HLA-DP and coeliac disease: family and population studies.

We investigated polymorphism of HLA-DP genes in three DR3 related diseases, confirming an association of coeliac disease with a Bgl II DP alpha polymorphism (a restriction fragment sized 3.5 kb present in 75% of patients compared to 34% of control subjects, p less than 0.001), and finding a weaker association with dermatitis herpetiformis (57% v 34%, p = 0.01) and no association with insulin dependent diabetes mellitus. The association with coeliac disease was further investigated. Msp I DP beta polymorphism was studied in 52 healthy subjects and 59 patients: a 4.9 kb fragment was present in 51% of patients with coeliac disease compared to 11.5% of control subjects (p less than 0.001). Furthermore, nearly all subjects with the DP alpha 3.5 kb fragment also had the DP beta 4.9 kb fragment. However, disease frequency was still increased in the DP alpha 3.5 positive/DP beta 4.9 negative group. In seven families, each with at least two affected members, while the DP alpha 3.5 fragment was frequently present in patients it did not preferentially segregate with any particular HLA haplotype--for example, those associated with DR3 or DR7--and therefore is not part of an extended haplotype associated with coeliac disease. We therefore conclude that a gene(s) in the HLA-DP region predisposes to coeliac disease independently of the HLA-DR/DQ regions.     

Neuroproliferation in the mucosa is a feature of coeliac disease and Crohn's disease.

The pathogenesis of villous damage in coeliac disease is unknown. Change to the delicate neuromuscular core may be significant and this study stained various categories of coeliac disease and controls with neuron-specific enolase (NSE) to examine neurofilaments in the mucosa. The amount of NSE staining was evaluated using computer image analysis. The first part of the study compared coeliac disease with Crohn's disease, carcinoma, and biopsy specimens from normal subjects. There was increased NSE staining in both the coeliac disease and Crohn's disease cases but not in carcinomas or normal controls. This difference was statistically significant. The average value for the coeliac disease patients was 50% higher than that of Crohn's disease patients. The second part of the study compared treated coeliac disease with untreated coeliac disease. Treated coeliac disease cases had normal amounts of NSE staining, which were the same as normal controls. These findings suggest that neuroproliferation is a feature of coeliac disease and Crohn's disease. Both share a common feature--namely chronic inflammation--which has been occasionally associated with neuroproliferation. The fact that neuroproliferation resolves with treatment is further evidence for its association with chronic inflammation. The extra neuroproliferation seen in coeliac disease compared with Crohn's disease may contribute to the architectural abnormalities seen in coeliac disease.     

HLA-DR expression, natural killer cells and IgE containing cells in the jejunal mucosa of coeliac children.

The expression of HLA-DR by surface and crypt epithelium and the numbers of cells of natural killer (NK) phenotype and of IgE containing cells were studied with monoclonal antisera using the peroxidase technique. We examined 48 jejunal biopsy specimens taken from 35 coeliac children before treatment (11), during gluten free diet (20) and after gluten challenge (17), and 13 control specimens. The luminal surface of the epithelial cells stained with HLA-DR antiserum in all specimens, but the cytoplasm of the surface epithelial cells took up the stain more frequently in the specimens from the controls (5/13) than those from the coeliacs (2/48) (p less than 0.01). In 21/28 specimens taken from coeliacs when on a gluten containing diet the crypt epithelium showed strong HLA-DR expression, while only 4/20 (p less than 0.01) specimens of coeliacs on a gluten free diet and 1/13 specimens of controls had similar staining. Among the intraepithelial lymphocytes no cells of NK phenotype were found in specimens from patients or controls. As compared with control specimens biopsy specimens from untreated coeliac patients showed smaller numbers of NK cells in the lamina propria. No difference was found in the numbers of IgE containing cells between the patients and controls. The strong expression of HLA-DR by the crypt epithelial cells in coeliac children on a normal diet suggest that these cells are involved in the presentation of the antigen.     

