In siblings of celiac children, rectal gluten challenge reveals gluten sensitization not restricted to celiac HLA

GASTROENTEROLOGY 1996;111:318–324

In Siblings of Celiac Children, Rectal Gluten Challenge Reveals Gluten Sensitization Not Restricted to Celiac HLA RICCARDO TRONCONE,* LUIGI GRECO,* MARINA MAYER,* GIUSEPPE MAZZARELLA,‡ LUIGI MAIURI,* MAURO CONGIA,§ FULVIA FRAU,§ STEFANO DE VIRGILIIS,§ and SALVATORE AURICCHIO* *Department of Pediatrics, University Federico II, Naples; ‡Istituto di Scienze dell’Alimentazione, Consiglio Nazionale delle Ricerche, Avellino; and §Istituto di Clinica e Biologia dell’Eta’Evolutiva, University of Cagliari, Cagliari, Italy

Background & Aims: Inflammatory changes in the rectum of patients with celiac disease after local instillation of gluten have been reported. The aim of this study was to examine rectal mucosa after local gluten challenge in children with celiac disease and their siblings. Methods: Rectal biopsy specimens were obtained before and 6 hours after rectal challenge with a peptictryptic digest of gliadin in 33 children with treated celiac disease, 12 controls, and 19 siblings of children with celiac disease. Epithelium and lamina propria volumes were determined, and CD3/ and gd/ lymphocytes were counted. Results: After local instillation of gliadin, a significant increment in the absolute number of intraepithelial lymphocytes was noted in patients with celiac disease but not in controls. Immunohistochemical analysis showed a significant increase in CD3/ and gd/ cells, with the gd/CD3 ratio remaining unchanged after challenge. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis was used to classify 6 of 13 siblings as having celiac disease. The positivity was not associated with the presence of the heterodimer encoded by the DQA*0501 DQB1*0201 alleles in any of the siblings. Conclusions: All patients with celiac disease were identified by rectal gluten challenge. Approximately half of the siblings reacted to rectal instillation of gluten. The genetic background of such sensitization to gluten remains to be elucidated.

C

eliac disease is sustained by an immunologic process that mainly affects the jejunal mucosa.1 Nonetheless, attention has been recently given to the presence of inflammatory changes in the rectal mucosa of patients with celiac disease. Quantitative histological and immunocytochemical studies of rectal mucosa from patients with untreated celiac disease have shown increased populations of plasma cells, mast cells, and CD3/ and gd/ lymphocytes.2 Other signs suggesting an activated T cell–mediated mucosal immune response were also noted, such as activated (CD25/) lymphocytes expressing interleukin 2 receptors.2 Furthermore, the expression of the adhesion / 5e10$$0030

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molecule vascular cell adhesion molecule 1 was higher in untreated than treated celiac disease or control mucosa.3 Withdrawal of gluten from the diet results in a reduction of the inflammatory response in the infiltrated cell populations and in the expression of activation molecules.2,3 These observations and the ease of access (compared with the jejunum) have favored the rectum as a site for gluten challenge. Significant immunopathologic, structural, and functional changes have been documented in adult patients with celiac disease during rectal challenge with gluten; in particular, an increased lamina propria volume and number of mast cells, neutrophils, and lymphocytes have been observed.4 An increase in intraepithelial lymphocyte numbers of more than 10% above baseline has been suggested as a test for the diagnosis of celiac disease both as a screening assay for untreated subjects and as a confirmatory test in patients with treated celiac disease.5 Recent studies have suggested that gluten sensitization is a condition of abnormal immunity to gluten that is restricted to genetically predisposed individuals. This condition is not exclusive of subjects on a gluten-containing diet who have a flat mucosa but may be suspected in subjects with milder changes of mucosal architecture and/or immunologic abnormalities, such as those in patients with celiac disease. These changes may include high gliadin antibody titer,6,7 increased g/d receptor– positive intraepithelial population,8,9 presence of serum endomysial antibody,10 and other signs suggesting activated mucosal cell–mediated immunity.11 The subjects may have gluten-sensitive signs, i.e., changes in gut pathology, and symptoms on gluten withdrawal and reintroduction. The aim of this study was to investigate the rectal mucosa after local instillation of gluten not only in patients with celiac disease but also in their siblings because 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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RECTAL GLUTEN CHALLENGE IN CELIACS AND SIBLINGS 319

previous observations7,9,11 have suggested the presence of immunologic and pathological abnormalities that could suggest sensitization to gluten in this population.

