Patent Grant
10544459
This invention relates to methods useful in the medical arts. In particular, various embodiments of the present invention relate to methods for diagnosis and treatment of Inflammatory Bowel Disease (IBD).
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Crohn's disease (CD) and ulcerative colitis (UC), collectively referred to as inflammatory bowel disease (IBD), are relatively common inflammatory diseases of the gastrointestinal (GI) tract, which are chronic, relapsing inflammatory disorders. Histopathologically and anatomically, these two conditions are distinct, with CD characterized by transmural inflammation that can occur throughout the GI tract, and UC characterized by more superficial inflammation confined to the colon and rectum. Interestingly, both diseases are dependent upon factors present within the complex intestinal microbiota. Indeed, a unifying hypothesis has emerged that proposes that IBD results from a dysregulated mucosal immune response to the intestinal microbiota in genetically susceptible individuals (Strober W, Fuss I J, Blumberg R S. The immunology of mucosal models of inflammation. Annu Rev. Immunol. 2002; 20:495-549. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 2003; 3:521-533.).
While the dependence of IBD on intestinal microbes is increasingly clear, the molecular mechanisms underlying this dependence are not. The intestinal mucosa is exposed to the largest concentration of foreign bacterial antigens of any tissue in the body, estimated to be up to 1012 organisms per gram of stool in the normal colon. An emerging concept is that there is an active “dialogue” between the microbiota, intestinal epithelial cells, and mucosal immune cells, with each partner communicating with the others (McCracken V J, Lorenz R G. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. Cell. Microbiol. 2001; 3:1-11.). In this context, “innate” immune responses, which recognize conserved microbial products such as lipopolysaccharide (LPS) and peptidoglycan (PG), are likely to be important in these microbial-host interactions and intestinal homeostasis. Critical to the host's “sensing” of microbes are members of the Toll-like receptor (TLR) family that, alone or in combination, recognize a wide array of microbe-associated molecular patterns on either pathogens or commensals (Kopp E, Medzhitov R. Recognition of microbial infection by Toll-like receptors. Curr. Opin. Immunol. 2003; 15:396-401. Akira S. Mammalian Toll-like receptors. Curr. Opin. Immunol. 2003; 15:5-11. Sieling P A, Modlin R L. Toll-like receptors: mammalian ‘taste receptors’ for a smorgasbord of microbial invaders. Current Opin. Microbiol. 2002; 5:70-75.). Various TLRs are expressed on intestinal epithelial cells (Cario E, Podolsky D K. Differential alteration in intestinal epithelial cell expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect. Immunol. 2000; 68:7010-7017. Gewirtz A T, Navas T A, Lyons S, Godowski P J, Madara J L. Cutting Edge: Bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 2001; 167:1882-1885. Abreu M T, et al. TLR4 and MD-2 expression is regulated by immune-mediated signals in human intestinal epithelial cells. J. Biol. Chem. 2002; 277:20431-20437. Hershberg R M. The epithelial cell cytoskeleton and intracellular trafficking V. Polarized compartmentalization of antigen processing and Toll-like receptor signaling in intestinal epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 2002; 283: G833-G839.) and more broadly on macrophages and dendritic cells in the lamina propria.
Given the involvement of innate immune mechanisms in the modulation of T cell responses, the bacterial dependence of IBD is likely to involve both bacterial products such as LPS, PG, and other TLR ligands, and specific bacterial antigens capable of stimulating CD4+ T cell responses. CD4+ T lymphocytes have been identified as the crucial effector cells in experimental models of IBD (Berg D J, et al. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4+ TH1-like responses. J. Clin. Invest. 1996; 98:1010-1020. Powrie F, et al Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity. 1994; 1:553-562. Cong Y, et al. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell Type 1 response and ability to transfer disease. J. Exp. Med. 1998; 187:855-864.), and these pathogenic CD4+ T cell responses are directed against the enteric microbiota. Enteric bacterial antigen-reactive CD4+ T cells are able to induce colitis when adoptively transferred into immunodeficient recipients (Cong Y, et al. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell Type 1 response and ability to transfer disease. J. Exp. Med. 1998; 187:855-864.). The in vitro data suggest that there is a relatively small number of immunodominant antigens that stimulate the pathogenic T cell responses (Brandwein S L, et al. Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora. J. Immunol. 1997; 159:44-52), but the complexity of the intestinal microflora has posed a significant challenge to their identification.
In humans, specific associations between particular bacterial species and the development of disease or its characteristics have not been established. Immune responses to commensal enteric organisms have been investigated in CD. It was been shown that CD patients have antibodies to specific bacterial antigens and that patients can be clustered into 4 groups depending on their antibody response patterns (Landers C J, Cohavy O, Misra R, Yang H, Lin Y C, Braun J, Targan S R. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123:689-99.). These clusters are (1) antibody responses against oligomannan (anti-Saccharomyces cerevisiae; ASCA), (2) antibody responses to both Escherichia coli outer membrane protein C (anti-OmpC) and a CD-related protein from Pseudomonas fluorescens (anti-CD-related bacterial sequence {I2}), (3) antibody responses to nuclear antigens (perinuclear antineutrophil cytoplasmic antibody; pANCA), or (4) low or no serological response to any of the tested antigens. These distinct antibody response patterns may indicate unique pathophysiological mechanisms in the progression of this complicated disease. In addition, phenotypic associations with specific serological response patterns have been discovered (Landers C J, Cohavy O, Misra R, Yang H, Lin Y C, Braun J, Targan S R. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123:689-99. Vasiliauskas E A, Plevy S E, Landers C J, Binder S W, Ferguson D M, Yang H, Rotter J I, Vidrich A, Targan S R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology 1996; 110:1810-9. Vasiliauskas E A, Kam L Y, Karp L C, Gaiennie J, Yang H, Targan S R. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics. Gut 2000; 47:487-96. Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24.).
Immunologic responses to bacterial products are key to the induction of inflammatory bowel disease in humans and in experimental models. The relationship of these immune responses to the underlying genetic and clinical phenotypes is just beginning to emerge. Thus, among patients with Crohn's disease, immune responses to different microbial antigens may be related to different pathophysiologic mechanisms, and may represent distinct genotypes and phenotypes.
Thus, there is need in the art to associate clinical phenotypes of Crohn's disease with various antigens, as such determination can enable more appropriate treatments for the disease. Furthermore, there exists a need for the diagnosis and treatment of Crohn's disease and subtypes of Crohn's disease.
The two common forms of IBD, CD and UC, are chronic, relapsing inflammatory disorders of the gastrointestinal tract. Each has a peak age of onset in the second to fourth decades of life and prevalences in European ancestry populations that average approximately 100-150 per 100,000 (D. K. Podolsky, N Engl J Med 347, 417 (2002); E. V. Loftus, Jr., Gastroenterology 126, 1504 (2004)). Although the precise etiology of IBD remains to be elucidated, a widely accepted hypothesis is that ubiquitous, commensal intestinal bacteria trigger an inappropriate, overactive, and ongoing mucosal immune response that mediates intestinal tissue damage in genetically susceptible individuals (D. K. Podolsky, N Engl J Med 347, 417 (2002). Genetic factors play an important role in IBD pathogenesis, as evidenced by the increased rates of IBD in Ashkenazi Jews, familial aggregation of IBD, and increased concordance for IBD in monozygotic compared to dizygotic twin pairs (S. Vermeire, P. Rutgeerts, Genes Immun 6, 637 (2005)). Moreover, genetic analyses have linked IBD to specific genetic variants, especially CARD15 variants on chromosome 16q12 and the IBD5 haplotype (spanning the organic cation transporters, SLC22A4 and SLC22A5, and other genes) on chromosome 5q31 (S. Vermeire, P. Rutgeerts, Genes Immun 6, 637 (2005); J. P. Hugot et al., Nature 411, 599 (2001); Y. Ogura et al., Nature 411, 603 (2001); J. D. Rioux et al., Nat Genet 29, 223 (2001); V. D. Peltekova et al., Nat Genet 36, 471 (2004)). CD and UC are thought to be related disorders that share some genetic susceptibility loci but differ at others.
The replicated associations between CD and variants in CARD15 and the IBD5 haplotype do not fully explain the genetic risk for CD. Thus, there is need in the art to determine other markers, genes, allelic variants and/or haplotypes that may assist in explaining the genetic risk, predicting disease progression, diagnosing, and/or predicting susceptibility for or protection against inflammatory bowel disease including but not limited to CD and/or UC.
The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.
Various embodiments of the present invention provide for methods for diagnosing Crohn's disease in a mammal. Additional embodiments provide for determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease. Further embodiments provide for treating Crohn's disease. In one embodiment, the mammal is a human.
In particular embodiments, diagnosing Crohn's disease may be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 expression indicates that the mammal has Crohn's disease. Determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease may also be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 indicates that the mammal has small bowel disease, internal penetrating/perforating disease or fibrostenosing disease.
Determining the presence of anti-CBir1 expression may be accomplished by various techniques. For example, determining the presence of anti-CBir1 expression may be performed by determining the presence of an RNA sequence or a fragment of an RNA sequence that encodes an anti-CBir1 antibody; for example, using Northern blot analysis or reverse transcription-polymerase chain reaction (RT-PCR). Determining the presence of anti-CBir1 expression may also be performed by determining the presence of anti-CBir1 antibodies; for example IgG anti-CBir1. Anti-CBir1 antibodies are not limited to IgG, as IgA, IgM, IgD and IgE are also contemplated in connection with various embodiments of the present invention. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate means for determining the presence of anti-CBir1 expression.
Determining the presence of anti-CBir1 antibodies may be accomplished by a number of ways. For example, the determination may be made by an enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, and mass spectrometric analysis.
In other embodiments of the invention, an immune complex can be detected with a labeled secondary antibody, for example, that has specificity for a class determining portion of an anti-CBir1 antibody. A signal from a detectable secondary antibody can be analyzed, and positive results indicate the presence of anti-CBir1 antibodies.
Additional embodiments of the present invention provide for methods of treating Crohn's disease by the use of antigen-directed therapy. The target antigen in this therapy may be flagellin, and particularly CBir1 or an immunoreactive fragment thereof.
In other embodiments, methods are provided to define a subset of CD patients that may have colitic disease, and/or colitic and small bowel disease. Defining this subset of CD patients may be performed by determining the presence of anti-CBir1 expression and determining the presence of perinuclear antineutrophil cytoplasmic antibodies (pANCA), where the presence of both is diagnostic of Crohn's disease with properties of colitic disease and/or colitic and small bowel disease. Determination of the presence of pANCA may also be accomplished using ELISA, SDS-PAGE, Western blot analysis, or mass spectrometric analysis. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate means for determining the presence of pANCA.
Further embodiments of the present invention provide for methods of treating the subset of CD patients with colitic disease and/or colitic and small bowel disease. Treating colitic disease and/or colitic and small bowel disease may be performed by manipulating the bacterial flora in the colon and/or colon and small bowel. Manipulation of the bacterial flora may be performed by administering antibiotics and/or probiotics.
Samples useful in various embodiments of the present invention can be obtained from any biological fluid having antibodies or RNA sequences or fragments of RNA sequences; for example, whole blood, plasma, serum, saliva, or other bodily fluid or tissue. The sample used in connection with various embodiments of the present invention may be removed from the mammal; for example, from drawn blood, aspirated fluids, or biopsies. Alternatively, the sample may be in situ; for example a tool or device may be used to obtain a sample and perform a diagnosis while the tool or device is still in the mammal.
A CBir1 antigen, or immunoreactive fragment thereof, useful in the invention can be produced by any appropriate method for protein or peptide synthesis.
Other embodiments of the present invention use anti-idiotypic antibodies specific to the anti-CBir1 antibody or other antibody of interest.
The present invention is also directed to kits for diagnosing and/or treating Crohn's disease and/or subtypes of Crohn's disease. The exact nature of the components configured in the inventive kits depends on their intended purpose. For instance, a quantity of CBir1 antigen may be included in the kit for determining the presence of anti-CBir1 antibodies. Instructions for use may be included in the kit.
Various embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual, comprising determining the presence or absence of at least one risk variant at the NOD2 locus selected from the group consisting of R702W, G908R and 1007fs, and determining the presence or absence of at least one risk serological marker, where the presence of at least one risk variant and at least one risk serological marker is diagnostic of susceptibility to Crohn's Disease.
In other embodiments, the presence of three of the risk variants at the NOD2 locus present a greater susceptibility than the presence of two, one or none of the risk variants at the NOD2 locus, and the presence of two of the risk variants at the NOD2 locus presents a greater susceptibility than the presence of one or none of the risk variants at the NOD2 locus but less than the presence of three risk variants at the NOD2 locus, and the presence of one of the risk variants at the NOD2 locus presents a greater susceptibility than the presence of none of the risk variants at the NOD2 locus but less than the presence of three or two of the risk variants at the NOD2 locus.
In other embodiments, the risk serological markers are selected from the group consisting of ASCA, I2, OmpC and Cbir. In another embodiment, the presence of four of the risk serological markers presents a greater susceptibility than the presence of three or two or one or none of the risk serological markers, and the presence of three of the risk serological markers presents a greater susceptibility than the presence of two or one or none of the risk serological markers but less than the presence of four risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of four or three risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of four or three or two of the risk serological markers.
In another embodiment, the invention further comprises the step of determining the presence or absence of one or more risk haplotypes at the TLR8 locus, wherein the presence of one or more risk haplotypes at the TLR8 locus is diagnostic of susceptibility to Crohn's Disease.
In another embodiment, the invention comprises the step of determining the presence or absence of one or more risk haplotypes at the TLR2 locus, wherein the presence of one or more risk haplotypes at the TLR2 locus is diagnostic of susceptibility to Crohn's Disease.
Other various embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual comprising determining the presence or absence of one or more risk haplotypes at the TLR8 locus in the individual, where the presence of one or more risk haplotypes is diagnostic of susceptibility to Crohn's Disease. In other embodiments, the individual is a female. In another embodiment, the method further comprises determining the presence of H3.
Other various embodiments provide methods of determining a low probability relative to a healthy individual of developing Crohn's Disease and/or ulcerative colitis in an individual, the method comprising determining the presence or absence of one or more protective haplotypes at the TLR8 locus in the individual, where the presence of one or more said protective haplotypes is diagnostic of a low probability relative to a healthy individual of developing Crohn's Disease and/or ulcerative colitis. In other embodiments, the individual is a female. In other embodiments, the method further comprises determining the presence of H2.
Further embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual comprising determining the presence or absence of one or more risk variants at the TLR2 locus in the individual, where the presence of one or more risk variants is diagnostic of susceptibility to Crohn's Disease. In another embodiment, the individual is Jewish. In another embodiment, the invention further comprises determining the presence of P631H at the TLR2 locus.
Various embodiments provide methods of diagnosing susceptibility to a subtype of Crohn's Disease in a child, comprising determining the presence or absence of at least one risk variant at the CARD15 locus selected from the group consisting of SNP8, SNP12, and SNP13, and determining the presence or absence of at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, and ASCA, where the presence of at least one variant and at least one risk serological marker is diagnostic of susceptibility to the subtype of Crohn's Disease in a child. In another embodiment, the subtype of Crohn's Disease in a child comprises an aggressive complicating phenotype, a small bowel disease phenotype, and/or an internal penetrating and/or fibrostenosing disease phenotype. In another embodiment, the presence of three of the risk serological markers presents a greater susceptibility than the presence of two, one or none of the risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of three of the risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of three or two of the risk serological markers. In another embodiment, the SNP8 comprises SEQ ID NO: 18. In another embodiment, the SNP12 comprises SEQ ID NO: 19. And in another embodiment, the SNP13 comprises SEQ ID NO: 20.
Other embodiments provide for methods of diagnosing susceptibility to a subtype of Crohn's Disease in a child, comprising determining the presence or absence of a high immune reactivity relative to a healthy individual for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, I2, and pANCA, where the presence of a high immune reactivity relative to a healthy individual to at least one risk serological marker is diagnostic of susceptibility to the subtype of Crohn's Disease in a child. In another embodiment, the subtype of Crohn's Disease in a child comprises an aggressive complicating phenotype. In another embodiment, a high immune reactivity comprises a high magnitude of expression for the risk serological marker. In another embodiment, the presence of four of the risk serological markers presents a greater susceptibility than the presence of three, two, one or none of the risk serological markers, and the presence of three of the risk serological markers presents a greater susceptibility than the presence of two, one or none of the risk serological markers but less than the presence of four of the risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of four or three of the risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of four or three or two of the risk serological markers.
Various embodiments also provide methods of treating Crohn's Disease in a child, comprising determining the presence of a high immune reactivity to a risk serological marker relative to a healthy individual, and administering a therapeutically effective amount of Crohn's Disease treatment.
Other embodiments provide methods of diagnosing ulcerative colitis in an individual, comprising determining the presence or absence of a risk variant at the CARD8 locus, where the presence of the risk variant at the CARD8 locus is diagnostic of susceptibility to ulcerative colitis. In other embodiments, the risk variant at the CARD8 locus comprises SEQ ID NO: 36. In other embodiments, the individual is a child.
Various embodiments provide methods of determining the prognosis of Crohn's Disease in an individual, comprising determining the presence or absence of a high immune reactivity relative to a healthy individual for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, and pANCA, where the presence of a high immune reactivity relative to a healthy individual to at least one risk serological marker is indicative of a prognosis of an aggressive form of Crohn's Disease. In other embodiments, the individual is a child. In other embodiments, the prognosis of an aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Other embodiments provide methods of determining the prognosis of Crohn's Disease in a pediatric subject, comprising determining the presence or absence of a high immune reactivity of Cbir1, OmpC, ASCA, and pANCA in the pediatric subject relative to a child who has and maintains a non-aggressive form of Crohn's Disease, where the presence of the high immune reactivity relative to a child who has and maintains a non-aggressive Crohn's Disease is indicative of a prognosis of an aggressive form of Crohn's Disease in the pediatric subject. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or stricturing disease phenotype.
Other embodiments provide methods of treating an aggressive form of Crohn's Disease in a pediatric subject, comprising determining the presence of a high immune reactivity of Cbir1, OmpC, ASCA and pANCA relative to a child who has and maintains a non-aggressive form of Crohn's Disease to prognose the aggressive form of Crohn's Disease, and treating the aggressive form of Crohn's Disease.
Other embodiments provide methods of determining the prognosis of Crohn's Disease in a subject, comprising determining the presence or absence of a high immune reactivity in the subject relative to an individual who has and maintains a non-aggressive form of Crohn's Disease for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, and pANCA, where the presence of the high immune reactivity relative to an individual who has and maintains a non-aggressive form of Crohn's Disease is indicative of a prognosis of an aggressive form of Crohn's Disease. In other embodiments, the subject is a pediatric subject. In other embodiments, the individual who has and maintains a non-aggressive form of Crohn's Disease is a child. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Various embodiments also provide methods of treating an aggressive form of Crohn's Disease in a subject, comprising determining the presence of a high immune reactivity relative to an individual who has and maintains a non-aggressive form of Crohn's Disease to prognose the aggressive form of Crohn's Disease, and treating the aggressive form of Crohn's Disease. In other embodiments, the subject is a pediatric subject. In other embodiments, the individual who has and maintains a non-aggressive form of Crohn's Disease is a child. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Various embodiments include a method of diagnosing susceptibility to a subtype of Crohn's disease in an individual, comprising determining the presence or absence of one or more risk variants at the Janus kinases 3 (JAK3) genetic locus in the individual, and determining the presence or absence of a positive expression of ASCA and/or anti-I2, where the presence of one or more risk variants at the JAK3 locus and the presence of ASCA and/or anti-I2 expression is indicative of susceptibility in the individual to the subtype of Crohn's Disease. In another embodiment, one of the one or more risk variants at the JAK3 locus comprises SEQ ID NO: 37. In another embodiment, one of the one or more risk variants at the JAK3 locus comprises SEQ ID NO: 38. In another embodiment, positive expression of ASCA and/or anti-I2 comprises a high level of expression relative to a healthy subject.
Other embodiments include a method of diagnosing a subtype of Crohn's disease in an individual, comprising obtaining a sample from the individual, assaying the sample for the presence or absence of a risk variant at the Janus kinases 3 (JAK3) genetic locus in the individual, and diagnosing the subtype of Crohn's disease based upon the presence of the risk variant at the JAK3 genetic locus. In another embodiment, the risk variant comprises SEQ ID NO: 37 and/or SEQ ID NO: 38. In another embodiment, the presence of the risk variant is associated with a positive expression of ASCA and/or anti-I2. In another embodiment, the positive expression of ASCA and/or anti-I2 comprises a high level of expression relative to a healthy subject.
Various embodiment of the present invention provide for a method of diagnosing Inflammatory Bowel Disease (IBD) in a subject, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants in genes selected from the group consisting of: NOD2, CARD15, CARD 8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent. In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38, or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the detection of the TLR8 risk variant in a female subject indicates an IBD diagnosis. In another embodiment, the detection of the TLR2 risk variant in a Jewish subject indicates an IBD diagnosis. In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an IBD diagnosis associated with a subtype of CD. In other embodiments, a subtype of CD comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype.
In various other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In various other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
In yet other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention provide for a process for predicting IBD susceptibility in a subject, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants in genes selected from the group consisting of: NOD2, CARD15, CARD 8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has increased susceptibility to IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject has a decreased susceptibility to IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent. In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC). In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID Nos: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38 or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii). In other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the detection of the TLR8 risk variant in a female subject indicates an increased susceptibility to IBD. In other embodiments, the detection of the TLR2 risk variant in a Jewish subject indicates an increased susceptibility to IBD. In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an increased susceptibility to IBD associated with a subtype of CD. In other embodiments, a subtype of Crohn's disease comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype.
In yet other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In yet other embodiment, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In another embodiment, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and/or risk serological factors and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention also provide for a method for treating a subject with IBD, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants selected from the group consisting of: NOD2, CARD15, CARD8, TLR8, TLR2 and JAK3; assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent; and prescribing a therapy to treat the subject diagnosed with IBD. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38 or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In yet other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the therapy is an antigen-directed therapy that targets Cbir-1 flagellin or an immunoreactive fragment thereof. In other embodiments, the therapy consists of manipulation of bacteria in the colon and/or small intestine.
