Intestinal Mucosa In Celiac Disease Research Articles

ПАТОГЕНЕТИЧЕСКИЕ АСПЕКТЫ ФОРМИРОВАНИЯ АНЕМИИ У ДЕТЕЙ С ЦЕЛИАКИЕЙ

Celiac disease (CD) is characterized by the formation of an anemic syndrome whose etiopathogenesis if of a multifactorial origin. The main types of anemias in children with celiac disease are iron deficiency anemia (IDA) and anemia of inflammation, also called anemia of chronic disease (ACD). This bibliographical review presents current information on the iron metabolism, morphological changes in the epithelium of the intestinal mucosa in celiac disease leading to the development of iron deficiency, pathogenesis of ACD formation in celiac disease, as well as the diagnosis of IDA and ACD in celiac disease.

Noninvasive Biomarkers of Gut Barrier Function in Patients Suffering from Diarrhea Predominant-IBS: An Update.

The intestinal barrier plays a crucial role in the absorption of nutrients and in preventing the entry of pathogenic microorganisms and toxic molecules. Several studies have shown a compromised intestinal barrier associated with low-grade inflammation in the small intestinal mucosa in celiac disease, inflammatory bowel disease, and irritable bowel syndrome (IBS), particularly in IBS with diarrhea (IBS-D). In light of these new data, IBS is no longer considered a functional disease but rather a heterogeneous syndrome that has yet to be carefully studied. Therefore, investigating the integrity and function of the intestinal barrier is now essential to improving knowledge of the pathophysiology of IBS-D and to improving the management of IBS-D patients. However, the study of the intestinal barrier must clarify some still unsolved methodological aspects and propose standardised assays before becoming a useful diagnostic tool. In this framework, this review will discuss data about the tests that noninvasively evaluate the integrity and functionality of the human intestinal barrier, paying particular attention to patients with IBS-D, in both clinical and research situations.

Structural conformation and self-assembly process of p31-43 gliadin peptide in aqueous solution. Implications for celiac disease.

Celiac disease (CeD) is a highly prevalent chronic immune-mediated enteropathy developed in genetically predisposed individuals after ingestion of a group of wheat proteins (called gliadins and glutenins). The 13mer α-gliadin peptide, p31-43, induces proinflammatory responses, observed by invitro assays and animal models, that may contribute to innate immune mechanisms of CeD pathogenesis. Since a cellular receptor for p31-43 has not been identified, this raises the question of whether this peptide could mediate different biological effects. In this work, we aimed to characterize the p31-43 secondary structure by different biophysical and insilico techniques. By dynamic light scattering and using an oligomer/fibril-sensitive fluorescent probe, we showed the presence of oligomers of this peptide in solution. Furthermore, atomic force microscopy analysis showed p31-43 oligomers with different height distribution. Also, peptide concentration had a very strong influence on peptide self-organization process. Oligomers gradually increased their size at lower concentration. Whereas, at higher ones, oligomers increased their complexity, forming branched structures. By CD, we observed that p31-43 self-organized in a polyproline II conformation in equilibrium with β-sheets-like structures, whose pH remained stable in the range of 3-8. In addition, these findings were supported by molecular dynamics simulation. The formation of p31-43 nanostructures with increased β-sheet structure may help to explain the molecular etiopathogenesis in the induction of proinflammatory effects and subsequent damage at the intestinal mucosa in CeD.

Cross-Talk Between Gluten, Intestinal Microbiota and Intestinal Mucosa in Celiac Disease: Recent Advances and Basis of Autoimmunity.

Celiac disease (CD) is an autoimmune disorder of the small intestine, caused by gluten induced inflammation in some individuals susceptible to genetic and environmental influences. To date, pathophysiology of CD in relation to intestinal microbiota is not known well. This review relies on contribution of intestinal microbiome and oral microbiome in pathogenesis of CD based on their interactions with gluten, thereby highlighting the role of upper gastrointestinal microbiota. It has been hypothesized that CD might be triggered by additive effects of immunotoxic gluten peptides and intestinal dysbiosis (microbial imbalance) in the people with or without genetic susceptibilities, where antibiotics may be deriving dysbiotic agents. In contrast to the intestinal dysbiosis, genetic factors even seem secondary in disease outcome thus suggesting the importance of interaction between microbes and dietary factors in immune regulation at intestinal mucosa. Moreover, association of imbalanced counts of some commensal microbes in intestine of CD patients suggests the scope for probiotic therapies. Lactobacilli and specific intestinal and oral bacteria are potent source of gluten degrading enzymes (glutenases) that may contribute to commercialization of a novel glutenase therapy. In this review, we shall discuss advantages and disadvantages of food based therapies along with probiotic therapies where probiotic therapies are expected to emerge as the safest biotherapies among other in-process therapies. In addition, this review emphasizes on differential targets of probiotics that make them suitable to manage CD as along with glutenase activity, they also exhibit immunomodulatory and intestinal microbiome modulatory properties.

