Distinction between coeliac disease and refractory sprue: a simple immunohistochemical method

AIMS: We recently showed that refractory sprue is distinct from coeliac disease, the former being characterized by abnormal intraepithelial T-lymphocytes expressing a cytoplasmic CD3 chain (CD3c), lacking CD3 and CD8 surface expression, and showing TCRgamma gene rearrangements. To take advantage of the abnormal phenotype of CD3c + CD8 - intraepithelial lymphocytes (IEL) in refractory sprue we developed a simple method to distinguish coeliac disease from refractory sprue. METHODS AND RESULTS: Comparative immunohistochemical studies using anti-CD3 and anti-CD8 antibodies were applied on paraffin-embedded and frozen biopsy specimens in refractory sprue (n = 6), coeliac disease (n = 10), healthy controls (n = 5) and suspected refractory sprue (n = 6). Comparable results were obtained on fixed and frozen biopsy specimens. In four of the six patients with suspected refractory sprue, abnormal CD3c + CD8 - IEL and TCRgamma gene rearrangements were found, as in refractory sprue; the remaining two patients had normal (CD3 + CD8 +) IEL and no TCRgamma gene rearrangements. Both patients had coeliac disease, as one failed to comply with a gluten-free diet, while the other was a slow responder. CONCLUSION: This simplified immunostaining method using anti-CD3 and anti-CD8 antibodies on paraffin sections can distinguish active coeliac disease from refractory sprue and should prove useful in clinical practice.     

An update in the diagnosis of coeliac disease

Walker M M & Murray J A
(2011) Histopathology 59 , 166–179 An update in the diagnosis of coeliac disease Coeliac disease is increasing in prevalence, which is currently estimated at one in 100 of the population and may occur de novo in adults. The diagnosis requires a joint clinicopathological approach; the recommended first‐line test is serology with immunoglobulin A (IgA) tissue transglutaminase and IgA endomysial antibodies. These serological tests show high levels of sensitivity and specificity, but biopsy is the gold standard to confirm the diagnosis. It is important that both tests are performed before the introduction of a gluten‐free diet. Although the classical histopathology changes of coeliac disease with partial or total villous atrophy are well recognized, the pathology classification of coeliac disease is changing, with recognition that coeliac disease may show minimal pathology (normal architecture and an intraepithelial lymphocyte count/100 enterocytes ≥ 25). This entity is also described as lymphocytic duodenosis, and recommendation of follow‐up serology testing is paramount in this condition. Follow‐up of patients with coeliac disease is warranted, as normal serology does not predict mucosal recovery. Failure to heal predicts risk of progression to refractory coeliac disease and malignancies. Refractory coeliac disease occurs in 1–2% of patients and this diagnosis requires a combined clinical and histopathology approach with immunocytochemistry.     

Anti-calreticulin immunoglobulin A (IgA) antibodies in refractory coeliac disease

Refractory coeliac disease (RCD) is a very rare and dangerous form of CD, in which gluten-free diet loses its therapeutic effect and the damage of intestinal mucosa persists. Because of the adherence to the diet, serological markers of CD [immunoglobulin A (IgA) antibodies against gliadin, tissue transglutaminase (tTG) and endomysium] are often missing in RCD patients. We found substantially elevated levels of IgA anti-calreticulin (CRT) antibodies in the sera of almost all RCD patients tested. These sera were negative for IgA antibodies to gliadin and tTG and only some of them showed IgA antibodies to enterocytes. Analysis of patients' IgA reactivity to CRT fragments (quarters and halves) by Western blotting revealed differences in the specificity of IgA antibodies between RCD and CD patients. We therefore used the Pepscan technique with synthetic overlapping decapeptides of CRT to characterize antigenic epitopes recognized by serum IgA antibodies of RCD patients. Employing this method we demonstrated several dominant antigenic epitopes recognized by IgA antibodies of RCD patients on the CRT molecule. Epitope GVTKAAEKQMKD was recognized predominantly by serum IgA of RCD patients. Our results suggest that testing for serum IgA antibodies against CRT and its selected peptide could be a very useful tool in RCD differential diagnosis.     

