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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Summary

Background

Reliable markers of early developing coeliac diseases are needed. Coeliac autoantibodies in the serum or Marsh I inflammation may be indicators of subsequent coeliac disease.

Aim

To investigate whether determination of intestinal transglutaminase 2-targeted autoantibody deposits would detect early developing coeliac disease better than previous methods.

Methods

The study investigated patients previously excluded for coeliac disease: 25 had positive serum coeliac autoantibodies (endomysial), 25 antibody-negative had Marsh I, and 25 antibody-negative had Marsh 0 finding. Seven (median) years after baseline investigation, new coeliac cases were recorded, and small bowel biopsy was offered to the rest of the patients. Serum and intestinal coeliac autoantibodies and intraepithelial lymphocytes were assessed as indicators of developing coeliac disease.

Results

Seventeen patients had developed coeliac disease: 13 in the autoantibody-positive group, three in the Marsh I group and one in the Marsh 0 group. At baseline, intestinal coeliac autoantibody deposits had a sensitivity and specificity of 93% and 93% in detecting subsequent coeliac disease, CD3+ 59% and 57%, γδ+ 76% and 60%, and villous tip intraepithelial lymphocytes 88% and 71%, respectively.

Conclusions

Endomysial antibodies with normal histology indicates early developing coeliac disease. Transglutaminase 2-targeted intestinal autoantibody deposits proved the best predictor of subsequent coeliac disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The diagnosis of coeliac disease requires the presence of small intestinal mucosal villous atrophy and crypt hyperplasia (Marsh III).1, 2 However, mucosal intraepithelial lymphocytosis evincing normal villous architecture (Marsh I) precedes this lesion. Marsh I lesion may thus indicate early developing coeliac disease, but this finding is also associated with other disorders. Only 2–10% of patients are eventually shown to suffer from genetic gluten intolerance.3, 4 Increased densities of γδ+5 or villous tip intraepithelial lymphocytes (IELs)6 have been of better positive predictive value than IELs in general in the diagnosis of early developing coeliac disease, but so far no single marker is considered a reliable indicator of this condition.

Serum immunoglobulin (Ig)A-class endomysial (EmA)7 and transglutaminase 2 (TG2)8, 9 antibodies are powerful tools in disclosing coeliac disease with overt villous atrophy. Some studies have suggested, however, that these autoantibodies might be less useful in the case of early developing coeliac disease,10–12 as the antibodies are thought to enter the circulation only after severe villous atrophy has developed. In contrast, there are also cases where coeliac autoantibodies have appeared in the serum before the development of villous atrophy, implying that they might be valuable in identifying patients with early developing coeliac disease.5, 13, 14

Previous studies have demonstrated that TG2 is the antigen for in vitro binding of coeliac IgA-class antibodies to intestinal and extraintestinal tissues.15, 16 Coeliac autoantibodies are produced in the small bowel mucosa,17–19 and it has been recognized that the small intestinal epithelial basement membrane region contains deposited IgA in untreated coeliac disease, though the target of this deposition remained unknown for decades.20, 21 We recently demonstrated in vivo that deposited extracellular IgA in the small intestinal mucosa targets TG2 in untreated coeliac disease patients.22 Further, our previous findings have suggested that TG2-targeted intestinal autoantibody deposits might be detectable before the development of overt villous atrophy and precede the appearance of serum coeliac autoantibodies.22, 23 These preliminary results raised the intriguing hypothesis that intestinal autoantibody deposits targeted against TG2 would precede manifest coeliac mucosal lesion, and further that this might furnish a method applicable in the diagnosis of early developing coeliac disease.

Recent evidence shows that coeliac disease is no longer restricted to severe enteropathy,24 and that patients can suffer from gluten-dependent clinical symptoms and complications of coeliac disease even before small bowel mucosal villous atrophy has developed.25, 26 Such patients benefit of gluten-free diet, and therefore, reliable markers of early developing coeliac disease are needed. However, previously published evidence of this condition has mainly consisted of case reports. This long-term follow-up study focused for the first time on early developing coeliac disease in a large, well-defined patient material. The aim was first to establish whether patients with normal villous architecture but positive serum coeliac autoantibodies, or Marsh I lesion in the small bowel mucosa, suffer from early developing coeliac disease. Secondly, we investigated whether intestinal TG2-targeted autoantibody deposits are superior in detecting early developing coeliac disease than the CD3+, γδ+ and villous tip IELs currently employed.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Patients and study design

Patients who initially had normal villous architecture but were subsequently shown to develop manifest coeliac disease with villous atrophy and crypt hyperplasia are referred as early developing coeliac disease patients in this study. This condition has also been regarded as latent coeliac disease in the literature.

