, thereby determining the level of the for treating IBS-A, IBS-C, IBS-D, IBS-M, or IBS-PI. Suit IBS marker present in the first sample; (c) contacting a second able drugs include, but are not limited to, tegaserod (Zel blood or serum sample taken from the Subjectata second time normTM), alosetron (Lotronex.R.), lubiprostone (AmitizatM), with an IBS marker binding moiety under conditions suitable rifamixin (XifaxanTM), MD-1100, probiotics, and a combina to transform the IBS marker present in the sample into a tion thereof. complex comprising the IBS marker and the IBS marker 0250 In instances where the sample is classified as an binding moiety; (d) determining the level of the complex, IBS-A or IBS-C sample and/or the individual is diagnosed thereby determining the level of the IBS marker present in the with IBS-A or IBS-C, a therapeutically effective dose of second sample; and (e) comparing the level of the IBS marker tegaserod or other 5-HT agonist (e.g., mosapride, renza present in the first sample to the level of the IBS marker pride, AG1-001, etc.) can be administered to the individual. In present in the second sample, wherein a higher level of the Some instances, when the sample is classified as IBS-C and/or IBS marker in the second sample relative to the first sample is the individual is diagnosed with IBS-C, a therapeutically indicative of the progression of IBS in the subject and a lower effective amount of lubiprostone or other chloride channel level of the IBS marker in the second sample relative to the activator, rifamixin or other antibiotic capable of controlling first sample is indicative of the regression of IBS in the sub intestinal bacterial overgrowth, MD-1100 or other guanylate ject. US 2012/03 15630 A1 Dec. 13, 2012 27
0256 In certain embodiments of the methods of monitor symptom profile, wherein the symptom profile is determined ing and assigning therapy, the method further comprises by identifying the presence or severity of at least one symp detecting the presence of level of at least one additional biom tom in the individual; and determining the presence or sever arker selected from the group consisting of Brain-Derived ity of IBS in the individual using an algorithm based upon the Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ diagnostic marker profile and the symptom profile. In certain ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo other embodiments, the method of monitoring IBS drug effi ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO cacy comprises determining a diagnostic marker profile O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi optionally in combination with a symptom profile, wherein siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), the symptom profile is determined by identifying the pres Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), ence or severity of at least one symptom in the individual; and Anti-Human Tissue Transglutaminase IgA (tTG). B-tryptase, determining the effectiveness of the drug using an algorithm prostaglandin E, histamine, and a combination thereof. In based upon the diagnostic marker profile and the symptom certain embodiments, the method comprises detecting the profile. One skilled in the art will appreciate that the diagnos presence or level of at least two, three, four, five, six, seven, tic marker profile and the symptom profile can be determined eight, nine, or all ten of the additional biomarkers provided simultaneously or sequentially in any order. above. 0260. In some embodiments, determining the presence or 0257. In yet other embodiments, the method my further severity of IBS or the effectiveness of an IBS drug is based comprises detecting the presence of level of at least one upon the diagnostic marker profile, alone or in combination biomarker selected from the group consisting of a cytokine with a symptom profile, in conjunction with a statistical algo (e.g., IL-8, IL-1 B, TWEAK, leptin, OPG, MIP-3 B, GROC, rithm. In certain instances, the statistical algorithm is a learn CXCL4/PF-4, and/or CXCL7/NAP-2), growth factor (e.g., ing statistical classifier system. The learning statistical clas EGF, VEGF, PEDF, BDNF, and/or SDGF), anti-neutrophil sifier system comprises any of the learning statistical antibody (e.g., ANCA, p-ANCA, cANCA, NSNA, and/or classifier systems described herein. SAPPA), ASCA (e.g., ASCA-IgA, ASCA-IgG, and/or 0261. In certain embodiments, the methods of the present ASCA-IgM), antimicrobial antibody (e.g., anti-Ompo anti invention can further comprise comparing the presence or body, anti-flagellin antibody, and/or anti-I2 antibody), lacto severity of IBS in the individual determined in step (b) to the ferrin, anti-tTG antibody, lipocalin (e.g., NGAL, NGAL/ presence or severity of IBS in the individual at an earlier time. MMP-9 complex), MMP (e.g., MMP-9), TIMP (e.g., TIMP As a non-limiting example, the presence or severity of IBS 1), alpha-globulin (e.g., alpha-2-macroglobulin, haptoglobin, determined for an individual receiving an IBS drug can be and/or orosomucoid), actin-severing protein (e.g., gelsolin), compared to the presence or severity of IBS determined for S100 protein (e.g., calgranulin), fibrinopeptide (e.g., FIBA), the same individual before initiation of use of the IBS drug or CGRP, tachykinin (e.g., Substance P), ghrelin, neurotensin, at an earlier time in therapy. In certain other embodiments, the corticotropin-releasing hormone, and combinations thereof. methods of the present invention can comprise determining In yet other embodiments, the presence or level of other the effectiveness of the IBS drug by comparing the effective diagnostic markers such as, for example, anti-lactoferrin anti ness of the IBS drug determined in step (b) to the effective body, L-selectin/CD62L, elastase, C-reactive protein (CRP), ness of the IBS drug in the individual at an earlier time in calprotectin, anti-U1-70 kDa autoantibody, Zona occludens 1 therapy. In additional embodiments, the methods can further (ZO-1), vasoactive intestinal peptide (VIP), serum amyloid comprise sending the IBS monitoring results to a clinician, A, gastrin, and a combination thereof may also be detected. e.g., a gastroenterologist or a general practitioner. 0258. In some embodiments, a panel for measuring one or 0262 F. Computer Readable Medium and Systems for more of the diagnostic markers described above may be con Classifying Samples structed and used for determining the presence or severity of 0263. In one aspect, the present invention provides a com IBS or for determining the effectiveness of an IBS drug. One puter-readable medium comprising code for controlling one skilled in the art will appreciate that the presence or level of a or more processors to classify whether a serum or blood plurality of diagnostic markers can be determined simulta sample from an subject is associated with irritable bowel neously or sequentially, using, for example, an aliquot or syndrome (IBS), the code comprising instructions to apply a dilution of the individual's sample. As described above, the statistical process to a data set comprising a diagnostic marker level of a particular diagnostic marker in the individual’s profile to produce a statistically derived decision classifying sample is generally considered to be elevated when it is at the sample as an IBS sample or non-IBS sample based upon least about 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, the diagnostic marker profile, wherein the diagnostic marker 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, profile indicates the level of at least one diagnostic marker 500%. 600%, 700%,800%,900%, or 1000% greater than the selected from the group consisting of carbohydrate deficient level of the same marker in a comparative sample or popula transferrin, urocortin, corticotrophin-releasing hormone tion of samples (e.g., greater than a median level). Similarly, binding protein, cortisol, adrenocorticotropic hormone, Sub the level of a particular diagnostic marker in the individual’s stance P. nerve growth factor, neurokinin A, neurokinin B, sample is typically considered to be lowered when it is at least vasoactive intestinal peptide, glucagon-like peptide 2, moti about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, lin, pituitary adenylate cyclase-activating peptide, Serotonin, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% tryptophan, serotonin O-sulfate, 5-hydroxyindoleacetic acid, less than the level of the same markerina comparative sample 5-HT glucuronide, tyrosine, phenylalanine, UDP-glucurono or population of Samples (e.g., less than a median level). Syltransferase 1-6, Serotonin reuptake transporter, tryptophan 0259. In certain embodiments, the method of monitoring hydroxylase 1, monoamine oxidase A, monoamine oxidase the progression or regression of IBS comprises determining a B, and hydroxytryptamine (serotonin) receptor 3A, and a diagnostic marker profile optionally in combination with a combination thereof. US 2012/03 15630 A1 Dec. 13, 2012 28
0264. In other embodiments, the computer-readable tively, the first and second statistical processes are imple medium for ruling in IBS comprises instructions to apply a mented in a single processor. In another embodiment, the first statistical process to a data set comprising a diagnostic marker statistical process is a learning statistical classifier system. profile optionally in combination with a symptom profile Examples of learning statistical classifier systems suitable for which indicates the presence or severity of at least one symp use in the present invention are described above. In certain tom in the individual to produce a statistically derived deci instances, the first and/or second statistical process is a single sion classifying the sample as an IBS sample or non-IBS learning statistical classifier system such as, for example, a sample based upon the diagnostic marker profile and the RF or C&RT. In certain other instances, the first and/or sec symptom profile. One skilled in the art will appreciate that the ond statistical process is a combination of at least two learn statistical process can be applied to the diagnostic marker ing statistical classifier systems. As a non-limiting example, profile and the symptom profile simultaneously or sequen the combination of learning statistical classifier systems com tially in any order. prises a RF and a NN or SVM, e.g., used in tandem. In some 0265. In a specific embodiment, the present invention pro instances, the data obtained from using the learning statistical vides a computer-readable medium including code for con classifier system or systems can be processed using a process trolling one or more processors to classify whether a sample ing algorithm. from an individual is associated with IBS, the code compris 0270. In another aspect, the present invention provides a ing instructions to apply a statistical process to a data set system for classifying whether a serum or blood sample from comprising a diagnostic marker profile to produce a statisti a subject is associated with irritable bowel syndrome (IBS), cally derived decision classifying the sample as an IBS the system comprising: (a) a data acquisition module config sample or non-IBS sample based upon the diagnostic marker ured to produce a data set comprising a diagnostic marker profile, wherein the diagnostic marker profile indicates the profile, wherein the diagnostic marker profile indicates the presence or level of at least one diagnostic marker in the presence or level of at least one diagnostic marker selected sample. from the group consisting of carbohydrate deficient transfer 0266. In a related aspect, the present invention provides a rin, urocortin, corticotrophin-releasing hormone-binding computer-readable medium including code for controlling protein, cortisol, adrenocorticotropic hormone, Substance P. one or more processors to classify whether a sample from an nerve growth factor, neurokinin A, neurokinin B. vasoactive individual is associated with IBS, the code comprising: (a) intestinal peptide, glucagon-like peptide 2, motilin, pituitary instructions to apply a first statistical process to a data set adenylate cyclase-activating peptide, serotonin, tryptophan, comprising a diagnostic marker profile to produce a statisti serotonin O-sulfate, 5-hydroxyindoleacetic acid, 5-HT glu cally derived decision classifying the sample as an IBD curonide, tyrosine, phenylalanine, UDP-glucuronosyltrans sample or non-IBD Sample based upon the diagnostic marker ferase 1-6, serotonin reuptake transporter, tryptophan profile, wherein the diagnostic marker profile indicates the hydroxylase 1, monoamine oxidase A, monoamine oxidase presence or level of at least one diagnostic marker in the B, and hydroxytryptamine (serotonin) receptor 3A, and a sample; and if the sample is classified as a non-IBD sample, combination thereof; (b) a data processing module configured (b) instructions to apply a second statistical process to the to process the data set by applying a statistical process to the same or different data set to produce a second statistically data set to produce a statistically derived decision classifying derived decision classifying the non-IBD sample as an IBS the sample as an IBS sample or non-IBS sample based upon sample or non-IBS sample. the diagnostic marker profile; and (c) a display module con 0267 In one embodiment, the computer-readable medium figured to display the statistically derived decision. for ruling in IBS comprises instructions to apply a statistical 0271 In certain embodiments, the system for classifying process to a data set comprising a diagnostic marker profile whether a serum or blood sample is associated with IBS, optionally in combination with a symptom profile which indi aiding in the diagnosis of IBS, or ruling in IBS comprises a cates the presence or severity of at least one symptom in the data acquisition module configured to produce a data set individual to produce a statistically derived decision classi comprising a diagnostic marker profile optionally in combi fying the sample as an IBS sample or non-IBS sample based nation with a symptom profile which indicates the presence or upon the diagnostic marker profile and the symptom profile. severity of at least one symptom in the individual; a data 0268. In other embodiments, the computer-readable processing module configured to process the data set by medium for first ruling out IBD and then ruling in IBS com applying a statistical process to the data set to produce a prises instructions to apply a first statistical process to a data statistically derived decision classifying the sample as an IBS set comprising a diagnostic marker profile optionally in com sample or non-IBS sample based upon the diagnostic marker bination with a symptom profile which indicates the presence profile and the symptom profile; and a display module con or severity of at least one symptom in the individual to pro figured to display the statistically derived decision. duce a statistically derived decision classifying the sample as 0272. In a related aspect, the present invention provides a an IBD sample or non-IBD sample based upon the diagnostic system for classifying whether a sample from an individual is marker profile and the symptom profile; and if the sample is associated with IBS, the system comprising: (a) a data acqui classified as a non-IBD Sample, instructions to apply a second sition module configured to produce a data set comprising a statistical process to the same or different data set to produce diagnostic marker profile, wherein the diagnostic marker pro a second statistically derived decision classifying the non file indicates the presence or level of at least one diagnostic IBD sample as an IBS sample or non-IBS sample. One skilled marker in the sample; (b) a data processing module config in the art will appreciate that the first and/or second statistical ured to process the data set by applying a first statistical process can be applied to the diagnostic marker profile and the process to the data set to produce a first statistically derived symptom profile simultaneously or sequentially in any order. decision classifying the sample as an IBD Sample or non-IBD 0269. In one embodiment, the first and second statistical sample based upon the diagnostic marker profile; if the processes are implemented in different processors. Alterna sample is classified as a non-IBD sample, a data processing US 2012/03 15630 A1 Dec. 13, 2012 29 module configured to apply a second statistical process to the 0276 G. Diseases and Disorders with IBS-Like Symp same or different data set to produce a second statistically tOmS derived decision classifying the non-IBD sample as an IBS 0277. A variety of structural or metabolic diseases and sample or non-IBS sample; and (c) a display module config disorders can cause signs or symptoms that are similar to IBS. As non-limiting examples, patients with diseases and disor ured to display the first and/or the second statistically derived ders such as inflammatory bowel disease (IBD), Celiac dis decision. ease (CD), acute inflammation, diverticulitis, ileal pouch 0273. In one embodiment, the system for first ruling out anal anastomosis, microscopic colitis, chronic infectious IBD and then ruling in IBS comprises a data acquisition diarrhea, lactase deficiency, cancer (e.g., colorectal cancer), a module configured to produce a data set comprising a diag mechanical obstruction of the Small intestine or colon, an nostic marker profile optionally in combination with a symp enteric infection, ischemia, maldigestion, malabsorption, tom profile which indicates the presence or severity of at least endometriosis, and unidentified inflammatory disorders of one symptom in the individual; a data processing module the intestinal tract can present with abdominal discomfort associated with mild to moderate pain and a change in the configured to process the data set by applying a first statistical consistency and/or frequency of stools that are similar to IBS. process to the data set to produce a first statistically derived Additional IBS-like symptoms can include chronic diarrhea decision classifying the sample as an IBD Sample or non-IBD or constipation or an alternating form of each, weight loss, sample based upon the diagnostic marker profile and the abdominal distention or bloating, and mucus in the stool. symptom profile; if the sample is classified as a non-IBD 0278 Most IBD patients can be classified into one of two sample, a data processing module configured to apply a sec distinct clinical Subtypes, Crohn's disease and ulcerative coli ond statistical process to the same or different data set to tis. Crohn's disease is an inflammatory disease affecting the produce a second statistically derived decision classifying the lower part of the ileum and often involving the colon and other non-IBD sample as an IBS sample or non-IBS sample; and a regions of the intestinal tract. Ulcerative colitis is character ized by an inflammation localized mostly in the mucosa and display module configured to display the first and/or the sec Submucosa of the large intestine. Patients suffering from ond statistically derived decision. these clinical subtypes of IBD typically have IBS-like symp 0274. In certain embodiments the diagnostic marker pro toms such as, for example, abdominal pain, chronic diarrhea, file indicates the level of at least one additional diagnostic weight loss, and cramping. marker selected from the group consisting of Brain-Derived 0279. The clinical presentation of Celiac disease is also Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ characterized by IBS-like symptoms such as abdominal dis ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo comfort associated with chronic diarrhea, weight loss, and ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO abdominal distension. Celiac disease is an immune-mediated O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of disorder of the intestinal mucosa that is typically associated Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi with villous atrophy, crypt hyperplasia, and/or inflammation siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), of the mucosal lining of the small intestine. In addition to the Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), malabsorption of nutrients, individuals with Celiac disease are at risk for mineral deficiency, Vitamin deficiency, Anti-Human Tissue Transglutaminase IgA (tTG), histamine, osteoporosis, autoimmune diseases, and intestinal malignan f-tryptase, prostaglandin E2 (PGE), and a combination cies (e.g., lymphoma and carcinoma). It is thought that expo thereof. In certain embodiments, the method comprises Sure to proteins such as gluten (e.g., glutenin and prolamine detecting the presence or level of at least two, three, four, five, proteins which are present in wheat, rye, barley, oats, millet, six, seven, eight, nine, or all ten of the additional biomarkers triticale, spelt, and kamut), in the appropriate genetic and provided above. environmental context, is responsible for causing Celiac dis 0275. In yet other embodiments, the diagnostic marker CaSC. profile indicates the level of at least one additional diagnostic 0280. Other diseases and disorders characterized by intes marker selected from the group consisting of a cytokine (e.g., tinal inflammation that present with IBS-like symptoms IL-8, IL-1 B, TWEAK, leptin, OPG, MIP-3|B, GROC, include, for example, acute inflammation, diverticulitis, ileal CXCL4/PF-4, and/or CXCL7/NAP-2), growth factor (e.g., pouch-anal anastomosis, microscopic colitis, and chronic EGF, VEGF, PEDF, BDNF, and/or SDGF), anti-neutrophil infectious diarrhea, as well as unidentified inflammatory dis antibody (e.g., ANCA, p-ANCA, cANCA, NSNA, and/or orders of the intestinal tract. Patients experiencing episodes SAPPA), ASCA (e.g., ASCA-IgA, ASCA-IgG, and/or of acute inflammation typically have elevated C-reactive pro ASCA-IgM), antimicrobial antibody (e.g., anti-Ompo anti tein (CRP) levels in addition to IBS-like symptoms. CRP is body, anti-flagellin antibody, and/or anti-I2 antibody), lacto produced by the liver during the acute phase of the inflam ferrin, anti-tTG antibody, lipocalin (e.g., NGAL, NGAL/ matory process and is usually released about 24 hours post MMP-9 complex), MMP (e.g., MMP-9), TIMP (e.g., TIMP commencement of the inflammatory process. Patients suffer 1), alpha-globulin (e.g., alpha-2-macroglobulin, haptoglobin, ing from diverticulitis, ileal pouch-anal anastomosis, and/or orosomucoid), actin-severing protein (e.g., gelsolin), microscopic colitis, and chronic infectious diarrhea typically S100 protein (e.g., calgranulin), fibrinopeptide (e.g., FIBA), have elevated fecal lactoferrin and/or calprotectin levels in CGRP, tachykinin (e.g., Substance P), ghrelin, neurotensin, addition to IBS-like symptoms. Lactoferrin is a glycoprotein corticotropin-releasing hormone, and combinations thereof. secreted by mucosal membranes and is the major protein in In yet other embodiments, the presence or level of other the secondary granules of leukocytes. Leukocytes are com diagnostic markers such as, for example, anti-lactoferrin anti monly recruited to inflammatory sites where they are acti body, L-selectin/CD62L, elastase, C-reactive protein (CRP), vated, releasing granule content to the Surrounding area. This calprotectin, anti-U1-70 kDa autoantibody, Zona occludens 1 process increases the concentration of lactoferrin in the stool. (ZO-1), vasoactive intestinal peptide (VIP), serum amyloid 0281 Increased lactoferrin levels are observed in patients A, gastrin, and a combination thereof may also be detected. with ileal pouch-anal anastomosis (i.e., a pouch is created US 2012/03 15630 A1 Dec. 13, 2012 30 following complete resection of colon in severe cases of cal algorithm, or by excluding (i.e., ruling out) those diseases Crohn's disease) when compared to other non-inflammatory and disorders that share a similar clinical presentation as IBS conditions of the pouch, like irritable pouch syndrome. and identifying (i.e., ruling in) IBS in a sample using, for Elevated levels of lactoferrin are also observed in patients example, a combination of statistical algorithms. with diverticulitis, a condition in which bulging pouches (i.e., (0285 H. Diagnostic Markers diverticula) in the digestive tract become inflamed and/or 0286 A variety of diagnostic markers are suitable for use infected, causing severe abdominal pain, fever, nausea, and a in the methods, systems, and code of the present invention for marked change in bowel habits. Microscopic colitis is a classifying a sample from an individual as an IBS sample or chronic inflammatory disorder that is also associated with for ruling out one or more diseases or disorders associated increased fecal lactoferrin levels. Microscopic colitis is char with IBS-like symptoms in a sample from an individual. acterized by persistent watery diarrhea (non-bloody), Examples of diagnostic markers include, without limitation, abdominal pain usually associated with weight loss, a normal cytokines, growth factors, anti-neutrophil antibodies, anti mucosa during colonoscopy and radiological examination, Saccharomyces cerevisiae antibodies, antimicrobial antibod and very specific histopathological changes. Microscopic ies, anti-tissue transglutaminase (tTG) antibodies, lipocalins, colitis consists of two diseases, collagenous colitis and lym matrix metalloproteinases (MMPs), complexes of lipocalin phocytic colitis. Collagenous colitis is of unknown etiology and MMP, tissue inhibitor of metalloproteinases (TIMPs), and is found in patients with long-term watery diarrhea and a globulins (e.g., alpha-globulins), actin-severing proteins, normal colonoscopy examination. Both collagenous colitis S100 proteins, fibrinopeptides, calcitonin gene-related pep and lymphocytic colitis are characterized by increased lym tide (CGRP), tachykinins, ghrelin, neurotensin, corticotro phocytes in the lining of the colon. Collagenous colitis is pin-releasing hormone (CRH), serine proteases (e.g., further characterized by a thickening of the sub-epithelial tryptases Such as B-tryptase, elastase, etc.), prostaglandin collagen layer of the colon. Chronic infectious diarrhea is an (e.g., PGE), histamine, C-reactive protein (CRP), lactofer illness that is also associated with increased fecal lactoferrin rin, anti-lactoferrin antibodies, calprotectin, hemoglobin, levels. Chronic infectious diarrhea is usually caused by a NOD2/CARD15, serotonin reuptake transporter (SERT), bacterial, viral, or protozoan infection, with patients present tryptophan hydroxylase-1,5-hydroxytryptamine (5-HT), ing with IBS-like symptoms such as diarrhea and abdominal lactulose, mast cell marker, stress marker, gastrointestinal pain. Increased lactoferrin levels are also observed in patients hormone, serotonin metabolite, serotonin pathway marker, with IBD. carbohydrate deficient transferrin (CDT), and combinations 0282. In addition to determining CRP and/or lactoferrin thereof. Additional diagnostic markers for predicting IBS in and/or calprotectin levels, diseases and disorders associated accordance with the present invention can be selected using with intestinal inflammation can also be ruled out by detect the techniques described in Example 14 from US Patent Pub ing the presence of blood in the stool. Such as fecal hemoglo lication No. 2008/0085524, filed Aug. 14, 2007, which is bin. Intestinal bleeding that occurs without the patient’s herein incorporated by reference in its entirety for all pur knowledge is called occult or hidden bleeding. The presence poses. One skilled in the art will also know of other diagnostic of occult bleeding (e.g., fecal hemoglobin) is typically markers suitable for use in the present invention. observed in a stool sample from the patient. Other conditions 0287. In particular embodiments, a diagnostic marker pro Such as ulcers (e.g., gastric, duodenal), cancer (e.g., Stomach file is determined by detecting the presence or level of at least cancer, colorectal cancer), and hemorrhoids can also present one, two, or all three of the following biomarkers: a serine with IBS-like symptoms including abdominal pain and a protease (e.g., a tryptase Such as B-tryptase); a prostaglandin change in the consistency and/or frequency of Stools. (e.g., PGE); and/or histamine. 