Small intestinal plasma cells in coeliac disease

Using a modified immunoperoxidase technique to achieve optimum staining and reproducible counts of plasma cells in paraffin embedded tissue, IgA, IgM, IgE, and IgG plasma cells were studied in small bowel biopsies from 20 controls, 23 untreated coeliac patients, 19 treated coeliac patients, and seven patients with Crohn's disease not involving duodenum or jejunum. In controls the ratio of the mean counts for IgA, IgM, IgE, and IgG plasma cells was 2.5:1:1:1 respectively. In patients with untreated coeliac disease, counts of all types of plasma cell were significantly increased approximately two-fold compared with controls although for IgG cells there was considerable overlap. The ratio of the mean plasma cell counts in the untreated coeliac patients was 3.5:1.5:2:1. Counts fell significantly after treatment with a gluten-free diet. There was no significant difference between counts in the controls and the Crohn's disease patients. The changes found in coeliac disease may simply be a non-specific response to mucosal damage. The increases in IgA and IgM plasma cells, however, suggest that the deposits of extracellular IgA and IgM observed in coeliac mucosa are locally produced, and the increase in IgE plasma cells raises the possibility that reaginic type hypersensitivity may be involved in coeliac disease.     

Morphometric electron and light microscope analysis of lymphoid cells in coeliac disease.

Lymphocytes in jejunal biopsies from normal subjects and from untreated, treated, and gluten-challenged coeliac patients were examined by accurate morphometric methods. There was no significant difference between lymphocytes from different sources and those in the jejunum. Transforming lymphocytes were ultrastructurally quite different from non-transforming lymphocytes. The appearance of transforming lymphocytes in the lamina propria in different groups of coeliac patients is in accordance with the increase in the number of plasma cells. In treated patients the results indicate a rapid humoral response to gluten challenge.     

Coeliac disease among healthy members of multiple case coeliac disease families

BACKGROUND: The main objective of the study was to assess the frequency of undetected coeliac disease among the first-degree relatives of families with two or more previously diagnosed coeliac disease patients. The value of the serum endomysial antibody test as a single means of detecting clinically silent coeliac disease was evaluated. The correlation of endomysial and tissue transglutaminase antibodies and the correlation of endomysial antibodies to the HLA typical for coeliac disease was determined. METHODS: A total of 137 multiple-case coeliac disease families with 872 family members were recruited; 466 healthy family members were simultaneously screened for gliadin and endomysial antibodies and thereafter for tissue tranglutaminase antibodies. Antibody-positive persons were typed for HLA-DQ2 and DQ8. The diagnosis of coeliac disease was based on the typical mucosal lesion on small-bowel biopsies. RESULTS: Forty-four (9.4%) of the healthy family members were positive for endomysial and 48 (10.3%) for gliadin antibodies; 42 biopsies revealed 29 new coeliac disease patients (6.2% of healthy individuals). Endomysial antibodies detected 97% and gliadin antibodies 52% of the new cases. All 44 endomysial-antibody-positive and 35 of 48 gliadin-antibody-positive individuals were positive for DQ2. Tissue transglutaminase antibodies corresponded well with endomysial antibodies. CONCLUSIONS: Undetected coeliac disease is common even among healthy first-degree relatives of multiple case families. The findings emphasize the value of serum endomysial antibodies in the detection of clinically silent coeliac disease. Endomysial-antibody-positive individuals, unlike gliadin-antibody-positive ones, share the coeliac disease-type HLA-DQ.     

Human intestinal mucosal mast cells: expanded population in untreated coeliac disease.

