Diagnostic Value of Anti-Saccharomyces cerevisiae and Antineutrophil Cytoplasmic Antibodies for Inflammatory Bowel Disease: High Prevalence in Patients with Celiac Disease

Diagnostic Value of Anti-Saccharomyces cerevisiae andAntineutrophil Cytoplasmic Antibodies for InflammatoryBowel Disease: High Prevalence in Patients withCeliac Disease

JAN G.M.C. DAMOISEAUX,1,5 BAS BOUTEN,1,2 ANNICK M.L.W. LINDERS,1,2 JOS AUSTEN,1

CAROLINE ROOZENDAAL,3 MAURICE G.V.M. RUSSEL,4 PIERRE-PHILIPPE FORGET,2 andJAN WILLEM COHEN TERVAERT1

Accepted: May 22, 2002

Both celiac disease and inflammatory bowel disease (IBD)are characterized by chronic diarrhea and the presence ofdistinct (auto)antibodies. In the present study we wanted todetermine the prevalence of serological markers for inflam-matory bowel disease, i.e., perinuclear antineutrophil cyto-plasmic antibodies (pANCA) and/or anti-Saccharomycescerevisiae antibodies (ASCA), in 37 patients with biopsy-confirmed celiac disease (Marsh IIIb/c). The majority of thepatients was positive for IgA (auto)antibodies typicallyassociated with celiac disease, i.e., antiendomysium antibod-ies (EMA) (86.5%), antigliadin antibodies (AGA) (73%),and antirecombinant human tissue transglutaminase antibod-ies (rh-tTGA) (86.5%). Four patients with selective IgAdeficiency could be identified by analyzing EMA, AGA, andrh-tTGA for the IgG isotype. The prevalence of pANCA andASCA, markers that are used for IBD, was unexpectedlyhigh in our cohort of patients with celiac disease: 8 patientswere positive for pANCA (IgG) and 16 patients werepositive for ASCA (IgG and/or IgA). These results indicatethat the presence of pANCA or ASCA in the serum ofpatients with chronic diarrhea does not exclude celiacdisease. A prospective study is required to determine

whether pANCA and/or ASCA identify patients at risk fordeveloping secondary autoimmune disease.

KEY WORDS: Inflammatory bowel disease; autoimmune disease;IgA deficiency.

INTRODUCTION

Celiac disease is defined as a disease of the proximalsmall intestine characterized by damage to the smallintestinal mucosa and is associated with permanentintolerance to gluten. Histologically celiac disease ischaracterized by a small bowel mucosal hyperplasticvillous atrophy with lowering of the villous height tocrypt ratio, an inflammatory infiltrate of the laminapropria, and an increase in intraepithelial lymphocytes(1). Clinical symptoms and abnormal small bowel his-tology resolve on removal of gluten from the diet (2).The wide spectrum of clinical manifestations rangesfrom asymptomatic to fatigue, vague abdominal symp-toms, weight loss, and diarrhea, to frank malabsorptionwith steatorrhea and nutrient deficiencies. Furthermore,prolonged celiac disease is associated with an increasedrisk for malignancy, especially intestinal T-cell lym-phoma (3, 4).

Whenever suspected, the diagnosis of celiac disease isverified with at least one intestinal biopsy specimenshowing the above described characteristic findings andan unequivocal clinical response to a gluten-free diet (5).Screening for elevated levels of several circulating (au-to)antibodies is helpful in selecting suspected patients forthe requirement of an intestinal biopsy for verification ofthe diagnosis. The level of these circulating (auto)anti-bodies is further useful in monitoring patients for com-

1Department of Clinical and Experimental Immunology, UniversityHospital Maastricht, Maastricht, The Netherlands.

2Department of Pediatrics, University Hospital Maastricht, Maastricht,The Netherlands.

3Department of Immunology, University Hospital Groningen, Gro-ningen, The Netherlands.

4Department of Gastroenterology, University Hospital Maastricht,Maastricht, The Netherlands.

