Anti-Saccharomyces cerevisiae antibodies in inflammatory bowel disease

Clinical and Applied Immunology Reviews 2 (2001) 45–63

1529-1049/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S1529-1049(01)00040-X

Anti-

Saccharomyces cerevisiae

antibodies ininflammatory bowel disease

Gary L. Norman, Ph.D.*

INOVA Diagnostics, Inc., 10180 Scripps Ranch Blvd., San Diego, CA 92131-1234, USA

Received 15 May 2001; Received in revised form 25 August 2001; Accepted 30 August 2001

Abstract

Crohn’s disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel dis-ease (IBD), a general term used to describe diseases that cause chronic inflammation of the intestine. Al-though there are many theories concerning the etiology of CD and UC, none have been proven. Sincemany symptoms of CD and UC are similar, diagnosis is often difficult and relies on detailed clinical eval-uation, the results of endoscopic, histologic and radiographic examination, and the exclusion of other dis-orders. Diagnosis of IBD in pediatric patients can often be particularly difficult because of nonspecificsymptoms early in the disease. While an accurate differential diagnosis is possible with most patients, 10–15% of patients have an indeterminate form with features common to both CD and UC. With time, manyof these patients are eventually classified as either CD or UC. The development of accurate, standardized,reproducible and non-invasive serological tests could provide clinicians with a valuable tool to assist in theassessment and diagnosis of IBD patients. Furthermore, accurate serological tests may assist cliniciansmake earlier diagnoses in pediatric patients. There has been a rapidly expanding interest and research intothe identification and development of serological markers to assist in the diagnosis and management of pa-tients with IBD. General markers of inflammation provide some information, but are not disease-specific.Autoantibodies to various antigens have been identified, but most have not proven to have adequate sensi-tivity, specificity or availability to be useful in the clinical laboratory. Two exceptions are ANCA (anti-

neutrophil cytoplasmic antibody and ASCA (anti-


antibodies). The first part ofthis article will present a general overview of IBD, followed by a review of some of the markers that havebeen evaluated for IBD. The second part will focus specifically on the development and current experi-ences with ASCA assays in the laboratory. © 2001 Elsevier Science Inc. All rights reserved.

Keywords:

Anti-


antibody; Crohn’s disease; Ulcerative colitis; Inflammatory bowel disease

Abbreviations: ANCA, anti-neutrophil cytoplasmic antibody; ANNA, anti-neutrophil nuclear antibody; ASCA,

anti-


antibody; cANCA, cytoplasmic ANCA; CD, Crohn’s disease; CRP, C-reactive protein;ECAC, epithelial cell-associated components; ESR, erythrocyte sedimentation rate; IBD, inflammatory bowel disease;IFA, indirect immunofluorescence assay; MPO, myeloperoxidase; PAB, pancreatic antibodies; pANCA, perinuclear

ANCA; PR3, proteinase 3; TNF-

�

, tumor necrosis factor-

�

; tTG, tissue transglutaminase; UC, ulcerative colitis.* Corresponding author. Tel.:

�

1-858-586-9900; fax:

�

1-858-586-9911.

E-mail address

: [email protected] (G.L. Norman).

46

G.L. Norman / Clin. Applied Immunol. Rev. 2 (2001) 45–63

1. Introduction

Like a thriving city, the intestine has a relatively stable and self-renewing population, aswell as a changing population of visitors and potential new residents, some welcome andsome not welcome. In the healthy gut, the immune system monitors the contents of the intes-tine through a complex balance of proinflammatory and anti-inflammatory controls exertedprimarily by cytokines and chemokines. The immune system must be tolerant of the normalintestinal microflora and incoming food, while being able to recognize and aggressively de-stroy harmful organisms. As the result of a variety of factors, including genetic susceptibil-ity, infection or other environmental factors, the immune system can become dysfunctional.Both self and non-self antigens can become targets of immune attack. If accurate and appro-priate controls are not regained, the dysfunctional attack on normal cells and tissues becomesself-perpetuating, with an amplification of the process and disease. In the case of the intes-tine, continuing abnormal regulation leads to further proinflammatory processes and tochronic inflammatory bowel disease.

Over a million individuals in the United States and Europe suffer from inflammatorybowel disease (IBD), a chronic, relapsing, inflammatory intestinal condition [1–3]. Individu-als with IBD may have a variety of symptoms including rectal bleeding, abdominal pain anddiarrhea. Crohn’s disease (CD) and ulcerative colitis (UC) are the two major forms of IBD.Both CD and UD are chronic and they affect men and women approximately equally. Theage of onset of CD and UC is usually between 15–30 years with a smaller peak of incidencebetween the ages of 50–70 [3]. Historically IBD was found to be most prevalent in popula-tions of Northern European and Anglo-Saxon ethnic derivation and in Ashkenazi Jews [4].In northern regions such as Northern Europe and the United States, the incidence of CD andUC was estimated as 5–7/100,000 and 6–12/100,000, respectively. In southern regions suchas Southern Europe, South Africa and Australia, the estimates were 0.1–4/100,000 for CDand 2–8/100,000 for UC [3]. Although there are conflicting results, it appears that while theincidence of both CD and UC has increased during most of this century, the incidence of UCand, in some regions CD, may have stabilized [1–3]. Increases in the incidence of CD havebeen noted in areas with previously low levels of disease, such as Southern Europe and in Is-rael [3–6]. This observation is often correlated with the increased westernization of the areasand as supporting the idea that some environmental factors connected to westernization areresponsible for the increased incidence rates [3].