Previous retrospective studies of intestinal mucosal mast cells in coeliac disease have given divergent results, and we have recently reported that inappropriate methodology could account for these discrepancies. In this prospective study, mucosal mast cell counts were performed in Carnoy fixed, peroral jejunal biopsy specimens from patients with coeliac disease, both untreated and treated with a gluten-free diet; and from controls (mainly irritable bowel syndrome). Mean mucosal mast cell count in 27 control subjects was 146/mm2, SD 29. Significantly higher values were obtained in untreated coeliac disease (mean 243, SD 41, p less than 0.001) returning to the normal range in coeliacs treated with a gluten-free diet with normal jejunal biopsy morphology. In seven patients mucosal mast cell counts were performed in multiple jejunal biopsies, and these showed that mucosal mast cell distribution was not patchy. There was no evidence of degranulation of intestinal mucosal mast cells under the conditions of routine biopsy (overnight fast). An increase in mucosal mast cells in untreated coeliac disease may be one explanation for the high number of IgE positive stained cells in the intestinal mucosa that has been reported by some authors.     

Jejunal lysozyme activity and the Paneth cell in coeliac disease.

The jejunal mucosa of patients with coeliac disease contains significantly fewer Paneth cells (PCC) per crypt (p less than 0.001) and tissue lysozyme activity (JLA) P less than 0.001) when compared with a group of subjects with normal jejunal mucosa. Neither PCC nor JLA return to normal with complete clinical recovery and otherwise complete histological recovery on a gluten free diet. There is a significant linear correlation between JLA and PCC suggesting that the Paneth cell is the principal source of jejunal lysozyme.     

Enumeration of Paneth cells in coeliac disease: comparison of conventional light microscopy and immunofluorescence staining for lysozyme.

By conventional light microscopy, a reduced number of Paneth cells per intestinal crypt was found in the jejunal mucosa of patients with untreated or gluten-challenged coeliac disease as compared with histologically normal control specimens. A much better detection sensitivity was obtained when Paneth cells were counted by fluorescence microscopy after immunostaining for lysozyme with a rhodamine-labelled rabbit IgG conjugate. This method showed that there was no numerical reduction of Paneth cells in coeliac disease, but that the proportion of cells with a low lysozyme content was increased. Most of these cells were probably missed by conventional microscopy in which identification of Paneth cells is principally based on a substantial cellular complement of acidophilic granules. A reduced number of lysozyme-containing granules in coeliac disease may reflect increased discharge enhanced secretory activity, or a raised turnover of the Paneth cells.     

Infertility and coeliac disease.

BACKGROUND: Coeliac women may suffer from gynaecological and obstetric complications. It is possible that these complications are the first symptom of coeliac disease. AIMS: To investigate the occurrence of subclinical coeliac disease in patients with infertility or recurrent miscarriages. SUBJECTS: Women of reproductive age who were attending the hospital because of either primary or secondary infertility, or two or more miscarriages. Women undergoing sterilisation served as control subjects. METHODS: The diagnostic investigation for infertility included the endocrine status, diagnostic laparoscopy, investigation of tubal patency, postcoital test, and semen analysis of the partner. Circulating antibodies against IgA class reticulin and gliadin were used in screening for coeliac disease. In positive cases, the diagnosis was confirmed by small bowel biopsy specimens. RESULTS: Four (2.7%) of 150 women in the infertility group, and none of the 150 control subjects were found to have coeliac disease (p = 0.06). All four women with coeliac disease suffered from infertility of unexplained origin. Altogether 98 women had no discoverable reason for infertility. Thus, in this subgroup the frequency of coeliac disease was 4.1% (four of 98), the difference from the control group being statistically significant (p = 0.02). None of the coeliac women had extensive malabsorption, but two had iron deficiency anaemia. One women with coeliac disease has had a normal delivery. None of the 50 women with miscarriage had coeliac disease. CONCLUSION: Patients having fertility problems may have subclinical coeliac disease, which can be detected by serological screening tests. Silent coeliac disease should be considered in the case of women with unexplained infertility.     