In yet other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In yet other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention also provide for a process for selecting a therapy for a subject with IBD comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants selected from the group consisting of: NOD2, CARD15, CARD8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent; and selecting a therapy for the subject with IBD. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38, or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In yet other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the therapy selected for a subject with IBD is an antigen-directed therapy. In other embodiments, the antigen-directed therapy targets Cbir-1 flagellin or an immunoreactive fragment thereof. In other embodiments, the therapy consists of manipulation of bacteria in the colon and/or small intestine. In other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In yet other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various other embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a female subject comprising: providing a sample from the female subject; assaying the sample to detect the risk and/or protective variants of TLR8, wherein TLR8 H3 is the risk variant and TLR8 H2 is the protective variant; and determining that the female subject has increased susceptibility to IBD if the TLR8 H3 risk variant is present and/or the TLR8 H2 protective variant is absent or determining that the subject has a decreased susceptibility to IBD if the TLR8 H2 protective variant is present and/or the TLR8 H3 risk variant is absent. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of risk variants and protective variants is relative to that detected in a healthy subject.
Various other embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a Jewish subject comprising: providing a sample from the Jewish subject; assaying the sample to detect the TLR2 risk variant, wherein P631H is the risk variant at the TLR2 locus; and determining that the Jewish subject has increased susceptibility to IBD if the P631H risk variant is present or determining that the subject has a decreased susceptibility to IBD if the P631H risk variant is absent. In other embodiments, the P631H risk variant comprises SEQ ID NO: 33. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of the risk variants is relative to that detected in a healthy subject.
Various embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a pediatric subject comprising: providing a sample from the pediatric subject; assaying the sample to detect the NOD2 and/or CARD15 risk variants; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the pediatric subject has increased susceptibility to IBD if one or more risk variants and/or risk serological factors are present or determining that the subject has a decreased susceptibility to IBD if the risk variants and/or risk serological factors are absent. In other embodiments, the risk variants at the NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, and at the CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively. In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC).
In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an IBD diagnosis associated with a subtype of CD. In other embodiments, a subtype of Crohn's disease comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype. In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and/or risk serological factors and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley & Sons (New York, N.Y. 1992); Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2001); and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor N.Y., 1988), provide one skilled in the art with a general guide to many of the terms used in the present application.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
“Fibrostenosis” as used herein refers to a classification of Crohn's disease characterized by one or more accepted characteristics of fibrostenosing disease. Such characteristics of fibrostenosing disease include, for example, documented persistent intestinal obstruction or intestinal resection for an intestinal obstruction. The fibrostenosis can be accompanied by other symptoms such as perforations, abscesses or fistulae, and can be further characterized by persistent symptoms of intestinal blockage such as nausea, vomiting, abdominal distention and inability to eat solid food.
“Immune complex” and “complex” as used herein refer to an aggregate of two or more molecules that result from specific binding between an antigen and an antibody.
“Secondary antibody” means an antibody or combination of antibodies, which binds to the antibody of interest (i.e., the primary antibody); for example an antibody that binds to a pANCA or binds an antibody that specifically binds a CBir1 flagellin antigen, or an immunoreactive fragment thereof.
“Labeled secondary antibody” means a secondary antibody, as defined above, that can be detected or measured by analytical methods. Thus, the term labeled secondary antibody includes an antibody labeled directly or indirectly with a detectable marker that can be detected or measured and used in an assay such as an enzyme-linked immunosorbent assay (ELISA), fluorescent assay, radioimmunoassay, radial immunodiffusion assay or Western blotting assay. A secondary antibody can be labeled, for example, with an enzyme, radioisotope, fluorochrome or chemiluminescent marker. In addition, a secondary antibody can be rendered detectable using a biotin-avidin linkage such that a detectable marker is associated with the secondary antibody. Labeling of the secondary antibody, however, should not impair binding of the secondary antibody to the CBir1 antigen.
“Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus adult and newborn subjects, as well as fetuses, whether male or female, are intended to be including within the scope of this term.
“Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down and/or lessen the disease even if the treatment is ultimately unsuccessful.
“CD” and “UC” as used herein refer to Crohn's Disease and Ulcerative colitis, respectively.
“Haplotype” as used herein refers to a set of single nucleotide polymorphisms (SNPs) on a gene or chromatid that are statistically associated.
“Risk” as used herein refers to an increase in susceptibility to IBD, including but not limited to CD and UC.
“Protective” and “protection” as used herein refer to a decrease in susceptibility to IBD, including but not limited to CD and UC.
“Risk variant” as used herein refers to an allele whose presence is associated with an increase in susceptibility to IBD, including but not limited to CD and UC, relative to a healthy individual.
“Protective variant” as used herein refers to an allele whose presence is associated with a decrease/low probability in susceptibility to IBD, including but not limited to CD and UC, relative to an individual diagnosed with IBD.
“Risk haplotype” as used herein refers to a haplotype sequence whose presence is associated with an increase in susceptibility to IBD, including but not limited to CD and UC, relative to a healthy individual, who does not have the risk haplotype.
“Protective haplotype” as used herein refers to a haplotype sequence whose presence is associated with a decrease in susceptibility to IBD, including but not limited to CD and UC, relative to an individual diagnosed with IBD.
“Risk serological marker” as used herein refers to a serological marker whose expression is associated with an increase in susceptibility to and/or risk for rapid disease progression of inflammatory bowel disease, including but not limited to Crohn's Disease and ulcerative colitis, relative to a healthy individual.
As used herein, the term “sero-reactivity” means positive expression of an antibody.
As used herein, “antibody sum (AS)” means the number of positive antibodies per individual, such as 0, or 1 or 2, or 3 positive.
As used herein, “antibody quartile score” means the quartile score for each antibody level (<25%=1, 25-50%=2, 51%-<75%=3, 75%-100%=4).
As used herein, “quartile sum score (QSS)” means the sum of quartiles score for all of the antibodies.
As described herein, the inventors regrouped patients based on a range of quartile sum scores, defined as “Quartile Sum Score (QSS) Group.” For example, quartile sum score 3-5=group 1, 6-7=group 2, 8-9=group 3 and 10-12=group 4.
As used herein, “ASCA” means anti-Saccharomyces cerevisiae antibodies.
As used herein, “pANCA” means perinuclear anti-neutrophil cytoplasmic antibodies.
As used herein, “OmpC” means outer membrane protein C.
As used herein, “I2” means Pseudomonas fluorescens-associated sequence.
As used herein, “OR” is an abbreviation for odds ratio.
As used herein, “CI” is an abbreviation for confidence interval.
As used herein, “OCTN” is an abbreviation for organic cation transporter.
As used herein, “IP” is an abbreviation for internal penetrating disease.
As used herein, “S” is an abbreviation of stricturing disease.
As used herein, “NPNS” is an abbreviation of non-penetrating, non-stricturing disease.
As used herein, “PP” is an abbreviation of perianal penetrating.
“Jak3” as used herein refers to Janus kinase 3.
As used herein, “CARD15” also means NOD2. As disclosed herein, an example of CARD15 is described as SEQ ID NO: 16.
As used herein, SNP 8, 12, and 13, are also described as R702W, G908R, and 1007fs, respectively, as well as R675W, G881R, and 3020insC, respectively. Examples of SNP 8, 12, and 13, are described herein as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, respectively.
An example of CARD8 is described herein as SEQ ID NO: 35.
An example of T10C variant at the CARD8 locus is described herein as SEQ ID NO: 36.
As used herein, the term of “TLR8 H3” is further described in
As used herein, the term of “TLR8 H2” is further described in
As used herein, examples of SNP variants at the Jak3 genetic locus are rs2302600 (SEQ ID NO: 37) and rs3212741 (SEQ ID NO: 38). However, as understood by one of skill in the art, additional risk variants the Jak2 genetic locus may be readily apparent to one of skill in the art and Jak3 risk variants are not limited to these specific SNP sequences. Similarly, SNP variants rs2302600 and rs3212741 themselves may also come in many additional versions, including for example, nucleotide probes encoding the complementary strands.
As used herein, the term “biological sample” means any biological material from which nucleic acid molecules can be prepared. As non-limiting examples, the term material encompasses whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acid.
As known to one of ordinary skill in the art, there are presently various treatments and therapies available for those diagnosed with Inflammatory Bowel Disease, including but not limited to surgery, anti-inflammatory medications, steroids, and immunosuppressants.
Serological Factors
Various embodiments of the present invention provide for methods for diagnosing Crohn's disease in a mammal. Additional embodiments provide for determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease. Further embodiments provide for treating Crohn's disease. In a one embodiment, the mammal is a human.
In particular embodiments, diagnosing Crohn's disease may be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 expression indicates that the mammal has Crohn's disease. Determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease may also be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 indicates that the mammal has small bowel disease, internal penetrating/perforating disease or fibrostenosing disease.
Determining the presence of anti-CBir1 expression may be accomplished by various means. For example, determining the presence of anti-CBir1 expression may be performed by determining the presence of an RNA sequence or a fragment of an RNA sequence that encodes an anti-CBir1 antibody; for example, using Northern blot analysis or reverse transcription-polymerase chain reaction (RT-PCR). Determining the presence of anti-CBir1 expression may also be performed by determining the presence of anti-CBir1 antibodies; for example IgG anti-CBir1. Anti-CBir1 antibodies are not limited to IgG, as IgA, IgM, IgD and IgE are also included in various embodiments of the present invention. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate techniques for determining the presence of anti-CBir1 expression.
Determining the presence of anti-CBir1 antibodies may be accomplished by a number of ways. For example, the determination may be made by an enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, and mass spectrometric analysis.
In other embodiments for the determination of the presence of anti-CBir1 antibodies, an immune complex can be detected with a labeled secondary antibody, for example, that has specificity for a class determining portion of an anti-CBir1 antibody. One skilled in the art understands that, preferably, a secondary antibody does not compete with the CBir1 flagellin antigen for binding to the primary antibody. A secondary antibody can bind any epitope of an anti-CBir1 antibody.
It is understood that a useful secondary antibody is specific for the species from which the sample was obtained. For example, if human serum is the sample to be assayed, mouse anti-human IgG can be a useful secondary antibody. A combination of different antibodies, which can be useful in the methods of the invention, also is encompassed within the meaning of the term secondary antibody, provided that at least one antibody of the combination reacts with an antibody that specifically binds a CBir1 antigen.
A secondary antibody can be rendered detectable by labeling with an enzyme such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase or urease. A secondary antibody also can be rendered detectable by labeling with a fluorochrome (such a fluorochrome emits light of ultraviolet or visible wavelength after excitation by light or another energy source), a chemiluminescent marker or a radioisotope.
A signal from a detectable secondary antibody can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a fluorometer to detect fluorescence in the presence of light of a certain wavelength; or a radiation counter to detect radiation, such as a gamma counter for detection of iodine-125. For detection of an enzyme-linked secondary antibody, for example, a quantitative analysis can be made using a spectrophotometer. If desired, the assays of the invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate techniques for determining the presence of anti-CBir1 antibodies.
Additional embodiments of the present invention provide for methods of treating Crohn's disease in a human by the use of antigen-directed therapy. The target antigen in this antigen-therapy may be flagellin, and particularly CBir1 or an immunoreactive fragment thereof.
In other embodiments, methods are provided to diagnose a subset of CD patients that may have colitic disease, and/or colitic and small bowel disease. Defining this subset of CD patients may be performed by determining the presence of anti-CBir1 expression and determining the presence of perinuclear antineutrophil cytoplasmic antibodies (pANCA), where the presence of both is diagnostic of Crohn's disease with properties of colitic disease and/or colitic and small bowel disease. Determination of the presence of pANCA may also be accomplished using ELISA, SDS-PAGE, Western blot analysis, or mass spectrometric analysis. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate means for determining the presence of pANCA.
Further embodiments of the present invention provide for methods of treating the subset of CD patients with colitic disease and/or colitic and small bowel disease. Treating colitic disease and/or colitic and small bowel disease may be performed by manipulating the bacterial flora in the colon and/or colon and small bowel. Manipulation of the bacterial flora may be performed by administering antibiotics and/or probiotics. Examples of probiotics include but are not limited to Bifidobacterium, including, B. bifidum, B. breve, B. infantis, and B. longum; Lactobacillus, including, L. acidophilus, L. bulgaricus, L. casei, L. plantarum, L. rhamnosus, L. reuiteri, and L. paracasei.
Samples useful in various embodiments of the present invention can be obtained from any biological fluid having antibodies or RNA sequences or fragments of RNA sequences; for example, whole blood, plasma, serum, saliva, or other bodily fluid or tissue. It is understood that a sample to be assayed according to the various embodiments of the present invention may be a fresh or preserved sample obtained from a subject to be diagnosed. Furthermore, the sample used in connection with various embodiments of the present invention may be removed from the mammal; for example, from drawn blood, aspirated fluids, or biopsies. Alternatively, the sample may be in situ; for example a tool or device may be used to obtain a sample and perform a diagnosis while the tool or device is still in the mammal.
A CBir1 antigen, or immunoreactive fragment thereof, useful in the invention can be produced by any appropriate method for protein or peptide synthesis.
Other embodiments of the present invention use anti-idiotypic antibodies specific to the anti-CBir1 antibody or other antibody of interest. An anti-idiotypic antibody contains an internal image of the antigen used to create the antibody of interest. Therefore, an anti-idiotypic antibody can bind to an anti-CBir1 antibody or other marker antibody of interest. One skilled in the art will know and appreciate appropriate methods of making, selecting and using anti-idiotype antibodies.
The present invention is also directed to kits for diagnosing and/or treating Crohn's disease and/or subtypes of Crohn's disease. The kit is useful for practicing the inventive methods of diagnosing and/or treating Crohn's disease and/or subtypes of Crohn's disease. The kit is an assemblage of materials or components.
The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of diagnosing Crohn's disease or subtypes of Crohn's disease. Subtypes include small bowel disease, internal perforating disease, fibrostenosing disease, colitic disease and colitic and small bowel disease. For instance, a quantity of CBir1 antigen may be included in the kit for determining the presence of anti-CBir1 antibodies in accordance with various embodiments of the present invention. Additional embodiments are configured for treating Crohn's disease or subtypes of Crohn's disease. Further embodiments are configured for treating Crohn's disease patients with colitic disease and/or colitic and small bowel disease. In one embodiment, the kit is configured particularly for the purpose of diagnosing human subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects.
Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to diagnose or treat Crohn's disease and/or subtypes of Crohn's disease. Optionally, the kit also contains other useful components, such as, secondary antibodies, enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase or urease), fluorochrome, chemiluminescent markers, radioisotopes, labeled secondary antibodies, tetramethylbenzidine substrates, multiple well plates, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, or other useful paraphernalia as will be readily recognized by those of skill in the art.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of anti-CBir1 and/or pANCA.
In one embodiment, Crohn's Disease is diagnosed when anti-CBir-1 is detected in the subject. In another embodiment, ulcerative colitis is diagnosed when pANCA is detected in the subject.
In one embodiment, antigen-directed therapy targeting CBir-1 flagellinis used to treat the subject diagnosed with IBD, CD and/or UC. In a\\nother embodiment, bacterial manipulation is used to treat the subject diagnosed with IBD, CD and/or UC.
Embodiments of the present invention provide for methods of diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease including but not limited to Crohn's Disease and/or ulcerative colitis. Other embodiments provide for methods of prognosing inflammatory bowel disease including but not limited to Crohn's Disease and/or ulcerative colitis. Other embodiments provide for methods of treating inflammatory bowel disease including but not limited to Crohn's Disease and/or ulcerative colitis.
The methods may include the steps of obtaining a biological sample containing nucleic acid from the individual and determining the presence or absence of a SNP and/or a haplotype in the biological sample. The methods may further include correlating the presence or absence of the SNP and/or the haplotype to a genetic risk, a susceptibility for inflammatory bowel disease including but not limited to Crohn's Disease and ulcerative colitis, as described herein. The methods may also further include recording whether a genetic risk, susceptibility for inflammatory bowel disease including but not limited to Crohn's Disease and ulcerative colitis exists in the individual. The methods may also further include a prognosis of inflammatory bowel disease based upon the presence or absence of the SNP and/or haplotype. The methods may also further include a treatment of inflammatory bowel disease based upon the presence or absence of the SNP and/or haplotype.
In one embodiment, a method of the invention is practiced with whole blood, which can be obtained readily by non-invasive means and used to prepare genomic DNA, for example, for enzymatic amplification or automated sequencing. In another embodiment, a method of the invention is practiced with tissue obtained from an individual such as tissue obtained during surgery or biopsy procedures.
NOD2 Variants
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility to Crohn's Disease in an individual by determining the presence or absence in the individual of R702W, G908, and/or 1000fs in the NOD2 gene. In another embodiment, the present invention provides methods of prognosis of Crohn's Disease in an individual by determining the presence or absence in the individual of R702W, G908, and/or 1000fs in the NOD2 gene. In another embodiment, the present invention provides methods of treatment of Crohn's Disease in an individual by determining the presence or absence in the individual of R702W, G908, and/or 1000fs in the NOD2 gene.
In another embodiment, sero-reactivity associated with NOD2 variants is diagnostic or predictive of susceptibility of Crohn's Disease. In another embodiment, the association of sero-reactivity of ASCA, I2, OmpC, or Cbir to variants R702W, G908R, or 1000fs, is diagnostic or predictive of susceptibility of Crohn's Disease. In another embodiment, the association of sero-reactivity of ASCA, I2, OmpC, or Cbir to variants R702W, G908R, or 1000fs provides methods of prognosis of Crohn's Disease. In another embodiment, the association of sero-reactivity of ASCA, I2, OmpC, or Cbir to variants R702W, G908R, or 1000fs provides methods of treatment of Crohn's Disease.
In another embodiment, the presence of R702W, G908R, or 1000fs NOD2 variant is diagnostic or predictive of an increased adaptive immune response.
TLR8 Variants
As disclosed herein, an example of a TLR8 genetic sequence is described as SEQ ID NO: 21. An example of a TLR8 peptide sequence is described herein as SEQ ID NO: 22.
H2 and H3 are further described herein by
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility for or protection against inflammatory bowel disease in an individual by determining the presence or absence in the individual of a haplotype in the TLR8 gene.
In one embodiment, the present invention provides a method of determining susceptibility and/or diagnosing Crohn's Disease in an individual by determining the presence or absence of a TLR8 risk haplotype. In another embodiment, the TLR8 risk haplotype includes H3. In another embodiment, the individual is a female.
In another embodiment, the present invention provides a method of determining protection against Crohn's Disease in an individual by determining the presence or absence of a TLR8 protective haplotype. In another embodiment, the TLR8 protective haplotype includes H2. In another embodiment, the individual is a female. In another embodiment, the presence of a H2 determines protection against ulcerative colitis.
In another embodiment, the presence of H3 and/or H2 may provide methods of prognosis of inflammatory bowel disease. In another embodiment, the presence of H3 and/or H2 may provide methods of treatment of inflammatory bowel disease.
TLR2 Variants
As disclosed herein, an example of a TLR2 genetic sequence is described as SEQ ID NO: 32. An example of a TLR2 peptide sequence is described herein as SEQ ID NO: 34.
The P631H variant of TLR2 is also described herein as SEQ ID NO: 33, wherein the position of the variant allele within the sequence listing is marked as M.
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility for or protection against Crohn's Disease in an individual by determining the presence or absence in the individual of a variant in the TLR2 gene.
In another embodiment, the P631H variant of the TLR2 gene is diagnostic or predictive of susceptibility to Crohn's Disease.
In another embodiment, sero-reactivity associated with TLR2 variants is diagnostic or predictive of susceptibility of Crohn's Disease. In another embodiment, the association of sero-reactivity of ASCA, I2, OmpC, or Cbir to the P631H variant of the TLR2 gene is diagnostic or predictive of susceptibility of Crohn's Disease. In another embodiment, the association of sero-reactivity of ASCA, I2, OmpC, or Cbir to the P631H variant of the TLR2 gene is diagnostic or predictive of susceptibility of Crohn's Disease in Jewish individuals.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of Haplotype 3 (H3) in TLR8. In another embodiment, a subject is not diagnosed with IBD if the subject has the presence of Haplotype 2 (H2) in TLR8.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of P631H in TLR2.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2); and (iv) the presence of P631H in TLR2 and/or a combination thereof.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2).
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of P631H in TLR2.
In one embodiment, a subject is at an increased risk of IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2) in TLR8; and (iv) the presence of P631H in TLR2 and/or a combination thereof.
In one embodiment, a subject is at a decreased risk of IBD if the subject has (i) the absence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the absence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the absence of Haplotype 3 (H3) in TLR8 and the presence of Haplotype 2 (H2); (iv) the absence of P631H in TLR2 and/or a combination thereof.
In one embodiment, a female subject is at an increased risk of IBD if the subject has the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2) in TLR8. In one embodiment, a female subject is at a decreased risk of IBD if the subject has the absence of Haplotype 3 (H3) in TLR8 and the presence of Haplotype 2 (H2) in TLR8.
In one embodiment, a Jewish subject is at an increased risk of CD if the subject has the presence of P631H in TLR2.
CARD 15 and CARD 8 Variants
As used herein, SNP's 8, 12, 13 are also referred to as R702W, G908R, 1007insC.
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility to a subtype of Crohn's Disease in an individual by determining the presence or absence of immune reactivity in the individual, where the presence of immune reactivity is diagnostic of the subtype of Crohn's Disease. In another embodiment, the present invention provides methods of prognosis of Crohn's Disease in an individual by determining the presence or absence of immune reactivity, wherein the presence of immune reactivity is indicative of a complicating Crohn's Disease prognosis. In another embodiment, the present invention provides methods of treatment of Crohn's Disease by administering a therapeutically effective amount of Crohn's Disease treatment wherein there is a presence of immune reactivity in the individual. In another embodiment, the subtype is complicating Crohn's Disease. In another embodiment, the subtype is small bowel disease, internal penetrating and/or fibrostenosing. In another embodiment, immune reactivity is a high expression of ASCA, OmpC, and/or Cbir1, relative to levels found in a healthy individual. In another embodiment, the individual is a child.