Subjects and Methods Patients Thirty-three patients with celiac disease (14 male and 19 female; median age, 12 years; age range, 7–19 years) were studied. All patients were diagnosed according to the criteria established in 1971 by the European Society for Paediatric Gastroenterology and Nutrition12; all had a subtotal villous atrophy while on a gluten-containing diet, a good clinical and histological response to a gluten-free diet, and histological relapse after gluten challenge. At the time of the study, all patients had been on a gluten-free diet for at least 2 years. Twelve control subjects (7 male and 5 female; median age, 8.3 years; age range, 5–10 years) were also studied; their final diagnoses were different from celiac disease (6 had constipation, 3 recurrent abdominal pain, and 3 failure to thrive). Finally, 19 healthy siblings of patients with celiac disease (7 male and 12 female; median age, 12.2 years; age range, 6–21 years) were investigated. Informed consent was obtained from all patients and a parent or guardian when appropriate. The study design was approved by the University ethical committee.

Challenge Protocol The subjects were challenged with 2 g of Frazer’s peptic-tryptic digest of gliadin (Sigma Chemical Co., St. Louis, MO).13 In 7 subjects, a peptic-tryptic digest of 2 g of bovine serum albumin (Sigma) was also used, obtained using the same protocol as for gliadin. The protein was dissolved in 20 mL sterile water (osmolality, 400 mOsm/L) and instilled into the lower rectum via a small catheter. Prechallenge and postchallenge biopsy specimens were obtained at 6 hours using a Crosby capsule positioned 10 cm from the anal verge.

Processing of Mucosal Biopsy Specimens Because of the limited (for ethical reasons) number of biopsy fragments that could be obtained from the same subject, both resin-embedded and frozen tissue were available for investigation from only a few subjects. Rectal biopsy specimens were resin-embedded in 27 patients with celiac disease, 12 controls, and 19 siblings. Rectal mucosa was quickly oriented on a card and fixed for 3 hours at room temperature in 0.1 mol/L cacodylate-buffered 2.5% ultrapure glutaraldehyde and cured in resin (Araldite; Reading, Berkshire, England). Sections of 1 mm thick were stained with 1% aqueous toluidine blue. Immunohistochemical analysis was performed in rectal biopsy specimens from 9 patients with celiac disease, 10 controls, and 13 siblings. The specimens were embedded in OCT compound (Tissue Tek; Miles Laboratories, Elkhart, IN), snap frozen, and stored in liquid nitrogen. Cryostat sections (5 mm) were air dried at room temperature and then fixed in acetone

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for 10 minutes. All sections were repeatedly washed at room temperature in Tris-buffered saline, incubated in normal rabbit serum (1:200 in Tris-buffered saline for 30 minutes), and stained according to the peroxidase-antiperoxidase method. The sections were individually tested with the monoclonal antibodies to CD3 (Dakopatts, Copenhagen, Denmark) and gd T-cell receptor (TCRd 1; T Cell Diagnostics, Cambridge, MA). After a 1-hour incubation at room temperature, the sections were incubated with rabbit anti-mouse serum (Dakopatts) for 30 minutes and with peroxidase antiperoxidase complex (Dakopatts) for another 30 minutes. Slides were washed in Trisbuffered saline for 10 minutes after incubation of each antibody. The immune reaction product was developed by a final incubation of 5 minutes with 2-amino-9-ethyl-carbazole. Omission of the primary antibody served as a control against nonspecific antibody binding. The sections were finally stained with Mayer’s hematoxylin and mounted.