As with many diseases, and especially so with long-term chronic conditions, early recog-nition, treatment and monitoring may help slow the progression of the disease and the devel-opment of more serious symptoms. In the case of IBD, diagnosis is often difficult, time-con-suming and invasive. Diagnosis relies on clinical examination supplemented with acombination of radiographic, endoscopic, histopathological and laboratory test results. CDand UC share common symptoms and even after a detailed diagnostic work-up, approxi-mately 10–12% of cases cannot be definitively categorized and are referred to as indetermi-nate colitis. Over time, about half of these patients are diagnosed as either CD or ulcerativecolitis [7–9]. The seriousness of IBD, the apparent increased prevalence, and heightenedawareness by both physicians and the public, has led to an active search for new laboratorytests to assist clinicians in making earlier, less invasive, and more definitive diagnoses. A va-


47

riety of serologic markers of IBD have been described. Most have been found to be of lim-ited value. Two markers, the presence of anti-


antibodies (ASCA)and a specific type of perinuclear anti-neutrophil cytoplasmic antibodies [referred to as atyp-ical pANCA, atypical ANCA, x-ANCA, or anti-neutrophil nuclear antibody (ANNA)] havebeen shown to be significantly more prevalent in patients with CD and UC, respectively[10,11]. This review will focus primarily on the status of ASCA as a marker of CD with abrief overview of pANCA and other serological markers.

2. IBD background

Although the predominant clinical forms of IBD are recognized as CD and UC, formswith overlapping features characteristic of both CD and UC are seen. The heterogeneity ofIBD and the observation of overlapping features has led to the suggestion that IBD might beviewed as encompassing a “continuous spectrum of diseases” (Fig. 1) [12].

Recognition of the heterogeneity of IBD patients and stratification of patients into defin-able subgroups is especially critical for studies to understand the diagnosis, pathology, prog-nosis and therapeutic management of IBD. Factors used for stratification of patients includedemographic features, anatomic location, disease behavior, disease course and serologicmarkers [12–14]. In the case of developing new laboratory assays for IBD, stratification ofIBD patients beyond the initial division into CD and UC may allow the assay to be targetedto specific clinical subgroups with a resultant higher sensitivity and specificity. Stratificationcan also assist the evaluation and targeting of therapeutic treatments and new drugs.

Clinically CD primarily affects the distal small intestine (although it may affect any partof the digestive tract), while UC primarily affects the colon. Inflammation in CD extendsdeep into the affected tissue and is typically asymmetrical and segmental, with areas of bothhealthy and diseased tissue. In contrast, inflammation and ulcers are localized to the superfi-cial tissue of the colon and rectum in UC and inflammation is usually symmetrical and unin-terrupted from the rectum proximally [13,15,16].

Increased permeability of the small intestine is found in most CD patients and in 25% ofhealthy relatives of these patients. The same defect is not found in UC patients or in theirhealthy relatives [17]. However, an impaired intestinal epithelial barrier resulting from al-tered tight junction structure may contribute to the diarrhea seen in UC [18]. The possibilitythat increased intestinal permeability in CD could lead to increased leakage of antigen acrossthe intestine and increased immune stimulation has been a recurrent theme in CD research.

Although the etiology of IBD is unknown, relevant components and a general scheme de-scribing possible pathogenetic pathways leading to IBD is emerging. One basic element isgenetic susceptibility. The risk of IBD in first-degree relatives of patients with IBD is in-creased 10–15 times over the general population [19,20]. Family studies have shown that

Fig. 1. IBD continuum.

48


10–35% of patients with IBD have affected family members [10,20–23]. Concordance ofmonozygotic twins with CD or UC compared to dizygotic twins also support a genetic com-ponent [24]. Other family studies have shown a high degree of concordance for the site andclinical type (inflammatory, structuring or fistulizing) of disease [22]. Extensive efforts toidentify genes responsible for IBD susceptibility are progressing. Work using establishedand emerging genomic techniques have suggested that 10 to 20 different genes may be in-volved in IBD. Regions of chromosomes 16 and 12 have been specifically linked to IBD[25]. DNA microarray analysis comparing the expression of 7306 genes in tissue taken fromCD, UC and healthy patient’s intestines found significant differences in the expression pro-files of 170 genes [26]. While genetics certainly makes some people more susceptible to thedevelopment of IBD, additional factors appear to be needed to trigger the host mucosal im-mune responses.

A role of environmental factors contributing to the development of IBD is supported bymany studies. Environmental factors probably play some role in the changing incidence ofCD in many developing geographic areas. Studies that have shown that people moving fromareas with low levels to areas with higher levels of IBD tend to develop higher IBD inci-dences in line with the local levels [27]. Smoking is an environmental factor that is recog-nized as increasing the incidence and severity of CD, while negatively influencing UC. How-ever, dissecting the particular element(s) influencing the two diseases is difficult because ofboth the presence of over 4000 different components in tobacco smoke and the confoundingeffect of lifestyle differences (diet, exercise, etc.) between smokers, ex-smokers and non-smokers [28]. A recent study showing an increased incidence of IBD among spouses of IBDpatients also suggested a possible role of environmental factors [29].

