Celiac Disease Mucosa Research Articles

21 P Enhanced expression of interferon regulatory factor-1 in celiac disease mucosa

21 P Enhanced expression of interferon regulatory factor-1 in celiac disease mucosa

Endoscopic appearance of jejunal mucosa in celiac disease: role of submucosal fibrosis

Endoscopic appearance of jejunal mucosa in celiac disease: role of submucosal fibrosis

Immunohistochemical analysis of candidate gene product expression in the duodenal epithelium of children with coeliac sprue.

Coeliac sprue is a chronic disease, in which there is a characteristic mucosal lesion of the small intestine and impaired nutrient absorption, which improves upon the withdrawal of wheat gliadins and related grain proteins from the diet. Biopsy specimens demonstrate diffuse enteritis with pronounced atrophy or total loss of villi. There is also a long term risk of malignant disease. To compare the immunoexpression of DCC (deleted in colon cancer), p53, E-cadherin, and beta-catenin in the duodenal mucosa of children with coeliac disease with that seen in children with no evidence of small intestinal disease. To gain more insight into the genetic and immunohistochemical alterations of the duodenal epithelium in coeliac disease, 21 endoscopic biopsies from children with coeliac disease and 10 duodenal biopsies from children without coeliac disease were immunohistochemically evaluated for p53, DCC, E-cadherin, and beta-catenin. DCC expression was not reduced in patients with coeliac disease compared with those without coeliac disease. p53 positive nuclear immunostaining was seen in seven of the 21 patients with coeliac disease. Positive nuclear staining was seen mainly in the deep and the lateral aspects of the crypts. All patients in the control group were negative for p53. In nine and three of the 21 patients with coeliac disease, respectively, the immunohistochemical expression of E-cadherin and beta-catenin was reduced. However, both E-cadherin and beta-catenin immunostaining in the control group was not altered. E-cadherin and beta-catenin were reduced in the duodenal epithelium of children with coeliac disease when compared with normal mucosa. p53 was overexpressed in the duodenal mucosa of patients with coeliac disease. The reduced expression of E-cadherin and beta-catenin and p53 overexpression may contribute to the morphological changes seen in the small intestinal mucosa in coeliac disease.

FAS engagement drives apoptosis of enterocytes of coeliac patients

BACKGROUND Villus atrophy is the most distinctive sign of untreated coeliac disease (CD) and epithelial apoptosis is considered to be involved in this stage of the coeliac lesion. The extent...

Local challenge on oral mucosa with an alpha-gliadin related synthetic peptide in patients with celiac disease.

Gluten-derived peptides (e.g., amino-acids 31-49 of alpha-gliadin) have been shown to cause changes typical of celiac disease in the gut. Gluten-derived peptides have mostly been used in in vitro studies. The easiest access to the gastrointestinal system may be the mouth. In the present study we were interested to see whether a synthetic peptide corresponding to amino-acids 31-49 of alpha-gliadin could induce inflammatory changes in the oral mucosa after a local challenge in celiac disease patients. The challenge was made by injecting the peptide solution at a concentration of 10 microg/ml submucosally into the oral mucosa of 10 celiac disease patients after a gluten-free diet (GFD) and 12 healthy control subjects. B and CD45RO+ T cells, mast cells, CD3+, CD4+, CD8+ lymphocytes, and alphabeta and gammadelta T-cell receptor-bearing (TcR alphabeta, TcR gammadelta) lymphocytes were counted and HLA DR expression was determined. The expression of CD25 and Ki-67 antigen was also examined. The peptide significantly increased the total number of T cells in the lamina propria of the celiac disease patients. The expression of T-cell activation marker CD25 (IL-2 receptor), but not that of cell proliferation marker Ki-67, was also significantly increased in the lamina propria after peptide challenge. Such a reaction was not observed in the controls. The numbers of CD3+ and CD4+ T cells in the lamina propria were also increased in celiac disease patients after the challenge. The count of TcR gammadelta+ cells was very small in the oral mucosa in celiac disease and showed no increase when the oral mucosa was challenged with the peptide. The expression of HLA DR staining was enhanced after the submucosal peptide challenge in celiac disease; however, the difference was not statistically significant. The results show that in the celiac disease patients after the peptide challenge the oral mucosal lamina propria responds with a nonproliferative increase of lymphocytes. Thus, submucosal challenge with the peptide 31-49 can be used as an aid in the diagnosis of celiac disease. However, further studies with optimized methodology, including various concentrations of the peptide, adjuvants, other peptides, etc., are warranted, especially because the oral mucosa provides the easiest access to an in vivo peptide challenge in celiac disease.

