Ibtissem Ghedira

Localization of tissue transglutaminase and N (epsilon)-(gamma) -glutamyl lysine in duodenal cucosa during the development of mucosal atrophy in coeliac disease

Expression and transamidation activity of tissue transglutaminase (tTG) may be involved in the morphological modifications leading to the mucosal atrophy observed in coeliac disease (CD). We aimed to investigate the localization of tTG within the duodenal mucosa during the development of villous atrophy. The localization and level of expression of Nɛ-(γ-glutamyl) lysine isopeptides which could reflect the transamidation activity of tTG were also analyzed. tTG and Nɛ-(γ-glutamyl) lysine were localized using an immunohistochemical technique on duodenal biopsies obtained from 75 patients with CD and 51 subjects with normal mucosa (control group). The number of cases displaying tTG-expressing cells in the basement membrane and lamina propria was significantly higher in CD patients than in the control group. Moreover, the intensity of tTG staining in these areas was higher in CD. In contrast, the number of biopsies with tTG-expressing enterocytes was significantly lower in CD than in the control group. There was no difference in Nɛ-(γ-glutamyl) lysine between the two populations. Tissue transglutaminase was differently expressed in the various areas of the mucosa according to the stage of atrophy, whereas the localization and the intensity of the labelling of Nɛ-(γ-glutamyl) lysine isopeptides did not show any modification. The preferential localization in the basement membrane and lamina propria may reflect the involvement of tTG in the development of mucosal atrophy in CD.

Anti‐Endothelial Cell Antibodies Determination by Cyto‐ELISA: A Comparative Study between Three Cell Types Used as Substrates

Abstract: Cyto‐ELISA has been widely used to investigate anti‐endothelial cell antibodies (AECAs); however, because various types of endothelial cells have been used, the results among studies differ. The aim of our study was to analyze and compare the results when determining AECAs in patients with connective tissue disease (CTD). We did so using a cyto‐ELISA with different cells as antigenic substrates: two different endothelial cells, one microvascular (HMEC‐1) and one from human bone marrow (HBMEC), and one epithelial cell line from breast adenocarcinoma as negative controls (MDA‐MB‐231). In this trial, we performed a retrospective study in 60 patients with CTD [46 with systemic lupus erythematosus, 8 with Sjögrens syndrome, and 6 with systemic sclerosis] and 32 healthy volunteers. Using cyto‐ELISA, the antibody against a cell was considered positive when the optical density (OD) obtained was higher than the mean OD obtained in the control group + 2 standard deviations (upper normal range). Patients were classified into three groups according to the OD obtained with the different cell lines: group 1: patients without any antibody; group 2: patients with specific AECAs; and group 3: patients with nonspecific AECAs. According to this classification, we found that 43.3% of patients with CTD have specific AECAs, and 28.3% have nonspecific antibodies. Our study delineates the heterogeneity of AECAs in patients with CTD. The use of HBMEC in cyto‐ELISA may increase the sensitivity of the test, and the use of nonendothelial cells as negative controls may improve its specificity.

Thrombopoietin Secretion by Human Ovarian Cancer Cells

The thrombopoietin (TPO) gene expression in human ovary and cancer cells from patients with ovarian carcinomatosis, as well as several cancer cell lines including MDA-MB231 (breast cancer), K562 and HL60 (Leukemic cells), OVCAR-3NIH and SKOV-3 (ovarian cancer), was performed using RT PCR, real-time PCR, and gene sequencing. Human liver tissues are used as controls. The presence of TPO in the cells and its regulation by activated protein C were explored by flow cytometry. TPO content of cell extract as well as plasma of a patient with ovarian cancer was evaluated by ELISA. The functionality of TPO was performed in coculture on the basis of the viability of a TPO-dependent cell line (Ba/F3), MTT assay, and Annexin-V labeling. As in liver, ovarian tissues and all cancer cells lines except the MDA-MB231 express the three TPO-1 (full length TPO), TPO-2 (12 bp deletion), and TPO-3 (116 pb deletion) variants. Primary ovarian cancer cells as well as cancer cell lines produce TPO. The thrombopoietin production by OVCAR-3 increased when cells are stimulated by aPC. OVCAR-3 cells supernatant can replace exogenous TPO and inhibited TPO-dependent cell line (Ba/F3) apoptosis. The thrombopoietin produced by tumor may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.

PO-13 - Production of a functional thrombopoietin by a human ovarian cancer cell line

INTRODUCTION Hemostatic abnormalities are frequently noticed in patients with malignant diseases. These complications include platelets disorders. The role of platelets in cancer extends beyond thrombocytosis and thrombosis, and also platelets promote cancer growth and metastatic dissemination. In the physiology, platelet production is regulated by thrombopoietin, which is mainly secreted by the liver. We, previously, reported that thrombopoietin could be secreted by the ovarian adenocarcinoma cell line, OVCAR-3. AIM Our main purpose is to analyze the gene expression of thrombopoietin in ovarian cancer cells and to assess its functionality. MATERIALS AND METHODS The thrombopoietin gene expression in ascitic cells from patients with ovarian carcinomatosis, as well as, in three cancer cell lines, including OVCAR-3 cells, performed using reverse transcription PCR, real-time PCR and gene sequencing, Normal human ovary and liver tissues are used as controls. The functionality of thrombopoietin on the basis of the viability of a thrombopoietin-dependent cell line (Ba/F3) using a co-culture method. RESULTS Similarly to liver and ovary tissues, all cancer cells lines express the three TPO-1 (full length TPO), TPO-2 (12bp deletion) and TPO-3 (116pb deletion) variants. By flow cytometry, we show that thrombopoietin production by OVCAR-3 could be increased when cells are stimulated by activated protein C. Lastly, Our results confirm that activated protein C may act, in a paracrine fashion, to boost thrombopoietin production. CONCLUSIONS We report, for the first time, that thrombopoietin secreted by ovarian cancer cells is functional. Hence, thrombopoietin produced by tumor cells may have a direct effect on thrombocytosis/thrombosis occurrence in patients with ovarian cancer.