0283. In addition, fecal calprotectin levels can also be 0288. In other embodiments, the presence or level of at assessed. Calprotectin is a calcium binding protein with anti least two, three, four, five, six, seven, eight, nine, ten, or more microbial activity derived predominantly from neutrophils diagnostic markers are determined in the individual's sample. and monocytes. Calprotectin has been found to have clinical In certain instances, the cytokine comprises one or more of relevance in cystic fibrosis, rheumatoid arthritis, IBD, col the cytokines described below. Preferably, the presence or orectal cancer, HIV, and other inflammatory diseases. Its level level of IL-8, IL-1B, TNF-related weak inducer of apoptosis has been measured in serum, plasma, oral, cerebrospinal and (TWEAK), leptin, osteoprotegerin (OPG), MIP-3|B, GROC, synovial fluids, urine, and feces. Advantages of fecal calpro CXCL4/PF-4, and/or CXCL7/NAP-2 is determined in the tectin in GI disorders have been recognized: stable for 3-7 individual's sample. In certain other instances, the growth days at room temperature enabling sample shipping through factor comprises one or more of the growth factors described regular mail; correlated to fecal alpha 1-antitrypsin in patients below. Preferably, the presence or level of epidermal growth with Crohn's disease; and elevated in a great majority of factor (EGF), vascular endothelial growth factor (VEGF), patients with gastrointestinal carcinomas and IBD. It was pigment epithelium-derived factor (PEDF), brain-derived found that fecal calprotectin correlates well with endoscopic neurotrophic factor (BDNF), and/or amphiregulin (SDGF) is and histological gradings of disease activity in ulcerative determined in the individual's sample. colitis, and with fecal excretion of indium-111-labelled neu 0289. In some instances, the anti-neutrophil antibody trophilic granulocytes, which is a standard of disease activity comprises ANCA, p-ANCA, cANCA, NSNA, SAPPA, and in IBD. combinations thereof. In other instances, the ASCA com 0284. In view of the foregoing, it is clear that a wide array prises ASCA-IgA, ASCA-IgG, ASCA-IgM, and combina of diseases and disorders can cause IBS-like symptoms, tions thereof. In further instances, the antimicrobial antibody thereby creating a substantial obstacle for definitively classi comprises an anti-Ompo antibody, anti-flagellin antibody, fying a sample as an IBS sample. However, the present inven anti-I2 antibody, and combinations thereof. tion overcomes this limitation by classifying a sample from 0290. In certain instances, the lipocalin comprises one or an individual as an IBS sample using, for example, a statisti more of the lipocalins described below. Preferably, the pres US 2012/03 15630 A1 Dec. 13, 2012 ence or level of neutrophil gelatinase-associated lipocalin precursor protein (NP 003285). The precursor protein is (NGAL) and/or a complex of NGAL and a matrix metallo then processed by the removal of a signal peptide (amino proteinase (e.g., NGAL/MMP-9 complex) is determined in acids 1-18) and activation peptide propeptide (amino acids the individual’s sample. In other instances, the matrix metal 19-30) resulting in the mature Tryptase beta-1 polypeptide loproteinase (MMP) comprises one or more of the MMPs (amino acids 31-275: UniProt: D15661). described below. Preferably, the presence or level of MMP-9 0296. In certain instances, the presence or level of a par is determined in the individual's sample. In further instances, ticular serine protease Such as tryptase is detected at the level the tissue inhibitor of metalloproteinase (TIMP) comprises of mRNA expression with an assay Such as, for example, a one or more of the TIMPs described below. Preferably, the hybridization assay or an amplification-based assay. In cer presence or level of TIMP-1 is determined in the individual’s tain other instances, the presence or level of a particular serine sample. In yet further instances, the alpha-globulin comprises protease Such as tryptase is detected at the level of protein one or more of the alpha-globulins described below. Prefer expression using, for example, an immunoassay (e.g., ably, the presence or level of alpha-2-macroglobulin, hapto ELISA) or an immunohistochemical assay. Suitable ELISA globin, and/or orosomucoid is determined in the individual’s techniques for determining the presence or level of tryptase in sample. a serum sample are described herein. In a particularly pre 0291. In certain other instances, the actin-severing protein ferred embodiment, the level of B-tryptase is detected in a comprises one or more of the actin-severing proteins blood or serum sample using a sandwich ELISA assay in described below. Preferably, the presence or level of gelsolin which the detecting antibody is an alkaline phosphatase con is determined in the individual's sample. In additional jugated anti-B-tryptase antibody, for example, commercial instances, the S100 protein comprises one or more of the antibody G3. A luminescent substrate, for example, CPSD S100 proteins described below including, for example, cal (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'- granulin. In yet other instances, the fibrinopeptide comprises chloro)tricyclo[3.3.1.13.7 decan-4-yl)phenyl phosphate), one or more of the fibrinopeptides described below. Prefer can then be used to enhance the sensitivity of the assay. ably, the presence or level of fibrinopeptide A (FIBA) is 0297 2. Prostaglandins determined in the individual’s sample. In further instances, 0298. The determination of the presence or level of at least the presence or level of a tachykinin such as Substance P. one prostaglandin in a sample is also useful in the present neurokinin A, and/or neurokinin B is determined in the indi invention. As used herein, the term “prostaglandin' includes vidual's sample. any member of a group of lipid compounds that are derived 0292. In some instances, the serine protease comprises enzymatically from fatty acids and have important functions one or more of the serine proteases described below. Prefer in the animal body. Every prostaglandin contains 20 carbon ably, the presence or level of a serine protease Such as tryptase atoms, including a 5-carbon ring. Prostaglandins, together (e.g., B-tryptase) is determined in the individual's sample. In with the thromboxanes and prostacyclins, form the pros other instances, the prostaglandin comprises one or more of tanoid class offatty acid derivatives. The prostanoid class is a the prostaglandins described below. Preferably, the presence Subclass of the eicosanoids. Non-limiting examples of pros or level of a prostaglandin such as PGE is determined in the taglandins include prostaglandin I2 (PGI2), prostaglandin E individual's sample. (PGE), prostaglandin F2 (PGF2), and combinations 0293. The presence or level of other diagnostic markers thereof. In a preferred embodiment, prostaglandin E. (PGE) Such as, for example, anti-lactoferrin antibody, L-selectin/ is detected in a blood or serum sample from a subject, for CD62L, elastase, C-reactive protein (CRP), calprotectin, example, a Subject Suspected of having IBS. In certain anti-U1-70 kDa autoantibody, Zona occludens 1 (ZO-1), embodiments, PGE may be detected by an ELISA or chemi vasoactive intestinal peptide (VIP), serum amyloid A, and/or luminescent assay. Suitable ELISA kits for determining the gastrin can also be determined. presence or level of PGE in a serum sample are available 0294 1. Serine Proteases from, e.g., Cayman Chemical Co. (Ann Arbor, Mich.). 0295 The determination of the presence or level of at least 0299 3. Histamine one serine protease in a sample is useful in the present inven 0300. The determination of the presence or level of hista tion. As used herein, the term "serine protease' includes any mine in a sample is also useful in the present invention. As member of a family of proteases in which one of the amino used herein, the term “histamine' includes a biogenic amine acids at the active site is serine. Non-limiting examples of involved in local immune responses as well as regulating serine proteases include tryptase (e.g., C.-tryptase, B-tryptase, physiological function in the gut and acting as a neurotrans Y-tryptase, and/or A-tryptase), elastase, chymotrypsin, mitter. Histamine triggers the inflammatory response. As part trypsin, Subtilisin, and combinations thereof. Tryptase is an of an immune response to foreign pathogens, histamine is abundant specific neutral protease of human mast cells that produced by basophils and by mast cells found in nearby can be measured in various biological fluids and can serve as connective tissues. Histamine increases the permeability of a useful marker for mast cell activation. In fact, tryptase can the capillaries to white blood cells and other proteins, in order be used as a marker for mast cell activation in IBS. It is to allow them to engage foreign invaders in the affected believed that tryptase, possibly by activation of protease tissues. It is found in virtually all animal body cells. In a activated receptor-2, is a good candidate to explain the pro preferred embodiment, histamine is detected in a blood or secretory and pro-inflammatory effects of mast cells, how serum sample from a subject, for example, a subject Sus ever other mast cell mediators could also involved. In a pected of having IBS. In certain embodiments, histamine may preferred embodiment, B-tryptase (TPBAB1) is detected in a be detected by an ELISA or chemiluminescent assay. Suitable blood or serum sample from a Subject. B-tryptase, also com ELISA kits for determining the presence or level of histamine monly referred to as Tryptase beta-1 or Tryptase I, is encoded in a serum sample are available from, e.g., Immunotech by the tryptase alpha/beta 1 gene (TPSABl; NM 003294), (Czech Republic) and Cayman Chemical Co. (Ann Arbor, which is translated to form a 275 amino acid tryptase beta-1 Mich.). US 2012/03 15630 A1 Dec. 13, 2012 32
0301 4. Cytokines IL-6/IL-9, IL-6/IL-10, IL-6/IL-12, IL-6/IL-13, IL-6/IL-15, 0302) The determination of the presence or level of at least IL-6/IL-17, IL-6/IL-23, IL-6/IL-27, IL-12/TWEAK, IL-12/ one cytokine in a sample is particularly useful in the present OPG, IL-12/IFN-O, IL-12/IFN-B, IL-12/IFN-y, IL-12/IL-1C., invention. As used herein, the term “cytokine' includes any of IL-12/IL-1B, IL-12/IL-1 ra, IL-12/IL-2, IL-12/IL-4, IL-12/ a variety of polypeptides or proteins secreted by immune cells IL-5, IL-12/IL-6, IL-12/SIL-6R, IL-12/IL-7, IL-12/IL-8, that regulate a range of immune system functions and encom IL-12/IL-9, IL-12/IL-10, IL-12/IL-13, IL-12/IL-15, IL-12/ passes Small cytokines Such as chemokines. The term “cytok IL-17, IL-12/IL-23, IL-12/IL-27, IL-10/TWEAK, IL-10/ ine also includes adipocytokines, which comprise a group of OPG, IL-10/IFN-O, IL-1B/IFN-B, IL-10/IFN-y, IL-10/IL-1C., cytokines secreted by adipocytes that function, for example, IL-1 B/IL-1 B, IL-10/IL-1ra, IL-10/IL-2, IL-10/IL-4, IL-10/ in the regulation of body weight, hematopoiesis, angiogen IL-5, IL-10/IL-6, IL-10/sIL-6R, IL-10/IL-7, IL-10/IL-8, esis, wound healing, insulin resistance, the immune response, IL-10/IL-9, IL-10/IL-12, IL-10/IL-13, IL-10/IL-15, IL-10/ and the inflammatory response. IL-17, IL-10/IL-23, IL-10/IL-27, IL-8/TWEAK, IL-8/OPG, 0303. In certain aspects, the presence or level of at least IL-8/IFN-O, IL-8/IFN-B, IL-8/IFN-y, IL-8/IL-1C., IL-8/IL one cytokine including, but not limited to, TNF-C, TNF 1B, IL-8/IL-1 ra, IL-8/IL-2, IL-8/IL-4, IL-8/IL-5, IL-8/IL-6, related weak inducer of apoptosis (TWEAK), osteoprote IL-8/sIL-6R, IL-8/IL-7, IL-8/IL-8, IL-8/IL-9, IL-8/IL-10, gerin (OPG), IFN-C, IFN-B, IFN-y, IL-1C., IL-1 B, IL-1 recep IL-8/IL-12, IL-8/IL-13, IL-8/IL-15, IL-8/IL-17, IL-8/IL-23, tor antagonist (IL-1 ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 IL-8/IL-27, or any other ratio of one cytokine to another. receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, 0305. In certain instances, the presence or level of a par IL-15, IL-17, IL-23, and IL-27 is determined in a sample. In ticular cytokine is detected at the level of mRNA expression certain other aspects, the presence or level of at least one withanassay Such as, for example, a hybridization assay oran chemokine such as, for example, CXCL1/GRO1/GROC. amplification-based assay. In certain other instances, the CXCL2/GRO2, CXCL3/GRO3, CXCL4/PF-4, CXCL5/ presence or level of a particular cytokine is detected at the ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL9/MIG, level of protein expression using, for example, an immunoas CXCL10/IP-10, CXCL11/1-TAC, CXCL12/SDF-1, say (e.g., ELISA) or an immunohistochemical assay. Suitable CXCL13/BCA-1, CXCL14/BRAK, CXCL15, CXCL16, ELISA kits for determining the presence or level of a cytokine CXCL17/DMC, CCL1, CCL2/MCP-1, CCL3/MIP-1o, such as IL-8, IL-1 B, MIP-3|B, GROC, CXCL4/PF-4, or CCL4/MIP-1B, CCL5/RANTES, CCL6/C10,CCL7/MCP-3, CXCL7/NAP-2 in a serum, plasma, saliva, or urine sample CCL8/MCP-2, CCL9/CCL10, CCL11/Eotaxin, CCL12/ are available from, e.g., R&D Systems, Inc. (Minneapolis, MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/MIP-5, Minn.), Neogen Corp. (Lexington, Ky.), Alpco Diagnostics CCL16/LEC, CCL17/TARC, CCL18/MIP-4, CCL19/MIP (Salem, N.H.). Assay Designs, Inc. (Ann Arbor, Mich.), BD 3 B, CCL20/MIP-3C, CCL21/SLC, CCL22/MDC, CCL23/ Biosciences Pharmingen (San Diego, Calif.), Invitrogen (Ca MPIF1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin marillo, Calif.), Calbiochem (San Diego, Calif.), CHEMI 3, CCL27/CTACK, CCL28/MEC, CL1, CL2, and CXCL1 is CON International, Inc. (Temecula, Calif.), Antigenix determined in a sample. In certain further aspects, the pres America Inc. (Huntington Station, N.Y.), QIAGEN Inc. (Va.- ence or level of at least one adipocytokine including, but not lencia, Calif.), Bio-Rad Laboratories, Inc. (Hercules, Calif.), limited to, leptin, adiponectin, resistin, active or total plasmi and/or Bender MedSystems Inc. (Burlingame, Calif.). nogen activator inhibitor-1 (PAI-1), visfatin, and retinol bind 0306 5. TWEAK ing protein 4 (RBP4) is determined in a sample. Preferably, (0307 TWEAK is a member of the TNF superfamily of the presence or level of IL-8, IL-1 B, TWEAK, leptin, OPG, structurally related cytokines. Full-length, membrane-an MIP-3|B, GROC, CXCL4/PF-4, and/or CXCL7/NAP-2 is chored TWEAK can be found on the surface of many cell determined. types and a smaller, biologically active form, generated via 0304. In yet other aspects, the ratio of cytokine levels is proteolytic processing, has also been detected in the extracel determined in a sample. For example, the ratio of the level of lular milieu (see, e.g., Chicheportiche et al., J. Biol. Chen., TNF-C. to the level of IL-10 (TNF-C/IL-10), or the ratio of the 272:32401-32410 (1997)). TWEAK acts via binding to a level of IL-10 to the level of IL-12 (IL-10/IL-12), may be TNF receptor superfamily member named fibroblast growth diagnostic of IBS. In yet other embodiments, the ratio of the factor-inducible 14 (Fn14, also known as tumor necrosis fac level any one cytokine to the level of any other one cytokine tor receptor superfamily member 12A or TNFRSF12A). may be useful. Non-limiting example of ratios that may be TWEAK has multiple biological activities, including stimu useful in the methods of the present invention include TNF lation of cell growth and angiogenesis, induction of inflam C/TWEAK, TNF-C/OPG, TNF-C/IFN-C, TNF-C/IFN-B, matory cytokines, and stimulation of apoptosis (see, e.g., TNF-C/IFN-y, TNF-C/IL-1C, TNF-C/IL-1 B, TNF-C/IL-1 ra. Wiley et al., Cytokine Growth Factor Rev., 14:241-249 TNF-C/IL-2, TNF-C/IL-4, TNF-C/IL-5, TNF-C/IL-6, TNF (2003)). In particular, TWEAK has been shown to induce the C/sIL-6R, TNF-C/IL-7, TNF-C/IL-8, TNF-C/IL-9, TNF-C/ expression of PGE, MMP-1, IL-6, IL-8, RANTES, and IL-10, TNF-C/IL-12, TNF-C/IL-13, TNF-C/IL-15, TNF-C/ IP-10 in fibroblasts and synoviocytes, and to upregulate IL-17, TNF-C/IL-23, TNF-C/IL-27, TWEAK/OPG, ICAM-1, E-selectin, IL-8, and MCP-1 expression in endot TWEAK/IFN-O, TWEAK/IFN-B, TWEAK/IFN-y, helial cells (see, e.g., Campbell et al., Front. Biosci., 9:2273 TWEAK/IL-1C, TWEAK/IL-1 B, TWEAK/IL-1ra, TWEAK/ 2284 (2004)). It has also been demonstrated that TWEAK IL-2, TWEAK/IL-4, TWEAK/IL-5, TWEAK/IL-6, binding to the Fn 14 receptor, or constitutive Fn 14 overexpres TWEAK/SIL-6R, TWEAK/IL-7, TWEAK/IL-8, TWEAK/ Sion, activates the NF-KB signaling pathway, which plays an IL-9, TWEAK/IL-10, TWEAK/IL-12, TWEAK/IL-13, important role in immune and inflammatory processes, onco TWEAK/IL-15, TWEAK/IL-17, TWEAK/IL-23, TWEAK/ genesis, cancer therapy resistance, and tumorigenesis (see, IL-27, IL-6/TWEAK, IL-6/OPG, IL-6/IFN-O, IL-6/IFN-B, e.g., Winkles et al., Cancer Lett., 235:11-17 (2006); and IL-6/IFN-y, IL-6/IL-1C., IL-6/IL-1B, IL-6/IL-1 ra, IL-6/IL-2, Winkles et al., Front. Biosci., 12:2761-2771 (2007)). One IL-6/IL-4, IL-6/IL-5, IL-6/sIL-6R, IL-6/IL-7, IL-6/IL-8, skilled in the art will appreciate that TWEAK is also known as US 2012/03 15630 A1 Dec. 13, 2012 tumor necrosis factor ligand superfamily member 12 (TN tems Laboratories, Inc. (Webster, Tex.), Immuno-Biological FSF12), APO3 ligand (APO3L), CD255, DR3 ligand, FN14, Laboratories, Inc. (Minneapolis, Minn.), and Cayman and UNQ181/PRO207. Chemical Co. (Ann Arbor, Mich.). 0308 Suitable ELISA kits for determining the presence or 0315 8. Growth Factors level of TWEAK in a biological sample such as a serum, 0316 The determination of the presence or level of one or plasma, Saliva, or urine sample are available from, e.g., Anti more growth factors in a sample is also useful in the present genix America Inc. (Huntington Station, N.Y.), Bender Med invention. As used herein, the term “growth factor” includes Systems Inc. (Burlingame, Calif.), Agdia Inc. (Elkhart, Ind.), any of a variety of peptides, polypeptides, or proteins that are American Research Products Inc. (Belmont, Mass.), capable of stimulating cellular proliferation and/or cellular Biomeda Corp. (Foster City, Calif.), BioVision, Inc. (Moun differentiation. tain View, Calif.), and Kamiya Biomedical Co. (Seattle, 0317. In certain aspects, the presence or level of at least Wash.). one growth factor including, but not limited to, epidermal 0309 6. Osteoprotegerin (OPG) growth factor (EGF), heparin-binding epidermal growth fac 0310. OPG is a 401-amino acid member of the TNF super tor (HB-EGF), vascular endothelial growth factor (VEGF), family of structurally related cytokines OPG, which is pigment epithelium-derived factor (PEDF; also known as homologous to the receptor activator of NFKB (RANK), SERPINF1), amphiregulin (AREG; also known as Schwan inhibits the differentiation of macrophages into osteoclasts noma-derived growth factor (SDGF)), basic fibroblast growth and regulates the resorption of osteoclasts by acting as a factor (bFGF), hepatocyte growth factor (HGF), transforming soluble decoy receptor for RANK ligand (RANKL.; also growth factor-O. (TGF-C.), transforming growth factor-13 known as OPG ligand (OPGL)). As a result, the OPG-RANK (TGF-3), bone morphogenetic proteins (e.g., BMP1 RANKL system plays a direct and essential role in the for BMP15), platelet-derived growth factor (PDGF), nerve mation, function, and survival of osteoclasts. The OPG growth factor (NGF), B-nerve growth factor (B-NGF), neu RANK-RANKL system has also been shown to modulate rotrophic factors (e.g., brain-derived neurotrophic factor cancer cell migration, thus controlling the development of (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), etc.), bone metastases. One skilled in the art will appreciate that growth differentiation factor-9 (GDF-9), granulocyte-colony OPG is also known as osteoprotegrin and osteoclastogenesis stimulating factor (G-CSF), granulocyte-macrophage colony inhibitory factor (OCIF). stimulating factor (GM-CSF), myostatin (GDF-8), erythro 0311 Suitable ELISA kits for determining the presence or poietin (EPO), and thrombopoietin (TPO) is determined in a level of OPG in a serum, plasma, saliva, or urine sample are sample. Preferably, the presence or level of EGF, VEGF, available from, e.g., Antigenix America Inc. (Huntington Sta PEDF, amphiregulin (SDGF), and/or BDNF is determined. tion, N.Y.), Immunodiagnostic Systems Ltd. (Boldon, United 0318. In certain instances, the presence or level of a par Kingdom), and BioVendor, LLC (Candler, N.C.). ticular growth factor is detected at the level of mRNA expres 0312 7. Leptin sion with an assay Such as, for example, a hybridization assay 0313 Leptin, a member of the adipocytokine family of oran amplification-based assay. In certain other instances, the cytokines, is a 16-kD peptide hormone that plays a critical presence or level of a particular growth factor is detected at role in the regulation of body weight by inhibiting food intake the level of protein expression using, for example, an immu and stimulating energy expenditure. It is predominantly Syn noassay (e.g., ELISA) or an immunohistochemical assay. thesized by adipocytes and circulates in the plasma in Suitable ELISA kits for determining the presence or level of amounts proportional to body fat content (see, e.g., Maffei et a growth factor such as EGF, VEGF, PEDF, SDGF, or BDNF al., Nat. Med., 1:1155-1161 (1995); Considine et al., Diabe in a serum, plasma, saliva, or urine sample are available from, tes, 45:992-994 (1996)). Leptin displays a high degree of e.g., Antigenix America Inc. (Huntington Station, N.Y.), homology among different species and it is also analogous in Promega (Madison, Wis.), R&D Systems, Inc. (Minneapolis, structure to other cytokines (see, e.g., Madei et al., FEBS Minn.), Invitrogen (Camarillo, Calif.), CHEMICON Interna Lett., 373:13-18 (1995)). Leptinacts through the leptin recep tional, Inc. (Temecula, Calif.), Neogen Corp. (Lexington, tor, a single-transmembrane-domain receptor of the class I Ky.), PeproTech (Rocky Hill, N.J.), Alpco Diagnostics (Sa cytokine Superfamily of receptors, which are characterized by lem, N.H.). Pierce Biotechnology, Inc. (Rockford, Ill.), and/ extracellular motifs of four cysteine residues and a number of or Abazyme (Needham, Mass.). fibronectin type III domains (see, e.g., Heim, Eur: J. Clin. 0319 9. Lipocalins Invest., 26:1-12 (1996)). The leptin receptor is known to exist 0320. The determination of the presence or level of one or as a homodimer and is activated by conformational changes more lipocalins in a sample is also useful in the present that occur following ligand binding to the receptor (see, e.g., invention. As used herein, the term “lipocalin' includes any of Devos et al., J. Biol. Chem., 272:18304-18310 (1997)). Six a variety of small extracellular proteins that are characterized leptin receptor isoforms, generated by alternate slicing, have by several common molecular recognition properties: the been identified to date (see, e.g., Wang et al., Nature, 393: ability to bind a range of small hydrophobic molecules; bind 684-688 (1998); Lee et al., Nature, 379:632-635 (1996)). ing to specific cell-surface receptors; and the formation of 0314 Suitable ELISA kits for determining the presence or complexes with Soluble macromolecules (see, e.g., Flowers, level of leptin in a biological sample Such as a serum, plasma, Biochem. J., 3 18:1-14 (1996)). The varied biological func saliva, or urine sample are available from, e.g., R&D Sys tions of lipocalins are mediated by one or more of these tems, Inc. (Minneapolis, Minn.), B-Bridge International properties. The lipocalin protein family exhibits great func (Mountain View, Calif.), Neogen Corp. (Lexington, Ky.), tional diversity, with roles in retinol transport, invertebrate Assay Designs, Inc. (Ann Arbor, Mich.), Invitrogen (Cama cryptic coloration, olfaction and pheromone transport, and rillo, Calif.), CHEMICON International, Inc. (Temecula, prostaglandin synthesis. Lipocalins have also been impli Calif.), Antigenix America Inc. (Huntington Station, N.Y.), cated in the regulation of cell homoeostasis and the modula LINCOResearch, Inc. (St. Charles, Mo.), Diagnostic Sys tion of the immune response, and, as carrier proteins, to act in US 2012/03 15630 A1 Dec. 13, 2012 34 the general clearance of endogenous and exogenous com loelastase), MMP-13 (collagenase-3), MMP-14, MMP-15, pounds. Although lipocalins have great diversity at the MMP-16, MMP-17, MMP-18 (collagenase-4), MMP-19, sequence level, their three-dimensional structure is a unifying MMP-20 (enamelysin), MMP-21, MMP-23, MMP-24, characteristic. Lipocalin crystal structures are highly con MMP-25, MMP-26 (matrilysin-2), MMP-27, and MMP-28 served and comprise a single eight-stranded continuously (epilysin) is determined in a sample. Preferably, the presence hydrogen-bonded antiparallel beta-barrel, which encloses an or level of MMP-9 is determined. internal ligand-binding site. 0326 In certain instances, the presence or level of a par 0321. In certain aspects, the presence or level of at least ticular MMP is detected at the level of mRNA expression with one lipocalin including, but not limited to, neutrophil gelati an assay Such as, for example, a hybridization assay or an nase-associated lipocalin (NGAL; also known as human neu amplification-based assay. In certain other instances, the trophil lipocalin (HNL) or lipocalin-2), von Ebner's gland presence or level of a particular MMP is detected at the level protein (VEGP; also known as lipocalin-1), retinol-binding of protein expression using, for example, an immunoassay protein (RBP), purpurin (PURP), retinoic acid-binding pro (e.g., ELISA) or an immunohistochemical assay. Suitable tein (RABP), C-globulin (A2U), major urinary protein ELISA kits for determining the presence or level of an MMP (MUP), bilin-binding protein (BBP), C.-crustacyanin, preg such as MMP-9 in a serum or plasma sample are available nancy protein 14 (PP14), f-lactoglobulin (Blg.), C-microglo from, e.g., Calbiochem (San Diego, Calif.), CHEMICON bulin (A1M), the gamma chain of C8 (C8), Apolipoprotein International, Inc. (Temecula, Calif.), and R&D Systems, Inc. D (Apod), lazarillo (LAZ), prostaglandin D2 synthase (Minneapolis, Minn.). (PGDS), quiescence-specific protein (QSP), choroid plexus 0327 11. Tissue Inhibitor of Metalloproteinases protein, odorant-binding protein (OBP), C-acid glycopro 0328. The determination of the presence or level of at least tein (AGP), probasin (PBAS), aphrodisin, orosomucoid, and one tissue inhibitor of metalloproteinase (TIMP) in a sample progestagen-associated endometrial protein (PAEP) is deter is also useful in the present invention. As used herein, the term mined in a sample. In certain other aspects, the presence or “tissue inhibitor of metalloproteinase” or “TIMP includes level of at least one lipocalin complex including, for example, proteins capable of inhibiting MMPs. a complex of NGAL and a matrix metalloproteinase (e.g., 0329. In certain aspects, the presence or level of at least NGAL/MMP-9 complex) is determined. Preferably, the pres one at least one TIMP including, but not limited to, TIMP-1, ence or level of NGAL or a complex thereof with MMP-9 is TIMP-2, TIMP-3, and TIMP-4 is determined in a sample. determined. Preferably, the presence or level of TIMP-1 is determined. 0322. In certain instances, the presence or level of a par 0330. In certain instances, the presence or level of a par ticular lipocalin is detected at the level of mRNA expression ticular TIMP is detected at the level of mRNA expression with withanassay Such as, for example, a hybridization assay oran an assay Such as, for example, a hybridization assay or an amplification-based assay. In certain other instances, the amplification-based assay. In certain other instances, the presence or level of a particular lipocalin is detected at the presence or level of a particular TIMP is detected at the level level of protein expression using, for example, an immunoas of protein expression using, for example, an immunoassay say (e.g., ELISA) or an immunohistochemical assay. Suitable (e.g., ELISA) or an immunohistochemical assay. Suitable ELISA kits for determining the presence or level of a lipoca ELISA kits for determining the presence or level of a TIMP lin Such as NGAL in a serum, plasma, or urine sample are such as TIMP-1 in a serum or plasma sample are available available from, e.g., AntibodyShop A/S (Gentofte, Den from, e.g., Alpco Diagnostics (Salem, N.H.), Calbiochem mark), LabClinics SA (Barcelona, Spain), Lucerna-Chem (San Diego, Calif.), Invitrogen (Camarillo, Calif.), CHEMI AG (Luzern, Switzerland), R&D Systems, Inc. (Minneapolis, CON International, Inc. (Temecula, Calif.), and R&D Sys Minn.), and Assay Designs, Inc. (Ann Arbor, Mich.). Suitable tems, Inc. (Minneapolis, Minn.). ELISA kits for determining the presence or level of the 0331 12. Globulins NGAL/MMP-9 complex are available from, e.g., R&D Sys 0332 The determination of the presence or level of at least tems, Inc. (Minneapolis, Minn.). Additional NGAL and one globulin in a sample is also useful in the present inven NGAL/MMP-9 complex ELISA techniques are described in, tion. As used herein, the term “globulin' includes any mem e.g., Kjeldsen et al., Blood, 83:799-807 (1994); and Kjeldsen ber of a heterogeneous series of families of serum proteins et al., J. Immunol. Methods, 198: 155-164 (1996). which migrate less than albumin during serum electrophore 0323 10. Matrix Metalloproteinases sis. Protein electrophoresis is typically used to categorize 0324. The determination of the presence or level of at least globulins into the following three categories: alpha-globulins one matrix metalloproteinase (MMP) in a sample is also (i.e., alpha-1-globulins or alpha-2-globulins); beta-globulins; useful in the present invention. As used herein, the term and gamma-globulins. “matrix metalloproteinase' or “MMP includes zinc-depen 0333 Alpha-globulins comprise a group of globular pro dent endopeptidases capable of degrading a variety of extra teins in plasma which are highly mobile in alkaline or elec cellular matrix proteins, cleaving cell Surface receptors, trically-charged solutions. They generally function to inhibit releasing apoptotic ligands, and/or regulating chemokines certain blood protease and inhibitor activity. Examples of MMPs are also thought to play a major role in cell behaviors alpha-globulins include, but are not limited to, alpha-2-mac Such as cell proliferation, migration (adhesion/dispersion), roglobulin (C2-MG), haptoglobin (Hp), orosomucoid, alpha differentiation, angiogenesis, and host defense. 1-antitrypsin, alpha-1-antichymotrypsin, alpha-2-antiplas 0325 In certain aspects, the presence or level of at least min, antithrombin, ceruloplasmin, heparin cofactor II, retinol one at least one MMP including, but not limited to, MMP-1 binding protein, and transcortin. Preferably, the presence or (interstitial collagenase), MMP-2 (gelatinase-A), MMP-3 level of C2-MG, haptoglobin, and/or orosomucoid is deter (stromelysin-1), MMP-7 (matrilysin), MMP-8 (neutrophil mined. In certain instances, one or more haptoglobin allo collagenase), MMP-9 (gelatinase-B), MMP-10 (stromelysin types such as, for example, Hp precursor, Hpf, HpC.1, and 2), MMP-11 (stromelysin-3), MMP-12 (macrophage metal Hpo.2, are determined. US 2012/03 15630 A1 Dec. 13, 2012
