Daniela Pizzuti

Toll-Like Receptor 2 Regulates Intestinal Inflammation by Controlling Integrity of the Enteric Nervous System

BACKGROUND & AIMS In the intestines, Toll-like receptor 2 (TLR2) mediates immune responses to pathogens and regulates epithelial barrier function; polymorphisms in TLR2 have been associated with inflammatory bowel disease phenotype. We assessed the effects of TLR2 signaling on the enteric nervous system (ENS) in mice. METHODS TLR2 distribution and function in the ileal neuromuscular layer of mice were determined by immunofluorescence, cytofluorimetric analysis, immunoprecipitation, and immunoblot analyses. We assessed morphology and function of the ENS in Tlr2(-/-) mice and in mice with wild-type Tlr2 (wild-type mice) depleted of intestinal microbiota, using immunofluorescence, immunoblot, and gastrointestinal motility assays. Levels and signaling of glial cell line-derived neurotrophic factor (GDNF) were determined using quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, and immunoprecipitation analyses. Colitis was induced by administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid to Tlr2(-/-) mice after termination of GDNF administration. RESULTS TLR2 was expressed in enteric neurons, glia, and smooth muscle cells of the intestinal wall. Tlr2(-/-) mice had alterations in ENS architecture and neurochemical profile, intestinal dysmotility, abnormal mucosal secretion, reduced levels of GDNF in smooth muscle cells, and impaired signaling via Ret-GFRα1. ENS structural and functional anomalies were completely corrected by administration of GDNF to Tlr2(-/-) mice. Wild-type mice depleted of intestinal microbiota had ENS defects and GDNF deficiency, similar to Tlr2(-/-) mice; these defects were partially restored by administration of a TLR2 agonist. Tlr2(-/-) mice developed more severe colitis than wild-type mice after administration of dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid; colitis was not more severe if Tlr2(-/-) mice were given GDNF before dextran sulfate sodium or 2,4 dinitrobenzensulfonic acid. CONCLUSIONS In mice, TLR2 signaling regulates intestinal inflammation by controlling ENS structure and neurochemical coding, along with intestinal neuromuscular function. These findings provide information as to how defective TLR2 signaling in the ENS affects inflammatory bowel disease phenotype in humans.

Lack of intestinal mucosal toxicity of Triticum monococcum in celiac disease patients

Objective. The treatment of celiac disease is based on lifelong withdrawal of foods containing gluten. Unfortunately, compliance with a gluten-free diet has proved poor in many patients (mainly due to its low palatability), emphasizing the need for cereal varieties that are not toxic for celiac patients. In evolutionary terms, Triticum monococcum is the oldest and most primitive cultivated wheat. The aim of this study was to evaluate the toxicity of T. monococcum on small intestinal mucosa, using an in vitro organ culture system. Material and methods. Distal duodenum biopsies of 12 treated celiac patients and 17 control subjects were cultured for 24 h with T. aestivum (bread) gliadin (1 mg/ml) or with T. monococcum gliadin (1 mg/ml). Biopsies cultured with medium alone served as controls. Each biopsy was used for conventional histological examination and for immunohistochemical detection of CD3 + intraepithelial lymphocytes (IELs) and HLA-DR. Secreted cytokine protein interferon-γ (IFN–γ) was measured in the culture supernatant using an enzyme-linked immunoadsorbent assay. Results. Significant morphological changes, HLA-DR overexpression in the crypt epithelium and an increased number of CD3 + IELs, found after bread gliadin exposure, were not observed in celiac biopsies cultured with T. monococcum gliadin. In contrast, with bread gliadin, there was no significant IFN-γ response after culture with monococcum gliadin. Similarly, biopsies from normal controls did not respond to bread or monococcum gliadin stimulation. Conclusions. These data show a lack of toxicity of T. monococcum gliadin in an in vitro organ culture system, suggesting new dietary opportunities for celiac patients.