Pathogenesis of Enteropathy-Associated T Cell Lymphoma.

To provide an update on the pathogenesis of enteropathy-associated T cell lymphoma (EATL) and its relationship with refractory celiac disease (RCD), in light of current knowledge of immune, genetic, and environmental factors that promote neoplastic transformation of intraepithelial lymphocytes (IELs). EATL frequently evolves from RCD type II (RCD II) but can occur "de novo" in individuals with celiac disease. Recurrent activating mutations in members of the JAK/STAT pathway have been recently described in EATL and RCD II, which suggests deregulation of cytokine signaling to be an early event in lymphomagenesis. Intraepithelial T cells are presumed to be the cell of origin of EATL (and RCD II). Recent in vitro molecular and phenotypic analyses and in vivo murine studies, however, suggest an origin of RCD II from innate IELs (NK/T cell precursors), which could also be the cell of origin of RCD II-derived EATL. The immune microenvironment of the small intestinal mucosa in celiac disease fosters the development of EATL, often in a multistep pathway.

Hormonal-metabolic patterns disorders of bone tissue mineralization in children with celiac disease

The review presents information about the pathophysiological mechanisms underlying disorders of the processes of mineralization of bone tissue in patients with celiac disease. The attention is focused on the role of vitamin D and its active metabolite calcitriol, and also parathyroid hormone in regulating calcium-phosphorus metabolism, which is carried out in target organs – small intestine, kidney, bone tissue. Atrophy of the intestinal mucosa in celiac disease is accompanied not only by malabsorption of calcium, and increased resorption of bone due to increased concentration of PTG. According to various studies on the background of developing the osteoporosis in celiac disease, the risk of bone fractures is significantly increased in patients. Strict adherence to a gluten-free diet allows to normalize both the absorption of calcium, and impaired hormonal mechanisms in bone remodeling in the acute phase, and can reduce the risks of osteoporosis and pathological fractures in patients with celiac disease.

Alteration of Tight Junction Gene Expression in Celiac Disease

The aim of the present study was to characterize the deregulation of epithelial tight junction genes and investigate its reversibility on removal of dietary gluten in small intestinal mucosa in celiac disease (CD). The expression levels of 23 genes related to tight junctions were studied in biopsies from 16 patients with active CD and compared with biopsies from the same patients taken after 2 years on gluten-free diet (GFD) and with 16 non-CD controls. Nine genes showed altered expression levels in patients with active disease (CLDN2, PARD6A, ZAK, SYMPK, MYH14, and ACTB were upregulated, whereas MAGI1, TJP1, and PPP2R3A were downregulated). Alterations were reversible after 2 years on treatment, except for PPP2R3A, implicated in the negative control of cell growth and division. At the biological network level, important dysfunctions in several processes within the pathway were observed, including intestinal permeability, apicobasal polarity, and cell proliferation. Our work confirms the involvement of tight junction genes related to permeability, polarity, and cell proliferation in the epithelial destruction observed in CD. Coexpression patterns of several genes support the idea of a common regulatory mechanism that seems to be altered in active CD. In general, GFD normalization confirms the reversibility of the process, except for the constitutive downregulation of PPP2R3A suggestive of a genetic implication. Further studies in proteins and cells or tissues are necessary to confirm these findings.