Recent advances in refractory coeliac disease: a review

Coeliac disease (CD) is an immune-mediated disease of the small intestine caused by intolerance to gluten. Removal of gluten from the diet results in a return to normal health for the majority of patients. A significant proportion of patients do not respond to a gluten-free diet and are considered to be suffering from refractory coeliac disease (RCD). Two types of RCD are now recognized: type 1 RCD is characterized by a polyclonal population of intraepithelial lymphocytes (IELs) with a normal immunophenotype, and type 2 RCD shows monoclonal IELs with an aberrant immunoprofile. Patients with RCD have a high risk of complications such as ulcerative jejunitis (UJ) and enteropathy-type T-cell lymphoma (ETTL). RCD2 may represent an early stage in the development of overt lymphoma. The diagnosis of RCD, therefore, has important implications, but remains a challenging area. In this paper we review the latest developments in RCD, including the diagnostic approach and a discussion of the key clinical, histological, immunohistochemical and molecular features of RCD and its complications.     

Enhanced interleukin-18 levels in the peripheral blood of children with coeliac disease

Coeliac disease (CoD) is a small intestinal disorder characterized by villous atrophy, crypt cell hyperplasia and an increased production of T helper cell type 1 (Th1) cytokines. Interleukin (IL)-18 is a pro-inflammatory cytokine that has a crucial role in maintaining the Th1 response. In this study, the serum levels of IL-18 were measured in children with CoD or other gastrointestinal diseases in order to evaluate the possibility of using IL-18 as a disease activity marker. IL-18 levels were higher in samples from CoD patients [median 443 pg/ml (148-885)] compared to healthy controls [median 205 pg/ml (11-379)], P <0.05. In contrast, the levels of IL-18 were not enhanced significantly in the serum from patients with inflammatory bowel disease (IBD) [median 324 pg/ml (207-546)] or in the disease control group [median 303 pg/ml (2-689)]. In CoD patients, after 2 weeks of gluten challenge (GC), serum IL-18 was unchanged [median 268 pg/ml (59-458)] compared to patients on a gluten-free diet [median 220 pg/ml (53-600)], while IL-18 was increased after 12 weeks of GC [median 551 pg/ml (94-952)], P <0.01. The IL-18 levels correlated with IgA anti-transglutaminase antibody levels (rs=0.59, P=0.016) in serum from untreated CoD patients, and IL-18 also followed the degree of small intestinal villous atrophy in 12 out of 19 CoD patients. Our results support the view that serum IL-18 concentrations in children with CoD follow disease activity, suggesting a role for IL-18 in the induction of an inflammatory Th1-response after gluten exposure.     

Serum cytokine pattern in young children with screening detected coeliac disease

Coeliac disease is an autoimmune disease characterized by inflammation localized to the small bowel, but less is known about systemic signs of inflammation. The aim was to measure cytokines of the T helper 1 (Th1) and T helper 2 (Th2) cell patterns in children with screening‐detected coeliac disease before and after treatment with a gluten‐free diet. Serum samples selected before and after the start of a gluten‐free diet from 26 3‐year‐old children diagnosed with biopsy‐proven coeliac disease and from 52 matched controls were assayed in an multiplex enzyme‐linked immunosorbent assay (ELISA) for the 10 cytokines: interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐8, IL‐10, IL‐12p70, IL‐13 and tumour necrosis factor (TNF)‐α. Among Th1 cytokines, IFN‐γ and IL‐12p70 were elevated significantly in children with coeliac disease compared to controls (P < 0·001 and P = 0·001, respectively). Similar findings were demonstrated for the Th2 cytokines IL‐5 (P < 0·001), IL‐10 (P = 0·001) and IL‐13 (P = 0·002). No difference in cytokine levels between the two groups was found for TNF‐α, IL‐1β, IL‐2, IL‐4 and IL‐8. After gluten‐free diet, levels of IL‐5, IL‐12 and IL‐10 decreased significantly (P < 0·001, P = 0·002 and P = 0·007) and IFN‐γ levels were reduced (P = 0·059). Young children with coeliac disease detected by screening demonstrate elevated levels of serum cytokines at time of diagnosis. A prolonged systemic inflammation may, in turn, contribute to long‐term complications known to be associated with untreated coeliac disease.     