Local challenge on oral mucosa with an α-gliadin related synthetic peptide in patients with celiac disease

OBJECTIVE: Gluten-derived peptides ( e.g., amino-acids 31–49 of α-gliadin) have been shown to cause changes typical of celiac disease in the gut. Gluten-derived peptides have mostly been used in in vitro studies. The easiest access to the gastrointestinal system may be the mouth. In the present study we were interested to see whether a synthetic peptide corresponding to amino-acids 31–49 of α-gliadin could induce inflammatory changes in the oral mucosa after a local challenge in celiac disease patients. METHODS: The challenge was made by injecting the peptide solution at a concentration of 10 μg/ml submucosally into the oral mucosa of 10 celiac disease patients after a gluten-free diet (GFD) and 12 healthy control subjects. B and CD45RO+ T cells, mast cells, CD3+, CD4+, CD8+ lymphocytes, and αβ and γδ T-cell receptor-bearing (TcRαβ, TcRγδ) lymphocytes were counted and HLA DR expression was determined. The expression of CD25 and Ki-67 antigen was also examined. RESULTS: The peptide significantly increased the total number of T cells in the lamina propria of the celiac disease patients. The expression of T-cell activation marker CD25 (IL-2 receptor), but not that of cell proliferation marker Ki-67, was also significantly increased in the lamina propria after peptide challenge. Such a reaction was not observed in the controls. The numbers of CD3+ and CD4+ T cells in the lamina propria were also increased in celiac disease patients after the challenge. The count of TcRγδ+ cells was very small in the oral mucosa in celiac disease and showed no increase when the oral mucosa was challenged with the peptide. The expression of HLA DR staining was enhanced after the submucosal peptide challenge in celiac disease; however, the difference was not statistically significant. CONCLUSIONS: The results show that in the celiac disease patients after the peptide challenge the oral mucosal lamina propria responds with a nonproliferative increase of lymphocytes. Thus, submucosal challenge with the peptide 31–49 can be used as an aid in the diagnosis of celiac disease. However, further studies with optimized methodology, including various concentrations of the peptide, adjuvants, other peptides, etc., are warranted, especially because the oral mucosa provides the easiest access to an in vivo peptide challenge in celiac disease.

Increased expression of mRNA for matrix metalloproteinases-1 and -3 and tissue inhibitor of metalloproteinases-1 in intestinal biopsy specimens from patients with coeliac disease

BackgroundExtracellular matrix (ECM) degradation may play a role in villus atrophy in coeliac disease (CD).AimsTo compare the cellular expression of mRNA transcripts for the two major matrix degrading proteases, matrix...

IL15 IS ABLE TO INDUCE MIGRATION OF CD3+CD8+ AND γ/δ+ T CELLS TO THE SURFACE EPITHELIUM IN COELIAC SMALL INTESTINAL MUCOSA

We have already demonstrated (1) that in vitro gliadin challenge of treated coeliac intestine, is effective in inducing intraepithelial migration of CD3+ cells. This migration is observed after 24 hours of organ culture and the CD3+ cells are apparently un-activated (CD25-). Therefore this T cell migration seems not to be caused by direct antigenic activation of the migrating CD3+ cells. In this study we have tested if IL15 may control this migration. This cytokine was chosen because of its reported effects on T cell migration in rheumatoid arthritis, where induces recruitment of T cells into the synovial membrane (2). Furthermore it is known that IL-15 is a growth factor for γ/δ T cells(3), a T cell sub-population which play a yet undefined role in coeliac disease (CD). Methods. 10 intestinal treated CD biopsies were cultured in vitro for 24 hours (1) in the presence of medium alone, or added with IL15 (10 ng/ml). Tissue specimens were therefore analysed by immunocytochemistry, by anti-CD3 (Dako, 1:200) and γ/δ (Dako 1:30) MoAbs by peroxidase staining technique (1). IL15+ cells in lamina propria were detected by anti-IL15 MoAb (M110, 1:100) in untreated CD mucosa (N=7), in 6 treated CD and in 6 controls Morphometric analysis was done as previously reported (1). Student's test as well as non parametric tests were used for analysis of the data. Results. The number of intraepithelial (IE) CD3+ cells was higher after IL15 treatment than after medium alone (mean 65.6/mm epithelim, ± 24, vs 27.2 ± 10.2 with medium alone, p<0.01). The number of CD8+ cells (44.5±15.9, vs 21.1±7.41, p<0.01) as well as of γ/δ+ cells (26.5±9.3, vs 8±2.7, p<0.01) was increased, whereas that of CD4+ cells was not(p = NS). The number of lamina propria mononuclear cells which express IL15 was higher in untreated CD intestine, than in treated CD or controls (17.4±10.8 per mm2 of lamina propria, vs 6.8±5.5 in controls, p<0.05 and vs 4.5±3 in treated CD, p<0.05). Conclusions. In treated CD intestine IL15 is effective in inducing migration of CD8+ and γ/δ+ cells to the surface epithelium. Moreover IL15 expression is higher in untreated CD than in treated and in controls. These results indicate that IL15 is the likely candidate cytokine modulating IE infiltration of T cells and therefore may play a role in the pathogenesis of CD.