There is abundant evidence that the intestinal flora is a critical component of the inflam-matory process. The human intestine contains 1–2 kg of bacteria made up of 200–500 differ-ent anaerobic and aerobic species [30,31]. A direct role of the luminal contents in IBD patho-genesis is supported by the observation that when the intestinal stream is diverted outthrough a stoma, inflammation disappears and reappears when the intestine is reconnected[32]. Additional evidence includes observations that the location of IBD lesions correspondto gut areas with the highest bacterial numbers and reports that treatment of CD patients withantibiotics can be efficacious in some cases [30]. Normal resident enteric bacteria are neces-sary for the development of spontaneous colitis in IL-10 deficient mice. When these mice areraised in a germ-free environment they do not develop colitis. These studies also have shownthat some resident bacteria, such as

Bacteriodes vulgatus

, have more proinflammatory capa-bilities than others [33–35]. The basic idea of probiotics is to modify and normalize the en-teric microflora with live microorganisms, such as lactobacilli, that do not have known in-flammatory propensities [30,31]. A role of bacteria in inflammation-induced fibrosis similarto that seen in CD was suggested by the intermural injection of purified bacterial cell wallpeptidoglycan-polysaccharide into rats, which induced chronic granulomatous enterocolitis,increased collagen synthesis, increased TGF-B1 and IL-6 expression [36]. Direct stimulationof myofibroblasts with the same cell wall preparation, also showed similar increased col-lagen synthesis and cytokine expression [36]. Animal models of IBD show that mucosal in-flammation can appear spontaneously as in the case of colitis in the cotton-top tamarin and aCD-like condition in the SAMP1/Yit mouse. Experimental IBD can also be induced by vari-


49

ous exogenous agents such as dextran sulfate or peptidoglycan polysaccharide in IL-10knockout animals or by transfer of specific cell populations into immunodeficient animalssuch as the SCID mouse [31,37].

At some stage in the host immune response, perhaps initially in some cases or during theprocess in other cases, the immune response becomes abnormal. While in the healthy intes-tine normal bacterial flora probably cause some degree of chronic immune stimulation, theprocess is under strict regulatory control and is tolerated by the immune system. In IBD,there appears to be a breakdown in tolerance to the normal flora. This concept is supportedby the experiments of Duchmann et al. showing that T cells from healthy individuals respondto heterologous microflora, but do not proliferate in response to organisms from their ownintestinal tract [38,39]. In contrast, T-cells from active CD lesions proliferate in response totheir normal microflora. Furthermore, the loss of tolerance to normal microflora can beshown to occur after the development of colitis in animal models [40]. In IBD, the balancebetween proinflammatory cytokines such as TNF-

�

, IL-1

�

, IL-2, IL-8, IL-12 and contrain-flammatory cytokines such as IL-10, IL-1ra, IL-4 and IL-13 becomes pathologically tiltedtoward the proinflammatory group [41]. Instead of controlling inflammation stimulated bythe normal enteric flora (and/or other abnormal flora, organisms or food material) by adown-regulation of the immune response, the inflammation continues, is amplified, andchronic inflammation ensues. While the precise alterations of various cytokines in IBD is not

Fig. 2. ASCA pathogenesis.

50


always consistent from study to study, there is a general consensus that CD is characterizedby a T-helper cell, type1 (Th1) response associated with IL-12, IFN-

�

and tumor necrosisfactor-

�

(TNF-

�

). Ulcerative colitis appears to be characterized by a modified Th2 responseand is associated with IL-5 and IL-10 [31,42,43].

It is probable that multiple paths can lead to IBD (Fig. 2). Alterations in the relative con-tributions of many elements and their subsequent interactions are likely responsible for thewide differences in the severity and clinical forms of IBD conditions.

3. Serological markers of IBD

3.1. General markers of inflammation

The heterogeneity of IBD complicates the identification of specific serological markers.Some markers may be primarily associated with CD, some with ulcerative colitis, and somecommon to both. Other markers may be associated with particular clinical phenotypes of CDor UC. Additionally, while correlations between IBD disease activity and the levels of somecommon laboratory analytes such as orosomucoid (

�

1

-acid glycoprotein), C-reactive proteinand neopterin have been observed, most of the changes are probably not specifically related toIBD, but rather reflect non-disease-specific inflammatory processes. Nevertheless, such mea-surements may have some value since they are inexpensive and provide evidence that somesort of inflammatory condition is present. There have been reports that markers such as eryth-rocyte sedimentation rate (ESR) and C-reactive protein (CRP) correlate with disease activityand can be predictive of relapse in UC [44,45].

The array of interrelated inflammatory cytokines and associated molecules active in IBDmake these logical targets for laboratory measurement. However, in order to justify expen-sive measurements of specific serum cytokines, the tests must provide a more direct assess-ment of specific disease than conventional inexpensive tests of acute phase response [43]. In-creased levels of soluble IL-2R

�

,

�

and

�

chains were reported to correlate with increasingCD activity, while increasing levels of IL-2

�

correlated with decreasing activity in UC [45].