0334. In certain instances, the presence or level of a par Examples of calgranulins include, without limitation, cal ticular globulin is detected at the level of mRNA expression granulin A (also known as S100A8 or MRP-8), calgranulin B withanassay Such as, for example, a hybridization assay oran (also known as S100A9 or MRP-14), and calgranulin C (also amplification-based assay. In certain other instances, the known as S100A12). presence or level of a particular globulin is detected at the 0341. In certain instances, the presence or level of a par level of protein expression using, for example, an immunoas ticular S100 protein is detected at the level of mRNA expres say (e.g., ELISA) or an immunohistochemical assay. Suitable sion with an assay Such as, for example, a hybridization assay ELISA kits for determining the presence or level of a globulin oran amplification-based assay. In certain other instances, the Such as C2-MG, haptoglobin, or orosomucoid in a serum, presence or level of a particular S100 protein is detected at the plasma, or urine sample are available from, e.g., GenWay level of protein expression using, for example, an immunoas Biotech, Inc. (San Diego, Calif.) and/or Immundiagnostik AG say (e.g., ELISA) or an immunohistochemical assay. Suitable (Bensheim, Germany) ELISA kits for determining the presence or level of an S100 0335 13. Actin-Severing Proteins protein such as calgranulin A (S100A8) or calgranulin B (S 0336. The determination of the presence or level of at least 100A9) in a serum, plasma, or urine sample are available one actin-severing protein in a sample is also useful in the from, e.g., Peninsula Laboratories Inc. (San Carlos, Calif.) present invention. As used herein, the term “actin-severing and Hycult biotechnology b.v. (Uden, The Netherlands). protein’ includes any member of a family of proteins (0342 Calprotectin, the complex of S100A8 and S100A9, involved in actin remodeling and regulation of cell motility. is a calcium- and zinc-binding protein in the cytosol of neu Non-limiting examples of actin-severing proteins include trophils, monocytes, and keratinocytes. Calprotectin is a gelsolin (also known as brevin or actin-depolymerizing fac major protein in neutrophilic granulocytes and macrophages tor), Villin, fragmin, and adseverin. For example, gelsolin is a and accounts for as much as 60% of the total protein in the protein of leukocytes, platelets, and other cells which severs cytosol fraction in these cells. It is therefore a Surrogate actin filaments in the presence of Submicromolar calcium, marker of neutrophil turnover. Its concentration in stool cor thereby solating cytoplasmic actingels. relates with the intensity of neutrophil infiltration of the intes 0337. In certain instances, the presence or level of a par tinal mucosa and with the severity of inflammation. In some ticular actin-severing protein is detected at the level of mRNA instances, calprotectin can be measured with an ELISA using expression with an assay Such as, for example, a hybridization Small (50-100mg) fecal samples (see, e.g., Johne et al., Scand assay or an amplification-based assay. In certain other J. Gastroenterol., 36:291-296 (2001)). instances, the presence or level of a particular actin-severing (0343 15. Tachykinins protein is detected at the level of protein expression using, for 0344) The determination of the presence or level of at least example, an immunoassay (e.g., ELISA) or an immunohis one tachykinin in a sample is also useful in the present inven tochemical assay. Suitable ELISA techniques for determin tion. As used herein, the term “tachykinin’ includes amidated ing the presence or level of an actin-severing protein Such as neuropeptides that share the carboxy-terminal sequence Phe gelsolin in a plasma sample are described in, e.g., Smith et al., X-Gly-Leu-Met-NH. Tachykinins typically bind to one or J. Lab. Clin. Med., 110:189-195 (1987); and Hiyoshi et al., more tachykinin receptors (e.g., TACR1, TACR2, and/or Biochem. Mol. Biol. Int., 32:755-762 (1994). TACR3). 0338 14. S100 Proteins 0345. In certain aspects, the presence or level of at least 0339. The determination of the presence or level of at least one tachykinin including, but not limited to, Substance P. one S100 protein in a sample is also useful in the present neurokinin A, and neurokinin B is determined in a sample. invention. As used herein, the term “S100 protein’ includes Preferably, the presence or level of substance P is determined. any member of a family of low molecular massacidic proteins Substance P is a peptide of 11 amino acids in length that is characterized by cell-type-specific expression and the pres released by nerve endings in both the central and peripheral ence of 2 EF-hand calcium-binding domains. There are at nervous systems. Among the numerous biological sites inner least 21 different types of S100 proteins in humans. The name vated by Substance P-releasing neurons are the skin, intes is derived from the fact that S100 proteins are 100% soluble in tines, stomach, bladder, and cardiovascular system. ammonium sulfate at neutral pH. Most S100 proteins are 0346. In certain instances, the presence or level of a par homodimeric, consisting of two identical polypeptides held ticular tachykinin is detected at the level of mRNA expression together by non-covalent bonds. Although S100 proteins are withanassay Such as, for example, a hybridization assay oran structurally similar to calmodulin, they differ in that they are amplification-based assay. In certain other instances, the cell-specific, expressed in particular cells at different levels presence or level of a particular tachykinin is detected at the depending on environmental factors. S-100 proteins are nor level of protein expression using, for example, an immunoas mally present in cells derived from the neural crest (e.g., say (e.g., ELISA) or an immunohistochemical assay. Suitable Schwann cells, melanocytes, glial cells), chondrocytes, adi ELISA kits for determining the presence or level of a tachy pocytes, myoepithelial cells, macrophages, Langerhans cells, kinin Such as Substance P in a serum, plasma, Saliva, or urine dendritic cells, and keratinocytes. S100 proteins have been sample are available from, e.g., MD Biosciences Inc. (St. implicated in a variety of intracellular and extracellular func Paul, Minn.), Assay Designs, Inc. (Ann Arbor, Mich.), R&D tions such as the regulation of protein phosphorylation, tran Systems, Inc. (Minneapolis, Minn.), Sigma-Aldrich Corp. scription factors, Ca" homeostasis, the dynamics of cytosk (St. Louis, Mo.), and Cayman Chemical Co. (Ann Arbor, eleton constituents, enzyme activities, cell growth and Mich.). differentiation, and the inflammatory response. 0347. 16. Ghrelin 0340 Calgranulin is an S100 protein that is expressed in 0348. The determination of the presence or level of ghrelin multiple cell types, including renal epithelial cells and neu in a sample is also useful in the present invention. As used trophils, and are abundant in infiltrating monocytes and herein, the term “ghrelin' includes a peptide of 28 amino granulocytes under conditions of chronic inflammation. acids that is an endogenous ligand for the growth hormone US 2012/03 15630 A1 Dec. 13, 2012 36 secretagogue receptor (GHSR) and is involved in regulating CRH is detected at the level of protein expression using, for growth hormone release. Ghrelin can be acylated, typically example, an immunoassay (e.g., ELISA) or an immunohis with an n-octanoyl group at serine residue three, to form tochemical assay. Suitable ELISA kits for determining the active ghrelin. Alternatively, ghrelin can exist as an unacy presence or level of CRH in a serum, plasma, Saliva, or urine lated form (i.e., desacyl-ghrelin). Ghrelin is primarily sample are available from, e.g., Alpco Diagnostics (Salem, expressed in specialized enterochromaffin cells located N.H.) and Cosmo Bio Co., Ltd. (Tokyo, Japan). mainly in the mucosa of the fundus of the stomach and has 0356. 19. Anti-Neutrophil Antibodies metabolic effects opposite to those of leptin. Ghrelin stimu 0357 The determination of ANCA levels and/or the pres lates food intake, enhances the use of carbohydrates and ence or absence of pANCA in a sample is also useful in the reduces fat utilization, increases gastric motility and acid present invention. As used herein, the term “anti-neutrophil secretion, and reduces locomotor activity. cytoplasmic antibody' or ANCA includes antibodies 0349. In certain instances, the presence or level of ghrelin directed to cytoplasmic and/or nuclear components of neu is detected at the level of mRNA expression with an assay trophils. ANCA activity can be divided into several broad Such as, for example, a hybridization assay or an amplifica categories based upon the ANCA staining pattern in neutro tion-based assay. In certain other instances, the presence or phils: (1) cytoplasmic neutrophil staining without perinuclear level of ghrelin is detected at the level of protein expression highlighting (cANCA); (2) perinuclear staining around the using, for example, an immunoassay (e.g., ELISA) or an outside edge of the nucleus (pANCA); (3) perinuclear stain immunohistochemical assay. Suitable ELISA kits for deter ing around the inside edge of the nucleus (NSNA); and (4) mining the presence or level of active ghrelin or desacyl diffuse staining with speckling across the entire neutrophil ghrelin in a serum, plasma, saliva, or urine sample are avail (SAPPA). In certain instances, p ANCA staining is sensitive able from, e.g., Alpco Diagnostics (Salem, N.H.). Cayman to DNase treatment. The term ANCA encompasses all vari Chemical Co. (Ann Arbor, Mich.), LINCO Research, Inc. (St. eties of anti-neutrophil reactivity, including, but not limited Charles, Mo.), and Diagnostic Systems Laboratories, Inc. to, cANCA, p-ANCA, NSNA, and SAPPA. Similarly, the term (Webster, Tex.). ANCA encompasses all immunoglobulin isotypes including, 0350. 17. Neurotensin without limitation, immunoglobulin A and G. 0351. The determination of the presence or level of neu 0358 ANCA levels in a sample from an individual can be rotensin in a sample is also useful in the present invention. As determined, for example, using an immunoassay Such as an used herein, the term “neurotensin' includes a tridecapeptide enzyme-linked immunosorbent assay (ELISA) with alcohol that is widely distributed throughout the central nervous sys fixed neutrophils. The presence or absence of a particular tem and the gastrointestinal tract. Neurotensin has been iden category of ANCA such as paNCA can be determined, for tified as an important mediator in the development and pro example, using an immunohistochemical assay Such as an gression of several gastrointestinal functions and disease indirect fluorescent antibody (IFA) assay. Preferably, the conditions, exerting its effects by interacting with specific presence or absence of pANCA in a sample is determined receptors that act directly or indirectly on nerves, epithelial using an immunofluorescence assay with DNase-treated, cells, and/or cells of the immune and inflammatory systems fixed neutrophils. In addition to fixed neutrophils, antigens (see, e.g., Zhao et al., Peptides, 27:2434-2444 (2006)). specific for ANCA that are suitable for determining ANCA 0352. In certain instances, the presence or level of neuro levels include, without limitation, unpurified or partially puri tensin is detected at the level of mRNA expression with an fied neutrophil extracts; purified proteins, protein fragments, assay Such as, for example, a hybridization assay or an ampli or synthetic peptides such as histone H1 or ANCA-reactive fication-based assay. In certain other instances, the presence fragments thereof (see, e.g., U.S. Pat. No. 6,074,835); histone or level of neurotensin is detected at the level of protein H1-like antigens, porin antigens, Bacteroides antigens, or expression using, for example, an immunoassay (e.g., ANCA-reactive fragments thereof (see, e.g., U.S. Pat. No. ELISA) or an immunohistochemical assay. Suitable ELISA 6,033,864); secretory vesicle antigens or ANCA-reactive techniques for determining the presence or level of neuro fragments thereof (see, e.g., U.S. patent application Ser. No. tensin in a sample are described in, e.g., Davis et al., J. 08/804,106); and anti-ANCA idiotypic antibodies. One Neurosci. Methods, 14:15-23 (1985); and Williams et al., J. skilled in the art will appreciate that the use of additional Histochem. Cytochem., 37:831-841 (1989). antigens specific for ANCA is within the scope of the present 0353. 18. Corticotropin-Releasing Hormone invention. 0354. The determination of the presence or level of corti 0359. 20. Anti-Saccharomyces cerevisiae Antibodies cotropin-releasing hormone (CRH; also known as corticotro 0360. The determination of ASCA (e.g., ASCA-IgA and/ pin-releasing factor or CRF) in a sample is also useful in the or ASCA-IgG) levels in a sample is also useful in the present present invention. As used herein, the term “corticotropin invention. As used herein, the term “anti-Saccharomyces cer releasing hormone.”“CRH.”“corticotropin-releasing factor.” evisiae immunoglobulin A' or ASCA-IgA' includes anti or “CRF includes a 41-amino acid peptide secreted by the bodies of the immunoglobulin A isotype that react specifi paraventricular nucleus of the hypothalamus that mediates cally with S. cerevisiae. Similarly, the term “anti the proximal part of the response to stress in mammals such as Saccharomyces cerevisiae immunoglobulin G’ or ASCA humans. CRH typically binds to one or more corticotropin IgG’ includes antibodies of the immunoglobulin G isotype releasing hormone receptors (e.g., CRHR1 and/or CRHR2). that react specifically with S. cerevisiae. CRH is expressed by the hypothalamus, spinal cord, stomach, 0361. The determination of whether a sample is positive spleen, duodenum, adrenal gland, and placenta. for ASCA-IgA or ASCA-IgG is made using an antigen spe 0355. In certain instances, the presence or level of CRH is cific for ASCA. Suchanantigen can be any antigen or mixture detected at the level of mRNA expression with an assay such of antigens that is bound specifically by ASCA-IgA and/or as, for example, a hybridization assay or an amplification ASCA-IgG. Although ASCA antibodies were initially char based assay. In certain other instances, the presence or level of acterized by their ability to bind S. cerevisiae, those of skill in US 2012/03 15630 A1 Dec. 13, 2012 37 the art will understand that an antigen that is bound specifi 0365. 21. Anti-Microbial Antibodies cally by ASCA can be obtained from S. cerevisiae or from a 0366. The determination of anti-Ompo antibody levels in variety of other sources so long as the antigen is capable of a sample is also useful in the present invention. As used binding specifically to ASCA antibodies. Accordingly, exem herein, the term “anti-outer membrane protein Cantibody” or plary sources of an antigen specific for ASCA, which can be “anti-Ompo antibody' includes antibodies directed to a bac used to determine the levels of ASCA-IgA and/or ASCA-IgG terial outer membrane porin as described in, e.g., PCT Patent in a sample, include, without limitation, whole killed yeast Publication No. WO 01/893.61. The term “outer membrane cells Such as Saccharomyces or Candida cells; yeast cell wall protein C or "Ompo” refers to a bacterial porin that is mannan Such as phosphopeptidomannan (PPM); oligosach immunoreactive with an anti-Ompo antibody. harides such as oligomannosides; neoglycolipids; anti 0367 The level of anti-OmpC antibody present in a ASCA idiotypic antibodies; and the like. Different species sample from an individual can be determined using an OmpC and strains of yeast, such as S. cerevisiae strainSul, Su2, CBS protein or a fragment thereof Such as an immunoreactive 1315, or BM 156, or Candida albicans strain VW32, are fragment thereof. Suitable OmpC antigens useful in deter Suitable for use as an antigen specific for ASCA-IgA and/or mining anti-Ompo antibody levels in a sample include, with ASCA-IgG. Purified and synthetic antigens specific for out limitation, an OmpC protein, an OmpC polypeptide hav ing Substantially the same amino acid sequence as the OmpC ASCA are also suitable for use in determining the levels of protein, or a fragment thereof Such as an immunoreactive ASCA-IgA and/or ASCA-IgG in a sample. Examples of puri fragment thereof. As used herein, an OmpC polypeptide gen fied antigens include, without limitation, purified oligosac erally describes polypeptides having an amino acid sequence charide antigens such as oligomannosides. Examples of syn with greater than about 50% identity, preferably greater than thetic antigens include, without limitation, synthetic about 60% identity, more preferably greater than about 70% oligomannosides such as those described in U.S. Patent Pub identity, still more preferably greater than about 80%, 85%, lication No. 20030105060, e.g., D-Man B(1-2) D-Man B(1-2) 90%. 95%, 96%, 97%, 98%, or 99% amino acid sequence D-Man B(1-2) D-Man-OR, D-Man C.(1-2) D-Man O.(1-2) identity with an OmpC protein, with the amino acid identity D-Man O.(1-2) D-Man-OR, and D-Man C.(1-3) D-Man O.(1-2) determined using a sequence alignment program Such as D-Man C.(1-2) D-Man-OR, wherein R is a hydrogen atom, a CLUSTALW. Such antigens can be prepared, for example, by C to Co alkyl, or an optionally labeled connector group. purification from enteric bacteria Such as E. coli, by recom 0362 Preparations of yeast cell wall mannans, e.g., PPM, binant expression of a nucleic acid such as Genbank Acces can be used in determining the levels of ASCA-IgA and/or sion No. K00541, by synthetic means such as solution or solid ASCA-IgG in a sample. Such water-soluble Surface antigens phase peptide synthesis, or by using phage display. can be prepared by any appropriate extraction technique 0368. The determination of anti-I2 antibody levels in a known in the art, including, for example, by autoclaving, or sample is also useful in the present invention. As used herein, can be obtained commercially (see, e.g., Lindberg et al., Gut, the term “anti-I2 antibody' includes antibodies directed to a 33:909-913 (1992)). The acid-stable fraction of PPM is also microbial antigen sharing homology to bacterial transcrip useful in the statistical algorithms of the present invention tional regulators as described in, e.g., U.S. Pat. No. 6,309, (Sendid et al., Clin. Diag. Lab. Immunol., 3:219-226 (1996)). 643. The term “12 refers to a microbial antigen that is immu An exemplary PPM that is useful in determining ASCA levels noreactive with an anti-I2 antibody. The microbial I2 protein in a sample is derived from S. uvarum strain ATCC #38926. is a polypeptide of 100 amino acids sharing some similarity 0363 Purified oligosaccharide antigens such as oligom weak homology with the predicted protein 4 from C. pasteur annosides can also be useful in determining the levels of ianum, Rv3557c from Mycobacterium tuberculosis, and a ASCA-IgA and/or ASCA-IgG in a sample. The purified oli transcriptional regulator from Aquifex aeolicus. The nucleic gomannoside antigens are preferably converted into neogly acid and protein sequences for the I2 protein are described in, colipids as described in, for example, Faille et al., Eur: J. e.g., U.S. Pat. No. 6,309,643. Microbiol. Infect. Dis., 11:438-446 (1992). One skilled in the 0369. The level of anti-I2 antibody present in a sample art understands that the reactivity of such an oligomannoside from an individual can be determined using an I2 protein or a antigen with ASCA can be optimized by varying the manno fragment thereof Such as an immunoreactive fragment syl chain length (Frosh et al., Proc Natl. Acad. Sci. USA, thereof. Suitable I2 antigens useful in determining anti-I2 82:1194-1198 (1985)); the anomeric configuration (Fuka antibody levels in a sample include, without limitation, an I2 Zawa et al. In “Immunology of Fungal Disease. E. Kurstak protein, an I2 polypeptide having Substantially the same (ed.), Marcel Dekker Inc., New York, pp. 37-62 (1989); Nish amino acid sequence as the I2 protein, or a fragment thereof ikawa et al., Microbiol. Immunol., 34:825-840 (1990); Pou Such as an immunoreactive fragment thereof. Such I2 lainet al., Eur: J. Clin. Microbiol., 23:46-52 (1993); Shibataet polypeptides exhibit greater sequence similarity to the I2 al., Arch. Biochem. Biophy.s., 243:338-348 (1985); Trinel et protein than to the C. pasteurianum protein 4 and include al., Infect. Immun., 60:3845-3851 (1992)); or the position of isotype variants and homologs thereof. As used herein, an I2 the linkage (Kikuchi et al., Planta, 190:525-535 (1993)). polypeptide generally describes polypeptides having an 0364 Suitable oligomannosides for use in the methods of amino acid sequence with greater than about 50% identity, the present invention include, without limitation, an oligom preferably greater than about 60% identity, more preferably annoside having the mannotetraose Man(1-3) Man(1-2) Man greater than about 70% identity, still more preferably greater (1-2) Man. Such an oligomannoside can be purified from than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% PPM as described in, e.g., Faille et al., supra. An exemplary amino acid sequence identity with a naturally-occurring I2 neoglycolipid specific for ASCA can be constructed by protein, with the amino acid identity determined using a releasing the oligomannoside from its respective PPM and sequence alignment program such as CLUSTALW. Such I2 Subsequently coupling the released oligomannoside to antigens can be prepared, for example, by purification from 4-hexadecylaniline or the like. microbes, by recombinant expression of a nucleic acid encod US 2012/03 15630 A1 Dec. 13, 2012
ing an 12 antigen, by Synthetic means such as Solution or Solid and increase the perceived sensation of abdominal pain. As phase peptide synthesis, or by using phage display. Such, the presence of increased levels of mast cells or mast 0370. The determination of anti-flagellin antibody levels cell metabolites in the gastrointestinal tract may be diagnostic in a sample is also useful in the present invention. As used of IBS. Thus, in one embodiment, biomarkers associated with herein, the term “anti-flagellin antibody' includes antibodies mast cell degranulation, such as tryptase, prostaglandin E2. directed to a protein component of bacterial flagella as histamine, and histamine metabolites (including 4-imida described in, e.g., PCT Patent Publication No. WO03/053220 Zoleacetic acid, t-methylhistamine, and t-methyl-4-imidazole and U.S. Patent Publication No. 2004.0043931. The term acetic acid) are useful for the methods of the present inven “flagellin' refers to a bacterial flagellum protein that is immu tion. In other embodiments, biomarkers that are differentially noreactive with an anti-flagellin antibody. Microbial flagel released from mast cells, including serotonin, nerve growth lins are proteins found in bacterial flagellum that arrange factor, vascular endothelial cell growth factor, TNF-C. TNF themselves in a hollow cylinder to form the filament. Y, IL-6, and IL-8 are useful for the methods of the present 0371. The level of anti-flagellin antibody present in a invention. sample from an individual can be determined using a flagellin 0375. In certain instances, the presence or level of a mast protein or a fragment thereof Such as an immunoreactive cell marker is detected at the level of mRNA expression with fragment thereof. Suitable flagellin antigens useful in deter an assay Such as, e.g., a hybridization assay, an amplification mining anti-flagellin antibody levels in a sample include, based assay, e.g. qPCR assay, RT-PCR assay, or a mass spec without limitation, a flagellin protein such as Cbir-1 flagellin, trometry based assay. In certain other instances, the presence flagellin X, flagellin A, flagellin B, fragments thereof, and or level of a mast cell marker is detected at the level of protein combinations thereof, a flagellin polypeptide having Substan expression using, e.g., an immunoassay (e.g., ELISA), an tially the same amino acid sequence as the flagellin protein, or immunohistochemical assay, or a mass spectrometry based a fragment thereof Such as an immunoreactive fragment assay. thereof. As used herein, a flagellin polypeptide generally 0376. In certain embodiments, the mast cell marker may describes polypeptides having an amino acid sequence with be tryptase, histamine, prostaglandin E2 (PGE), or a combi greater than about 50% identity, preferably greater than about nation thereof. In yet other embodiments, the presence or 60% identity, more preferably greater than about 70% iden increased abundance of mast cells may be detected by using tity, still more preferably greater than about 80%, 85%, 90%, an anti-Ki antibody, such as a Ki-MC1 or Ki-M1P mono 95%, 96%, 97%, 98%, or 99% amino acid sequence identity clonal antibody (Hamann K. et al., Br. J Dermatol. (1995)), or with a naturally-occurring flagellin protein, with the amino with a fluorescent berberine binding assay (Berlin and Ener acid identity determined using a sequence alignment program back, Int Arch Allergy Appl Immunol. (1983)). such as CLUSTALW. Such flagellin antigens can be prepared, 0377. 23. Gastrointestinal Hormones e.g., by purification from bacterium Such as Helicobacter 0378. The determination of the presence or level of at least Bilis, Helicobacter mustelae, Helicobacter pylori, Butyriv one gastrointestinal hormone in a sample is also useful in the ibrio fibrisolvens, and bacterium found in the cecum, by present invention. As used herein, the term 'gastrointestinal recombinant expression of a nucleic acid encoding a flagellin hormone' includes a chemical or peptide present in the intes antigen, by synthetic means such as Solution or solid phase tinal tract that is released by a cell, which alters the metabo peptide synthesis, or by using phage display. lism of a second cell located in the organism. Generally, 0372 22. Mast Cell Markers hormones promote a response in a target cell after binding to 0373 The determination of the presence or level of mast a specific receptor located on the Surface of the cell. In ani cell markers in a sample is also useful in the present invention. mals, hormones are commonly transported throughout the As used herein, the term “mast cell marker' includes any body in the bloodstream, however, they may also be translo protein or metabolite that is indicative of the presence or cated by diffusion. In this fashion, endocrine hormone mol increased abundance of mast cells, or mastocytes. In certain ecules are secreted directly into the bloodstream, while exo embodiments, these mast cell markers may be present on the crine hormones are secreted directly into a duct, from which cell surface of a mast cell and/or be differentially expressed or they traverse into the bloodstream or alternatively diffuse present within a mast cell. Mast cells are generally found in throughout the body via paracrine signaling. Non-limiting connective tissue and harbor large granules containing high examples of gastrointestinal hormones suitable for use as IBS levels of histamine and heparin. These cells are believed to be markers include calcitonin gene-related peptide (CGRP), derived from CD34+ bone marrow precursor cells and func substance P nerve growth factor (NGF), neurokinin A, neu tion in several biological processes including wound healing, rokinin B. vasoactive intestinal peptide (VIP), glucagon-like defense against pathogens, allergy, and anaphylaxis. Non peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), motilin, limiting examples of mast cell markers include tryptase, his pituitary adenylate cyclase-activating peptide (PACAP), tamine, prostaglandin E2 (PGE), prostaglandin D2 (PGD). secretin, cholescystokinin, neuropeptide Y, pancreatic carboxypeptidase A, chymase, CD25, CD34, CD117 (c-Kit), polypeptide, peptide YY. enteroglucagon, and glucose-de LAMP-1 (CD107a), LAMP-2 (CD107b), and the like. pendent insulinotropic peptide. 0374. It has been postulated that inflammatory mast cells 0379. In certain instances, the presence or level of a gut proximal to intestinal innervation may contribute to abdomi peptide hormone marker is detected at the level of mRNA nal pain experienced in IBS (Rijnierse A. et al., Pharmacol expression with an assay Such as, e.g., a hybridization assay, Ther. (2007)). Consistent with this, increased numbers and an amplification-based assay, e.g. qPCR assay, RT-PCR activation of mast cells are found at intestinal sites in ISB assay, or a mass spectrometry based assay. In certain other patients. After being localized to the gastrointestinal tract, instances, the presence or level of a peptide hormone marker mast cells may be stimulated by allergens, microbial prod is detected at the level of protein expression using, e.g., an ucts, and/or neurotransmitters, to release a wide variety of immunoassay (e.g., ELISA), an immunohistochemical assay, bioactive mediators that stimulate the secreto-motor reflexes or a mass spectrometry based assay. US 2012/03 15630 A1 Dec. 13, 2012 39
0380 24. Calcitonin Gene-Related Peptide (CGRP) man Chemical Co. (Ann Arbor, Mich.), Bachem Holding 0381 CGRP is a member of the calcitonin family of pep AG/Peninsula Laboratories, LLC (San Carlos, Calif.), and tides, which in humans exists in two mature forms, C.-CGRP MD Biosciences Inc. (St. Paul, Minn.). and B-CGRP. C.-CGRP is a 37 amino acid peptide: ACD (0387 26. Nerve Growth Factor (NGF) TATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF (0388 NGF is a member of the NGF-beta family and is a NH. (SEQID NO: 1) formed from the alternative splicing of secreted protein which homodimerizes and is incorporated the calcitonin/CGRP gene. B-CGRP is a related 37 amino into a larger complex. NGF has nerve growth stimulating acid polypeptide ACNTATCVTHRLAGLLSRSGGMVK activity and the complex is involved in the regulation of SNFVPTNVGSKAF-NH. (SEQID NO:2) formed from the growth and the differentiation of sympathetic and certain calcitonin-related polypeptide, beta precursor gene. In certain sensory neurons. a small secreted protein which induces the instances, CGRP may consist of a calcitonin isoform CGRP differentiation and survival of particular target neurons. NGF preproprotein (NP 001029125), or variant thereof (such as binds to at least two receptors on the surface of cells which are NP 001029124 or NP 001732), CGRP propeptide capable of responding to this growth factor, TrkA and (CAA34070), or calcitonin-related polypeptide, beta precur LNGFR. NGF is a small polypeptide (residues 122-241 of sor (NP 000719). NP 002497) that is derived from a precursor polypeptide 0382 CGRP is one of the most abundant peptides pro (NP 002497) encoded by the NGF gene (NM 002506). In duced in both peripheral and central neurons (Rosenfeld MG certain embodiments, NGF, an NGF precursor polypeptide, et al., Nature (1983)), and is the most potent peptide vasodi or NGF propeptide (residues 19-121 of NP 002497) may be lator and can function in the transmission of pain (Brian SD useful as an IBS marker. et al., Nature (1985); McCullochet al., PNAS (1986)). CGRP 0389. In certain instances, the presence or level of NGF or is thought to play a role in cardiovascular homeostasis and a precursor thereof, is detected at the level of mRNA expres nociception by mediating its effects though a heteromeric sion with an assay Such as, e.g., a hybridization assay, an receptor composed of a G protein-coupled receptor called amplification-based assay, e.g. qPCR assay, RT-PCR assay, calcitonin receptor-like receptor (CLR) and a receptor activ ora mass spectrometry based assay. In certain other instances, ity-modifying protein (RAMP1) (Poyner D R et al., Pharma the presence or level of NGF, a precursor thereof, or a pro col Rev. (2002)). protein thereof, is detected at the level of protein expression 0383. In certain instances, the presence or level of a CGRP using, e.g., an immunoassay (e.g., ELISA), an immunohis or precursor thereof, is detected at the level of mRNA expres tochemical assay, or a mass spectrometry based assay. Suit Sion with an assay Such as, e.g., a hybridization assay, an able ELISA kits for determining the presence or level of NGF amplification-based assay, e.g. qPCR assay, RT-PCR assay, in a serum, plasma, saliva, or urine sample are available from, ora mass spectrometry based assay. In certain other instances, e.g., Millipore (Billerica, Mass.), CUSABIO Biotech Co., the presence or level of a peptide hormone marker is detected Ltd. (Newark, Del.), Emelca Biosciences (Belgium), and at the level of protein expression using, e.g., an immunoassay Signosis, Inc. (Sunnyvale, Calif.). (e.g., ELISA), an immunohistochemical assay, or a mass spectrometry based assay. Suitable ELISA kits for determin 0390 27. Neurokinin A ing the presence or level of a CGRP in a serum, plasma, Saliva, 0391 Neurokinin A is a peptide of 10 amino acids in or urine sample are available from, e.g., Cayman Chemical length (HKTDSFVGLM: SEQ ID NO:4) derived from a Co. (Ann Arbor, Mich.), Bachem Holding AG/Peninsula polypeptide precursor after differential splicing of the pre Laboratories, LLC (San Carlos, Calif.), and BioPorto Diag protachyknin A gene (TAC1; NM 003182). The peptide is a nostics (Denmark). member of the tachykininfamily of neuropeptide neurotrans 0384. 25. Substance P mitters. In certain embodiments, neurokinin A or a neuroki 0385 Substance P is a peptide of 11 amino acids in length nin A precursor protein may be useful as a marker of IBS, for (RPKPQQFFGLM-NH; SEQID NO:3) that is released by example a TAC1 polypeptide (NP 003173 and nerve endings in both the central and peripheral nervous NP 054703) or transcript thereof. systems. Among the numerous biological sites innervated by 0392. In certain instances, the presence or level of neuro Substance P-releasing neurons are the skin, intestines, stom kinin A or precursor thereof, is detected at the level of mRNA ach, bladder, and cardiovascular system. Substance P is expression with an assay Such as, e.g., a hybridization assay, derived from a polypeptide precursor after differential splic an amplification-based assay, e.g. qPCR assay, RT-PCR ing of the preprotachyknin A gene (TAC1; NM 003182). In assay, or a mass spectrometry based assay. In certain other certain embodiments, substance P or a substance P precursor instances, the presence or level of neurokinin A is detected at protein may be useful as a marker of IBS, for example a TAC1 the level of protein expression using, e.g., an immunoassay polypeptide (NP 003173; NP 054704; NP 054702; and (e.g., ELISA), an immunohistochemical assay, or a mass NP 054703) or transcript thereof. spectrometry based assay. Suitable ELISA kits for determin 0386. In certain instances, the presence or level of sub ing the presence or level of neurokinin A in a serum, plasma, stance Porprecursor thereof, is detected at the level of mRNA saliva, or urine sample are available from, e.g., Abcam (Cam expression with an assay Such as, e.g., a hybridization assay, bridge, Mass.), Fitzgerald Industries (North Acton, Mass.), an amplification-based assay, e.g. qPCR assay, RT-PCR and RayBiotech, Inc (Norcross, Ga.). assay, or a mass spectrometry based assay. In certain other 0393. 