Expression of Microbiota, Toll‐like Receptors, and Their Regulators in the Small Intestinal Mucosa in Celiac Disease

Less than one-tenth of the carriers of the risk genes HLA-DQ2 or HLA-DQ8 develop celiac disease, suggesting that other genetic and environmental factors are important in the pathogenesis. The role of gut microbiota has been addressed previously with inconsistent findings. Our aim was to evaluate microbiota, its receptors (Toll-like receptors [TLRs]), and regulators of the TLRs in the small intestinal mucosa in celiac disease. Microbiota was analyzed by quantitative polymerase chain reaction (total bacteria and 10 bacterial group- and species-specific primers) and gene expression of interleukin-8 (IL-8), TLR2, TLR3, TLR4, TLR5, TLR9, and regulators of TLRs, Toll-interacting protein (TOLLIP), and single immunoglobulin IL-1R-related molecule, by relative quantitative reverse transcription-polymerase chain reaction in 10 children with celiac disease (untreated celiacs), 9 children with normal small intestinal mucosa (controls), and 6 adults with celiac disease with normal small intestinal mucosa after following a gluten-free diet (treated celiacs). Small intestinal microbiota was comparable among controls, untreated celiacs, and treated celiacs. Expression of IL-8 mRNA, a marker of intestinal inflammation, was significantly increased in untreated celiacs as compared with treated celiacs (P=0.002) and controls (P=0.001). Expression of TLR-2 mRNA was significantly decreased in untreated (P=0.001) and treated (P=0.03) celiacs, whereas expression of TLR-9 mRNA was increased in untreated celiacs (P=0.001) as compared with controls. Expression of TOLLIP mRNA was downregulated in untreated celiacs as compared with controls (P=0.02). Altered gene expression of TLR2, TLR9, and TOLLIP in small intestinal biopsies in celiac disease suggests that microbiota-associated factors may be important in the development of the disease.

Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking.

Background and ObjectivesDamage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation.Methods/Principal FindingsCell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells.Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor.ConclusionIn this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

Infiltration of forkhead box P3-expressing cells in small intestinal mucosa in coeliac disease but not in type 1 diabetes

Because the role of regulatory T cells in the intestinal inflammation is unknown in coeliac disease (CD) and type 1 diabetes (T1D), the expression of forkhead box P3 (FoxP3), CD25, transforming growth factor-beta, interferon (IFN)-gamma, interleukin (IL)-4, IL-8, IL-10, IL-15 and IL-18 was measured by quantitative reverse transcription-polymerase chain reaction in the small intestinal biopsies from paediatric patients with active or potential CD, T1D and control patients. The numbers of FoxP3- and CD25-expressing cells were studied with immunohistochemistry. Enhanced intestinal expressions of FoxP3, IL-10 and IFN-gamma mRNAs were found in active CD when compared with controls (P-values < 0.001, 0.004, <0.001). In potential CD, only the expression of IFN-gamma mRNA was increased. The numbers of FoxP3-expressing cells were higher in active and potential CD (P < 0.001, P = 0.05), and the ratio of FoxP3 mRNA to the number of FoxP3-positive cells was decreased in potential CD when compared with controls (P = 0.007). The ratio of IFN-gamma to FoxP3-specific mRNA was increased in active and potential CD (P = 0.001 and P = 0.002). Patients with T1D had no changes in regulatory T cell markers, but showed increased expression of IL-18 mRNA. The impaired up-regulation of FoxP3 transcripts despite the infiltration of FoxP3-positive cells in potential CD may contribute to the persistence of inflammation. The increased ratio of IFN-gamma to FoxP3 mRNA in active and potential CD suggests an imbalance between regulatory and effector mechanisms. The increased intestinal expression of IL-18 mRNA in patients with T1D adds evidence in favour of the hypothesis that T1D is associated with derangements in the gut immune system.

Relationship between IL-18 and sICAM-1 serum levels in patients affected by coeliac disease: preliminary considerations

Objectives: Cytokine production from activated T cells play a pathogenetic role in mucosal lesions of coeliac disease (CD). Active interleukin (IL)-18 is expressed in the small intestinal mucosa in CD but not in healthy controls. IL-18 enhances intercellular adhesion molecule-1 (ICAM-1) expression. We analyzed IL-18 serum levels in CD patients before and after gluten-free diet and the possible correlation with soluble ICAM-1 (sICAM-1) serum levels. Methods: The study comprises ten CD patients before and after gluten free diet and ten healthy controls. Serum IL-18 and sICAM-1 levels were assayed by immunoenzymatic methods. Results: Serum IL-18 and sICAM-1 levels were significantly higher in patients with CD before diet with respect to healthy controls ( P<0.05), with a highly significant correlation between the two parameters (Rho=0.800, P=0.0167). Gluten free diet significantly reduces IL-18 and sICAM-1. Conclusion: Our findings show that IL-18 serum concentrations correlated with the clinical status of CD patients suggesting a role for this cytokine in the pathophysiology of this disease.