3.2. Autoantibodies

Speculation that autoantibodies to intestinal mucosal components might have a patho-genic role in IBD has a long history. While such a role is still unproven, it is clear that certainautoantibodies are found more frequently in some forms of IBD. The significance of ele-vated levels of most of the antibodies remains unclear. Over 40 years ago, Broberger andPerlmann found increased levels of anti-goblet cell antibodies in sera from UC patients com-pared to controls [47]. More recent studies found about 36% of UC and 30% of CD patientshad anti-goblet cell antibodies, which appeared to react with mucin components secreted bythe goblet cells present primarily in the colon [48]. Antibodies to intestinal epithelial cell-associated components (ECAC) have been found to be more frequent in patients with IBDand their first-degree relatives than in controls [49]. Using cultured human colon cell lines,36% of UC, 13% of CD and 0% of control sera had anti-colon cell antibodies [50]. Elevatedlevels of antibodies to an erythrocyte membrane protein were found in patients with CD, UCand

Campylobacter jejeuni

enterocolitis [51].


51

A 40 kDa colonic protein recognized by UC, but not CD sera, was recently identified as tro-pomysin [52]. Tropomyosin, a cytoskeletal microfilamental protein, appears as multiple iso-forms some of which may be organ- and function-specific. Isoforms 5 and 1 (out of at least eightpossible isoforms) have been identified as the distinct isoforms recognized by UC sera [52].

Many CD patients produce a characteristic speckled immunofluorescence pattern over theacini of the exocrine, but not the endocrine portion of pancreas tissue sections. The specifictarget of the reactivity is unknown but it appears to be a large protein complex with multiplesubunits [53]. Pancreatic antibodies (PAB) are very specific (97–100%) for CD compared toUC and normal patients, but show a sensitivity of only 27–38% for CD [54,55].

Detection of ANCA is routinely used for identification of various vasculitides. On etha-nol-fixed human neutrophil slides a cytoplasmic ANCA (cANCA) pattern is characteristic ofWegener’s granulomatosis and proteinase 3 (PR3) reactivity. A perinuclear ANCA(pANCA) pattern is characteristic of microscopic vasculitities, polyarteritis, polyangiitis andcresentic glomeronephritis. Sera from patients with these conditions are usually myeloperox-idase (MPO) positive. An atypical pANCA pattern is characteristic of specimens from manyUC, primary sclerosing cholangitits and autoimmune hepatitis, type 1 patients [56–60]. Theatypical nomenclature is derived from the observation that the specimens give a strongpANCA pattern on ethanol-fixed neutrophil slides that disappears when the specimens aretested on formalin-treated slides and are MPO negative. This is in contrast to typical pANCAspecimens, which show a cANCA pattern on formalin slides and are usually MPO positive.An alternative method used by some laboratories to identify UC associated-pANCA reactiv-ity involves initial identification of ANCA reactive specimens on methanol-fixed neutrophilsfollowed by indirect immunofluorescence assay (IFA) on DNase-treated neutrophils. The ul-cerative colitis-pANCA sera no longer produce a pANCA pattern following digestion[46,57]. Approximately 60–80% UC, 65–75% primary sclerosing cholangitis and 50– 96%of AIH-1 specimens demonstrate atypical pANCA reactivity [56–61). Intense efforts by nu-merous laboratories to define the primary target(s) of atypical pANCA reactivity have yet tosucceed. It is likely that atypical pANCA reactivity may be directed toward several differenttarget antigens. Possible targets of atypical pANCA reactivity such as lactoferrin, bacterial/permeability-increasing protein, cathepsin G, elastase, lysozyme,

�

-glucuronidase, catalaseand

�

-enolase have been individually investigated as markers for UC and CD. Most showonly modest ability to discriminate UC from CD [58]. Additional studies are needed to deter-mine if they recognize specific clinical subgroups of IBD or are nonspecific markers with lit-tle specific relevance to IBD. A 50 kDa myeloid-specific protein localized to the inner side ofthe nuclear membrane was recently isolated by Terjung et al. and appears to be a major targetof atypical pANCA reactivity [59]. Sera from 92% of patients with inflammatory bowel orhepatobiliary disease and atypical pANCA reactivity recognized the purified protein.

3.3. ASCA background

A role of dietary components in the pathogenesis of IBD has long been suspected. Rela-tive to normal patients, increased levels of antibodies to maize and bovine serum albuminwere reported in both CD and UC patients [62]. Increased IgG antibodies to a variety of bo-vine milk proteins (

�

-casein,

�

-lactoglobulin a & b) was primarily found in CD patients

52


compared to UC or control patients [63,64]. IgA antibodies to

�

-casein were higher in CDand UC patients compared to healthy controls [64]. While these studies suggested that the in-creased levels of antibodies maybe a result of the increased intestinal permeability or inflam-mation and stimulation of immune responses, other studies did not confirm these results[65,66]. If increased permeability was responsible for the increased antibodies to these di-etary components, one might expect to see increased antibodies to the common egg proteinovalbumin, however this was not found [65]. Numerous bacterial and viral organisms havebeen investigated as possible etiologic agents for CD. Increased antibodies to