Hypogalactosylation of serum IgG in patients with coeliac disease

Coeliac disease (CD) is described as an autoimmune enteropathy associated with the presence of IgG and IgA antigliadin and antitransglutaminase autoantibodies. While of diagnostic significance, the role of these autoantibodies in the immunopathogenesis of CD is elucidated. An inappropriate T cell immune response to gluten is also involved in the pathogenesis of CD, as evidenced by autoantibody switching. The N-glycans released from serum IgG of CD patients and three groups of healthy controls, of differing age ranges, were analysed by NH2-high performance liquid chromatography (HPLC). The fucosylated biantennary N- glycans were the most abundant neutral oligosaccharides; in particular, the agalacto form (G0F) showed a mean value of 42% (s.d. +/- 7.4), 30% (s.d. +/- 5.9), 26% (s.d. +/- 4.2) and 35% (s.d. +/- 6.8) for CD patients, healthy children, healthy adults under 40 and healthy adults over 40 years old, respectively. The ratio of asialo agalacto fucosylated biantenna to asialo monogalacto fucosylated biantenna (G0F)/(G1F) for CD patients showed a significant increase compared to healthy children (P < 0.0002), healthy adults under 40 (P < 0.0002) and healthy adults over 40 years old (P < 0.01). Hypogalactosylation was more pronounced for CD patients than for the patients with other autoimmune diseases such as rheumatoid arthritis or psoriatic arthritis.     

Cytokine-producing cells in peripheral blood of children with coeliac disease secrete cytokines with a type 1 profile.

Coeliac disease (CoD) is a small intestinal disorder characterized by crypt cell hyperplasia and villous atrophy, and the production of cytokines from T cells and macrophages are of importance for the histological changes seen in CoD. A peroral immunization with an antigen, which gives rise to a mucosal immune response, may increase the levels of circulating cytokine-producing cells, and we wanted to obtain a better picture of an eventual emergence of activated circulating T cells in the peripheral blood in children with CoD. The cytokine expression of interferon-gamma (IFN-gamma), IL-4, IL-6 and IL-10 was measured at the single-cell level by an ELISPOT method in 38 children with CoD. The numbers of IFN-gamma-producing cells in the peripheral blood was increased in children with untreated CoD (P < 0.01) and after gluten challenge (P < 0.05) compared with healthy controls. Also, the numbers of IL-6-producing cells were increased (P < 0.05) after gluten challenge compared with the healthy controls. A paired comparison showed that the numbers of IFN-gamma-producing cells increased after gluten challenge (P < 0.05), whereas no such change was seen for IL-4- or IL-10-producing cells. There were no differences in the numbers of IFN-gamma-producing cells between the group of children with treated CoD and the groups of untreated or challenged CoD children. IL-4 production correlated with serum levels of total IgE. These results show that circulating mononuclear cells in children with active CoD secrete cytokines compatible with a type 1 response.     

Mucosal reactivity to cow's milk protein in coeliac disease

Patients with coeliac disease (CD) on a gluten-free diet may still have gastrointestinal symptoms. On clinical grounds cow's milk (CM) protein sensitivity may be suspected. Here, using rectal protein challenge, we investigated the local inflammatory reaction to gluten and CM protein in adult patients with CD in remission. Rectal challenges with wheat gluten and dried CM powder were performed in 20 patients with CD and 15 healthy controls. Fifteen hours after challenge the mucosal reaction was recorded by the mucosal patch technique with measurements of local release of neutrophil and eosinophil granule constituents; myeloperoxidase (MPO) and eosinophil cationic protein (ECP). We measured the mucosal production of nitric oxide (NO) simultaneously. Six of the patients who reacted to CM were also challenged with alpha-lactalbumin and casein. In 18 of 20 patients gluten challenge induced neutrophil activation defined as increased MPO release and increased NO synthesis. Ten of these 20 patients showed a similarly strong inflammatory reaction to CM challenge. Six of the CM sensitive patients were challenged with specific CM proteins: casein and alpha-lactalbumin. Casein, in contrast to alpha-lactalbumin, induced an inflammatory response similar to that produced by CM. A mucosal inflammatory response similar to that elicited by gluten was produced by CM protein in about 50% of the patients with coeliac disease. Casein, in particular, seems to be involved in this reaction.     