Morphometric Analysis One-micrometer-thick toluidine blue–stained Epon sections (TAAB Laboratories, Reading, Berkshire, England) were quantitated by computerized image analysis (Kontron, Eching, England). Surface epithelium, crypt epithelium, and lamina propria were measured with respect to a constant test area (104 mm2) of muscularis mucosa. The cellularity of each fragment was assessed by determining the number of crypt and surface epithelial lymphocytes. The total number of sectioned profiles observed within the unit tissue volume overlying 104 mm2 muscularis mucosa was divided by effective section thickness (mean diameter of nuclei plus actual section thickness, or 5 mm / 1 mm) to obtain the absolute number of cells present within each tissue volume compartment. For immunostained cells, crypt and surface epithelial and lamina propria lymphocytes were considered. Absolute populations were also determined with reference to a standard reference area of muscularis mucosae (104 mm2). These counts were obtained through a microscope with a calibrated ocular graticule aligned parallel to the muscularis mucosae. In 9 subjects (3 patients with celiac disease, 3 controls, and 3 siblings), all measurements were repeated by two independent observers. The coefficient of variation (i.e., the mean absolute difference between observers times 100 divided by the mean intraobserver pooled value) was 7.5% and 4% for CD3/ cells in the epithelium and lamina propria, respectively, and 10.3% and 11.5% for gd/ cells in the epithelium and lamina propria, respectively.

HLA Typing Molecular typing was performed as described previously.14 Briefly, DNA was obtained by a rapid extraction method. Amplification of the polymorphic second exon of DRB1, DQA1, and DQB1 genes and dot blot analysis of amplified DNA with sequence-specific oligonucleotide probes was performed. DR4 specific amplification and sequence-specific oligonucleotide probe hybridization for DRB1*04 alleles was performed in DR4-positive patients.

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Table 1. Compartment Volume and Intraepithelial Lymphocyte Number in Rectal Biopsy Specimens Taken Before and After Local Instillation of a Peptic-Tryptic Digest of Gliadin Controls (n Å 12)

Surface epithelial volume (106 mm3) Crypt epithelial volume (106 mm3) Lamina propria volume (106 mm3) Intraepithelial lymphocytes/ 104 mm2 muscularis mucosae

Patients with celiac disease (n Å 27)

Prechallenge

Postchallenge

Pa

Prechallenge

Postchallenge

Pa

0.32 { 0.09 1.83 { 0.35 2.22 { 0.43

0.34 { 0.10 1.74 { 0.71 2.70 { 0.57

NS NS NS

0.32 { 0.15 1.56 { 0.53 2.71 { 1.08

0.29 { 0.10 1.79 { 0.50 2.63 { 0.78

NS NS NS

44.82 { 8.14

38.72 { 11.4

NS

61.42 { 32.86

87.89 { 43.54

õ0.01

NOTE. Results are mean { SD. a Wilcoxon matched-pairs signed rank test.

Intestinal Permeability and Serological Assays Cellobiose and mannitol permeability tests were performed according to Strobel et al.15 Serum gliadin antibody and endomysial antibody were detected by enzyme-linked immunosorbent assays and indirect immunofluorescence, respectively, as already described.16

Jejunal Biopsy Jejunal biopsies were performed with a Watson capsule under fluoroscopic control. Each biopsy specimen was sliced in two parts. One fragment was fixed in 10% formalin, embedded in paraffin wax, sectioned at 5-mm thickness, and stained with H&E. The second fragment was embedded in OCT compound, snap frozen, and stored in liquid nitrogen. Immunohistochemistry on 5-mm cryostat sections was performed as described above. Intraepithelial CD3/ and gd/ intraepithelial lymphocytes were counted per millimeter of epithelium.

Statistics Data were edited, corrected, and assessed by descriptive analysis using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL). Multivariate analysis was performed by a stepwise discriminant function including only variables contributing with P values of õ0.001 to the discrimination of groups.