5To whom correspondence should be addressed at Department ofClinical and Experimental Immunology, University Hospital Maas-tricht, P. O. Box 5800, 6202 AZ Maastricht, The Netherlands. Tel:�31-43-3881433; fax: �31-43-3884164; e-mail: [email protected].

Journal of Clinical Immunology, Vol. 22, No. 5, September 2002 (©2002)

2810271-9142/02/0900-0281/0 © 2002 Plenum Publishing Corporation

pliance with the gluten-free diet and/or a gluten chal-lenge. These serological tests include the detection ofantiendomysium antibodies (EMA) and antigliadin anti-bodies (AGA). The endomysial antigen has been identi-fied as tissue transglutaminase (tTG) (6), and recentlyrecombinant human tTG (rh-tTG) has become availablefor testing in solid-phase assays (7). In clinical practiceantibodies are primarily tested for the IgA isotype.However, due to the relatively high incidence of IgA-deficiency in patients with celiac disease, additionaltesting for the IgG isotype is recommended (8, 9). EMA,and consequently anti-tTG antibodies (tTGA), are con-sidered highly specific and sensitive for celiac disease.AGA have been reported to be less sensitive and specificfor celiac disease than either EMA or tTGA (10, 11). Thedifferential diagnosis of chronic diarrhea includes, be-sides celiac disease, inflammatory bowel disease (IBD).IBD consists of two entities, ulcerative colitis andCrohn’s disease, conditions that are associated withperinuclear antineutrophil cytoplasmic antibodies(pANCA) and/or anti-Saccharomyces cerevisiae anti-bodies (ASCA) (12, 13).

The aim of the present study was to determine theprevalence of serological markers for IBD in our patientswith biopsy-confirmed celiac disease. Hereto we haveanalyzed the prevalence of the (auto)antibodies that arerelevant for celiac disease and IBD (EMA, AGA, rh-tTGA, pANCA, and ASCA) in a cohort of 37 untreatedpatients with biopsy-confirmed celiac disease (MarshIIIb/c). The results obtained reveal that rh-tTGA but notAGA have similar high sensitivity for celiac disease asEMA, and that testing for both IgA and IgG improves thesensitivity of all three serological tests. The prevalenceof pANCA and ASCA in our cohort of 37 patients withceliac disease was found to be 22% and 43%, respec-tively, suggesting that these latter antibodies do notdiscriminate celiac disease from IBD.

MATERIALS AND METHODS

Patients

Patients included in the present study were selectedretrospectively via the department of pathology using alist of duodenal biopsies that were evaluated for histo-logical characteristics of celiac disease during the periodJanuary 1996 to June 2000. Next, the clinical history andfinal diagnosis made by the departments of gastroenter-ology and pediatrics were evaluated. Only patients thatfulfilled the following diagnostic criteria for celiac dis-ease were included: (a) clinical suspicion of celiac

disease at presentation, (b) the presence of (sub)totalvillous atrophy (Marsh score of IIIb/c) at duodenalbiopsy, and (c) clinical remission on a gluten-free diet.At the time of diagnosis serum was sampled and thepresence of (auto)antibodies were tested in the immunol-ogy laboratory.

As controls, the sera of 35 healthy controls (UniversityHospital Maastricht) and of 40 IBD controls were in-cluded. The IBD controls consisted of 20 patients withulcerative colitis and 20 patients with Crohn’s disease.These IBD patients were diagnosed in the UniversityHospital Groningen as based on accepted clinical, endo-scopic, and radiological criteria supported by histopa-thology (14, 15).

Antiendomysium Antibodies

Serum EMA levels of the IgA and IgG isotype weredetermined by means of indirect immunofluorescence(IIF) using fixed cryostat sections of the distal part ofmonkey esophagus (Scimedx, Densville, NJ). Briefly,the sections were incubated for 30 min with the patientserum diluted 1:10 in phosphate buffered saline (PBS).After extensive washing the sections were incubatedwith fluorescein–isothiocyanate (FITC)-conjugated anti-human IgA or IgG antibodies (Scimedx) for another 30min. The slides were washed and examined by fluores-cent microscopy by two independent observers; in caseof a difference of opinion a third observer was decisive.Staining of endomysial areas surrounding the sarco-lemma of smooth muscle fibers showing a pattern ofinterconnecting networks was considered positive.