In one embodiment, the present invention provides a method of diagnosing susceptibility to a subtype of Crohn's Disease by determining the presence of immune reactivity, and determining the presence of CARD15 variants, wherein the presence of immune reactivity and one or more CARD15 variants is diagnostic of susceptibility to the subtype of Crohn's Disease. In another embodiment, the present invention provides a method of prognosis of Crohn's Disease in an individual by determining the presence of immune reactivity, and determining the presence of CARD 15 variants, wherein the presence of immune reactivity and one or more CARD 15 variants is indicative of a complicating Crohn's Disease prognosis. In another embodiment, the present invention provides a method of treatment of Crohn's Disease by administering a therapeutically effective amount of Crohn's Disease treatment wherein there is a presence of immune reactivity and CARD15 variants in the individual. In another embodiment, the CARD15 variants comprise SNPs 8, 12, and/or 13. In another embodiment, immune reactivity is a high expression of ASCA, OmpC, and/or Cbir1, relative to levels found in a healthy individual. In another embodiment, the individual is a child. In another embodiment, the subtype of Crohn's Disease is small bowel disease, internal penetrating and/or fibrostenosis.
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility to a subtype of Crohn's Disease in an individual by determining the presence or absence of immune reactivity in the individual, where the presence of immune reactivity is diagnostic of the subtype of Crohn's Disease. In another embodiment, the present invention provides methods of prognosis of Crohn's Disease in an individual by determining the presence or absence of immune reactivity, wherein the presence of immune reactivity is indicative of a complicating Crohn's Disease prognosis. In another embodiment, the present invention provides methods of treatment of Crohn's Disease by administering a therapeutically effective amount of Crohn's Disease treatment wherein there is a presence of immune reactivity in the individual. In another embodiment, the subtype is complicating Crohn's Disease. In another embodiment, the subtype is small bowel disease, internal penetrating and/or fibrostenosing. In another embodiment, immune reactivity is a high expression of ASCA, OmpC, Cbir1, and/or I2 relative to levels found in a healthy individual. In another embodiment, the individual is a child.
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility to ulcerative colitis in an individual by determining the presence or absence of a CARD8 risk variant in the individual, where the presence of the CARD8 risk variant is diagnostic of ulcerative colitis. In another embodiment, the present invention provides methods of treatment of ulcerative colitis by administering a therapeutically effective amount of ulcerative colitis treatment wherein there is a presence of a CARD8 risk variant in the individual. In another embodiment, the CARD8 variant is T10C. In another embodiment, the individual is a child.
In one embodiment, the present invention provides methods of diagnosing and/or predicting susceptibility to inflammatory bowel disease in a child by determining the presence or absence of high expression of anti-Cbir1 relative to a healthy individual, wherein the presence of the high expression of anti-Cbir1 relative to a healthy individual is indicative of susceptibility to inflammatory bowel disease in the child. In another embodiment, the present invention provides methods of treatment for inflammatory bowel disease in a child by administering a therapeutically effective amount of inflammatory bowel disease treatment in a child with a high expression of anti-Cbir1 relative to a healthy individual.
In one embodiment, the present invention provides a method of predicting Crohn's Disease progression in an individual by determining the presence or absence of a high immune reactivity relative to a healthy individual. In another embodiment, the present invention provides a method of treatment of Crohn's Disease by administering a therapeutically effective amount of Crohn's Disease treatment in an individual with immune reactivity relative to a healthy individual. In another embodiment, the present invention provides a method of treating an aggressive form of Crohn's Disease in a pediatric subject by determining the presence of a high immune reactivity and treating the aggressive form of Crohn's Disease. In another embodiment, the present invention provides a method of determining the prognosis of Crohn's Disease in a subject by determining the presence or absence of a high immune reactivity relative to a child with a non-aggressive form of Crohn's Disease. In another embodiment, immune reactivity includes OmpC, ASCA, Cbir1 and/or pANCA. In another embodiment, the individual is a child. In another embodiment, the subject is a pediatric subject. In another embodiment, immune reactivity is determined by time to complication or surgery. In another embodiment, the immune reactivity is associated with disease phenotype, such as disease location, behavior and/or surgery. In another embodiment, the presence of the high immune reactivity is indicative of a prognosis of an aggressive form of Crohn's Disease.
As described herein, various embodiments provide methods of prognosis of Crohn's Disease by determining a high immune reactivity of various markers, such as OmpC, ASCA, Cbir1 and/or pANCA, where a high immune reactivity of one or more markers is associated with a prognosis of developing an aggressive form of Crohn's Disease. Immune reactivity is determined by comparing both the presence and magnitude of markers to a standard set by those marker levels found in a subject who has and maintains a non-aggressive form of Crohn's Disease.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of T10C in CARD8.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of anti-CBir1, pANCA, anti-OmpC, ASCA and/or anti-I2.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8 and/or a combination thereof.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8 and/or a combination thereof, (vi) the presence of anti-CBir1, pANCA, anti-OmpC, ASCA and/or a combination thereof.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of T10C in CARD8.
In one embodiment, a subject is at an increased risk for IBD if the subject has the presence of anti-CBir1, pANCA, anti-OmpC and/or ASCA.
In one embodiment, a subject is at an increased risk of IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8 and/or a combination thereof.
In one embodiment, a subject is at an increased risk of IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8; the presence of anti-CBir1, pANCA, anti-OmpC, ASCA, and/or a combination thereof.
In one embodiment, a subject is at a decreased risk of IBD if the subject has (i) the absence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the absence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the absence of T10C in CARD8 and/or a combination thereof.
JAK3 Variants
In one embodiment, the present invention provides a method of diagnosing susceptibility to a subtype of Crohn's Disease by determining the presence or absence of a risk variant at the JAK3 locus, where the presence of the risk variant at the JAK3 locus is indicative of susceptibility to the subtype of Crohn's Disease. In another embodiment, the risk variant is associated with ASCA and/or anti-I2 expression. In another embodiment, the risk variant at the JAK3 locus comprises SEQ ID NO: 37. In another embodiment, the risk variant at the JAK3 locus comprises SEQ ID NO: 38.
In one embodiment, the present invention provides a method of diagnosing Crohn's Disease by determining the presence or absence of a risk variant at the JAK3 locus, where the presence of the risk variant at the JAK3 locus is indicative of Crohn's Disease. In another embodiment, the risk variant is associated with ASCA and/or anti-I2 expression. In another embodiment, the risk variant at the JAK3 locus comprises SEQ ID NO: 37. In another embodiment, the risk variant at the JAK3 locus comprises SEQ ID NO: 38.
In another embodiment, the present invention provides a method of treating Crohn's Disease by determining the presence of a risk variant at the JAK3 locus and treating the Crohn's Disease.
In one embodiment, the present invention provides a method of determining protection against inflammatory bowel disease in an individual by determining the presence or absence of a protective haplotype at the JAK3 locus, where the presence of a protective haplotype at the JAK3 locus is indicative of a decreased likelihood of inflammatory bowel disease.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of JAK3 risk variants.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of ASCA and/or anti-I2.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of JAK3 risk variants; and (ii) ASCA, anti-I2 and/or a combination thereof.
In one embodiment, a subject is at an increased risk for IBD if the subject has the presence of JAK3 risk variants.
In one embodiment, a subject is at an increased risk for IBD if the subject has the presence of ASCA and/or anti-I2.
In one embodiment, a subject is at an increased risk for IBD if the subject has (i) the presence of JAK3 risk variants; and (ii) ASCA, anti-I2 and/or a combination thereof.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of T10C in CARD8.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of Haplotype 3 (H3) in TLR8. In another embodiment, a subject is not diagnosed with IBD if the subject has the presence of Haplotype 2 (H2) in TLR8.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of P631H in TLR2.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of JAK3 risk variants.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of anti-CBir1, pANCA, anti-OmpC, ASCA and/or anti-I2.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8; (iv) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2); (v) the presence of P631H in TLR2; (vi) the presence of JAK3 risk variants and/or a combination thereof.
In one embodiment, a subject is diagnosed with IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8; (iv) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2); (v) the presence of P631H in TLR2; (vi) the presence of JAK3 risk variants; (vi) the presence of anti-CBir1, pANCA, anti-OmpC, ASCA, anti-I2 and/or a combination thereof.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R702W, G908R, 1007insC or a combination thereof in NOD2.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of R675W, G881R, 3020incC or a combination thereof in CARD15.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of T10C in CARD8.
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2).
In one embodiment, a subject is at an increased risk of IBD if the subject has the presence of P631H in TLR2.
In one embodiment, a subject is diagnosed with IBD if the subject has the presence of JAK3 risk variants.
In one embodiment, a subject is at an increased risk for IBD if the subject has the presence of anti-CBir1, pANCA, anti-OmpC, ASCA and/or anti-I2.
In one embodiment, a subject is at an increased risk of IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8; (iv) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2); (v) the presence of JAK3 risk variants; (vi) the presence of P631H in TLR2 and/or a combination thereof.
In one embodiment, a subject is at an increased risk of IBD if the subject has (i) the presence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the presence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the presence of T10C in CARD8; (iv) the presence of Haplotype 3 (H3) in TLR8 and the absence of Haplotype 2 (H2); (v) the presence of JAK3 risk variants; (vi) the presence of P631H in TLR2 and/or a combination thereof; (vi) the presence of anti-CBir1, pANCA, anti-OmpC, ASCA, anti-I2 and/or a combination thereof.
In one embodiment, a subject is at a decreased risk of IBD if the subject has (i) the absence of R702W, G908R, 1007insC or a combination thereof in NOD2; (ii) the absence of R675W, G881R, 3020incC or a combination thereof in CARD15; (iii) the absence of T10C in CARD8; (iv) the absence of Haplotype 3 (H3) in TLR8 and the presence of Haplotype 2 (H2); (v) the absence of P631H in TLR2 and/or a combination thereof.
Various embodiments of the present invention provide for methods for diagnosing Crohn's disease in a mammal. Additional embodiments provide for determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease. Further embodiments provide for treating Crohn's disease. In one embodiment, the mammal is a human.
In particular embodiments, diagnosing Crohn's disease may be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 expression indicates that the mammal has Crohn's disease. Determining a subtype of Crohn's disease, such as a phenotypic feature associated with Crohn's disease may also be performed by determining the presence of anti-CBir1 expression, where the presence of anti-CBir1 indicates that the mammal has small bowel disease, internal penetrating/perforating disease or fibrostenosing disease.
Determining the presence of anti-CBir1 expression may be accomplished by various techniques. For example, determining the presence of anti-CBir1 expression may be performed by determining the presence of an RNA sequence or a fragment of an RNA sequence that encodes an anti-CBir1 antibody; for example, using Northern blot analysis or reverse transcription-polymerase chain reaction (RT-PCR). Determining the presence of anti-CBir1 expression may also be performed by determining the presence of anti-CBir1 antibodies; for example IgG anti-CBir1. Anti-CBir1 antibodies are not limited to IgG, as IgA, IgM, IgD and IgE are also contemplated in connection with various embodiments of the present invention. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate means for determining the presence of anti-CBir1 expression.
Determining the presence of anti-CBir1 antibodies may be accomplished by a number of ways. For example, the determination may be made by an enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, and mass spectrometric analysis.
In other embodiments of the invention, an immune complex can be detected with a labeled secondary antibody, for example, that has specificity for a class determining portion of an anti-CBir1 antibody. A signal from a detectable secondary antibody can be analyzed, and positive results indicate the presence of anti-CBir1 antibodies.
Additional embodiments of the present invention provide for methods of treating Crohn's disease by the use of antigen-directed therapy. The target antigen in this therapy may be flagellin, and particularly CBir1 or an immunoreactive fragment thereof.
In other embodiments, methods are provided to define a subset of CD patients that may have colitic disease, and/or colitic and small bowel disease. Defining this subset of CD patients may be performed by determining the presence of anti-CBir1 expression and determining the presence of perinuclear antineutrophil cytoplasmic antibodies (pANCA), where the presence of both is diagnostic of Crohn's disease with properties of colitic disease and/or colitic and small bowel disease. Determination of the presence of pANCA may also be accomplished using ELISA, SDS-PAGE, Western blot analysis, or mass spectrometric analysis. These examples are not intended to be limiting, as one skilled in the art will recognize other appropriate means for determining the presence of pANCA.
Further embodiments of the present invention provide for methods of treating the subset of CD patients with colitic disease and/or colitic and small bowel disease. Treating colitic disease and/or colitic and small bowel disease may be performed by manipulating the bacterial flora in the colon and/or colon and small bowel. Manipulation of the bacterial flora may be performed by administering antibiotics and/or probiotics.
Samples useful in various embodiments of the present invention can be obtained from any biological fluid having antibodies or RNA sequences or fragments of RNA sequences; for example, whole blood, plasma, serum, saliva, or other bodily fluid or tissue. The sample used in connection with various embodiments of the present invention may be removed from the mammal; for example, from drawn blood, aspirated fluids, or biopsies. Alternatively, the sample may be in situ; for example a tool or device may be used to obtain a sample and perform a diagnosis while the tool or device is still in the mammal.
A CBir1 antigen, or immunoreactive fragment thereof, useful in the invention can be produced by any appropriate method for protein or peptide synthesis.
Other embodiments of the present invention use anti-idiotypic antibodies specific to the anti-CBir1 antibody or other antibody of interest.
The present invention is also directed to kits for diagnosing and/or treating Crohn's disease and/or subtypes of Crohn's disease. The exact nature of the components configured in the inventive kits depends on their intended purpose. For instance, a quantity of CBir1 antigen may be included in the kit for determining the presence of anti-CBir1 antibodies. Instructions for use may be included in the kit.
Various embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual, comprising determining the presence or absence of at least one risk variant at the NOD2 locus selected from the group consisting of R702W, G908R and 1007fs, and determining the presence or absence of at least one risk serological marker, where the presence of at least one risk variant and at least one risk serological marker is diagnostic of susceptibility to Crohn's Disease.
In other embodiments, the presence of three of the risk variants at the NOD2 locus present a greater susceptibility than the presence of two, one or none of the risk variants at the NOD2 locus, and the presence of two of the risk variants at the NOD2 locus presents a greater susceptibility than the presence of one or none of the risk variants at the NOD2 locus but less than the presence of three risk variants at the NOD2 locus, and the presence of one of the risk variants at the NOD2 locus presents a greater susceptibility than the presence of none of the risk variants at the NOD2 locus but less than the presence of three or two of the risk variants at the NOD2 locus.
In other embodiments, the risk serological markers are selected from the group consisting of ASCA, I2, OmpC and Cbir. In another embodiment, the presence of four of the risk serological markers presents a greater susceptibility than the presence of three or two or one or none of the risk serological markers, and the presence of three of the risk serological markers presents a greater susceptibility than the presence of two or one or none of the risk serological markers but less than the presence of four risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of four or three risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of four or three or two of the risk serological markers.
In another embodiment, the invention further comprises the step of determining the presence or absence of one or more risk haplotypes at the TLR8 locus, wherein the presence of one or more risk haplotypes at the TLR8 locus is diagnostic of susceptibility to Crohn's Disease.
In another embodiment, the invention comprises the step of determining the presence or absence of one or more risk haplotypes at the TLR2 locus, wherein the presence of one or more risk haplotypes at the TLR2 locus is diagnostic of susceptibility to Crohn's Disease.
Other various embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual comprising determining the presence or absence of one or more risk haplotypes at the TLR8 locus in the individual, where the presence of one or more risk haplotypes is diagnostic of susceptibility to Crohn's Disease. In other embodiments, the individual is a female. In another embodiment, the method further comprises determining the presence of H3.
Other various embodiments provide methods of determining a low probability relative to a healthy individual of developing Crohn's Disease and/or ulcerative colitis in an individual, the method comprising determining the presence or absence of one or more protective haplotypes at the TLR8 locus in the individual, where the presence of one or more said protective haplotypes is diagnostic of a low probability relative to a healthy individual of developing Crohn's Disease and/or ulcerative colitis. In other embodiments, the individual is a female. In other embodiments, the method further comprises determining the presence of H2.
Further embodiments provide methods of diagnosing susceptibility to Crohn's Disease in an individual comprising determining the presence or absence of one or more risk variants at the TLR2 locus in the individual, where the presence of one or more risk variants is diagnostic of susceptibility to Crohn's Disease. In another embodiment, the individual is Jewish. In another embodiment, the invention further comprises determining the presence of P631H at the TLR2 locus.
Various embodiments provide methods of diagnosing susceptibility to a subtype of Crohn's Disease in a child, comprising determining the presence or absence of at least one risk variant at the CARD15 locus selected from the group consisting of SNP8, SNP12, and SNP13, and determining the presence or absence of at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, and ASCA, where the presence of at least one variant and at least one risk serological marker is diagnostic of susceptibility to the subtype of Crohn's Disease in a child. In another embodiment, the subtype of Crohn's Disease in a child comprises an aggressive complicating phenotype, a small bowel disease phenotype, and/or an internal penetrating and/or fibrostenosing disease phenotype. In another embodiment, the presence of three of the risk serological markers presents a greater susceptibility than the presence of two, one or none of the risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of three of the risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of three or two of the risk serological markers. In another embodiment, the SNP8 comprises SEQ ID NO: 18. In another embodiment, the SNP12 comprises SEQ ID NO: 19. And in another embodiment, the SNP13 comprises SEQ ID NO: 20.
Other embodiments provide for methods of diagnosing susceptibility to a subtype of Crohn's Disease in a child, comprising determining the presence or absence of a high immune reactivity relative to a healthy individual for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, I2, and pANCA, where the presence of a high immune reactivity relative to a healthy individual to at least one risk serological marker is diagnostic of susceptibility to the subtype of Crohn's Disease in a child. In another embodiment, the subtype of Crohn's Disease in a child comprises an aggressive complicating phenotype. In another embodiment, a high immune reactivity comprises a high magnitude of expression for the risk serological marker. In another embodiment, the presence of four of the risk serological markers presents a greater susceptibility than the presence of three, two, one or none of the risk serological markers, and the presence of three of the risk serological markers presents a greater susceptibility than the presence of two, one or none of the risk serological markers but less than the presence of four of the risk serological markers, and the presence of two of the risk serological markers presents a greater susceptibility than the presence of one or none of the risk serological markers but less than the presence of four or three of the risk serological markers, and the presence of one of the risk serological markers presents a greater susceptibility than the presence of none of the risk serological markers but less than the presence of four or three or two of the risk serological markers.
Various embodiments also provide methods of treating Crohn's Disease in a child, comprising determining the presence of a high immune reactivity to a risk serological marker relative to a healthy individual, and administering a therapeutically effective amount of Crohn's Disease treatment.
Other embodiments provide methods of diagnosing ulcerative colitis in an individual, comprising determining the presence or absence of a risk variant at the CARD8 locus, where the presence of the risk variant at the CARD8 locus is diagnostic of susceptibility to ulcerative colitis. In other embodiments, the risk variant at the CARD8 locus comprises SEQ ID NO: 36. In other embodiments, the individual is a child.
Various embodiments provide methods of determining the prognosis of Crohn's Disease in an individual, comprising determining the presence or absence of a high immune reactivity relative to a healthy individual for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, and pANCA, where the presence of a high immune reactivity relative to a healthy individual to at least one risk serological marker is indicative of a prognosis of an aggressive form of Crohn's Disease. In other embodiments, the individual is a child. In other embodiments, the prognosis of an aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Other embodiments provide methods of determining the prognosis of Crohn's Disease in a pediatric subject, comprising determining the presence or absence of a high immune reactivity of Cbir1, OmpC, ASCA, and pANCA in the pediatric subject relative to a child who has and maintains a non-aggressive form of Crohn's Disease, where the presence of the high immune reactivity relative to a child who has and maintains a non-aggressive Crohn's Disease is indicative of a prognosis of an aggressive form of Crohn's Disease in the pediatric subject. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or stricturing disease phenotype.
Other embodiments provide methods of treating an aggressive form of Crohn's Disease in a pediatric subject, comprising determining the presence of a high immune reactivity of Cbir1, OmpC, ASCA and pANCA relative to a child who has and maintains a non-aggressive form of Crohn's Disease to prognose the aggressive form of Crohn's Disease, and treating the aggressive form of Crohn's Disease.
Other embodiments provide methods of determining the prognosis of Crohn's Disease in a subject, comprising determining the presence or absence of a high immune reactivity in the subject relative to an individual who has and maintains a non-aggressive form of Crohn's Disease for at least one risk serological marker, selected from the group consisting of Cbir1, OmpC, ASCA, and pANCA, where the presence of the high immune reactivity relative to an individual who has and maintains a non-aggressive form of Crohn's Disease is indicative of a prognosis of an aggressive form of Crohn's Disease. In other embodiments, the subject is a pediatric subject. In other embodiments, the individual who has and maintains a non-aggressive form of Crohn's Disease is a child. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Various embodiments also provide methods of treating an aggressive form of Crohn's Disease in a subject, comprising determining the presence of a high immune reactivity relative to an individual who has and maintains a non-aggressive form of Crohn's Disease to prognose the aggressive form of Crohn's Disease, and treating the aggressive form of Crohn's Disease. In other embodiments, the subject is a pediatric subject. In other embodiments, the individual who has and maintains a non-aggressive form of Crohn's Disease is a child. In other embodiments, the aggressive form of Crohn's Disease further comprises a rapid complicating internal penetrating and/or fibrostenosing disease phenotype.
Various embodiments include a method of diagnosing susceptibility to a subtype of Crohn's disease in an individual, comprising determining the presence or absence of one or more risk variants at the Janus kinases 3 (JAK3) genetic locus in the individual, and determining the presence or absence of a positive expression of ASCA and/or anti-I2, where the presence of one or more risk variants at the JAK3 locus and the presence of ASCA and/or anti-I2 expression is indicative of susceptibility in the individual to the subtype of Crohn's Disease. In another embodiment, one of the one or more risk variants at the JAK3 locus comprises SEQ ID NO: 37. In another embodiment, one of the one or more risk variants at the JAK3 locus comprises SEQ ID NO: 38. In another embodiment, positive expression of ASCA and/or anti-I2 comprises a high level of expression relative to a healthy subject.
Other embodiments include a method of diagnosing a subtype of Crohn's disease in an individual, comprising obtaining a sample from the individual, assaying the sample for the presence or absence of a risk variant at the Janus kinases 3 (JAK3) genetic locus in the individual, and diagnosing the subtype of Crohn's disease based upon the presence of the risk variant at the JAK3 genetic locus. In another embodiment, the risk variant comprises SEQ ID NO: 37 and/or SEQ ID NO: 38. In another embodiment, the presence of the risk variant is associated with a positive expression of ASCA and/or anti-I2. In another embodiment, the positive expression of ASCA and/or anti-I2 comprises a high level of expression relative to a healthy subject.