28. Neurokinin B instances, the presence or level of a peptide hormone marker 0394 Neurokinin B is a peptide of 10 amino acids in is detected at the level of protein expression using, e.g., an length (DMHDFFVGLM-NH.; SEQID NO:5) derived from immunoassay (e.g., ELISA), an immunohistochemical assay, the tachykinin 3 precursor (NP 037383), which is encoded or a mass spectrometry based assay. Suitable ELISA kits for by the tachykinin 3 gene (TAC3; NM 003182). The peptide determining the presence or level of Substance P in a serum, is a member of the tachykinin family of neuropeptide neu plasma, saliva, or urine sample are available from, e.g., Cay rotransmitters. In certain embodiments, neurokinin B or a US 2012/03 15630 A1 Dec. 13, 2012 40 neurokinin B precursor protein may be useful as a marker of immunoassay (e.g., ELISA), an immunohistochemical assay, IBS, for example a TAC3 polypeptide (NP 037383) or tran or a mass spectrometry based assay. Suitable ELISA kits for script thereof. determining the presence or level of GLP-2 in a serum, 0395. In certain instances, the presence or level of neuro plasma, Saliva, or urine sample are available from, e.g., Ray kinin B or precursor thereof, is detected at the level of mRNA Biotech, Inc (Norcross, Ga.), BioSupply UK (England), and expression with an assay Such as, e.g., a hybridization assay, BioCat GmbH (Germany). an amplification-based assay, e.g. qPCR assay, RT-PCR 0402. 31. Motilin assay, or a mass spectrometry based assay. In certain other 0403 Motilin is a 22 amino acid peptide hormone instances, the presence or level of neurokinin B is detected at (FVPIFTYGELQRMQEKERNKGQ; SEQID NO:8) that is the level of protein expression using, e.g., an immunoassay secreted from endocrine M cells located in the small intes (e.g., ELISA), an immunohistochemical assay, or a mass tines. Once released, motilin stimulates the production of spectrometry based assay. Suitable ELISA kits for determin pepsin and regulates migrating motor complexes, which are ing the presence or level of neurokinin B in a serum, plasma, responsible for the transportation of indigestible substances saliva, or urine sample are available from, e.g., Abcam (Cam through the digestive tract. Motilin is derived from promoti bridge, Mass.), Bachem Holding AG/Peninsula Laboratories, lin, a precursor polypeptide encoded by the motilin gene LLC (San Carlos, Calif.), and RayBiotech, Inc (Norcross, (MLN; Entrez GeneID: 4295). Transcription of the MLN Ga.). gene undergoes differential splicing into one of two isoforms 0396. 29. Vasoactive Intestinal Peptide (VIP) (NM 002418 and NM 001040109), which result in the 0397) VIP is a 28 amino acid peptide hormone (HS translation of two different precursor polypeptides DAVFTDNYTRLRKQMAVKKYLNSILN; SEQID NO:6) (NP 002409 and NP 001035198). In certain embodiments, found in the gut, pancreas, and hypothalamus of humans. This motilin, a motilin precursor polypeptide, a motilin propeptide peptide induces Smooth muscle relaxation, causes inhibition (such as motilin-associated peptide), or a transcript encoding of gastric acids secretion and absorption from the intestinal motilin may be useful as a marker of IBS. lumen, and stimulates the secretion of water into pancreatic 04.04. In certain instances, the presence or level of motilin juice and bile. VIP is derived from a polypeptide precursor is detected at the level of mRNA expression with an assay after differential splicing of the vasoactive intestinal peptide Such as, e.g., a hybridization assay, an amplification-based gene (Entrez, GeneID: 7432). In certain embodiments, VIP, a assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom VIP precursor protein (such as NP919416 or NP 003372), a etry based assay. In certain other instances, the presence or VIP propeptide, or a transcript encoding a VIP peptide (such level of motilin, a precursor thereof, or a propeptide thereof is as NM 003381 or NM 194435) may be useful as a marker detected at the level of protein expression using, e.g., an of IBS. immunoassay (e.g., ELISA), an immunohistochemical assay, 0398. In certain instances, the presence or level of VIP is or a mass spectrometry based assay. Suitable ELISA kits for detected at the level of mRNA expression with an assay such determining the presence or level of motilin in a serum, as, e.g., a hybridization assay, an amplification-based assay, plasma, saliva, or urine sample are available from, e.g., e.g. qPCR assay, RT-PCR assay, or a mass spectrometry Abcam (Cambridge, Mass.), CUSABIO Biotech Co., Ltd. based assay. In certain other instances, the presence or level of (Newark, Del.), and Phoenix Biotech (China). VIP, a precursor thereof, or a propeptide thereof is detected at 04.05 32. Pituitary Adenylate Cyclase-Activating Peptide the level of protein expression using, e.g., an immunoassay (PACAP) (e.g., ELISA), an immunohistochemical assay, or a mass (0406 PACAP is a vasoactive neuropeptide (VN) found spectrometry based assay. Suitable ELISA kits for determin throughout the nervous system, gastrointestinal tract, and ing the presence or level of VIP in a serum, plasma, Saliva, or lymphatic system. VNs, such as PACAP and VIP function as urine sample are available from, e.g., Phoenix Pharmaceuti neurotransmitters, neuroregulators, neurotropic stimulators, cals, Inc. (Burlingame, Calif.), BioPorto Diagnostics (Den hormonal regulators, vasodilators, and immune and nocicep mark), and RayBiotech, Inc (Norcross, Ga.). tion modulators. PACAP is a precursor protein (NP 001108) 0399 30. Glucagon-Like Peptide 2 (GLP-2) encoded by the Adenylate Cyclase Activating Polypeptide 1 04.00 GLP-2 is a 33 amino acid peptide hormone (pituitary) gene (ADCYAP1; Entrez GeneID: 1271617). (HADGSFSDEMNTILDNLAARDFINWLIQTKITD, SEQ Transcription of the ADCYAP1 gene undergoes differential ID NO:7) that is expressed in the intestines, pancreas, and splicing into one of two isoforms (NM 001099733 and brain. GLP-2 is known to be co-secreted with GLP-1 into the NM 001117), which result in the translation of two different intestines, where it functions to regulate gastric motility, precursor polypeptides (NP 001093203 and NP 001108). secretion, Sugar transport, and cellular differentiation and The expressed PACAP polypeptide is further processed into proliferation. GLP-2 is derived from proglucagon, a polypep various smaller peptides, including two propeptides, PACAP tide precursor encoded by the glucagon gene (CGC: related peptide (DVAHGILNEAYRKVLDQLSAGKHLQS NM 002054). In certain embodiments, GLP-2, a GLP-2 pre LVARGVGGSLGGGAGDDAEPLS: SEQ ID NO:9), pitu cursor protein (such as NP 002045), a GLP-2 propeptide, or itary adenylate cyclase-activating polypeptide 38 a transcript encoding a GLP-2 peptide (such as NM 002054) (HSDGIFTDSYSRYRKQMAVKKYLAAV may be useful as a marker of IBS. LGKRYKQRVKNK: SEQID NO:10), and pituitary adeny 04.01. In certain instances, the presence or level of GLP-2 late cyclase-activating polypeptide 27 (HSDGIFTDSYS is detected at the level of mRNA expression with an assay RYRKQMAVKKYLAAVL: SEQ ID NO:11). In certain Such as, e.g., a hybridization assay, an amplification-based embodiments, PACAP, a PACAP precursor polypeptide, a assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom PACAP propeptide (such as PACAP-related peptide, pituitary etry based assay. In certain other instances, the presence or adenylate cyclase-activating polypeptide 38, and pituitary level of GLP-2, a precursor thereof, or a propeptide thereof is adenylate cyclase-activating polypeptide 27), or a transcript detected at the level of protein expression using, e.g., an encoding PACAP may be useful as a marker of IBS. US 2012/03 15630 A1 Dec. 13, 2012
0407. In certain instances, the presence or level of PACAP 0413. In certain instances, the presence or level of an is detected at the level of mRNA expression with an assay enteric nervous system serum metabolite is detected with a Such as, e.g., a hybridization assay, an amplification-based mass spectrometry based assay, a proton magnetic resonance assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom spectroscopy based assay, a chromatographic assay (e.g., liq etry based assay. In certain other instances, the presence or uid chromatographic assay or HPLC), an immunoassay, (e.g., level of PACAP, a precursor thereof, or a propeptide thereof is ELISA), and the like. detected at the level of protein expression using, e.g., an 0414 I. Serotonin Pathway Markers immunoassay (e.g., ELISA), an immunohistochemical assay, 0415. The determination of the presence or level of at least or a mass spectrometry based assay. Suitable ELISA kits for one serotonin pathway marker in a sample is also useful in the determining the presence or level of PACAP in a serum, present invention. As used herein, the term "serotonin path plasma, Saliva, or urine sample are available from, e.g., Santa way marker” includes genes, proteins, and enzymes involved Cruz, Biotechnology, Inc. (Santa Cruz, Calif.), US Biological in the biosynthesis or metabolism of serotonin, or the regu (Swampscott, Mass.), and Novus Biologicals (Littleton, lation of serotonin levels in the gastrointestinal tract. Non Colo.). limiting examples of serotonin pathway markers suitable for 0408. 33. Serotonin Metabolites use as IBS markers include UDP-glucuronosyltransferase 1-6 04.09. The determination of the presence or level of at least (uGT1A6), serotonin reuptake transporter (SERT), tryp one serotonin (5-hydroxytryptamine; 5-HT) metabolite in a tophan hydroxylase 1 (TPH1), monoamine oxidase A (MAO sample is also useful in the present invention. As used herein, A), monoamine oxidase B (MAO-B), and hydroxytryptamine the term "serotonin metabolite' includes serotonin, serotonin receptor 3A (5-HT3A, 5-HT3R). biosynthesis intermediates, and serotonin metabolites. Sero 0416) 1. UDP-Glucuronosyltransferase 1-6 (UGT1A6) tonin is primarily found in the gastrointestinal tract, where it 0417 UGT1A6 is a member of UDP-glucuronosyl/UDP functions to regulate intestinal movements, and to a lesser glucosyltransferase Superfamily of membrane-bound extent in the central nervous systems, where it participates in enzymes of the ER, which catalyze the glucuronidation of the regulation of mood, appetite, sleep, muscle contraction, various substrates. The UDP-glucuronosyl/UDP-glucosyl and various cognitive functions. Serotonin levels are upregu transferase Superfamily of enzymes catalyzes the addition of lated in the gastrointestinal tracts of IBS patients. Further, the glycosyl group from a UTP-Sugar to a small hydrophobic serotonin concentrations are higher in IBS(D) than IBS(C). molecule. Mammalian UDP-glucuronosyltransferases are However, fluctuations in serotonin levels are not easily mea described in, e.g., Burchellet al., DNA Cell Biol., 10(7):487 sured in plasma due to the tight regulation of re-uptake into 94 (1991). These enzymes share a conserved domain of about blood platelets by the serotonin transporter (SERT). As such, 50 amino acid residues in their C-terminal region and play a in one embodiment provided herein, serotonin metabolites major role in the detoxification and Subsequent elimination of may be detected in biological samples, such as blood and Xenobiotics such as drugs (e.g., topoisomerase I inhibitors) urine, in order to reflect the concentration of serotonin in the and carcinogens. It was previously discovered that serotonin gut, for the diagnosis of IBS. Non-limiting examples of sero is a specific endogenous substrate of UGT1A6 (King C. et al., tonin metabolites suitable for use as IBS markers include Arch Biochem Biophys (1999)). UGT1A6, an E.C.2.4.1.17 tryptophan, 5-HT-O-sulfate, serotonin O-sulfate (5-Hydrox enzyme, is encoded by the UDPglucuronosyltransferase 1 ytryptamine O-sulfate; 5-HT-O-sulfate), 5-Hydroxyin family, polypeptide A6 gene (UGT1A6: Entrez, GeneID: doleacetic acid (5-HIAA), 5-HT glucuronide (5-HT-GA), 54578) and matures from one of two precursor polypeptides and 5-hydroxytrytophol (5-HTOL). (NP 001063 and NP995584) after alternative splicing of the 0410. In certain instances, the presence or level of a sero transcript (NM 001072 and NM 205862). Several alleles tonin metabolite is detected with a mass spectrometry based exist for the UGT1A6 gene, including UGT1A6*1, assay, a proton magnetic resonance spectroscopy based assay, UGT1A6*2, UGT1A6*3, and UGT1A6*4 (Nagar S. et al., a chromatographic assay (e.g., liquid chromatographic assay Pharmacogenetics (2004)). These haplotypes contain various or HPLC), an immunoassay, (e.g., ELISA), and the like. combinations of known UGT1A6 SNPs, which include 0411 34. Enteric Nervous System Serum Metabolites rs675.9892 (158 Ts-G), rs1042708 (348 CA), rs2070959 0412. The determination of the presence or level of at least (680 A>G), rs1105879 (691 A>G), and rs1042709 (1667 one enteric nervous system serum metabolite in a sample is G>C). In certain embodiments, UGT1A6, a precursor protein also useful in the present invention. As used herein, the term thereof, a UGT1A6 mRNA, or a UGT1A6 SNP, haplotype, or “enteric nervous system serum metabolite' includes dopam genotype may be useful as a marker of IBS. ine, norepinephrine, epinephrine, tyrosine, phenylalanine, 0418. In certain instances, the presence or level of dopamine, norepinephrine, epinephrine biosynthesis inter UGT1A6 is detected at the level of mRNA expression with an mediates, and dopamine, norepinephrine, epinephrine assay Such as, e.g., a hybridization assay, an amplification metabolites. In one embodiment provided herein, enteric ner based assay, e.g. qPCR assay, RT-PCR assay, or a mass spec Vous system serum metabolites may be detected in biological trometry based assay. In certain other instances, the presence samples, such as blood and urine, in order to diagnose, Sub or level of UGT1A6 or a precursor thereof is detected at the type, or provide a prognosis for IBS. In certain embodiments, level of protein expression using, e.g., an immunoassay (e.g., an elevated level of tyrosine is associated with or indicative of ELISA), an immunohistochemical assay, or a mass spectrom IBS or associated with an increased likelihood of IBS. In etry based assay. Suitable ELISA kits for determining the other embodiments, an elevated level of tyrosine is associated presence or level of UGT1A6 in a serum, plasma, saliva, or with or indicative of IBS-D or IBS-M. In another embodi urine sample are available from, e.g., Abnova (Walnut Calif.) ment, an elevated level of phenylalanine is associated with or and Santa Cruz, Biotechnology, Inc. (Santa Cruz, Calif.). In indicative of IBS or associated with an increased likelihood of yet other instances, a SNP or particular haplotype or genotype IBS. In other embodiments, an elevated level of phenylala of the UGT1A6 gene is detected with an assay Such as, e.g., a nine is associated with or indicative of IBS-D or IBS-M. hybridization assay, an amplification-based assay, e.g. qPCR US 2012/03 15630 A1 Dec. 13, 2012 42 assay, RT-PCR assay, a single nucleotide sequencing assay, or (117+1840 C-T), rs503964 (723 C>T), rs490895 (789 C>T), a mass spectrometry based assay. rs41274350 (820 C>A), rs41274348 (929 C>T), and 0419 2. Serotonin-Selective Reuptake Transporter rs56151798 (1095 TDC). Several TPH1 SNPs are associated (SERT) with psychiatric disorders, including impulsive-aggression 0420 SERT is a member of the monoamine transporter measures (New A S et al., Am J Med. Genet. (1998)) and family of membrane-bound receptors that facilitate the trans schizophrenia (Allen N C et al., Nat. Genet. (2008)). In cer port of monoamine neurotransmitters into and out of cells. tain embodiments TPH1, a TPH1 mRNA, or a TPH1 SNP, The serotonin receptor (SERT) mediates the uptake of sero haplotype, polymorphic variant, or genotype may be useful as tonin into neurons and platelets, thereby regulating serotonin a marker of IBS. levels in vivo. Polymorphisms in the SERT gene have previ 0424. In certain instances, the presence or level of TPH1 is ously been associated with various diseases, including Sud detected at the level of mRNA expression with an assay such den infant death syndrome (SIDS), Alzheimer's disease, post as, e.g., a hybridization assay, an amplification-based assay, traumatic stress disorder, depression, pulmonary e.g. qPCR assay, RT-PCR assay, or a mass spectrometry hypertension, myocardial infarction, and IBS (for review, see, based assay. In certain other instances, the presence or level of Murphy D L et al., Mol. Interv. (2004)). SERT is a 70 kDa TPH1 is detected at the level of protein expression using, e.g., transmembrane receptor (NP 001036) encoded by the solute an immunoassay (e.g., ELISA), an immunohistochemical carrier family 6 (neurotransmitter transporter, serotonin), assay, or a mass spectrometry based assay. Suitable ELISA member 4 gene (SLC6A4: Entrez, GeneID: 6532: kits for determining the presence or level of TPH1 in a serum, NM 001045). Various SNPs have been identified in the plasma, saliva, or urine sample are available from, e.g., Novus SLC6A4 gene, including rs6355 (472 G>C), rs2228673 (908 Biologicals (Littleton, Colo.) and Abcam (Cambridge, G>C), rs28914832 (1578 A>C), rs28914833 (1698 TDC), Mass.). In yet other instances, a SNP polymorphism, or par rs28914834 (1953 C>G), rs6352 (2120 A>C), and 1425V. ticular haplotype or genotype of the TPH1 gene is detected Furthermore, polymorphisms in the promoter region of the with an assay Such as, e.g., a hybridization assay, an amplifi SLC6A4 gene, including an A-G polymorphism (rs25531) cation-based assay, e.g. qPCR assay, RT-PCR assay, a single and a deletion of 44 base pair polymorphism, which results in nucleotide sequencing assay, or a mass spectrometry based the short variant (rs4795541; Heils A et al., J Neurochem. assay. (1996)), have been described. In certain embodiments SERT, 0425 4. Monoamine oxidase A (MAO-A) an SLC6A4 mRNA, or an SLC6A4 SNP, haplotype, poly 0426 MAO-A, an E.C. 1.4.3.4 enzyme, is a member of the morphic variant, or genotype may be useful as a marker of enzyme family of flavin-containing amine oxidoreductases. IBS. This enzyme, which is localized to the outer mitochondrial 0421. In certain instances, the presence or level of SERT is membrane, catalyzes the deamination of neuroactive and detected at the level of mRNA expression with an assay such vasoactive amines, including serotonin, norepinephrine, and as, e.g., a hybridization assay, an amplification-based assay, epinephrine, with the aid of a FAD cofactor. MAO-A is 59.7 e.g. qPCR assay, RT-PCR assay, or a mass spectrometry kDa protein (NP 000231) encoded by the monoamine oxi based assay. In certain other instances, the presence or level of dase A gene (MAOA: Entrez GeneID: 4128; NM 000240). SERT is detected at the level of protein expression using, e.g., Several SNPs have been identified in the MAOA gene, includ an immunoassay (e.g., ELISA), an immunohistochemical ing rs1800464 (566 A>C), rs58524323 (696 G>A), assay, or a mass spectrometry based assay. Suitable ELISA rs 1137068 (818 G>T), rs72554.632 (1067 C>T), rs6323 kits for determining the presence or level of SERT in a serum, (1072 G>T), rs61730725 (1074 Ts-C), rs1799835 (1121 plasma, saliva, or urine sample are available from, e.g., T>G), rs1800465 (1207 A>T), rs1803987 (1324 G>T), Abgent, Inc. (San Diego, Calif.), OriCiene Technologies rs7065428 (1342 A>G), rs1803986 (1516 GDT), rs1137070 (Rockville, Md.), and Abcam (Cambridge, Mass.). In yet (1591 TYC), rs1800466 (1740 ADG), rs3788862 (NT other instances, a SNP polymorphism, or particular haplo 079573.3, position 6369131 A/C), and rs1465108 (NT type or genotype of the SLC6A4 gene is detected with an 079573.3, position 6389976 A/G). Several MAOA SNPs assay Such as, e.g., a hybridization assay, an amplification have been associated with disorders, including autism spec based assay, e.g. qPCR assay, RT-PCR assay, a single nucle trum disorder (Yoo H J et al., Neurosci Res. (2009)), bipolar otide sequencing assay, or a mass spectrometry based assay. affective disorder (Lin et al., Behav Brain Funct. (2008)), and 0422. 3. Tryptophan Hydroxylase 1 (TPH1) Attention-deficit/hyperactivity disorder (Rommelse N N et 0423 TPH1 is an E.C.1.14.16.4 enzyme that catalyzes the al., Am J Med Genet B Neuropsychiatr Genet. (2008)). In hydroxylation of L-tryptophanto form 5-hydroxytryptophan, certain embodiments MAO-A, an MAOA mRNA, or an an intermediate of serotonin biosynthesis. Two isoforms of MAOA SNP, haplotype, polymorphic variant, or genotype tryptophan hydroxylase (TPH1 and TPH2) are found in the may be useful as a marker of IBS. gut and central nervous systems of mammals. TPH1 is the 0427. In certain instances, the presence or level of MAOA predominant isoform found in the gut and TPH2 is the pre is detected at the level of mRNA expression with an assay dominant isoform expressed in the central nervous system. Such as, e.g., a hybridization assay, an amplification-based Specifically, TPHs are expressed in several cell lineages, assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom including raphe neurons, pinealocytes, mast cells, mono etry based assay. In certain other instances, the presence or nuclear leukocytes, beta-cells of the islets of Langerhans, and level of MAO-A is detected at the level of protein expression intestinal and pancreatic enterochromaffin cells. TPH1 is a 50 using, e.g., an immunoassay (e.g., ELISA), an immunohis kDa protein (NP 004.170) encoded by the tryptophan tochemical assay, or a mass spectrometry based assay. Suit hydroxylase 1 gene (TPH1; Entrez GeneID: 7166: able ELISA kits for determining the presence or level of NM 004179). Several SNPs have been identified in the MAO-A in a serum, plasma, saliva, or urine sample are avail TPH1 gene, including rs172423 (301+1281 G>A), rs211102 able from, e.g., Novus Biologicals (Littleton, Colo.), Abcam (930+629 A>G), rs10488683 (402+1276 A>G), rs684302 (Cambridge, Mass.), and Abnova (Walnut, Calif.). In yet US 2012/03 15630 A1 Dec. 13, 2012 other instances, a SNP polymorphism, or particular haplo precursor polypeptide (NP 000860, NP 001155244, and type or genotype of the MAOA gene is detected with an assay NP 998786) after alternative splicing of the transcript Such as, e.g., a hybridization assay, an amplification-based (NM 000869, NM 001161772, and NM 213621). Sev assay, e.g. qPCR assay, RT-PCR assay, a single nucleotide eral SNPs have been identified in the HTR3A gene, including sequencing assay, or a mass spectrometry based assay. rs33940208 (168C>T), rs72466464 (170 C>T), rs72466465 0428 5. Monoamine Oxidase B (MAO-B) (205 GDA), rs72466467 (705 GDT), rs34327364 (714 GDA), 0429 MAO-B, an E.C. 1.4.3.4 enzyme, is a member of the rs4938063 (896 GDA), rs56232120 (969 G>A), rs35815285 enzyme family of flavin-containing amine oxidoreductases. (1169 G>A), rs12285005 (1198 C>T), rs35592083 (1320 MAO-B shares about 70% sequence identity with MAO-A, C>T), rs3411 1946 (1471 GDA), rs3594.4954 (1479 C>T), and like MAO-A is localized to the outer mitochondrial mem rs 1176713 (1515 A>G), rs1150220 (NT 033899.7, position brane and catalyzes the deamination of neuroactive and vaso 17420302C/T), and rs1062613 (97 C>T). Several HTR3A active amines. MAO-B preferentially deaminates benzy SNPs have been associated with disorders, including treat lamine, phenylethylamine, and dopamine. Due to the fact that ment-resistant schizophrenia (Ji X et al., Neurosci Lett. MAO-A deaminates serotonin and epinephrine with higher (2008)) and autism (Anderson B M et al., Neurogenetics affinity than MAO-B, MAO-B inhibitors are preferred over (2009)). In certain embodiments 5-HT3R, an HTR3A MAO-A inhibitors for first-line treatment of depression. mRNA, an HTR3A microRNA, or an HTR3A SNP haplo MAO-B is 58.8 kDa protein (NP 000889) encoded by the type, polymorphic variant, or genotype may be useful as a monoamine oxidase B gene (MAOB: Entrez, GeneID: 4129: marker of IBS. NM 000898). Several SNPs have been identified in the 0433. In certain instances, the presence or level of HTR3A MAO-B gene, including rs12010260 (260 G>T), rs17856663 is detected at the level of mRNA expression with an assay (289 GDA), rs17852046 (565 C>A), rs12845783 (767 A>C), Such as, e.g., a hybridization assay, an amplification-based rs 12850496 (775 CA), rs12845773 (777 Al-C), rs7879356 assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom (782 A>G), rs55815323 (1087 C>T), rs3.952696 (1229A>G), etry based assay. In certain other instances, the presence or rs6324 (1637 C>T), rs1040399 (NT 079573.3, position level of 5-HT3R is detected at the level of protein expression 6477166 C/T), and rs6651806 (NT 079573.3, position using, e.g., an immunoassay (e.g., ELISA), an immunohis 6540731 A/C). Several MAOB SNPs have been associated tochemical assay, or a mass spectrometry based assay. Suit with disorders, including attention deficit hyperactivity dis able ELISA kits for determining the presence or level of order (Li J et al., Am J Med Genet B Neuropsychiatr Genet. 5-HT3R in a serum, plasma, saliva, or urine sample are avail (2008) and vulnerability of major depression (Dlugos A Met able from, e.g., RayBiotech, Inc (Norcross, Ga.) and Santa al., J Neural Transm. (2009)). In certain embodiments MAO Cruz, Biotechnology, Inc. (Santa Cruz, Calif.). In yet other B, an MAOB mRNA, oran MAOBSNP, haplotype, polymor instances, a SNP polymorphism, or particular haplotype or phic variant, or genotype may be useful as a marker of IBS. genotype of the HTR3A gene is detected with an assay such 0430. In certain instances, the presence or level of MAOB as, e.g., a hybridization assay, an amplification-based assay, is detected at the level of mRNA expression with an assay e.g. qPCR assay, RT-PCR assay, a single nucleotide sequenc Such as, e.g., a hybridization assay, an amplification-based ing assay, or a mass spectrometry based assay. assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom 0434. J. Stress Markers etry based assay. In certain other instances, the presence or 0435 The determination of the presence or level of at least level of MAO-B is detected at the level of protein expression one stress marker in a sample is also useful in the present using, e.g., an immunoassay (e.g., ELISA), an immunohis invention. As used herein, the term “stress marker” includes tochemical assay, or a mass spectrometry based assay. Suit any peptide, hormone, metabolite, protein or gene that func able ELISA kits for determining the presence or level of tions to mediate the central nervous system's response to MAO-B in a serum, plasma, saliva, or urine sample are avail stress. These markers include hormones released in response able from, e.g., Novus Biologicals (Littleton, Colo.), Abcam to stress, metabolites that are differentially produced in (Cambridge, Mass.), and Santa Cruz, Biotechnology, Inc. response to stress, genes and proteins that are differentially (Santa Cruz, Calif.). In yet other instances, a SNP polymor expressed in response to stress, and cellular receptors that phism, or particular haplotype or genotype of the MAOB bind stress-dependent hormones and metabolites. Non-limit gene is detected with an assay Such as, e.g., a hybridization ing examples of stress markers suitable for use as IBS mark assay, an amplification-based assay, e.g. qPCR assay, RT ers include corticotrophin-releasing hormone (CRH; CRF), PCR assay, a single nucleotide sequencing assay, or a mass Urocortin (Ucn), Corticotropin-releasing hormone-binding spectrometry based assay. protein (CRFBP), and Adrenocorticotropic hormone (ACTH, 0431 6. Hydroxytryptamine Receptor 3A (5-HT3R) corticotrophin). 0432 5-HT3R is a member of the ligand-gated ion chan 0436 1. Corticotrophin-Releasing Hormone (CRH; CRF) nel receptor superfamily. The 5-HT3R polypeptide is a sub 0437. CRH is a neuroendocrine hormone involved in the unit of the pentameric transmembrane 5-HT receptors, stress response of the central nervous system. This hormone which may consist of homopentameric 5-HT3R or heteropen mediates many of the stress-related peripheral effects and tameric structures containing at least one 5-HT3R subunit. behavioral responses. CRH has been shown to be involved in The 5-HT receptor is an ion conducting pore that when stress-induced motility in both the upper and lower gas bound by serotonin elicits a conformational change resulting trointestinal tract. CRH is a 41-amino acid peptide hormone in an excitatory response in neurons. It has been recently (SEEPPISLDLTFHLLREVLEMA shown that microRNAs contribute to the regulation of HT3R RAEQLAQQAHSNRKLMEII: SEQ ID NO:12) that is receptors (Kapeller J et al., Hum Mol. Genet. (2008)). The derived from a precursor polypeptide (corticotrophin releas 5-HT3R protein is encoded by the 5-hydroxytryptamine (se ing hormone precursor: NP 000747) encoded by the corti rotonin) receptor 3A gene (HTR3A: Entrez GeneID: 3359) cotropin releasing hormone gene (CRH: Entrez, GeneID: and is processed by removal of a signal peptide from the 1392: NM 000756). Several SNPs have been identified in US 2012/03 15630 A1 Dec. 13, 2012 44 the CRH gene, including rs12721511 (189 C>G), qPCR assay, RT-PCR assay, a single nucleotide sequencing rs72556398 (236 G->C), rs3832590 (330-CCG>CGCCCG), assay, or a mass spectrometry based assay. rs6159 (473 A>C), rs283.64016 (692 GDC), rs283.64017 0442. 3. Corticotropin-Releasing Hormone-Binding Pro (NT 008183.18, position 18943997 A/G), and rs283.64014 tein (CRHBP) (NT 008183.18, position 18943683 C/G). In certain 0443 CRHBP binds with high affinity to both CRH and embodiments CRH, a CRH precursor polypeptide, a CRH Ucn, preventing CRF receptor activation and thus modulating propeptide, a CRH mRNA, or a CRH SNP, haplotype, poly the bioactivity and bioavailability of these peptide hormones. morphic variant, or genotype may be useful as a marker of Strikingly, these interactions are mediated by distinct binding IBS. surfaces on CRHBP (Huising et al., J Biol. Chem. (2008)). 0438. In certain instances, the presence or level of CRH is CRHBP is a secreted glycoprotein derived from a precursor detected at the level of mRNA expression with an assay such polypeptide (NP 001873) encoded by the corticotropin as, e.g., a hybridization assay, an amplification-based assay, releasing hormone binding protein gene (CRHBP: Entrez e.g. qPCR assay, RT-PCR assay, or a mass spectrometry GeneID: 1393; NM 001882). Several SNPs have been iden based assay. In certain other instances, the presence or level of tified in the CRHBP gene, including rs1133082 (372 C>A), CRH is detected at the level of protein expression using, e.g., rs 1053967 (529 C>T), rs34347676 (544 G>A), rs26844303 an immunoassay (e.g., ELISA), an immunohistochemical (547 C>G), rs3792738 (NT 006713.14, position 26842142 assay, or a mass spectrometry based assay. Suitable ELISA A/C), rs41272246 (NT 006713.14, position 26843420 kits for determining the presence or level of CRH in a serum, A/C), and rs2135078 (NT 006713.14, position 26859826 plasma, saliva, or urine sample are available from, e.g., IBL C/T). Several CRHBP SNPs have been associated with dis America (Minneapolis, Minn.) and Abbiotec (San Diego, orders, including anxiety and alcohol abuse disorders (Enoch Calif.). In yet other instances, a SNP polymorphism, or par ticular haplotype or genotype of the CRH gene is detected M A et al., PLoS One (2008)) and spontaneous preterm birth with an assay Such as, e.g., a hybridization assay, an amplifi (Velez D Ret al., PLoS One (2008)). In certain embodiments cation-based assay, e.g. qPCR assay, RT-PCR assay, a single CRHBP, a CRHBP mRNA, or a CRHBP SNP, haplotype, nucleotide sequencing assay, or a mass spectrometry based polymorphic variant, or genotype may be useful as a marker assay. of IBS. 0439 2. Urocortin 1 (Ucn) 0444. In certain instances, the presence or level of CRHBP 0440 Ucn is a peptide hormone and member of the sau is detected at the level of mRNA expression with an assay vagine?corticotropin-releasing factor/urotensin I family. Uro Such as, e.g., a hybridization assay, an amplification-based cortin has high affinity for both the CRF-1 and CRF-2 recep assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom tors and is a more potent anorectic than CRH. Unlike CRH, etry based assay. In certain other instances, the presence or which is predominantly produced in the hypothalamus, UCN level of CRHBP is detected at the level of protein expression is produced in the gastrointestinal tract and as such, is more using, e.g., an immunoassay (e.g., ELISA), an immunohis easily detected in plasma than is CRH. UCN is a 40-amino tochemical assay, or a mass spectrometry based assay. Suit acid peptide hormone (DNPSLSIDLTFHLLRTLLE able ELISA kits for determining the presence or level of LARTQSQRERAEQNRIIFDSV: SEQ ID NO:13) that is CRHBP in a serum, plasma, saliva, or urine sample are avail derived from a precursor polypeptide (urocortin prepropro able from, e.g., Abgent, Inc. (San Diego, Calif.), Abbiotec tein: NP 003344) encoded by urocortin gene (UCN; Entrez (San Diego, Calif.), and Alpco (Salem, N.H.). In yet other GeneID:7349; NM 003353). Several SNPs have been iden instances, a SNP polymorphism, or particular haplotype or tified in the CRH gene, including rs11557508 (NT 022184. genotype of the CRHBP gene is detected with an assay such 14; position 6347221 CIT), rs283.64046 (NT 022184.14; as, e.g., a hybridization assay, an amplification-based assay, position 6346948 A/G), rs35403114 (NT 022184.14; posi e.g. qPCR assay, RT-PCR assay, a single nucleotide sequenc tion 6346902 C/T), rs6709781 (NT 022184.14; position ing assay, or a mass spectrometry based assay. 6346798 C/G), rs28364045 (525 C-T), and rs11557507 (NT 022184.14; position 6346118 G/T). In certain embodi 0445. 4. Cortisol ments UCN, a UCN precursor polypeptide, a UCN propep 0446 Cortisol is a corticosteroid hormone produced in the tide, a UCN mRNA, or a UCNSNP, haplotype, polymorphic adrenal gland in response to stress and anxiety. Production of variant, or genotype may be useful as a marker of IBS. the glucocorticoid is regulated by CRH and the adrenocorti 0441. In certain instances, the presence or level of UCN is cotropic hormone (ACTH). After being released in response detected at the level of mRNA expression with an assay such to stress or anxiety, cortisol functions to help restore homeo as, e.g., a hybridization assay, an amplification-based assay, stasis, and affects the levels of multiple metabolites in the e.g. qPCR assay, RT-PCR assay, or a mass spectrometry body, including insulin, collagen free amino acids in serum, based assay. In certain other instances, the presence or level of gastric acid secretion, Sodium, potassium, water, and copper. UCN is detected at the level of protein expression using, e.g., In certain instances, the presence or level of cortisol is an immunoassay (e.g., ELISA), an immunohistochemical detected with a mass spectrometry based assay, a proton assay, or a mass spectrometry based assay. Suitable ELISA magnetic resonance spectroscopy based assay, a chromato kits for determining the presence or level of UCN in a serum, graphic assay (e.g., liquid chromatographic assay or HPLC), plasma, saliva, or urine sample are available from, e.g., Novus an immunoassay, (e.g., ELISA), a fluorescence resonance Biologicals (Littleton, Colo.), Abcam (Cambridge, Mass.), energy transfer (FRET) based assay (Yu et al., Assay Drug and US Biological (Swampscott, Mass.). In yet other Dev Technol. (2007)), an enzyme fragment complementation instances, a SNP polymorphism, or particular haplotype or based assay (Kumar A et al., Biosens Bioelectron. (2007)), a genotype of the UCN gene is detected with an assay Such as, fluorimetric assay (Appel D et al., Anal Bioanal Chem. e.g., a hybridization assay, an amplification-based assay, e.g. (2005)), and the like. US 2012/03 15630 A1 Dec. 13, 2012