Results Rectal Gluten Challenge in Patients With Celiac Disease and Controls Morphometric analysis on resin-embedded rectal mucosa. No significant differences were noted be-

tween patients with celiac disease and controls when compartment volumes (surface and crypt epithelial volumes and lamina propria volume) were measured in prechallenge biopsy specimens (Table 1). In the same specimens, the absolute number of intraepithelial lymphocytes was slightly higher (P Å 0.044) in rectal biopsy specimens from patients with celiac disease. Although no differences were noted between the volume compartments before and 6 hours after the local / 5e10$$0030

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instillation of gluten, a significant (P õ 0.05) increment in the absolute number of intraepithelial lymphocytes was noted after challenge in patients with celiac disease (Table 1). Such an increase was absent in controls. Six hours after challenge, 22 of 27 patients with celiac disease (81.5%) had an absolute number of intraepithelial lymphocytes greater than 50 per 104 mm2 muscularis mucosae (i.e., ú1SD above the mean of controls). Twenty of 27 (74.1%) showed a percentage increment above 32% (i.e., ú1SD above the mean of controls). Rectal challenge with bovine serum albumin. Seven patients with celiac disease also underwent

rectal challenge with a peptic-tryptic digest of bovine serum albumin under conditions identical to those of the gluten challenge. No significant differences were noted in the number of intraepithelial lymphocytes in baseline (69.0 { 19.5) and postchallenge biopsy specimens (60.6 { 25.4). A highly significant difference was noted between prechallenge and postchallenge specimens in the same subjects challenged with gluten. Immunohistochemistry. Immunohistochemical analysis showed results similar to those of the histological analysis. In the prechallenge biopsy specimens, absolute numbers of CD3/ intraepithelial and lamina propria lymphocytes were not significantly different in patients with celiac disease compared with controls. The same was observed when gd/ cells were counted. After local instillation of gliadin, the absolute number of intraepithelial CD3/ and gd/ lymphocytes was significantly (P õ 0.05) higher than prechallenge values in patients with celiac disease, but not in controls; the gd/ CD3 ratio after challenge was unchanged (Table 2). The proportion of subjects with high absolute values of intraepithelial and lamina propria CD3/ and gd/ lymphocytes after challenge is shown in Table 3. Discriminant analysis. A discriminant score was computed on the basis of all immunohistochemical data, with complete data available from 9 celiacs and 10 control subjects (Table 4). Classification analysis allowed a WBS-Gastro

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RECTAL GLUTEN CHALLENGE IN CELIACS AND SIBLINGS 321

Table 2. CD3 and gd/ Lymphocyte Numbers in Rectal Biopsy Specimens Taken Before and After Local Instillation of a PepticTryptic Digest of Gliadin Controls (n Å 10) Prechallenge CD3/ epithelium gd/ epithelium gd/CD3 epithelium CD3/ lamina propria gd/ lamina propria gd/CD3 lamina propria

30.3 3.2 0.13 104.5 5.2 0.05

{ { { { { {

13.0 1.4 0.09 25.9 1.9 0.02

Patients with celiac disease (n Å 9)

Postchallenge

Pa

{ { { { { {

NS NS NS NS NS NS

30.3 4.4 0.17 81.3 3.7 0.05

12.9 1.9 0.12 19.8 1.8 0.03

Prechallenge 34.8 4.4 0.14 131.2 6.0 0.07

{ { { { { {

13.6 1.9 0.08 130.0 4.1 0.05

Postchallenge

Pa

{ { { { { {

õ0.05 õ0.05 NS NS NS NS

58.8 6.5 0.15 157.1 11.9 0.06

34.4 2.2 0.10 76.7 11.1 0.04

NOTE. Data are expressed as lymphocytes/104 mm2 (mean { SD). a Wilcoxon matched-pairs signed rank test.

correct identification of 100% of subjects (9 of 9 celiacs and 10 of 10 controls) (Figure 1). A simplified equation with a reduced number of parameters was attempted, but the efficacy of the classification was reduced. Rectal Gluten Challenge and HLA Typing in Siblings of Children With Celiac Disease Morphometric analysis and immunohistochemistry. Taken as a group, all the parameters studied after

challenge (both in the computer-assisted morphometric analysis on resin-embedded material and in the counts obtained in immunohistochemistry) were not significantly different from prechallenge values in siblings. The proportion of subjects with high absolute values of intraepithelial and lamina propria CD3/ and gd/ lymphocytes is shown in Table 3. Ten of 19 subjects had high lymphocyte counts after rectal gluten challenge in at least two