From 1995 to 1999 altogether 707 adult patients underwent small intestinal biopsy at the Department of Gastroenterology and Alimentary Tract Surgery in Tampere University Hospital due to suspicion of coeliac disease. Small bowel mucosal villous morphology and the densities of CD3+ and γδ+ IELs were determined in all. A coeliac disease diagnosis was established when villous atrophy and crypt hyperplasia (Marsh III lesion) were present in the biopsy specimens.2 In 545 patients the villous architecture was normal and coeliac disease was hence excluded. However, 29 of them had positive coeliac autoantibodies in the serum and either Marsh 0 or Marsh I findings in the mucosa; in addition, 69 antibody-negative patients had Marsh I lesions in the small bowel mucosa.

According to power calculations the number of patients in each study group was set at 25. The study groups consisted of age- and sex-matched patients with normal villous architecture: a random sample of 25 of 29 patients with positive serum coeliac autoantibodies, 25 antibody-negative with Marsh I and 25 antibody-negative with Marsh 0 finding in the small bowel mucosa. The demographic data and primary reasons for coeliac disease suspicion in the 75 study patients are shown in Table 1.

Table 1.   Demographic data and primary reason for coeliac disease suspicion in study patients at baseline when coeliac disease was excluded upon normal small bowel biopsy finding
 Patients with positive coeliac autoantibodies in the serum (n = 25)Patients with Marsh I finding in the small bowel mucosa* (n = 25)Patients with Marsh 0 finding in the small bowel mucosa* (n = 25)
  1. * Age- and sex-matched to coeliac autoantibody-positive patients (patients had no measurable coeliac autoantibodies in the serum).

  2. † Diarrhoea, flatulence, indigestion, abdominal distension or pain.

  3. ‡ Arthritis, skin symptoms, mouth ulcers, dental enamel defects, neurological symptoms, elevated liver enzymes, alopecia, gynaecological disorders.

  4. § Autoimmune thyroid disorders, Sjögren's disease, insulin-dependent diabetes mellitus, family history of coeliac disease.

Female, n (%)19 (76)19 (76)19 (76)
Age, median (range)39 (18–67)39 (17–73)39 (18–68)
Primary reason for coeliac disease suspicion, n (%)
 Abdominal symptoms†8 (32)18 (72)14 (56)
 Anaemia or malabsorption2 (8)2 (8)6 (24)
 Extraintestinal symptoms‡7 (28)5 (20)5 (20)
 Coeliac disease at-risk groups§8 (32)

The follow-up investigation took place in 2004. Altogether 69 of the original 75 subjects could be traced. The emergence of coeliac disease during the follow-up was recorded in patient files, and all patients without prior development of coeliac disease were invited for a follow-up visit a median of 7.3 years after baseline investigation. At the follow-up, clinical symptoms were recorded. The daily consumption of gluten was analysed by a dietician according to a 4-day record of food intake to ascertain that patients had not reduced their gluten intake. Patients were asked to undergo upper gastrointestinal endoscopy and small bowel biopsy.

Finally, baseline (Figure 1) histological and serological findings in patients who were shown to have developed mucosal lesion compatible with coeliac disease during the whole follow-up were compared with patients again excluded for the disease by small bowel biopsy.

image

Figure 1.  The development of coeliac disease (CD) in study patients with prior CD suspicion but normal villous architecture. Study groups consisted of 25 serum coeliac autoantibody-positive patients with Marsh 0 or with Marsh I finding in the small bowel mucosa and age- and sex-matched autoantibody-negative patients; 25 with Marsh I finding and 25 with Marsh 0 finding in the small bowel mucosa. Baseline indicates the time when CD was first suspected but excluded based on normal small bowel biopsy finding in all study patients; all of the statistical calculations in this study were carried out from baseline findings (GFD = gluten-free diet, * CD diagnosed during the follow-up on clinical grounds, † CD diagnosed at the follow-up visit).

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Serum coeliac autoantibodies

EmA (primate-type reticulin) and reticulin antibodies (ARA, rodent-type reticulin) have been shown to be directed exclusively to TG2 in both rodent and primate tissues.15, 16 During the study period, EmA replaced ARA in clinical practice. These two coeliac autoantibody tests have proved to be virtually identical in our laboratory,27 and are thus termed here coeliac autoantibodies. At the follow-up visit EmA was used together with the TG2 antibody test.

Serum IgA-class ARA and EmA were determined by an indirect immunofluorescence (IF) method; a serum dilution of 1:≥5 was considered positive in both. In ARA, a typical R1 pattern was required in rat kidney and liver sections;28 in EmA human umbilical cord was used as a substrate.7 Assessment of serum IgA-class TG2 was carried out by enzyme-linked immunosorbent assay (ELISA; Celikey, Pharmacia Diagnostics, GmbH, Freiburg, Germany) using human recombinant TG2 as antigen, and the lower limit of positivity was a unit value of 5.29

Small bowel mucosal morphology and IELs

Upon endoscopy, seven forceps biopsy specimens were taken from the distal part of the duodenum. Five of these were processed, stained with haematoxylin and eosin (H & E) and studied under light microscopy. The villous height–crypt depth ratios (Vh/CrD) were determined from several well-oriented biopsy samples from multiple sites in order to detect patchy forms of villous atrophy, as described previously.30 Vh/CrD < 2 was considered compatible with coeliac disease (Marsh III).