0447 5. Adrenocorticotropic Hormone (ACTH, Corti lactoferrin is detected at the level of protein expression using, cotrophin) for example, an immunoassay (e.g., ELISA) or an immuno 0448 ACTH is a 39 amino acid tropic hormone (SYSME histochemical assay. A lactoferrin ELISA kit available from HFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF; Calbiochem (San Diego, Calif.) can be used to detect human SEQID NO:14) produced and secreted by the anterior pitu lactoferrin in a plasma, urine, bronchoalveolar lavage, or itary, which functions to stimulate production and release of cerebrospinal fluid sample. Similarly, an ELISA kit available thyroid hormone by the thyroid gland. ACTH is commonly from U.S. Biological (Swampscott, Mass.) can be used to produced in response to biological stress and affects the determine the level of lactoferrin in a plasma sample. U.S. increased production of androgens and cortisol from the adre Patent Publication No. 2004.0137536 describes an ELISA nal cortex. ACTH is derived from the mature form of the assay for determining the presence of elevated lactoferrin pro-opiomelanocortin preprotein (NP 000930; levels in a stool sample. Likewise, U.S. Patent Publication NP 001030333) which is translated after alternative splicing No. 20040033537 describes an ELISA assay for determining of the proopiomelanocortin gene (POMC: Entrez, GeneID: the concentration of endogenous lactoferrin in a stool, mucus, 5443; NM 000939; NM 001035256). Pro-opiomelanocor or bile sample. In some embodiments, then presence or level tin preprotein is heavily processed into 11 potential polypep of anti-lactoferrin antibodies can be detected in a sample tides, including NPP (residues 27-102), Melanotropin using, e.g., lactoferrin protein or a fragment thereof. gamma (residues 77-87), a potential peptide (residues (105 0452. The determination of the presence or level of carbo 134), corticotrophin (residues 138-176), melanotropin alpha hydrate deficient transferrin (CDT) in a sample is also useful (residues 138-150), corticotropin-like intermediary peptide in the present invention. CDT is a diagnostic test currently (residues 156-176), lipotropin beta (residues 179-267), lipo used to detect the presence of heavy alcohol consumption. tropin gamma (residues 179-234), melanotropin beta (resi The test measures the level of carbohydrate deficient trans dues 217-234), beta-endorphin (residues 237-267), and Met ferrin proteins, i.e. transferrin polypeptides modified by Zero, enkephalin (residues 237-241). Several SNPs have been one, or two sialic acid chains, generally in a blood, serum, identified in the POMC gene, including rs8192605 (281 hair, or urine sample. As such, CDT tests are used to identify C>T), rs28932470 (421 A>G), rs28932471 (448 C-T), individuals with alcohol abuse problems or alcoholism. In rs28930368 (545 C>T), rs35254395 (552-insAG certain embodiments CDT tests or CDT levels may be useful CAGCGGC), rS10654394 (560insGCCGCTGCT), as a marker of IBS. In certain other instances, the presence or rs34650613 (609 C>T), rs3192606 (657 CDG), rs45463492 level of CDT is detected by an electrophoretic assay, as sold (697 G>T), rs2071345 (848 C>T), rs28932472 (969 C>G), by Consumer Genetics, Inc. (Sunnyvale, Calif.) and Sebia rs3754860 (NT 022184.14, position 4209.187 C/T), electrophoresis (Norcross, Ga.). In certain other instances, rs1009388 (NT 022184.14, position 4207034 C/G), and the presence or level of CDT is detected using, e.g., an immu rs 1042571 (NT 022184.14, position 4199820 C/T). Several noassay (e.g., ELISA), an isoelectric focusing assay, an POMC SNPs have been associated with disorders, including immunohistochemical assay, or a mass spectrometry based Parkinson's disease (Shadrina M et al., Neurosci Lett. assay. (2006)), obesity (Ahituv Net al., Am J Hum Genet. (2007), 0453 Immunoassays such as ELISA are also particularly and substance dependence (Zhang H et al., Biol Psychiatry useful for determining the presence or level of C-reactive (2009)). In certain embodiments ACTH, a POMC mRNA, or protein (CRP) in a sample. For example, a sandwich colori a POMC SNP, haplotype, polymorphic variant, or genotype metric ELISA assay available from Alpco Diagnostics (Sa may be useful as a marker of IBS. lem, N.H.) can be used to determine the level of CRP in a 0449 In certain instances, the presence or level of POMC serum, plasma, urine, or stool sample. Similarly, an ELISA is detected at the level of mRNA expression with an assay kit available from Biomeda Corporation (Foster City, Calif.) Such as, e.g., a hybridization assay, an amplification-based can be used to detect CRP levels in a sample. Other methods assay, e.g. qPCR assay, RT-PCR assay, or a mass spectrom for determining CRP levels in a sample are described in, e.g., etry based assay. In certain other instances, the presence or U.S. Pat. Nos. 6,838,250 and 6,406,862; and U.S. Patent level of ACTH is detected at the level of protein expression Publication NOS. 2006.0024682 and 200600 19410. using, e.g., an immunoassay (e.g., ELISA), an immunohis 0454. In addition, hemoccult, fecal occult blood, is often tochemical assay, or a mass spectrometry based assay. Suit indicative of gastrointestinal illness and various kits have able ELISA kits for determining the presence or level of been developed to monitor gastrointestinal bleeding. For ACTH in a serum, plasma, Saliva, or urine sample are avail example, Hemoccult SENSA, a Beckman Coulter product, is able from, Calbiotech (Spring Valley, Calif.), GenWay Bio a diagnostic aid for gastrointestinal bleeding, iron deficiency, tech, Inc. (San Diego, Calif.), and MD Bioproducts (St. Paul, peptic ulcers, ulcerative colitis, and, in some instances, in Minn.). In yet other instances, a SNP polymorphism, or par screening for colorectal cancer. This particular assay is based ticular haplotype or genotype of the POMC gene is detected on the oxidation of guaiac by hydrogen peroxide to produce a with an assay Such as, e.g., a hybridization assay, an amplifi blue color. A similar colorimetric assay is commercially cation-based assay, e.g. qPCR assay, RT-PCR assay, a single available from Helena Laboratories (Beaumont, Tex.) for the nucleotide sequencing assay, or a mass spectrometry based detection of blood in stool samples. Other methods for detect assay. ing occult blood in a stool sample by determining the pres 0450 K. Other Diagnostic Markers ence or level of hemoglobin or heme activity are described in, 0451. The determination of the presence or level of lacto e.g., U.S. Pat. Nos. 4.277,250, 4,920,045, 5,081,040, and ferrin in a sample is also useful in the present invention. In 5,310,684. certain instances, the presence or level of lactoferrin is 0455 The determination of the presence or level of detected at the level of mRNA expression with an assay such fibrinogen or a proteolytic product thereof such as a fibrin as, for example, a hybridization assay or an amplification opeptide in a sample is also useful in the present invention. based assay. In certain other instances, the presence or level of Fibrinogen is a plasma glycoprotein synthesized in the liver US 2012/03 15630 A1 Dec. 13, 2012 46 composed of 3 structurally different subunits: alpha (FGA); present invention to aid in classifying IBS (see, e.g., Gershon, beta (FGB); and gamma (FGG). Thrombin causes a limited J. Clin. Gastroenterol., 39(5 Suppl.):5184-193 (2005)). proteolysis of the fibrinogen molecule, during which fibrin 0460. In certain aspects, the level of tryptophan hydroxy opeptides A and B are released from the N-terminal regions of lase-1 mRNA is a diagnostic marker. For example, tryp the alpha and beta chains, respectively. Fibrinopeptides A and tophan hydroxylase-1 mRNA has been shown to be signifi B, which have been sequenced in many species, may have a cantly reduced in IBS (see, e.g., Coats, Gastroenterology, physiological role as vasoconstrictors and may aid in local 126:1897-1899 (2004)). In certain other aspects, a lactulose hemostasis during blood clotting. In one embodiment, human breath test to measure methane, which is indicative of bacte fibrinopeptide A comprises the sequence: Ala-Asp-Ser-Gly rial overgrowth, can be used as a diagnostic marker for IBS. Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg 0461 Additional diagnostic markers include, but are not limited to, L-selectin/CD62L, anti-U1-70 kDa autoantibod (SEQID NO:15). In another embodiment, human fibrinopep ies, Zona occludens 1 (ZO-1), vasoactive intestinal peptide tide B comprises the sequence: Glp-Gly-Val-Asn-Asp-ASn (VIP), serum amyloid A, gastrin, NB3 gene polymorphisms, Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg (SEQ ID NO:16). An NCI1 gene polymorphisms, fecal leukocytes, C2A and C2C ELISA kit available from American Diagnostica Inc. (Stam adrenoreceptor gene polymorphisms, IL-10 gene polymor ford, Conn.) can be used to detect the presence or level of phisms, TNF-C. gene polymorphisms, TGF-B1 gene poly human fibrinopeptide A in plasma or other biological fluids. morphisms, C.-adrenergic receptors, G-proteins, 5-HT2 gene 0456. In certain embodiments, the determination of the polymorphisms, 5-HTTLPR gene polymorphisms, 5-HT presence or level of calcitonin gene-related peptide (CGRP) receptor gene polymorphisms, Zonulin, and the 33-mer pep in a sample is useful in the present invention. Calcitonin is a tide (Shanet al., Science, 297:2275-2279 (2002); PCT Patent 32-amino acid peptide hormone synthesized by the parafol Publication No. WO 03/068170). licular cells of the thyroid. It causes reduction in serum cal 0462. L. Classification Markers cium, an effect opposite to that of parathyroid hormone. 0463) A variety of classification markers are suitable for CGRP is derived, with calcitonin, from the CT/CGRP gene use in the methods, systems, and code of the present invention located on chromosome 11. CGRP is a 37-amino acid peptide for classifying IBS into a category, form, or clinical Subtype and is a potent endogenous vasodilator. CGRP is primarily such as, for example, IBS-constipation (IBS-C), IBS-diar produced in nervous tissue; however, its receptors are rhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating (IBS-A), expressed throughout the body. An ELISA kit available from or post-infectious IBS (IBS-PI). Examples of classification Cayman Chemical Co. (Ann Arbor, Mich.) can be used to markers include, without limitation, any of the diagnostic detect the presence or level of human CGRP in a variety of markers described above (e.g., leptin, serotonin reuptake samples including plasma, serum, nervous tissue, CSF, and transporter (SERT), tryptophan hydroxylase-1,5-hydrox culture media. ytryptamine (5-HT), tryptase, PGE, histamine, and the like), 0457. In other embodiments, the determination of the as well as antrum mucosal protein 8, keratin-8, claudin-8, presence or level of an anti-tissue transglutaminase (tTG) Zonulin, corticotropin-releasing hormone receptor-1 antibody in a sample is useful in the present invention. As (CRHR1), corticotropin-releasing hormone receptor-2 used herein, the term “anti-tTG antibody' includes any anti (CRHR2), and the like. body that recognizes tissue transglutaminase (tTG) or a frag 0464 For instance. Examples 1 and 2 below illustrate that ment thereof. Transglutaminases area diverse family of Ca"- measuring B-tryptase levels is particularly useful for distin dependent enzymes that are ubiquitous and highly conserved guishing IBS-C patient samples from IBS-A and IBS-D across species. Of all the transglutaminases, tTG is the most patient samples. Similarly, Example 1 from US Patent Pub widely distributed. In certain instances, the anti-tTG antibody lication No. 2008/0085524, filed Aug. 14, 2007, which is is an anti-tTG IgA antibody, anti-tTG IgG antibody, or mix herein incorporated by reference in its entirety for all pur tures thereof. An ELISA kit available from ScheBo Biotech poses, illustrates that measuring leptin levels is particularly USA Inc. (Marietta, Ga.) can be used to detect the presence or useful for distinguishing IBS-C patient samples from IBS-A level of human anti-tTG IgA antibodies in a blood sample. and IBS-D patient samples. In addition, mucosal SERT and 0458. The determination of the presence of polymor tryptophan hydroxylase-1 expression have been shown to be phisms in the NOD2/CARD15 gene in a sample is also useful decreased in IBS-C and IBS-D (see, e.g., Gershon, J. Clin. in the present invention. For example, polymorphisms in the Gastroenterol., 39(5 Suppl):5184-193 (2005)). Furthermore, NOD2 gene such as a C2 107T nucleotide variant that results IBS-C patients show impaired postprandial 5-HT release, in a R703W protein variant can be identified in a sample from whereas IBS-PI patients have higher peak levels of 5-HT (see, an individual (see, e.g., U.S. Patent Publication No. e.g., Dunlop, Clin Gastroenterol Hepatol., 3:349-357 20030190639). In an alternative embodiment, NOD2 mRNA (2005)). Furthermore, as can be seen in FIG. 11, serum levels levels can be used as a diagnostic marker of the present of histamine and PGE is also particularly useful for distin invention to aid in classifying IBS. guishing IBS-D patient samples from IBS-A, IBS-C, and 0459. The determination of the presence of polymor healthy control patient samples. phisms in the serotonin reuptake transporter (SERT) gene in 0465 M. Statistical Algorithms a sample is also useful in the present invention. For example, 0466. In some aspects, the present invention provides polymorphisms in the promoter region of the SERT gene have methods, assays, systems, and code for classifying whether a effects on transcriptional activity, resulting in altered 5-HT sample is associated with IBS using a statistical algorithm or reuptake efficiency. It has been shown that a strong genotypic process to classify the sample as an IBS sample or non-IBS association was observed between the SERT-P deletion/dele sample. In other aspects, the present invention provides meth tion genotype and the IBS phenotype (see, e.g., Yeo Gut, ods, systems, and code for classifying whether a sample is 53:1396-1399 (2004)). In an alternative embodiment, SERT associated with IBS using a first statistical algorithm or pro mRNA levels can be used as a diagnostic marker of the cess to classify the sample as a non-IBD sample or IBD US 2012/03 15630 A1 Dec. 13, 2012 47 sample (i.e., IBD rule-out step), followed by a second statis cessed using a processing algorithm. Such a processing algo tical algorithm or process to classify the non-IBD Sample as rithm can be selected, for example, from the group consisting an IBS sample or non-IBS sample (i.e., IBS rule-in step). of a multilayer perceptron, backpropagation network, and Preferably, the statistical algorithms or processes indepen Levenberg-Marquardt algorithm. In other instances, a com dently comprise one or more learning statistical classifier bination of such processing algorithms can be used, such as in systems. As described herein, a combination of learning sta a parallel or serial fashion. tistical classifier systems advantageously provides improved 0470 The term “statistical algorithm' or “statistical pro sensitivity, specificity, negative predictive value, positive pre cess' includes any of a variety of statistical analyses used to dictive value, and/or overall accuracy for classifying whether determine relationships between variables. In the present a sample is associated with IBS. In a preferred embodiment, invention, the variables are the presence or level of at least one the methods, assays, systems, and code provided herein use a marker of interest and/or the presence or severity of at least combination of at least two statistical algorithms. one IBS-related symptom. Any number of markers and/or 0467. In some embodiments, the first statistical algorithm symptoms can be analyzed using a statistical algorithm is a learning statistical classifier system selected from the described herein. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, group consisting of a random forest (RF), classification and 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 60, regression tree (C&RT), boosted tree, neural network (NN), or more biomarkers and/or symptoms can be included in a Support vector machine (SVM), general chi-squared auto statistical algorithm. In one embodiment, logistic regression matic interaction detector model, interactive tree, multiadap is used. In another embodiment, linear regression is used. In tive regression spline, machine learning classifier, and com certain instances, the statistical algorithms of the present binations thereof. In certain instances, the first statistical invention can use a quantile measurement of a particular algorithm is a single learning statistical classifier system. marker within a given population as a variable. Quantiles are Preferably, the single learning statistical classifier system a set of "cut points' that divide a sample of data into groups comprises a tree-based statistical algorithm such as a RF or containing (as far as possible) equal numbers of observations. C&RT. In certain other instances, the first statistical algorithm For example, quartiles are values that divide a sample of data is a combination of at least two learning statistical classifier into four groups containing (as far as possible) equal numbers systems, e.g., used in tandem or parallel. As a non-limiting of observations. The lower quartile is the data value a quarter example, a RF can first be used to generate a prediction or way up through the ordered data set; the upper quartile is the probability value based upon the diagnostic marker profile, data value a quarter way down through the ordered data set. alone or in combination with a symptom profile, and a NN Quintiles are values that divide a sample of data into five (e.g., artificial NN) can then be used to classify the sample as groups containing (as far as possible) equal numbers of obser a non-IBD sample or IBD sample based upon the prediction Vations. The present invention can also include the use of or probability value and the same or different diagnostic percentile ranges of marker levels (e.g., tertiles, quartile, marker profile or combination of profiles. The hybrid RF/NN quintiles, etc.), or their cumulative indices (e.g., quartile sums learning statistical classifier system of the present invention of marker levels, etc.) as variables in the algorithms (just as typically classifies the sample as a non-IBD Sample with a with continuous variables). sensitivity, specificity, positive predictive value, negative pre 0471 Preferably, the statistical algorithms of the present dictive value, and/or overall accuracy of at least about 75%, invention comprise one or more learning statistical classifier 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, systems. As used herein, the term “learning statistical classi 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, fier system” includes a machine learning algorithmic tech 96%, 97%, 98%, or 99%. nique capable of adapting to complex data sets (e.g., panel of 0468. In certain embodiments, the second statistical algo markers of interest and/or list of IBS-related symptoms) and rithm comprises any of the learning statistical classifier sys making decisions based upon Such data sets. In some embodi tems described above. In certain instances, the second statis ments, a single learning statistical classifier system such as a tical algorithm is a single learning statistical classifier system classification tree (e.g., random forest) is used. In other Such as, for example, a tree-based statistical algorithm (e.g., embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or RF or C&RT). In certain other instances, the second statistical more learning statistical classifier systems are used, prefer algorithm is a combination of at least two learning statistical ably in tandem. Examples of learning statistical classifier classifier systems, e.g., used in tandem or parallel. As a non systems include, but are not limited to, those using inductive limiting example, a RF can first be used to generate a predic learning (e.g., decision/classification trees such as random tion or probability value based upon the diagnostic marker forests, classification and regression trees (C&RT), boosted profile, alone or in combination with a symptom profile, and trees, etc.), Probably Approximately Correct (PAC) learning, aNN (e.g., artificial NN) or SVM can then be used to classify connectionist learning (e.g., neural networks (NN), artificial the non-IBD sample as a non-IBS sample or IBS sample neural networks (ANN), neuro fuzzy networks (NFN), net based upon the prediction or probability value and the same or work structures, perceptrons such as multi-layer perceptrons, different diagnostic marker profile or combination of profiles. multi-layer feed-forward networks, applications of neural The hybrid RF/NN or RF/SVM learning statistical classifier networks, Bayesian learning in belief networks, etc.), rein system described herein typically classifies the sample as an forcement learning (e.g., passive learning in a known envi IBS sample with a sensitivity, specificity, positive predictive ronment such as naive learning, adaptive dynamic learning, value, negative predictive value, and/or overall accuracy of at and temporal difference learning, passive learning in an least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, unknown environment, active learning in an unknown envi 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, ronment, learning action-value functions, applications of 93%, 94%, 95%, 96%, 97%, 98%, or 99%. reinforcement learning, etc.), and genetic algorithms and evo 0469. In some instances, the data obtained from using the lutionary programming. Other learning statistical classifier learning statistical classifier system or systems can be pro systems include Support vector machines (e.g., Kernel meth US 2012/03 15630 A1 Dec. 13, 2012 48 ods), multivariate adaptive regression splines (MARS), Lev using the SVM'software developed by Thorsten Joachims enberg-Marquardt algorithms, Gauss-Newton algorithms, (Cornell University) or using the LIBSVM software devel mixtures of Gaussians, gradient descent algorithms, and oped by Chih-Chung Chang and Chih-Jen Lin (National Tai learning vector quantization (LVO). wan University). 0472 Random forests are learning statistical classifier 0476. The learning statistical classifier systems described systems that are constructed using an algorithm developed by herein can be trained and tested using a cohort of samples Leo Breiman and Adele Cutler. Random forests use a large (e.g., serological samples) from healthy individuals, IBS number of individual decision trees and decide the class by patients, IBD patients, and/or Celiac disease patients. For choosing the mode (i.e., most frequently occurring) of the example, samples from patients diagnosed by a physician, classes as determined by the individual trees. Random forest and preferably by a gastroenterologist as having IBD using a analysis can be performed, e.g., using the RandomForests biopsy, colonoscopy, oran immunoassay as described in, e.g., software available from Salford Systems (San Diego, Calif.). U.S. Pat. No. 6,218,129, are suitable for use in training and See, e.g., Breiman, Machine Learning, 45:5-32 (2001); and testing the learning statistical classifier systems of the present http://stat-www.berkeley.edu/users/breiman/RandomFor invention. Samples from patients diagnosed with IBD can ests/cc home.htm, for a description of random forests. also be stratified into Crohn's disease or ulcerative colitis 0473 Classification and regression trees represent a com using an immunoassay as described in, e.g., U.S. Pat. Nos. puter intensive alternative to fitting classical regression mod 5,750,355 and 5,830,675. Samples from patients diagnosed els and are typically used to determine the best possible model with IBS using a published criteria such as the Manning, for a categorical or continuous response of interest based Rome I, Rome II, or Rome III diagnostic criteria are suitable upon one or more predictors. Classification and regression for use in training and testing the learning statistical classifier tree analysis can be performed, e.g., using the CART Software systems of the present invention. Samples from healthy indi available from Salford Systems or the Statistica data analysis viduals can include those that were not identified as IBD software available from StatSoft, Inc. (Tulsa, Okla.). A and/or IBS samples. One skilled in the art will know of description of classification and regression trees is found, additional techniques and diagnostic criteria for obtaining a e.g., in Breiman et al. “Classification and Regression Trees.” cohort of patient samples that can be used in training and Chapman and Hall, New York (1984); and Steinberg et al., testing the learning statistical classifier systems of the present “CART: Tree-Structured Non-Parametric Data Analysis.” invention. Salford Systems, San Diego, (1995). 0477. As used herein, the term “sensitivity” refers to the 0474 Neural networks are interconnected groups of arti probability that a diagnostic method, system, or code of the ficial neurons that use a mathematical or computational present invention gives a positive result when the sample is model for information processing based on a connectionist positive, e.g., having IBS. Sensitivity is calculated as the approach to computation. Typically, neural networks are number of true positive results divided by the sum of the true adaptive systems that change their structure based on external positives and false negatives. Sensitivity essentially is a mea or internal information that flows through the network. Spe sure of how well a method, system, or code of the present cific examples of neural networks include feed-forward neu invention correctly identifies those with IBS from those with ral networks such as perceptrons, single-layer perceptrons, out the disease. The statistical algorithms can be selected Such multi-layer perceptrons, backpropagation networks, ADA that the sensitivity of classifying IBS is at least about 60%, LINE networks, MADALINE networks, Learnmatrix net and can be, for example, at least about 65%, 70%, 75%, 76%, works, radial basis function (RBF) networks, and self-orga 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, nizing maps or Kohonen self-organizing networks; recurrent 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, neural networks such as simple recurrent networks and 97%, 98%, or 99%. In preferred embodiments, the sensitivity Hopfield networks; stochastic neural networks such as Bolt of classifying IBS is at least about 90% when a combination Zmann machines; modular neural networks such as commit of learning statistical classifier systems is used (see, Example tee of machines and associative neural networks; and other 10 from US Patent Publication No. 2008/0085524, which is types of networks Such as instantaneously trained neural net incorporated herein by reference in its entirety for all pur works, spiking neural networks, dynamic neural networks, poses) or at least about 85% when a single learning statistical and cascading neural networks. Neural network analysis can classifier system is used (see, Example 11 from US Patent be performed, e.g., using the Statistica data analysis Software Publication No. 2008/0085524, which is incorporated herein available from StatSoft, Inc. See, e.g., Freeman et al. In by reference in its entirety for all purposes). “Neural Networks: Algorithms, Applications and Program 0478. The term “specificity” refers to the probability that a ming Techniques. Addison-Wesley Publishing Company diagnostic method, system, or code of the present invention (1991); Zadeh, Information and Control, 8:338-353 (1965); gives a negative result when the sample is not positive, e.g., Zadeh, “IEEE Trans. on Systems, Man and Cybernetics.” not having IBS. Specificity is calculated as the number of true 3:28-44 (1973); Gersho et al. In “Vector Quantization and negative results divided by the sum of the true negatives and Signal Compression.” Kluywer Academic Publishers, Bos false positives. Specificity essentially is a measure of how ton, Dordrecht, London (1992); and Hassoun, “Fundamentals well a method, system, or code of the present invention of Artificial Neural Networks.” MIT Press, Cambridge, excludes those who do not have IBS from those who have the Mass., London (1995), for a description of neural networks. disease. The statistical algorithms can be selected Such that 0475 Support vector machines are a set of related super the specificity of classifying IBS is at least about 70%, for vised learning techniques used for classification and regres example, at least about 75%, 80%, 85%, 86%, 87%, 88%, sion and are described, e.g., in Cristianini et al., “An Intro 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or duction to Support Vector Machines and Other Kernel-Based 99%. In preferred embodiments, the specificity of classifying Learning Methods. Cambridge University Press (2000). IBS is at least about 86% when a combination of learning Support vector machine analysis can be performed, e.g., statistical classifier systems is used (see, Example 10 from US US 2012/03 15630 A1 Dec. 13, 2012 49