Antigliadin Antibodies

The presence of AGA of the IgA and IgG isotype wasdetermined by means of fluorescent enzyme immunoas-say (FEIA)-Unicap (Pharmacia Diagnostics, Freiburg,Germany). The assay was performed according to themanufacturers protocol. In this assay the sera werediluted 1:100 in diluent. The results were presented inmg/liter and were calculated on a standard curve. Thecutoff values for the IgA AGA were for all ages 3.0mg/liter, whereas for the IgG AGA the cutoff value forpatients below 3 years was 30 mg/liter and for patients of3 years and older 18 mg/liter. Cutoff values wereprovided by the manufacturer and were based on theoptimal combination of clinical sensitivity and specific-ity in a cohort of patients with celiac disease and patientsfree from celiac disease but with gastrointestinal symp-toms.

282 DAMOISEAUX ET AL.


Antibodies to Recombinant Human TissueTransglutaminase

Serum levels of IgA and IgG rh-tTGA were quanti-tated by the Celikey-ELISA (Pharmacia Diagnostics)according to the manufacturers protocol. Serum wasdiluted 1:100 in diluent. The results were presented inarbitrary units (AU)/ml and were calculated on a stan-dard curve. The cutoff value for both the IgA and IgGrh-tTGA was 5 AU/ml. Cutoff values were provided bythe manufacturer and were based on the optimal combi-nation of clinical sensitivity and specificity in a cohort ofpatients with celiac disease and patients free from celiacdisease but with gastrointestinal symptoms.

Antineutrophil Cytoplasmic Antibodies

Detection of ANCA was performed by an IIF tech-nique on ethanol-fixed granulocytes (INOVA Diagnos-tics, San Diego, CA). Samples were initially diluted 1:16in PBS. A FITC conjugated goat anti-human IgG anti-body (INOVA) was used for detection of bound IgGantibodies. All slides were evaluated by two independentobservers; in case of a difference in opinion a thirdobserver was decisive. Three staining patterns wereconsidered ANCA positive: perinuclear (pANCA), finegranular cytoplasmic (cANCA), and diffuse homoge-neous cytoplasmic (atypical ANCA) staining of theneutrophils. Samples that were scored positive werefurther analyzed in dilutions of 1:32 and 1:64. In addi-tion, ANCA specificities were tested in all sera byELISA for seven different antigens: proteinase 3 (PR3),myeloperoxidase (MPO), bactericidal permeability-increasing protein (BPI), elastase, cathepsin G, ly-sozyme, and lactoferrin (Orgentec Diagnostika, Mainz,Germany). Patient samples were diluted 1:100 withdiluent. Next the ELISA was performed and evaluatedaccording to the manufacturers protocol. For evaluationeach patient’s optical density was corrected by a factorspecific for each distinct antigen. These correction fac-tors were provided by the manufacturer. A sample wasconsidered positive for a specific ANCA antigen whenthe corrected optical density of the patient sample forthat specific antigen was higher than the optical densityof the cutoff control.

Anti-Saccharomyces cerevisiae Antibodies

Serum levels of IgA ASCA were quantitated by theMedizym-ASCA IgA ELISA (Medipan, Selchow, Ger-many) according to the manufacturers protocol. Serumwas diluted 1:50 in diluent. The results were presented in

AU/ml and were calculated on a standard curve. Thecutoff value for the IgA ASCA was 20 AU/ml asprovided by the manufacturer. For the detection of IgGASCA the Medizym-ASCA IgG ELISA (Medipan) wasused. According to the manufacturers protocol the serumwas diluted 1:50 in diluent. The binding index wascalculated on dividing the optical density of the sampleby the optical density of the cutoff control (20 AU/ml) assupplied by the manufacturer. The sera were consideredpositive in case the binding index was �1.0.