Possible Anandamide and Palmitoylethanolamide involvement in human stroke

BackgroundEndocannabinoids (eCBs) are ubiquitous lipid mediators that act on specific (CB1, CB2) and non-specific (TRPV1, PPAR) receptors. Despite many experimental animal studies proved eCB involvement in the pathogenesis of stroke, such evidence is still lacking in human patients. Our aim was to determine eCB peripheral levels in acute stroke patients and evaluate their relationship with clinical disability and stroke volume.MethodsA cohort of ten patients with a first acute (within six hours since symptoms onset) ischemic stroke and a group of eight age- and sex-matched normal subjects were included. Groups were also matched for metabolic profile. All subjects underwent a blood sample collection for anandamide (AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA) measurement; blood sampling was repeated in patients on admission (T0), at 6 (T1) and 18 hours (T2) thereafter. Patients neurological impairment was assessed using NIHSS and Fugl-Meyer Scale arm subitem (FMSa); stroke volume was determined on 48 h follow-up brain CT scans. Blood samples were analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.Results1)T0 AEA levels were significantly higher in stroke patients compared to controls. 2)A significant inverse correlation between T0 AEA levels and FMSa score was found. Moreover a positive correlation between T0 AEA levels and stroke volume were found in stroke patients. T0 PEA levels in stroke patients were not significantly different from the control group, but showed a significant correlation with the NIHSS scores. T0 2-AG levels were lower in stroke patients compared to controls, but such difference did not reach the significance threshold.ConclusionsThis is the first demonstration of elevated peripheral AEA levels in acute stroke patients. In agreement with previous murine studies, we found a significant relationship between AEA or PEA levels and neurological involvement, such that the greater the neurological impairment, the higher were these levels.

Cingulin is dispensable for epithelial barrier function and tight junction structure, and plays a role in the control of claudin-2 expression and response to duodenal mucosa injury

Summary Cingulin (CGN) is a 140 kDa protein, which is localized to the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signaling in cultured cells. To investigate the function of CGN at the organism level, we generated CGN knockout (CGN−/−) mice by homologous recombination. CGN−/− mice are viable and fertile, and are born at the expected mendelian ratios. Immunohistochemistry, immunofluorescence, electron microscopy and permeability assays of epithelial tissues of CGN−/− mice show no cingulin labeling at junctions, a normal localization of TJ proteins, and normal TJ structure and barrier function. Microarray analysis of intestinal cells does not show significant changes in gene expression between CGN−/− and CGN+/+ mice, whereas immunoblotting analysis shows a twofold increase in the levels of claudin-2 protein in the duodenum and the kidney of CGN−/− mice, compared to CGN+/+ littermates. Furthermore, CGN−/− mice show an exacerbated response to the ulcerogenic action of cysteamine, whereas acute injury of the colon by dextran sodium sulfate elicits undistinguishable responses in CGN−/− and CGN+/+ mice. We conclude that at the organism level cingulin is dispensable for the structure and barrier function of TJ, and is embedded in signaling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum.

Oral Propranolol Decreases Intestinal Permeability in Patients With Cirrhosis: Another Protective Mechanism Against Bleeding?

Oral Propranolol Decreases Intestinal Permeability in Patients With Cirrhosis: Another Protective Mechanism Against Bleeding?

In vitro model for IgE mediated food allergy

Abstract Background. In intestinal food allergy, the non-specificity of gastrointestinal symptoms and the limited access to the reacting organ are the reasons for the limited understanding of the pathophysiology of this disease and the difficulties in establishing an appropriate diagnosis in the individual patient. Objective. To develop an in vitro model reproducing pathophysiological mechanisms of IgE mediated food allergy. Methods. Distal duodenum biopsies of nine patients with food allergy and 10 control subjects were cultured for 3 h with medium alone and with 1mg/ml of peptic-tryptic digest of wheat gliadin, wheat albumins, and apple proteins. Each biopsy was used for conventional histological examination and for immunohistochemical detection of IgE-positive cells. We have also analyzed the expression of tight junction proteins, occludin, claudin-1, and ZO-1 by immunoconfocal microscopy. Histamine and tryptase release were measured in the culture medium and collected at 0, 30 min, and 3 h of culture using an enzyme and radio immunoassay, respectively. Results. Exposure of small intestinal biopsy specimens of patients with food allergy to food allergens led to a significative increase of IgE-positive cells with a significative increase of histamine and tryptase release and an altered expression of tight junction proteins. No differences were found in intestinal biopsies of controls, cultured with or without food antigens. Conclusions. Small intestinal organ culture is a functional model of food allergy and could be considered as an in vitro oral food challenge, with evident reduction of costs and risks for the patients.

