10 Antibodies in the exploration of inflammatory bowei disease pathogenesis and disease stratification

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10 Antibodies in the exploration of inflammatory bowei disease pathogenesis and disease stratification

JONATHAN BRAUN, OFFER COHAVYAND MARK EGGENA

Introduction

Normal mucosal homeostasis is the interdependent relationship of epithelium, luminal microorganisms, and the regional immune system (Fig. 1). This tripartite relationship has particularly evolved during the eutherian period, due to the emergence of a dynamic, antigen-specific immune system [1, 2]. The mammalian immune system has an exceptional capacity for specific antigen recognition and immunologic memory to microibal antigens, and for powerful amplification of effector mechanisms against such antigenic targets [3, 4]. These properties obviously require special adaptation to preserve the mucosal-microbial interrelationship essential for normal intestinal function. It is widely understood that inflammatory bowel disease (IBD) involves a chronic disturbance in this homeostasis.

In recent years there has been a particular effort to understand this homeostatic disturbance in the context of immunologic activation and tissue damage. In particular, this effort has been informed by the emerging understanding of Helicobacter pylori pathogenesis in peptic ulcer disease. From this precedent we have learned that the manifestation of clinical disease reflects the interplay of commensal bacterial and host traits, which together affect levels of colonization, and the nature and intensity of inflammation and tissue damage [5, 6]. Our review focuses on the insights provided by IBD-associated antibodies on disease pathogenesis and clinical stratification. We will first consider the nature of immunologic quiescence in the mucosa. We will then assess the concepts and experimental issues regard- ing disease-related antibodies and their antigenic targets. This will be followed by an analysis of the

A.

Luminal Bacteria

Epitheliunni ^ 3 Immune System Homeostasis of Intestine

I

B.

Inflammatory Bowel Disease

Figure 1. Immune activation as a phenotype of disturbed mucosal homeostasis in IBD.

Stephan R. Targan, Fergus Shanahan andLoren C. Karp (eds.), Inflammatory Bowel Disease: From Bench to Bedside, 2nd Edition, 211-222.

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microbial antigens and autoantigens identified by IBD-related antibodies. Finally, we will discuss how these antibodies and antigens are being applied to issues of disease diagnosis, clinical stratification, and strategies for treatment.

The quiescence of the mucosal immune system to locai microbial antigens and autoantigens

The immune system is classically understood to launch strong responses upon encounter with microorganisms. It is likely that such proinflammatory responses reflect programming of nascent lymphocyte and antigen-presenting populations by microbial products [3]. The mechanism of this programming is multifaceted, but clearly includes elements of the innate immunity [4, 7-9]. In contrast, immune responses to microbial products by gastrointestinal lymphocytes are remarkably curtailed. For example, in the human it is difficult to detect native or induced T cell or antibody responses to abundant commensal bacteria, either in the mucosal or peripheral sites [10-14]. Moreover, such studies indicate that this immunologic quiescence may be relatively restricted to autologous bacterial strains, since experimental challenge with heterologous (but not autologous) strains is relatively immunogenic. These findings suggest a state of immune tolerance to antigens encountered in the gastrointestinal environment, including those expressed by commensal bacteria.

Indeed, the apparent tolerogenic consequence of gastrointestinal antigen exposure has been inten- sively studied from the standpoint of therapeutic applications of oral tolerance [15].

Table 1. Autoantigens in IBD Antigen

The mechanism of immunologic quiescence in the mucosa is a fascinating and still unresolved issue (see Chapter 5). Conceptually, this quiescence probably reflects the coordinated contribution of each of the three mucosal elements. For example, certain chemokines and the Goci2-linked signaling cascade suppress IL-12 expression by macrophages and dendritic cells, hence polarizing immune responses away from T H l diff'erentiation [16-19]. Among these, MCP-1 and MIP-loc are constitutively produced by colonic epithelial cells [20], and may thereby contribute to the blunted THl mucosal phenotype. Within the lymphoid compartment itself, subsets of dendritic cells and intraepithelial lymphocytes are likely to play a role, through their multifaceted regulation of ocP T cell function, and production of growth factors preserving epithelial integrity [21-23].

Bacterial traits also contribute to this quiescent environment, presumably reflecting the coevolution of intestinal colonists with immunologic discretion.

Thus Bacteroides and Bifidobactcr species, while the numerically most abundant genera of intestinal (colonic) colonists, are subject to minimal or modest T cell and antibody activity in healthy individuals.