3 The humoral immune system in coeliac disease

IgA is transported into intestinal secretions to perform exclusion of luminal antigens. The prerequisites are antigen sampling by the Peyer's patch M cells, antigen processing by antigen-presenting cells, and presentation of antigenic peptides by HLA class II molecules to immunocompetent T cells. The basis for intestinal immunity is the maturation cycle of specifically primed T and B cells from the gut-associated lymphoid tissue via mesenteric lymph nodes and peripheral blood back to the intestinal lamina propria. In coeliac disease, patients are sensitized against gluten and serum gliadin antibodies are often detected. Gliadin antibodies are also found in other gastrointestinal diseases, other disorders and in healthy individuals not carrying the coeliac disease-specific DQA/DQB alleles. On the other hand, serum reticulin and endomysium autoantibodies are both sensitive and highly disease-specific. Positivity in patients with normal jejunal morphology indicates latency of coeliac disease. These tissue autoantibodies are directed against fibroblast-derived extracellular matrix proteins. The immune system is involved in the amplification and perpetuation of the abnormalities of the intestinal mucosa in coeliac disease. The role of antibody in the pathogenesis remains unknown. The author hypothesizes gluten-triggered autoimmune mechanism to be operative.

Raised pro-inflammatory cytokines interleukin 6 and tumour necrosis factor alpha in coeliac disease mucosa detected by immunohistochemistry.

The levels of two pro-inflammatory cytokines, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha), in coeliac disease were studied by immunohistochemistry. Jejunal biopsy specimens from patients with untreated disease, (n = 11), treated disease (n = 9), and normal controls, (n = 11) were stained to detect IL-6, TNF-alpha, CD45 (pan-leukocyte), and CD68 (macrophage surface antigen). Positive cells were identified in the epithelium (per 100 enterocytes) and in the lamina propria (per unit area). There was a significant increase in median IL-6 and TNF-alpha staining in both the lamina propria and the epithelium of untreated coeliac disease patients (lamina propria, 16.2 and 13.0 respectively; epithelium, 0.86 and 1.21, all p < 0.05) when compared with treated coeliac disease patients (lamina propria; 6.0 and 6.2, epithelium; 0.60 and 0.60) and controls (lamina propria; 6.5 and 7.5, epithelium; 0.58 and 0.60). A significant increase in the number of CD45 positive cells was found in the untreated coeliac disease lamina propria and epithelium (p < 0.05) but this was accompanied by a significant rise in CD68 positive cells in the lamina propria only (p < 0.05). Increased IL-6 and TNF-alpha in the lamina propria and epithelium of patients with untreated coeliac disease further supports their role in the immune pathogenesis of this disorder.

Gliadin induced changes in the expression of MHC-class II antigens by human small intestinal epithelium. Organ culture studies with coeliac disease mucosa.

Jejunal biopsies from 16 treated coeliac disease patients and from nine controls were cultured with and without a peptic-tryptic digest of gliadin. Cultures with a peptic-tryptic digest of maize prolamins were also undertaken. Frozen sections of baseline and cultured mucosa were stained by immunofluorescence with an anti-HLA-DR monoclonal antibody. Before culture the villous epithelium from both controls and treated coeliac disease expressed DR molecules while the crypt epithelium did not. When biopsies from treated coeliac disease were cultured with gliadin the expression of DR was enhanced in the crypt epithelium in eight of 14 cultures and in 11 of 14 was reduced or absent on the villous epithelium. No change was observed in control cultures. We conclude that gliadin is capable of inducing HLA-DR on the crypt epithelium of in vitro cultured coeliac disease mucosa, providing indirect evidence that gliadin may activate cell mediated immune mechanisms within the small bowel mucosa. This model could prove useful in identifying the immunogenic sequence(s) of gliadins and related prolamins.