Various embodiment of the present invention provide for a method of diagnosing Inflammatory Bowel Disease (IBD) in a subject, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants in genes selected from the group consisting of: NOD2, CARD15, CARD 8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent. In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38, or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the detection of the TLR8 risk variant in a female subject indicates an IBD diagnosis. In another embodiment, the detection of the TLR2 risk variant in a Jewish subject indicates an IBD diagnosis. In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an IBD diagnosis associated with a subtype of CD. In other embodiments, a subtype of CD comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype.
In various other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis.
In various other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
In yet other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention provide for a process for predicting IBD susceptibility in a subject, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants in genes selected from the group consisting of: NOD2, CARD15, CARD 8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has increased susceptibility to IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject has a decreased susceptibility to IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent. In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC). In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID Nos: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38 or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii). In other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the detection of the TLR8 risk variant in a female subject indicates an increased susceptibility to IBD. In other embodiments, the detection of the TLR2 risk variant in a Jewish subject indicates an increased susceptibility to IBD. In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an increased susceptibility to IBD associated with a subtype of CD. In other embodiments, a subtype of Crohn's disease comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype.
In yet other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In yet other embodiment, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In another embodiment, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and/or risk serological factors and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention also provide for a method for treating a subject with IBD, comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants selected from the group consisting of: NOD2, CARD15, CARD8, TLR8, TLR2 and JAK3; assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent; and prescribing a therapy to treat the subject diagnosed with IBD. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38 or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In yet other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the therapy is an antigen-directed therapy that targets Cbir-1 flagellin or an immunoreactive fragment thereof. In other embodiments, the therapy consists of manipulation of bacteria in the colon and/or small intestine.
In yet other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In yet other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various embodiments of the present invention also provide for a process for selecting a therapy for a subject with IBD comprising: providing a sample from the subject; assaying the sample to detect risk and/or protective variants selected from the group consisting of: NOD2, CARD15, CARD8, TLR8, TLR2 and JAK3; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the subject has IBD if one or more risk variants and/or risk serological factors are present and the protective variants are absent or determining that the subject does not have IBD if one or more protective variants are present and the risk variants and/or risk serological factors are absent; and selecting a therapy for the subject with IBD. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, expression of any one or more of anti-CBir1, NOD2, TLR2 or a combination thereof is indicative of CD and wherein expression of any one or more of pANCA, CARD8 or a combination thereof is indicative of UC.
In other embodiments, the risk variants are NOD2, CARD15, CARD 8, TLR2, TLR 8 and JAK3, wherein the TLR8 locus is H3 and comprises SEQ ID NOs: 23-31. In other embodiments, the risk variants located at the: NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively, CARD8 locus is T10C and comprises SEQ ID NO: 36, TLR8 locus is H3 and comprises SEQ ID NOs: 23-31, TLR2 locus is P631H and comprises SEQ ID NO: 33, and JAK3 comprises SEQ ID NO: 37, SEQ ID NO: 38, or a combination thereof. In other embodiments, the subject is diagnosed with IBD if the subject expresses any one or more of (i) NOD2, CARD15, CARD 8, TLR8, TLR2, JAK3 risk variants or a combination thereof or if the subject expresses any one or more of (ii) anti-Cbir1 antibody, pANCA, anti-OmpC, ASCA, anti-I2 serological risk factors or a combination thereof or (iii) if the subject expresses the combination of (i) and (ii).
In yet other embodiments, TLR8 comprises a protective variant and the protective variant located at the TLR8 locus is H2 and comprises SEQ ID NOs: 23-31. In other embodiments, the therapy selected for a subject with IBD is an antigen-directed therapy. In other embodiments, the antigen-directed therapy targets Cbir-1 flagellin or an immunoreactive fragment thereof. In other embodiments, the therapy consists of manipulation of bacteria in the colon and/or small intestine.
In other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis.
In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In yet other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject. In other embodiments, the presence of twelve risk haplotypes presents a greater susceptibility than the presence of eleven, ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eleven risk haplotypes presents a greater susceptibility than the presence of ten, nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, wherein the presence of ten risk haplotypes presents a greater susceptibility than the presence of nine, eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of nine risk haplotypes presents a greater susceptibility than the presence of eight, seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of eight risk haplotypes presents a greater susceptibility than the presence of seven, six, five, four, three, two, one or none of the risk haplotypes, and the presence of seven risk haplotypes presents a greater susceptibility than the presence of six, five, four, three, two, one or none of the risk haplotypes, and the presence of six risk haplotypes presents a greater susceptibility than the presence of five, four, three, two, one or none of the risk haplotypes, and the presence of five risk haplotypes presents a greater susceptibility than the presence of four, three, two, one or none of the risk haplotypes, and the presence of four risk haplotypes presents a greater susceptibility than the presence of three, two, one or none of the risk haplotypes, and the presence of three risk haplotypes presents a greater susceptibility than the presence of two, one or none of the risk haplotypes, and the presence of two risk haplotypes presents a greater susceptibility than the presence of one or none of the risk haplotypes, and the presence of one risk haplotype presents a greater susceptibility than the presence of none of the risk haplotypes.
Various other embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a female subject comprising: providing a sample from the female subject; assaying the sample to detect the risk and/or protective variants of TLR8, wherein TLR8 H3 is the risk variant and TLR8 H2 is the protective variant; and determining that the female subject has increased susceptibility to IBD if the TLR8 H3 risk variant is present and/or the TLR8 H2 protective variant is absent or determining that the subject has a decreased susceptibility to IBD if the TLR8 H2 protective variant is present and/or the TLR8 H3 risk variant is absent. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of risk variants and protective variants is relative to that detected in a healthy subject.
Various other embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a Jewish subject comprising: providing a sample from the Jewish subject; assaying the sample to detect the TLR2 risk variant, wherein P631H is the risk variant at the TLR2 locus; and determining that the Jewish subject has increased susceptibility to IBD if the P631H risk variant is present or determining that the subject has a decreased susceptibility to IBD if the P631H risk variant is absent. In other embodiments, the P631H risk variant comprises SEQ ID NO: 33. In other embodiments, IBD comprises Crohn's Disease (CD) and ulcerative colitis (UC). In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). In other embodiments, the detection of the risk variants is relative to that detected in a healthy subject.
Various embodiments of the present invention also provide for a method of diagnosing susceptibility to IBD in a pediatric subject comprising: providing a sample from the pediatric subject; assaying the sample to detect the NOD2 and/or CARD 15 risk variants; optionally, assaying the sample to detect risk serological factors selected from the group consisting of: anti-CBir1, pANCA, anti-OmpC, ASCA and anti-I2; and determining that the pediatric subject has increased susceptibility to IBD if one or more risk variants and/or risk serological factors are present or determining that the subject has a decreased susceptibility to IBD if the risk variants and/or risk serological factors are absent. In other embodiments, the risk variants at the NOD2 locus are R702W, G908R and 1007insC and comprise SEQ ID NO: 18, 19 and 20, respectively, and at the CARD15 locus are R675W, G881R and 3020insC and comprise SEQ ID NO: 18, 19 and 20, respectively. In other embodiments, IBD comprises Crohn's disease (CD) and ulcerative colitis (UC). In other embodiments, the detection of the NOD2 and/or CARD15 risk variants and/or risk serological factors in a pediatric subject indicates an IBD diagnosis associated with a subtype of CD. In other embodiments, a subtype of Crohn's disease comprises aggressive complicating phenotype, small bowel disease phenotype, internal penetrating and/or fibrostenosing disease phenotype. In other embodiments, there is a greater susceptibility to IBD when an increased number of risk variants and/or risk serological factors and a decreased number of protective variants are present and a decreased susceptibility when an increased number of protective variants and a decreased number of risk variants are present. In other embodiments, the detection of risk serological factors comprises using a technique selected from the group consisting of Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbant assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western Blot and mass spectrometric analysis. In other embodiments, the detection of risk variants comprises using a technique selected from the group consisting of allelic discrimination assay, sequence analysis, allele-specific oligonucleotide hybridization assay, heteroplex mobility assay (HMA), single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE).
In other embodiments, the detection of risk variants, risk serological factors and protective variants is relative to that detected in a healthy subject.
Variety of Methods and Materials
A variety of methods can be used to determine the presence or absence of a variant allele or haplotype. As an example, enzymatic amplification of nucleic acid from an individual may be used to obtain nucleic acid for subsequent analysis. The presence or absence of a variant allele or haplotype may also be determined directly from the individual's nucleic acid without enzymatic amplification.
Analysis of the nucleic acid from an individual, whether amplified or not, may be performed using any of various techniques. Useful techniques include, without limitation, polymerase chain reaction based analysis, sequence analysis and electrophoretic analysis. As used herein, the term “nucleic acid” means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.
The presence or absence of a variant allele or haplotype may involve amplification of an individual's nucleic acid by the polymerase chain reaction. Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).
A TaqmanB allelic discrimination assay available from Applied Biosystems may be useful for determining the presence or absence of an IL23R variant allele. In a TaqmanB allelic discrimination assay, a specific, fluorescent, dye-labeled probe for each allele is constructed. The probes contain different fluorescent reporter dyes such as FAM and VICTM to differentiate the amplification of each allele. In addition, each probe has a quencher dye at one end which quenches fluorescence by fluorescence resonant energy transfer (FRET). During PCR, each probe anneals specifically to complementary sequences in the nucleic acid from the individual. The 5′ nuclease activity of Taq polymerase is used to cleave only probe that hybridize to the allele. Cleavage separates the reporter dye from the quencher dye, resulting in increased fluorescence by the reporter dye. Thus, the fluorescence signal generated by PCR amplification indicates which alleles are present in the sample. Mismatches between a probe and allele reduce the efficiency of both probe hybridization and cleavage by Taq polymerase, resulting in little to no fluorescent signal. Improved specificity in allelic discrimination assays can be achieved by conjugating a DNA minor grove binder (MGB) group to a DNA probe as described, for example, in Kutyavin et al., “3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperature,” Nucleic Acids Research 28:655-661 (2000)). Minor grove binders include, but are not limited to, compounds such as dihydrocyclopyrroloindole tripeptide (DPI).
Sequence analysis also may also be useful for determining the presence or absence of an IL23R variant allele or haplotype.
Restriction fragment length polymorphism (RFLP) analysis may also be useful for determining the presence or absence of a particular allele (Jarcho et al. in Dracopoli et al., Current Protocols in Human Genetics pages 2.7.1-2.7.5, John Wiley & Sons, New York; Innis et al., (Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990)). As used herein, restriction fragment length polymorphism analysis is any method for distinguishing genetic polymorphisms using a restriction enzyme, which is an endonuclease that catalyzes the degradation of nucleic acid and recognizes a specific base sequence, generally a palindrome or inverted repeat. One skilled in the art understands that the use of RFLP analysis depends upon an enzyme that can differentiate two alleles at a polymorphic site.
Allele-specific oligonucleotide hybridization may also be used to detect a disease-predisposing allele. Allele-specific oligonucleotide hybridization is based on the use of a labeled oligonucleotide probe having a sequence perfectly complementary, for example, to the sequence encompassing a disease-predisposing allele. Under appropriate conditions, the allele-specific probe hybridizes to a nucleic acid containing the disease-predisposing allele but does not hybridize to the one or more other alleles, which have one or more nucleotide mismatches as compared to the probe. If desired, a second allele-specific oligonucleotide probe that matches an alternate allele also can be used. Similarly, the technique of allele-specific oligonucleotide amplification can be used to selectively amplify, for example, a disease-predisposing allele by using an allele-specific oligonucleotide primer that is perfectly complementary to the nucleotide sequence of the disease-predisposing allele but which has one or more mismatches as compared to other alleles (Mullis et al., supra, (1994)). One skilled in the art understands that the one or more nucleotide mismatches that distinguish between the disease-predisposing allele and one or more other alleles are preferably located in the center of an allele-specific oligonucleotide primer to be used in allele-specific oligonucleotide hybridization. In contrast, an allele-specific oligonucleotide primer to be used in PCR amplification preferably contains the one or more nucleotide mismatches that distinguish between the disease-associated and other alleles at the 3′ end of the primer.
A heteroduplex mobility assay (HMA) is another well known assay that may be used to detect a SNP or a haplotype. HMA is useful for detecting the presence of a polymorphic sequence since a DNA duplex carrying a mismatch has reduced mobility in a polyacrylamide gel compared to the mobility of a perfectly base-paired duplex (Delwart et al., Science 262:1257-1261 (1993); White et al., Genomics 12:301-306 (1992)).
The technique of single strand conformational, polymorphism (SSCP) also may be used to detect the presence or absence of a SNP and/or a haplotype (see Hayashi, K., Methods Applic. 1:34-38 (1991)). This technique can be used to detect mutations based on differences in the secondary structure of single-strand DNA that produce an altered electrophoretic mobility upon non-denaturing gel electrophoresis. Polymorphic fragments are detected by comparison of the electrophoretic pattern of the test fragment to corresponding standard fragments containing known alleles.
Denaturing gradient gel electrophoresis (DGGE) also may be used to detect a SNP and/or a haplotype. In DGGE, double-stranded DNA is electrophoresed in a gel containing an increasing concentration of denaturant; double-stranded fragments made up of mismatched alleles have segments that melt more rapidly, causing such fragments to migrate differently as compared to perfectly complementary sequences (Sheffield et al., “Identifying DNA Polymorphisms by Denaturing Gradient Gel Electrophoresis” in Innis et al., supra, 1990).
Other molecular methods useful for determining the presence or absence of a SNP and/or a haplotype are known in the art and useful in the methods of the invention. Other well-known approaches for determining the presence or absence of a SNP and/or a haplotype include automated sequencing and RNAase mismatch techniques (Winter et al., Proc. Natl. Acad. Sci. 82:7575-7579 (1985)). Furthermore, one skilled in the art understands that, where the presence or absence of multiple alleles or haplotype(s) is to be determined, individual alleles can be detected by any combination of molecular methods. See, in general, Birren et al. (Eds.) Genome Analysis: A Laboratory Manual Volume 1 (Analyzing DNA) New York, Cold Spring Harbor Laboratory Press (1997). In addition, one skilled in the art understands that multiple alleles can be detected in individual reactions or in a single reaction (a “multiplex” assay). In view of the above, one skilled in the art realizes that the methods of the present invention for diagnosing or predicting susceptibility to or protection against CD in an individual may be practiced using one or any combination of the well known assays described above or another art-recognized genetic assay.
There are many techniques readily available in the field for detecting the presence or absence of antibodies, polypeptides or other biomarkers, including protein microarrays. For example, some of the detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
Similarly, there are any number of techniques that may be employed to isolate and/or fractionate antibodies or protein biomarkers. For example, a biomarker and/or antibody may be captured using biospecific capture reagents, such as aptamers or other antibodies that recognize the antibody and/or protein biomarker and modified forms of it. This method could also result in the capture of protein interactors that are bound to the proteins or that are otherwise recognized by antibodies and that, themselves, can be biomarkers. The biospecific capture reagents may also be bound to a solid phase. Then, the captured proteins can be detected by SELDI mass spectrometry or by eluting the proteins from the capture reagent and detecting the eluted proteins by traditional MALDI or by SELDI. One example of SELDI is called “affinity capture mass spectrometry,” or “Surface-Enhanced Affinity Capture” or “SEAC,” which involves the use of probes that have a material on the probe surface that captures analytes through a non-covalent affinity interaction (adsorption) between the material and the analyte. Some examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these.
Alternatively, for example, the presence of biomarkers such as polypeptides and antibodies may be detected using traditional immunoassay techniques. Immunoassay requires biospecific capture reagents, such as antibodies, to capture the analytes. The assay may also be designed to specifically distinguish protein and modified forms of protein, which can be done by employing a sandwich assay in which one antibody captures more than one form and second, distinctly labeled antibodies, specifically bind, and provide distinct detection of, the various forms. Antibodies can be produced by immunizing animals with the biomolecules. Traditional immunoassays may also include sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays.
Prior to detection, antibodies and/or biomarkers may also be fractionated to isolate them from other components in a solution or of blood that may interfere with detection. Fractionation may include platelet isolation from other blood components, sub-cellular fractionation of platelet components and/or fractionation of the desired biomarkers from other biomolecules found in platelets using techniques such as chromatography, affinity purification, 1D and 2D mapping, and other methodologies for purification known to those of skill in the art. In one embodiment, a sample is analyzed by means of a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD was used. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4 T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn's disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.
A molecular technique known as serological expression cloning (SEC) was used to identify specific bacterial antigens driving experimental IBD. SEC involves the screening of DNA expression libraries in lambda phage with defined antisera.
Molecular cloning of antigens by SEC using sera from colitic C3H/HeJBir mice is described. The dominant antigens identified were a family of related, novel flagellins. Strong reactivity against specific flagellins was seen in multiple models of experimental colitis across several distinct MHC haplotypes. There was a clear IgG2a predominance to the anti-flagellin response, suggesting a concurrent Th1-biased T cell response against flagellin. Indeed, marked reactivity against flagellin was seen in mesenteric and splenic T cell cultures from colitic animals, and flagellin-specific T cells were able to induce colitis when adoptively transferred into immunodeficient animals. Surprisingly, the reactivity against these flagellins (but not against the dissimilar Salmonella flagellin) was also seen in human IBD sera, with significant reactivity in patients with CD but not UC or control patients.
Using an unbiased molecular screen to search for bacterial antigens relevant to IBD, the dominant antigens identified were a family of related, novel flagellins. A strong, IgG2a-biased serological response to these specific flagellins was seen in multiple models of experimental colitis across several distinct MHC haplotypes. In addition, marked reactivity against these flagellins was seen at the T cell level, and flagellin-specific T cells were able to induce colitis when adoptively transferred into immunodeficient animals. Interestingly, while these flagellins were identified from mouse cecal bacteria, there was clear, specific reactivity against these molecules in patients with CD (but not in patients with UC or in NCs).
It has been observed that full-length flagellin Fla-X (endotoxin free) is capable of stimulating TNF-α production by human macrophages in vitro (M. J. Lodes and R. M. Hershberg). It is tempting to speculate that the intrinsic “adjuvanticity” of flagellin is likely to contribute to its antigenicity. While flagellin molecules clearly have the capacity to stimulate the production of proinflammatory cytokines via TLR5, and while not wishing to be bound to any particular theory, it is believed that the B and T cell responses to flagellin contribute more directly to the chronic intestinal inflammation seen in IBD.
The clinical data (
In general, the data are consistent with the belief that IBD is associated with a defect in tolerance to commensal organisms (Duchmann R, et al. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease. Clin. Exp. Immunol. 1995; 102:448-455.). The IgG2a-biased antibody against Fla-X and CBir1 highlights the Th1 bias of the T cell responses seen. The broad recognition of these flagellins in several different mouse models and in humans with CD indicates that these flagellins are among the immunodominant antigens of the microbiota. However, the exact role of these flagellins in the pathogenesis of IBD (e.g., whether they are predominant or obligatory for disease) compared with that of other microbial antigens remains to be defined. While not wishing to be bound to any particular theory, it is believed that a T cell regulatory response to specific flagellins (and/or other bacterial antigens) may be selectively impaired in IBD. In this context, specific flagellin molecules may represent novel targets for antigen-directed therapy in IBD.
As observed with the specific flagellins identified here, only a subset of patients with CD show specific seroreactivity against I2 (an antigen derived from Pseudomonas fluorescens).
Antibody responses to certain microbial antigens define heterogeneous groups of Crohn's patients; multiple and high-level responses to these antigens are associated with aggressive clinical phenotypes. The flagellin, CBir1, identified by the inventors in the C3H/HeJBir mouse model, is a dominant antigen capable of inducing colitis in mice and eliciting antibody responses in a subpopulation of patients with Crohn's disease. Serum response to CBir1 flagellin in Crohn's disease patients was evaluated and compared to previously defined responses to oligomannan (ASCA), I2, OmpC and neutrophil nuclear autoantigens (pANCA), and to determine anti-CBir1 associated phenotypes.
It was found that the presence and level of IgG anti-CBir1 were associated with Crohn's disease, independently. Anti-CBir1 was present in all antibody subgroups and expression increases in parallel with increases in the number of antibody responses. pANCA+ Crohn's patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is independently associated with small bowel, internal-penetrating and fibrostenosing disease features.
Thus, serum responses to CBir1 independently identify a unique subset of patients with complicated Crohn's disease. This is the first bacterial antigen identified in a murine model with a similar pattern of aberrant reactivity in a subset of Crohn's disease patients.
Serologic expression cloning was used to identify an immunodominant antigen, CBir1 flagellin, to which strong B cell and CD4+ T cell responses occur in colitic mice. Transfer of CBir1 specific CD4+ Th1 T cells to C3H/SCID mice generated a severe colitis dependent on endogenous expression of CBir1 flagellin in the cecum and colon. These findings prove that CBir1 flagellin is an immunodominant antigen of the enteric microbial flora. Of note, approximately 50% of patients with CD had serum reactivity to CBir1, whereas patients with ulcerative colitis, patients with other inflammatory GI diseases, and control subjects had little or no reactivity to this flagellin. The inventors determined the relationship of serum reactivity to CBir1 and the previously defined responses to oligomannan (ASCA), OmpC, I2 and pANCA in patients with CD and to define distinct clinical phenotypes. Results show that antibodies to CBir1 are independently associated with CD, have no correlation to levels of previously defined antibodies, are expressed in ASCA-negative and pANCA+ CD patients and are independently associated with aspects of complicated CD.
Investigations have yielded compelling evidence that serum antibody to CBir1 flagellin, marks for an independent subset of patients with CD. It is shown that the level of response can vary widely, that these responses are relatively stable over time and do not correspond with active or remission disease states. It is believed that anti-CBir1 expression is independent of serologic responses to previously defined bacterial antigens and is independently associated with complicated CD. It is also the first antigen to be discovered with a role as ligand for activation of the innate immune response via Toll-like receptors and a strong immunogen for adaptive immunity. This dual effect provides a focus for investigations of the role of anti-CBir1 in the pathogenesis of this subset of patients with CD.