Patent Publication No. 2008/0085524, which is incorporated in a patient population having a disease prevalence is at least herein by reference in its entirety for all purposes) or at least about 60%, and can be, for example, at least about 65%, 70%, about 84% when a single learning statistical classifier system 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, is used (see, Example 11 from US Patent Publication No. 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 2008/0085524, which is incorporated herein by reference in 95%,96%.97%.98%, or 99%. In preferred embodiments, the its entirety for all purposes). overall accuracy of classifying IBS is at least about 80% when 0479. As used herein, the term “negative predictive value' a combination of learning statistical classifier systems is used or “NPV refers to the probability that an individual identified (see, Example 10 from US Patent Publication No. 2008/ as not having IBS actually does not have the disease. Negative 0085524, which is incorporated herein by reference in its predictive value can be calculated as the number of true entirety for all purposes). negatives divided by the sum of the true negatives and false 0483 N. Disease Classification System negatives. Negative predictive value is determined by the 0484 FIG. 13 illustrates a disease classification system characteristics of the diagnostic method, system, or code as (DCS) (200) according to one embodiment of the present well as the prevalence of the disease in the population ana invention. As shown therein, a DCS includes a DCS intelli lyzed. The statistical algorithms can be selected such that the gence module (205). Such as a computer, having a processor negative predictive value in a population having a disease (215) and memory module (210). The intelligence module prevalence is in the range of about 70% to about 99% and can also includes communication modules (not shown) for trans be, for example, at least about 70%, 75%, 76%, 77%, 78%, mitting and receiving information over one or more direct 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, connections (e.g., USB, Firewire, or other interface) and one 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or or more network connections (e.g., including a modem or 99%. In preferred embodiments, the negative predictive value other network interface device). The memory module may of classifying IBS is at least about 87% when a combination include internal memory devices and one or more external of learning statistical classifier systems is used (see, Example memory devices. The intelligence module also includes a 10 from US Patent Publication No. 2008/0085524, which is display module (225). Such as a monitor or printer. In one incorporated herein by reference in its entirety for all pur aspect, the intelligence module receives data Such as patient poses). test results from a data acquisition module such as a test 0480. The term “positive predictive value” or “PPV system (250), either through a direct connection or over a refers to the probability that an individual identified as having network (240). For example, the test system may be config IBS actually has the disease. Positive predictive value can be ured to run multianalyte tests on one or more patient samples calculated as the number of true positives divided by the sum (255) and automatically provide the test results to the intelli of the true positives and false positives. Positive predictive gence module. The data may also be provided to the intelli value is determined by the characteristics of the diagnostic gence module via direct input by a user or it may be down method, system, or code as well as the prevalence of the loaded from a portable medium Such as a compact disk (CD) disease in the population analyzed. The statistical algorithms or a digital versatile disk (DVD). The test system may be can be selected such that the positive predictive value in a integrated with the intelligence module, directly coupled to population having a disease prevalence is in the range of the intelligence module, or it may be remotely coupled with about 80% to about 99% and can be, for example, at least the intelligence module over the network. The intelligence about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, module may also communicate data to and from one or more 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In preferred client systems (230) over the network as is well known. For embodiments, the positive predictive value of classifying IBS example, a requesting physician or healthcare provider may is at least about 90% when a combination of learning statis obtain and view a report from the intelligence module, which tical classifier systems is used (see, Example 10 from US may be resident in a laboratory or hospital, using a client Patent Publication No. 2008/0085524, which is incorporated system (230). herein by reference in its entirety for all purposes). 0485 The network can be a LAN (local area network), 0481 Predictive values, including negative and positive WAN (wide area network), wireless network, point-to-point predictive values, are influenced by the prevalence of the network, star network, token ring network, hub network, or disease in the population analyzed. In the methods, systems, other configuration. As the most common type of network in and code of the present invention, the statistical algorithms current use is a TCP/IP (Transfer Control Protocol and Inter can be selected to produce a desired clinical parameter for a net Protocol) network such as the global internetwork of clinical population with a particular IBS prevalence. For networks often referred to as the “Internet with a capital “I. example, learning statistical classifier systems can be selected that will be used in many of the examples herein, but it should for an IBS prevalence of up to about 1%, 2%. 3%, 4%, 5%, be understood that the networks that the present invention 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, might use are not so limited, although TCP/IP is the currently 45%, 50%, 55%, 60%. 65%, or 70%, which can be seen, e.g., preferred protocol. in a clinician's office Such as a gastroenterologists office or a 0486 Several elements in the system shown in FIG.2 from general practitioner's office. US Patent Publication No. 2008/0085524 may include con 0482. As used herein, the term “overall agreement’ or ventional, well-known elements that need not be explained in “overall accuracy” refers to the accuracy with which a detail here. For example, the intelligence module could be method, system, or code of the present invention classifies a implemented as a desktop personal computer, workstation, disease state. Overall accuracy is calculated as the Sum of the mainframe, laptop, etc. Each client system could include a true positives and true negatives divided by the total number desktop personal computer, workstation, laptop, PDA, cell of sample results and is affected by the prevalence of the phone, or any WAP-enabled device or any other computing disease in the population analyzed. For example, the statisti device capable of interfacing directly or indirectly to the cal algorithms can be selected Such that the overall accuracy Internet or other network connection. A client system typi US 2012/03 15630 A1 Dec. 13, 2012 50 cally runs an HTTP client, e.g., a browsing program, Such as tomassociated with IBS that the patient is experiencing or has Microsoft's Internet ExplorerTM browser, Netscape's Naviga recently experienced. In one aspect, a statistical process pro torTM browser, Opera's browser, or a WAP-enabled browser in duces a statistically derived decision classifying the patient the case of a cell phone, PDA or other wireless device, or the sample as an IBS sample or non-IBS sample based upon the like, allowing a user of the client system to access, process, diagnostic marker profile and/or symptom profile. In another and view information and pages available to it from the intel aspect, a first statistical process produces a first statistically ligence module over the network. Each client system also derived decision classifying the patient sample as an IBD typically includes one or more user interface devices, such as sample or non-IBD sample based upon the diagnostic marker a keyboard, a mouse, touch screen, pen or the like, for inter profile and/or symptom profile. If the patient sample is clas acting with a graphical user interface (GUI) provided by the browser on a display (e.g., monitor screen, LCD display, etc.) sified as a non-IBD sample, a second statistical process is (235) in conjunction with pages, forms, and other information applied to the same or a different data set to produce a second provided by the intelligence module. As discussed above, the statistically derived decision classifying the non-IBD sample present invention is suitable for use with the Internet, which as an IBS sample or non-IBS sample. The first and/or the refers to a specific global internetwork of networks. However, second statistically derived decision may be displayed on a it should be understood that other networks can be used display device associated with or coupled to the intelligence module, or the decision(s) may be provided to and displayed instead of the Internet, Such as an intranet, an extranet, a at a separate system, e.g., a client system (230). The displayed virtual private network (VPN), a non-TCP/IP based network, results allow a physician to make a reasoned diagnosis or any LAN or WAN, or the like. prognosis. 0487. According to one embodiment, each client system and all of its components are operator configurable using 0490 O. Therapy and Therapeutic Monitoring applications, such as a browser, including computer code run 0491. Once a sample from an individual has been classi using a central processing unit Such as an Intel(R) Pentium R fied as an IBS sample, the methods, systems, and code of the processor or the like. Similarly, the intelligence module and present invention can further comprise administering to the all of its components might be operator configurable using individual atherapeutically effective amount of a drug useful application(s) including computer code run using a central for treating one or more symptoms associated with IBS (i.e., processing unit (215) such as an Intel Pentium processor or an IBS drug). For therapeutic applications, the IBS drug can the like, or multiple processor units. Computer code for oper be administered alone or co-administered in combination ating and configuring the intelligence module to process data with one or more additional IBS drugs and/or one or more and test results as described herein is preferably downloaded drugs that reduce the side-effects associated with the IBS and stored on a hard disk, but the entire program code, or drug. portions thereof, may also be stored in any other volatile or 0492 IBS drugs can be administered with a suitable phar non-volatile memory medium or device as is well known, maceutical excipient as necessary and can be carried out via such as a ROM or RAM, or provided on any other computer any of the accepted modes of administration. Thus, adminis readable medium (260) capable of storing program code, tration can be, for example, intravenous, topical. Subcutane Such as a compact disk (CD) medium, digital versatile disk ous, transcutaneous, transdermal, intramuscular, oral, buccal, (DVD) medium, a floppy disk, ROM, RAM, and the like. Sublingual, gingival, palatal, intra-joint, parenteral, intra-ar 0488. The computer code for implementing various teriole, intradermal, intraventricular, intracranial, intraperito aspects and embodiments of the present invention can be neal, intralesional, intranasal, rectal, vaginal, or by inhala implemented in any programming language that can be tion. By “co-administer it is meant that an IBS drug is executed on a computer system Such as, for example, in C, administered at the same time, just prior to, or just after the C++, C#, HTML, Java, JavaScript, or any other scripting administration of a second drug (e.g., another IBS drug, a language. Such as VBScript. Additionally, the entire program drug useful for reducing the side-effects of the IBS drug, etc.). code, orportions thereof, may be embodied as a carrier signal, 0493 A therapeutically effective amount of an IBS drug which may be transmitted and downloaded from a software may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, Source (e.g., server) over the Internet, or over any other con or more times, or the dose may be administered by continuous ventional network connection as is well known (e.g., extranet, infusion. The dose may take the form of solid, semi-solid, VPN, LAN, etc.) using any communication medium and pro lyophilized powder, or liquid dosage forms, such as, for tocols (e.g., TCP/IP, HTTP, HTTPS, Ethernet, etc.) as are well example, tablets, pills, pellets, capsules, powders, Solutions, known. Suspensions, emulsions, Suppositories, retention enemas, 0489. According to one embodiment, the intelligence creams, ointments, lotions, gels, aerosols, foams, or the like, module implements a disease classification process for ana preferably in unit dosage forms suitable for simple adminis lyzing patient test results and/or questionnaire responses to tration of precise dosages. determine whether a patient sample is associated with IBS. 0494. As used herein, the term “unit dosage form” refers to The data may be stored in one or more data tables or other physically discrete units Suitable as unitary dosages for logical data structures in memory (210) or in a separate Stor human Subjects and other mammals, each unit containing a age or database system coupled with the intelligence module. predetermined quantity of an IBS drug calculated to produce One or more statistical processes are typically applied to a the desired onset, tolerability, and/or therapeutic effects, in data set including test data for a particular patient. For association with a suitable pharmaceutical excipient (e.g., an example, the test data might include a diagnostic marker ampoule). In addition, more concentrated dosage forms may profile, which comprises data indicating the presence or level be prepared, from which the more dilute unit dosage forms of at least one marker in a sample from the patient. The test may then be produced. The more concentrated dosage forms data might also include a symptom profile, which comprises thus will contain Substantially more than, e.g., at least 1, 2, 3, data indicating the presence or severity of at least one symp 4, 5, 6, 7, 8, 9, 10, or more times the amount of the IBS drug. US 2012/03 15630 A1 Dec. 13, 2012
0495 Methods for preparing such dosage forms are a buffer, e.g., bicarbonate, for reconstitution prior to admin known to those skilled in the art (see, e.g., REMINGTON'S istration, or the buffer may be included in the lyophilized PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publish dosage form for reconstitution with, e.g., water. The lyo ing Co., Easton, Pa. (1990)). The dosage forms typically philized dosage form may further comprise a suitable vaso include a conventional pharmaceutical carrier or excipient constrictor, e.g., epinephrine. The lyophilized dosage form and may additionally include other medicinal agents, carriers, can be provided in a syringe, optionally packaged in combi adjuvants, diluents, tissue permeation enhancers, solubiliz nation with the buffer for reconstitution, such that the recon ers, and the like. Appropriate excipients can be tailored to the stituted dosage form can be immediately administered to an particular dosage form and route of administration by meth individual. ods well known in the art (see, e.g., REMINGTON'S PHAR MACEUTICAL SCIENCES, supra). (0502. In therapeutic use for the treatment of IBS, an IBS 0496 Examples of suitable excipients include, but are not drug can be administered at the initial dosage of from about limited to, lactose, dextrose, Sucrose, Sorbitol, mannitol, 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of Starches, gum acacia, calcium phosphate, alginates, traga from about 0.01 mg/kg to about 500 mg/kg, from about 0.1 canth, gelatin, calcium silicate, microcrystalline cellulose, mg/kg to about 200 mg/kg, from about 1 mg/kg to about 100 polyvinylpyrrolidone, cellulose, water, Saline, syrup, methyl mg/kg, or from about 10 mg/kg to about 50 mg/kg, can be cellulose, ethylcellulose, hydroxypropylmethylcellulose, used. The dosages, however, may be varied depending upon and polyacrylic acids such as Carbopols, e.g., Carbopol 941, the requirements of the individual, the severity of IBS symp Carbopol980, Carbopol981, etc. The dosage forms can addi toms, and the IBS drug being employed. For example, dos tionally include lubricating agents such as talc, magnesium ages can be empirically determined considering the severity Stearate, and mineral oil; wetting agents; emulsifying agents; of IBS symptoms in an individual classified as having IBS Suspending agents; preserving agents such as methyl-, ethyl-, according to the methods described herein. The dose admin and propyl-hydroxy-benzoates (i.e., the parabens); pH istered to an individual, in the context of the present inven adjusting agents such as inorganic and organic acids and tion, should be sufficient to affect a beneficial therapeutic bases; Sweetening agents; and flavoring agents. The dosage response in the individual over time. The size of the dose can forms may also comprise biodegradable polymer beads, dex also be determined by the existence, nature, and extent of any tran, and cyclodextrin inclusion complexes. adverse side-effects that accompany the administration of a 0497 For oral administration, the therapeutically effective particular IBS drug in an individual. Determination of the dose can be in the form of tablets, capsules, emulsions, sus proper dosage for a particular situation is within the skill of pensions, solutions, syrups, sprays, lozenges, powders, and the practitioner. Generally, treatment is initiated with smaller sustained-release formulations. Suitable excipients for oral dosages which are less than the optimum dose of the IBS administration include pharmaceutical grades of mannitol, drug. Thereafter, the dosage is increased by Small increments lactose, starch, magnesium Stearate, sodium saccharine, tal until the optimum effect under circumstances is reached. For cum, cellulose, glucose, gelatin, Sucrose, magnesium carbon convenience, the total daily dosage may be divided and ate, and the like. administered in portions during the day, if desired. 0498. In some embodiments, the therapeutically effective (0503 As used herein, the term “IBS drug includes all dose takes the form of a pill, tablet, or capsule, and thus, the pharmaceutically acceptable forms of a drug that is useful for dosage form can contain, along with an IBS drug, any of the treating one or more symptoms associated with IBS. For following: a diluent such as lactose. Sucrose, dicalcium phos example, the IBS drug can be in a racemic or isomeric mix phate, and the like; a disintegrant such as starch orderivatives ture, a Solid complex bound to an ion exchange resin, or the thereof a lubricant Such as magnesium Stearate and the like; like. In addition, the IBS drug can be in a solvated form. The and a binder Such a starch, gum acacia, polyvinylpyrrolidone, term “IBS drug is also intended to include all pharmaceuti gelatin, cellulose and derivatives thereof. An IBS drug can cally acceptable salts, derivatives, and analogs of the IBS also be formulated into a Suppository disposed, for example, drug being described, as well as combinations thereof. For in a polyethylene glycol (PEG) carrier. example, the pharmaceutically acceptable salts of an IBS 0499 Liquid dosage forms can be prepared by dissolving drug include, without limitation, the tartrate. Succinate, tart or dispersing an IBS drug and optionally one or more phar arate, bitartarate, dihydrochloride, salicylate, hemisuccinate, maceutically acceptable adjuvants in a carrier Such as, for citrate, maleate, hydrochloride, carbamate, Sulfate, nitrate, example, aqueous saline (e.g., 0.9% w/v Sodium chloride), and benzoate salt forms thereof, as well as combinations aqueous dextrose, glycerol, ethanol, and the like, to form a thereof and the like. Any form of an IBS drug is suitable for Solution or Suspension, e.g., for oral, topical, or intravenous use in the methods of the present invention, e.g., a pharma administration. An IBS drug can also be formulated into a ceutically acceptable salt of an IBS drug, a free base of an IBS retention enema. drug, or a mixture thereof. 0500 For topical administration, the therapeutically 0504 Suitable drugs that are useful for treating one or effective dose can be in the form of emulsions, lotions, gels, more symptoms associated with IBS include, but are not foams, creams, jellies, Solutions, Suspensions, ointments, and limited to, serotonergic agents, antidepressants, chloride transdermal patches. For administration by inhalation, an IBS channel activators, chloride channel blockers, guanylate drug can be delivered as a dry powder or in liquid form via a cyclase agonists, antibiotics, opioids, neurokinin antagonists, nebulizer. For parenteral administration, the therapeutically antispasmodic or anticholinergic agents, belladonna alka effective dose can be in the form of sterile injectable solutions loids, barbiturates, glucagon-like peptide-1 (GLP-1) analogs, and sterile packaged powders. Preferably, injectable solu corticotropin releasing factor (CRF) antagonists, probiotics, tions are formulated at a pH of from about 4.5 to about 7.5. free bases thereof, pharmaceutically acceptable salts thereof, 0501. The therapeutically effective dose can also be pro derivatives thereof, analogs thereof, and combinations vided in a lyophilized form. Such dosage forms may include thereof. Other IBS drugs include bulking agents, dopamine US 2012/03 15630 A1 Dec. 13, 2012 52 antagonists, carminatives, tranquilizers, dextofisopam, phe GTP-010 may be useful for treating IBS symptoms such as nyloin, timolol, and diltiazem. constipation. CRF antagonists such as astressin and probiot 0505 Serotonergic agents are useful for the treatment of ics such as VSL#3(R) may be useful for treating one or more IBS symptoms such as constipation, diarrhea, and/or alter IBS symptoms. One skilled in the art will know of additional nating constipation and diarrhea. Non-limiting examples of IBS drugs currently in use or in development that are suitable serotonergic agents are described in Cash et al., Aliment. for treating one or more symptoms associated with IBS. Pharmacol. Then, 22:1047-1060 (2005), and include 5-HT, 0509 An individual can also be monitored at periodic time receptoragonists (e.g., MKC-733, etc.), 5-HT, receptorago intervals to assess the efficacy of a certain therapeutic regi nists (e.g., tegaserod (ZelnormTM), prucalopride, AG1-001, men once a sample from the individual has been classified as etc.), 5-HT receptor antagonists (e.g., alosetron an IBS sample. For example, the levels of certain markers (LotroneXR), cilansetron, ondansetron, granisetron, dolas change based on the therapeutic effect of a treatment such as etron, ramosetron, palonosetron, E-3620, DDP-225, DDP a drug. The patient is monitored to assess response and under 733, etc.), mixed 5-HT receptor antagonists/5-HT, receptor stand the effects of certain drugs or treatments in an individu agonists (e.g., cisapride, mosapride, renzapride, etc.), free alized approach. Additionally, patients may not respond to a bases thereof, pharmaceutically acceptable salts thereof, drug, but the markers may change, Suggesting that these derivatives thereof, analogs thereof, and combinations patients belong to a special population (not responsive) that thereof. Additionally, amino acids like glutamine and can be identified by their marker levels. These patients can be glutamic acid which regulate intestinal permeability by discontinued on their current therapy and alternative treat affecting neuronal or glial cell signaling can be administered ments prescribed. to treat patients with IBS. 0506 Antidepressants such as selective serotonin IV. Examples reuptake inhibitor (SSRI) or tricyclic antidepressants are par 0510. The following examples are offered to illustrate, but ticularly useful for the treatment of IBS symptoms such as not to limit, the claimed invention. abdominal pain, constipation, and/or diarrhea. Non-limiting 0511. The Examples from US Patent Publication No. examples of SSRI antidepressants include citalopram, flu 2008/0085524, filed Aug. 14, 2007, are herein incorporated Voxamine, paroxetine, fluoxetine, Sertraline, free bases by reference in their entirety for all purposes. thereof, pharmaceutically acceptable salts thereof, deriva tives thereof, analogs thereof, and combinations thereof. Example 1 Examples of tricyclic antidepressants include, but are not limited to, desipramine, nortriptyline, protriptyline, amitrip A Tryptase ELISA for Predicting IBS tyline, clomipramine, doxepin, imipramine, trimipramine, 0512. This example describes a sensitive ELISA for maprotiline, amoxapine, clomipramine, free bases thereof, detecting the presence or level of mast cell B-tryptase. See pharmaceutically acceptable salts thereof, derivatives also, FIGS. 1-7. thereof, analogs thereof, and combinations thereof. 0513 Background: Mast cells play an important role in the 0507 Chloride channel activators are useful for the treat pathogenesis of irritable bowel syndrome (IBS). Increased ment of IBS symptoms such as constipation. A non-limiting mast cell infiltration and activation in distal gut segments are example of a chloride channel activator is lubiprostone (Am associated with symptom onset and severity of IBS. Mast itizatM), a free base thereof, a pharmaceutically acceptable cells have been implicated in the elevated response of visceral salt thereof, a derivative thereof, or an analog thereof. In afferent nerves to mucosal stimulus in IBS patients. Measure addition, chloride channel blockers such as crofelemer are ment of mast cell markers can have important implication in useful for the treatment of IBS symptoms such as diarrhea. clinical diagnosis of IBS. However this effort was hindered Guanylate cyclase agonists such as MD-1100 are useful for due to lack of sensitive assays. the treatment of constipation associated with IBS (see, e.g., 0514 Methods: Here we report the development and vali Bryant et al., Gastroenterol., 128:A-257 (2005)). Antibiotics dation of a highly sensitive two-side ELISA assay to measure Such as neomycin can also be suitable for use in treating tryptase level in human serum samples (detection limit 0.019 constipation associated with IBS (see, e.g., Park et al., Gas ng/ml). The assay is precise, robust, and reproducible. Serum troenterol., 128:A-258 (2005)). Non-absorbable antibiotics tryptase concentration in healthy controls and IBS patients like rifaximin (XifaxanTM) are suitable to treat small bowel was measured using this assay. bacterial overgrowth and/or constipation associated with IBS 0515 Results: The average serum tryptase level in healthy (see, e.g., Sharara et al., Am. J. Gastroenterol., 101:326-333 controls was 9.32+2.1 ng/ml (n=156). IBS-D and IBS-A (2006)). patients showed significant higher serum tryptase concentra 0508 Opioids such as kappa opiods (e.g., asimadoline) tion (12.71 ng/ml (n=209) for IBS-D and 11.94 ng/ml (n=57) may be useful for treating pain and/or constipation associated for IBS-A, p<0.01); while the average tryptase level is 9.34 with IBS. Neurokinin (NK) antagonists such as talnetant, ng/ml (n=118) for IBS-C, which has no significant difference saredutant, and other NK2 and/or NK3 antagonists may be from healthy controls for IBS-C. useful for treating IBS symptoms such as oversensitivity of 0516 Conclusion: This is the first biomarker developed so the muscles in the colon, constipation, and/or diarrhea. Anti far that differentiates IBS-D and IBS-A patients from IBS-C spasmodic oranticholinergic agents such as dicyclomine may patients and healthy controls. Combining serum tryptase be useful for treating IBS symptoms such as spasms in the level with other Prometheus IBS biomarkers, we were able to muscles of the gut and bladder. Other antispasmodic or anti improve the accuracy of diagnosing IBS-D and IBS-A cholinergic agents such as belladonna alkaloids (e.g., atro patients. pine, Scopolamine, hyoscyamine, etc.) can be used in combi 0517. An ELISA assay was developed for the determina nation with barbiturates such as phenobarbital to reduce tion of serum mast cell B-tryptase level using rabbit anti bowel spasms associated with IBS. GLP-1 analogs such as tryptase as capture antibody and alkaline phosphatase conju US 2012/03 15630 A1 Dec. 13, 2012
