The current diagnostic criteria for coeliac disease are based on small bowel mucosal villous atrophy with crypt hyperplasia (1). The gluten-triggered mucosal lesion develops gradually from mucosal inflammation to elongation of crypts and finally to overt villous atrophy (2,3), the atrophy thus covering only the end stage of the disease. Evidence suggests that coeliac patients may suffer from gluten-sensitive symptoms and signs even before villous atrophy has developed (4–7). Minor small bowel mucosal morphological and inflammatory changes such as increased densities of CD3+ and γδ+ intraepithelial lymphocytes (IELs) are unspecific and subject to false diagnosis (8–11), and new reliable tools to detect mild enteropathy coeliac disease are therefore warranted. A specific test also should encompass proof of gluten dependency when patients are placed on gluten-free dietary treatment.
Apart from mucosal changes, a typical feature of untreated coeliac disease is the presence of serum immunoglobulin (Ig)A–class autoantibodies against transglutaminase-2 (TG2) (12,13), which offer high sensitivity and specificity (14,15). In fact, positive autoantibodies in patients having normal small bowel mucosal villous morphology do not necessarily constitute a false positive finding because they may be a predictive sign of forthcoming mucosal villous atrophy and coeliac disease (9,16–18). These autoantibodies are produced in the small intestinal mucosa (19,20), where they may be present even when there are no measurable levels in the sera (5,21,22). Intestinal TG2-specific IgA deposits may offer a diagnostic tool for detecting early-stage coeliac disease without villous atrophy (9). These intestinal antibodies (with absence in serum) also have been found in patients with diabetes mellitus and in first-degree relatives of coeliac patients, without full certainty as to whether these antibodies are gluten induced or related to coeliac disease (23–25). The demonstration of gluten dependency in intestinal TG2-specific IgA deposits would further enhance the specificity of the test.
Our aim was to evaluate the gluten dependency and diagnostic value of small bowel mucosal TG2-specific IgA autoantibody deposits in overt and mild enteropathy coeliac disease. Our series included children and adults with latent coeliac disease, who initially showed normal small bowel mucosal villous architecture but subsequently developed mucosal villous atrophy and celiac disease while continuing on a normal gluten-containing diet. In addition, we found individuals evincing small bowel mucosal TG2-specific IgA autoantibody deposits in intact villi who were directly placed on an experimental gluten-free diet to find evidence of gluten sensitivity. The data were compared with those from patients with overt coeliac disease and noncoeliac controls.
PATIENTS AND METHODS
The study group comprised 48 patients suspected of coeliac disease but found to have normal small bowel mucosal villous architecture. Of these, 28 had latent coeliac disease because they developed mucosal villous atrophy during the follow-up when continuing on a gluten-containing diet (Table 1). In these patients, the median follow-up time from normal small bowel mucosal architecture to villous atrophy was 1.7 years (range 0.2–7.4 years). After the mucosal deterioration, the patients were placed on a gluten-free diet, the median duration of the diet being 1.4 years (range 0.8–5.9 years). The remaining 20 patients in the study group were found to have small bowel mucosal IgA deposits in intact villi (defined in this study as potential coeliac disease) (Table 1); to find evidence for their gluten dependency, these individuals were placed on an experimental gluten-free diet even if they did not fullfil the current diagnostic criteria for coeliac disease. After 1 year, the response to the dietary treatment was evaluated.
The controls comprised 13 subjects with overt coeliac disease with small bowel mucosal villous atrophy and crypt hyperplasia and 42 patients with suspicion of coeliac disease but no evidence of villous atrophy in 2 successive biopsy samples on a gluten-containing diet with a median interval of 6.0 years (range 0.8–10.0 years) (Table 1). The study protocol was approved by the Ethical Committee of Tampere University Hospital. All of the subjects gave written informed consent.
Small Bowel Mucosal Morphology and Intraepithelial Lymphocytes
Small bowel mucosal biopsies were taken upon upper gastrointestinal endoscopy and in small children with a Watson capsule. For morphometrical analysis, the samples were paraffin embedded, processed, and stained with hematoxylin-eosin. The villous height–crypt depth ratio was counted in well-oriented biopsy samples as previously described (26) and a ratio <2.0 was considered compatible with coeliac disease.
