Diamine oxidase plasma activities after treatment with heparin and jejunal morphometry in untreated coeliac disease.

Diamine oxidase plasma concentrations after treatment with heparin were measured and compared with the surface to volume ratio of jejunal biopsy samples assessed by a morphometric technique in patients with untreated and treated coeliac disease and in biopsied controls. As expected, enzyme activity was significantly lower in patients with untreated coeliac disease than in patients on a gluten-free diet and in biopsied controls. No difference was found between treated patients and biopsied controls. There was a significant overall correlation between plasma enzyme activity and surface to volume ratio of jejunal mucosa, although two untreated patients without an overt malabsorption syndrome but with a very low surface to volume ratio had normal enzyme activity. This study shows that in coeliac disease plasma diamine oxidase activity after treatment with heparin does not always mirror the extent of the jejunal lesions, particularly in those patients with minimal or unrelated symptoms who would benefit most from a valid screening test to identify their condition.     

Timing of infiltration of T lymphocytes induced by gluten into the small intestine in coeliac disease.

Jejunal biopsy was performed on control subjects and patients with treated and untreated coeliac disease. Monoclonal antibodies to T lymphocyte surface markers were used to quantify T cell phenotypic subsets in the jejunal mucosa. The patients with untreated coeliac disease had significantly more of both suppressor/cytotoxic and helper/inducer T cells in the surface epithelium than either the control subjects or the treated patients. Serial jejunal biopsy specimens were taken from five treated coeliac patients for six hours after a gluten challenge. In four of these five infiltration of the surface epithelium by both T cell phenotypes, together with deterioration in the villus architecture, had occurred both within two hours of having started the challenge. This suggests that T lymphocytes may have a role in the pathogenesis of coeliac disease.     

Computer card morphometry of jejunal biopsies in childhood coeliac disease.

The histological changes in 95 jejunal biopsy specimens from children have been analyzed by a new mporphometric technique. The microscope image of the specimen is traced directly onto computer data cards. A simple sketch records accurate quantitative data in a matrix of 840 points, retaining the spatial arrangement of the tissue components. The data are fed via an optical mark data card reader, into a mini-computer. FORTRAN IV programs allow calculation of surface area, villous heights, and component volumes in metric units, and of volume proportions, volume-to-volume ratios, and surface-to-volume ratios. Pictorial and numerical printouts are produced, which are suitable for inclusion in the patient's notes. Jejunal biopsies from 37 controls and 26 untreated coeliac patients were clearly distinguished morphometrically. Sixteen pairs of biopsies from coeliac patients on long-term gluten-free diets before, and 12 weeks after, the reintroduction of dietary gluten significantly reflected the effects of gluten challenge. Comparison of control and abnormal biopsies showed a spatial redistribution of the components, more than a change in their absolute amounts. There was no significant differences in the total epithelial volumes in controls, treated or untreated patients, suggesting that the mucosal lesion in coeliac disease is not a true atrophy.     

Brush border enzymes in coeliac disease: histochemical evaluation.

Two hundred and ninety four duodenal and jejunal mucosal biopsy specimens from patients with coeliac disease, treated and untreated, and other conditions were examined histologically and by histochemical staining for five peptidase and three disaccharidase enzymes to determine profiles of activity. Suppression of activity paralleled the histology with the following enzymes: lactase, trehalase, brush border endopeptidase, dipeptidyl peptidase II and isomaltase. Lactase, trehalase, and brush border endopeptidase were specifically suppressed in untreated coeliac disease and were diagnostically useful. Examination of a combination of enzymes is recommended.     

HLA-DR, DP and DQ expression in the small intestine of patients with coeliac disease

Frozen sections of jejunal mucosa from control subjects and patients with both treated and untreated coeliac disease were examined for HLA class II DR, DP and DQ expression. Different staining patterns with monoclonal antibodies to the different class II subgroups were observed with the control subjects. There was some inter-subject variation but in general DR greater than DP greater than DQ staining was observed with the villous enterocytes staining most strongly with the staining decreasing towards the crypt bases. The patients with treated coeliac disease gave a similar pattern to the controls. The patients with untreated coeliac disease generally gave a more intense and relatively uniform staining of both surface and crypt enterocytes for all class II subgroups.     