Deficiency of invariant natural killer T cells in coeliac disease

BACKGROUND: Immunoregulatory invariant natural killer (iNK) T cells rapidly produce interleukin (IL)-4 and other cytokines that suppress a Th1 response and are deficient in some autoimmune diseases. AIM: The aim of this study was to investigate any deficiency of iNK T cells in coeliac disease. METHODS: Blood was collected from 86 subjects with coeliac disease and from 152 healthy control subjects for investigation of Valpha24+ T cells by flow cytometry. iNK T cells were assessed by Valpha24 and alpha-galactosylceramide/CD1d tetramer markers in 23 normal controls and 13 subjects with coeliac disease. Intracellular IL-4 was measured after anti-CD3 antibody stimulation. Duodenal biopsies were obtained in a subgroup of subjects with coeliac disease and control subjects for Valpha24 mRNA expression using relative PCR and for Valpha24+ T cells by immunofluorescence. RESULTS: The mean numbers of circulating Valpha24+ T cells and iNK T cells in coeliac disease were 27% (p<0.001) and 16% (p<0.001), respectively, of levels in control subjects. After in vitro anti-CD3 stimulation, numbers of IL-4+ producing iNK T cells from subjects with coeliac disease were unchanged but increased by 21% in control subjects. In subjects with coeliac disease, Valpha24 mRNA intestinal expression was reduced to 17% (p<0.001) by relative PCR and numbers of intestinal Valpha24+ T cells were 16% (p<0.01) of levels in control subjects. CONCLUSIONS: We conclude that Valpha24+ T cells and iNK T cells are deficient in coeliac disease. We speculate that this deficiency could contribute to the failure of immunological oral tolerance that seems to underlie this disease.     

Seroconversion of reticulin autoantibodies predicts coeliac disease in insulin dependent diabetes mellitus.

Serum IgA class reticulin autoantibody test was performed prospectively once a year on 238 children and adolescents with insulin dependent diabetes mellitus (IDDM). At the initial testing, within one year after onset of IDDM, five were positive and 233 were negative. During follow up a further 11 of the initially antibody negative children became positive (6.7%). Jejunal biopsy was performed at the appearance of the autoantibodies and silent coeliac disease was shown in nine (3.8%). One of these children showed on initial biopsy after the onset of IDDM to have normal jejunal mucosal architecture deteriorating later to a flat lesion. Jejunal immunohistochemical studies of another of the patients positive for reticulin autoantibodies but normal on routine biopsy showed an increased density of intraepithelially located gamma/delta T cells and aberrant HLA-DR expression in the crypts pointing to ongoing mucosal inflammation and potential coeliac disease. This study shows that in IDDM patients, reticulin autoantibody negative subjects become antibody positive, which may be followed by coeliac disease. Repeated serological screening and rebiopsy should be considered to detect late developing clinically silent coeliac disease among patients with IDDM.     

Tyrosine phosphorylation in the human duodenum.

Many growth factor receptors including the epidermal growth factor receptor function through tyrosine kinase activity. The aim of this study was to examine the constitutive level of tyrosine phosphorylation in the normal duodenum and in the hyperproliferative coeliac duodenum. A flow cytometric assay was devised using monoclonal antibody to phosphorylated (but not native) tyrosine residues to determine the levels of tyrosine phosphorylation in both CD3 positive intraepithelial lymphocytes and CD3 negative epithelial cells obtained by EDTA treatment of endoscopically obtained duodenal biopsy specimens. In addition, immunohistochemistry was performed on 18 formalin fixed coeliac duodenal biopsy specimens and eight control specimens. Tyrosine phosphorylation could be detected by flow cytometry on duodenal enterocytes and this expression was up regulated by pretreatment with epidermal growth factor. Tyrosine phosphorylation decreased with progression from the villus to the crypt, however, and was virtually undetectable on crypt enterocytes. Immunohistochemistry of the coeliac duodenum showed virtually absent tyrosine phosphorylation in the crypt. Increased tyrosine phosphorylation was detected in the infiltrating T cells. In conclusion, tyrosine phosphorylation in the duodenum is confined to the non-proliferative villous epithelium and is virtually undetectable in the proliferative crypt compartment. These findings suggest that tyrosine kinase activity is not a significant factor in the regulation of crypt cell proliferation in the human duodenum either in normal subjects or in coeliac disease patients.     