Such responses are typically genus- rather than species-specific, further indicating the absence of specific immune activity elicited by these intestinal colonists [24-27]. Secretory IgA activity can be substantial to some abundant colonic species, and this appears in part to reflect a primitive innate immune recognition [28, 29].

Stable colonization indicates that commensal bacteria are well adapted to such recognition, and emerging evidence indicates that this adaptation includes active bacterial regulation of the innate bacterial recognition process [30-34]. These observa-

Disease

UC, CD subset UC

UC, CD subset

UC, hepatobiliary disease Various

UC Various UC subset CD CD subset

References

7 5 , 8 3 , 1 6 1 - 1 6 3 164-168 169-171 172 173-178

179,180 181-183 184,185 186,187 188,189 pANCA

Tropomyosin Anti-endothelial IgG

50 kDa myeloid-specific nuclear protein Neutrophil granule proteins (cathepsin G,

lactoferrin, BPI, catalase, alpha-enolase) HMG-1 and HMG-2

Histone HI 24,28 kDa protein Hsp60

Pancreatic acinar cell antigen

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tions focus on three important issues for further investigation: (a) bacterial products involved in host recognition; (b) intrinsic bacterial resistance mechanisms to recognition-induced host effector processes; (c) extrinsic bacterial traits which modify specific and innate immune recognition in the intestine. Resolution of these issues is central to important new anti-inflammatory therapeutic strategies, notably probiotics [35].

Tolerance to autoantigens is a parallel immunologic issue to such antibacterial quiescence. A variety of processes contribute to self-tolerance, including central and peripheral clonal deletion, blunted activity of stimulatory receptor pathways, and deviation of effector function [36-38]. These processes relate to IBD pathogenesis in two general ways. First, mechanisms of these tolerance processes should encompass the mechanisms of immune quiescence to commensal bacteria. Indeed, there is substantial evidence that peripheral tolerance in many cases arises through activation and differentiation of auto- reactive lymphocytes in the mucosal environment [39-41]. Second, the recurrent observation of autoimmunity to mucosal antigens in IBD suggests a direct contribution of such activity to intestinal damage.

Disordered mucosal immunity in IBD-susceptibiiity, stimuli, and maricer antibodies

At the tissue level the hallmark of ulcerative colitis (UC) and Crohn's disease (CD) is aberrant immune activity, and the mucosal damage caused by the resultant inflammatory processes (Fig. 2). Accord- ingly, the core issues in IBD pathogenesis are the identity of the stimuli driving the disordered immune function, and the host traits promoting aberrant responses to these stimuli. Genetic approaches have identified a series of loci and candidate genes mod- ifying host susceptibility [42-50]. While the identity of the relevant genes at these loci is unknown, it is likely that some will function directly or indirectly to modify the recognition or effector processes induced by the immunologic stimuli. CD4"^ T cell activity (and its disease-specific effector heterogeneity) is presently accepted as the focal point of the immuno- pathogenic process in IBD, initiating and organizing the effector processes leading to mucosal damage [51-55]. However, T cells pose a formidable chal-

Stimulatory Bacteria Invaders Commensals

\ Damaged IViucosa

Anti-Bacterial T Cells ^ j Cellular Effectors

/

Anti-Bacterial B Cells i ^

Marker Antibodies

Figure 2. Relationship between stimulatory bacteria, tissue- destructive effector mechanisms, and marker antibody production in IBD.

lenge in the identification of their cognate antigenic stimuli. Identification approaches generally require cell-based assays, with the attendant complexities of clonal heterogeneity and assay insensitivity with mixed populations. Some innovations have been developed, using TCR polymorphism analysis and expression cloning technologies [56-59]. However, these have not yet reached the robustness required for evaluation of undefined antigenic systems.

Fortunately, immune responses typically include recruitment of antigen-specific B lymphocytes (Fig.

2). Such recruitment is a consequence of membrane immunoglobulin expression by the B cell, and the highly efficient antigen uptake and presentation mediated by this receptor in antigen-specific B cells [60, 60-65]. As a result, antibodies specific for the major antigenic stimuli of an immune response are commonly produced, even when the humoral response is not central to the immune eff*ector mechanism (e.g. responses predominating with T H l - or CTL-dependent processes). Such antibodies are initially recognized as disease-associated 'marker' antibodies, often detected adventitiously with crossreactive laboratory antigens. However, antibodies are superb analytic reagents, providing a powerful tool for identification of disease-related tissue antigen. The precedent of diabetes mellitus is a striking case where the isolation and use of disease- specific marker antibodies propelled the characterization of antigenic stimuli central to this complex immunologic disease [66, 67].