The villous tip IEL score/20 enterocytes was calculated from the mean value of five random villi. The reference value was set at 4.2 IELs/20 enterocytes.31 The correlation coefficients for intraobserver variation for villous tip IELs were 0.89 and for interobserver variation 0.87 in our laboratory.

Two small bowel biopsy specimens were freshly embedded in optimal cutting temperature compound (OCT; Tissue-Tec, Miles Inc, Elkhart, IN, USA), snap-frozen in liquid nitrogen and stored at −70 °C. Immunohistochemical stainings were carried out on 5-μm-thick frozen sections. CD3+ IELs were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA, USA) and γδ+ IELs with T-cell receptor (TCR)-γ antibody (Endogen, Woburn, MA, USA). Positive IELs were counted with a ×100 flat field light microscope objective throughout the surface epithelium; at least 30 fields measuring 1.6 mm in epithelial length were counted and IEL density expressed as cells/mm of epithelium.32 For comparison, in a case report the values of IELs are also given as IELs/100 enterocytes. CD3+ IELs correlated well with the total IEL density in the small bowel mucosa32 and thus a normal density of these cells indicated a Marsh 0 and increased density of a Marsh I finding.2 The reference values were set at 37 cells/mm for CD3+ IELs and 4.3 for γδ+ IELs.33 In our laboratory the correlation coefficients for intraobserver variation for CD3+ and γδ+ IELs were 0.95 and 0.98, and those for interobserver variation were 0.92 and 0.98 respectively. Evaluations of the specimens and calculations of the IELs were carried out by one investigator and without prior knowledge of disease history or laboratory findings.

Small bowel mucosal TG2-specific IgA deposits

In earlier studies we have shown that untreated coeliac disease patients have in vivo in situ IgA deposits on TG2 in their small bowel mucosa. When this IgA was eluted from the tissues, it targeted purified TG2 both in ELISA and Western blot. In addition, when TG2 binding to fibronectin was disrupted by chloroacetic acid, disappearance of extracellular IgA deposits was demonstrated in coeliac small bowel samples.22 The method used here was based on our previous experiments to detect TG2-specific antibodies in situ in tissue sections by their co-localization with TG2 when double-labelled by IF.

In the present study, small bowel mucosal TG2-specific IgA deposits were investigated in each patient with an available frozen small bowel specimen at baseline. From each of these cases altogether six unfixed, 5-μm-thick sections from frozen small bowel specimens were processed, three for investigating IgA deposits and three for double-colour labelling for both IgA and TG2 by direct IF. IgA was detected using fluorescein isothiocyanate-labelled rabbit antibody against human IgA (Dako AS, Glostrup, Denmark) at a dilution of 1:40 in phosphate-buffered saline (PBS, 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; in contrast in normal small bowel samples IgA is detected inside the plasma and epithelial cells only.22, 23 The evaluation was carried out blindly without knowledge of the disease history or laboratory findings. For the double labelling, sections were stained for human IgA (green, as above) and for TG2 (red) using monoclonal mouse antibodies against TG2 (CUB7402, NeoMarkers, Fremont, CA, USA) followed by rhodamine-conjugated antimouse immunoglobulin antibodies (Dako), both diluted 1:200 in PBS. More than 500 small bowel specimens have been investigated for IgA deposits in our laboratory so far, and intraobserver and interobserver variations have both been 98% in the detection of the presence or the absence of TG2-targeted IgA deposits between five investigators.

HLA typing

The study patients were genotyped for human leucocyte antigen (HLA)-DQB1*02, DQB1*0302 and DQA1*05 alleles using the DELFIA Coeliac Disease Hybridization Assay (Perkin-Elmer Life and Analytic Sciences, Wallac Oy, Turku, Finland). DQB1*02 and DQA1*05 are associate alleles for HLA DQ2 and DQB1*0302 for HLA DQ8, and these haplotypes are found in 96–100% of coeliac disease patients.34, 35

Statistics

The size of the study groups was determined by power calculations: the assumption was that at least 50% of patients with positive coeliac autoantibodies in the serum and at most 10% of patients with negative autoantibodies having a Marsh I or a Marsh 0 finding at baseline would develop overt coeliac disease during the study. α was given the value of 0.05 and the power was 90%. In order to elicit significant differences between the study groups the number of patients in each group had to be at least 23, and was finally set at 25.