Increase in gamma/delta T cell receptor bearing lymphocytes in normal small bowel mucosa in latent coeliac disease.

A jejunal biopsy specimen from an asymptomatic 35 year old man was studied because of a low serum titre of reticulin antibody and the finding of coeliac disease in his son. In this specimen villous structure was quite normal as was the total number of intraepithelial lymphocytes, but the number of gamma/delta T cell receptor bearing lymphocytes was 10 times higher than the mean in control subjects. Two years later a further biopsy specimen was obtained because of clinical symptoms and an increased titre of reticulin antibody. This specimen showed villous atrophy with crypt hyperplasia and increased infiltration of intraepithelial lymphocytes compatible with coeliac disease. A control biopsy specimen taken during gluten free diet showed normalisation of the villous architecture. Latent coeliac disease may be characterised by an increase in gamma/delta positive cells similar to that seen in established coeliac disease.     

Defective synthesis of apolipoprotein A-I in jejunal mucosa in coeliac disease

Apolipoprotein A-I was studied in biopsy specimens from the duodenojejunal junction by means of an indirect immunoperoxidase technique. In normal control subjects apolipoprotein A-I was immunohistochemically detected only in the absorptive cells at the tips of villi and was mainly localized supranuclearly. In 14 patients with coeliac disease apolipoprotein A-I was virtually undetectable. This implies that the inflammatory lesion in coeliac disease decreases the intestinal mass of apolipoprotein A-I, because it destroys the villous absorptive cells. Apolipoprotein A-I concentrations in plasma were also decreased (by 40%) in patients with coeliac disease, suggesting that the intestine has a role in maintaining plasma apolipoprotein A-I levels.     

Compliance of adolescents with coeliac disease with a gluten free diet.

A cohort of 123 patients with coeliac disease, diagnosed in the first three years of life and followed up for at least 10 years, was reevaluated during the teenage period in terms of compliance with the diet and clinical state. Mucosal structure and lymphocytes were assessed in small intestinal biopsy specimens obtained from 36 subjects, by computerised image analysis. Of these adolescents with coeliac disease, 65% were adhering to a strict gluten free diet, 11.4% were on a gluten free diet but with occasional gluten intake, and 23.6% were on a gluten containing diet. Clinical symptoms occurred more frequently in patients on a gluten containing diet, but not in patients on a semi-strict diet. Occasional intake of small amounts (0.06-2 g/day) of gluten did not produce increased concentrations of antigliadin antibodies but resulted in an appreciably increased crypt epithelial volume and expanded crypt intraepithelial lymphocyte population.     

Histology of the terminal ileum in coeliac disease

BACKGROUND: The histological lesion of gluten sensitivity primarily affects the proximal small bowel. The purpose of this study was to assess whether there were features of gluten-sensitive enteropathy in biopsies taken from the terminal ileum during colonoscopy/ileoscopy. Specific and sensitive abnormalities might facilitate diagnosis of coeliac disease in patients undergoing colonoscopy as their initial procedure or help select those who should proceed to upper gastrointestinal endoscopy and duodenal biopsy. METHODS: Terminal ileal biopsies, taken from 30 patients with duodenal villous atrophy consistent with coeliac disease and from 60 control patients with no evidence of coeliac or inflammatory bowel disease, were reviewed blindly and compared. Biopsies were assessed for the presence or absence of villous atrophy and crypt hyperplasia, and counts were made of intraepithelial lymphocytes (IELs). RESULTS: One patient only, in the coeliac group, had partial villous atrophy with crypt hyperplasia in the terminal ileum. IEL counts were significantly higher (P< 0.005) in the coeliac group than among controls (mean per 100 enterocytes 26 versus 10). An ileal IEL count > or =25 had a sensitivity for duodenal villous atrophy (VA) of 60% and specificity of 100%. CONCLUSIONS: Coeliac disease may affect the entire small bowel. Increased IEL density in the terminal ileum is associated with duodenal VA and should prompt a search for coeliac disease by serology and duodenal biopsy. Conversely, a normal IEL count does not allow the exclusion of coeliac disease with confidence.     