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Technologically, there are several issues to consider in isolating disease-related marker antibodies. First, the antibody composition of patient sera is complex, and the relevant marker antibodies typically must be isolated to reduce background seroreactivity and increase titer to analytically useful levels. This is most commonly achieved by affinity purification of antigen-specific antibodies from individuals with high-titer antibody levels. The conventional approach is hampered by limited antibody yield (particularly with low-titer antibodies), and reproducibility with different donors due to heterogeneity in fine antigen specificity.

Ahernatively, monoclonal antibodies can be produced, using hybridoma or phage-display technology [68-73]. Such reagents are analytically superior because of renewability and consistency. Use of these clonal approaches requires efficient localization and isolation of marker antibody-producing cells. These requirements are significant challenges for hybridoma preparation, and are compounded by the need for cells which are biologically suitable as fusion partners. The powerful cloning and selection features of phage-display technology overcome some of these difficulties. However, phage-display methods can suffer from unfavorable reassortment of heavy and light chain genes, and donor and avidity skewing of fine specificity in the course of clonal selection.

In the following sections we will review the use of such isolated marker antibodies to identify and characterize their cognate autoantigens and microbial antigens. Together, these molecules and cell types comprise the set of candidate immunologic stimuli predominant in IBD-associated immunity.

Several criteria have been used to assess the signifi- cance of these antigens in disease pathogenesis:

specificity and sensitivity of expression in IBD;

expression in relevant tissue sites; and targeting by regulatory and effector T cells. While these criteria can associate the antigens with IBD, they leave open the critical issue of causation. This issue is addressed by demonstration of these criteria in animal disease models, and direct studies in which immune responses induced by these antigens might elicit disease. At this writing, no antigens have reached this level of experimental validation. We will denote those antigens which fulfill multiple criteria of disease association, and thus can be considered high-priority candidates for an immunologic role in disease.

Autoantigens

pANCA

Perhaps the most widely studied marker antibody in IBD is pANCA - antibodies to a perinuclear neutrophil antigen [74-76]. These antibodies are distinguished from other cANCA and pANCA by sensitivity of their antigen to DNAse I treatment, and localization to the inner nuclear membrane leaflet [77, 78]. Reliable detection of this UC-associated pANCA depends on proper fixation procedures, IgG isotype specificity, and DNAse I sensitivity.

Using these criteria, 60-70% of UC patients are antibody-positive. Antibody levels are relatively stable over time regardless of disease activity, and uncommonly occur in patients with other gastrointestinal diseases. This indicates that they do not represent non-specific markers of general colonic inflammation. In fact, pANCA expression is elevated in unaffected family members, concordant in monozygotic twins, and associated with a distinct MHC II haplotype [79-83]. These observations indicate that pANCA expression is a phenotype associated with immunogenetic susceptibility for UC.

The close association of pANCA expression with disease susceptibility is emphasized by their occurrence m theTCRa '' and C3H/HeJBir colitis -prone mouse strains [84, 85]. It is also notable that pANCA antibodies are characteristic of patients with primary sclerosing cholangitis, in which UC is highly concordant [77, 86, 87]. About 20-25% of CD patients also express pANCA, and analyses of these individuals reveal a CD subset with distinctive genetics and UC-like clinical features [83, 88-93]. The use of pANCA for disease stratification is addressed in the final section.

pANCA-related autoantigens

The search for the pANCA antigen(s) has been a challenging one. The first candidates were granule proteins of the neutrophil, particularly cathepsin G and lactoferrin [88, 89, 94, 95]. Some reports have also described other granule proteins, including bactericidal/permeability-increasing protein (BPI), catalase, and alpha-enolase [96,97]. Disease association studies of these antigens using ELISA and Western analysis indicate a distinct p a t t e r n compared with pANCA, notably their discordance with pANCA levels, correlation with disease activity,

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relatively high occurrence in CD compared to UC, and association with other rheumatologic diseases.

Biochemically, these antigens also would not appear to fulfill the subcellular localization criterion of the pANCA antigen [78]. However, it is notable that antibody levels to granule proteins appear to identify patient subsets with distinct clinical courses, and may thus be a phenotype of the corresponding immunologic traits underlying these clinical manifestations.

A recent study employed high-titer pANCA sera to isolate a myeloid-specific 50 kDa nuclear envelope protein [98]. Western analysis of a large patient serum panel discriminated most patients with UC, primary sclerosing cholangitis, and autoimmune hepatitis. It will be important for this intriguing report to be validated for disease specificity in other laboratories, and further defined biochemically and in colitis model systems.

