Combined RNAscope and immunohistochemistry staining on duodenal paraffin sections as a new tool to reveal cytolytic potential of intraepithelial lymphocytes

: Immunohistochemistry (IHC) is a consolidated technique for the identification of surface and cytoplasmic antigens in cells or tissue sections using specific antibodies, yet simultaneous detection of two markers on the same cell may be difficult to achieve. Here we develop a protocol to perform a double staining using RNAscope, a new in-situ hybridization (ISH) technology, to visualize perforin transcripts, and classical IHC to visualize either CD8 or TcRγδ positive intraepithelial lymphocytes (IELs) in small intestinal paraffin sections of celiac disease (CD) patients. This double assay will allow to investigate the cytotoxic properties of two subsets of IELs in different stages of CD, thus contributing to understand the events leading to tissue destruction and healing

Enterocyte Proliferation and Signaling Are Constitutively Altered in Celiac Disease

Celiac disease (CD) occurs frequently, and is caused by ingestion of prolamins from cereals in subjects with a genetic predisposition. The small intestinal damage depends on an intestinal stress/innate immune response to certain gliadin peptides (e.g., A-gliadin P31-43) in association with an adaptive immune response to other gliadin peptides (e.g., A-gliadin P57-68). Gliadin and peptide P31-43 affect epithelial growth factor receptor (EGFR) signaling and CD enterocyte proliferation. The reason why the stress/innate immune and proliferative responses to certain gliadin peptides are present in CD and not in control intestine is so far unknown. The aim of this work is to investigate if, in CD, a constitutive alteration of enterocyte proliferation and signaling exists that may represent a predisposing condition to the damaging effects of gliadin. Immunofluorescence and immunohistochemistry were used to study signaling in CD fibroblasts and intestinal biopsies. Western blot (WB) analysis, immunoprecipitation, and quantitative PCR were also used. We found in CD enterocytes enhancement of both proliferation and Epidermal Growth Factor Receptor (EGFR)/ligand system. In CD enterocytes and fibroblasts we found increase of the phosphorylated downstream signaling molecule Extracellular Signal Regulated Kinase (ERK); block of the ERK activation normalizes enterocytes proliferation in CD mucosa. In conclusion the same pathway, which gliadin and gliadin peptide P31-43 can interfere with, is constitutively altered in CD cells. This observation potentially explains the specificity of the damaging effects of certain gliadin peptides on CD intestine.</p

Intestinal production of anti-tissue transglutaminase 2 antibodies in patients with diagnosis other than celiac disease

Abstract: It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25+, CD3+, and TCR- + was assessed in subjects with positive (n = 32) and negative (n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all (n = 8) positive subjects. Lamina propria CD25+ cell count was significantly (p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR- +/CD3+ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives: Damage 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 Findings: Cell 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. Conclusion: In 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

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies

Controllo neuroendocrino della secrezione del pancreas endocrino di un teleosteo: blennius Gattorugine

Dottorato di ricerca in biologia evoluzionistica e del differenziamento. 8. ciclo. Tutore Rosalba Putti. Coordinatore Silvana FilosaConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Discriminant score for celiac disease based on immunohistochemical analysis of duodenal biopsies

OBJECTIVES: Celiac disease (CD) represents a spectrum, which includes cases with minor histological abnormalities (potential CD). The aim of this work is to evaluate the contribution of immunohistochemical analysis of duodenal biopsies to the diagnosis of gluten-related minor enteropathy. METHODS: Duodenal biopsies from 56 patients with untreated CD and 56 controls were analyzed for CD3 and γδ intraepithelial lymphocyte number, γδ/CD3 ratio, and density of CD25+ lamina propria cells. A discriminant equation was obtained by which 61 more biopsies with normal villous architecture were blindly evaluated. RESULTS: All of the immunohistochemical parameters were significantly different between patients with CD and controls. None of the single parameters showed sufficient specificity for CD. The combination of all of the 4 markers resulted in the following discriminant equation: discriminant score (Dscore) = (CD3 × 0.06) - (γδ × 0.119) + (CD25 × 0.012) + (γδ/CD3 × 0.131) - 4.709. Using this Dscore, patients were correctly classified as celiac or controls in 97.3% of the cases. When this equation was applied to a validation set of 61 patients with normal villous architecture and unknown diagnosis, 92.9% of those with a positive score turned out to be patients with potential CD. A normal score, however, did not exclude this condition. CONCLUSIONS: Immunohistochemistry represents a specific tool for the diagnosis of CD, but does lack sensitivity in detecting all of the potential CD cases

Mucosal Healing in Celiac Disease: Villous Architecture and Immunohistochemical Features in Children on a Long-Term Gluten Free Diet

Considerable heterogeneity exists across studies assessing intestinal mucosal recovery in celiac (CD) patients on a gluten-free diet (GFD). We aimed at investigating histological and immunohistochemical features in CD patients on a long-term GFD and to correlate them to the GFD duration. Morphometrical and immunohistochemical analysis were retrospectively performed on duodenal biopsies in three groups of children: 33 on a long-term (&gt;2 years) GFD (GFD-group), four of which remained seropositive despite dietary adherence, 31 with villous atrophy (ACD-group) and 76 heathy, non-celiac (CTR-group). Moreover, in the GFD-group, we correlated immunohistochemical alterations to the GFD duration. The villous to crypt (V/C) ratio significantly improved after the GFD and completely normalized in all patients, becoming even higher than in the CTR-group (median value 3.2 vs. 3, p = 0.007). In parallel, the number of CD3+ and TCR&gamma;&delta;+ cells in the epithelium were significantly reduced in the GFD compared to ACD patients, even if they remained higher than in the CTR-group (p &lt; 0.05). In contrast, CD25+ cells in the lamina propria significantly decreased after the GFD (p &lt; 0.05) and become comparable to the CTR-group (p = 0.9). In the GFD-group there was no difference in the immunohistochemical parameters between seropositive and seronegative patients and alterations did not correlate to GFD length. In conclusion, a GFD is able to both restore a normal V/C ratio and reduce inflammation, but the epithelium maintains some stigmata of the disorder, such as an increased number of CD3+ and TCR&gamma;&delta;+ cells. These alterations persist regardless of the duration of the GFD

Intestinal deposits of anti-tissue transglutaminase IgA in childhood celiac disease

Background: High serum levels of anti-tissue-transglutaminase-2 IgA antibodies (anti-TG2), which are produced and deposited in the intestine, characterize celiac disease. Aim: To assess the diagnostic value of intestinal deposits of anti-TG2 IgA for celiac disease in a paediatric population. Methods: 344 children underwent duodenal biopsy for the suspicion of CD, and were divided into 3 groups: group A, 144 celiac subjects with villous atrophy (Marsh 3b–c); group B, 109 subjects with high serum levels of anti-TG2 but normal intestinal mucosa (Marsh 0–1) (potential celiac disease patients); group C, 91 subjects with normal levels of serum anti-TG2: 70 with Marsh 0–1 and 21 with Marsh 3a mucosa. All duodenal sections were evaluated for the presence of intestinal deposits of anti-TG2 IgA by double immunofluorescence. Results: Deposits of anti-TG2 IgA were present in 96%, 68%, 12% of patients from groups A, B, C, respectively. Diagnostic sensitivity and specificity for celiac disease were 96% and 88% vs. 97% and 100% for serum anti-TG2, respectively. The degree of concordance with serum anti-TG2 was 85%. Conclusion: Detection of intestinal deposits of anti-TG2 IgA is a useful diagnostic tool. Further research is needed regarding their ability to predict evolution to villous atrophy in potential celiac disease