One piece of capsule biopsy or 2 forceps biopsy specimens were freshly embedded in optimal cutting temperature compound (OTC, Tissue-Tec, Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at −70°C. Immunohistochemical studies were carried out on 5-μm-thick frozen sections. CD3+ IELs were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA) and γδ+ IELs with T-cell receptor bearing cell γ antibody (T Cell Diagnostics, Woburn, MA). Positive IELs were counted from immunohistochemically stained specimens with a ×100 light microscope objective as described elsewhere (27). The reference value for CD3+ IELs was set at 37 cells per millimeter of epithelium and for γδ+ cells at 4.3 cells per millimeter of epithelium (27).
Small bowel Mucosa TG2-specific IgA Deposits
Small bowel mucosal TG2-specific IgA deposits were investigated in unfixed, 5-μm-thick, frozen sections of small bowel specimens by direct immunofluorescence using fluorescein isothiocyanate–labelled rabbit antibody against human IgA (Dako, Glostrup, Denmark) at a dilution of 1:40 in phosphate-buffered saline, pH 7.4. In coeliac disease a clear subepithelial IgA deposition can be found below the basement membrane along the villous and crypt epithelium and around mucosal vessels; this is in contrast to normal small bowel samples, in which IgA is detected only inside the plasma and epithelial cells (21). The IgA deposits were graded semiquantitatively from 0 to 3 according to their intensity along the basement membrane in the villous crypt area, as previously described (5). It has been shown that these mucosal IgA deposits are specifically targeted against TG2 (21,22). For double labelling, sections were stained for human IgA (green, as above, this paragraph) and for TG2 (red) using monoclonal mouse antibodies against TG2 (CUB7402, NeoMarkers, Fremont, CA), followed by rhodamine-conjugated anti-mouse immunoglobulin antibodies (Dako), both diluted 1:200 in phosphate-buffered saline. In our laboratory, intraobserver and interobserver agreement in the detection of present or absent TG2-specific IgA deposits has been 98% among 5 investigators. All of the evaluations were carried out blindly without knowledge of disease history or laboratory findings.
Serum IgA-class reticulin (ARA) or endomysial antibodies were determined by an indirect immunofluorescence method as described earlier (14). A positive staining pattern seen in a serum dilution of 1:5 or more was considered positive in both tests. During the study period, endomysial antibodies replaced ARA in clinical practice. If the endomysial antibodies result was not available, then the ARA result was used instead. These 2 coeliac autoantibody tests have proved in our laboratory to be almost identical (28), and both have been shown to be directed against TG2 (13). In this study, these antibodies are indicated as serum coeliac autoantibodies. In retrospect, it was impossible here to determine serum TG2-antibodies by enzyme-linked immunosorbent assay in all of the patients and controls.
HLA typing was based on polymerase chain reaction with allele-specific primers identifying HLA DQ2 and DQ8, and performed with a Dynal DQ low-resolution SSp kit (Dynal, Oslo, Norway). In coeliac disease, 90% to 95% of patients carry the HLA DQ2-haplotype and most of the rest carry HLA DQ8 (29).
Quantitative data are expressed as medians and ranges and qualitative data as percent of abnormal values. Statistical differences were evaluated using the Mann-Whitney U, Wilcoxon, and McNemar tests, as appropriate. All of the testing was 2-sided, and P < 0.05 were considered statistically significant. All of the calculations were performed with the Statistical Package for Social Sciences version 14.0 (SPSS, Chicago, IL).