Statistics

Results are presented as the absolute number andpercentage of patients that are positive for the respectivebiological marker. Overall differences between variableswere evaluated by a chi square test. P values �0.05 wereconsidered statistically significant.

RESULTS

EMA and rh-tTGA IgA Have Similar Sensitivity forCeliac Disease

A total of 37 patients (15 males, 22 females; medianage 33, range, 1–91 years) diagnosed with celiac diseaseduring the period January 1996 to June 2000 in thedepartments of gastroenterology (adults; n � 23; 10males, 13 females) and pediatrics (children; n � 14; 5boys, 9 girls) were included in the present study. EMA,AGA, and rh-tTGA of the IgA isotype were detected in32 of 37 patients (86.5%), 27 of 37 patients (73.0%), and32 of 37 patients (86.5%), respectively. Four of fivepatients in which no IgA autoantibodies were detectedwere IgA deficient (see next paragraph). All patients whowere identified by positive EMA were also positive forrh-tTGA. Furthermore, none of the patients who werenegative for EMA were identified by positive AGA(Table I). Finally, no difference in prevalence of EMA,AGA, or rh-tTGA was found between adults and chil-dren (not shown). Altogether, in our cohort of 37 celiacdisease patients there was a 100% correlation betweenthe presence of IgA EMA and IgA rh-tTGA. Further-more, positive tests were found in 86.5% of our patientsand additional testing for IgA AGA did not improve thesensitivity.

Additional Analysis of the IgG Isotype Improves theDiagnostic Sensitivity for Celiac Disease

In five patients with celiac disease no IgA EMA,AGA, or rh-tTGA were found (Table I; patients 12, 13,

AUTOANTIBODIES IN CELIAC DISEASE 283


18, 25, and 37). These five patients did not cluster into aspecific subgroup in terms of age or sex. The serum levelof total IgA in four of these five patients was below thedetection threshold (patients 12, 13, 18, and 37). Weanalyzed the sera of these five patients for autoantibodiesof the IgG isotype in the EMA, AGA, and rh-tTGAassays. All four IgA-deficient patients were positive inall these three tests when analyzing for the IgG isotype(not shown). The remaining patient (# 25) was seroneg-ative for both IgA and IgG isotypes. Altogether, thesensitivity of a specific assay increased on combinedanalysis for the IgA and IgG isotypes and 36 of 37patients (97.3%) were positive both in the EMA andrh-tTGA assays.

The Presence of pANCA or ASCA Is Not an ExclusionCriterion for Celiac Disease

Eight of 37 patients with celiac disease (21.6%) werepositive for ANCA (Table II). In all eight patients aperinuclear pattern (pANCA) was observed. Upon titra-tion of the sera, three sera (all adults) were positive in adilution of 1:16, whereas five sera (4 children and 1adult) were positive in a dilution of 1:64 or more.Remarkably, three of four patients with IgA deficiencywere pANCA positive. In only one of the pANCA-positive patients a condition that has been previouslyassociated with ANCA (16) was diagnosed, i.e., autoim-mune hepatitis. Since we have used a relatively low

Table I. Prevalence of Autoantibodies in 37 Patients with biopsy-confirmed celiac disease

Patientnumber

Agea

(year) SexEMAIgA

AGAIgA

rh-tTGAIgA

ANCA ASCA

IgG Specb IgA IgG

1 1 F � � � � B � �2 1 F � � � � � � �3 1 F � � � � � � �4 1 F � � � � � � �5 1 F � � � � � � �6 1 F � � � � Ly � �7 2 F � � � � � � �8 2 F � � � � � � �9 3 M � � � � P � �

10 5 M � � � � � � �11 6 M � � � � � � �12c 8 M � � � � B,C,E,Ly � �13c 10 M � � � � � � �14 16 F � � � � � � �15 22 F � � � � � � �16 25 F � � � � � � �17 30 F � � � � B � �18c 32 F � � � � C,Ly � �19 38 M � � � � P � �20 42 F � � � � � � �21 43 M � � � � � � �22 45 F � � � � � � �23 46 M � � � � � � �24 46 M � � � � Ly � �25c 49 F � � � � � � �26 51 M � � � � � � �27 52 M � � � � B � �28 53 F � � � � B � �29 57 F � � � � B � �30 59 M � � � � Ly � �31 62 F � � � � � � �32 62 M � � � � � � �33 64 M � � � � La � �34 67 M � � � � � � �35 68 F � � � � B � �36 68 F � � � � P � �37c 91 F � � � � B,Ly � �

aAge refers to the age at the time of diagnosis.bANCA specificities include B, BPI; C, cathepsin G; E, elastase; La, lactoferrin; Ly, lysozyme; P, proteinase 3.cRespective patients are IgA deficient (�0.06 g/liter).



serum dilution (1:16) for the detection of pANCA by theIIF technique we have validated this cutoff using healthycontrols (n � 35). Only 1 of 35 normal controlsrevealed a pANCA reactivity indicating that the preva-lence of pANCA, as detected in our system, is signifi-cantly increased as compared to healthy controls (P �0.05; chi square test). Next, we incorporated 40 IBDcontrols as positive controls. As shown in Table II, 17/20(85%) patients with ulcerative colitis had pANCA intheir serum, whereas pANCA was detected in the serumof only 4 of 20 (20%) patients with Crohn’s disease.

ANCA specificities were analyzed only in the sera ofthe patients with celiac disease. By ELISA 16 of 37patients were positive for one (n � 13) or more (n � 3)ANCA antigens: PR3 (n � 3), MPO (n � 0), BPI (n �8), elastase (n � 1), cathepsin G (n � 2), lysozyme(n � 6), and lactoferrin (n � 1). No correlationbetween ANCA as detected by IIF and by ELISA wasfound (Table I).

The presence of ASCA was determined for both theIgA and IgG isotype. Eleven of 37 patients with celiacdisease (29.7%) were positive for IgA ASCA and 12patients (32.4%) were positive for IgG ASCA (Table II).Seven of 37 patients (18.9%) were positive for both IgAand IgG ASCA resulting in a positive correlation be-tween IgA and IgG ASCA (P � 0.05; chi square test).Within the group of four patients with IgA deficiencyonly one patient was IgA ASCA negative and IgG ASCApositive. There was no correlation between the presenceof pANCA and ASCA. Also, the cutoff of the ASCAassay was validated by analyzing healthy controls (n �35). Only 1 of 35 normal controls revealed ASCAreactivity with both IgG as well as IgA. This indicatesthat the prevalence of ASCA (IgA and/or IgG) issignificantly increased in patients with celiac diseasecompared to healthy controls (P � 0.001; chi squaretest). Next, 40 sera of IBD patients were analyzed aspositive controls. As shown in Table II, only 1 of 20patients (5%) with ulcerative colitis had ASCA (IgA) inhis serum. More surprising, only 2 of 20 patients (10%)

with Crohn’s disease revealed ASCA (both IgA and IgG)reactivity in their serum.

DISCUSSION

In the present study we have analyzed the prevalenceof (auto)antibodies that are known to be relevant for thedifferential diagnosis of celiac disease and IBD in acohort of 37 untreated patients with biopsy-confirmedceliac disease. The results obtained reveal that the assayfor rh-tTGA has similar high sensitivity (86.5%) forceliac disease as EMA and that testing for both IgA andIgG improves the sensitivity (97.3%) of both serologicaltests. The prevalence of pANCA and ASCA (IgG orIgA), markers that are associated with IBD, was unex-pectedly high in our cohort of patients with celiacdisease: 22% and 43%, respectively. This was signifi-cantly more than in 35 healthy controls. These resultsconfirm that EMA and rh-tTGA are sensitive markers forceliac disease and this enables the use of these markersfor screening in order to select suspected patients for therequirement of an intestinal biopsy for verification of thediagnosis. Second, our results indicate that the presenceof pANCA and ASCA does not exclude celiac disease.