Table 3. Proportion of Subjects With High Lymphocyte Counts 6 Hours After Local Instillation of a PepticTryptic Digest of Gliadin

Cutoffa

Variables Total IELs

b

50

Epithelium CD3/c

d

44d

Lamina propria CD3/c

101d

Epithelium gd/c

6.3d

Lamina propria gd/c

Controls (%)

5.3d

Patients with celiac disease ( %)

3/12 (25) 2/10 (20)

22/27 (81.5)

1/10 (10) 1/10 (10)

8/9 (88.9)

1/10 (10)

6/9 (77.8)

6/9 (66.7)

4/9 (44.4)

Variables contributing to discrimination

6/19 (31.6) 1/13 (7.7)

Postchallenge (epithelium / lamina propria gd/) % Increase of lamina propria CD3/ Postchallenge-prechallenge (epithelium / lamina propria CD3/) Postchallenge (epithelium / lamina propria CD3/) Postchallenge epithelium CD3/ % Increase (epithelium / lamina propria CD3/) Postchallenge 0 prechallenge (epithelium / lamina propria gd/) Postchallenge epithelium gd/

6/13 (46.2) 6/13 (46.2) 5/13 (38.5)

IEL, intraepithelial lymphocyte. 1SD above the control mean. b Counts on resin-embedded biopsy specimens. c Immunohistochemistry on frozen biopsy specimens. d Lymphocytes/104 mm2 muscularis mucosae.

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Table 4. Discriminant Function Parameters: Patients With Celiac Disease Versus Controls

Siblings (%)

a

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of the following parameters: total intraepithelial lymphocyte number (measured in resin embedded material) and numbers of lamina propria and intraepithelial CD3/ and gd/ cells. It was then clear that two subgroups were present in such a population. The discriminant score was calculated for each sibling, and 6 of 13 (those for whom complete equation data were available) were classified as having celiac disease (Figure 1). Jejunal biopsy specimens of these subjects showed a normal villous/crypt ratio. Immunohistochemical analysis showed a high count in 1 subject and slightly increased number of CD3/ intraepithelial lymphocytes in 2 subjects. Similarly, 3 of 5 siblings with a positive response to rectal gluten challenge had an increased number of gd/ intraepithelial lymphocytes in their jejunal biopsy specimens (Table 5). All subjects had normal values of cellobiose and mannitol sugar permeability test, as well as normal values of serum antigliadin antibodies and absence of serum endomysial antibodies.

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Wilks’ l

Discriminant function coefficients (unstandardized)

0.621

0.6393

0.522

0.0727

0.304

0.0237

0.272 0.221

0.0599 00.08

0.208

0.0778

0.192 0.146 Constant

00.5536 00.3829 010.34

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Figure 1. Distribution of discriminant scores in controls (䊐), patients with celiac disease (䊏), and siblings of patients with celiac disease ( ).

HLA typing. Six patients with celiac disease underwent HLA molecular typing; 4 of them encoded the DQA1*0501 DQB1*0201 heterodimer, and 2 had the DQA1*0101, DQB1*0501 haplotype, also associated with celiac disease.14 None of the siblings classified as patients with celiac disease according to the rectal challenge results encoded the DQA1*0501 DQB1*0201 heterodimer, but 1 was DR4, DQB1* 0302 (serologically DQ8) positive and 1 had the DQA1*0101, DQB1*0501 haplotype. Finally, of the siblings classified as ‘‘nonceliac’’ patients, 3 encoded the DQA1*0501 DQB1*0201 heterodimer and 1 the DQA1*0101, DQB1*0501 heterodimer. Data are summarized in Table 6.