Salivary antigliadin and antiendomysium antibodies in coeliac disease

The definitive diagnosis of coeliac disease is based on typical changes in the small intestine biopsy specimens. To screen individuals for coeliac disease serum IgA and IgG antigliadin (AGA), IgA antireticulin (ARA) and IgA antiendomysium (EmA) antibodies are used. The aim of this study was to investigate whether these antibodies can also be detected in saliva as diagnostic markers of coeliac disease. The study population comprised 30 patients with coeliac disease treated with a gluten-free diet, 14 patients with untreated coeliac disease and 13 healthy control subjects. Sera and saliva were tested simultaneously for the presence of IgA and IgG AGA and IgA EmA. None of patients studied had a selective IgA deficiency. There was no significant difference in salivary IgA AGA levels between the three groups tested and there was no correlation between the individual serum and salivary values of IgA AGA. Salivary IgG AGA levels were very low or undetectable. Serum IgA AGA showed a low sensitivity (36.4%) to detect an untreated patient with coeliac disease. All salivary samples, regardless of the study group were negative for IgA EmA. Serum IgA EmAs were universally detected in the sera of patients with newly diagnosed coeliac disease and also in the sera of five of 30 patients with treated coeliac disease. No IgA EmA was detected in the sera of controls. None of the patients studied had a selective IgA deficiency either. Serum IgA EmA is the most sensitive, and IgA and IgG AGA are good indicators for coeliac disease, but salivary IgA or IgG AGA and salivary IgA EmA are not helpful for the diagnosis or follow-up of coeliac disease patients.     

Gluten affects epithelial differentiation-associated genes in small intestinal mucosa of coeliac patients

In coeliac disease gluten induces an immunological reaction in genetically susceptible patients, and influences on epithelial cell proliferation and differentiation in the small-bowel mucosa. Our aim was to find novel genes which operate similarly in epithelial proliferation and differentiation in an epithelial cell differentiation model and in coeliac disease patient small-bowel mucosal biopsy samples. The combination of cDNA microarray data originating from a three-dimensional T84 epithelial cell differentiation model and small-bowel mucosal biopsy samples from untreated and treated coeliac disease patients and healthy controls resulted in 30 genes whose mRNA expression was similarly affected. Nine of 30 were located directly or indirectly in the receptor tyrosine kinase pathway starting from the epithelial growth factor receptor. Removal of gluten from the diet resulted in a reversion in the expression of 29 of the 30 genes in the small-bowel mucosal biopsy samples. Further characterization by blotting and labelling revealed increased epidermal growth factor receptor and beta-catenin protein expression in the small-bowel mucosal epithelium in untreated coeliac disease patients compared to healthy controls and treated coeliac patients. We found 30 genes whose mRNA expression was affected similarly in the epithelial cell differentiation model and in the coeliac disease patient small-bowel mucosal biopsy samples. In particular, those genes involved in the epithelial growth factor-mediated signalling pathways may be involved in epithelial cell differentiation and coeliac disease pathogenesis. The epithelial cell differentiation model is a useful tool for studying gene expression changes in the crypt-villus axis.     

Secretion of gliadin antibody by coeliac jejunal mucosal biopsies cultured in vitro.

The role of gliadin antibodies in coeliac disease is unknown. We have investigated this by maintaining jejunal biopsies from control subjects, treated and untreated coeliac patients in organ culture for 24 h. The tissue culture supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for total IgG, IgM and IgA, anti-gliadin and casein antibodies. Jejunal biopsies from untreated coeliac patients secreted more total IgM and IgA but not IgG, than biopsies from control subjects: values for treated coeliac patients were similar to those from control subjects. There was increased secretion of IgG and IgM gliadin antibody, but not casein antibody, by jejunal biopsies from untreated coeliac patients compared with those from control subjects.     

Antigliadin antibody measurement by chemiluminescence ELISA in the diagnosis of coeliac disease.

AIMS--To develop a chemiluminescence enzyme linked immunosorbent assay (ELISA) for the detection of circulating gliadin antibodies in the diagnosis of coeliac disease. To compare this method for linearity and sensitivity with an established colorimetric method. METHODS--Three sets of age and sex matched patient groups were studied: normal controls (patients with no clinical signs of intestinal disorders); gastrointestinal controls (patients with a known gastrointestinal disorder other than coeliac disease); and patients in whom suspected coeliac disease had been confirmed by positive jejunal biopsy. IgG antigliadin antibody (IgG-AGA) and IgA antigliadin antibody (IgA-AGA) titres were determined. RESULTS--Comparison of the colorimetric and chemiluminescence methods showed close correlation of measured antibody levels for both control patient groups. In the coeliac patients correlation of antibody levels measured by both methods was not possible because the colorimetric assay is limited by the spectrophotometer's limits of detection. This problem was overcome by the chemiluminescence method which was linear over a greater range and to far higher values. CONCLUSIONS--The chemiluminescence ELISA performs as well as the colorimetric assay at low and average antibody levels and has the advantage of also giving a numerical value to higher antibody titres. The method was accurate and reproducible in confirming the diagnosis of coeliac disease in patients with positive jejunal biopsy and was capable of monitoring progress of the disease.     