It is well documented that EMA and tTGA aresensitive and specific for celiac disease (7, 17). It also isestablished that AGA are less sensitive and specific forceliac disease than EMA and tTGA (17, 18). Finally,since selective IgA deficiency is frequently associatedwith celiac disease, with an incidence 10 to 15-foldhigher than in the general population (8, 9, 19), com-bined testing for autoantibodies of the IgA and IgGisotypes further increases the sensitivity (7, 17). Due tothe high sensitivity and specificity of the EMA test, thisIIF test is recognized as the gold standard for serologicaldetecting celiac disease. Since the ELISA test for rh-tTGA reveals similar results as the EMA IIF test, it isanticipated that the ELISA will replace the IIF becausethe ELISA is easier to handle, less time consuming, andless subject to interoperator variation.

Table II. Prevalence of pANCA and ASCA in Patients with Celiac Disease and Controlsa

pANCA(IgG)

ASCA(IgA)

ASCA(IgG)

ASCA(IgA and/or IgG)

Celiac diseaseChildren 3/14 (21.4%) 3/14 (21.4%) 2/14 (14.3%) 4/14 (28.6%)Adults 5/23 (21.7%) 8/23 (34.8%) 10/23 (43.5%) 12/23 (52.2%)Total 8/37 (21.6%) 11/37 (29.7%) 12/37 (32.4%) 16/37 (43.2%)

Healthy controls 1/35 (2.9%) 1/35 (2.9%) 1/35 (2.9%) 1/35 (2.9%)IBD controls

Ulcerative colitis 17/20 (85.0%) 1/20 (5.0%) 0/20 (0.0%) 1/20 (5.0%)Crohn’s disease 3/20 (20.0%) 2/20 (10.0%) 2/20 (10.0%) 2/20 (10.0%)

apANCA, Perinuclear antineutrophil cytoplasmic antibodies; ASCA, anti-Saccharomyces cerevisiae antibodies; IBD, inflammatory bowel disease.



ASCA and pANCA are well recognized markers forCrohn’s disease and ulcerative colitis (13, 20–23). In thepresent study, we first confirmed these results in a cohortof 40 IBD patients in order to validate the assays andrespective cutoff values used in our laboratory. Next, weobserved a relatively high prevalence of both pANCA aswell as ASCA in patients with celiac disease. Theprevalence of pANCA has also been described by Free-man (24) but not by others (22, 23, 25). However, five ofsix patients with pANCA and celiac disease described byFreeman (24) had concomitant other autoimmune disor-ders, such as colitis or primary sclerosing cholangitis,that have previously been found to be associated withANCA (16). In our study, only one of eight patients withpANCA had an additional ANCA-associated autoim-mune disease, i.e., autoimmune hepatitis. The discrep-ancy between the present study and the studies that didnot observe pANCA in patients with celiac disease maybe explained by the fact that the latter studies weredesigned to examine the prevalence of ANCA in IBD(22–25). In these studies the patients with celiac diseasewere used as disease controls and it was not describedwhich diagnostic criteria were used and whether serumsamples were obtained at the time of diagnosis or at thetime patients were already treated with a gluten-free diet.Like in IBD (15, 26, 27), the specificity of pANCA inceliac disease is quite diverse. In our small cohort ofpatients with pANCA, the majority had antibodies tolysozyme. Antibodies to lysozyme, however, are notspecific for celiac disease, since these antibodies can befound in a variety of inflammatory disorders, includingIBD (28, 29). The high prevalence of ASCA in celiacdisease also was previously reported by Giaffer et al.(30), who reported high levels of IgG ASCA but not IgAASCA. Darroch et al. (31), however, did not observeincreased levels of either IgG ASCA or IgA ASCA inpatients with celiac disease compared to healthy controls.Again, in contrast to the present study, both studies weredesigned to examine the prevalence of ASCA in patientswith IBD resulting in similar drawbacks as mentionedabove with respect to the population of patients withceliac disease. Indeed, the first study included 11 of 14patients with celiac disease who were on a gluten-freediet (30).

