No abstract

We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.

We aimed to study the expression and localization of the molecular components of enterocyte junctions in celiac disease together with the level of tyrosine phosphorylation, a phenomenon known to affect their cellular distribution and function, and to explore the influence of proinflammatory cytokines. Duodenal biopsy specimens from patients with celiac disease and control subjects were used for immunoprecipitation, immunoblotting, and immunolocalization by using antioccludin, anti-zonula occludens (ZO)-1, anti-E-cadherin, anti-beta-catenin, and antiphosphotyrosine antibodies. The same procedures were carried out on filter-grown Caco-2 cells incubated in the absence or presence of interferon g and tumor necrosis factor a. In active celiac disease, the absence of a phosphorylated ZO-1 and the extensive phosphorylation of beta-catenin might be responsible for the absence of membranous localization of occludin and E-cadherin, respectively. The in vitro system showed an influence of the cytokines on the assembly of these complexes that proved the opposite to celiac samples as far as tight junctions were concerned because the presence of a phosphorylated ZO-1 enables occludin to localize in the membrane.

SUMMARYTissue transglutaminase (tTG) seems to be the target self-antigen for endomysial antibodies in coeliac disease (CD) and to catalyse the critical deamidation of gliadin which strengthens its recognition by HLA-restricted gut-derived T cells. To date, it has not been demonstrated whether gliadin is crosslinked to tTG within the gut wall, a phenomenon known to occur in vitro . We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa. The immunoprecipitation and Western blotting experiments were performed on mucosal biopsies obtained from untreated, treated CD patients and biopsied controls, by using either anti-tTG or anti-gliadin antibodies, in both denaturating/reducing or nondenaturating/nonreducing conditions. A subset of experiments was performed by using anti-tTG antibodies purified by affinity chromatography from sera of untreated coeliac patients. The localization of tTG and gliadin was studied by immunofluorescence at confocal laser microscopy on seriate sections of diseased and normal duodenal mucosa by using the same antibodies of the coimmunoprecipitation section. The amounts of tTG and gliadin coimmunoprecipitated with anti-tTG monoclonal antibody in untreated CD mucosa were significantly increased compared to those of the other two groups. When performing the experiments in nondenaturating/nonreducing conditions, a high molecular weight band formed by both molecules, was evidenciated. Also the anti-tTG antibodies purified from patients' sera turned out to be able to coimmunoprecipitate the two molecules. The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa. Our findings firstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls, and the ability of circulating tTG-autoantibodies to recognize and immunoprecipitate the tTG-gliadin complexes.

Gap junctional communication permits the direct exchange of small molecules and ions and has been implicated in tissue homeostasis/metabolite exchange. The lack of gap junctional intercellular communication (GJIC) plays important roles in the promotion and progression of carcinogenesis. In the present study, we demonstrate that treatment of human hepatoma Hep G2 cells with retinoic acid (RA) results in increased amounts and phosphorylation of connexins, their stabilisation in plasma membrane plaques and enhanced GJIC. In cultured fetal hepatocytes, which represent a non-transformed, proliferating and incompletely differentiated liver system, the effects of RA are limited to the establishment of connexin in areas of cell-cell contact and the improvement of GJIC. This suggests that modulation of cell-cell channel communication by RA occurs differently in these two experimental models: while RA is able to revert cell transformation in Hep G2 cells, in fetal hepatocytes it may induce the expression of a more differentiated phenotype.

Tumor size correlates with lymph node metastasis in breast cancer. In multifocal lesions there is controversy about considering the summation of the largest diameter of each tumor. A total of 122 patients with multifocal breast cancer were compared in a retrospective study with 177 patients with unifocal tumors, correlating tumor size with lymph node metastasis. In multifocal tumors, two sizes were considered: the diameter of the largest tumor and the combined diameter of all lesions. Relationship was established by three different logistic models using variables such as age, number of lesions, histologic type, and grade. At a same size of the largest diameter of a unifocal or multifocal lesions and the combined diameter of a multifocal lesion, the latter shows less probability of nodal metastasis indicating that combined diameter is an overestimation of the lesion size. Our results indicate that in multifocal breast cancer, only the diameter of the largest tumor breast cancer has relationship with lymph node metastasis.

HighlightsBreast sarcoma is a rare but aggressive entity.Core biopsy will be the procedure of choice for the diagnosis.Lymph node metastases are uncommon and surgery represents the only potentially curative therapy.Tumor size and, specially, an adequate resection margin are the most important prognostic factors and determinant of long-term survival.There is no consensus in the use of adjuvant therapy, it will depend mainly on the risk of tumor recurrence.

The usefulness of cultured hepatocytes is limited by the gradual loss of their typical physiological functions that occurs in vitro, mainly due to the absence of microenviromental conditions found in vivo. In this study we describe the effect of retinoic acid on the re-establishment of morphological characteristics and on the reorganization of the cytoskeletal network in cultured rat hepatocytes. Results obtained demonstrate that retinoic acid can influence hepatocyte differentiation, as regards the recovery of cell polarity, polyhedric shape and reformation of bile canaliculi and junctional complexes. The main target of this action appears to be the cytoarchitecture of cytoskeletal components, particularly cytokeratin filaments, which regain the configuration present in intact liver. The reorganization of the intermediate filaments does not seem to be dependent on the induction of higher levels of cytokeratin proteins, but rather appears to be due to post-translational regulation. The effect of retinoic acid on the cytoskeletal organization could determine the stabilization of intercellular contacts by means of junctions, leading to the appearance of morpho-functional characteristics typical of well-differentiated hepatocytes.