Discussion In this study, rectal biopsy specimens from patients with treated celiac disease were analyzed morphometrically and by immunohistochemical techniques and compared with disease control mucosa. No statistically significant differences were noted when volumes and total lymphocyte populations were compared in patients with treated celiac disease and controls. The results confirm previous observations in adults,2 and a trend was observed toward an increased absolute number of intraepithelial lymphocytes in patients with celiac disease. Data obtained by computerized histological analysis on resinembedded mucosa were confirmed by immunohistochemistry. More interesting are the observations made after local instillation of a gliadin digest. On the basis of previous reports4,5 and of a pilot study, a single postchallenge biopsy specimen was taken 6 hours after a gliadin enema. A substantial infiltration of lymphocytes was noted in the epithelium. These data were paralleled by the immunohistochemical analysis showing a recruitment of CD3 / 5e10$$0030

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and gd/ lymphocytes and confirm previous observations in adult subjects.4,5 Only one study has addressed the same issue in a pediatric population, failing to show any difference in histological parameters (including intraepithelial lymphocyte counts) between prechallenge and postchallenge biopsy specimens.17 There are several possible explanations for the difference with our results (smaller number of subjects, different counting techniques); the most likely explanation is the short duration of the challenge (1 hour). The CD3-positive population infiltrating the mucosa after challenge was characterized in terms of the T-cell receptor expressed. It has been shown that patients with celiac disease have a significantly increased number of jejunal epithelial cells bearing the gd form of the Tcell receptor,18 irrespective of the gluten content of their diet.19 This phenomenon is quite specific for celiac disease, but the role of these cells remains unknown. The number of gd/ cells in the rectum of patients with treated celiac disease was comparable with that measured in nonceliac subjects, in contrast to the number in the jejunum. Nevertheless, after a local challenge, the marked increase in CD3-positive cells was matched by a significant increase in the gd/ population, with the gd/CD3 ratio remaining unchanged. Discrepancies with a previous report,20 which failed to show an increase of gd/ cells, could be ascribed to the smaller number of observations performed by these investigators and the different counting techniques. Because the functional role of these cells is unknown, it is difficult to interpret such changes. Gliadin-specific cells probably belong to the T-cell receptor ab/ subset,21 and it cannot be excluded that gd/ cells are recruited because of the epithelial stress induced by gluten. All phenomena observed in celiac disease seem quite specific for the antigen gliadin, with a rectal challenge using an unrelated protein producing no change. In terms of diagnostic value, none of the parameters, when used Table 5. Morphometric and Immunohistochemical Data on Jejunal Biopsy Specimens of 5 Siblings With a Positive Response to Rectal Gluten Challenge Sibling

Villous/ crypt ratio

CD3/ IELb

gd/ IELb

1 2 3 4 5 Normal valuesa

2.8 3.4 2.6 2.8 3.0 ú2.0

27.5 37.8 102.0 42.6 21.7 õ34.0

7.8 0.9 3.9 0.8 3.3 õ3.2

IEL, intraepithelial lymphocyte. a 2SD above the mean of a group of 28 age-matched controls. b Per millimeter of epithelium.

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Table 6. HLA Molecular Typing of Siblings of Children With Celiac Disease DRB1

DQA1

DQB1

DRB1

DQA1

DQB1

0402 0101 1101-4 1401 1301 0408

0301 0101 0501 0101 0103 0301

0302 0501 0503a 0503 0603 0304

0701 1101 0405 1101-4 1101-4 0701

0201 0501 0301 0501 0501 0201

0201 0301 0201 0301 0301 0303

Siblings with a positive rectal gluten challenge response classified as patients with celiac disease 1 2 3 4 5 6

0701 1101-4 0701 1104 0701 0102

0201 0501 0201 0501 0201 0101

0201 0301 03032 0503 0201 0501

Siblings with a negative rectal gluten challenge response classified as controls 1 1102-3 0501 0301 2 0301 0501 0201 3 0101 0101 0501 4 1501 0101 0602 5 0701 0201 0201 6 1102-3 0501 0301 a

In this subject, the assignment of the DQB1 alleles to their respective chromosomes is presumptive because the DRB1-DQB1 association has not been reported previously.