Small bowel mucosal TG2-specific IgA deposits already were seen in the first biopsy in all but 1 of the patients with latent coeliac disease, when mucosal villous architecture was still intact (Table 2, Fig. 1). When the patients continued on a normal gluten-containing diet, small bowel mucosal villous atrophy and crypt hyperplasia developed; in parallel, the intensity of mucosal IgA deposits increased, and the deposits were present in all of the patients at the time of the diagnosis of coeliac disease (Figs. 1 and 2). The intensity of mucosal IgA deposits again decreased significantly when the patients were placed on a gluten-free diet, albeit minor depositions still were seen in many. A total of 7 patients in the latent coeliac disease group were initially serum autoantibody negative; 6 of them still had positive small bowel mucosa IgA deposits, 2 had family history for coeliac disease, and all 6 cases tested had coeliac-type HLA. Sixteen of the 20 patients having TG2-specific small bowel mucosal IgA deposits in intact villi (potential coeliac disease) agreed to a follow-up biopsy after 1 year on a gluten-free diet. The intensity of the deposits decreased in 15 (94%) of the 16 on a gluten-free diet (Figs. 1 and 2). Parallel to these changes, abdominal symptoms and signs of malabsorption resolved in 13 (65%) and improved in 5 (25%) of the patients whilst on the gluten-free diet (Table 3). TG2-specific small bowel mucosal IgA deposits were present in all of the patients with untreated overt coeliac disease, and the intensity of the deposits decreased with the diet. In noncoeliac controls the deposits were seen initially in 1 of 22 (5%) and in 3 of 38 (8%) patients on a gluten-containing diet in the follow-up biopsies (Fig. 1); these deposit-positive cases did not present with any particular clinical sign and had no family history of coeliac disease. The histological, serological, and HLA DQ findings are set out in Table 2 and Figure 1. At the first biopsy in the noncoeliac control group, densities of CD3+ IELs were increased in 41% and γδ+ IELs in 48%, and during the study the cell densities decreased significantly with time without any dietary intervention (Table 3, Fig. 1). At the outset, 4 (10%) of the noncoeliac controls were coeliac autoantibody positive, but all of them seroconverted negatively during the study (Table 2).
The present study confirms earlier data indicating that the borders of coeliac disease clearly extend beyond small bowel mucosal villous atrophy (30). The current diagnostic criteria have been applied for many years and small intestinal mucosal atrophy has been sine qua non for the diagnosis (1). To revise the criteria of coeliac disease beyond villous atrophy requires the demonstration of gluten dependency of the symptoms and histology in genetically susceptible individuals. We have shown in this article that by careful examination we are able to find coeliac disease in patients with normal villous structure. Once the criteria have been revised, the inevitable consequence is that these patients should be treated with a gluten-free diet. In this study, mucosal TG2-specific IgA deposits were accurate markers for gluten sensitivity; they were detected early on in the disease process, their intensity increased as enteropathy progressed on a gluten-containing diet, and their intensity decreased along with mucosal recovery on a gluten-free diet. These intestinal coeliac autoantibody deposits proved to be better initial markers for gluten sensitivity than small bowel mucosal IEL densities, and they also were able to detect serum coeliac autoantibody-negative cases having mild enteropathy coeliac disease (Table 2). This was demonstrated in patients with latent coeliac disease for whom the development of villous atrophy had been confirmed by follow-up on a gluten-containing diet. Furthermore, in patients with TG2-specific IgA deposits but no evidence of progression to overt coeliac disease, the deposits were shown to be gluten dependent (Fig. 1). The process was akin to that of overt coeliac disease and latent coeliac disease. In this study, the majority of patients with latent coeliac disease had experienced gluten-dependent symptoms before subsequent diagnostic enteropathy had developed (Table 1).
Furthermore, 18 (90%) of the 20 patients found to have small bowel mucosal TG2-specific IgA deposits, but considered in view of normal small bowel histology not to have celiac disease, benefited from gluten-free dietary treatment (Table 3). Similar cases have been reported in the literature (6,31,32); some subjects have even been diagnosed as having osteopenia or osteoporosis (4,7,33). Of note, in this study group the nonresponsive symptoms were refractory epilepsy and arthritis, indicating that these extraintestinal manifestations were not gluten dependent. Assuming that the diagnostic criteria for the disease should indeed be widened to cover mild enteropathy coeliac disease, some issues should be stressed to avoid overdiagnosis of the disorder. Marsh 1-type small bowel mucosal lymphocytosis is an unspecific finding (8–10), as also seen in the present study; the densities of CD3+ and γδ+ IELs also decreased with time in noncoeliac controls without any dietary intervention (Fig. 1). Some noncoeliac patients evinced negative seroconversion during the follow-up; these cases were ARA positive without HLA DQ2 or DQ8.