Glycoprotein synthesis and secretion by cultured small intestinal mucosa in coeliac disease.

Glycoprotein biosynthesis by jejunal mucosa was examined during culture in vitro in 26 patients with coeliac disease and 19 controls. The incorporation rates of tritiated glucosamine into tissue and secreted glycoproteins were determined using established techniques. The total glucosamine incorporation in untreated coeliac patients was significantly greater than that of histologically normal mucosa (p less than 0.001) and jejunal tissue from patients with treated coeliac disease (p less than 0.01). Enhanced secretion of in vitro labelled glycoproteins was observed in untreated coeliac patients. The total incorporation of tritiated glucosamine in intestinal tissues was correlated with goblet cell numbers. These results indicate that quantitative changes in glycoprotein synthesis and secretion occur in coeliac disease.

Increased tissue concentration of neuropeptide Y in the duodenal mucosa in coeliac disease.

Neuropeptide Y (NPY) is localized to intestinal nerve fibres, of which there are few in normal duodenal mucosa. In the duodenal mucosa of 10 patients with coeliac disease and in a control group of 21 patients with other gastrointestinal symptoms but with normal function of the small intestine we studied the frequency of such fibres by immunohistochemistry and the tissue concentration of NPY by radioimmunoassay. Patients with coeliac disease had an increased number of NPY nerve fibres and significantly elevated tissue concentrations compared with the control group. The eluted fractions obtained by high-pressure liquid chromatography of duodenal extracts showed the same immunoreactive components in the two groups. This study therefore suggested proliferation of the peptide-containing nerve system in coeliac disease. The increased NPY levels in the duodenal mucosa may be of functional significance for the disease symptoms.

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.

In vitro (Organ culture) studies of the toxicity of specific A-gliadin peptides in celiac disease

Specific peptides of known amino acid sequence were prepared from α-gliadin (A-gliadin) by cleavage of the protein with cyanogen bromide and chymotrypsin and purification of the resulting peptides. The three peptides derived from the cyanogen bromide cleavage spanned the complete sequence of A-gliadin (266 residues). Four peptides derived from chymotryptic digestion covered the N-terminal sequence through residue 68. These peptides were tested for toxicity in celiac disease by organ culture of biopsied small intestinal tissues taken from patients with active celiac disease. Enterocyte height was used as a measure of peptide effect on cultured tissues. Five of seven peptides tested significantly inhibited increase of enterocyte height in the cultures and were considered toxic on this basis. The largest common sequences among the toxic peptides were -pro-ser-gln-gln- and -gln-gln-gln-pro-; these sequences were absent from the non-toxic peptides. The relationship of these sequences to the damaging effect of gliadins on the small intestinal mucosa in celiac disease remains to be investigated.

Gastrointestinal peptide profile in children with celiac disease.

In order to investigate if a plasma profile of gastrointestinal peptides reflects changes in jejunal mucosa in celiac disease, we studied basal and postprandial plasma levels of gastrin, secretin, somatostatin, vasoactive intestinal polypeptide (VIP), and neurotensin in children with untreated and treated celiac disease and in a control group of children. Basal and 30-min postprandial secretin concentrations were statistically significantly lower in untreated celiac children compared to both treated celiac (p less than 0.01 and p less than 0.05, respectively) and control children (p less than 0.001). Plasma secretin levels 30 min after a breakfast meal were also statistically significantly lower (p less than 0.001) in treated celiac children with respect to the control group. In both untreated and treated celiac groups, basal and postprandial plasma levels of somatostatin and VIP were statistically significantly decreased (p less than 0.001) compared to control children. Moreover, there was a significant rise in postprandial levels of neurotensin after a breakfast meal in untreated celiac children. On the contrary, there was no rise of neurotensin in healthy children. These findings seem to indicate that determination of plasma profile of gastrointestinal peptides in children with celiac disease may be useful in monitoring the development of this disease and, thus, the number of jejunal biopsies could be decreased.