With respect to the etiology of ASCA it has beenhypothesized that these antibodies are the consequenceof increased permeability of the small intestine. Thisphenomenon is observed in both Crohn’s disease as wellas celiac disease. The impaired intestinal barrier functionresults in increased antigen leakage and immune stimu-lation. Typically, either the intestinal permeability or theimmune response generated is selective for yeast anti-

gens, since not all dietary components induce antibodyproduction. This selectivity may be genetically deter-mined. Indeed, a significant proportion of healthy first-degree relatives of Crohn’s disease patients have abnor-mal intestinal permeability (32) and ASCA (33),suggesting a primary defect of the tight junctions. On theother hand, a selective immune response directs towardgenes that are involved in antigen processing, presenta-tion, and/or recognition. Since ASCA are not restrictedto patients with Crohn’s disease, but as shown in thepresent study, also are apparent in patients with celiacdisease, candidate genes should be searched for ingenetic regions that reveal linkage with both diseases.Besides linkage to the MHC region, this also holds forthe regions 16q12 and 5q31 (34–36). Further studiesmay reveal whether these regions are indeed involved inthe etiology of ASCA. Whether a genetic factor also isinvolved in the etiology of pANCA is unlikely, sincestudies on the occurrence of pANCA in relatives ofpatients with ulcerative colitis are not conclusive. It isgenerally believed that ANCA are the result of chronicinflammation with extensive infiltration of neutrophilsand release of granula content. The concomitant presenceof reactive metabolites may alter the proteins releasedfrom the neutrophilic granules rendering these proteinsimmunogenic (16). Although this may be true for ulcer-ative colitis where pANCA tend to occur mainly inpatients with a severe course of disease, it will notexplain the occurrence of pANCA in celiac disease.Indeed, in celiac disease lymphocytes predominate in thelesions, whereas neutrophils are hardly present. Finally,although ASCA are considered to be more prevalent insmall bowel disease, whereas pANCA are more preva-lent in colonic disease, we observed 3 of 37 (8%) patientswith celiac disease who were positive for both pANCAand ASCA. Similar results have been obtained in IBDpatients where 24 of 582 (4%) were positive for bothantibody specificities (37).

An increased prevalence of a wide variety of autoim-mune disorders in celiac disease has been reported bothin children and in adults. These include insulin-dependent diabetes mellitus, connective tissue disease,Hashimoto’s thyroiditis, Graves’ disease, and IBD (38–41). Furthermore, the prevalence of autoimmune disor-ders in celiac disease appears to be related to the durationof exposure to gluten (42). Apart from shared or adjacentHLA loci in these diseases, as is the case for celiacdisease, insulin-dependent diabetes mellitus and thyroidautoimmunity, posttranslational modification of potentialautoantigens by tTG could play a role in the pathogenesisof these with celiac disease-associated autoimmune dis-orders (43–45). From our data the question arises



whether patients who have pANCA and/or ASCA are atrisk for developing IBD. A prospective study is requiredto answer this question. Moreover, it has to be deter-mined whether these autoantibodies will disappear or notwhen patients are on a gluten-free diet.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Y. Knapen-Portz forher excellent technical assistance and Dr. J.W. Arends,head of the pathology department of the UniversityHospital Maastricht, for providing the pathological eval-uations of the duodenal biopsies. We thank Dr. S.Vermeire (Department of Gastroenterology, UniversityHospital Gasthuisberg, Leuven, Belgium) for helpfuldiscussions.

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Diagnostic Value of Anti-Saccharomyces cerevisiae and Antineutrophil Cytoplasmic Antibodies for Inflammatory Bowel Disease: High Prevalence in Patients with Celiac Disease