Bacteroidesfragilis

,

Streptococcus facealis

,

peptostreptococcus

,

eubacterium

and

enterbacteriaceae

spe-cies have been reported [67,68]. While increased seroreactivity to enteric bacterial pathogenssuch as

Campylobacter

jejuni

and

fetus

,

Listeria monocytogenes

,

Yersina enterocolitica

, se-rotype 3 and 9,

Yersina pseudotuberculosis

,

Brucella abortus

, and

Mycobacteria arabi-nomannan

were reported, the fact that increased seroreactivity in U.S. patients was seen to

Y.enterocolitita

type 3, a serotype rarely isolated in the United States, suggested that the re-activity to all of these organisms may reflect a general hyperreactivity of CD patients [68].Blaser et al. suggested that the reactivity results from cross-reacting bacterial antigenspresent in the normal gut flora [68]. The similarity of bovine Johne’s disease and human CDhas maintained interest in the possible role for mycobacteria

,

especially

Mycobacteria avium

subsp.

paratuberculosis,

the etiologic agent of Johne’s disease, in CD pathogenesis. Molecu-lar evidence of

M. paratuberculosis

sequences in CD lesions and serological evidence of re-activity to specific proteins of

M. tuberculosis

and

paratuberculosis

has been demonstratedby multiple groups [69–72]. Considering the number of studies with similar findings, someassociation of a mycobacterial-like organism with CD is reasonable in at least some sub-groups [72]. Whether the presence of

M. paratuberculosis

or related organisms and antimy-cobacterial antibodies are relevant to the development of CD or are secondary effects of in-testinal dysfunction still remains to be determined [69].

Beginning in the late 1980s, several studies examined sera from patients with CD or UCfor antibodies to


(baker’s or brewers yeast). This organism iscommonly used in the preparation of many foods and some people, especially those occupa-tionally exposed such as bakers, are known to develop sensitivity to the organism [73]. Thesestudies found that ASCA of both the IgG and IgA isotypes were more frequently found inCD patients compared to normal healthy patients and to patients with UC [7–10,65,66,73–75,78–81]. Baking, brewing (ale, lager) and wine-making strains of

S. cerevisiae

were allshown to be targets of ASCA [66,74–76]. In contrast, levels of antibodies directed against

Candida albicans

, a common yeast found in about 50% of healthy individuals, were similarin CD, UC and normal patient groups [74,75]. The lack of increased antibodies in CD pa-tients to

C. albicans

, like the similar observation with egg ovalbumin, suggested that the in-creased antibodies to

S. cerevisiae

were probably not a result of an indiscriminate increasedintestinal permeability [74]. Immunological exposure to ingested and disrupted

S. cerevisiae

in contrast to the infectious colonization by

C. albicans

was suggested as possibly relevant tothe differing response [76]. The finding of increased antibodies to the enteric pathogens suchas

Campylobacter

,

Listeria

,

Yersina

and

Brucella,

which are orally ingested and all have atropism for intestinal tissue, suggests that the difference between the digested

S. cerevisiae

and the colonizing organisms is not the critical difference. Biochemical studies identified the


53

predominant immunogenic ASCA antigen as a 200 kDa water-soluble, heat-stable phospho-peptidomannan component of the cell wall of

S. cerevisiae

[75–77]. Oligomannosides arewidely distributed among yeasts, viruses, bacteria and human glycoproteins and ASCA reac-tivity could be a result of cross-reacting antibodies to antigens found in non-yeast organisms[75]. Fine structure analysis suggests that specific O-linked side chains consisting ofMan

�

1

→

3Man

�

1

→

2Man1

→

2 are overrepresented in mannan fractions most reactive withsera from CD patients compared to other yeast mannans [77] and may account for the speci-ficity of discriminating

S. cerevisiae strains.

3.4. Assay designs

Most ASCA assays use standard ELISA methodology. Baking, brewing (both ale and la-ger) and wine-making strains of S. cerevisiae have all been shown to be targets of ASCA, al-though some strains demonstrate better sensitivity and specificity than others. A variety ofantigen preparations have been used for construction of ASCA ELISA assays. These includeantigen derived from disrupted or boiled S. cerevisiae and purified oligopeptidomannansfrom the S. cerevisiae cell wall [10,66,75,78,79]. Isotype-specific assays for ASCA IgG orASCA IgA and polyvalent assays for the combined detection of IgG, IgA and IgM antibod-ies are all in use [80].

3.5. ASCA frequency in IBD

The frequency of ASCA in CD patients ranges from approximately 50–80% for IgG and35–50% for IgA. In contrast, approximately 2–14% of UC and 1–7% of healthy controlshave IgG or IgA ASCA antibodies [7,10,11,65,66,73–76,78–88]. A combination of differentassay methods, antigen preparations, specific or polyvalent conjugates, and different choicesbalancing sensitivity and specificity contribute to the wide variation in reported results (Fig. 3).In addition, the selection and characterization of the patient populations is particularly criti-cal in the case of ASCA and other assays focused on IBD serodiagnostics because of the het-erogeneity of IBD patients. Differing inclusion criteria can make comparison of test resultsdifficult or misleading. Careful chart review and analysis of each patient’s history and statusat the point when the serum sample was obtained is essential for deriving optimal informa-tion and understanding from a study. Side by side evaluations of assays on the same group ofspecimens, such as the recent study by Vermeire et al. [80] is necessary to meaningfullycompare assay performances.