It has been previously shown that groups of patients with unique disease characteristics can be distinguished by the presence and level of serum antibodies to one, two or all of the following antigens: oligomannan (ASCA); the novel Crohn's related bacterial sequence, I2; and E. coli outer-membrane porin-C(OmpC). While each of these reactivities may serve to subclassify phenotypes within CD, none of them have yet been shown to have any direct pathophysiologic significance. The dominant serologic immune response to CBir1 flagellin was found by serologic expression cloning using sera from colitic mice to screen a DNA phage library derived from mouse cecal bacteria. CBir1 flagellin was then used to generate a specific CD4+ Th1 cell line. Transfer of this Th1 cell line into SCID mice induced a colitis due to reactivity to endogenous CBir1 flagellin in the microbial flora indicating that CBir1 is an immunodominant antigen in mouse colitis. CBir1 is the first bacterial antigen capable of inducing colitis in animals that demonstrates a similar aberrant immune response in patients with CD.
Interesting findings resulted from the examination of the relationship of anti-CBir1 to the previously defined antibodies to microbial antigens in patients with CD. The level of response to CBir1 is greater in patients who have increasing levels of reactivity to ASCA, OmpC, and I2 (with a peak occurring in those who respond to all three), which is consistent with the concept that this subset of patients has a propensity to respond to multiple bacterial antigens. However, high CBir1 reactivity was seen across all antibody-defined subsets, which is consistent with it being independent of the other antibody responses.
The data presented herein show that the serotypic and phenotypic associations with anti-CBir1 expression, (small bowel, internal penetrating, and fibrostenosing disease) differ from those associated with any or a combination of responses to I2, OmpC, oligomannan, or neutrophil nuclear antibodies. The lack of relationship to small bowel surgery and to ulcerative colitis-like suggest that to define the true phenotype associated with this antibody response may require further more precise clinical groupings.
Another seroreactivity that defines a subgroup of patients is pANCA, which is predominantly associated with ulcerative colitis and may reflect cross reactivity to bacteria (Seibold F, Brandwein S, Simpson S, Terhorst C, Elson C O. pANCA represents a cross-reactivity to enteric bacterial antigens. J Clin Immunol 1998; 18:153-60.); however, there is a subset of patients with CD who also express pANCA (Vasiliauskas E A, Plevy S E, Landers C J, Binder S W, Ferguson D M, Yang H, Rotter J I, Vidrich A, Targan S R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology 1996; 110:1810-9. Vasiliauskas E A, Kam L Y, Karp L C, Gaiennie J, Yang H, Targan S R. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics. Gut 2000; 47:487-96.). pANCA+ CD patients have both colitic and left-sided disease with features similar to ulcerative colitis. Among the population of CD patients who express pANCA but do not react to the other known antigens, 40-44% expressed anti-CBir1, while anti-CBir1 expression was found in only 4% of pANCA+ ulcerative colitis patients. Because anti-CBir1 expression appears to be associated with a specific CD subtype, it may prove to be useful in distinguishing among patients with indeterminate colitis; i.e, those that may be more Crohn's-like compared to those that may be more ulcerative colitis-like. Used in combination with pANCA, anti-CBir1 expression may also be used diagnose a subset of patients with colitic and/or colitic and small bowel disease, perhaps defining those patients potentially likely to respond to manipulation of bacteria using either antibiotics or probiotics.
The expression of antibodies to CBir1 is indicative of an adaptive immune response to this antigen. Antibody reactivity to flagellin may provide an important tool to define potential differences in pathophysiologic immune mechanisms in innate and adaptive immunity in a subset of patients with CD. Anti-CBir expression defines a subgroup of CD patients not previously recognized by other serologic responses and is independently associated with aspects of the complicated CD phenotype. These results represent the first example of discovery from animal models having direct correlates in human disease.
Isolation of Genomic DNA of Mouse Cecal Bacterium
Pelleted bacteria from C3H/HeJBir mouse ceca were inactivated at 80° C. for 20 minutes and then were treated with 2 ml lysozyme (20 mg/ml in Tris-EDTA [TE] buffer) for 1 hour at 37° C. This solution was rocked at room temperature for 10 minutes with 40 μl proteinase K (10 mg/ml) and 140 μl 20% SDS (Sigma-Aldrich, St. Louis, Mo., USA) and then incubated for 15 minutes at 65° C., then 0.4 ml of 5 M NaCl and 0.32 ml of a 10% cetyltrimethylammonium bromide (CTAB) solution (1 g CTAB [Sigma-Aldrich], 1.4 ml 5M NaCl, and 8.6 ml distilled H2O) was added, followed by incubation at 65° C. for 10 minutes. DNA was then extracted twice with phenol, followed by extraction with phenol/chloroform/isoamyl alcohol (24:24:2), and then with chloroform. Finally the DNA was precipitated with 0.6 volumes of isopropanol and resuspended in TE buffer.
Genomic Expression Library Construction
A detailed description of library construction can be found in the following references: Lodes, M. J., Dillon, D. C., Houghton, R. L., and Skeiky, Y. A. W. 2004. Expression cloning. In Molecular diagnosis of infectious diseases. 2nd edition. J. Walker, series editor; J. Decker and U. Reischl, volume editors. Humana Press. Totowa, N.J., USA. 91-106. Briefly, 20 μg of genomic DNA of mouse cecal bacterium was resuspended in 400 μl of TE buffer and was sonicated for five seconds at 30% continuous power with a Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pa., USA) to generate fragments of approximately 0.5-5.0 kb. DNA fragments were blunted with T4 DNA polymerase (Invitrogen, Carlsbad, Calif., USA) and were ligated to EcoRI adaptors (Stratagene, La Jolla, Calif., USA) with T4 DNA ligase (Stratagene). Adapted inserts were then phosphorylated with T4 polynucleotide kinase (Stratagene) and were selected by size with a Sephacryl 400-HR column (Sigma-Aldrich). Approximately 0.25 μg of insert was ligated to 1.0 μg Lambda ZAP Express Vector treated with EcoRI and calf intestinal alkaline phosphatase (Stratagene), and the ligation mix was packaged with Gigapack III Gold packaging extract (Stratagene) following the manufacturer's instructions.
Expression Screening
Immunoreactive proteins were screened from approximately 6×105 plaque-forming units (PFU) of the unamplified cecal bacterium expression lambda library. Briefly, twenty 150-mm petri dishes were plated with E. coli XL1-Blue MRF′ host cells (Stratagene) and approximately 3×104 PFU of the unamplified library and were incubated at 42° C. until plaques formed. Dry nitrocellulose filters (Schleicher and Schuell, Keene, N.H., USA), pre-wet with 10 mM isopropyl β-thiogalactopyranoside (IPTG), were placed on the plates, which were then incubated overnight at 37° C. Filters were removed and washed three times with PBS containing 0.1% Tween 20 (PBST) (Sigma-Aldrich), blocked with 1.0% BSA (Sigma-Aldrich) in PBST, and washed three times with PBST. Filters were next incubated overnight with E. coli lysate-adsorbed C3H/HeJ Bir mouse serum (1:200 dilution in PBST), washed three times with PBST, and incubated with a goat anti-mouse IgG+IgA+IgM (heavy and light chain) alkaline phosphatase-conjugated secondary antibody (diluted 1:10,000 with PBST; Jackson Laboratories, West Grove, Pa., USA) for 1 hour. Filters were finally washed three times with PBST and two times with alkaline phosphatase buffer (pH 9.5) and were developed with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Invitrogen). Reactive plaques were then isolated and a second or third plaque purification was performed. Excision of phagemid followed the Stratagene Lambda ZAP Express protocol, and the resulting plasmid DNA was sequenced with an automated sequencer (ABI, Foster City, Calif., USA) using M13 forward, reverse, and sequence-specific internal DNA sequencing primers. Nucleic acid and predicted protein sequences were used to search the GenBank nucleotide and translated databases. Protein analysis was performed with the PSORT program (National Institute for Basic Biology, Okazaki, Japan) and with the IDENTIFY program of EMOTIF (Department of Biochemistry, Stanford University). Sequence alignments were produced with the MegAlign program (Clustal) of DNAStar (Madison, Wis., USA). Note that 20 random clones from the lambda library were picked and sequenced prior to serolological expression cloning. None of the 20 were found to be derived from mouse DNA and no flagellins were identified.
Cloning of Full-length Flagellins Representing Clones CBir1 and Fla-X
Clone CBir1 contains the conserved NH2 and variable regions of an unknown immunoreactive flagellin. The full-length sequence was obtained by first amplifying the unknown CBir1 carboxy terminus from total genomic cecal bacterium DNA with Expand polymerase (Roche, Indianapolis, Ind., USA) and the primers CBir1var1 (designed from the variable region of CBir1; CACAATCACAACATCTACCCAG; SEQ ID NO: 1) and CBir1 Carb Z (designed from the carboxy terminus of the related flagellin B of Butyrivibrio fibrisolvens, GenBank accession number AF026812; 5′-TTACTGTAAGAGCTGAAGTACACCCTG-3′; SEQ ID NO: 2). This PCR product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), digested with EcoRI, and gel-isolated (carboxy end of CBir1). Clone CBir1 plasmid DNA, which represents the NH2 terminus plus flagellin central variable region and overlaps with the cloned carboxy region, was digested with ScaI and then gel-isolated. Both overlapping (181-bp) DNA fragments (approximately 20 ng each) were added to a PCR reaction with the primers CBir1 HIS and CBir1 TERMX (see below), and the amplification product was cloned and expressed as described below.
Fla-X is an immunoreactive full-length flagellin sequence with no known identity in the public databases. Full-length flagellin Fla-X was cloned from total cecal bacterium genomic DNA by PCR amplification with the primers CBir Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATC-3′; SEQ ID NO: 3) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′; SEQ ID NO: 4), and was expressed as described below. The amplification product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and several clones were sequenced.
Recombinant Protein
Recombinant Salmonella muenchen flagellin (≥95% pure by SDS-PAGE) was obtained from Calbiochem (La Jolla, Calif., USA). Expression of other recombinant flagellin proteins and deletion constructs was accomplished by amplification from the cloned plasmid or genomic DNA (full length Fla-X) with Pfu polymerase (Stratagene) and the following primer pairs: for full-length CBir1, CBir1 HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATTTACAGGC-3′; SEQ ID NO: 5) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′; SEQ ID NO: 6); for the CBir1 NH2 plus variable regions, CBir1 HIS and CBir1 AV TERM (5′-ATAGACTAAGCTTAAGAAACCTTCTTGATAGCGCCAG-3′; SEQ ID NO: 7); for the CBir1 NH2 terminus, CBir1 HIS and CBir1 A TERM (5′-TAGACTGAATTCTAGTCCATAGCGTCAACGTTCTTTGTGTC-3′; SEQ ID NO: 8); for the CBir1 carboxy terminus, CBir1 C HIS (5′-CAATTACATATGCATCACCATCACCATCACAAGATGAACTTCCATGTAGGTGC-3′; SEQ ID NO: 9) and CBir1 TERMX; for full-length Fla-X, CBir Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATC-3′; SEQ ID NO: 10) and CBir1 TERMX (ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′; SEQ ID NO: 11); for the Fla-X NH2 plus variable regions, Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATCTTAGAGC-3′; SEQ ID NO: 12) and Fla-X AV TERM (5′-ATAGACTAAGCTTAGAGGCTGAAATCAATGTCCTCG-3′; SEQ ID NO: 13); for the Fla-X NH2 terminus, Fla-X HIS and Fla-X A TERM (5′-ATAGACTAAGCTTAATGTGCTGAAAGATATCTTGTCAC-3′; SEQ ID NO: 14); and for the Fla-X carboxy terminus, Fla-X C HIS (5′-CAATTACATATGCATCACCATCACCATCACTTCAGCCTCCATGTAGGTGCAGATGC-3′; SEQ ID NO: 15) and CBir1 TERMX. Primers contained restriction sites for cloning (in bold) and a six-histidine tag (in italics) for protein purification (NH2 terminus). The amplification products were digested with the restriction enzymes NdeI and HindIII or EcoRI, depending on the primer set used, gel-isolated, and ligated to a pET 17b plasmid vector (Novagen, Madison, Wis., USA) previously cut with NdeI and with HindIII or EcoRI and dephosphorylated with alkaline phosphatase (MB grade; Roche). The ligation mix was transformed into XL1 Blue competent cells (Stratagene) and plasmid DNA was prepared for sequencing (Qiagen, Valencia, Calif., USA). Recombinant protein was expressed by transformation of plasmid DNA into BL21 pLysS competent cells (Novagen) and induction of a single-colony cell culture with 2 mM IPTG (Sigma-Aldrich). Recombinant protein was recovered from cell lysate with nickel-nitrilotriacetic acid agarose beads (Qiagen), following the manufacturer's instructions, and was dialyzed in 10 mM Tris, pH 4-11 depending on predicted recombinant pI characteristics. Recombinant proteins were “quality-checked” for purity by SDS-PAGE followed by staining with Coomassie blue and by NH2-terminal protein sequencing, and were quantified with a Micro BCA assay (Pierce, Rockford, Ill., USA). Recombinants were assayed for endotoxin contamination with the Limulus assay (Bio Whittaker, Walkersville, Md., USA). Production of the Mycobacterium tuberculosis 38-kDa protein has been described previously (Lodes M J, et al. Serodiagnosis of human granulocytic ehrlichiosis by using novel combinations of immunoreactive recombinant proteins. J. Clin. Microbiol. 2001; 39:2466-2476.).
ELISA
Ninety-six-well EIA/RIA microtiter plates (3369; Corning Costar, Cambridge, Mass., USA) were coated overnight at 4° C. with 100 ng/well of the recombinant proteins. Solutions were then aspirated from the plates, which were then blocked for 2 hours at room temperature with PBS containing 1% (weight/volume) BSA. This was followed by washing in PBST. Serum diluted in PBS containing 0.1% BSA was added to wells and incubated for 30 minutes at room temperature, followed by washing six times with PBST and then incubation with secondary antibody-HRP conjugate (1:10,000 dilution) for 30 minutes. Plates were then washed six times in PBST and then were incubated with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry, Gaithersburg, Md., USA) for 15 minutes. The reaction was stopped by the addition of 1 N sulfuric acid, and plates were “read” at 450 nm using an ELISA plate reader (Biotek instrument EL311, Hyland Park Va.). Background values were determined by reading of reactions that lacked the primary antibody step.
Western Blot Analysis
Recombinant antigens (50-200 ng/lane) were subjected to SDS-PAGE analysis using 15% polyacrylamide “minigels.” The antigens were transferred to nitrocellulose BA-85 (Schleicher & Schuell, Keene, N.H., USA) and were blocked for 1 hour at room temperature with PBS containing 1% Tween 20. Blots were then washed three times, 10 minutes each wash, in PBST. Next, blots were probed for 1 hour at room temperature with serum diluted 1:500 in PBST followed by washing three times, 10 minutes each wash, in PBST. Blots were then incubated for 30 minutes at room temperature with secondary antibody-HRP diluted 1:10,000 in wash buffer and were again washed three times for 10 minutes each wash in PBST containing 0.5 M sodium chloride. Finally, blots were incubated in chemiluminescent substrate for ECL (Amersham Plc, Little Charlton, UK) for about 1 minute and then were exposed to X-ray film (XAR5) for 10-60 seconds, as required.
CD4+ T Cell Isolation and Culture, and Generation of a Cbir1-Specific T Cell Line
CD4+ T cells were isolated from mesenteric lymph nodes (MLNs) of mice with BD IMAG anti-mouse CD4 beads according to the manufacturer's instructions (BD Biosciences Pharmingen, San Diego, Calif., USA). Briefly, MLN cells were labeled with anti-CD4 beads and then were placed within the magnetic field of the BD Imagnet. The unlabeled cells in suspension were removed and the cells binding to beads were washed and used in the CD4+ T cell culture. More than 99% of cells were CD4+, as shown by flow cytometry. For the generation of a T cell line reactive to CBir1, CD4+ T cells were isolated from MLNs of C3H/HeJBir mice as described above and were cultured with splenic APCs that were pulsed with CBir1 (100 mg/ml) overnight. The cells were restimulated every 10-14 days.
Antigen-Specific Proliferation of T Cells
Spleen and MLN CD4+ T cells, isolated as described above, or a CBir1 flagellin-specific T cell line (4×105 cells/well) were incubated in triplicate in the presence of antigen-pulsed, irradiated APCs (4×105 cells per well; treated with 1-100 μg/ml antigen for 18 hours at 37° C.) in 96-well flat-bottomed tissue culture plates (Falcon, Lincoln Park, N.J., USA) at 37° C. in 5% CO2 humidified air. [3H]thymidine (0.5 μCi) (New England Nuclear, Boston, Mass., USA) was added at day 3 of culture and the cells were harvested at 16 hours after the pulse. The cells were harvested on glass fiber filters on a PHD cell harvester (Cambridge Technology Inc., Watertown, Mass., USA), washed with distilled water, and dried. Proliferation was assessed as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman Instruments, Palo Alto, Calif., USA) of the harvested samples, and was expressed as cpm±SD. The preparation of epithelial cell proteins and food antigens has been described previously (Cong Y, et al. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell Type 1 response and ability to transfer disease. J. Exp. Med. 1998; 187:855-864.). Ethical approval for animal studies was obtained from the Institutional Animal Care and Use Committee at the University of Alabama (Birmingham, Ala.) and from Corixa Corporation.
Specificity of CD4+ T Cell Stimulation
APCs were BALB/c spleen cells that were pulsed for 24 hours with nothing, OVA peptide at 2 μg/ml, CBir1 at 100 μg/ml, or Fla-X at 100 μg/ml, alone or in combinations as shown in Table 5. These APCs were washed and irradiated with 3,000 rads prior to culture. CD4+ T cells were isolated from DO11.10 mice and were cultured at a density of 1×105 with 4×105 prepulsed APCs. [3H]TdR was added at day 3 of culture and the cells were harvested after 16 hours.
Adoptive Transfer
CD4+ T cells were cultured with cecal bacterial antigen-pulsed and irradiated C3H/HeJ splenic cells in complete medium at 37° C. for 4 days in 5% CO2 air before being transferred intravenously into C3H/HeSnJ scid/scid recipients. Three months later, the recipients were killed and then the cecum and the proximal, medial, and distal portions of the colon were fixed in formalin. Fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin for histological examination. All slides were “read” by an experienced pathologist (A. Lazenby, Department of Pathology, University of Alabama at Birmingham) without knowledge of their origin.
Human Subjects
Serum samples from 212 subjects (50 UC patients, 100 CD patients, 22 DCs, and 40 NCs) were obtained from the serum archive of the Cedars-Sinai IBD Research Center. Sera were produced from standard phlebotomy blood specimens and were given an “anonymous” number code, divided into aliquots, and stored at −80° C. until use. The UC and CD patient specimens were obtained from a genetic case-control study (Toyoda H, et al. Distinct associations of HLA class II genes with inflammatory bowel disease. Gastroenterology. 1993; 104:741-748. Yang H-Y, et al. Ulcerative colitis: a genetically heterogeneous disorder defined by genetic (HLA class II) and subclinical (antineutrophil cytoplasmic antibodies) markers. J. Clin. Invest. 1993; 92:1080-1084.). Each patient's diagnosis was confirmed by clinical history, endoscopic and radiologic examination, and histopathology findings. The NC group is a collection of environmental controls that contain sera from individuals with no symptoms/signs of disease (i.e., spouses). DC samples include sera from patients with presumed infectious enteritis (stool culture negative for specific pathogens), blastocystis, celiac disease, collagenous colitis, irritable bowel syndrome, radiation proctitis, and acute schistosomiasis. The UC group includes both pANCA-positive and -negative specimens, while the CD group contains samples that are marker-negative, ASCA+; I2+; OmpC+(I2-positive); OmpC+, I2+, and ASCA+; and pANCA+. Ethical approval for human studies was obtained from the institution review board at Cedars-Sinai Medical Center.
Nucleotide Sequence Accession Numbers
The nucleotide sequence data for the flagellins CBir1 and Fla-X have been assigned GenBank accession numbers AY551005 and AY551006, respectively.
Seroreactivity in Mice is Directed Mainly Against a Specific Group of Flagellins
Serologic expression cloning resulted in 55 clones that were sequenced and identified. Using the basic local alignment search tool to search the GenBank databases demonstrated that 15 (26.8%) of these clones were flagellin-like sequences. None of the sequences directly matched any flagellin in the GenBank database, and all flagellin sequences identified represented unique clones. Given the average insert size of 0.8 kb in the library, no full-length flagellin clones were identified. However, all of the flagellin clones contained sequences derived from the conserved NH2 terminus, with varying amounts of the hypervariable central domain, and only two clones contained partial sequence from the conserved carboxy domain. Sequences from the 15 flagellin clones identified (CBir1-CBir15) were aligned at the protein level to flagellin sequences available in the public domain using the Clustal program in DNAStar. As shown in
Number of clones with a similar homology (No. clones). BspA, bacteroides surface protein A; ParB, chromosome partitioning protein B; WrbA, tryptophan-repressor-binding protein A; dnaA, chromosome replication initiator A.