Quantitative data were expressed as medians and ranges. Statistical differences between study groups were evaluated using the Pearson chi-square test, Fisher's exact test or Mann–Whitney U-test, as appropriate. P < 0.05 was considered statistically significant. Sensitivities and specificities of different markers in detecting early developing coeliac disease were calculated. All calculations were performed with spss (version 12.0.1).

Ethical considerations

The study protocol was approved by the Ethics Committee of Tampere University Hospital. Informed consent was obtained from all study subjects.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Coeliac disease diagnosed on clinical grounds during the follow-up

In 2004, altogether 15 with initially normal villous architecture had developed small bowel mucosal villous atrophy and crypt hyperplasia, and hence were diagnosed with coeliac disease by routine clinical follow-up before study enrolment. Twelve were in the autoantibody-positive group, two in Marsh I and one in Marsh 0 group (Figure 1, Table 2). In addition, two patients from the original study group had adopted a complete gluten-free diet during the follow-up without proper histological verification of the disease. They were excluded from further evaluations and considered not to have coeliac disease.

Table 2.   Clinical, histological and serological findings in patients who later developed villous atrophy compatible with CD; investigations were carried out at baseline when CD was excluded
Number and study groupGenderAge (years)Primary reason for CD suspicionHLA DQ2/ DQ8Serum IgA-class coeliac autoantibodiesIntestinal TG2- specific IgA depositsCD3+ IELs (Marsh I)γδ+ IELsVillous tip IELsFollow-up time before the development of villous atrophy (years)Clinical manifestations at the time of the CD diagnosis
  1. Ab, serum coeliac autoantibodies; ND, no data; TG2, transglutaminase; IEL, intraepithelial lymphocyte; IgA, immunoglobulin A; CD, coeliac disease.

  2. + = positive, present or increased density; − = negative, absent or normal density.

  3. * Coeliac disease diagnosed at the follow-up visit.

1. Ab+F64Mouth ulcersND+++++0.3Mouth ulcers
2. Ab+F36Family historyND+++++1.0None
3. Ab+F27Family history+/−++++1.0Abdominal pain
4. Ab+M44Neurological symptoms+/−++++ND1.4Neurological symptoms
5. Ab+F62Abdominal distensionND++++1.5Abdominal distension
6. Ab+F47Abdominal distension+/−+++2.0Abdominal distension
7. Ab+F42Autoimmune thyroid disease+/−+++++2.2Fatigue
8. Ab+F50Abdominal painND+ND++2.6Abdominal pain
9. Ab+M32Diarrhoea+/−++++4.0Abdominal pain
10. Ab+F39Loose stools+/−+++++4.3Loose stools
11. Ab+F67Mouth ulcersND++++4.3Loose stools
12. Ab+F26Elevated liver enzymes+/−++++4.7Abdominal pain
13. Ab+F66Type 1 diabetes+/−+++5.2*Mouth ulcers
14. Marsh IF67Loose stools+/−+++1.7Loose stools
15. Marsh IM32Arthritis+/−ND+++3.4Arthritis
16. Marsh IM34Diarrhoea+/+++7.4*Diarrhoea
17. Marsh 0F68Abdominal pain+/−+++1.3Abdominal pain

Follow-up investigation

At the follow-up visit two new coeliac disease patients with typical small bowel histological findings were detected: one in the autoantibody-positive group and one in the Marsh I group (Table 2, Figures 1 and 2). At follow-up, both new coeliac disease patients had positive serum EmA and TG2 antibodies. During the follow-up, three patients had developed severe heart disease and endoscopy was considered unethical (Figure 1).

image

Figure 2.  Patient with early developing coeliac disease having positive coeliac autoantibodies in the serum at baseline when coeliac disease was excluded by biopsy. At baseline the density of CD3+ intraepithelial lymphocytes (IELs) was 12 cells/mm (9 IELs/100 enterocytes) and the density of γδ+ IELs was 0.0 cells/mm (0 IELs/100 enterocytes). Subepithelial coeliac-type small bowel mucosal immunoglobulin (Ig)A deposits (a, green, arrow) and transglutaminase 2 (TG2; c, red) in the early developing stage of coeliac disease and after villous atrophy has developed (e and g respectively). Intestinal IgA deposits were detected both at baseline and at the time coeliac disease was diagnosed. Yellow colour in composite pictures (b and f) indicates co-localization of coeliac-type IgA deposits and TG2. In haematoxylin and eosin (H & E)-stained sections normal villous architecture at baseline (d) followed by villous atrophy compatible with coeliac disease 5.2 years later (h) is shown. The density of CD3+ IELs was 76 cells/mm (52 IELs/100 enterocytes) and the density of γδ+ IELs was 26.0 cells/mm (18 IELs/100 enterocytes) at the time coeliac disease was diagnosed.