The liver in coeliac disease

The pathology of the liver in 19 cases of malabsorption is reported. Five of these were proven to have adult coeliac disease, in the others that diagnosis was presumed by exclusion of other causes of malabsorption and by the coincidence of other conditions known to be associated with coeliac disease. Of these cases, three had liver changes of chronic hepatitis and two of these were in the proven coeliac group, including a case with cirrhosis and a hepatoma. In addition, less severe liver changes such as portal tract fibrosis and portal tract infiltration by inflammatory cells were present greatly in excess to that of the controls. The reasons for the occurrence of liver damage in coeliac disease are outlined and discussed in relation to the liver disorders associated with jejunoileal bypass used in the treatment of obesity. Possible mechanisms of liver injury in coeliac disease are described.     

Speeding up coeliac disease diagnosis in cardiological settings

Introduction

High prevalence of coeliac disease (CD) has been reported among patients with idiopathic dilated cardiomyopathy (DCM). We evaluated the feasibility and diagnostic accuracy of screening for CD by rapid test of anti-transglutaminase antibodies in the cardiology outpatients’ clinic.

Material and methods

We screened the blood samples of 104 patients with DCM, 44 of their first-degree relatives, 63 diseased controls and 101 healthy controls for the presence of anti-transglutaminase antibodies in a drop of whole blood using a rapid assay. This test was compared to the enzyme-linked immunosorbent assay and the anti-endomysium antibody test.

Results

Our rapid test was positive in three (2.9%) DCM patients, in one (2%) relative and in one (1%) healthy control. These subjects were positive at both control assays. Two DCM patients had iron-deficient anaemia. The healthy relative was asymptomatic, while the healthy control experienced extreme asthenia. The relative refused intestinal biopsy, while the others showed histological evidence of CD. During the gluten-free diet, the patient with the worst left ventricular ejection fraction (LVEF) underwent heart transplant, and LVEF values improved in the other two. Anaemia and tiredness resolved in all patients.

Conclusion

Early detection of CD in a cardiological setting allows prompt treatment with a gluten-free diet of gluten-dependent complaints with potential benefits for the course of DCM.     

The immune recognition of gluten in coeliac disease

Coeliac disease, the most common intestinal disorder of western populations, is an autoimmune enteropathy caused by an abnormal immune response to dietary gluten peptides that occurs in genetically susceptible individuals carrying the HLA-DQ2 or -DQ8 haplotype. Despite the recent progresses in understanding the molecular mechanisms of mucosal lesions, it remains unknown how increased amounts of gluten peptides can enter the intestinal mucosa to initiate the inflammatory cascade. Current knowledge indicates that different gluten peptides are involved in the disease process in a different manner, some fragments being 'toxic' and others 'immunogenic'. Those defined as 'toxic' are able to induce mucosal damage either when added in culture to duodenal endoscopic biopsy or when administered in vivo, while those defined as 'immunogenic' are able to specifically stimulate HLA-DQ2- or DQ8-restricted T cell clones isolated from jejunal mucosa or peripheral blood of coeliac patients. These peptides are able to trigger two immunological pathways: one is thought to be a rapid effect on the epithelium that involves the innate immune response and the other represents the adaptive immune response involving CD4+ T cells in the lamina propria that recognize gluten epitopes processed and presented by antigen presenting cells. These findings are the subject of the present review.     