Mucosal IgA defect in primary biliary cirrhosis.

The pathogenetic mechanism of the association between PV and UC is not clear. Although the antigenic determinant in PV, desmosomal-associated 130-kd glycoprotein, is limited to the stratified squamous epithelium (3, 10), cross-reactivity between antigens in the skin or oral and bowel mucosa has been considered (8). Recently, various serum antibodies against distinct adhesion molecules of the epidermis or dermoepidermal basement membrane zone and T-cell responses have been detected (2, 11). In autoimmune-based disorders genetic predisposition is important. It is well known that HLAs A1, B8, DR3, and DR4 are associated with various autoimmune disorders such as gluten enteropathy, dermatitis herpetiformis, UC, and autoimmune hepatitis. Our patient has the HLA DR4 genotype, which may be significant in the pathogenesis of these disorders in her. In conclusion, we consider that the association of PV and UC may be coincidental or due to cross-reactivity between antigens in the oral mucosa and bowel. It can be considered that an autoimmune disease predisposes to the other autoimmune diseases.

A reply to Donald D. Kasarda: Lack of intestinal mucosal toxicity of Triticum monococcum in celiac disease patients

TO THE EDITOR: We thank Dr. Kasarda for his thought-provoking comments regarding Triticum monococcum toxicity in celiac disease. I appreciate the opportunity to provide further clarification. Gliadins were extracted as described by De Ritis et al. [1]; 10 g flour was dissolved in 10 ml of 70% ethanol and left for 1 h at room temperature. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected, dialyzed against acetic acid 0.04 M and lyophilized. Peptic tryptic digest of gliadin was prepared following a two-step procedure: gliadin (1 g) pool was digested in 10 ml HCl 0,2 M (pH 1.8), with 20 mg purified pepsin, at 378C for 2 h. The resultant peptic digest was further digested by adding 20 mg purified trypsin after pH adjustment to 8.0 with NaOH 2 M, at 378C for 4 h. Inactivation of enzymes was achieved by pH adjustment to pH 7 and by heating at 1008C for 15 min. Insoluble material was removed by centrifugation at 10,000 g for 30 min. The peptic tryptic digest of gliadin was freeze dried and stored at 208C. Previous studies showed that the A-genome donor of the hexaploid wheats is T. urartu [2 4]. Restriction fragment length polymorphism analysis using single-copy or repetitive DNA sequences indicated that the T. monococcum genome is differentiated from that of T. urartu [4,5]. This was confirmed by analysing the gliadin fractions of T. monococcum that differ substantially from those of T. urartu , resembling the gliadin pattern of polyploid wheats [6]. These differences could explain the different toxicity potential between T. monococcum and hexaploid species. Other investigators, using a different in vitro system, have also shown that T. monococcum gliadins are less harmful than gliadins of hexaploid species [7,8]. Dr. Kasarda rightly indicates the relevance of the 31 49 peptide. This peptide display a direct cytotoxic effect on the small-intestine mucosa of celiac patients, but it is unable to bind HLA DQ2/DQ8 molecules or to stimulate CD4 T cells. Several studies have clearly suggested a strong association between celiac disease and HLA DQ2 or the DQ8 haplotype, which is able to present to T cell gliadin peptides, deamidated by tissue transglutaminase, the target of celiac disease-specific autoantibody [9,10]. This is a very strong indication that the glutenspecific, HLA-DQ2/8-restricted, T-cell response is required for disease development. As healthy controls lack gluten-specific T cells, a possible explanation for this observation is that the gluten-specific T-cell response sets the stage for the effects induced by the 31 49 peptide. In our study we found low levels of IFN-g and absence of HLA-DR expression in duodenal epithelium, which strongly suggests a reduced immunogenicity of T. monococcum . We agree with Dr. Kasarda that further work is needed to determine the identity of gliadins and their