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All patients except those with histologically proven coeliac disease were consuming a normal, gluten-containing diet throughout the study period. The remaining 30 patients who underwent endoscopy were again excluded for coeliac disease; all showed normal villous architecture in the follow-up biopsy specimens and were negative for serum EmA and TG2 antibodies. This means that five patients who were autoantibody-positive at baseline had undergone negative seroconversion; at baseline, one of three with a frozen sample available had detectable intestinal IgA deposits, while at the end of the follow-up it was one of four. All five patients undergoing negative seroconversion had clinical symptoms suggesting coeliac disease at baseline, and all had Marsh 0 finding in the small bowel mucosa.

Characteristics of early developing coeliac disease patients

Baseline symptoms, serological and histological findings in the 17 patients with established early developing coeliac disease (at the time coeliac disease was excluded) are shown in Table 2. Fifty-two percentage (13 of 25) of patients with baseline-positive autoantibodies in the serum, 12% (three of 25) of patients with Marsh I and 4% (one of 25) of patients with Marsh 0 at baseline were later shown to have developed villous atrophy compatible with coeliac disease (P < 0.001). Of the autoantibody-positive patients with early developing coeliac disease, seven had Marsh I and six had Marsh 0 findings in the small bowel mucosa at baseline. The presence of intestinal IgA deposits in patients with early developing coeliac disease and in patients again excluded for coeliac disease at baseline and at the end of the follow-up is shown in Table 3. Baseline serum TG2 antibody results were available in five patients developing coeliac disease during the follow-up. Two of these were positive for these antibodies, and also for EmA; three were negative for TG2 antibodies (one of whom had positive EmA).

Table 3.   The presence of intestinal transglutaminase 2-specific IgA deposits in study groups at baseline when CD was excluded and at the end of the follow-up period
 Baseline, n (%)End of the follow-up, n (%)
  1. * All available intestinal deposit results are shown.

  2. † Small bowel mucosal villous atrophy and crypt hyperplasia was demonstrated in all patients.

  3. ‡ Normal small bowel mucosal morphology.

  4. IgA, immunoglobulin A; CD, coeliac disease.

Early developing CD patients, all14/15 (93)*15/15 (100)*†
 CD autoantibody-positive group12/12 (100)12/12 (100)
 Marsh I group1/2 (50)2/2 (100)
 Marsh 0 group1/1 (100)1/1 (100)
Patients excluded for CD by biopsy, all1/14 (7)*1/28 (4)*‡
 CD autoantibody-positive group1/3 (33)1/4 (25)
 Marsh I group0/8 (0)0/12 (0)
 Marsh 0 group0/3 (0)0/12 (0)

There were no differences in median age or gender distribution between patients with early developing coeliac disease and patients excluded for coeliac disease. Forty-four percentage of patients with early developing coeliac disease and 10% of those excluded for coeliac disease had first-degree relatives affected by coeliac disease (P = 0.021). HLA DQ2 or DQ8 was detected in every early developing coeliac disease patient with an available sample (n = 12), and in 10 of 29 (35%) patients excluded for coeliac disease (P < 0.001).

Counting of total IELs (CD3+) at baseline failed to detect early developing coeliac disease (Table 4). Intestinal TG2-specific IgA deposits had the best sensitivity and specificity for the condition, 93% in both. The percentages of γδ+ and villous tip IELs were lower: the sensitivity of γδ+ IELs was 76% and specificity was 60%, and the corresponding values for villous tip IELs were 88% and 71% respectively (Table 5). Nonetheless, even γδ+ and villous tip IELs were statistically significantly more often elevated in patients with early developing coeliac disease than in patients excluded for the disease (Table 4). We also tested different combinations of the tests shown in Table 5. The combination of HLA DQ2/DQ8 and intestinal TG2-specific IgA deposits achieved the sensitivity of 91% and the specificity of 100% and HLA and serum autoantibody testing gave a 67% sensitivity and a 97% specificity. In other combinations, the maximal sensitivity was with villous tip IELs and HLA DQ2/DQ8, 82%, with the specificity of 89%. Some other combinations yielded the 100% specificity, but the sensitivity was at its best only 79% (villous tip IELs and intestinal TG2-specific IgA deposits). Thus, the combination did not provide any additional benefit.

Table 4.   Histological findings in patients with prior coeliac disease suspicion at baseline when coeliac disease was excluded upon normal small bowel biopsy finding
 Early developing coeliac disease (n = 17)Coeliac disease excluded by biopsy (n = 30)P-value
  1. Vh/CrD, villous height–crypt depth ratio; TG2, transglutaminase; IEL, intraepithelial lymphocyte; IgA, immunoglobulin A.