Differential upregulation of intercellular adhesion molecule-1 in coeliac disease.

The expression of intercellular adhesion molecule-1 (ICAM-1) was studied on peroral jejunal biopsies from patients with coeliac disease. The biopsies from untreated patients exhibited greater staining of the superficial lamina propria cells compared with treated patients and controls. A gluten challenge in treated patients produced an altered staining pattern within 2 h. The results demonstrate the role of ICAM-1 expression in coeliac disease, providing further evidence for the role of lamina propria cells in the pathogenesis of this condition.     

Expression of Major Histocompatibility Complex Class II Subregion Products by Jejunal Epithelium in Patients with Coeliac Disease

The MHC class II subregion products (HLA-DR), HLA-DP, and HLA-DQ) were located by immunofluorescence in serial sections of ethanol-fixed, paraffin-embedded jejunal mucosa from control subjects and patients with coeliac disease (CD). DR staining was seen in a granular luminal distribution and basolaterally on surface epithelial cells in both untreated and treated CD patients and in controls. In untreated CD the crypt epithelium was positive for DR almost to the bottom of the glands. This contrasted with virtually absent glandular DR staining in controls and weak staining including only the upper part of the crypts in 5 out of 11 treated patients. HLA-DP was present apically in the surface epithelium in all untreated patients, in 5 out of 11 treated patients, and in 4 out of 11 controls. HLA-DQ appeared only in three untreated patients and was restricted to patches of surface epithelium. The number of intraepithelial T lymphocytes per millimetre of surface epithelium was significantly higher in untreated than in treated CD patients or controls; it was also significantly higher in specimens with epithelial DP expression than in those without. This suggested that intraepithelial lymphocytes modulate epithelial class II expression.     

Abnormal pancreolauryl tests in coeliac disease: lack of correlation with the degree of intestinal mucosal damage.

AIMS: To determine the frequency of abnormal pancreolauryl tests in untreated and treated adults with coeliac disease and to see whether abnormalities in treated coeliac patients correlate with the degree of recovery of intestinal morphology or brush border enzyme activity. METHODS: Pancreolauryl tests were performed in a study population of 57 adult coeliac patients (25 on gluten containing diets and 32 on gluten free diets), 59 symptomatic controls, and eight patients with pancreatic disease. Brush border enzyme activity and morphological assessment were performed on small intestinal biopsies in 27 of the treated coeliac patients. RESULTS: Forty per cent of untreated coeliac patients and 18% of treated coeliac patients had abnormal tests. In treated coeliac patients, no significant correlation was detected between the pancreolauryl test result and either brush border enzyme activity or morphological parameters. CONCLUSION: Abnormal pancreolauryl test results are common in untreated and treated adult coeliac disease patients. Abnormalities in treated coeliac patients do not correlate with the degree of recovery of small intestinal morphology or brush border enzymes.     

Increase in T‐Cell Subsets of Oral Mucosa: a Late Immune Response in Patients with Treated Coeliac Disease?*

BACKGROUND AND AIMS: In coeliac disease, the gut involvement is gluten-dependent. Following the introduction of a gluten-free diet, inflammatory cell infiltration decreases in the small intestinal mucosa. Our hypothesis was that the oral mucosa might mirror the changes found in coeliac disease similarly to the mucosa of the small intestine. Thus, the number of inflammatory cells in the oral mucosa would decrease in patients with coeliac disease on a gluten-free diet. METHODS: The distribution CD45RO+ and CD3(+) T cells, T-cell subpopulations (CD4(+), CD8(+), T-cell receptor (TCR)alpha beta+ and TCR gamma delta+ cells) and HLA DR expression were studied in the buccal mucosa of 15 untreated and 44 gluten-free diet treated coeliac disease patients, and of 19 controls. All 15 patients with untreated coeliac disease were immunglobulin (Ig)A endomysial antibody positive and all 44 patients on gluten-free diet except one were endomysial antibody negative, as were all control subjects. RESULTS: Untreated coeliac disease patients did not differ from controls in the densities of CD45RO+ cells, CD3(+) cells or of T-cell subsets. In contrast, in treated coeliac disease patients, a significant increase in the numbers of mast cells, CD3(+) and CD4(+) lymphocytes was found in the lamina propria of oral mucosa as compared with patients with untreated coeliac disease and controls. The increase in CD3(+) T cells was in part owing to an increase in lymphocytes expressing no TCR. No differences were found in the expression of human leucocyte antigen (HLA) DR in the epithelium or in the lamina propria in the patient groups studied or in the controls. In treated coeliac disease patients only a few TCR gamma delta+ T cells were found intraepithelially and in the lamina propria, but these cells were not detected in the lamina propria of oral mucosa of patients with untreated coeliac disease or in the controls. CONCLUSIONS: The infiltration of T cells into oral mucosa was increased in treated coeliac disease patients in spite of adherence to a gluten-free diet. Because the CD3(+) T cell count was higher than those of the TCR alpha beta+ and TCR gamma delta+ T cells, there must be other cells involved, probably natural killer (NK) cells. The increase in T-cell subsets in the treated coeliac disease patients seems not to result from poor dietary compliance, but might occur as a late immune response in coeliac disease and reflect chronic immunologic stimulation followed by regeneration of memory T cells.     