gated G3 as detecting antibody. Luminescent substrate CPSD added to the plate. After incubating at RT for another 2 hours, (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'- the plate was washed, and then incubated with 100 ml of chloro)tricyclo[3.3.1.13.7 decan-4-yl)phenyl phosphate), AP-conjugated anti-human tryptase at an optimized dilution was used to enhance the assay sensitivity. Linear dose-re in assay buffer. The plate was incubated for 2 hours with sponse curve was observed over the standard range of 1-1000 gentle agitation and then washed. 100 ml of Tropix. CSPD ng/ml for B-tryptase in buffer with 5% BSA and 10% normal substrate was added to each well and incubated in the dark for human serum. The low limit of the assay was 0.019 ng/ml, the 30 minutes before reading the luminescence with a lumines intra-assay and inter-assay coefficients of variation were cence plate reader. The Relative Luminescent Unit (RLU) and below 15% at 1 ng/ml and 1000 ng/ml tryptase concentra the tryptase concentration was plotted with the Prism Graph tions. The recovery of known amounts of purified tryptase pad Program. Tryptase detection range=0.019-5000 ng/ml. added to serum was 88%. The immunoassay was utilized to examine serum levels of tryptase from healthy controls, EC50=65 ng/ml. Recovery was 81.5% with 20 ng spiked in IBS-C and IBS-D patients. Average serum tryptase level in normal pooled serum. healthy control was 7.0+2. 1 ng/ml (n=113). The average 0523 FIG. 9 shows the optimization of the tryptase tryptase level in IBS-C and IBS-D were 9.6 ng/ml (n=116) ELISA described herein. FIG. 10 shows tryptase levels in and 12.7 (n=209) respectively. Using the cutoff value of 11.4 serum from healthy controls (n=139) and from subjects with ng/ml (average+2SD), the assay specificity for GI healthy IBS (n=378). Tryptase levels were measured by ELISA in control was 82% (n=156), the assay sensitivity for IBS-C and serum samples diluted 10 times with assay buffer. IBS IBS-D were 21.5% and 24.9% respectively. Combine with patients are also shown according to its subtypes: IBS-D symptoms of the patient and other markers for IBS, serum (n=206): diarrhea predominant; IBS-C (n=116): constipation tryptase levels may help differentiate IBS-D from other types predominant; IBS-A (n=56): alternating symptoms. Each of irritable bowel syndrome. value is the average of duplicate determinations. Solid lines are the median value from each group. The cutoff value of 12 Example 2 ng/ml was calculated from healthy Subjects with median plus Evaluation of the Diagnostic Utility of Serum 2SD (dotted line). *p-0.0001 vs healthy subjects; **p-0. Tryptase Levels in IBS 0001 vs IBS-D; Mann Whitney U test. Table 2 provides a summary of tryptase levels in IBS Subtypes and healthy sub 0518 Background: Mast cells play an important role in the jects (cutoff=12.0 ng/ml). pathogenesis of irritable bowel syndrome (IBS). Increased mast cell infiltration and activation in distal gut segments are TABLE 2 associated with symptom onset and severity of IBS. Mast cells have been implicated in the elevated response of visceral Tryptase levels (ng/ml) in IBS Subtypes and afferent nerves to mucosal stimulus in IBS patients. Measure healthy subjects (cutoff = 12.0 ng/ml). ment of mast cell markers can have important implications in Healthy clinical diagnosis of IBS. However, this effort was hindered Control IBS IBS-D IBS-C IBS-A due to lack of sensitive assays. Subjects (n) 139 381 209 116 56 0519 Methods: Here we report the development and vali Mean (ng ml) 7.1 10.3 10.3 9.6 12.1 dation of a highly sensitive two-site ELISA assay to measure STDEV 2.4 9.2 8.7 9.4 10.7 tryptase level in human serum samples (detection limit 0.019 Positive 9 82 44 22 16 ng/ml). The assay is precise, robust, and reproducible. Serum tryptase concentrations in healthy controls and IBS patients was measured using this assay. 0524 FIG. 11 shows the increased serum level of hista 0520 Results: The average serum tryptase level in healthy mine and PGE in IBS patients vs healthy controls. PGE was controls was 7.1+2.5 ng/ml. IBS-D and IBS-A patients measured in 50 ul of serum by ELISA using a kit from showed higher serum tryptase concentrations (10.3+8.7 Cayman. Histamine was measured in 10 ul of serum using an ng/ml for IBS-D and 12.1+10.7 ng/ml for IBS-A) than the Immunotech EIA kit. The samples were tested in duplicate. healthy control subjects, while the average tryptase level was The Solid line is the median of each group. As such, in certain 9.6+9.4 ng/ml for IBS-C. There was a statistical difference of aspects, IBS-D may be diagnosed or distinguished from other serum tryptase levels between the IBS-C and IBS-D groups clinical subtypes of IBS by detecting a higher level of PGE (p<0.01). relative to healthy control, IBS-A, and/or IBS-C samples or 0521. Conclusion: Serum tryptase concentration is the standards. In certain other aspects, IBS-D or IBS-A may be first biomarker developed so far that differentiates IBS-D diagnosed or distinguished from IBS-C by detecting a higher from IBS-C patients. Serum tryptase levels can be combined level of histamine relative to healthy control and/or IBS-C with other IBS biomarkers to improve the accuracy of diag samples or standards. nosing IBS. 0525 Conclusions: (1) A sandwich ELISA method was 0522 FIG. 8 shows the dose-dependent responses of developed that can measure serum tryptase level with high human tryptase in the tryptase ELISA described herein. Pro sensitivity, accuracy, and precision. The assay has a high tocol: A 96-well microtiter plate was coated with 100 ml of 2 degree of reproducibility and is suitable for routine testing of mg/ml of anti-human tryptase antibody in Sodium carbonate a large number of human sera. (2) Using this ELISA, we (pH 9.6) at 4°C. overnight. After washing with PBST, the found significant differences in serum tryptase levels between plate was incubated with 300 ml/well of blocking/assay healthy controls and IBS patients. Among the IBS patients, buffer (5% BSA in PBS) at room temperature (RT) for 30 IBS-D and IBS-A patients had statistically higher tryptase minutes with gentle agitation. After washing, 100 ml/well of levels compared to IBS-C patients. (3) Additional mast cell serially diluted (1:8 in assay buffer) human tryptase were markers, histamine and PGE, were also found to be aberrant US 2012/03 15630 A1 Dec. 13, 2012 54 in IBS patient serum samples. Combining these markers with tryptase resulted in improved diagnostic accuracy of IBS. TABLE 3-continued Exemplary first section of a questionnaire for identifying Example 3 the presence or severity of IBS-related symptoms. Questionnaire for Identifying the Presence or Sever- 3. In the last 3 months, O) Never -> ity of Symptoms Associated with IBS how often did you feel CD Less than one day a month uncomfortably full after (2) One day a month 0526. This example illustrates a questionnaire that is use- a regular-sizedgll meal? 8 ().Two toiy three ity d 8 Oll ful for identifying the presence or severity of one or more GS) More than one day a week IBS-related symptoms in an individual. The questionnaire (6) Every day can be completed by the individual at the clinic or physician's 4. In the last 3 months, O) Never -> office, or can be brought home and submitted when the indi-- 0 howunable often to finishwere ayou CD(2) LessOne daythan a onemonth day a month vidual returns to the clinic or physicians office, e.g., to have regular size meal? G.) Two to three days a month his or her blood drawn. (4) One day a week 0527. In some embodiments, the questionnaire comprises Mr. y one day a week a first section containing a set of questions asking the indi- 5. In the last 3 months, (O) Never -> vidual to provide answers regarding the presence or severity how often did you have O Less than one day a month of one or more symptoms associated with IBS. The question- pain or burning in the (2) One day a month naire generallylv includincludes questionsti directed to identifvingIdenulying thune middleabdomen, of yourabove your G.)(4) TwoOne dayto three a week days a month presence, severity, frequency, and/or duration of IBS-related belly button but not in GS) More than one day a week symptoms such as chest pain, chest discomfort, heartburn, your chest? (6) Every day uncomfortable fullness after having a regular-sized meal, 6. the R d ents Never --> inability to finish a regular-sized meal, abdominal pain, GSMR Sh 8We (2) 5. yon y 8 Oll abdominal discomfort, constipation, diarrhea, bloating, and/ anywhere in your G.) Two to three days a month or abdominal distension. abdomen? (4) One day a week 0528. In certain instances, the first section of the question- 8 Mr. . one day a week naire includes all or a subset of the questions from a ques- 7. In the last 3 months, O) S. barely tionnaire developed by the Rome Foundation Board based on how often did you have (1) Sometimes the Rome III criteria, available at http://www.romecriteria. (We l8l this bowel el org/questionnaires/. For example, the questionnaire can ents (0-2) a to the time include all or a subset of the 93 questions set forth on pages 8. In the last 3 months, O) Never or rarely 920-936 of the Rome III Diagnostic Questionnaire for the how often did you have O Sometimes (25% of the time) Adult Functional GI Disorders (Appendix C), available at hard or lumpy stools? en (50% of the time) http://www.romecriteria.org/pdfs/AdultFunctGIQ.pdf. Pref- of the time (75%0. of the time) erably, the first section of the questionnaire contains 16 of the 9. In the last 3 months, O) N.Y., rarely 93 questions set forth in the Rome III Diagnostic Question- how often did you strain (1) Sometimes naire (see, Table 3). Alternatively, the first section of the during bowl el questionnaire can contain a Subset (e.g., 1,2,3,4,5,6,7,8,9, movements: (3) to the time 10, 11, 12, 13, 14, or 15) of the 16 questions shown 1n Table 10. In the last 3 months, O) Never or rarely 3. As a non-limiting example, the following 10 questions set how often did you have (1) Sometimes forth in Table 3 can be included in the questionnaire: Question a feeling ofspite el Nos. 2, 3, 5, 6, 9, 10, 11, 13, 15, and 16. One skilled in the art syster bowel . the time will appreciate that the first section of the questionnaire can 11. In the last 3 months, O) Never or rarely comprise questions similar to the questions shown in Table 3 how often did you have (1) Sometimes regarding pain, discomfort, and/or changes in stool consis- a sensation that the stool el tenc could not be passed, G) Most of the time y. (i.e., blocked), when ways having a bowel TABLE 3 movement? 12. In the last 3 months, O) Never or rarely Exemplary first section of a questionnaire for identifying how often did you press Sometimes the presence or severity of IBS-related symptoms. on or around your Often bottom or remove stool G) Most of the time 1. In the last 3 months, O) Never in order to complete a (4) Always how often did you have O Less than one day a month bowel movement? pain or discomfort in the (2) One day a month 13. Did any of the O) No middle of your chest G.) Two to three days a month symptoms of O Yes (not related to heart (4) One day a week constipation listed in problems)? G) More than one day a week ip (6) Every day questions 27-32 above 2. In the last 3 months, O) Never begin more than 6 how often did you have (1) Less than one day a month months ago. heartburn (a burning (2) One day a month 14. In the last 3 months, O) Never or rarely --> discomfort or burning G.) Two to three days a month how often did you have CD Sometimes (25% of the time) pain in your chest)? (4) One day a week oose, mushy or watery (2) Often (50% of the time) GS) More than one day a week stools? (3) Most of the time (75% of the time) (6) Every day (4) Always US 2012/03 15630 A1 Dec. 13, 2012 55
0533. Alternatively, a “Yes” or “No” answer from the indi TABLE 3-continued vidual to the following question: “Are you currently experi encing any symptoms?’ can be combined with the analysis of Exemplary first section of a questionnaire for identifying one or more of the biomarkers described herein and processed the presence or severity of IBS-related symptoms. using a single statistical algorithm or a combination of statis 15. In the last 3 months, O Newer --> tical algorithms to increase the accuracy of predicting IBS. how often did you have O Less than one day a month bloating or distension? (2) One day a month G.) Two to three days a month Example 4 (4) One day a week GS) More than one day a week Blood based diagnostic assay for the diagnosis of (6) Every day Irritable Bowel Syndrome (IBS) 16. Did your symptoms of (0) No bloating or distention OYes 0534. The present example describes the first blood-based begin more than 6 biomarker test for Irritable Bowel Syndrom (IBS). This test months ago? can aid clinicians in the diagnosis of IBS. The IBS Diagnostic described below was validated using well-characterized IBS 0529. In other embodiments, the questionnaire comprises samples collected from recognized IBS experts and GI clin a second section containing a set of questions asking the ics. The samples were either Rome II or Rome III positive and the patients had a diagnosis of IBS for greater than one year. individual to provide answers regarding the presence or The test has a sensitivity of 50%, specificity of 88% with an severity of negative thoughts or feelings associated with hav overall accuracy of 70%. ing IBS-related pain or discomfort. For example, the ques 0535 The total cohort used to develop the present assay tionnaire can include questions directed to identifying the consisted of 1,721 serum samples. The validation cohort used presence, severity, frequency, and/or duration of anxiety, fear, consisted of 516 serum samples, of which 50% were diag nervousness, concern, apprehension, worry, stress, depres nosed with IBS according to Rome II or Rome III criteria: Sion, hopelessness, despair, pessimism, doubt, and/or nega 36% were Non-IBS disease controls; and 14% were normal tivity when the individual is experiencing pain or discomfort healthy controls. The sensitivity of the assay is 50% and the associated with one or more symptoms of IBS. specificity is 88%. When ruling in IBS, wherein the physician 0530. In certain instances, the second section of the ques has determined that there is about a 75% probability of dis tionnaire includes all or a subset of the questions from a ease in the patient, 94% of the positive test results are true questionnaire described in Sullivan et al., The Pain Catastro positives, while 38% of the negative test results are true phizing Scale: Development and Validation, Psychol. Assess., negatives. When ruling out IBS, wherein the physician has 7:524–532 (1995). For example, the questionnaire can determined that there is about a 25% probability of disease in include a set of questions to be answered by an individual the patient, 86% of the negative test results are true negatives, according to a Pain Catastrophizing Scale (PCS), which indi while 61% of the positive test results are true positives. cates the degree to which the individual has certain negative 0536 The assay involves the quantitative analysis of bio thoughts and feelings when experiencing pain: 0 not at all; markers combined with a two learning statistical classifier 1 to a slight degree; 2 to a moderate degree; 3 to a great system consisting of a random forest classifier and a neural degree; 4 all the time. The second section of the question network classifier. naire can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 0537. The specimen requirements for the assay consist of or more questions or statements related to identifying the the physician obtaining 2.0 ml of separated serum in, for presence or severity of negative thoughts or feelings associ example, an SST tube. For best results, the sample should be ated with having IBS-related pain or discomfort. As a non centrifuges and refrigerates within 2 hours of collection. limiting example, an individual can be asked to rate the When samples need to be shipped prior to assay detection, the degree to which he or she has one or more of the following samples should be refrigerated or frozen. For best results, the thoughts and feelings when experiencing pain: "I worry all samples should be stored, prior to use, for no more than 7 days the time about whether the pain will end: “I feel I can’t stand at 4°C. or 30 days if frozen. it anymore”: “I become afraid that the pain will get worse”: “I 0538 Briefly, enzyme-linked immunosorbent assays anxiously want the pain to go away'; and “I keep thinking (ELISAs) are performed with antibodies specific for the IBS about how much it hurts.” One skilled in the art will under biomarkers ASCA-IgA, CBirl, ANCA, and tTG. Addition stand that the questionnaire can comprise similar questions ally, chemiluminescent assays are performed for the IBS regarding negative thoughts or feelings associated with hav biomarkers BDNF, NGAL, TWEAK, GRO-O, IL-1 B, and ing IBS-related pain or discomfort. TIMP-1. Reference values for the normal range of these 0531. In some embodiments, the questionnaire includes markers in healthy subjects are provided in Table 4. only questions from the first section of the questionnaire or a subset thereof (see, e.g., Table 3). In other embodiments, the TABLE 4 questionnaire includes only questions from the second sec tion of the questionnaire or a Subset thereof. Reference values for the markers detected in the IBS assay. 0532. Upon completion of the questionnaire by the indi Marker Reference Level vidual, the numbers corresponding to the answers to each question can be Summed and the resulting value can be com BDNF (Brain-Derived Neurotrophic Factor) 7536.5-31324.4 pg/ml NGAL (Neutrophil Gelatinase-Associated 28.3-272.5 ng/ml bined with the analysis of one or more diagnostic markers in Lipocalin) a sample from the individual and processed using the statis TWEAK (TNF-related Weak Inducer of 351.6-1751.7 pg/ml tical algorithms described herein to increase the accuracy of Apoptosis predicting IBS. US 2012/03 15630 A1 Dec. 13, 2012 56
Subjects using high performance liquid chromatography TABLE 4-continued (HPLC). We developed and optimized a high throughput HPLC method using a Diphenyl column which enabled us to Reference values for the markers detected in the IBS assay. separate over 20 peaks in IBS and healthy control samples. Marker Reference Level Plasma, serum and urine samples from the same Subjects GRO-C. (Growth-Regulated Oncogene Alpha) 26.4-499.3 pg/ml were compared for levels of 5-HT and its metabolites. IL-1B (Interleukin-1 Beta) 279.5-1358.6 fg/ml 0543 Briefly, serum samples were collected in SST tubes TIMP-1 (Tissue Inhibitor of 156.1-410.6 ng/ml Metalloproteinase-1) according to the approved IRB (Prometheus 08IBS04 proto ASCA-IgA (Anti-Saccharomyces cerevisiae <20.0 EUml col). 300 ul of human serum was deproteinated with 60 ul of Antibody) CBirl (Anti-CBirl Antibody) <21.0 EUml isopropanol and 360 ul of acetonitrile. The mixture was then ANCA (Anti-Human Neutrophil Cytoplasmic <12.1 EUml centrifuged at 2000xg for 15 minutes. The supernatant con Antibody) taining metabolites was transferred to a second tube and tTG (Anti-Human Tissue Transglutaminase <4.0 Uml IgA) concentrated using a Speed Vac Plus (model SC 110A). The dry pellet was reconstituted with 160ul of water, of which 100 ul is injected into the HPLC system (Agilant 1200). 0539. In certain instances, information from the detection of the IBS biomarkers described above can be combined with 0544 Liquid chromatography was performed using an symptom information provided by the patient. For example, Agilent 1200 HPLC system consisting of a quaternary pump, the patient may fill out a checklist of their symptoms, the an autosampler, a DAD and a FLD. Chromatographic sepa information from which can be combined with the informa ration was performed on a Varian Pursuit Diphenyl 5 um tion from the IBS biomarkers to further aid the clinician in the 4.6x150 mm column, protected by a Metaguard Pursuit XR diagnosis of IBS. An example of a checklist that may be used 10 um C18 guard column, as well as, a SunFire 5um 4.6x20 in this fashion is provided in Table 5. mm C18 guard column. For the first 20 minutes of the analysis a gradient from 100% buffer A to 100% buffer B was TABLE 5 employed. After 55 minutes, the mobile phase was changed Example patient checklist of symptoms commonly associated with IBS. from 100% buffer B to 100% buffer A over 1 minute. Buffer IBS Symptom Checklist A was then kept at 100% for 10 minutes in preparation for the Fill out and bring to your next appointment. next injection. Mobile phase A consisted of a 10 mM sodium Your Name acetate buffer, pH 4 using glacial acetic acid. Mobile phase B Appointment Date Place a check mark next to any symptoms you are currently having or is the same as buffer A but with 12% methanol. 5-HT, as well have had in the past. Note how often these symptoms are present. as other indoles, were detected using the FLD set at an exci Recurrent abdominal pain or discomfort tation of 225 and emission of 342. Tryptophan and Kynure Abnormal stool frequency (greater than 3 bowel movements day or less than 3 bowel movements/week) nine are detected with the UV set at the wavelengths of Abnormal stool form (lumpy/hard or loosef watery stool) respectively, 278 and 360. Abnormal stool passage (straining, urgency, or feeling of incomplete bowel movement) 0545. Measurements were carried out at 25°C. at Eox=0. Passage of mucus 65 V. The calibration curves are linear through the range of Bloating or feeling of abdominal distension 10-200 pg/mL. Method validation was performed according Gassiness Feelings of urgency (the need to find a restroom fast) to internationally accepted criteria. Blood was collected from healthy controls and IBS subjects. 0546. Upon analysis, several known analytes were identi Example 5 fied, such as tryptophan and 5-HT and its metabolites, 5-HT O-S and Kynurenine, as well as several novel metabolites Identification of Novel Metabolites in Serum of with retention time at 7.5 minute, 13.5 minute, 52 minute by Patients with Irritable Bowel Syndrome fluorescent detector and at 20 minute by UV detector, respec 0540 5-Hydroxytryptamine is converted from tryptophan tively. Representative chromatograms from this analysis are catalyzed by tryptophan dehydrogenase. Over 95% of 5-HT shown in FIG. 14. The level of each biomarker is shown in in the body is released from the enterohromaffin cells of the FIG. 15. gastrointestinal tract. 5-HTacts through various receptor Sub (0547 As can be seen in Table 6, the serum 5-HT concen types located on Submucosal and myenteric neurons of the trations in all three IBS subtypes were lower than those of enteric nerve system is known to play a key role in the normal functioning of the GI tract. Abnormal level of 5-HT is asso healthy controls after normalized by internal standards, a ciated with pathogenesis of irritable bowel syndrome. median AUC of 94.5 for healthy subjects, 66.85, 48.3, and 0541 5-HT (Hydroxytryptamines) plays an important 46.25 for IBS-C, IBS-D, and IBS-M respectively. The area role in GI motility, secretion, and sensation. Serotonergic under curve for the peaks at 7.5 min and 13.5 were higher in therapies act on 5-HT receptors effectively ameliorate symp all three IBS subgroups after normalized by 5-HT. IBS-D toms that IBS patients experience. Study 5-HT metabolites patients had higher level of 20 min UV peaks compared to may lead to identification of biologically relevant biomarkers IBS-C. IBS-M, and healthy volunteers. These findings sug that can be used for accurate diagnosis of IBS. gest serum metabolites may serve as biomarkers for diagnosis 0542 Serum metabolites were investigated in samples of IBS. The predictive power of the serum metabolites is from 12 IBS-C, 24 IBS-D, 14 IBS-M patients and 38 healthy illustrated in FIG. 16. US 2012/03 15630 A1 Dec. 13, 2012 57
TABLE 6 Results of serum metabolite analysis via HPLC. Mean Median S.E.
Peak IBS-C IBS-D IBS-M HC IBS-C IBS-D IBS-M HC IBS-C IBS-D IBS-M HC
20 Min. UV 88.5 114.1 90.8 83.0 66.3 108.9 75.9 67.8 22.2 13.9 12.9 11.1 (Unknown) 30 Min. UV 380.3 569.O 466.7 359.3 267.2 388.4 372.5 256.6 124.8 112.2 7O.O 51.7 (Unknown) 5-HTIS 89.0 51.3 52.1 97.5 66.9 48.3 46.3 94.1 23.9 8.7 8.3 9.6 7.5 Min. 7486.O 44130 3233.O. 1148.0 948.O 1583.0 2219.O 806.2 466O.O 1242.O 814.6 1492 FLD,S-HT 13.5 Min. 699.2 299.2 1933 75.6 76.4 97.8 1638 42.1 524.5 94.5 43.5 13.9 FLD,S-HT
Example 6 extracted by isoproponol and acitonitrile. Metabolites were Effect of Inflammatory Cytokine Treatment on 5-HT separated by HPLC using a diphenol column (Aligant sys Metabolism and Mast Cell Activation in Human tem). The machine was run at a gradient of 0-12% methanol Blood Cells for 20 minutes, followed by an isocratic run at 12% methanol for 55 minutes. 5-HT and other indoles are detected using the 0548 Irritable bowel syndrome is a multi-factorial func FLD set at an excitation of 225 and emission of 342. Tryp tional GI disorder. Inflammatory stimulation in local gut tophan and Kynurenine were detected with the UV set at the mucosahas been implicated as a triggerfor IBS disease onset. wavelengths of 278 and 360 respectively. Representative Serotonin dysregulation, mast cell activation and stress response all contribute to pathogenesis of the disease. The chromatograms from this analysis are shown in FIG. 17. As present example provides a study of the interactions of those seen in FIG. 18 and Table 7, TNF-C. treatment of blood pathways in inflammatory cytokine treated blood samples samples significantly stimulated tryptophan and 5-HT from healthy volunteers. Changes in 5-HT metabolism and metabolism, resulting in elevated kynurenin and 5-HT-O-S plasma level of mast cell and stress markers were measured. production. It was found that inflammatory stimulation alter tryptophan and 5-HT metabolism. Similarly, plasma kynurenine and TABLE 7 5-HT-O-S level were up-regulated by TNF-Citreatment. IL-1B stimulation resulted in increase level of PGE, which is a Effect of inflammatory cytokine treatment on the levels marker of mast cell activation. Quantification of 5-HT bio of tryptophan, kynurenine, and 5-HT-O-S in blood samples. synthesis enzyme (TPH-1) and metabolic enzyme (MAO) led to finding of change in transcript of those enzymes by cytok Treatment Tryptophan Kynurenine 5-HTO S ines Understanding of the interactions between those path ways help us to identify biology relevant biomarkers to assist Blank 2,851.90 17.10 56.50 clinical diagnosis. IL1b 3,865.96 S4.90 86.70 0549. Irritable bowel syndrome is a heterogenous disor TIMP1 3,903.20 71.10 85.00 der. There is increasing evidence for involvement of the TNFa. 3,471.10 147.56 124.67 immune system in its pathogenesis. Gastrointestinal infection may serve as a triggering factor for onset of symptoms. IBS is 2 Plex 3,950.35 22.70 91.20 often described as a “brain-get disorder. Alterations in gas S Plex 3,857.94 26.30 92.60 trointestinal motility and secretion may underlie irregularities in bowel habits that may be mediated by dysregulation of serotonin signaling system. Activated mast cells in proximity 0551 MAO, TPH1, and SERT expression profiling: 2.4 ml to colonic nerves correlated with abnormal pain in patients of each treated blood sample was added into Paxigen tubes, with IBS. Mast cells are well known for capable of releasing total RNA was prepared according to manufacture's protocol. and producing a variety of inflammatory mediators upon 200 ng of total RNA was reversely transcribed into cDNA, activation. It remains unclear how those pathways communi 200 ng of cDNA was used for quantification subsequently by cate with each other and whether the interactions behave the qPCR reaction (ABI 7000 systems). same in IBS patients as in healthy Subjects. 0552 Measurement of immune markers: Mast cell mark 0550 Blood samples were collected and treated as fol ers, 5-HT, and Urocortin (Ucn) were measured by ELISA. lows. Fresh human blood samples from healthy volunteers Tryptase and Ucn were measured using assays developed by were collected into heparin tubes. The samples were ali quoted into seven 15 ml colloquial tubes and treated with Prometheus. PGE, histamine, and 5-HT were measured inflammatory cytokines, TNF-C., IL-1B, TIMP1, NGAL, using the commercial assays. As seen in Table 8 and FIG. 19. 2-Plex (BDNF and TWEAK), 5-Plex (Gro-a, ASCA, ANCA, Ucn levels were significantly elevated in response to TNF-C. tTG, CBirl) for 2 day at 37° C. in cell culture incubator. In TIMP1, and NGAL treatment. As seen in Table 9 and FIG.20, order to measure the levels of metabolites in the samples, the mast cell marker PGE was significantly elevated in chemicals from 300 ul of treated plasma samples were response to IL-1B treatment. US 2012/03 15630 A1 Dec. 13, 2012 58
tion and HPLC purification of pooled human serum samples TABLE 8 was performed to isolate those markers and used mass spec trometric analysis to establish their chemical structures. One Effect of inflammatory cytokine treatment of the marker was identified to be 5-HT itself, while two of the on the levels of Ucn release. other isolated markers were identified to be tyrosine and Treatment Ucn ng/ml phenylalanine and the identity of those marker peaks in the analytical HPLC were subsequently confirmed by spiking Blank NA 5-plex NA synthetic tyrosine and phenylalanine into human serum 2 plex NA samples. Serum level of 5-HT was found to be higher in HC IL-1b NA subjects compared to IBS patients, whereas IBS patient sub NGAL 14.7 - 8.5 types have varied levels of tyrosine and phenylalanine. Mea TIMP1 37.8 - 21 Surement of tyrosine and phenylalanine levels is relevant to TNFalpha 42.8 + 1.1 IBS diagnosis since these two amino acids are precursors for the biosynthesis of dopamine, norepinephrine and epineph rine, which are important regulators of the enteric nervous TABLE 9 system. Effect of inflammatory cytokine treatment 0555. While the etiology of IBS remains obscure, there is on the levels of mast cell markers. a body of evidence Suggesting several pathophysiological pathways that are dysregulated in IBS, including serotonin Mast cells markers (5-HT) biosynthesis and metabolism, mast cell infiltration, Treatment Tryptase PGE, Histamine visceral hypersensitivity, stress response and bacteria infec tion (post infectious IBS). Of these, the 5-HT system plays a Blk 19.8 + 1.1 1621.7 - 89.1 294.9 - 43.3 significant role in GI physiology and pathophysiology and 5plex 20.5 - 1.3 1505.8 - 105.5 269.2 - 18.2 2plex 21.7 - 0.7 1916.8 - 126.4 279.7 - 29.8 several 5-HT receptor agonists and antagonists are either IL-1B 20.7 - 0.74 5588.6 - S60.8 257.3 - 35.3 approved or under clinical development for the management NGAL 20.3 + 1 17OO.3 - 23.6 2655 - 20.0 of IBS. For this reason, identification of 5-HT and its metabo TIMP-1 21.2 - 0.27 1818.5 - 1851 2616 - 30.5 lites as potential biomarkers in IBS patient serum samples TNF-ct, 20.4 + 2.0 2394.3 - 834 208.8 - 28.9 may facilitate an accurate diagnosis of IBS and provide a better treatment. Towards this goal, a high performance liquid 0553. The present example demonstrates that TNF-C. chromatography (HPLC) method has been developed to iden treatment leads to increase release of tryptophan metabolites, tify potential biomarkers in serum samples from IBS patients kynurenin, and 5-HT metabolite, 5-HT-O-S in healthy human and healthy control (HC) subjects. blood cells. This alteration in tryptophan and 5-HT metabo 0556 Preparation of Serum Samples for HPLC Analysis: lism may be due to change in 5-HT MAO and TPH1 expres IBS patient serum samples were collected in SST tubes sion in blood cells induced by cytokine treatment. Further according to an IRB approved protocol and healthy control more, it was found that the level of the mast cell marker PGE serum samples were obtained from Volunteers. To prepare a was significantly increased upon IL-1B treatment of fresh serum sample for HPLC analysis, 300 uL of serum was whole blood. Further studies of cytokine response in IBS deproteinated by vortexing with 60LL ofisopropanol and 360 patient blood cells may shed some light on the mechanistic uL of acetonitrile in an Eppendorf tube and the mixture cen interaction in the pathways contributing the IBS pathogen trifuged at 2000xg for 15 min. The supernatant containing the CS1S. serum metabolites was transferred to a second Eppendorf tube and the solvent evaporated using a Speed Vac Plus con Example 7 centration (model SC 110A). The dry pellet is reconstituted in Discovery of Tyrosine, Phenylalanine and Serotonin 160 u, of water, of which 100 uL is injected into the HPLC as Useful Metabolite Biomarkers for the Identifica system for analysis. 0557. Analytical HPLC Procedure: The HPLC system tion of IBS and Subtypes Thereof employed for the analysis is an Agilent model 1200 HPLC 0554 Serotonin (5-Hydroxy-tryptamine, 5-HT) plays an system, consisting of a binary pump, an autosampler, a DAD important role in gastrointestinal (GI) motility, secretion and detector and a FLD detector. Separation of the metabolites sensation. A 5-HT3 receptor antagonist, Alosetron, was was performed on a 4.6x150mm, 5um, Varian Pursuit Diphe approved for the management of severe diarrhea-predomi nyl column, protected by a 10um, C18 Metaguard Pursuit XR nant female irritable bowel syndrome (IBS) patients and a guard column as well as a 4.6x20 mm, 5um, C18 Sun Fire 5-HT4 receptor agonist, Tegaserod, was approved for the guard column connected in series. Mobile phase A consisted management of constipation-predominant IBS patients. Due of a 10 mM sodium acetate solution, buffered to pH 4.0 with to the prominent role 5-HT plays in IBS, analysis of 5-HT and glacial acetic acid and mobile phase B was 12% methanol by its metabolites in serum may lead to the identification of volume in mobile phase A. The HPLC analysis was carried biologically relevant biomarkers that can be used for accurate out at room temperature by using a gradient that changed diagnosis of IBS. For this purpose, high performance liquid from 100% mobile phase A to 100% mobile phase B in 20 min chromatography (HPLC) has been employed to analyze at a flow-rate of 1 mL/min and then stayed at 100% mobile serum metabolites from IBS patients and healthy control phase B for 35 min. At 55 min, the mobile phase was changed (HC) subjects, looking for novel biomarkers. Indeed, several from 100% B to 100% A in 1 min and maintained at 100% A potential biomarkers were discovered, which exhibited dif for the next 10 min in preparation for the next analysis. 5-HT ferent concentrations in IBS patients versus HC subjects. In as well as other indole-containing molecules were detected order to identify those novel biomarkers, a large Scale extrac by using the FLD detector set at an excitation wave-length of US 2012/03 15630 A1 Dec. 13, 2012 59