Vh/CrD, median (range)2.7 (2.0–5.0)3.1 (2.4–4.3)0.063
Increased density of CD3+ IELs (Marsh I), n (%)10/17 (59)13/30 (43)0.371
Increased density of γδ+ IELs, n (%)13/17 (76)12/30 (40)0.032
Increased density of villous tip IELs, n (%)14/16 (88)8/28 (29)<0.001
Intestinal TG2-specific IgA deposits present14/15 (93)1/14 (7)<0.001
Table 5.   Sensitivities and specificities of different markers in detecting coeliac disease before the development of villous atrophy
 Sensitivity (95% CI)Specificity (95% CI)
  1. Patients excluded for coeliac disease at re-evaluation served as controls.

  2. CI, confidence interval; TG2, transglutaminase; IEL, intraepithelial lymphocyte; IgA, immunoglobulin A.

Intestinal TG2-specific IgA deposits present0.93 (0.70–0.99)0.93 (0.69–0.99)
Serum IgA-class coeliac autoantibodies present0.76 (0.53–0.90)0.83 (0.66–0.93)
Increased density of villous tip IELs0.88 (0.64–0.97)0.71 (0.53–0.85)
Increased density of γδ+ IELs0.76 (0.53–0.90)0.60 (0.42–0.75)
Increased density of CD3+ IELs (Marsh I)0.59 (0.36–0.78)0.57 (0.39–0.73)
HLA DQ2/DQ8 present1.0 (0.76–1.0)0.66 (0.47–0.80)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study elucidated the natural history of coeliac disease. Altogether 17 patients with normal villous architecture at baseline, while consuming a gluten-containing diet, subsequently developed mucosal damage diagnostic for coeliac disease. Majority of cases had initially positive coeliac autoantibodies in the serum. When these TG2-targeted autoantibodies were examined where they are produced, in the small bowel mucosa, 93% of all patients with early developing coeliac disease were identified in the absence of villous atrophy.

Occasionally coeliac disease diagnosis can be problematic; small bowel biopsy is considered the gold standard in coeliac disease diagnosis despite involving some obvious difficulties in interpretation. Even in the diagnostics of overt coeliac disease, a false diagnosis can be made, because at least 10% of biopsy specimens are of poor quality36 and further, villous atrophy may occasionally be patchy.37 In this study multiple small bowel biopsies were taken from several sites in order to detect also patients with patchy mucosal lesion. There is still always a possibility that a patchy form of villous atrophy is missed, and hence new methods are needed.

The diagnosis of early developing coeliac disease is even more challenging; minor histological abnormalities are unspecific, subjective and difficult to interpret. According to the recent studies focusing on early developing coeliac disease it has become evident that the recognition of coeliac disease without villous atrophy is important,24–26 especially because the patients benefit of dietary treatment. In agreement with this also our patients with early developing coeliac disease suffered from various symptoms typical of coeliac disease before the development of overt villous atrophy (Table 2). However, we demonstrated that Marsh classification could not be utilized reliably in the detection of early developing coeliac disease. Therefore, patients with Marsh I lesion in the small bowel mucosa should not be advised to adhere to a gluten-free diet in the absence of additional evidence.

The value of villous tip IELs in discovering patients with early developing coeliac disease was superior to Marsh I lesion, as has also previously been shown in smaller series.6, 31, 38 The clear advantage of villous tip IELs is that they can be studied in routine H & E-stained sections, whereas other histological methods applied here required frozen samples. In this study increased densities of γδ+ IELs were not only indicative of early developing coeliac disease, but also 40% of patients excluded for coeliac disease also on re-evaluation had increased densities of these cells at baseline when coeliac disease was first suspected. False-positive γδ+ cells have similarly been reported elsewhere.13, 23

The diagnosis of both overt and early developing coeliac disease should be based on reliable evidence. Histology has its limitations, as stated above. Nor are coeliac autoantibodies 100% specific. Five autoantibody-positive patients in this study did not proceed to villous atrophy during the follow-up, though it is possible that a longer follow-up might reveal progression of the disease in these individuals. Antibody testing was changing during the study period, and TG2 antibody test was not available at the time of baseline investigations in most patients. However, EmA and TG2 antibody tests correlate closely,8, 29 and the sensitivity and specificity values of these tests have been equal,39 even though some occasional patients remain negative for EmA despite positive for TG2 antibodies, and vice versa. DQ is mandatory in the identification of patients with genetic gluten intolerance, and patients with HLA DQ2 or DQ8 having positive coeliac autoantibodies in the serum are highly likely to suffer from coeliac disease. However, HLA DQ2 and DQ8 alone can only be used when patients are excluded for coeliac disease, because they are common in the population in general.40