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.

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.

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.

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.

Small intestinal mucosa in coeliac disease and cow's milk protein intolerance: morphometric and immunofluorescent studies.

One hundred and twenty-five small intestinal biopsies from children with coeliac disease (CD), cow's milk protein intolerance (CMPI) and controls were compared by morphometric analysis, by counting intraepithelial lymphocytes (IEL) and by quantitative evaluation of immunoglobulin-containing cells of lamina propria. The double-stain immunofluorescent technique of Brandtzaeg and Baklien [2] was used, based on defined mucosal tissue units. A patchy enteropathy was found in 60% of CMPI, and in 14% of nonspecific changes, but never in CD. There was a significant difference in crypt depth between CD (360 +/- 80 microns) and CMPI (217 +/- 92 microns), even when lesions of equal grade were compared (P less than 0.015). IEL counts per 1000 epithelial cells showed even better discrimination between the groups (CD: 793 +/- 173, CMPI: 320 +/- 143, P less than 0.001). In CD, there was a relatively greater increase of IgM-cells (X 4.9) and of IgG-cells (X 4.2) than of IgA-cells (X 2.6). Ig-cell changes outlasted the morphological lesion in CD on a gluten-free diet. In CMPI, IgM-cells (X 2.3), IgG-cells (X 2.5), and IgA-cells (X 1.7) were proportionately increased, compared to controls. A special increase of IgE-cells in CMPI could not be substantiated. By computerized stepwise discriminant analysis based upon crypt depth, villus/crypt-ratio, IEL-count, and counts of IgM-, IgG-, IgA-containing cells of lamina propria, accurate classification of patient groups was accomplished, even when only the initial biopsy data were analysed.

Cell-mediated immunity to gluten within the small intestinal mucosa in coeliac disease.

Jejunal biopsies from controls and coeliac patients were maintained in organ culture in the presence of gluten fraction III. The culture media were assayed for evidence of lymphokine activity in a migration inhibition test using normal peripheral blood leucocytes. Significant inhibition of migration was produced by media from untreated coeliac patients compared with controls (P less than 0.005) or treated coeliac patients (P less than 0.001), indicating the production of a leucocyte migration inhibition factor (LIF) by untreated coeliac mucosa in response to gluten fraction III. The degree of inhibition correlated with the preculture interepithelial lymphocyte count in the coeliac biopsies (P less than 0.02). In six coeliac patients studied when on a normal diet and on a gluten-free diet, LIF was produced while on a normal diet, but not while on a gluten-free diet. These results suggest that a local cell-mediated immune reaction to gluten is present in the mucosa of patients with untreated coeliac disease but that this is reversed by treatment with a gluten-free diet.

Small gut mucosal activities of pyrimidine precursor enzymes in celiac disease

Summary The increased mitotic cell count in the small intestinal mucosa in celiac disease has given rise to question as to whether increased cell turnover or prolonged mitosis were occurring. Two enzymes in the pyrimidine biosynthetic pathway, aspartate carbamoyl transferase and dihydroorotate dehydrogenase, have been measured in rat and human intestinal mucosa. Activities of aspartate carbamoyl transferase in normal mucosa obtained at surgery were 247 ± 114 nmoles per g per min. No inhibition of activity of this enzyme was noted after addition of cytidine mono-, di-, or triphosphate. Dihydroorotate dehydrogenase activity was 18.1 ± 9.2 nmoles per g per min from intestinal mucosa obtained at surgery. Peroral biopsy samples from normal subjects showed aspartate carbamoyl transferase activity of 282 ± 142 nmoles and dihydroorotate dehydrogenase activity of 9.9 ± 8.6 nmoles per g per min. Mucosa obtained from patients with celiac disease snowed normal or elevated enzyme activities; the more severe lesions showed the higher levels. These results provide indirect evidence to support increased cell turnover rather than maturation arrest in mitosis in the small intestinal epithelial cells in celiac disease.