Nuclear high-mobility group proteins (HMG-1 and 2) [99, 100] and members of the histone HI family [81, 101, 102] are a second category of pANCA antigen candidates. These structurally related nuclear proteins associate with distinct chromatin domains that are localized to the nuclear envelope region, explaining their pANCA staining pattern, and fulfilling this pANCA antigen criterion [103-105]. A core epitope (PKKAK) was identified using a UC-related anti-histone HI monoclonal (phage-display) antibody [102]. Structurally, this epitope is present in both families of nuclear proteins, and is distinct from the C-terminal histone HI epitopes predominating in anti-Hl antibodies associated with SLE, HIV, and rheumatoid arthritis [106-108]. Anti-Hl and anti-HMG antibodies are each expressed in 15-25% of pANCA"^ ulcerative patients, suggesting that these two specificities could account for as much as half of the pANCA antibody response. However, these antibodies do not correlate well with pANCA levels or UC specificity, indicating that they are probably distinct from the predominant UC-pANCA seroreactivity. It is interesting to note that antibodies to HMG-1 and -2 are highly correlated with autoimmune hepatitis, and an epitopically distinct pANCA typical of this disease [77,109].

A 24,28 kDa antigen has been identified with the 5-3 pANCA human monoclonal antibody [110], and found to be expressed in mast cells (including those of mucosal origin), certain ganglionic cells, and pancreatic islets [111, 112]. Seroreactivity to these antigens was observed by Western analysis in UC patients, but this evaluation was limited by the lack

of biochemically isolated and defined antigen for comprehensive studies. This protein(s) does not appear to represent the predominant p A N C A antigen, since it is localized intracellularly as a cytoplasmic granule.

Other autoantigens

Antiepithelial antibodies have been a recurrent focus of investigation in IBD [113]. In recent years tropomyosin has received substantial support as a candidate epithelial antigen [114-118]. Antibodies to this antigen are expressed in the majority of UC patients, and are uncommon in control gastrointestinal disease patients. A tropomyosin epitope, H I A E - DADRK, provided excellent discrimination for this disease-related antibody activity, and such antibodies mediated antibody-dependent cytotoxicity in a tissue culture cell line [114, 118]. Antitropomyosin antibodies have also been identified in TCRoc'^" and G(xi2"^~ colitis-prone mice, and in the latter case precede clinical disease [52, 117, 119]. Using a representative monoclonal antibody, a crossreactive epitope of the appropriate molecular weight (40 kDa) was detected in epithelial cells of tissues involved with extracolonic manifestations of UC (skin, biliary). These observations are striking, and the role of this autoantigenic target in UC pathogenesis clearly deserves broader investigation.

Antibodies to heat-shock proteins, notably hsp60, are elevated in certain CD patients [120]. In the mouse a clonal hsp60-specific CD8 T cell line induced colitis when transferred to recipient mice.

The disease process was TNF-oc-dependent, and inflamed m u c o s a s h o w e d i n c r e a s e d h s p 6 0 expression. These findings are provocative, due to the following implications: hsp60 may be a colitis autoantigen, an amplification loop may occur for pathogenesis through inflammation-mediated antigen up-regulation; and CD8 T cells may function in regulatory or effector roles for chronic colitis [121].

An anti-acinar cytoplasmic granule pancreatic autoantibody has been reported by Seibold and colleagues in about 25% of CD patients [122, 123].

While no biochemical definition of this antigen has been reported, it is apparently distinguished from the 24,28 kDa antigen by pancreatic cell type and IBD subtype (CD versus UC). Antiendothelial antibodies have been reported in about 75% of UC patients and 25% of CD patients [124-126]. Antibody levels were correlated with disease activity, although the antibodies themselves did not appear to be cytotoxic.

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While the antigen remains undefined, the appropriate microanatomic localization and strong disease association recommend them for further study.

Microbial antigens

For more than 15 years investigators have observed elevated IgG seroreactivity in CD patients to a variety of bacterial taxa [10, 127-130]. This is also an immunologic feature of at least one IBD animal model C3H/HeJBir [24]. In that model the antibody response cecal bacteria is accompanied by corresponding antibacterial CD4"^ T cell activity, and such T cell lines can transfer colitis [131]. It is interesting to note that absorption experiments revealed crossreactivity between antibacterial IgG and pANCA activity [85].