We consider that intestinal IgA deposits targeted against TG2 are currently the best method in revealing early developing coeliac disease. Although serological tests predicted the development of villous atrophy relatively well, four (24%) of 17 patients with histologically confirmed coeliac disease had negative serology at baseline. Furthermore, there were five patients who underwent spontaneous negative seroconversion. Only one of them had intestinal IgA deposits at baseline, again showing that the method is more specific than serology. Investigation of intestinal IgA deposits is a special method requiring frozen small bowel biopsy specimens, which limits its utility. This method should be available at least in special centres, because it is clearly beneficial in cases where the conventional histology is ambiguous, especially in cases where a second investigation and biopsy is considered to confirm the diagnosis of coeliac disease. By investigating these deposits in small bowel biopsy specimens, when coeliac disease was first suspected, we were able in the majority of cases to diagnose early developing coeliac disease before the development of forthcoming villous atrophy. However, patients with early developing coeliac disease do not yet fulfil the traditional European Society for Pediatric Gastroenterology and Nutrition diagnostic criteria for coeliac disease. We have now shown that the criteria are no longer valid; many patients suffer from gluten-dependent symptoms before the development of villous atrophy, and the criteria should thus be revised.

In conclusion, the detection of intestinal TG2-specific IgA deposits proved a powerful diagnostic tool in coeliac disease without villous atrophy. We recommend that these deposits be invariably investigated when early developing coeliac disease is suspected. The presence of these intestinal TG2-targeted deposits strengthens the diagnosis of coeliac disease and dietary treatment should be considered at least in symptomatic patients. Further, patients with coeliac disease suspicion but normal villous architecture having positive coeliac autoantibodies in the serum or increased density of γδ+ or villous tip IELs in the small bowel mucosa should be followed up, whereas patients with Marsh I alone do not require routine surveillance. The detection of intestinal TG2-specific IgA deposits provides a reliable means to discover patients with early developing coeliac disease, and thus the results of this study should be taken into consideration when the diagnostic criteria for coeliac disease are revised.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by the Research Fund of the Finnish Coeliac Society, the Medical Research Fund of Tampere University Hospital, the Finnish Medical Foundation, the Foundation for Pediatric Research in Finland, the National Graduate School of Clinical Investigation, the Finnish Foundation of Gastroenterological Research, the Yrjo Jahnsson foundation and the Finnish Medical Society Duodecim.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
  • 1
    Walker-Smith JA, Guandalini S, Schmitz J, Shmerling DH, Visakorpi JK. Revised criteria for diagnosis of coeliac disease. Arch Dis Child 1990; 65: 90911.
  • 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: 33054.
  • 3
    Lähdeaho ML, Kaukinen K, Collin P, et al. Celiac disease: from inflammation to atrophy: a long-term follow-up study. J Pediatr Gastroenterol Nutr 2005; 41: 448.
  • 4
    Kakar S, Nehra V, Murray JA, Dayharsh GA, Burgart LJ. Significance of intraepithelial lymphocytosis in small bowel biopsy samples with normal mucosal architecture. Am J Gastroenterol 2003; 98: 202733.
    Direct Link:
  • 5
    Iltanen S, Holm K, Ashorn M, Ruuska T, Laippala P, Mäki M. Changing jejunal gamma/delta T cell receptor (TCR)-bearing intraepithelial lymphocyte density in coeliac disease. Clin Exp Immunol 1999; 117: 515.
  • 6
    Goldstein NS, Underhill J. Morphologic features suggestive of gluten sensitivity in architecturally normal duodenal biopsy specimens. Am J Clin Pathol 2001; 116: 6371.
  • 7
    Ladinser B, Rossipal E, Pittschieler K. Endomysium antibodies in coeliac disease: an improved method. Gut 1994; 35: 7768.
  • 8
    Sulkanen S, Halttunen T, Laurila K, et al. Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease. Gastroenterology 1998; 115: 13228.
  • 9
    Dieterich W, Laag E, Schöpper H, et al. Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology 1998; 115: 131721.
  • 10
    Rostami K, Kerckhaert J, Tiemessen R, Von Blomberg ME, Meijer JWR, Mulder CJJ. Sensitivity of antiendomysium and antigliadin antibodies in untreated celiac disease: disappointing in clinical practice. Am J Gastroenterol 1999; 94: 88894.
    Direct Link:
  • 11