Regardless of assay methodologies or study designs, the general observation that ASCAantibodies are found much more frequently in CD patients compared to UC and control pa-tients is similar in all assays.

All assays show that ASCA IgG and IgA levels in CD patients are highly variable. Thedistribution of ASCA IgG and IgA results on a panel of well-characterized clinical samplesis shown in Fig. 4. In this figure, the ASCA results obtained on panel of well-characterizedclinical samples from the University of Vienna [81] and University Hospital of Gathuisberg,Belgium [80] were combined, the patients within each clinical group were sorted by increas-ing ASCA IgG values, and finally both the ASCA IgG and IgA results of each patient wereplotted. The figure shows that: (a) ASCA IgG and IgA values for CD patients range from in-

54 G.L. Norman / Clin. Applied Immunol. Rev. 2 (2001) 45–63

significant to very high levels; (b) there is a subgroup of CD patients that do not show eitherASCA IgG or IgA antibodies; (c) there is a subpopulation of CD patients with low to moder-ate ASCA IgG values and strikingly high ASCA IgA values; and (d) most UC and normalcontrols have low levels of both ASCA IgG or IgA antibodies. Barnes et al. [78] found thatASCA IgA was 100% specific for CD. While the study represented by Fig. 4 supported thisconclusion, our studies of additional IBD and normal individuals have identified a few UCand normal individuals with positive ASCA IgA results. Vermeire et al. reported a similarfinding using four different ASCA assays [80].

Although almost every study of ASCA measures both ASCA IgG and IgA either sepa-rately or in a polyvalent formulation, some have questioned the diagnostic benefit of measur-ing ASCA IgA in addition to ASCA IgG. In our experience, we find that approximately twothirds of the CD patients with ASCA IgG are also positive for ASCA IgA. Using the same

Fig. 3. Range of reported ASCA values from the literature.

Fig. 4. Profile of ASCA IgG and IgA values in clinical groups. Within each clinical group, paired ASCA IgG andASCA IgA values for each specimen were first sorted by increasing ASCA IgG value. Both the ASCA IgG and thecorresponding ASCA IgA value were then plotted. As a result of the large number of patients, it is not possible toresolve the individual IgG results. However, the range of ASCA IgG values, as well as spikes of very high ASCAIgA values associated with lower ASCA IgA results, is strikingly apparent. Data extracted from references [80,81]

G.L. Norman / Clin. Applied Immunol. Rev. 2 (2001) 45–63 55

ASCA IgG and IgA kits, five different clinical sites found that from 0 to 19% of their CD pa-tients had only ASCA IgA antibodies. Overall for the 291 CD patients tested at the five sites,4.1% of the CD patients had ASCA IgA but not ASCA IgG antibodies. Combining theASCA IgG and ASCA IgA results increased the overall sensitivity of some ASCA assaysabout 10% in the study by Vermeire et al. [80]. No particular clinical feature has been associ-ated with the ASCA IgA positive-IgG negative phenotype. The existence of CD specimensthat are only positive for ASCA IgA suggests that both ASCA IgG and IgA antibodiesshould be measured in the laboratory, either using separate ASCA IgG and IgA assays or apolyvalent assay configuration. The most striking result of the separate measurement and thecombined interpretation of ASCA IgA and IgG results is the enhancement of specificity.Studies consistently show that specimens with both high ASCA IgG and high ASCA IgAvalues are almost always CD patients [7,79,81–83]. Specificities of up to 100% for CD havebeen reported for specimens positive for both ASCA IgG and IgA [7]. Analysis of the com-bined data set shown in Fig. 4, showed that of 102 patients positive for both ASCA IgG andIgA antibodies, 100 were CD patients and two were UC patients. None of 148 control pa-tients were both ASCA IgG and IgA positive. Subsequent studies have identified a fewhealthy controls, which were positive for both ASCA IgG and IgA antibodies.

3.6. Clinical significance of ASCA antibodies

Diagnosis of IBD relies on clinical examination, the results of endoscopic, radiologic, his-topathologic and laboratory tests, patient and family medical histories, and exclusion of othergastrointestinal infections or conditions. For about 10% of patients, definitive classificationis not possible and optimum therapeutic treatment may be delayed because of the diagnosticuncertainty. ASCA testing offers the clinician another tool to use in the initial diagnosticevaluation of patients with possible IBD or with indeterminate colitis. While not diagnostic,the test only requires the drawing of blood, is relatively inexpensive, can be done at the ini-tial patient visit, and is clearly a desirable option for evaluation of pediatric populations. Astrong positive result, together with high clinical suspicion, may help in decisions for oragainst additional invasive testing. Clinicians must understand and clearly convey to patientshowever, that a positive ASCA result does not mean the patient has CD or IBD. A strongpositive result, especially when both ASCA IgG and IgA levels are high however, is a strongindicator of CD, but this result has also been seen in some other clinical conditions as will bediscussed later. Similarly, a negative ASCA result does not mean the patient does not haveCD, since some well-characterized CD patients do not have ASCA antibodies.