Furthermore, 8% of these controls had minor small bowel mucosal IgA deposits without any signs of gluten sensitivity in the follow-up biopsy. Long-term follow-up is needed to show whether these subjects will eventually develop overt coeliac disease (7,34,35). To conclude, no single test alone can reliably detect early-stage coeliac disease without villous atrophy, but gluten-dependent TG2-specific IgA deposits offer a good diagnostic tool. Based on the findings in this study, we suggest that the deposits should be investigated when coeliac disease is suspected but the small bowel mucosal villous morphology is equivocal. In symptomatic patients (as was the case in the present series) having signs of minor enteropathy coeliac disease without villous atrophy, gluten-free dietary treatment should be considered. In asymptomatic cases, the benefits of early treatment are more ambiguous and subject to further studies. In the meantime, follow-up with a normal gluten-containing diet is recommended. In coeliac disease, however, gluten-induced symptoms may occur outside the intestine (30), and during the follow-up the wide clinical spectrum of the disease should be kept in mind.
1. Walker-Smith JA, Guandalini S, Schmitz J, et al
. Revised criteria for diagnosis of coeliac disease. Arch Dis Child 1990; 65:909–911.
2. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“celiac sprue”). Gastroenterology 1992; 102:330–354.
3. Mäki M, Holm K, Koskimies S, et al
. Normal small bowel biopsy followed by coeliac disease. Arch Dis Child 1990; 65:1137–1141.
4. Kaukinen K, Mäki M, Partanen J, et al
. Celiac disease without villous atrophy. Revision of criteria called for. Dig Dis Sci 2001; 46:879–887.
5. Kaukinen K, Peräaho M, Collin P, et al
. Small bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized study. Scand J Gastroenterol 2005; 40:564–572.
6. Troncone R. Latent coeliac disease
in Italy. Acta Paediatr 1995; 84:1252–1257.
7. Matysiak-Budnik T, Malamut G, de Serre NP, et al
. Long-term follow-up of 61 patients diagnosed in childhood: evolution towards latency is possible on a normal diet. Gut 2007; 56:1379–1386.
8. Kakar S, Nehra V, Murray JA, et al
. Significance of intraepithelial lymphocytosis in small bowel biopsy samples with normal mucosal architecture. Am J Gastroenterol 2003; 98:2027–2033.
9. Salmi TT, Collin P, Järvinen O, et al
. Immunoglobin A autoantibodies against transglutaminase 2 in the small intestinal mucosa predict forthcoming coeliac disease. Aliment Pharmacol Ther 2006; 24:541–552.
10. Lähdeaho ML, Kaukinen K, Collin P, et al
. Celiac disease: from inflammation to atrophy. A longterm follow-up study. J Pediatr Gastroenterol Nutr 2005; 41:44–48.
11. Paparo F, Petrone E, Tosco A, et al
. Clinical, HLA, and small bowel immunohistochemical features of children with positive serum antiendomysium antibodies and architecturally normal small intestinal mucosa. Am J Gastroenterol 2005; 100:2294–2298.
12. Dieterich W, Ehnis T, Bauer M, et al
. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3:797–801.
13. Korponay-Szabo IR, Laurila K, Szondy Z, et al
. Missing endomysial and reticulin binding of coeliac antibodies in transglutaminase 2 knockout tissues. Gut 2003; 52:199–204.
14. Sulkanen S, Halttunen T, Laurila K, et al
. Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease. Gastroenterology 1998; 115:1322–1328.
15. Rostom A, Dube C, Cranney A, et al
. The diagnostic accuracy of serologic tests for celiac disease: a systematic review. Gastroenterology 2005; 128:S38–46.