Several interpretations have been suggested to explain the diversity of antibacterial recognition in CD. First, formation of such antibodies might be a secondary consequence of disease-related epithelial permeability and excessive luminal bacterial exposure [132, 133]. As will be illustrated in the following examples, detailed analysis of antibody levels and familial patterns does not support this model of antibody formation. Second, bacteria-dependent inflammation may be elicited in IBD by innate immunity to conserved bacterial products such as LPS and cell wall peptidoglycans [134]. However, this mechanism is not a sufficient factor, since mono- association studies with bacteria expressing proinflammatory molecules have in almost all cases been unsuccessful. A provisional conclusion is that the pertinent colitigenic traits of luminal bacteria, whether targeting antigenic or innate eff'ector processes, are expressed in only a subset of bacterial species. A plausible goal of the marker antibody studies is to identify candidate bacteria to be evaluated for these traits.

Table 2. Microbial and environmental antigens in IBD

Microorganism Antigen

ASCA

Antibodies to the cell wall mannan polysaccharide of Saccharomyces cerevisiae (ASCA) are detectable in about 60% of patients with CD and may fluctuate with disease activity [83, 135-140]. ASCA is highly specific for CD, with minimal seroreactivity of patients with UC, other colitides, or non-gastrointestinal diseases. To our knowledge ASCA levels have not been evaluated in IBD animal models. The origin of the antigenic stimulus for this ASCA response is uncertain, since the core epitope of this response is recurrent among taxa of plants and bacteria [141]. Thus, while ASCA levels are consistent with a CD-related bacterial cell wall- specific response, it is conceivable that dietary antigens may also play a stimulatory role [142, 143].

A substantial fraction of CD patients are seronegative despite similar clinical activity. This observation suggests that mucosal disruption and antigenic overexposure is not in itself a predominant factor. Concordance and intraclass correlation of ASCA levels in affected and unaffected first-degree relatives indicate that seronegative and seropositive phenotypes are distinct familial and perhaps genetic traits [144, 145]. The use of ASCA for disease stratification and preclinical risk assessment is discussed in the final section.

Mycobacterium

Antibodies to mycobacterial antigens are selectively associated with CD [11, 146, 147]. Recombinant proteins (p35 and p36) have been characterized from M. paratuberculosis, which in a tandem IgG immunoassay showed high specificity and sensitivity to CD ( ^ 75% and ~ 90%) compared to normals and UC patients [148-150]. This species specificity has been independently confirmed using a conventional absorption strategy [147]. IgA ELISA seroreactivity was also observed with a conserved mycobacterial

Disease References Undefined

M. paratuberculosis

B. caccae, B. thetaiotaomicron E. coli

P. fluorescens

ASCA p35, p36, HupB OmpW, SusC (?) OmpC 12 Dietary

CD CD CD

CD, UC subset CD

CD

190-194 195-200 201,202 201 203 204-207

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protein, HupB [151]. It is interesting to add that the latter antigen was initially identified by its antigenic crossreactivity with a pANCA histone HI-related epitope.

These observations comprise one line of evidence implicating mycobacteria, particularly M. paratuber- culosis, in CD pathogenesis. This hypothesis stems from the role of this organism as an etiologic agent in Johne's disease, a granulomatous colitis of cattle [152]. To this point, studies with species-specific PCR are inconsistent in localizing mycobacterial sequences to CD lesions; mycobacterial antibiotic therapy also has not yet demonstrated efficacy (reviewed in reference 153). Several mycobacterial antigens appear useful for CD serodiagnosis, but further lines of experimentation are required to resolve or refine the pathogenesis hypothesis.

OmpC and OmpW

Using a monoclonal pANCA antibody, libraries of colonic bacteria were generated and screened for crossreactive antigens by immunoblot analysis [154]. Three major bacterial species were identified:

Escherichia coli, Bacteroides caccae, and B. thetaio- taomicron. The E. coli protein was cloned and confirmed by recombinant expression and genetic analysis as the outer membrane porin OmpC. IgG ELISA demonstrated elevated IgG anti-OmpC in high-titered pANCA UC patients, and IgA anti- OmpC in approximately half of CD patients. Simi- larly, a newly described outer membrane protein, OmpW, was identified as the antigen for B. caccae, and IgA anti-OmpW was elevated in the same subset of CD patients [155]. Based on size and close sequence homology, the antigen in B. thetaiotaomi- cron is expected to be SusC. These findings identify a set of antigenically related bacterial outer membrane proteins as immunologic targets in CD, and reveal a structural relationship between them. Notably, these proteins are homologous to Rag A of Porphyromonas gingivalis, and hence reveal their structural relation- ship to a bacterial virulence factor in periodontal disease.