    Tursi A, Brandimarte G, Giorgetti G, Gigliobianco A, Lombardi D, Gasbarrini G. Low prevalence of antigliadin and anti-endomysium antibodies in subclinical/silent celiac disease. Am J Gastroenterol 2001; 96: 150710.
    Direct Link:
  • 12
    Abrams JA, Diamond B, Rotterdam H, Green PHR. Seronegative celiac disease: increased prevalence with lesser degrees of villous atrophy. Dig Dis Sci 2004; 49: 54650.
  • 13
    Iltanen S, Holm K, Partanen J, Laippala P, Mäki M. Increased density of jejunal gamma/delta+ T cells in patients having normal mucosa – marker of operative autoimmune mechanisms? Autoimmunity 1999; 29: 17987.
  • 14
    Collin P, Helin H, Mäki M, Hällström O, Karvonen AL. Follow-up of patients positive in reticulin and gliadin antibody tests with normal small bowel biopsy findings. Scand J Gastroenterol 1993; 28: 5958.
  • 15
    Korponay-Szabo IR, Sulkanen S, Halttunen T, et al. Tissue transglutaminase is the target in both rodent and primate tissues for celiac disease-specific autoantibodies. J Pediatr Gastroenterol Nutr 2000; 31: 5207.
  • 16
    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: 199204.
  • 17
    Marzari R, Sblattero D, Florian F, et al. Molecular dissection of tissue transglutaminase autoantibody response in celiac disease. J Immunol 2001; 166: 41706.
  • 18
    Picarelli A, Maiuri L, Frate A, Greco M, Auricchio S, Londei M. Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease. Lancet 1996; 348: 10657.
  • 19
    Sblattero D, Ventura A, Tommasini A, et al. Cryptic gluten intolerance in type 1 diabetes: identifying suitable candidates for a gluten free diet. Gut 2006; 55: 1334.
  • 20
    Shiner M, Ballard J. Antigen-antibody reactions in jejunal mucosa in childhood coeliac disease after gluten challenge. Lancet 1972; 1: 12025.
  • 21
    Karpati S, Kosnai I, Török E, Kovacs J. Immunoglobulin A deposition in jejunal mucosa of children with dermatitis herpetiformis. J Invest Dermatol 1988; 91: 3369.
  • 22
    Korponay-Szabo IR, Halttunen T, Szalai Z, et al. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut 2004; 53: 6418.
  • 23
    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 clinical study. Scand J Gastroenterol 2005; 40: 56472.
  • 24
    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: 22948.
    Direct Link:
  • 25
    Kaukinen K, Mäki M, Partanen J, Sievänen H, Collin P. Celiac disease without villous atrophy: revision of criteria called for. Dig Dis Sci 2001; 46: 87987.
  • 26
    Tursi A, Brandimarte G. The symptomatic and histologic response to a gluten-free diet in patients with borderline enteropathy. J Clin Gastroenterol 2003; 36: 137.
  • 27
    Mäki M. The humoral immune system in coeliac disease. Baillieres Clin Gastroenterol 1995; 9: 23149.
  • 28
    Hällström O. Comparison of IgA-class reticulin and endomysium antibodies in coeliac disease and dermatitis herpetiformis. Gut 1989; 30: 122532.
  • 29
    Mäki M, Mustalahti K, Kokkonen J, et al. Prevalence of celiac disease among children in Finland. N Engl J Med 2003; 348: 251724.
  • 30
    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: 52531.
  • 31
    Järvinen TT, Collin P, Rasmussen M, et al. Villous tip intraepithelial lymphocytes as markers of early-stage coeliac disease. Scand J Gastroenterol 2004; 39: 42833.
  • 32
    Arranz E, Bode J, Kingstone K, Ferguson A. Intestinal antibody pattern of coeliac disease: association with gamma/delta T cell receptor expression by intraepithelial lymphocytes, and other indices of potential coeliac disease. Gut 1994; 35: 47682.
  • 33
    Järvinen TT, Kaukinen K, Laurila K, et al. Intraepithelial lymphocytes in celiac disease. Am J Gastroenterol 2003; 98: 13327.
    Direct Link:
  • 34
    Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. Evidence for a primary association of celiac disease to a particular HLA-DQ α/β heterodimer. J Exp Med 1989; 169: 34550.
  • 35
    Polvi A, Arranz E, Fernandez-Arquero M, et al. HLA-DQ2-negative celiac disease in Finland and Spain. Hum Immunol 1998; 59: 16975.
  • 36
    Collin P, Kaukinen K, Vogelsang H, et al. Anti-endomysial and anti-human recombinant tissue transglutaminase antibodies in the diagnosis of coeliac disease: a biopsy-proven European multicentre study. Eur J Gastroenterol Hepatol 2005; 17: 8591.
  • 37
    Scott BB, Losowsky MS. Patchiness and duodenal-jejunal variation of the mucosal abnormality in coeliac disease and dermatitis herpetiformis. Gut 1976; 17: 98492.
  • 38
    Biagi F, Luinetti O, Campanella J, et al. Intraepithelial lymphocytes in the villous tip: do they indicate potential coeliac disease? J Clin Pathol 2004; 57: 8359.
  • 39
    Hill ID. What are the sensitivity and specificity of serological tests for celiac disease? Do sensitivity and specificity vary in different populations? Gastroenterology 2005; 128: S2532.
  • 40
    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: 46977.