The effects of serotonin on crypt cell proliferation in the human duodenal mucosa in vitro

Cell kinetic studies on the small bowel mucosa in coeliac disease have shown an increased number of proliferating cells per crypt due to a reduction in the crypt cell cycle time. Similar findings occur in rats after the intraperitoneal injection of serotonin. In vitro studies have therefore been performed to determine whether serotonin influenced cell proliferation in the human duodenal mucosa. Normal mucosal biopsies from 18 patients were cultured for 22 hours, using an organ culture technique. Specimens from 9 patients were cultured in a basic medium and from 9 patients in the basic medium with added serotonin (2mg/ml). After 18 hours a stathmokinetic technique was used to determine the crypt cell proliferation rate (CCPR) by adding vincristine sulphate (1μ/ml) to the culture medium. Tissue samples were removed at hourly intervals for A hours and the mean number of metaphase arrests in 10 whole crypts was determined. The CCPR was derived by drawing a regression line between the mean values obtained on each patient. The results indicate that serotonin significantly increases the CCPR in cultured tissues (p 0.001). Described abnormalities of serotonin synthesis jn coeliac disease may be involved in the pathogenesis of this disorder.

Enhanced synthesis of cholesterol and its precursors in jejunal mucosa in coeliac disease.

Patients with coeliac disease have a greatly enhanced whole body synthesis of cholesterol, but its origin is not known. In this paper the synthesis and concentrations of cholesterol and its precursors, squalene and methyl sterols, were studied in jejunal biopsies from healthy volunteers and adult patients with coeliac disease. The incorporation rates of 14C-acetate and 3H-mevalonate into non-saponifiable lipids (sum of squalene, methyl sterols and cholesterol) were increased 14 and six times in the damaged mucosa, respectively. The cyclisation rate of mucosal squalene and the conversion of squalene to cholesterol were also increased, indicating that cholesterol synthesis was activated before mevalonate, probably at the step of HMG-CoA reductase, and also after mevalonate and squalene. The steps from squalene to cholesterol were apparently not rate limiting because the mucosal concentrations and the percentage distribution of squalene and sterols were similar in the patients and in the controls. A positive correlation of the cholesterol synthesis with the number of crypt cells, suggests that the expanded crypt cell population contributed to the enhanced intestinal cholesterologenesis. Furthermore, the serum cholesterol level was negatively correlated with the 14C- and 3H-counts in the mucosal cholesterol and with the cyclisation rate of squalene probable signs of regulatory role of serum low density lipoprotein cholesterol in intestinal cholesterol synthesis. Consequently, provided that the synthesis of mucosal cholesterol is as high in vivo as in vitro it could contribute to the highly increased overall synthesis of cholesterol in the patients with coeliac disease.

Duodenal bulb plasma cells in duodenitis and duodenal ulceration.

Using an immunoperoxidase technique IgA, IgM, IgE and IgG plasma cells were studied in endoscopic duodenal bulb biopsies taken from 14 controls, 25 patients with grade 1 duodenitis (Whitehead classification), 12 patients with grade 2 duodenitis and three with grade 3 duodenitis. The control counts were compared with those in the jejunum and rectum. In addition cell counts were compared in 16 pairs of patients, with and without duodenal ulcer, exactly matched for grade of duodenitis. The control counts were not significantly different from counts in jejunum or rectum except for IgG which were higher in the jejunum (p = 0.03). IgA plasma cell counts were significantly increased in both grade 1 and grade 2 duodenitis compared with controls (p less than 0.05 and p less than 0.01). There was no significant difference for the other plasma cells. All plasma cell counts were decreased in the small group of grade 3 duodenitis compared with the other groups. There was no significant difference between counts in duodenitis whether or not there was associated duodenal ulceration. The isolated IgA plasma cell response of the duodenal bulb mucosa in duodenitis is very different from that of the jejunal mucosa in coeliac disease, and the rectal mucosa in inflammatory bowel disease and bacterial colitis and probably represents the basic response to any mucosal damage.

Quantification of enterochromaffin cells with serotonin immunoreactivity in the duodenal mucosa in coeliac disease.

Enterochromaffin cells in the duodenal mucosa were stained by using a monoclonal antibody against serotonin immunoreactive sites and an indirect immunoperoxidase technique. A semi-automatic image analysing system showed increased numbers of these cells in patients with untreated coeliac disease compared with a control group. The number of serotonin positive granules in individual enterochromaffin cells also seemed to be increased in patients with coeliac disease, a finding which may be related to the pathogenesis of this disorder.