Intestinal permeability in patients with coeliac disease and relatives of patients with coeliac disease.

The functional integrity of the small bowel is impaired in coeliac disease. Intestinal permeability, as measured by the sugar absorption test probably reflects this phenomenon. In the sugar absorption test a solution of lactulose and mannitol was given to the fasting patient and the lactulose/mannitol ratio measured in urine collected over a period of five hours. The sugar absorption test was performed in nine patients with coeliac disease with an abnormal jejunum on histological examination, 10 relatives of patients with coeliac disease with aspecific symptoms but no villous atrophy, six patients with aspecific gastrointestinal symptoms but no villous atrophy, and 22 healthy controls to determine whether functional integrity is different in these groups. The lactulose/mannitol ratio (mean (SEM) is significantly higher in both coeliac disease (0.243 (0.034), p < 0.0001)) and relatives of patients with coeliac disease (0.158 (0.040), p < 0.005)) v both healthy controls (0.043 (0.006)) and patients with aspecific gastrointestinal symptoms (0.040 (0.011)). The lactulose/mannitol ratio in relatives of coeliac disease patients was significantly lower than in the coeliac disease patient group (p = 0.04). The lactulose/mannitol ratio was the same in healthy controls and patients with aspecific gastrointestinal symptoms. It is concluded that the sugar absorption test is a sensitive test that distinguishes between patients with coeliac disease and healthy controls. The explanation for the increased permeability in relatives of patients with coeliac disease is uncertain. Increased intestinal permeability may be related to constitutional factors in people susceptible to coeliac disease and may detect latent coeliac disease. The sugar absorption test may therefore be helpful in family studies of coeliac disease.     

Genetic influences on splenic function in coeliac disease.

Splenic function was assessed using 'pitted' erythrocyte counts in 61 first degree relatives of patients with coeliac disease. 'Pitted' erythrocyte counts were normal in 12 parents, but were raised in 20% of 49 siblings and/or children of coeliac patients. First degree relatives had higher 'pitted' erythrocyte counts than normal controls (p = 0.002). The counts were lower in coeliac relatives than in age matched coeliacs (p = 0.0001), but no difference was present between the relatives and coeliac patients whose small bowel mucosa was morphologically normal. Considerable interfamily variation was found in 'pitted' erythrocyte counts, both in the coeliac patients and first degree relatives, and the pattern tended to 'run true' within families. The genetic factor influencing splenic function in coeliac disease is not HLA-linked but seems to be associated with a second, probably recessive, gene influencing the inheritance of coeliac disease.     

Histology of the terminal ileum in coeliac disease

Background: The histological lesion of gluten sensitivity primarily affects the proximal small bowel. The purpose of this study was to assess whether there were features of gluten‐sensitive enteropathy in biopsies taken from the terminal ileum during colonoscopy/ileoscopy. Specific and sensitive abnormalities might facilitate diagnosis of coeliac disease in patients undergoing colonoscopy as their initial procedure or help select those who should proceed to upper gastrointestinal endoscopy and duodenal biopsy. Methods: Terminal ileal biopsies, taken from 30 patients with duodenal villous atrophy consistent with coeliac disease and from 60 control patients with no evidence of coeliac or inflammatory bowel disease, were reviewed blindly and compared. Biopsies were assessed for the presence or absence of villous atrophy and crypt hyperplasia, and counts were made of intraepithelial lymphocytes (IELs). Results: One patient only, in the coeliac group, had partial villous atrophy with crypt hyperplasia in the terminal ileum. IEL counts were significantly higher (P?Conclusions: Coeliac disease may affect the entire small bowel. Increased IEL density in the terminal ileum is associated with duodenal VA and should prompt a search for coeliac disease by serology and duodenal biopsy. Conversely, a normal IEL count does not allow the exclusion of coeliac disease with confidence.