Pseudomonas 12

Representional difference analysis was used to isolate microbial D N A segments specific for CD lesional mucosa versus adjacent uninvolved mucosa [156]. This search resulted in the isolation of 12, a tetR bacterial transcription factor family member.

derived from Pseudomonas fluorescens (Wei et al., in preparation). In the large intestine, quantitative PCR established that the 12 sequence was present in

^ 50% of CD lesions (compared to 5-10% of histo- logically uninvolved CD mucosa, or other inffamma- tory controls). In the small intestine, 12 was detectable in the ileum of both patients and healthy controls, indicating commensal colonization of this compartment. Serum IgA anti-I2 ELISA with recombinant 12 detected ^ 60% of CD patients, and

^ 5%o of non-CD controls. In the mouse the 12 sequence is also localized at sites homologous to the human (distal small intestine). Immunologically, 12 is the target of a strong proliferative and IL-10 cytokine response mediated by murine CD4"^ splenic T cells. Several lines of evidence indicate that this response to 12 is a T cell superantigen [157]. In contrast, the T cell response to 12 in colitis-prone mouse strains predominated with IFN-y production.

The microanatomic localization of 12 and its unique immunostimulatory activity reflect traits of P. fluor- escens which may be pertinent to proinflammatory activity in susceptible hosts.

Diagnosis and disease stratification

The strong association of the pANCA and ASCA expression with UC and CD, respectively, has prompted efforts to use these marker antibodies to define more biologically homogeneous patient subsets for IBD diagnosis, prognosis, and treatment planning. With regard to diagnosis, combined testing for these two analytes increases sensitivity for overall IBD serodiagnosis, due to the occurrence of an ASCA^/pANCA"" C D subset, and the greater lability of ASCA but not pANCA levels to disease activity [83, 90, 91]. High pANCA levels predict a more aggressive disease phenotype, including elevated disease activity and pouchitis [88, 93, 158- 160]. Conversely, pANCA"^ CD patients are distinguished by later onset and more UC-like features, including resistance to anti-TNF-oc therapy. This distinctive biology is correlated with diff'erential allelism at the MHC locus, indicating that pANCA expression is an intermediate marker for the genetics of disease susceptibility. In CD, high ASCA levels are independently associated with aggressive disease (early onset, perforation, and fibrostenosing disease [91], although this was not observed in the pediatric population [90].

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As noted previously, seronegative and seropositive ASCA phenotypes are each distinct familial traits in CD, and are observed in clinical unaffected family members [144, 145]. Similarly, pANCA-positive and negative family phenotypes define UC and CD subsets [79, 80, 83, 91]. This presumably reflects biologically important differences in host genetics, bacterial exposure, or both. Accordingly, ASCA and pANCA are promising immunologic parameters to identify family members at risk for disease for early, preventative intervention. No prospective studies have yet addressed whether any of the known ser- omarkers is indeed a precHnical disease susceptibility marker. However, this issue is an important oppor- tunity to develop, in view of the clinical progress on early intervention in immunologic diseases such as diabetes mellitis [67].

As elaborated in this chapter, distinctive genetics and microbiology are likely to underlie the expression of the emerging panel of antigen-defined, disease-related autoantibodies and microbial antibodies. Moreover, levels of these antibodies are generally independent of ASCA and pANCA, indicating that they may c o m p l e m e n t these established disease markers in patient stratification.

In the near term, realization of these opportunities will most critically require further validation of disease-related antigens and organisms, using animal model systems. Moreover, mechanistic characterization of their action should permit the design of novel therapies at an antigen-specific or microbial level. At the clinical level systematic studies will be required to identify new combinations of marker antibodies, in concert with emerging genetic markers which most effectively discriminate patient subpopu- lations. This homogenization of patient subsets will be useful for empirically refining diagnosis and treatment planning. In addition, homogeneous subsets should permit more powerful analysis of genetic traits and biologic processes responsible for disease pathogenesis, and ultimately the incorporation of s o p h i s t i c a t e d t h e r a p e u t i c and p r e v e n t a t i v e interventions.

Acknowledgements

This work was supported by NIH DK46763, the UCLA Clinical and Fundamental Immunology Training Grant (AI 07126-23), the Crohn's and Colitis Foundation, UCLA CURE, and the Jonsson Comprehensive Cancer Center.

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