alone, can be used to predict the diagnosis of celiac disease, in contrast to data in adults.5 Nevertheless, a combination of immunohistochemical data in a discriminatory equation has allowed the correct classification of 100% of cases, both patients with celiac disease and controls. When this discriminant equation was applied to a third ‘‘unknown’’ population (siblings of patients with celiac disease), part of the population was classified as having celiac disease; these subjects can be considered sensitized to gluten. It is increasingly recognized that a condition of abnormal immunity to gluten and related proteins on a definite genetic HLA background is not restricted to patients on a gluten-containing diet who have a flat mucosa.8,9 In gluten-sensitive enteropathy, a spectrum of dynamically related pattern of mucosal damage has been described from infiltrative to hypoplastic atrophic picture.1 Moreover, not only subtle pathological abnormalities but also immunologic tests have been proposed to identify such subjects with signs of sensitization to gluten but without ‘‘overt’’ celiac disease. The abnormalities detected by such tests include high gliadin antibody titer6,7 and presence of endomysial antibody,10 increased gd/ T-cell receptor expression by intraepithelial lymphocytes,8,9 and high concentrations of immunoglobulin (Ig) M antigliadin antibody, other IgM class antibodies, and IgA antigliadin antibodies (the ‘‘celiac-like intestinal antibody’’ pattern) in specimens of jejunal fluid and whole gut lavage fluid.6,22 It should be considered that a significant association has already been found between the celiac-like intestinal antibody pattern and high density of gd/ intraepithelial lymphocytes in the jejunal mucosa.23 In this regard, first-degree relatives of patients with celiac disease represent a special population. A recent Finnish study showed that 41% of healthy relatives / 5e10$$0030

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had an increased density of gd/ intraepithelial lymphocytes in the small intestinal mucosa.9 A dose effect of DQA and DQB genes was found; the same genetic background was present in relatives with an apparently normal small intestinal mucosa, but presence of inflammatory changes detectable by immunohistochemistry, such as presence of interleukin 2 receptor–positive cells and strong staining of epithelial cells with DP and DQ antibodies, increased percentage of crypt cells in mitosis.11 On the other hand, gliadin-positive relatives with normal jejunal mucosa were genetically different from probands.24 In our study, sensitization to gluten, measured by rectal gluten challenge, was not paralleled by serological findings. Furthermore, only 3 of 5 siblings with a positive response to rectal gluten challenge had an increased number of intraepithelial gd/ lymphocytes in the jejunal biopsy specimen. Finally, no linkage was found between the positive response to rectal gluten challenge and the presence of genes known to be the most strongly associated with celiac disease.25 In the Campania region, similarly to what was found in other populations, 92% of patients with celiac disease encode the DQA1*0501, DQB1*0201 heterodimer. In fact, none of the siblings investigated in this study encoded such heterodimer conferring susceptibility to celiac disease; one was DR4 positive and another had the DQA1*0101, DQB1*0501 haplotype, also associated with celiac disease.14 On the other hand, patients with low-grade enteropathy whose symptoms and mucosal signs of activated cell-mediated immunity are clearly gluten-sensitive and whose HLA is not typical of celiac disease have been observed.26 These data support previous suggestions, based on studies of mice,27 that separate factors may be responsible for sensitization to gluten and for susceptibility to develop severe WBS-Gastro

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mucosal lesions. A different set of genes could favor the progression to villous atrophy, influencing the intensity of cell-mediated immune response in the gut mucosa. In conclusion, our data show that approximately half of the siblings of patients with celiac disease show signs of sensitization to gluten as they mount an inflammatory local response to rectal gluten challenge. The genetic background and the clinical meaning of such gluten sensitivity need to be established. Further studies, particularly at the jejunal level, are necessary before deciding if any action is to be taken in this subset of first-degree relatives.

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Received April 12, 1995. Accepted March 11, 1996. Address requests for reprints to: Salvatore Auricchio, M.D., Dipartimento di Pediatria, via Sergio Pansini 5, I-80131 Naples, Italy. Fax: (39) 81-5469811. Supported by grant 95.04648.ST75 (Utilizzo dei Cereali e Legumi della Dieta Mediterranea) by Consiglio Nazionale delle Ricerche. The authors thank Drs. Maria Micillo and Maria Cucciardi for performing the serum endomysial and gliadin antibody measurements and Drs. Francesco Paparo and Patrizia Mugione for morphometric and immunohistochemical analysis of the jejunal biopsy specimens.

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