Because of strong serum antibody reactivity to one particular flagellin clone, called CBir1, it was cloned and expressed its full-length gene. During this effort, the inventors also cloned a second, highly homologous and reactive flagellin (83.5% similarity to CBir1 at the NH2 conserved domain) and refer to it here as Fla-X (
Antibody Reactivity to Flagellin Directed Against the NH2 Terminus is of the IgG2a Subclass and Correlates with Disease
Western blot analyses using these purified recombinant flagellins and fragments with sera from the diseased C3H/HeJBir mice demonstrated the strong reactivity to flagellin, predominantly to the NH2-terminal fragments (
In order to generate more quantitative data with multiple IBD models at various time points in the course of disease, an antibody subclass ELISA against full-length or fragments of CBir1 or Fla-X was developed (
Anti-CBir1 Reactivity in CD Patient Sera but not Normal or UC Patient Sera
Subsequently, a large panel of sera from controls and patients with IBD for reactivity against CBir1 and Fla-X using antigen-specific ELISAs was tested. It was found that a significantly higher level of serum anti-CBir1/Fla-X flagellin in CD patients than in NCs, disease controls (DCs), and UC patients (
Reactivity to the Salmonella muenchen flagellin (which is highly similar to the flagellin of Eschericia coli [84-91% at the conserved NH2 end]), however, showed no significant correlation to disease (
Marked reactivity against flagellin is seen at the T cell level, and flagellin-specific T cells are able to induce colitis when adoptively transferred. Given the strong IgG2a bias seen in the antibody response in the mouse IBD strains, and while not wishing to be bound by any particular theory, it is believed that flagellin-specific Th1 T cells would be present in mice with IBD. To address this possibility, CD4+ T cells from pooled spleens and mesenteric lymph nodes from colitic mdr1a−/−, C3H/HeJBir, and C3H/HeJ.IL-10−/− mice (and haplotype-matched, noncolitic FVB and C3H/HeJ mice) were purified and tested the cells for reactivity against purified CBir1 and Fla-X in vitro in the presence of antigen-presenting cells (APCs). CD4+ T cells from the colitic mdr1a−/−, C3H/HeJBir, and C3H/HeJ.IL-10−/− mice, but not from age-matched control FVB or C3H/HeJ mice raised in the same mouse facility, responded to CBir1, as assessed by proliferation (
OVA-specific T cell line DO11.10 proliferates specifically in the presence of OVA peptide, but not nonspecifically in the presence of recombinant proteins CBir1 or Fla-X.
It has been previously shown that a T cell line specific for cecal bacterial protein/antigen (CBA), but not CD4+ T cells polyclonally activated by anti-CD3, could induce mucosal inflammation when adoptively transferred into H-2-matched immunodeficient scid/scid mice (Cong Y, et al. CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell Type 1 response and ability to transfer disease. J. Exp. Med. 1998; 187:855-864.). To address the potentially pathogenic role of flagellin-specific T cells in the initiation of mucosal inflammation, a CD4+ T cell line reactive with CBir1 flagellin from C3H/HeJBir mice was generated by repeated stimulation with antigen and APCs. This CD4+ T cell line strongly responded to CBir1 but not to Fla-X or a variety of other microbial, food, and epithelial antigens (
Human Subjects
Serum samples from 484 subjects (40 normal controls (NC), 21 disease controls (DC), 50 UC patients, and 373 CD patients) were selected from the serum archive of the Cedars-Sinai IBD Research Center. All research related activities were approved by the Cedars-Sinai Medical Center, Institutional Review Board. Diagnosis for each patient was based on standard endoscopic, histologic, and radiographic features. The normal control (NC) group is a collection of environmental controls that contain sera from individuals with no symptoms/signs of disease (i.e. spouses of patients). Disease controls (DC) include sera from patients with presumed infectious enteritis (stool culture negative for specific pathogens), blastocystis, celiac disease, collagenous colitis, irritable bowel syndrome, radiation proctitis, and acute schistosomiasis. For UC, groups chosen were pANCA− (n=25, seronegative) and pANCA+ (n=25, pANCA EU>45, no other antibody reactivity present). For CD, two cohorts were chosen: Cohort 1 (Lodes M J, Cong Y, Elson C O, Mohamath R, Landers C J, Targan S R, Fort M, Hershberg R M. Bacterial flagellin is a dominant antigen in Crohn disease. J Clin Invest 2004; 113:1296-306.) (n=100) was comprised of patients with select antibody expression to test CBir-1's specificity for CD and its relationship with other CD associated antibodies, Cohort 2 (n=303) was unbiased, previously well clinically and serologically characterized (Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24.), with an overlap of 30 patients between the two. Within Cohort 1, groups chosen were seronegative (n=40), ASCA+ (n=15, IgG ASCA EU>40, IgA ASCA EU>45, and no other antibody reactivity present), I2+ (n=15, anti-I2 EU>40, and no other antibody reactivity present), I2/OmpC+ (n=15, OmpC EU>30, and anti-I2 reactivity present), I2+/OmpC+/ASCA+ (n=15, anti-I2, anti-OmpC, IgG and IgA ASCA all positive, but no ANCA reactivity allowed), and pANCA+ (n=25, ANCA EU>35, no other antibody reactivity present). Cohort 2 was used for determining antibody groups as well as for phenotype analysis using definitions of clinical subgroup previously reported (Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24.). Serum samples from 44 CD patients diagnosed as above were analyzed for changes in antibody expression. Twenty of these patients were under treatment with infliximab and had experienced a CD Activity Index (CDAI) change of at least 70 (mean=Δ181) at time points at least 4 months apart with serum drawn at both times. The other 24 patients were drawn at the time of surgery and once at least 6 months following surgery.
ELISA
ELISA analysis of anti-CBir1 was performed as described in Lodes, et al. (Lodes M J, Cong Y, Elson C O, Mohamath R, Landers C J, Targan S R, Fort M, Hershberg R M. Bacterial flagellin is a dominant antigen in Crohn disease. J Clin Invest 2004; 113:1296-306.) but using NH2-terminal fragment of CBir1 (147aa) without knowledge of diagnosis or other serology results. Briefly, ELISA plates were coated overnight with 100 ng/well of CBir1, then blocked with 1% BSA in PBS for 2 hours. Plates were washed and serum was added at a 1:200 dilution in 1% BSA-PBS for a 30 minute incubation. After washing, horseradish peroxidase conjugated anti-human IgG at a 1:10,000 dilution was added and incubated for 30 minutes. After another wash, the plates were incubated with tetramethylbenzidine substrate for 15 minutes. The reaction was stopped with 1 N sulfuric acid and read at 450 nm. Positive was defined as the mean+2 SD of the healthy controls. For Cohort 2 and the longitudinal cohorts and phenotype cohorts, this assay was modified to be more similar to the ANCA, OmpC and I2 protocols: alkaline phosphatase was substituted as the secondary conjugate and incubated for 1 hour followed by paranitrophenyl phosphate as substrate for 30 minutes.
Statistical Analysis
Differences between disease groups were tested with non-parametric (Wilcoxon signed rank) statistics for quantitative levels. To determine the associations between antibody responses (positivity) toward microbial antigens, auto-antigens, and disease phenotype characteristics, univariate analyses utilizing χ2 tests were performed. The Cochran-Armitage test for trend was utilized to test if there is a linear trend in the proportion of patients with positive anti-CBir1 expression as the number of antibody responses increased. A p-value (p trend) <=0.05 suggests that the linear trend is statistically significant. A stratified Cochran-Mantel-Haenszel test was used to determine the association between anti-CBir1 and disease phenotypes. Multivariate analysis with logistic regression modeling was also performed to determine the primary associations among qualitative serological responses with disease phenotypes. All statistic tests were preformed using Statistical Analysis Software (Version 8.02; SAS Institute, Inc., Cary, N.C.).
Serum Reactivity to CBir1 Defines a Subset of Patients with Crohn's Disease
Serologic expression cloning of a cecal bacterial antigen phage library identified the flagellin, CBir1, as an immunodominant antigen recognized by colitic mice and by approximately half of patients with CD. Serum from two separate cohorts was used to investigate subgroups of CD patients. Cohort 1 consisted of sera from 100 CD patients selected on the basis of antibody profile. Newly tested sera from a group of 303 unselected patients that were studied and reported on in Mow et al (Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24.) comprised Cohort 2. For antigen, the amino domain of CBir1 flagellin was used because most of the IgG reactivity was to this region of the molecule. In addition, this form of CBir1 had a lower baseline reactivity among inflammatory controls, patients with UC or CD, and healthy control subjects, compared to the full length construct. As shown in
Levels of Antibodies to CBir1 do not Correlate with Disease Activity
As had been done with the previously defined CD-related antigens (I2/OmpC, oligomannan; Landers C J, Cohavy O, Misra R, Yang H, Lin Y C, Braun J, Targan S R. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123:689-99. Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24.), determining whether the level of anti-CBir1 expression changed in association with disease activity was sought. Serum samples from medically-resistant patients with CD, who were undergoing surgical removal of active disease were collected. Samples were taken again 6 months post-operatively and analyzed for differences in response. In general, there was very little change before and 6 months post surgery, when patients were in clinical and endoscopic remission (
Antibody Response to CBir1 and Other Crohn's Disease-Associated Anti-microbial Immune Responses
To determine the relationship of expression and level of anti-CBir1 expression to the previously defined antibodies to microbial antigens (anti-I2, anti-OmpC, ASCA), multiple logistic regression analysis with Cohort 1 was used, it was found that anti-CBir1 relates independently to CD when controlled for anti-I2, anti-OmpC and ASCA (p<0.001). In addition, there is no relationship between the level of response to CBir1 and any one of the other four antibodies (
As previously described by Landers et al (Landers C J, Cohavy O, Misra R, Yang H, Lin Y C, Braun J, Targan S R. Selected loss of tolerance evidenced by Crohn's disease-associated immune responses to auto- and microbial antigens. Gastroenterology 2002; 123:689-99.), homogeneous groups of CD patients based on selective antibody responses to specific microbial antigens and associated clinical features were defined. Thus far, the largest subgroup of CD has been stratified based on expression of ASCA. Among patients selected for study based on their antibody profiles (Cohort 1), anti-CBir1 is expressed in bothASCA-negative (46%) and ASCA+(60%) CD patients (
Antibodies to CBir1 and pANCA Expression
A small percentage of CD patients are solely pANCA-positive. pANCA is associated with ulcerative colitis, and in CD, pANCA marks for left-sided disease with ulcerative colitis-like features (Vasiliauskas E A, Plevy S E, Landers C J, Binder S W, Ferguson D M, Yang H, Rotter J I, Vidrich A, Targan S R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology 1996; 110:1810-9. Vasiliauskas E A, Kam L Y, Karp L C, Gaiennie J, Yang H, Targan S R. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics. Gut 2000; 47:487-96. Esters N, Vermeire S, Joossens S, Noman M, Louis E, Belaiche J, De Vos M, Van Gossum A, Pescatore P, Fiasse R, Pelckmans P, Reynaert H, Poulain D, Bossuyt X, Rutgeerts P. Serological markers for prediction of response to anti-tumor necrosis factor treatment in Crohn's disease. Am J Gastroenterol 2002; 97:1458-62. Peeters M, Joossens S, Vermeire S, Vlietinck R, Bossuyt X, Rutgeerts P. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease. Am J Gastroenterol 2001; 96:730-4.). pANCA does not differentiate between UC and UC-like CD. Determine whether anti-CBir1 expression had any bearing on this subgroup was sought. Of the pANCA+ patients with CD, 40-44% (Cohort 2 and Cohort 1, respectively) expressed anti CBir1 and none of other antibodies v. only 4% in pANCA+ ulcerative colitis (
Crohn's Disease Phenotypic Associations with Anti-CBir1 Expression
It was previously determined that antibody responses to the microbial antigens, OmpC, I2, oligomannan and neutrophil nuclear antigen(s) is associated with anatomical location as well as disease expression (Vasiliauskas E A, Plevy S E, Landers C J, Binder S W, Ferguson D M, Yang H, Rotter J I, Vidrich A, Targan S R. Perinuclear antineutrophil cytoplasmic antibodies in patients with Crohn's disease define a clinical subgroup. Gastroenterology 1996; 110:1810-9. Vasiliauskas E A, Kam L Y, Karp L C, Gaiennie J, Yang H, Targan S R. Marker antibody expression stratifies Crohn's disease into immunologically homogeneous subgroups with distinct clinical characteristics. Gut 2000; 47:487-96. Mow W S, Vasiliauskas E A, Lin Y C, Fleshner P R, Papadakis K A, Taylor K D, Landers C J, Abreu-Martin M T, Rotter J I, Yang H, Targan S R. Association of antibody responses to microbial antigens and complications of small bowel Crohn's disease. Gastroenterology 2004; 126:414-24. Arnott I D R, Landers C J, Nimmo E J, Drummond H E, Targan S R, Satsangi J. Reactivity to microbial components in Crohn's disease is associated with severity and progression. Am J Gastroenterol 2004; 99:2376-84.). Anti-CBir1 expression appears to be independently associated with CD. Therefore, to determine whether a clinical phenotype is independently associated with anti-CBir1 expression, the serotypically- and phenotypically-defined Cohort 2 was used to assess the overall and specific phenotypes associated with anti-CBir1 expression. It was found that 61% of patients with complicated (internal penetrating, fibrostenosing disease features, and those with or without history of surgery) were anti-CBir1+, compared to 42% of patients with inflammatory-only CD (p=0.002). Anti-CBir1 expression was positively associated with small bowel disease, fibrostenosing and internal penetrating disease (see Table 3), regardless of the presence of antibodies to 1, 2, or all 3 other antigens. Unlike some of the other antibody responses, anti-CBir1 expression was neither associated with small bowel surgery, nor was it negatively associated with the UC-like CD population. To further assess the independent relationship of anti-CBir1 expression to CD phenotypes, the inventors performed a multivariate logistic regression model analysis with the four CD-associated antibodies. The results in Table 4 show that anti-CBir1 expression is independently associated with small bowel, internal penetrating and fibrostenosing disease. Consistent with this finding is the lower frequency of anti-CBir1 expression in the cohort of patients treated with infliximab (30%,
While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The accompanying claims are intended to cover such modifications as would fall within the true spirit and scope of the invention. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description. All changes that come within the meaning of and range of equivalency of the claims are intended to be embraced therein.
The inventors performed a genome-wide association study testing autosomal single nucleotide polymorphisms (SNPs) on the Illumina HumanHap300 Genotyping BeadChip. Based on these studies, the inventors found single nucleotide polymorphisms (SNPs) and haplotypes that are associated with increased or decreased risk for inflammatory bowel disease, including but not limited to CD and UC. These SNPs and haplotypes are suitable for genetic testing to identify at risk individuals and those with increased risk for complications associated with serum expression of Anti-Saccharomyces cerevisiae antibody, and antibodies to I2, OmpC, and Cbir. The detection of protective and risk SNPs and/or haplotypes may be used to identify at risk individuals, predict disease course and suggest the right therapy for individual patients. Additionally, the inventors have found both protective and risk allelic variants for Crohn's Disease and Ulcerative Colitis.
As disclosed herein, the inventors examined a case-control cohort consisting of 763 Crohn's Disease patients, 351 ulcerative colitis patients, and 254 control patients. The patients were genotyped using Illumina technology. SNPs were chosen to tag common Caucasian haplotypes using information from the Innate Immunity PGA.
NOD2: Serologic Analysis and Classification
Sera were analyzed for expression of ASCA, anti-I2, anti-OmpC, in a blinded fashion by enzyme-linked immunosorbent assay (ELISA). Antibody levels were determined and results expressed as ELISA units (EU/ml) that are relative to a Cedars-Sinai laboratory (IgA-I2, IgA-OmpC) or a Prometheus Laboratory standard (San Diego, Calif., IgA and IgG ASCA) derived from a pool of patient sera with well-characterized disease found to have reactivity to these antigens. Quantitation of IgG anti-Cbir1 reactivity was expressed in ELISA units derived based on a proportion of reactivity relative to a standardized positive control. As ASCA can be expressed in both an IgA and IgG class, positivity to ASCA was determined if either class of antibody was above the reference range. In determining a quantitative measure of ASCA, the reactivity was first log-transformed and standardized. The higher of two standardized units was then used to determine the quartile of reactivity. With the exception of determining variance (see statistical analysis), the magnitude of reactivity to the other three antigens was not standardized as each is represented by a single class of antibody. The magnitude of the serologic response to each antigen was divided into four equal quartiles in CD patients, unaffected relatives and healthy controls, evaluated as three separate cohorts, to determine quartile sum scores.
NOD2: Genotyping
Three NOD2 variants (R702W, G908R, and 1007fs), were adapted to the TaqMan MGB (Applied Biosystems, Foster City, Calif.) genotyping platform.
NOD2: Statistical Analysis
The inventors assessed the relationship between carriage of a NOD2, TLR2, TLR4, and TLR9 variant and collective sero-reactivity to microbial antigens both qualitatively and semi-quantitatively. The inventors then determined if any particular NOD2 variant was predominant and examined whether any particular antibody or combinations of antibodies was predominant in determining the relationship between NOD2 variants and sero-reactivity. The contribution of NOD2 to collective sero-reactivity was evaluated by calculating the percent of variance that could be attributed to the presence of NOD2 variants. Finally, the inventors examined whether the presence of a NOD2 variant was related to sero-reactivity to microbial antigens in unaffected relatives of CD patients and healthy controls.
To determine the significance of increasing frequency of carriage of any NOD2 variants with increasing numbers of qualitatively positive antibodies and with increasing quartile sum (range, 4-16), the Cochran-Armitage trend test was performed. To test for differences in the mean quartile sum between those individuals with no NOD2 variant versus those with any variant, the student's t-test was used since the distribution was approximately a normal distribution. One-way ANOVA analysis was done to test the linear trend of mean quartile sum among those with 0, 1, and 2 NOD2 variants. One-way ANOVA analysis was used to test for a difference in sero-reactivity associated with specific NOD2 variants and similarly when comparing mean quartile sum between differing TLR genotypes.
The non-parametric Mann-Whitney test was used to compare the level of seroreactivity between those individuals who carried versus those who did not carry a NOD2 variant for each antibody. To identify whether there is a significant difference in the frequency of carriage of a NOD2 variant among groups within each set with single, double and triple antibody positivity, chi-square analysis was performed.
To determine what proportion of the variation in the sero-reactivity to microbial antigens was attributable to the presence of a NOD2 variant, a coefficient of determination (R2), defined as 1−SS (regression)/SS (total) in ANOVA was used. Sero-reactivity was defined, for this analysis, as the sum of the 4 standardized antibodies, where anti-OmpC=[log(anti-OmpC)−mean(log(anti-OmpC))]/SD(log(anti-OmpC)) and similarly for the other antibodies.
All analyses were performed using SAS computer software (version 8.2; SAS institute, Inc., Cary, N.C., USA, 1999).
NOD2
As disclosed herein, the inventors studied the serologic and genetic (NOD2) characteristics of a 732 patient cohort (Table 5). ASCA is detected in 50.4%, anti-I2 in 58.1%, anti-OmpC in 37.2% and anti-Cbir1 in 56.4% (Table 5). Simple heterozygosity for a disease-predisposing NOD2 variant is detected in 194 patients (26.5%), compound heterozygosity for two NOD2 variants is detected in 23 patients (3.1%), and homozygosity for two NOD2 variants is detected in 16 patients (2.2%) (Table 5).
As disclosed herein, an example of a NOD2 genetic sequence is described as SEQ ID NO: 16. An example of a NOD2 peptide sequence is described herein as SEQ ID NO: 17. R702W, G908R, and 1007fs variant alleles are also described herein as SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively, wherein the position of the variant allele is marked within the sequence listing as a letter other than A, C, G or T.
As further disclosed herein, a Crohn's Disease patient cohort was divided into five groups based on the number of antibodies (from zero to four) for which they are qualitatively positive and the proportion of patients with NOD2 variant in each group is determined. NOD2 variants are present with increasing frequency in patients with reactivity to an increasing number of microbial antigens, especially when there is reactivity to two or more antibodies (
As further disclosed herein, the inventors compared the serologic response of patients carrying a NOD2 variant to those carrying no variant. In patients carrying any NOD2 variant, the mean number of positive antibodies is higher than in those carrying no variant (2.24+/−versus 1.92+/−1.24, respectively; P=0.0008) (Table 6). Patients carrying any NOD2 variant have a higher mean quartile sum than those carrying no variant (10.60+/−3.03 versus 9.72+/−3.01, respectively; P=0.0003) (Table 6).
As disclosed herein, the inventors compared the serologic response of patients with two defective alleles versus having only one. The mean quartile sum increases in parallel with increasing number of NOD2 variants (P trend=0.002) (
As further disclosed herein, the inventors examined the absolute level of response to each antibody individually rather than collectively. For each of the four antibodies, the magnitude of sero-reactivity is higher when a NOD2 variant is present (Table 7).
As further disclosed herein, the inventors divided Crohn's Disease patients into 16 mutually exclusive groups based on all possible permutations of antibody positivity: no positive antibodies, single antibody positivity (4 groups in set 1), double antibody positivity (6 groups in set 2), triple antibody positivity (4 groups in set 3), and all antibodies positive. The inventors tested whether there is a significant difference among groups within each set where the groups had the same number of antibody positivity. There is no statistically significant difference in the frequency of NOD2 variants among groups within each set, and no single antibody or combination of antibody positivity is wholly responsible for the association between sero-reactivity and variant status (
As further disclosed herein, the inventors calculated 2.7% as the proportion of variability in sero-reactivity that was attributable to the presence of a NOD2 variant.
As further disclosed, a quartile sum was derived in Crohn's Disease patients, unaffected relatives, and healthy controls, based on the distribution of the magnitude of sero-reactivity within each cohort, with the same quartile sum in a Crohn's Disease patient or in a relative or healthy control not representative of the same absolute magnitude of response and not directly comparable. The magnitude of serologic response is significantly lower in unaffected relatives and healthy controls, compared to cases, and generally fell within the normal range. Sera was utilized from 220 unaffected relatives of Crohn's Disease patients (92% first degree). In the unaffected relatives the mean quartile sum in those individuals carrying any NOD2 variant is higher than those carrying no variant (10.67+/−2.73 vs. 9.75+/−2.52; P=0.02) (
NOD2 is a member of a family of intracellular cytosolic proteins important in mediating the host response to bacterial antigens and is found in epithelial cells of the small and large intestine as well as monocytes, macrophages, T and B cells, Paneth cells and dendritic cells (39-42). NOD2 senses MDP, a highly conserved component of bacterial peptidoglycan, which leads to the secretion of anti-bacterial substances such as alpha-defensins and the activation of nuclear factor kappa B (NF-kB) (43-44).
The inventors examined serologic and genetic data in 748 Crohn's Disease patients. ASCA and antibodies of I2, OmpC, and Cbir were measured by ELISA. Antibody sums (AS) and overall quartile sums (QS) (ranging from 4-16) of levels for all four antibodies were calculated as previously described (Mow et al Gastro 2004; 126:414). Genotyping (TaqmanMGB) was performed for 3 CD-associated variants of the NOD2 gene, R702W, G908R, and 1007fs.