Transglutaminase and coeliac disease: endomysial reactivity and small bowel expression

This study was aimed at verifying whether tissue transglutaminase (tTG) is the sole autoantigen eliciting anti-endomysial antibodies in coeliac disease (CoD) and investigating tTG expression in normal and coeliac mucosa. Twelve anti-endomysial-positive coeliac sera and 12 anti-endomysial-negative control sera (10 microl, diluted 1:5-1:400 in PBS pH 7.3) were preincubated with 10, 20 or 50 microg guinea pig liver tTG at 4 degrees C overnight. Monkey oesophagus tissue slides were then tested with tTG-preincubated and non-preincubated sera to search for IgA anti-endomysial reactivity by indirect immunofluorescence. Moreover, six sections of monkey oesophagus were incubated with an anti-tTG mouse MoAb, six sections with an anti-cytokeratin mouse MoAb and six sections with only 3% bovine serum albumin. Finally, endoscopic duodenal biopsy sections obtained from 12 patients affected by untreated CoD, six patients affected by treated CoD and 10 biopsied controls were immunohistochemically stained with a peroxidase-conjugated anti-tTG MoAb. Our results show that (i) preincubation with tTG abolished endomysial immunofluorescence in most, but not in all, coeliac sera; (ii) the incubation of anti-tTG MoAb with sections of monkey oesophagus resulted in an immunofluorescence staining pattern similar but not identical to that of anti-endomysial-positive coeliac sera; (iii) although tTG expression was present at muscularis mucosae and pericryptal fibroblast in both normal and coeliac mucosa, it was slightly more marked and evident in the latter. Although our absorption experiment was performed with guinea pig liver tTG, we confirm that tTG is the predominant antigen of endomysial antibodies, but we speculate that, at least in some patients, it is not the only one.     

Prevalence of anaemia among rural pre-school children of West Bengal,India

Background: Micronutrient malnutrition is a major public health nutritional problem in India, and iron deficiency anaemia (IDA) continues to be a major nutritional problem of public health significance, affecting all physiological groups, of which rural pre-school children are the most vulnerable.

Aim: The main aim of the present study was to assess the prevalence of anaemia among rural pre-school children.

Subjects and methods: A community-based cross-sectional study was carried out in rural areas of West Bengal State during 2002–2003. A total of 437 pre-school children were covered for the estimation of blood haemoglobin levels.

Results: A majority (81%) of the rural children of West Bengal were anaemic, and the prevalence was significantly (p<0.001) higher among 1–3-year-old (91%) as compared to 4–5-year-old (74.6%) children. A significantly (p<0.01) higher proportion of 1+ (OR=7.7; 95% CI: 2.6–22.4) and 2+ year children (OR=3.0; 95% CI: 1.5–6.0) and those belonging to lower socio-economic Scheduled Caste and Scheduled Tribe communities were at risk for anaemia (OR=2.3; 95% CI 1.3–3.9).

Conclusions: The prevalence of anaemia is a severe nutritional problem of public health significance. Therefore, iron supplementation and health and nutrition education programmes should be strengthened. The community needs to be encouraged to diversify their diets by consuming iron-fortified and iron-rich foods.     

Avidity progression of dietary antibodies in healthy and coeliac children

In most individuals minute amounts of food proteins pass undegraded across the intestinal mucosa and trigger antibody formation. Children with coeliac disease have enhanced antibody production against gliadin as well as other dietary antigens, e.g. beta-lactoglobulin, in cow's milk. Antibody avidity, i.e. the binding strength between antibody and antigen, often increases during antibody responses and may be related to the biological effectiveness of antibodies. The aim of the present study was to determine the avidity of serum IgG antibodies against beta-lactoglobulin and gliadin in healthy children during early childhood and compare these avidities to those found in children with coeliac disease. The average antibody avidity was analysed using a thiocyanate elution assay, whereas the antibody activity of the corresponding sera was assayed by ELISA. The avidity of serum IgG antibodies against beta-lactoglobulin as well as gliadin increased with age in healthy children, even in the face of falling antibody titres to the same antigens. Children with untreated coeliac disease had IgG anti-beta-lactoglobulin antibodies of significantly higher avidity than healthy children of the same age, and the same trend was observed for IgG antigliadin antibodies. The present data suggest that the avidities of antibodies against dietary antigens increase progressively during early childhood, and that this process seems to be accelerated during active coeliac disease.     

Contribution of the MHC region to the familial risk of coeliac disease.