A solid-phase radioimmunoassay for measurement of circulating antibody titres to wheat gliadin and its subfractions in patients with adult coeliac disease

A solid-phase radioimmunoassay for the measurement of circulating antibody titres to wheat gliadin is described. Using this assay, we have measured antibody titres to unfractionated gliadin in normal healthy controls, in coeliac patients on a gluten-free or a normal diet, and in patients with ulcerative colitis and Crohn's disease. High titres of antibodies to unfractionated gliadin were observed only in the patients with untreated coeliac disease. Antibody titres to alpha, beta, gamma and omega gliadin subfractions were measured in patients with untreated coeliac disease and compared with titres in normal controls. Patients with untreated coeliac disease had higher antibody titres to the gliadin subfractions. No specific pattern of circulating antibody titres to gliadin subfractions was observed in the untreated coeliac patients which would provide a diagnostic profile. These results suggest shared antigenicity between the gliadin subfractions.     

Jejunal plasma cells and in vitro immunoglobulin production in adult coeliac disease.

IgA, IgE, IgG and IgM plasma cells in small bowel mucosal biopsies from 15 controls, 16 untreated and 14 treated coeliac patients and five patients with selective serum IgA deficiency (four of whom also had coeliac disease) were quantified using an indirect immunoperoxidase technique. The IgA, IgG and IgM plasma cell counts were significantly increased in the untreated coeliac patients. The cell counts were intermediate in the treated coeliac group. These changes were in parallel to production in vitro of IgA and sIgA, IgG, and IgM by cultured mucosal biopsies from the same patients. The IgA deficient patients had very few mucosal IgA cells but elevated IgG and IgM plasma cell numbers; again these changes were reflected in the production in vitro of immunoglobulins. IgE plasma cell counts were very low in all patients and there were no differences between patient groups. The changes in cell counts and mucosal immunoglobulin production were not reflected in serum IgA, IgM and IgG concentrations but serum secretory IgA was significantly elevated in the untreated coeliac patients compared with controls, with the treated coeliac patients being intermediate. The raised mucosal plasma cell counts reflect the local mucosal production of immunoglobulin but not the immunoglobulin concentrations of serum, emphasising the importance of studying the immune function of the gut itself in coeliac disease rather than immunological abnormalities in serum.     

Intraepithelial lymphocytes in the villous tip: do they indicate potential coeliac disease?

BACKGROUND: The counting of intraepithelial lymphocytes (IELs) in the villous tips of architecturally normal small bowel biopsy specimens was proposed as a method to measure mucosal infiltration in gluten sensitive patients. AIMS: To apply this straightforward method in duodenal biopsy specimens from patients affected by potential coeliac disease (PCD) to verify whether it can discriminate these patients from controls. METHODS: Paraffin wax embedded duodenal sections from 11 patients affected by PCD were stained with an antihuman CD3 antibody. Sections from 19 patients affected by treated coeliac disease (TCD) and 17 patients in whom coeliac disease was excluded were stained with the same antibody to serve as controls. The slides were examined blindly. IELs/20 enterocytes in five randomly chosen villous tips were counted. Patients affected by PCD were all on a gluten containing diet. They had an architecturally normal duodenal mucosa and were positive for endomysial antibody. Both TCD and non-coeliac controls were negative for endomysial antibody. RESULTS: The mean villous tip IEL scores were 4.6 (SD, 1.5; range, 1.4-7.8) in non-coeliac controls, 7.9 (SD, 4.0; range, 2.0-18.6) in TCD, and 9.2 (SD, 4.7; range, 5.8-21.8) in patients with PCD. The difference between PCD and non-coeliac controls was significant. CONCLUSIONS: This is a very simple and sufficiently reliable method to count IELs. In patients with an architecturally normal duodenal mucosa, the IEL count in villous tips helps to distinguish between patients with PCD and non-coeliac controls.     