ASCA was detected in 51%, anti-I2 in 58%, anti-OmpC in 38%, and anti-Cbir1 in 56%. 250 of 748 Crohn's Disease patients (33.4%) had at least one NOD2 variant; 206 (27.5%) having one and 44 (5.9%) having two. NOD2 variants were present at increasing frequency in patients with reactivity to increasing numbers of antigens. Variants were present in those with 0, 1, 2, 3, or 4 positive antibodies in 24%, 25%, 36%, 36%, and 46%, respectively (p for trend, 0.0001). NOD2 variants were present at increasing frequency in patients with increasing cumulative quantitative immune response as reflected by individual QS (p for trend, 0.0001). QS were also clustered into four groups by increasing cumulative quantitative immune response (group 1=4-6, group 2=7-9, group 3=10-13, and group 4=14-16). The frequency of having at least one NOD2 variant in each of the four groups was 22%, 29%, 35%, and 49% in groups 1, 2, 3, and 4, respectively (p for trend, 0.0001). The mean AS (number of positive antibodies) and QS was higher for patients with at least one NOD2 variant versus those with no variant (2.28+/−1.21 and 10.70+/−2.99 vs. 1.90+/−1.23 and 9.68+/−2.97, respectively. P, 0.0001).
Individuals with Crohn's disease who have variants of the NOD2 gene as a marker of abnormal innate immunity are more likely to have an increased adaptive immune response to multiple enteric organisms. The data provides a pathophysiologic link to similar findings in rodent mucosal inflammation. This allows disease relevant crossover genetic and functional studies.
TLR8
The inventors examined a case-control cohort consisting of 763 Crohn's Disease patients, 351 ulcerative colitis patients, and 254 control patients. The patients were genotyped using Illumina technology. SNPs were chosen to tag common Caucasian haplotypes using information from the Innate Immunity PGA.
Both a “risk” and a “protective” TLR8 haplotype were associated with CD in females (risk haplotype (H3): 18% of CD subjects had H3 compared with 8.9% of control subjects; protective haplotype (H2): 59% of CD subjects had H2 compared to 72% of control subjects). No significant association with TLR8 and CD in males was observed. H2 was also associated with UC in females (59% of UC females had H2 compared with 72% of controls, p=0.024) as well as males (32% of UC males had H2 compared with 47% of controls, p=0.009).
TLR8 haplotypes as described herein utilize data from the published Innate Immunity PGA collaboration.
TLR8 is an X-linked IBD susceptibility gene, with common haplotypes predisposing and protecting. The associations further emphasize the importance of gene variation in innate immunity as genetic determinants, not only of CD, but of UC as well.
TLR2
The inventors studied if the relationship between variants in innate immune receptors and sero-reactivity to microbial antigens differed in Jewish (J) versus non-Jewish (NJ) patients with CD. Sera from 731 CD patients (282 J, 449 NJ) was tested for ASCA, anti-I2, anti-OmpC, and anti-CBir1 by ELISA while DNA was tested for five TLR2, two TLR4, and two TLR9 variants. The magnitude of responses to microbial antigens was examined according to variant status. Overall quartile sums (QS) (ranging from 4-16) of levels for all four antibodies were calculated as previously described (Mow et at Gastro 2004; 126:414).
There is no association between any TLR4 or 9 variant and sero-reactivity to microbial antigens in Jewish or non-Jewish patients with CD. There is an association between the non-synonymous, non-conservative P631H variant of TLR2 and ASCA positivity in Jewish patients (OR 2.75, p for interaction=0.01). There is an association between the P631H variant of TLR2 and cumulative quantitative response to microbial antigens in Jewish patients with CD. QS were clustered into four groups by increasing cumulative quantitative immune response (group 1=4-6, group 2=7-9, group 3=10-13, and group 4=14-16). The frequency of carriage of the P631H variant of TLR2 increased in parallel with QS cluster in Jewish patients; 2.86%, 3.70%, 7.02%, and 13.46% in groups 1, 2, 3, and 4, respectively (p for trend=0.03). No similar association is found in non-Jewish patients; 7.14%, 10.42%, 6.67%, and 5.45% in groups 1, 2, 3, and 4, respectively (p for trend=0.40).
Jewish, but not non-Jewish patients with CD who carry the P631H variant of TLR2 have increased sero-reactivity to microbial antigens. The data adds evidence to the paradigm that, in CD, innate immune defects lead to enhanced adaptive immune response to microbial antigens. The differential response to the same genetic variant in two different populations shows a possible gene-gene interaction consistent with the multigenic nature of CD.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children
Crohn's disease (CD) is a heterogeneous disorder characterized by diverse clinical phenotypes (inflammatory, fibrostenosing [FS], internal penetrating [IP]) that appear to be influenced by genetic and immune factors. Children frequently manifest an aggressive disease course, and the ability to identify those at risk for complicated disease at diagnosis would be invaluable in guiding initial therapy.
The inventors examined the association of serological immune responses and CARD15 with CD phenotype in a large well-characterized pediatric collaborative cohort. Sera were collected from 797 prospectively followed pediatric CD cases and tested for immune responses to microbial antigens: anti-Cbir1 (flagellin), anti-outer membrane protein C (anti-OmpC) and anti-Saccharomyces-cerevisiae (ASCA) using ELISA. Genotyping (TaqmanMGB) was performed for 3 CD-associated variants of CARD15 (SNPs 8, 12, 13). Disease phenotypes were determined blinded to genotype and immune responses. Associations between immune responses, CARD15 and clinical phenotype were evaluated.
CARD15 variants and immune responses were present in 34% and 78%, respectively. Small bowel (SB) location, IP and/or FS disease behavior were present in 68% (n=542) and 20% (n=152) of children after a median follow-up of 31 months. The odds of developing IP and/or FS disease were highest in patients positive for all 3 immune responses (Table 9). The highest level for each individual antibody was associated with IP and/or FS with the odds being highest when using the sum of all immune response levels (Table 10). Multivariate analysis confirmed the Anti-OmpC (p<0.0002) and anti-Cbir1 (p=0.005) association with IP as well as ASCA (p=0.02) and anti-Cbir1 (p=0.04) with FS. CARD15 was associated with small bowel disease (OR=1.7; p<0.0001) only, not with disease behavior. The rate of complicated CD increases in children as the number and magnitude of immune reactivity increases. Baseline immune response assessment may identify children at risk for complicating IP/FS phenotypes, for whom early, aggressive immunomodulatory therapy could be of benefit.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression
Crohn's disease (CD) is a heterogeneous disorder characterized by diverse clinical phenotypes. Childhood-onset CD has been described as a more aggressive phenotype. Genetic and immune factors may influence disease phenotype and clinical course. The inventors examined the association of immune responses to microbial antigens with disease behavior and prospectively determined the influence of immune reactivity on disease progression in pediatric CD patients.
Sera were collected from 196 pediatric CD cases and tested for immune responses: anti-I2, anti-outer membrane protein C (anti-OmpC), anti-Cbir1 flagellin (anti-CBir1), and anti-Saccharomyces-cerevisiae (ASCA) using ELISA. Associations between Immune responses and clinical phenotype were evaluated.
Fifty-eight patients (28%) developed internal penetrating and/or stricturing (IP/S) disease after a median follow-up of 18 months. Both anti-OmpC (p<0.0006) and anti-12 (p<0.003) were associated with IP/S disease. The frequency of IP/S disease increased with increasing number of immune responses (p trend=0.002). The odds of developing IP/S disease were highest in patients positive for all four immune responses (OR (95% CI): 11 (1.5-80.4); p=0.03). Pediatric CD patients positive for ≥1 immune response progressed to IP/S disease sooner after diagnosis as compared to those negative for all immune responses (p<0.03).
The presence and magnitude of Immune responses to microbial antigens are significantly associated with more aggressive disease phenotypes among children with CO. This demonstrates that the time to develop a disease complication in children is significantly faster in the presence of immune reactivity, thereby predicting disease progression to more aggressive disease phenotypes among pediatric CD patients.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression: Patient Population
Pediatric CD patients were enrolled from participating sites of the Western Regional Pediatric IBD Research Alliance. In order to be eligible, all CD patients must have undergone complete colonoscopy with ileal intubation or complete colonoscopy and small bowel follow through. A diagnosis of CD for this study required at least two of the following: (1) history of abdominal pain, weight loss, short stature, malaise, rectal bleeding, or diarrhea; (2) characteristic endoscopic findings of discontinuous ulcerations, cobblestoning, fistula, or severe perianal disease; (3) radiologic features of stricture, fistula, or evidence of cobblestoning, or ulceration of the mucosa; (4) macroscopic appearance at laparotomy of typical bowel wall induration, mesenteric lymphadenopathy, or serosal involvement showing creeping fat, or other inflammatory changes; (5) histopathology showing transmural inflammatory cell infiltrate or epithelial granulomas and absence of identifiable infectious agents (16). Blood for serological analysis was drawn at each of the participating sites and sent via overnight FedEx to the Genotyping Core Facility of the Medical Genetics Institute/GCRC and the Immunobiology Institute at Cedars-Sinai Medical Center (CSMC). This study was approved by the Ethics Review Board at each participating site.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression: Data Collection
Subjects and their families completed patient demographic forms at the time of blood draw and physicians completed clinical information forms in reference to both date of diagnosis and date of last follow-up. Once collected, all data were then transferred and stored in a secure relational (Oracle) database for analysis. For the purpose of this study, key variables included date of diagnosis, age at diagnosis, date of last follow-up and duration of disease as of last follow-up, ethnicity, family history, disease location, disease behavior, granulomas, and surgical procedures.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression: Phenotype
All phenotype assessments were performed by clinical investigators blinded to genetic and immune response analysis and based on the following uniform definitions:
Disease location at diagnosis was defined by the extent of the disease involvement at the time of initial presentation. Disease extent was based on endoscopic, histologic, and radiographic evidence of inflammation.
Disease location as of last follow-up was defined by the maximal extent of the disease involvement at the point of last follow-up or before a patient underwent first resection. Other than anal/perianal disease, location change was documented when clinically indicated investigations were performed anytime from diagnosis until the date of last follow-up. For the purpose of analysis, disease location as of last follow-up was used for all genotype/immune response-phenotype associations.
There were five disease locations that patients were categorized into (1) small bowel only: disease of the small bowel proximal to the cecum and distal to the ligament of treitz; (2) large bowel only: any colonic location between the cecum and rectum with no small bowel disease; (3) small and large bowel: disease of the small bowel and any location between the cecum and rectum; (4) upper digestive tract disease involving at least one of the following sites: esophagus, stomach. and duodenum; (5) anal: perianal and anal lesions including skin tags and anal ulcers. Patients could have been in more than one category such that patients with small and/or large bowel disease may also have concomitant upper tract and/or anal disease.
Disease behavior at diagnosis was defined by the behavior of the disease at presentation.
Disease behavior as of last follow-up was defined by the disease behavior observed as of last follow-up. At both time points, data may have been obtained after a patient underwent a surgical resection, as reliable data are often obtained at the time of surgery for defining complicated disease behaviors.
Disease behavior was divided into two broad categories: noncomplicating and complicating disease behaviors. Noncomplicating behavior referred to uncomplicated inflammatory disease without evidence of stricturing or penetrating disease behaviors (nonpenetrating nonstricturing [NPNS]). Complicating behaviors referred to penetrating and stricturing disease. (1) Stricturing disease was defined as the occurrence of constant Iuminal narrowing demonstrated by radiologic, endoscopic, or surgical examination combined with pre-stenotic dilatation and/or obstructive signs or symptoms. (2) Penetrating disease was defined as either IP if patients had evidence of entero-enteric or entero-vesicular fistulae, intraabdominal abscesses, or intestinal perforation, or perianal penetrating (PP) if patients developed either perianal fistulae or abscesses or recto-vaginal or ano-vaginal fistulae.
For the purpose of analysis, stricturing and IP complications were grouped into one outcome. PP and patients without complications (NPNS) comprised the other two comparison groups.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression: Immune Responses
All blood samples were taken at the time of consent and enrollment. Sera were analyzed for expression of ASCA, antiOmpC, anti-12, and anti-CBir1 antibodies in a blinded fashion by ELISA. Analysis and IgG and IgA ASCA were performed at Cedars-Sinai Medical Center or Prometheus Laboratories using the same technology. All assays for anti-OmpC, anti-I2, and anti-CBir1 were performed at Cedars-Sinai. Antibody levels were determined and results expressed as ELISA units (EU/mL), which are relative to a Cedars-Sinai Laboratory (IgA-12, IgA-OmpC, and IgG CBir1) or a Prometheus Laboratories Standard (IgA and IgG ASCA), which is derived from a pool of patient sera with well-characterized disease found to have reactivity to this antigen.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Predict Disease Progression: Statistical Analysis
To determine the associations between disease phenotype characteristics and antibody responses toward microbial antigens, univariate analyses using chi-squared tests were performed. Odds ratios (OR) and 95% confidence intervals were calculated to compare the odds of positive serum reactivity toward the microbial antigens (CBir1,I2. OmpC. and ASCA) in the group of patients with a certain disease characteristic with the group of patients without such a characteristic. Quantitative comparison of immune response levels between groups (IP/S+vs IP/S−) for each antibody was performed using nonparametric Wilcoxin rank test. Multivariate analysis with logistic regression modeling was also performed to determine the primary associations among qualitative serological responses with disease phenotypes. To compare the length of time to the development of a disease complication between groups, Kaplan-Meier estimator of survival probability was calculated to construct survival curves. The log-rank test was used to test if the survival curves were significantly different between subgroups of patients. All analyses were performed by using Statistical Analysis Software (Version 8.02. SAS Institute, Inc., Cary N.C.).
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Patient Population Results
A total of 196 pediatric CD patients were eligible for analysis. Eighty-five percent (168/196) were Caucasians and 28% were of Jewish background. The median age at diagnosis was 12 yr (1-18) and the median age at study was 13 yr (4-19). The cohort comprised 47% males and 53% females. A positive family history of IBD was reported in 29% of patients.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Clinical Phenotypes Results
A total of 38 (19%) patients had either a stricturing and/or penetrating complication at the time of diagnosis. After a median follow-up time (median disease duration as of last follow-up) of 18 months (1-200), the total number of pediatric CD patients who experienced a disease complication increased to 58 (30%). Table 11 details the clinical phenotypes of the pediatric CD cohort. Of the 35 patients with internal penetrating and/or stricturing (IP/S) disease, 18 had isolated stricturing disease, 11 had IP and 6 had both complications. Thirty-two of the 58 patients (55%) underwent a combined total of 53 surgeries related to disease complications, 38 (72%) of which were small bowel surgeries for IP/S disease complications. The remaining surgeries were for perianal perforating diseases. All but two patients (15/17) with IP disease and 45% of patients with isolated stricturing disease underwent small bowel surgery as of last follow-up.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses Results
Serum was collected at a median of 9.4 months (0-211.7) after diagnosis, 18% of patients (35/196) had serum collected at the time of diagnosis or within 1 month of diagnosis and 33% (64/196) within 3 months of diagnosis. A total of 77.0% of patients were positive for at least one immune response, 23.7% of which were positive for a combination of any two immune responses. 16.4% of patients were positive for all three responses, and 3.4% were positive for al 1 four responses. ASCA anti-12, anti-OmpC, and anti-CBir1 were present in 43%, 26%, 22%, and 53%, respectively.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Immune Responses and CD Phenotypes Results
Presence and magnitude of immune responses influence disease behavior. A statistically significant association was not found for any of the immune responses with family history, ethnicity, or the presence of granulomas. ASCA was the only antibody significantly associated with small bowel disease location; yet was not associated with disease behavior. Both anti-12 (p=0.0034) and anti-OmpC (p=0.0006) were associated with complicating disease behaviors, more specifically IP/S disease. The frequency of isolated perianal perforating disease was similar between immune response groups (±) for all four antibodies. In addition to the qualitative associations observed for anti-OmpC and anti-12, the magnitude of the immune response to OmpC and I2 also had an association with internal perforation and/or stricturing disease (p=0.008 and p=0.002 for anti-OmpC and anti-I2, respectively). The anti-OmpC association continued to be significant in the multivariate logistic regression, which showed that anti-OmpC (p<0.02) was independently associated with IP/S disease. ASCA, anti-I2, and anti-Cbir1 did not show any independent association with disease behavior.
Cumulative influence of immune responses on disease behavior. Individually there is a clear association with individual immune responses I2 and OmpC with IP/S. The inventors then examined whether there was a cumulative influence of immune responses on disease behavior and determined if the odds of having complicating IP/S disease were greater in the presence of multiple immune responses. As demonstrated, the frequency of IP/S disease significantly increased (p trend=0.002) as the number of immune responses increased. The OR demonstrate that the odds of having IP/S disease was significantly increased in children positive for a combination of any three immune responses (OR [95% CI]; OR=5.5 [1.3-23.6]; p=0.02) and even more so in children positive for all four immune responses (OR [95% CI]; OR=11.0 [1.5-80.4]; p=0.03) as compared to those patients negative for all immune responses (baseline group).
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Disease Progression Results
Based on the cross-sectional data, immune responses are associated with the presence of disease complications. For the second aim of the study, the inventors set out to examine whether seropositive patients (≥1 immune response) have a greater risk to progress to IP/S as compared to seronegative patients (0 immune responses). The inventors used a longitudinal study to answer this question which included only those patients who did not have IP/S at diagnosis (NPNS±PP) and continued to be uncomplicated (NPNS±PP) at the time the serum was collected for immune response measurement so that we could be certain that when clinically recognizable IP/S occurred it was after the sera were collected for antibody measurement. The median time from diagnosis to serum draw was 9.2 months (0-142.3). Among those who developed IP/S (10/167) during the follow-up, the median time from diagnosis to the onset of IP/S was 48 months. As of last follow-up, 8.2% (8/97) of the seropositive group had IP/S versus only 2.9% (2./70) in the seronegative group. Because longer disease duration increases the chance of developing IP/S and not all patients are followed for the same amount of time, the inventors performed survival analysis to take the length of follow-up into consideration. The inventors first evaluated survival with OmpC, I2, and ASCA. Given the same length of follow-up, among those patients positive for at least one serology, more progressed to IP/S than those negative for the three serologies (p=0.03). Saying it differently, those patients positive for at least one serology progressed to IP/S faster than those negative for all three serologies. We then examined whether the addition of Cbir1 changed the survival outcome. Of significance is that the two patients who developed IP/S in the presumptive seronegative group, when measuring 12, OmpC, and ASCA only, were actually CBir1 positive. The inventors have fewer patients followed out long enough in those who had all four antibodies measured. Thus, when the inventors have adequate such numbers these anti-CBIR positive patients would be reclassified to the seropositive group. As of last follow-up, all seronegative patients remained complication free.
Serum Immune Responses Predict Rapid Disease Progression Among Children with Crohn's Disease: Conclusion
The inventors have demonstrated that immune reactivity to specific microbial antigens is associated with complicating disease behaviors. This study demonstrates that immune responses to an increasing number of microbial antigens are associated with complicating IP/S disease behaviors in pediatric CD patients. Moreover, disease progression to a more aggressive disease phenotype in children is accelerated in the presence of immune reactivity. Serum immune responses predict a more rapid disease progression from uncomplicated to complicated disease.
CARD8: A Novel Association with Childhood-Onset Ulcerative Colitis (UC)
CARD proteins play an important role in apoptosis and cytokine regulation, including NfKB, processes which are important in the pathogenesis of IBD. CARD15/NOD2 was the first novel gene reported to confer Crohn's disease (CD) susceptibility and influence disease phenotype. CARD4 has not been found to be associated with CD. McGovern et al reported a significant CD association with the CARD8/TUCAN/CARDINAL gene toured at 19q13.3 in adult patients.
The inventors investigated the association of the CARD8-T10C polymorphism with susceptibility to UC and CD in children. DNA was collected from 342 subjects (75 CD trios, 39 UC trios). Both parents and the affected child were genotyped for 3 allelic variants of the CARD15 gene (R702W. G908R, 1007insC, also referred to as SNP 8, 12 and 13) as an association control and 1 variant of the CARD gene (T10C) using Taqman technology. The transmission disequilibrium test (TDT) was used to test association with either UC or CD using GENEHUNTER 2.0.
CARD8 allele T was present in 63% of CD patients and 77% of UC patients. CARD15 frequency (any variant) was 25% and 11% in CD and in UC, respectively. Similar frequencies were observed for parents for both genes. As expected, transmission distortion was seen for all CARD15 variants in CD, but not in UC. No association was observed between CARD8 and CD, however, in contrast, TDT showed a highly significant association with UC, with over transmission of the CARD8 common allele (Table 12).
This shows a CARD8 association with childhood-onset UC. The over transmission of the common allele in this analysis is similar to that which is seen with PPARgamma in type 2 diabetes and the insulin gene polymorphism in type 1 diabetes. These findings are in contrast to the adult CD association showing different mechanisms for pediatric IBD.
Antibodies to a Novel Flagellin (CBIR1) Adds Clinical Utility to the Diagnosis and Differentiation of Pediatric IBD
Approximately ⅔ of IBD patients are positive for antibodies to microbial and auto-antigens. A novel antibody, anti-Cbir1, may have unique diagnostic properties and phenotypic associations in children. The inventors examined the added utility of anti-Cbir1 in the diagnosis and differentiation of pediatric IBD patients as compared to previously defined antibodies: ASCA, OmpC, 12 and pANCA.
Sera from 331 pediatric IBD patients (111 UC, 220 CD) were tested by ELISA for anti-OmpC, anti-I2, ASCA, anti-Cbir1 and pANCA. Quantitative and qualitative expression of antibody markers was evaluated. Anti-Cbir1 was present in 55% of CD vs. 15% of UC (p<0.001). 41% of anti-Cbir1 (+) UC patients were also positive for >1 CD-related antibody. Anti-Cbir1 was present in 53% of ASCA(−) CD patients and in 52% (31/60) of patients negative for all antibodies. The most Cbir1 reactive CD subset was OmpC+/I2+(74% median=49) and least reactive was ASCA+(56%, median=31). 13.5% of pANCA (+) only UC patients were anti-CBir1 (+) as compared to 35% of pANCA(+) only CD patients (p=0.03). Both pANCA and anti-Cbir1 levels were higher in pANCA (+) CD vs. UC (median pANCA: 46.6 vs. 70.0: p=0.003, and median anti-Cbir1: 21 vs. 12 p<0.0001).