Susceptibility to coeliac disease is genetically determined by possession of specific HLA-DQ alleles, acting in concert with one or more non-HLA linked genes. The pattern of risk seen in sibs and twins in coeliac disease is most parsimonious with a multiplicative model for the interaction between the two classes of genes. Based on a sib recurrence risk for coeliac disease of 10% and a population prevalence of 0.0033, the sib relative risk is 30. To evaluate the contribution of the MHC region to the familial risk of coeliac disease, we have examined haplotype sharing probabilities across this region in 55 coeliac disease families. Based on these probabilities the sib relative risk of coeliac disease associated with the MHC region is 3.7. Combining these results with published data on allele sharing at HLA, the estimated sib relative risk associated with the MHC region is 3.3. Therefore, the MHC genes contribute no more than 40% of the sib familial risk of coeliac disease and the non-HLA linked gene (or genes) are likely to be the stronger determinant of coeliac disease susceptibility.     

Anti-endomysial antibody of IgG1 isotype detection strongly increases the prevalence of coeliac disease in patients affected by type I diabetes mellitus

A strong association between type 1 insulin-dependent diabetes mellitus (IDDM1) and coeliac disease (CD) is well documented, but it is known that prevalence values are underestimated. Serum anti-endomysial antibodies (EMA), considered diagnostic for CD because of their high sensitivity and specificity, belong to the IgA class, but the existence of EMA of IgG1 isotype in the presence or absence of IgA deficiency was reported. In order to re-evaluate the occurrence of CD in IDDM1 patients we performed a screening in IDDM1 patients using EMA of both isotypes. Ninety-four adults affected by IDDM1 (unaffected by CD before enrolling) were enrolled and 83 blood donors as controls. All subjects were on a gluten-containing diet. Histology and biopsy culture were performed. EMA IgA and IgG1 in sera and culture supernatants were detected. Serum EMA were positive in 13 of 94 IDDM1 patients (13.8%). Six of 13 presented IgA-EMA, seven of 13 presented IgG1-EMA. No EMA were found in the control population. Total intestinal atrophy was found in all six patients with serum IgA-EMA and in five of seven with serum IgG1-EMA. Diagnosis of CD was confirmed by histology and organ culture in all 13 patients with serum EMA. The prevalence of CD in the patients affected by IDDM1 was 6.4% for IgA-EMA-positive and 7.4% for IgG1-EMA-positive patients. We confirmed the prevalence of CD in the IDDM1 population obtained with IgA-EMA screening only (6.4%). This prevalence value increases dramatically to 13.8% when IgG1-EMA are also used in the screening. We conclude that IgG1-EMA should also be sought whenever an IDDM1 patient undergoes screening for CD.     

Interleukin 18 maintains a long-standing inflammation in coeliac disease patients

Dietary gluten induces an early response in the intestine of coeliac disease patients (CD), within a few hours, and this is driven by high levels of proinflammatory cytokines, including IFNgamma and IL-15, as has been thoroughly shown by gluten stimulation of biopsy explants. Our aim was to identify the immune mediators involved in the long-standing inflammation in untreated CD patients at diagnosis. mRNA and protein levels of TNFalpha, IL-12(p35), IL-12(p40), IL-15, IL-18 and IL-23(p19) were quantified in biopsies from active CD patients, CD patients on a gluten-free diet (GFD), healthy controls, and patients with non-CD inflammation and mild histological changes in the intestine. Biopsies from CD patients on a GFD were also stimulated in vitro with gliadin, and protein expression of IL-15 and IL-18 was analysed. Levels of IL-12 and IL-23 mRNA are nearly absent, and TNFalpha levels remain unchanged among different groups. Both the active and inactive forms of IL-18 protein have been found in all samples from active CD, and protein expression was only localized within the crypts. Levels of IL-15 mRNA remain unchanged, and protein expression, localized within the lamina propria, is found in a small number of samples. In vitro stimulation with gluten induces the expression of IL-15 and IL-18. In active CD, the early response following gluten intake characterized by high IFNgamma levels is driven by IL-18, and probably IL-15, and this alternates with periods of long-standing inflammation with moderate IFNgamma levels, maintained by IL-18 alone.