16. Collin P, Helin H, Mäki M, et al
. Follow-up of patients positive in reticulin and gliadin antibody tests with normal small bowel biopsy findings. Scand J Gastroenterol 1993; 28:595–598.
17. Corazza GR, Andreani ML, Biagi F, et al
. Clinical, pathological, and antibody pattern of latent celiac disease: report of three adult cases. Am J Gastroenterol 1996; 91:2203–2207.
18. Dickey W, Hughes DF, McMillan SA. Patients with serum IgA endomysial antibodies and intact duodenal villi: clinical characteristics and management options. Scand J Gastroenterol 2005; 40:1240–1243.
19. Marzari R, Sblattero D, Florian F, et al
. Molecular dissection of tissue transglutaminase autoantibody response in celiac disease. J Immunol 2001; 166:4170–4176.
20. Picarelli A, Maiuri L, Frate A, et al
. Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease. Lancet 1996; 348:1065–1067.
21. Korponay-Szabo IR, Halttunen T, Szalai Z, et al
. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004; 53:641–648.
22. Salmi TT, Collin P, Korponay-Szabo I, et al
. Endomysial antibody–negative coeliac disease: clinical characteristics and intestinal autoantibody deposits. Gut 2006; 55:1746–1753.
23. Ziberna F, Baldas V, Martelossi S, et al
. Intestinal mucosa anti-transglutaminase antibodies in early stage of gluten intolerance. J Pediatr Gastroenterol Nutr 2006; 42:E19–20.
24. Sblattero D, Ventura A, Tomassini A, et al
. Cryptic gluten intolerance in type 1 diabetes: identifying suitable candidates for gluten free diet. Gut 2006; 55:133–134.
25. Maglio M, Paparo F, Franzese A, et al
. Anti-tissue transglutaminase antibodies are deposited in the small intestinal mucosa of patients with type 1 diabetes. Gastroenterology 2006; 130(Suppl 2):A70.
26. Kuitunen P, Kosnai I, Savilahti E. Morphometric study of the jejunal mucosa in various childhood enteropathies with special reference to intraepithelial lymphocytes. J Pediatr Gastroenterol Nutr 1982; 1:525–531.
27. Järvinen TT, Kaukinen K, Laurila K, et al
. Intraepithelial lymphocytes in celiac disease. Am J Gastroenterol 2003; 98:1332–1337.
28. Mäki M. The humoral immune system in coeliac disease. Baillieres Clin Gastroenterol 1995; 9:231–249.
29. Karell K, Louka AS, Moodie SJ, et al
. HLA types in celiac disease patients not carrying the DQA1*05-DQB1*02 (DQ2) heterodimer: results from the European genetics cluster on celiac disease. Hum Immunol 2003; 64:469–477.
30. Kaukinen K, Collin P, Mäki M. Latent coeliac disease
or coeliac disease beyond villous atrophy? Gut 2007; 56:1339–1340.
31. Wahab PJ, Crusius JBA, Meijer JWR, et al
. Gluten challenge in borderline gluten-sensitive enteropathy. Am J Gastroenterol 2001; 96:1464–1469.
32. Tursi A, Brandimarte G. The symptomatic and histologic response to a gluten-free diet in patients with borderline enteropathy. J Clin Gastroenterol 2003; 36:13–17.
33. Esteve M, Rosinach M, Fernandez-Banares F, et al
. Spectrum of gluten-sensitive enteropathy in first-degree relatives of patients with coeliac disease: clinical relevance of lymphocytic enteritis. Gut 2006; 55:1739–1745.
34. Hogberg L, Stenhammar L, Falth-Magnusson K, et al
. Anti-endomysium and anti-gliadin antibodies as serological markers for a very late mucosal relapse in a coeliac girl. Acta Paediatr 1997; 86:335–336.
35. Simell S, Hoppu S, Hekkala A, et al
. Fate of five celiac disease–associated antibodies during normal diet in genetically at-risk children from birth in a natural history study. Am J Gastroenterol 2007; 102:2026–2035.