The presence of ASCA appears to be a familial trait and has been found in approximately20% of the first-degree relatives of CD patients [83–86]. The prevalence of ASCA has alsobeen found to be much higher in cases of sporadic CD (63%) and in families with only CD(62%) compared to families with both CD and UC (33%) [85]. Evidence of vertical trans-mission of the ASCA marker from mother to child was reported in one study, while anotherfound evidence of both mother to child and father to child transmission [84–89]. Whetherthe presence of ASCA in unaffected individuals predicts an increased risk is still unclear.Additional retrospective and prospective longitudinal studies are necessary to answer thisquestion.


Since ASCA values are not uniform among CD patients, studies have attempted to corre-late clinical observations with ASCA levels (Table 1) In general, IBD patients who areASCA positive are more likely to have a CD-like phenotype, while ASCA negative speci-mens are more likely to have a UC-type phenotype. The specificity of the evaluation is en-hanced when ASCA and ANCA test results are considered together. An ASCA�/atypicalpANCA� result is strongly associated with a CD phenotype and ASCA�/atypicalpANCA� is strongly associated with a UC phenotype [10,11,88,90]. The use of ASCA, es-pecially when combined with ANCA results, has proven useful to help resolve cases of inde-terminate colitis. ASCA positive-atypical pANCA negative specimens tended to resolve toCD, while ASCA negative-atypical pANCA positive specimens tended to predict UC or UC-like CD [7,91]. An examination of 28 patients with indeterminate colitis, found that of sevenpatients who were eventually assigned a definitive diagnosis, two patients with ASCA�/atypical pANCA� had CD, two patients with ASCA�/atypical pANCA� had UC, one pa-tient with ASCA�/atypical pANCA� had UC, one patient with ASCA�/atypicalpANCA� had UC-like CD, and one patient negative for both ASCA and atypical pANCAwas diagnosed with CD [88]. Although this was a small sample set, the results indicate thatmeasurement of ASCA and ANCA antibodies is of value for evaluating indeterminate colitispatients. This same study reported combined ASCA�/atypical pANCA� results were 94%specific for IBD compared to controls and had a positive predictive value of 91%. Similarly,the combined result of ASCA�/atypical pANCA� was 92% specific for CD and ASCA�/atypical pANCA� was 98% specific for UC [88].

The presence of ASCA is strongly associated with small bowel disease, while the pres-ence of atypical pANCA (and the absence of ASCA antibody) is associated with colonic dis-ease [81,82,85]. The presence of ASCA, like ANCA, appears to be independent of diseaseactivity [7,75,81,85]. However, high levels of ASCA—especially when levels of both ASCAIgG and IgA are high—have been shown to be associated with particular disease featuressuch as earlier age of onset [10,82,85] fibrostenosing and penetrating disease [81,82], moreaggressive disease, more frequent surgeries and less UC-like disease [82]. Following resec-

Table 1ASCA and reported clinical observations

General• Crohn’s disease ��ulcerative colitis �normal• Small bowel disease ��small & large, � large• Generally independent of disease activity• Familial trait—20% 1st-degree relatives• ↑↓ or no change following resection?• Predictive of response to anti-TNF-�?• ASCA� ulcerative colitis patients more treatment resistant?

High levels ASCA, especially high IgG and IgA• Increased fibrostenosing & penetrating disease• More aggressive disease• Increased frequency of surgery• Earlier age of onset


tion, ASCA levels have been reported both to remain unchanged or to decrease [7,10,78].Approximately 30% of CD patients that do not respond to conventional treatment also do notrespond to treatment with anti-TNF-� (infliximab) [92]. Kam et al. [93] reported that thehigher the levels of ASCA, the greater the response to infliximab treatment. Vermeire et al.[92], in contrast, did not find a significant correlation with ASCA values and response to in-fliximab. Both groups, however, suggested that an ASCA negative-atypical pANCA positivephenotype might be associated with non-response and that further study was needed. Thepresence of ASCA antibodies in UC patients was found to be associated with treatment-re-sistant disease in a preliminary report and may help clinicians in managing some patients ifthe report is confirmed [94].

3.7. ASCA frequency in normal individuals and other clinical conditions

In healthy normal individuals, the frequency of ASCA antibodies ranges from 1–7% forASCA IgG, 0–8% for IgA ASCA, 2–15% for IgG or IgA, and 0–8% for IgG and IgA. Whenthe ASCA test results on normal healthy individuals tested in our laboratory were combinedwith results from other groups using the same ASCA IgG and IgA ELISA kits, we found thatof 501 specimens, 4.8% were positive for ASCA IgG, 1% were positive for ASCA IgA, and0% were positive for both ASCA IgG and IgA. The high specificity of dual positive resultsextends to UC patients where only two of 161 UC patients were positive for both ASCA IgGand IgA. High specificity of dual positive results has been reported by other studies [7,82].However, in subsequent studies we have found a few sera obtained from presumed normalindividuals that were positive for both ASCA IgG and IgA. Usually either the IgG or IgAASCA result was a borderline positive. These patients could have subclinical disease, unrec-ognized disease, a familial history of IBD or they may just have nonspecific cross-reactingantibodies with no clinical significance. We have found that some of these specimens werealso positive for gliadin IgG antibodies, although none were gliadin IgA or tissue trans-glutaminase (tTG) IgA positive. Gaiffer et al. [79] found that half of the celiac patients intheir study were ASCA IgG, but not ASCA IgA positive, while Ruemmele et al. [7] reportedthat none of their celiac patients were ASCA positive. Gliadin IgG is known to be less spe-cific than gliadin IgA or tTG IgA. Nevertheless, in view of the specificity seen with com-bined ASCA IgG/IgA positive specimens, the positive gliadin IgG result might define a sub-group with some type of subclinical inflammation or intestinal disease.