225 nm and emission wave-length of 342 nm. Kynurenine Example 8 and other metabolites were detected by using the DAD detec tor with the UV wave-length set at 278 and 360 nm, respec TNFC. Treatment Results in Increased Release of the tively. Tryptophan Metabolite, Kynurenin, and the 5-HT Metabolite, 5-HT-O-SO3H. IL-1 (B Treatment 0558 Large Scale Isolation and Identification of the Increases PGE and TNFC. and TIMP1 Treatment Metabolites: To establish the chemical structure of the biom Elevates Urocortin Levels in Whole Blood Cells arker peaks identified in the HPLC analysis, a large scale purification of a pooled serum sample was performed by 3D 0561. Irritable bowel syndrome (IBS) is a multi-factorial BioCptima and the structure of the isolated metabolite mark functional gastrointestinal (GI) disorder. Inflammatory ers was determined by mass spectrometry. stimulation in local gut mucosa has been implicated as one trigger for IBS disease onset. Pathways involved with sero 0559 FIG. 22 shows the representative chromatograms tonin (5-HT) dysregulation, mast cell activation and stress obtained from the HPLC analysis of a serum sample from an response all contributed to the pathogenesis of the disease. In IBS patient (FIG. 22, panels a, b and c) and a healthy control an attempt to find biomarkers for the diagnosis of IBS, the Subject (panels d, e and f). The peaks in panels a and d were interplay of these pathways has been studied in inflammatory detected by UV absorbance at 278 nm, while the peaks in cytokine treated whole blood samples from healthy volun panels band e were detected by UV absorbance at 360 nm. teers. Changes in 5-HT metabolism and levels of mast cell The peaks in panels c and f were monitored by fluorescence activation and stress response markers were determined. detection. As seen in FIG.22, three prominent peaks (denoted Inflammatory cytokine stimulation was found to alter tryp in boxes), eluting at 7.5, 13.5 and 17.2 minutes showed a tophan and 5-HT metabolism. Plasma kynurenine and 5-HT difference in peak area between the IBS and HC serum O-SO3H levels were up-regulated by TNFC. treatment. IL-1B samples. Difference in peak area between IBS and HC serum stimulation resulted in increased level of PGE, which is a samples was also noted at other eluted peaks but they were not marker for mast cell activation. Measurement of 5-HT bio as prominent. Due to the significant difference in the 7.5, 13.5 synthesis enzyme (TPH1) and metabolism enzyme (MAO) and 17.2 minute peak areas between IBS and HC subjects, all mRNAs levels indicated that the transcripts of these enzymes of the collected serum samples were evaluated by the HPLC were affected by cytokine treatment. Thus, understanding the method. FIG. 23 shows the statistical distribution of the three interactions between these pathways could help us to identify peak levels in the serum samples obtained from the IBS-C, biological relevant markers to assist the clinical diagnosis of IBS-D, IBS-M and HC subjects. As seen in FIG. 23, serum IBS. levels of the 17.2 minute peak were found to be higher in HC 0562 Blood Sample Collection and Treatment: Fresh subjects compared to IBS patients, whereas IBS patient sub human blood samples from healthy volunteers were collected types have varied levels in the 7.5 and 13.5 minute peaks. The into heparin tubes. The collected samples were aliquoted into peak eluting at 17.2 minute was readily identified as 5-HT by seven 15 mL Colloquial tubes and treated with inflammatory co-elution with a spiked in 5-HT standard in the HPLC analy cytokines, TNF-C. IL-1B, TIMP1 (tissue inhibitor of metal sis but the identity of the 7.5 and 13.5 minute peaks was not loproteinases-1), NGAL (neutrophil gelatinase-associated known. In order to establish the chemical structure of the lipocalin) for 2 day at 37°C. in a cell culture incubator. these two peaks, a large scale purification of a pooled serum 0563. Measurement of Metabolites: The product from sample was performed to isolate the compounds present in each 300 u, of treated plasma sample was extracted with these two peaks and determine their structure by mass spec isopropanol and acetonitrile and the extract analyzed by using trometry. FIG. 24 shows the structure elucidation of the 7.5 a diphenyl column on an Agilent HPLC system. Separation of minute peak by mass spectrometry to establish its identity as the metabolites was performed with a linear gradient from the amino acid tyrosine and FIG. 25 shows the structure 0-12% methanol in 10 mM sodium acetate buffer, pH 4.0, for elucidation of the 13.5 minute peak by mass spectrometry to 20 min, followed by an isocratic run at 12% methanol in the establish its identity as the amino acid phenylalanine FIG. 26 same buffer for 35 minutes. Indole metabolites and 5-HT shows the HPLC analysis results from the spiking of standard were detected using the FLD detector set at an excitation D-tyrosine to the serum samples to generate a linear dose wave length of 225 nm and emission wave length of 342 nm. dependent response curve and FIG. 27 shows the HPLC Tryptophan and Kynurenine were detected with the UV set at analysis results from the spiking of standard L-phenylalanine wave lengths of 278 nm and 360 nm, respectively. to the serum samples to generate a linear dose-dependent 0564). MAO, TPH1 and SERT Expression Profiling: Ali response curve. quots of 2.4 mL from each of the treated blood samples were 0560. In this study, an HPLC method has been developed added into PAXgen tubes and total RNA was prepared to analyze the metabolites present in serum samples obtained according to manufacturer's protocol. Aliquots of 200ng total from IBS patients and healthy control subjects, looking for RNA were reversely transcribed into cDNA and 200ng of the potential biomarkers for the diagnosis of IBS. Three promi resulting cDNA was used for quantification by qPCR reaction nent markers were discovered, which corresponded to (ABI 7000 systems). tyrosine, phenylalanine and serotonin. Serum levels of sero 0565 Measurement of Immune Markers: Tryptase and tonin was found to be higher in healthy control subjects Ucn (urocortin) were measured using assays developed by compared to IBS patients, whereas IBS patient subtypes have Prometheus. PGE, histamine, and 5-HT were measured varied levels of tyrosine and phenylalanine Measurement of using the commercially available ELISA assays. tyrosine and phenylalanine levels, in addition to serotonin 0566 FIG. 28 shows the 5-HT pathway and its participa level, is relevant to IBS diagnosis since these two amino acids tion in the pathophysiology of IBS. A 5-HT3 receptor antago are precursors for the biosynthesis of dopamine, norepineph nist, Lotronex, was approved for the management of severe rine and epinephrine, which are important regulators of the diarrhea-predominant female IBS patients because it blocks enteric nervous system. the activation of the 5-HT3 receptors that results in stimula US 2012/03 15630 A1 Dec. 13, 2012 60 tion of gut secretion, propulsion and pain. Table 10 shows the effects of cytokine treatment on the levels of the 5-HT path TABLE 11-continued way metabolites in whole blood cells while FIG.29 shows the effects of cytokine treatment on the transcript levels of 5-HT Levels of mast cell activation markers produced pathway metabolic enzymes in whole blood cells. As seen in by cytokine treatment of whole blood cells. Table 10, TNFC. treatment greatly increased the relative levels Tryptase PGE, Histamine of the metabolites kynurenine and 5-HT-O-SO3H, indicating Cytokine Treatment (ng/mL) (pg/mL) (pg/mL) that this cytokine stimulated tryptophan and 5-HT metabo lism, which resulted in the increase of kynurenine and 5-HT NGAL 2O3 + 1 1700.3 23.6 2655 - 20.0 O-SO3H synthesis. No distinct effects on 5-HT pathway TIMP1 21.2 - 0.27 1818.5 - 1851 261.6 30.5 metabolites were noted by treatment with IL-1B and TIMP1. TNFC 20.4 2.O 2394.3 83.4 208.8 28.9 0567 By contrast, TNFC. treatment of whole blood cells inhibited the transcript of MAObut not TPH1, whereas IL-1 B 0568. This example demonstrates that TNFC. treatment treatment greatly increased the transcript level of TPH1 but resulted in increased release of the tryptophan metabolite, not that of MAO (see, FIG. 29). TIMP1 treatment also slightly kynurenin, and the 5-HT metabolite, 5-HT-O-SO3H, in increased the transcript level of TPH1 but inhibited the tran healthy human whole blood cells. Without being bound by script level of MAO whereas NGAL treatment did not seem to theory, the alteration seen in tryptophan and 5-HT metabo have much effect on these two transcripts. The transcript level lism could be caused by changes in the expression of the of SERT was not detectable in any of the treated cells. Table enzymes MAO and TPH1 in blood cells induced by the cytok 11 and FIG.30 show the levels of mast cell activation markers ine treatment. produced by cytokine treatment of whole blood cells. Treat 0569. As seen in FIG. 30, the level of the mast cell activa ment with IL-1B greatly increase the level of the marker tion marker PGE was greatly increased upon IL-1B treat PGE2, whereas no distinct changes in other markers levels ment of whole blood cells. Furthermore, as shown in FIG.31, were noted by treatment with NGAL, TIMP1 and TNFC. TNFC. and TIMP1 treatment also greatly elevated the level of FIG.31 shows the level of the stress response marker urocor the stress response marker urocortin in whole blood cells. tin (Ucn) produced by cytokine treatment of whole blood Further studies of the responses in cytokine treatment of IBS cells. Urocortin level was greatly elevated by treatment with patient blood cells may shed some light on the mechanistic TNFO, TIMP1 and NGAL but not by IL-1 B. FIG. 19 shows a interaction in the pathways that contribute to the pathogenesis diagram illustrating the interplay between leukocytes, entero of IBS. chromaffin cells, mast cells and platelets in the gastrointesti REFERENCES nal system mediated by some of the markers determined 0570) 1. Barbara G and Cremon C. Serine proteases: new herein. players in diarrhea-predominant irritable bowel syndrome. Gut. 2008 May: 57(5):591-9. TABLE 10 (0571) 2. Barbara G. Wang B, Stanghellini V. et al. Mast Cell-Dependent Excitation of Visceral-Nociceptive Sen Levels of 5-HT pathway metabolites produced sory Neurons in Irritable Bowel Syndrome. Gastroenterol by cytokine treatment of whole blood cells. ogy 2007; 132:26-37. Relative (0572. 3. Foley K F. Pantano C, Ciolino A, Mawe G. M. Cytokine Relative Relative 5-HT-O-SOH IFN-Y and TNF-C. decrease serotonin transporter function Treatment Tryptophan Level Kynurenine Level Level and expression in Caco2 cells. Am J Physiol Gastrointest Untreated 2,851.90 17.10 56.50 Liver Physiol, 292: G779-G784, 2007. IL-1B 3,865.96 S4.90 86.70 (0573 4. Burczynski M. E. Peterson R L., Twine N C, TIMP1 3,903.20 71.10 85.00 Zuberek KA, Brodeur B.J. Casciotti L, Maganti V. Reddy TNFC 3,471.10 147.56 124.67 PS, Strahs A, Immermann F. Spinelli W. Schwertschlag U. Slager AM, Cotreau MM, Dorner A.J. Molecular classi fication of Crohn's disease and ulcerative colitis patients using transcriptional profiles in peripheral blood mono TABLE 11 nuclear cells. J Mol Diagn, 8:51-61, 2006. Levels of mast cell activation markers produced 0574 Although the foregoing invention has been by cytokine treatment of whole blood cells. described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art Tryptase PGE, Histamine will appreciate that certain changes and modifications may be Cytokine Treatment (ng/mL) (pg/mL) (pg/mL) practiced within the scope of the appended claims. In addi Untreated 19.81.1 1621.7 - 89.1 294.9 43.3 tion, each reference provided herein is incorporated by refer IL-1B 20.7 - 0.74 5588.6 S60.8 257.335.3 ence in its entirety to the same extent as if each reference was individually incorporated by reference.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS : 17
<21 Os SEQ ID NO 1 &211s LENGTH: 37 US 2012/03 15630 A1 Dec. 13, 2012 61
- Continued
212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human alpha- calcitonin gene-related peptide (alpha-CGRP) 22 Os. FEATURE: <221s NAME/KEY: AMIDATION <222s. LOCATION: (37) . . . (37 <223> OTHER INFORMATION: phenylalaninamide
<4 OOs, SEQUENCE: 1 Ala Cys Asp Thir Ala Thr Cys Val Thir His Arg Lieu Ala Gly Lieu. Lieu 1. 5 1O 15 Ser Arg Ser Gly Gly Val Val Lys Asn Asin Phe Val Pro Thr Asn Val 2O 25 3O Gly Ser Lys Ala Phe 35
<210s, SEQ ID NO 2 &211s LENGTH: 37 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human beta- calcitonin gene-related peptide (beta- CGRP) 22 Os. FEATURE: <221s NAME/KEY: AMIDATION <222s. LOCATION: (37) . . . (37 <223> OTHER INFORMATION: phenylalaninamide <4 OOs, SEQUENCE: 2 Ala Cys Asn. Thir Ala Thr Cys Val Thir His Arg Lieu Ala Gly Lieu. Lieu 1. 5 1O 15 Ser Arg Ser Gly Gly Met Val Lys Ser Asin Phe Val Pro Thr Asn Val 2O 25 3O Gly Ser Lys Ala Phe 35
<210s, SEQ ID NO 3 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human Substance P peptide 22 Os. FEATURE: <221s NAME/KEY: AMIDATION <222s. LOCATION: (11) . . . (11) <223> OTHER INFORMATION: methioninamide
<4 OOs, SEQUENCE: 3 Arg Pro Llys Pro Glin Glin Phe Phe Gly Lieu Met 1. 5 1O
<210s, SEQ ID NO 4 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human neurokinin A peptide <4 OOs, SEQUENCE: 4 His Llys Thr Asp Ser Phe Val Gly Lieu Met 1. 5 1O US 2012/03 15630 A1 Dec. 13, 2012 62
- Continued <210s, SEQ ID NO 5 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human neurokinin B peptide 22 Os. FEATURE: <221s NAME/KEY: AMIDATION <222s. LOCATION: (10) . . . (10) <223> OTHER INFORMATION: methioninamide
<4 OOs, SEQUENCE: 5 Asp Met His Asp Phe Phe Val Gly Lieu Met 1. 5 1O
<210s, SEQ ID NO 6 &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human vasoactive intestinal peptide (VIP) <4 OOs, SEQUENCE: 6 His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Lieu. Arg Lys Glin 1. 5 1O 15 Met Ala Wall Lys Llys Tyr Lieu. Asn. Ser Ile Lieu. Asn 2O 25
<210s, SEQ ID NO 7 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human glucagon-like peptide 2 (GLP-2) <4 OO > SEQUENCE: 7 His Ala Asp Gly Ser Phe Ser Asp Glu Met Asn. Thir Ile Lieu. Asp Asn 1. 5 1O 15 Lieu Ala Ala Arg Asp Phe Ile Asn Trp Lieu. Ile Glin Thr Lys Ile Thr 2O 25 3O Asp
<210s, SEQ ID NO 8 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human motilin peptide <4 OOs, SEQUENCE: 8 Phe Val Pro Ile Phe Thr Tyr Gly Glu Lieu. Glin Arg Met Glin Glu Lys 1. 5 1O 15 Glu Arg Asn Lys Gly Glin 2O
<210s, SEQ ID NO 9 &211s LENGTH: 48 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human pituitary adenylate cyclase-activating peptide (PACAP) - related peptide, PACAP propeptide
<4 OOs, SEQUENCE: 9 US 2012/03 15630 A1 Dec. 13, 2012 63
- Continued
Asp Wall Ala His Gly Ile Lieu. Asn. Glu Ala Tyr Arg Llys Val Lieu. Asp 1. 5 1O 15 Glin Lieu. Ser Ala Gly Llys His Lieu. Glin Ser Lieu Val Ala Arg Gly Val 2O 25 3O Gly Gly Ser Lieu. Gly Gly Gly Ala Gly Asp Asp Ala Glu Pro Lieu. Ser 35 4 O 45
<210s, SEQ ID NO 10 &211s LENGTH: 38 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human pituitary adenylate cyclase-activating polypeptide 38, PACAP propeptide
<4 OOs, SEQUENCE: 10 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Glin 1. 5 1O 15 Met Ala Wall Lys Llys Tyr Lieu Ala Ala Val Lieu. Gly Lys Arg Tyr Lys 2O 25 3O Glin Arg Val Lys Asn Llys 35
<210s, SEQ ID NO 11 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human pituitary adenylate cyclase-activating polypeptide 27, PACAP propeptide
<4 OOs, SEQUENCE: 11 His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Glin 1. 5 1O 15 Met Ala Wall Lys Llys Tyr Lieu Ala Ala Val Lieu 2O 25
<210s, SEQ ID NO 12 &211s LENGTH: 41 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human corticotropin-releasing hormone (CRH, CRF) peptide
<4 OOs, SEQUENCE: 12 Ser Glu Glu Pro Pro Ile Ser Lieu. Asp Lieu. Thir Phe His Lieu. Lieu. Arg 1. 5 1O 15 Glu Val Lieu. Glu Met Ala Arg Ala Glu Gln Lieu Ala Glin Glin Ala His 2O 25 3O Ser Asn Arg Llys Lieu Met Glu Ile Ile 35 4 O
<210s, SEQ ID NO 13 &211s LENGTH: 4 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human urocortin 1 (Ucn)
<4 OOs, SEQUENCE: 13 US 2012/03 15630 A1 Dec. 13, 2012 64
- Continued
Asp ASn Pro Ser Lieu. Ser Ile Asp Lieu. Thir Phe His Lieu. Lieu. Arg Thr 1. 5 1O 15
Lieu. Lieu. Glu Lieu Ala Arg Thr Glin Ser Glin Arg Glu Arg Ala Glu Glin 2O 25 3O
Asn Arg Ile Ile Phe Asp Ser Val 35 4 O
<210s, SEQ ID NO 14 &211s LENGTH: 39 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human adrenocorticotropic hormone (ACTH, corticotrophin)
<4 OOs, SEQUENCE: 14 Ser Tyr Ser Met Glu. His Phe Arg Trp Gly Llys Pro Val Gly Lys Lys 1. 5 1O 15
Arg Arg Pro Val Llys Val Tyr Pro Asn Gly Ala Glu Asp Glu Ser Ala 2O 25 3O
Glu Ala Phe Pro Leu Glu Phe 35
<210s, SEQ ID NO 15 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human fibrinopeptide A, N-terminal region of fibrinogen alpha chain (FGA)
<4 OOs, SEQUENCE: 15 Ala Asp Ser Gly Glu Gly Asp Phe Lieu Ala Glu Gly Gly Gly Val Arg 1. 5 1O 15
<210s, SEQ ID NO 16 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: human fibrinopeptide B, N-terminal region of fibrinogen beta chain (FGB)
<4 OOs, SEQUENCE: 16
Glu Gly Val Asn Asp Asn. Glu Glu Gly Phe Phe Ser Ala Arg 1. 5 1O
<210s, SEQ ID NO 17 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic amidated neuropeptide tachykinin shared carboxy-terminal sequence 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: Xaa = any amino acid US 2012/03 15630 A1 Dec. 13, 2012
- Continued
22 Os. FEATURE: <221s NAME/KEY: AMIDATION <222s. LOCATION: (5) . . . (5) <223> OTHER INFORMATION: methioninamide
<4 OOs, SEQUENCE: 17 Phe Xaa Gly Lieu Met 1. 5
1. A method for aiding in the diagnosis of irritable bowel to the control level or concentration is indicative of an syndrome (IBS) in a Subject, said method comprising: increased likelihood of said subject not having IBS. (a) contacting a blood or serum sample from the Subject 9-38. (canceled) with an IBS marker binding moiety under conditions 39. The method of claim 1, wherein the method further suitable to transform the IBS marker present in the comprises determining the level or concentration of at least sample into a complex comprising the IBS marker and one additional biomarker in a biological sample from the the IBS marker binding moiety; and Subject, the biomarker selected from the group consisting of (b) determining the level of said complex, thereby deter Brain-Derived Neurotropic Factor (BDNF), Neutrophil mining the level or concentration of the IBS marker Gelatinase-Associated Lipocalin (NGAL). TNF-related present in the sample, Weak Inducer of Apoptosis (TWEAK), Growth-Related wherein the IBS marker is selected from the group consist Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-13), Tis ing of carbohydrate deficient transferrin, urocortin, cor Sue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Saccha ticotrophin-releasing hormone-binding protein, corti romyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 Sol, adrenocorticotropic hormone, Substance P. nerve Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic growth factor, neurokinin A, neurokinin B. vasoactive Antibody (ANCA), Anti-Human Tissue Transglutaminase intestinal peptide, glucagon-like peptide 2, motilin, pitu IgA (tTG), histamine, B-tryptase, prostaglandin E2 (PGE), itary adenylate cyclase-activating peptide, serotonin, and a combination thereof. tryptophan, serotonin O-sulfate, 5-hydroxyindoleacetic 40. The method of claim 1, wherein the method further acid, 5-HT glucuronide, tyrosine, phenylalanine, UDP comprises: glucuronosyltransferase 1-6, Serotonin reuptake trans (d) determining a symptom profile for the Subject, wherein porter, tryptophan hydroxylase 1, monoamine oxidase said symptom profile is determined by identifying the A monoamine oxidase B, and hydroxytryptamine (sero presence or severity of at least one symptom in said tonin) receptor 3A. Subject; and 2. The method of claim 1, wherein the method further (e) diagnosing the Subject as having IBS or not having IBS comprises: using an algorithm based upon the level of an IBS biom (c) comparing the level or concentration of the IBS marker arker and the symptom profile. present in the sample to a control level or concentration, 41. (canceled) wherein a difference in the level or concentration of the 42. (canceled) IBS marker present in the sample relative to the control 43. The method of claim 1 wherein the method comprises level or concentration is indicative of an increased like the use of an algorithm based upon the level of an IBS biom lihood of said subject having IBS. arker. 3. The method of claim 2, wherein the control level or 44-50. (canceled) concentration is the level or concentration of the IBS marker 51. A method for monitoring the progression or regression present in a blood or serum sample from a healthy Subject. of irritable bowel syndrome (IBS) in a subject, the method 4. The method of claim 3, wherein an increased level or comprising: concentration of the IBS marker present in the sample relative (a) contacting a first blood or serum sample taken from the to the control level or concentration is indicative of an subject at a first time with an IBS marker binding moiety increased likelihood of said subject having IBS. under conditions suitable to transform the IBS marker 5. The method of claim 3, wherein the same or a reduced present in the sample into a complex comprising the IBS level or concentration of the IBS marker present in the sample marker and the IBS marker binding moiety; relative to the control level or concentration is indicative of an (b) determining the level of said complex, thereby deter increased likelihood of said subject not having IBS. mining the level of the IBS marker present in the first 6. The method of claim 2, wherein the control level or sample: concentration is the level or concentration of the IBS marker (c) contacting a second blood or serum sample taken from present in a blood or serum sample from a subject with IBS. the subjectata second time with the IBS marker binding 7. The method of claim 6, wherein the same oran increased moiety under conditions suitable to transform the IBS level or concentration of the IBS marker present in the sample marker present in the sample into a complex comprising relative to the control level or concentration is indicative of an the IBS marker and the IBS marker binding moiety; increased likelihood of said subject having IBS. (d) determining the level of said complex, thereby deter 8. The method of claim 6, wherein a reduced level or mining the level of the IBS marker present in the second concentration of the IBS marker present in the sample relative sample; and US 2012/03 15630 A1 Dec. 13, 2012 66
(e) comparing the level of the IBS marker present in the tide, serotonin, tryptophan, serotonin O-sulfate, 5-hy first sample to the level of the IBS marker present in the droxyindoleacetic acid, 5-HT glucuronide, tyrosine, second sample, phenylalanine, UDP-glucuronosyltransferase 1-6, Sero wherein a higher level of the IBS marker in the second tonin reuptake transporter, tryptophan hydroxylase 1, sample relative to the first sample is indicative of the monoamine oxidase A, monoamine oxidase B, and progression of IBS in the subject and a lower level of the hydroxytryptamine (serotonin) receptor 3A. IBS marker in the second sample relative to the first 92-117. (canceled) sample is indicative of the regression of IBS in the sub 118. The computer-readable medium of claim 91, wherein ject, and the diagnostic marker profile indicates the level of at least one wherein the IBS marker is selected from the group consist additional diagnostic marker in a biological sample from the ing of carbohydrate deficient transferrin, urocortin, cor subject selected from the group consisting of Brain-Derived ticotrophin-releasing hormone-binding protein, corti Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ Sol, adrenocorticotropic hormone, Substance P. nerve ated Lipocalin (NGAL), TNF-related Weak Inducer of Apo growth factor, neurokinin A, neurokinin B. vasoactive ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO intestinal peptide, glucagon-like peptide 2, motilin, pitu O), Interleukin-1 Beta (IL-f3), Tissue Inhibitor of itary adenylate cyclase-activating peptide, serotonin, Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi tryptophan, serotonin O-sulfate, 5-hydroxyindoleacetic siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), acid, 5-HT glucuronide, tyrosine, phenylalanine, UDP Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), glucuronosyltransferase 1-6, Serotonin reuptake trans Anti-Human Tissue Transglutaminase IgA (tTG), histamine, porter, tryptophan hydroxylase 1, monoamine oxidase B-tryptase, prostaglandin E2 (PGE2), and a combination A monoamine oxidase B, and hydroxytryptamine (sero thereof. tonin) receptor 3A. 119. The computer-readable medium of claim 91, wherein 52-80. (canceled) said computer-readable medium comprises instructions to 81. The method of claim 51, wherein the method further apply a statistical process to a data set comprising said diag comprises determining the level of at least one additional nostic marker profile in combination with a symptom profile biomarker in a biological sample from the Subject, the biom which indicates the presence or severity of at least one symp arker selected from the group consisting of Brain-Derived tom in said Subject to produce a statistically derived decision Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ classifying said sample as an IBS sample or non-IBS sample ated Lipocalin (NGAL). TNF-related Weak Inducer of Apo based upon said diagnostic marker profile and said symptom ptosis (TWEAK), Growth-Related Oncogene Alpha (GRO profile. O), Interleukin-1 Beta (IL-13), Tissue Inhibitor of 120-122. (canceled) Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi 123. A system for classifying whether a serum or blood siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), sample from a subject is associated with irritable bowel syn Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), drome (IBS), said system comprising: Anti-Human Tissue Transglutaminase IgA (tTG), histamine, (a) a data acquisition module configured to produce a data B-tryptase, prostaglandin E2 (PGE2), and a combination set comprising a diagnostic marker profile, wherein said thereof. diagnostic marker profile indicates the presence or level 82. The method of claim 51, wherein the method further of at least one diagnostic IBS marker selected from the comprises determining a symptom profile for the Subject, group consisting of carbohydrate deficient transferrin, wherein said symptom profile is determined by identifying urocortin, corticotrophin-releasing hormone-binding the presence or severity of at least one symptom in said protein, cortisol, adrenocorticotropic hormone, Sub Subject. stance P. nerve growth factor, neurokinin A, neurokinin 83. (canceled) B. vasoactive intestinal peptide, glucagon-like peptide 84. The method of claim 51, wherein the method comprises 2, motilin, pituitary adenylate cyclase-activating pep the use of an algorithm based upon the level of an IBS biom tide, serotonin, tryptophan, serotonin O-sulfate, 5-hy arker. droxyindoleacetic acid, 5-HT glucuronide, tyrosine, 85-90. (canceled) phenylalanine, UDP-glucuronosyltransferase 1-6, Sero 91. A computer-readable medium comprising code for tonin reuptake transporter, tryptophan hydroxylase 1, controlling one or more processors to classify whether a monoamine oxidase A, monoamine oxidase B, and serum or blood sample from an Subject is associated with hydroxytryptamine (serotonin) receptor 3A. irritable bowel syndrome (IBS), said code comprising: (b) a data processing module configured to process the data instructions to apply a statistical process to a data set com set by applying a statistical process to the data set to prising a diagnostic marker profile to produce a statisti produce a statistically derived decision classifying said cally derived decision classifying said sample as an IBS sample as an IBS sample or non-IBS sample based upon sample or non-IBS sample based upon said diagnostic said diagnostic marker profile; and marker profile, (c) a display module configured to display the statistically wherein said diagnostic marker profile indicates the level derived decision. of at least one diagnostic IBS marker selected from the 124-149. (canceled) group consisting of carbohydrate deficient transferrin, 150. The system of claim 123, wherein the diagnostic urocortin, corticotrophin-releasing hormone-binding marker profile indicates the level of at least one additional protein, cortisol, adrenocorticotropic hormone, Sub diagnostic marker in a biological sample from the Subject stance P. nerve growth factor, neurokinin A, neurokinin selected from the group consisting of Brain-Derived Neuro B. vasoactive intestinal peptide, glucagon-like peptide tropic Factor (BDNF), Neutrophil Gelatinase-Associated 2, motilin, pituitary adenylate cyclase-activating pep Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis US 2012/03 15630 A1 Dec. 13, 2012 67
(TWEAK), Growth-Related Oncogene Alpha (GRO-O.), a data set comprising said diagnostic marker profile in com Interleukin-1 Beta (IL-B), Tissue Inhibitor of Metalloprotein bination with a symptom profile which indicates the presence ase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Antibody or severity of at least one symptom in said Subject to produce (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti-Human a statistically derived decision classifying said sample as an Neutrophil Cytoplasmic Antibody (ANCA), Anti-Human IBS sample or non-IBS sample based upon said diagnostic Tissue Transglutaminase IgA (tTG), histamine, B-tryptase, marker profile and said symptom profile. prostaglandin E2 (PGE2), and a combination thereof. 152-154. (canceled) 151. The system of claim 123, wherein said data processing module comprises instructions to apply a statistical process to c c c c c