T cell proliferation, MHC class II restriction and cytokine products of gliadin-stimulated peripheral blood mononuclear cells (PBMC).

The immune response of PBMC to gliadin was investigated in patients with coeliac disease (CoD) by examining proliferation, MHC restriction and cytokine production. Gliadin induced low levels of proliferation in 63% of eight untreated patients, 32% of 28 treated patients and 35% of 31 healthy control subjects. In MHC restriction studies, the proliferative response to gliadin was inhibited (range 47-98% inhibition) in the presence of a MoAb to HLA-DR in each of three coeliac and three control donors studied. Using flow cytometry, increased expression of activation markers (HLA-DR and IL-2R) was demonstrated on gliadin-stimulated T cells from four of nine coeliac patients and three of seven healthy control donors. Cytokines were studied in culture supernatants using ELISA. Gliadin was a potent inducer of IL-6 and IL-10 in 100% of coeliac patients and controls, whereas IL-4 was not produced in either subject group. Gliadin induced IL-2 production in 40% of untreated patients, 42% of treated patients and 35% of healthy control donors. Interferon-gamma (IFN-gamma) in gliadin-stimulated cultures was found only in coeliac patients, observed in 33% of untreated patients and 25% of treated patients. Spontaneous secretion of both IL-2 and IFN-gamma was found more frequently in patients with untreated disease (87% of cases versus 21% of controls for IFN-gamma and 40% versus 0% for IL-2). These results suggest, as manifest by IFN-gamma production, that gliadin stimulates a Th1/Th0-like response in coeliac patients and a Th0-like response in healthy controls.     

Pitfalls in diagnosing coeliac disease.

AIMS--To highlight the pitfalls in the diagnosis of coeliac disease and to make recommendations for its diagnosis and the management of refractory cases with equivocal histology. METHODS--Six patients, referred since 1989 with a diagnosis of coeliac disease based on duodenal biopsy specimens taken at endoscopy, and who failed to respond to a gluten-free diet were studied. All patients were subjected to peroral jejunal biopsy. Morphometric analysis of villus height:crypt depth ratios, surface enterocyte cell heights, and intraepithelial counts was used to aid in the assessment of equivocal histology. RESULTS--Subsequent small intestinal biopsy specimens both taken when the patients were following a gluten-free diet and after gluten challenge were normal in all cases. Morphometric analysis and intraepithelial counts were normal. CONCLUSIONS--Misinterpretation of the original slides was often due to poor sample quality and tangential sectioning. Failure to respond to a gluten-free diet should always raise doubt regarding the initial diagnosis, especially when the findings are normal. For correct diagnosis at least three distal duodenal biopsy specimens should be taken simultaneously, and these should be of an adequate size and correctly orientated. Review by a histopathologist experienced in gastrointestinal diagnosis is essential in difficult cases. Quantitative morphometric analysis is helpful in equivocal cases, and jejunal suction biopsy, following a gluten challenge, may be necessary in patients refractory to treatment.     

Flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease

There is increasing evidence that proinflammatory cytokines contribute to many of the small intestinal features in coeliac disease. The aim of the study was to investigate the expression of two proinflammatory cytokines, migration inhibition factor (MIF) and tumour necrosis factor alpha (TNF-alpha) in duodenal biopsy specimens from patients with coeliac disease on a gluten-free diet and normal control subjects. A flow cytometric system was used to analyse intracellular protein levels of MIF and TNF-alpha in freshly isolated cells from duodenal biopsies taken from 12 patients with treated coeliac disease and 10 healthy control subjects. From the biopsy specimens, single cell suspensions of the epithelium and lamina propria were prepared using EDTA/DTT and enzymes. Intracellular cytokine expression was studied in intraepithelial lymphocytes (IELs), lamina propria T cells (LP T) and intestinal epithelial cells using different surface labelling antibodies. MIF protein was constitutively expressed in IELs, LP T cells and epithelial cells from normal intestinal mucosa. In contrast, although TNF-alpha was found in LP T cells, this cytokine was virtually undetectable in either IELs or epithelial cells. In coeliac disease, intracellular levels of MIF were significantly higher in epithelial cells compared with control subjects (P = 0.005). Raised levels of TNF-alpha were found in epithelial cells (P = 0.03) as well as IELs (P = 0.045) from coeliac patients compared with controls. The findings from this study show up-regulated expression of MIF and TNF-alpha in IELs and epithelial cells of histologically normal mucosa in patients with coeliac disease. Increased expression of proinflammatory cytokines in cells occupying the epithelial layer could help explain the rapidity with which the coeliac mucosa may respond to gluten challenge.