In our own experience, we have found a low frequency of IgG or IgA ASCA in diseasesera from non-IBD groups. No specimens positive for gliadin IgA, antinuclear antibody,H.pylori, or tTG IgA were found to be positive for ASCA IgG or IgA antibody. We did finda few specimens positive for gliadin IgG (2/8), AIH type 1 (4/15), and HCV (1/6) antibodiesthat were also positive for ASCA [87]. Similar findings of ASCA in specimens from liverdisease patients have been reported by others [66,95].

A particularly interesting observation was that two of two alcoholic hepatitis patients werepositive for IgG and IgA ASCA [87]. Is there a link between the leaky gut of the alcoholichepatitis and CD patients and the presence of ASCA?

The finding of ASCA antibodies in 20% of the healthy first-degree relatives of CD patientsraises the question if ASCA may be a marker of increased risk or subclinical disease


[83,85,86]. Although there is no evidence of predictive value at this time, studies to answer thisquestion by following ASCA positive unaffected relatives of CD patients are underway [85].

3.8. ASCA testing in the clinical laboratory

Until recently ASCA testing was available at only a few research and reference laborato-ries, usually using in-house testing procedures and reagents. ASCA testing is now availableat a growing number of reference laboratories. It is critical that laboratories carefully evalu-ate the performance characteristics and validate the performance of ASCA assays they arecontemplating adding to their menus. As was shown in Fig. 3, there is a wide variation in thesensitivity and specificity of various assays and laboratory directors must be certain to care-fully address this issue. ASCA IgG and IgA ELISA kits cleared for in vitro diagnostics use inthe United States are produced by one manufacturer (INOVA Diagnostics, San Diego, CA).

When might ASCA testing be most useful and when might it be of questionable value?The difficulty in establishing guidelines for the clinical laboratory are a result of the widevariability of results obtained with different assays (Fig. 3) and the lack of large detailed stud-ies using standardized assays. While many studies have suggested that ASCA antibody levelsmay prove useful in a variety of situations, the same studies often conclude with the state-ment “more study is needed to confirm this observation.” Studies underway will clarify manyof these observations and testing recommendations will subsequently evolve. Meanwhilewhat guidance can be given to physicians and clinical laboratories offering ASCA testing?Table 2 provides some guidance on this issue. First it is critical that physicians understandthat neither ASCA, nor ANCA testing is diagnostic for CD or UC, respectively. There is gen-eral agreement that screening unselected populations for ASCA antibodies is not useful.There is also general agreement that ASCA testing may be of value when the diagnosis is not

Table 2ASCA testing in the clinical laboratory

Situation Usefulness Refsa

Screening unselected patients Not useful 80,88,97Definitive diagnosis of Crohn’s disease Not useful. ASCA is adjunctive

test to aid in diagnosis88,97

Indeterminate colitis May be useful 91,97,98 Aid in assessing a symptomatic

individual’s likelihood of IBD May be useful 97,98

Pediatric patients with chronic, nonspecificGI symptoms

May be useful 7,11,96–98

Aid in differentiating Crohn’s disease andulcerative colitis

May be useful insome cases

7,10,80,88,97

Adjunctive testing for hard to diagnosepatients

May be useful 97

Prediction of response to anti-TNF-� Maybe, more study needed,ANCA should also be done

92,93,97

Stratification of patients Useful for research, additionalapplications still under study

12,13

aSelected references, these points are made in many papers


clear and where additional test information may help clarify the diagnosis. Screening of pa-tients with persistent abdominal complaints may help identify which patients are more likelyto have IBD and help decrease long diagnostic delays experienced by some patients (96–98).

4. Conclusions

Why the majority of CD, but not UC, patients have elevated levels of antibodies to thecommon yeast S. cerevisiae is fascinating, but still unexplained. CD and UC appear to be atopposite ends of an intestinal inflammation continuum. ASCA and atypical pANCA corre-late with CD and UC phenotypes, respectively. New studies will help refine the significanceof the presence of ASCA antibodies, the relevance of ASCA IgG and IgA isotypes, the pre-dictive value of ASCA antibodies, and the use of ASCA for patient monitoring and manage-ment. In the future, as new markers are identified, they can be combined with ASCA andANCA results to produce test profiles to address the heterogeneity of IBD patients. Correla-tion of ASCA measurements with careful analysis and substratification of patients by clinicalfeatures will help our understanding of both the basis for ASCA reactivity and suggest howto best utilize the results of ASCA testing.

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Anti-Saccharomyces cerevisiae antibodies in inflammatory bowel disease