Anti-Cbir1 increased detection of CD cases negative for all other antibodies. Cbir1 reactivity added to the differentiation of pANCA+ CD from pANCA+ UC and can minimize misdiagnosed CD colitis patients. Both the presence and magnitude of anti-Cbir1 reactivity adds to the clinical utility of presently known antibodies in pediatric IBD.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children
The inventors determined whether immune responses and/or CARD 15 variants are associated with complicated disease phenotypes and predict disease progression. Sera were collected prospectively from 796 pediatric CD cases and tested for anti-Cbir1 (flagellin), anti-outer membrane protein C (anti-OmpC), anti-Saccharomyces-cerevisiae (ASCA) and perinuclear anti-neutrophil cytoplasmic antibody (pANCA) using ELISA. Genotyping (TaqmanMGB) was performed for 3 CARD15 variants (SNPs 8, 12, 13). Associations between immune responses (antibody sum (AS) and quartile sum score (QSS), CARD15, and clinical phenotype were evaluated. All phenotype assessments were performed by clinical investigators blinded to genetic and immune response analysis.
32% of patients developed at least one disease complication within a median of 32 months and 18% underwent surgery. 73% of patients were positive for at least 1 immune response. The frequency of IP, S and surgery significantly increased (p trend<0.0001 for all 3 outcomes) with increasing AS and QSS. 9% of seropositive groups had IP/S vs. 2.9% in the seronegative group (p=0.01). 12% of seropositive groups underwent surgery vs. 2% in the seronegative group (p=0.0001). The highest AS group and QSS group demonstrated the most rapid disease progression (p<0.0001). Increased hazard ratio was observed for AS group 3 (7.8 [2.2-28.7] p<0.002 and QSS group 4 (11.0 [1.5,83.0] p<0.02).
The inventors found that the rate of complicated CD increases in children as the number and magnitude of immune reactivity increases. Disease progression is significantly faster in children expressing immune reactivity. Baseline immune response assessment predict children at risk for complicating IP/S phenotypes, in whom early effective therapy would be of benefit.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Patient Population
Pediatric CD patients were enrolled from 21 participating sites of the Western Regional Pediatric IBD Research Alliance, The Pediatric IBD Collaborative Research Group and the Wisconsin Pediatric IBD Alliance.
In order for pediatric CD patients to be eligible, all CD patients must have undergone complete colonoscopy with ileal intubation or complete colonoscopy and small bowel follow through. A diagnosis of CD was based on standard diagnostic criteria. Blood for serological analysis was drawn and sent to The Immunobiology Institute at Cedars-Sinai Medical Center (CSMC) for all sites in the Western Regional and Wisconsin Alliance. Serological analyses were run at Prometheus Laboratories (San Diego, Calif.) for all patients drawn at sites of the Pediatric IBD Collaborative Research Group. Genotyping was performed by the Genotyping Core Facility of the Medical Genetics Institute/GCRC at CSMC for all Western Regional sites, at the Children's Hospital of Wisconsin (SK) for the Wisconsin Alliance, and at Prometheus Laboratories for all sites of The Pediatric IBD Collaborative Research Group.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Disease Location
Disease location was defined by the extent of the disease involvement at the time of initial presentation. Disease extent was based on endoscopic, histologic and radiographic evidence of inflammation.
There were 5 disease locations that patients were categorized into: 1) Small bowel only: disease of the small bowel proximal to the cecum and distal to the ligament of treitz; 2) Large bowel only: any colonic location between cecum and rectum with no small bowel disease; 3) Small and large bowel: disease of the small bowel and any location between cecum and rectum; 4) Upper digestive tract: disease involving at least one of the following sites: esophagus, stomach, duodenum; 5) Anal: perianal and anal lesions including skin tags and anal ulcers. Patients could have been in more than one category such that patients with small and/or large bowel disease may also have concomitant upper tract and/or anal disease.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Disease Behavior
Disease behavior at diagnosis was defined by the behavior of the disease at presentation. Disease behavior as of last follow-up was defined by the disease behavior observed as of last follow-up. At both time points, data may have been obtained after a patient underwent a surgical resection, as reliable data is often obtained at the time of surgery for defining complicated disease behaviors.
Disease behavior was divided into 2 broad categories: non-complicating and complicating disease behaviors: non-complicating behavior: referred to uncomplicated inflammatory disease without evidence of stricturing or penetrating disease behaviors (non-stricturing non-penetrating [NPNS]). Complicating behaviors referred to penetrating and stricturing disease. 1) Stricturing disease (S): was defined as the occurrence of constant luminal narrowing demonstrated by radiologic, endoscopic or surgical examination combined with pre-stenotic dilatation and/or obstructive signs or symptoms. 2) Penetrating disease: was defined as either internal penetrating (IP) if patients had evidence of entero-enteric or entero-vesicular fistulae, intra-abdominal abscesses or intestinal perforation or perianal penetrating (PP) if patients developed either perianal fistulae or abscesses or recto-vaginal or ano-vaginal fistulae.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Immune Responses
All blood samples were taken at the time of consent and enrollment. Sera were analyzed for expression of pANCA, ASCA, anti-OmpC, and anti-CBir1 antibodies in a blinded fashion by ELISA. Serological analyses were performed at CSMC or Prometheus Laboratories using the same technology. Antibody levels were determined and results expressed as ELISA units (EU/ml), which are relative to a Cedars-Sinai Laboratory or a Prometheus Laboratories Standard which is derived from a pool of patient sera with well-characterized disease found to have reactivity to this antigen.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Definitions of Immune Responses
The following definitions were used for all analyses involving ASCA, anti-OmpC and anti-CBir1 immune responses. pANCA was analyzed separately given that pANCA has been shown to be negatively associated with the majority of disease phenotypes except large bowel disease location.
Antibody sum (AS): number of positive antibodies per individual: 0, or 1 or 2, or 3 positive.
Antibody Quartile Score: quartile score for each antibody level (<25%=1, 25-50%=2, 51%-<75%=3, 75%-100%=4).
Quartile Sum Score(QSS): sum of quartiles score for all 3 antibodies (ASCA (A or G, anti-OmpC and anti-CBir1). Minimum score of 3 (all antibodies had a quartile score of 1) and maximum score of 12 (all antibodies had a quartile score of 4).
Quartile Sum Score (QSS) Group: In order to minimize the number of patient subsets i.e. quartile sum score 3-12, the inventors regrouped patients based on a range of quartile sum scores: Quartile sum score 3-5=group 1, 6-7=group 2, 8-9=group 3 and 10-12=group 4.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Genotyping
Three single nucleotide polymorphisms (SNP's) in the CARD15 gene have been associated with CD. CARD15 SNP's R675W (rs2066844, CEPH-IBD1-snp8), G881R (rs2066845, CEPH-IBD1-snp12), and 3020insC (rs2066847, CEPH-IBD1-snp13) were adapted to the TaqMan MGB genotyping platform following the manufacturer's instructions and using PrimerExpress design software (Applied Biosystems, Foster City, Calif.). The TaqMan MGB platform is a two-probe, 5′-exonuclease PCR assay that employs a minor groove binder on the 3′-end of the probes in order to give greater allele discrimination.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Statistical Analysis
To determine the associations between disease phenotype characteristics and antibody responses toward microbial antigens, univariate analyses using χ2 tests were performed. Odds ratios (OR) and 95% confidence intervals were calculated to compare the odds of positive serum reactivity (antibody sum, quartile sum score, quartile sum score group) towards the microbial antigens (CBir1, OmpC, and ASCA) in the group of patients with a certain disease characteristic with the group of patients without such a characteristic. For the OR calculations the minimum antibody sum of 0, the minimum quartile sum score of 3 and the minimal quartile sum score group 1 were set as baseline, i.e. OR of 1.0 Quantitative comparison of immune response levels between groups (IP/S+ vs. IP/S−) for each antibody was performed using non-parametric Wilcoxin Rank test. Stepwise multivariable analysis using logistic regression modeling was also performed to determine the primary associations among qualitative serological responses with disease phenotypes. To compare the length of time to the development of a disease complication between groups, Kaplan-Meier estimator of survival probability was calculated to construct survival curves. The log-rank test was used to test if the survival curves were significantly different between subgroups of patients. The hazard ratio (HR) of occurrence of complication or surgery among patients who were sera positive compared to those who were sera negative as well as who were in higher antibody sum or quartile sum group compared to those who were in baseline group were estimated from Cox's proportional hazards model and adjusted for all other covariates. All HRs were expressed as a point estimate with 95% confidence interval. Patients who only had sera data after the occurrence of complications or surgery were not included in the survival analysis. Age at diagnosis and gender were included as covariates in all the multivariable analyses. The OR/HR for age at diagnosis was explained as the times of odds/hazards increase (e.g. OR-1) per one year older at diagnosis. All analyses were performed by using Statistical Analysis Software (Version 9.1; SAS Institute, Inc., Cary, N.C.).
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Patient Demographics
A total of 796 pediatric CD patients were eligible for analysis. Eighty-seven percent (694/796) were Caucasians and 28% were of Jewish background. The median age at diagnosis was 12 [0.6-18] years and the median disease duration as of last follow up was of 32 [1-235] months. The cohort was comprised of 56% males and 44% females.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Clinical Phenotypes
A total of 236 (30.3%) patients presented with (96/796 [12%]) or developed (140/796 [18%]) at least one disease complication within the median follow up time of 32 months: 116 stricturing disease, 70 internal penetrating, and 115 perianal penetrating disease. Ten patients had all 3 complications and 45 had a combination of 2 of the 3 complications. One hundred and forty patients (18%) underwent a CD related surgery of which 89 were small bowel resections. Of the remaining surgeries: a total of 42 were involving perianal penetrating disease; 24 patients underwent colectomy and 3 patients a limited colonic resection. Fifteen patients had more than one surgery.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Immune Response and Genotype Frequencies
Serum was collected at the time of diagnosis or within 1 month of diagnosis in 18% (146/796) of patients and 30% (241/796) within 3 months of diagnosis. The remaining patients had serum collected greater than 3 months from time of diagnosis. A total 73% of patients were positive for at least one microbial driven immune response (ASCA, anti-OmpC or anti-Cbir1), 27% of whom were positive for a combination of any 2 of these immune responses and 8% of patients were positive for all 3 responses. ASCA, anti-OmpC, anti-CBir1 and pANCA were present in 45%, 18%, 52%, and 19% respectively. NOD2/CARD15 (any variant) was observed in 34% of patients (25% heterozygote and 9% homozygote or compound heterozygote).
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Cross Sectional Analyses
Univariate analysis of immune responses and NOD2/CARD15 genotype demonstrated that NOD2/CARD15 (all variants individually or any variant) was only associated with small bowel disease location (OR [95% CI]1.9 [1.4-2.7] p<0.0001) and had no association with disease behavior. ASCA was associated with small bowel disease (2.9 [2.1-4.0] P<0.0001) and perianal disease (1.5 [1.1-2.2]<0.02). C Bir1 was also associated with small bowel disease (1.6 [1.2-2.3] p=0.002) and OmpC had no significant association with any disease location. pANCA was associated with large bowel disease (4.0 [1.8-8.8] p<0.0001). ASCA, anti-CBir1 and anti-OmpC were negatively associated with non-penetrating non stricturing disease (NPNS); in contrast all showed a positive association with complicating disease and surgery. The odds of having internal penetrating (IP), perianal penetrating (PP), stricturing (S) disease and surgery were highest in the presence of anti-OmpC. As disclosed herein, there was a cumulative influence of number of immune responses (antibody sum) as well as the magnitude of the immune response (quartile sum score group) on disease behavior. The frequency of internal penetrating, stricturing disease and surgery significantly increased (p trend<0.0001) as the number of immune responses increased (antibody sum 0-3) and magnitude of immune response (quartile sum score group 1-4) increased. The odds ratios for the 3 disease behaviors and surgery associated with antibody sum and quartile sum score groups are disclosed herein.
Multivariable analysis confirmed the association of small bowel location with ASCA (OR [95% CI]:2.3 [1.6-3.2]; p<0.0001), anti-CBir1 (OR 1.5 [1-1.2]; p=0.03), pANCA (OR: 0.6 [0.4-0.9]; p=<0.007); and NOD2/CARD15 (OR; 1.7 [1.1-2.4]; p=0.007). Large bowel location was associated with pANCA (OR: 2.8 [1.4-5.4]; p<0.004). Results of the multivariable analysis for the independent associations with disease behavior and surgery are disclosed herein. All individual antibodies were included in the model as well as a single unit change in antibody quartile sum score as a co-variable (e.g. increase in score of 3 to 4). There was a significant association seen with quartile sum score change and complicating disease behaviors as well as surgery, such that for each unit of quartile sum increase the OR increased by 1.3 for internal penetrating and stricturing disease and 1.2 for surgery. The difference between a score of the minimum 3 and the maximum score of 12 equates to an OR of 10.6 (=1.3)9 and 5.2 (=1.2)9, respectively. Quartile sum score was not independently associated with small bowel disease location as compared to the presence of the individual antibodies as noted above. These results show that disease location is associated more so with the presence of the immune responses and less so by the antibody levels, whilst disease behavior and surgery are more significantly associated with the magnitude of the immune response. Additional independent associations were found between female gender and older age at diagnosis.
Increased Immune Reactivity Predicts Aggressive Complicating Crohn's Disease in Children: Predictors of Disease Progression
The inventors' cross-sectional data demonstrate that both single and multiple immune responses are associated with the presence of disease complications and surgery. For the second aim of the study, the inventors set out to examine whether seropositive patients (1, 2, or 3 positive for ASCA, anti-OmpC and/or anti-CBir1) had a greater risk to progress to internal penetrating and/or stricturing (IP/S) disease as well as to surgery, as compared to seronegative patients (0 such immune responses). The inventors used a longitudinal study to answer this question which included only those patients who did not have IP/S or surgery at diagnosis (NPNS+/−PP) and continued to have uncomplicated disease status at the time the serum was collected for immune response measurement. Thus the inventors could be certain that in these individuals, when clinically recognizable IP/S or surgery occurred, it did so after the serum was collected. A total of 536 patients met these inclusion criteria. The median time from diagnosis to serum draw was 10 [0-211] months for the 536 patients included in the prospective analysis. A total of 90 of the entire prospective cohort of patients (n=536) developed IP/S in follow up; however 59% (53 patients) were eliminated from this analysis as they had immune responses collected after the complication occurred. Among the 37 patients who developed IP/S during the follow-up after serum was drawn, the median [range] time from diagnosis to the onset of IP/S was 26 [4-108] months. Thirty two of the 363 seropositive patients (9%) had IP/S vs. only 2.9% (5/173). in the seronegative group (p=0.01). Among the 61 patients who underwent surgery (any CD related surgery after serum was drawn) the median [range] time from diagnosis to surgery was 30 [1-105] months. Twelve percent (57/464) of the seropositive (at least one positive) patients had undergone surgery vs. only 2% (4/189) in the seronegative group (p=0.0001). Because longer disease duration increases the chance of developing IP/S as well as surgery, and not all patients were followed for the same amount of time, we performed survival analysis to take the length of follow-up into consideration. The Kaplan-Meier survival analysis, followed by the log-rank test for the different antibody sum and quartile sum score group comparisons, showed that overall survival times for IP/S and CD-related surgery were significantly lower for those positive for immune responses, and this was true when both the quantity of immune responses and magnitude of those responses were assessed. The first analyses examined antibody sum: 0 vs. 1 vs. 2 vs. 3 and time to development of IP and/or S as well as time to surgery. Given the same length of follow up, among those patients with antibody sum greater than 1, more progressed to IP/S than those negative for all 3 or positive for only 1 antibody (p=<0.0001). In other words, those patients positive for at least 2 immune responses (antibody sum 2 or 3) progressed to IP/S faster that those negative for all or positive for only 1 antibody. The group positive for all 3 antibodies demonstrated the most rapid disease progression with a median [range] time to disease progression of 20 [4-65] months. The same rapid progression to surgery was seen among the higher antibody sum group. Like antibody sum, those patients in the highest quartile sum score group (group 4=Quartile sum score 10-12) progressed faster to IP/S and surgery and the median [range] time to IP/S and surgery was 21 [4-65] months and 27 [1-93] months, respectively. The survival curves were very similar when evaluating intestinal resection only (n=48) as compared to any CD surgery (n=61) (Log Rank: p<0.0001 for the 4 antibody sum groups and p=0.001 when comparing survival among the 4 quartile sum groups). The most conservative way to evaluate the predictive abilities of immune response was to limit inclusion in the survival analysis to only patients whose serum was drawn before a complication or surgery. The inventors also performed survival analysis on all 90 patients who developed IP and/or S in follow up regardless of when serologies were drawn. For both antibody sum and quartile sum score group, the results showed a significantly higher number of patients progressing to complication faster in the face of seropositivity.
The predictive ability of immune responses for rapid progression to the first IP/S or surgical event was further evaluated by fitting Cox-proportional hazards models. OmpC (HR [95% CI]; p value) (2.4 [1.2-4.9]; p=0.01) and CBir1 (2.5 [1.2-5.2]; p<0.02), but not ASCA, were associated with increased hazard of IP/S, as was older age at diagnosis (1.2 [1.1-1.3]; p=0.004). Lower hazards were observed with pANCA positivity (0.16 [0.04-0.70]; p<0.02). Antibody sums 2 and 3 as well as quartile sum score groups 3 and 4 were associated with an increased hazard for developing disease complications (IP/S). Hazard Ratios for all CD related surgeries as well as for intestinal resections only were calculated controlling for both disease location and disease complication (IP, S and PP). OmpC was associated with increased hazard of any CD related surgery (2.2 [1.3-3.8;] p=0.004 or intestinal resection surgery (3.5 [1.9-6.4]; p=0.001). The Cox proportional hazard model also tested the predictive ability of antibody sum groups and quartile sum score groups for surgery. Results of any CD related surgery are disclosed herein. When examining intestinal resection surgery, an increased hazard was observed for antibody sum 3 (7.8 [2.2-28.7]; p<0.002 and quartile sum score group 4 (11.0 [1.5-83.0]; p<0.02).
The inventors investigated the role genetic variants in the gene JAK3 may have in the development of Crohn's Disease. The inventors performed an antibody genome wide association study using patients diagnosed with Crohn's Disease, and found an association of JAK3 variants with expression of anti-I2 and ASCA for Crohn's Disease. The results of these studies are described in Tables 13-31 herein.
Results demonstrating the association of anti-I2 as positive/negative expression with JAK3 SNP rs2302600 (SEQ ID NO: 37) as a result of GWAS. Mantel-Haenszel Chi-Square statistics for the degree of freedom (DF), value and probability of anti-I2 antibody expression associated with genotype alleles AA, CA and CC for SEQ ID NO: 37 at the JAK3 genetic locus (Table 13).
Results demonstrating the association of anti-I2 with JAK3 SNP rs2302600 (SEQ ID NO: 37) under dominant genetic model (Table 14).
Results demonstrating the association of ASCA with JAK3 SNP rs2302600 (SEQ ID NO: 37) under dominant genetic model (Table 15).
Results demonstrating the association of JAK3 variant rs2302600 (SEQ ID NO: 37) with anti-I2 level in Crohn's Disease patients (Table 16).
Results demonstrating the association of JAK3 variant rs2302600 (SEQ ID NO: 37) with ASCA level in Crohn's Disease patients (Table 17).
Results demonstrating the association of ASCA as positive/negative expression with JAK3 SNP rs3212741 (SEQ ID NO: 38) as a result of GWAS. Mantel-Haenszel Chi-Square statistics for the degree of freedom (DF), value and probability of ASCA antibody expression associated with genotype alleles CC, TC, and TT for SEQ ID NO: 38 at the JAK3 genetic locus (Table 18).
Results demonstrating the association of JAK3 SNP rs3212741 (SEQ ID NO: 38) under dominant genetic model (Table 19).
Results demonstrating the association of JAK3 variant rs3212741 (SEQ ID NO: 38) with ASCA level in Crohn's Disease patients (Table 20).
While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should typically be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, typically means at least two recitations, or two or more recitations).
Accordingly, the invention is not limited except as by the appended claims.
This application is a continuation-in-part of U.S. Ser. No. 11/720,785 filed on Sep. 17, 2007, currently pending, which is a U.S. national stage application of PCT/US2005/044335 filed on Dec. 8, 2005, now expired, which claims priority to U.S. Ser. No. 60/634,339 filed on Dec. 8, 2004; and is a continuation-in-part of U.S. Ser. No. 12/527,376 filed on Sep. 1, 2009, currently pending, which is a U.S. national stage application of PCT/US2008/054033 filed on Feb. 14, 2008, now expired, which claims priority to U.S. Ser. No. 60/889,806 filed on Feb. 14, 2007; and is a continuation-in-part of U.S. Ser. No. 12/529,106 filed on Aug. 28, 2009, currently pending, which is a U.S. national stage application of PCT/US2008/056103 filed on Mar. 6, 2008, now expired, which claims priority to U.S. Ser. No. 60/893,308 filed on Mar. 6, 2007; and is a continuation-in-part of U.S. Ser. No. 13/124,311 filed on Apr. 14, 2011, currently pending, which is a U.S. national stage application of PCT/US2009/061698 filed on Oct. 22, 2009, now expired, which claims priority to U.S. Ser. No. 61/107,590 filed on Oct. 22, 2008; and is a continuation-in-part of U.S. Ser. No. 13/410,881 filed on Mar. 2, 2012, currently pending, which is a U.S. divisional application of U.S. Ser. No. 12/599,549 filed on Dec. 18, 2008 and issued as U.S. Pat. No. 8,153,443 on Apr. 10, 2012, which was a national stage application of PCT/US2008/63202 filed on May 9, 2008, now expired, which claims priority to U.S. Ser. No. 60/917,254 filed on May 10, 2007; and is a continuation-in-part of U.S. Ser. No. 12/196,505 filed on Aug. 22, 2008, currently pending, which is a U.S. continuation application of U.S. Ser. No. 12/032,442 filed on Feb. 15, 2008, now abandoned, which claims priority to U.S. Ser. No. 60/890,429 filed on Feb. 16, 2007, the contents of each of which are herein incorporated by reference in their entirety.
This invention was made with government support under Grant Nos. DK046763, DK071176 and DK066248 awarded by the National Institutes of Health. The U.S. Government may have certain rights in this invention.
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| 60890429 | Feb 2007 | US |
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