1,720,969 research outputs found
ISOLATION AND CULTURE OF FIBROBLASTS FROM ENDOSCOPIC DUODENAL BIOPSIES OF CELIAC PATIENTS
No full textIntroduction and Aim.Esophagogastroduodenoscopy is widely used to investigate the diseases of the upper gastrointestinal tract as celiac disease. Beside its diagnostic and therapeutic potential, it can be useful as source of primary intestinal fibroblasts through the collection of bioptic specimens. Aim of the study is to isolate and culture primary fibroblasts from endoscopic duodenal biopsies of celiac and non-celiac subjects, to analyze their growth patterns and the morphometric characteristics.
Patients and Methods. 60 duodenal bioptic specimens from 20 celiac patients and 114 from 38 non-celiac subjects were mechanically chopped and enzymatically digested in order to obtain primary cell cultures. Growth patterns, karyotype (Q-banding analysis), expression of typing proteins (fibroblast surface protein and cytokeratin 20) and morphometric parameters (diameters and their ratio, perimeter, area and perimeter/area ratio at computerised image analysis) were investigated on cultured cells.
Results. Primary cells were successfully cultured in 78% of the collected duodenal biopsies. Cultured cells, expressing the fibroblast surface protein, were negative for cytokeratine 20 and maintained a normal kariotype. Cells grew slowly without differences between the celiac and the non celiac group. Morphometric analysis of celiac fibroblasts revealed significantly increased dimensions, with a preserved diameters ratio, and a reduced perimeter/area ratio.
Conclusion. For the first time this study demonstrates the feasibility of culturing primary fibroblast cell from endoscopic duodenal biopsies in celiac and non-celiac subjects, opening a new window of opportunity in studies intended to establish the role of fibroblasts as a possible partaker in the pathogenesis of the celiac mucosal damage
Transglutaminase 2 (TG2) mediates the cytotoxic effect of resveratrol in cholangiocarcinoma (CC) cell lines
No full textBackground and aim: Cholangiocarcinoma (CC) is characterized by a poor prognosis. As curative medical regimen is currently unavailable for patient unsuitable for surgery, new drugs are urgently needed. Resveratrol (RES) has previously been reported to
play a cytotoxic effect on CC cell cultures and the related increase of type 2 transglutaminase (TG2) involved in carcinogenesis and apoptosis. Present study was aimed at evaluating if TG2 inhibition could reduce the cytotoxic effect of RES on CC cell lines. Material and methods: CC cell lines SK-CHA-1 and MZ-CHA-1, grown on a three dimensional cell culture model, were treated for 72 hours with RES (64 microM, after a dose finding preliminary study) alone or combined with TG2 inhibitors (cystamine and two selectives ones, B003 and T101). The following points were investigated: cell viability (clonigenic test), cell morphology (light microscopy -LM, transmission electron microscopy -TEM and immunoistochemistry-IMC), Q-banding (karyotype analysis), and TG2 analysis (colorimetric method and Western Blotting). Results: RES treatment induced a significant inhibition of cell growth. The co-treatment RES/TG2 inhibitors prevented growth inhibition in both cell lines; the cell growth for SK-CHA-1 with RES 64 microM and the three different inhibitors (respectively cystamine, B003 and T101) increased of the 24%, 42% and 76% vs. percentage of colony growth from cells treated only with RES; for MZ-CHA-1 the cell growth increase was of 75%, 33% and 83% (cystamine, B003 and T101) vs. percentage of colony growth from cells treated only with RES. LM, TEM and IHC results demonstrated a partial protection with T101. The normalization of cell growth was associated to an inhibition of TG2 activity both in MZ-CHA-1 (85% with cystamine, 60% with B003 and 45% with T101 vs.100% controls) and SK-CHA-1 (95% with cystamine, 30% with B003 and 15% with T101 vs. 100% controls). Conclusions: Our data demonstrated that the cytotoxic effect of RES in SK-CHA-1 and MZ-CHA-1 is TG2 mediated
Immunoreactivity of antibodies against transglutaminase-deamidated gliadins in adult celiac disease
No full textBackground The significance of the presence of anti-gliadin antibodies in patients affected by celiac disease is still unclear. It is hypothesized that gliadin deamidation, catalysed by transglutaminase, plays a role in favoring the antigen presentation. Aim To determine the immunoreactivity of anti-gliadin antibodies from untreated celiac patients to transglutaminase deamidated gliadins. Materials and methods Gliadins from wheat flour underwent enzymatic digestion and were deamidated or cysteamine-transamidated by transglutaminase. Immunoreactivity of anti-gliadin antibodies from untreated adult celiac patients sera was evaluated by means of a competitive enzyme-linked immunosorbent assay (ELISA) method. Results Gliadin deamidation increased antibodies immunoreactivity from 25% to 50% while cysteamine incorporation into the gliadin peptides resulted in an immunoreactivity decrease. Conclusions Increased immunoreactivity of transglutaminase deamidated gliadins tested with anti-gliadin antibodies from untreated adult celiac patients supports the hypothesis of a pivotal role of gliadin deamidation in the pathomechanism of celiac disease
Resveratrol impairs the formation of MDA-MD-231 multicellular tumor spheroids concomitant with ceramide accumulation
No full textResveratrol exerts a drastic growth inhibitory effect on human breast cancer MDA-MB-231 cells grown both in vitro and in vivo. Here we show that resveratrol affects the aggregation properties of MDA-MB-231 cells into multicellular tumor spheroids (MCTSs), in association with induction of de novo synthesis of ceramide. After 9 days of 3D growth in the presence of resveratrol (64 microM), MDA-MB-231 cells formed significantly smaller MCTSs. Further, cells from these aggregates failed to form colonies. Addition of resveratrol (64 microM) to preformed MDA-MB-231 MCTSs caused no significant size change, consistent with lack of ceramide induction. Only some apoptotic blebs were found on the MCTSs surface. Altogether these findings indicate that resveratrol might be effective for prevention of breast cancer cell growth
Evaluation of transglutaminase expression in the hepatobiliary tract
No full textINTRODUCTION/OBJECTIVES: Transglutaminases [1] are an enzymatic family widely present in different tissues. New insights showed that transglutaminase type 2 (TG2) plays a pivotal role in several human diseases as liver fibrosis and viral hepatitis. Although the relevant pathogenetic involvement, its hepatic metabolism and homeostasis are not clearly understood.
AIMS & METHODS: Our aim was to investigate in a combined model (in vitro and ex vivo) the presence and expression of TG2 in the human hepatobiliary tract. We studied TG2 expression in cell cultures of hepatocytes (Hep-G2) and cholangiocytes
(SK-CHA-1 and MZ-CHA-1), with and without the choleretic stimulation of bile acid
(glycochenodeoxycholic acid 0.5, 0.75mM for 48 hours) and in human bile from
endoscopic retrograde cholangiographic catheterism (7 patients, 4M, mean age 47).
We evaluated in cell lysates or culture medium TG2 protein (western blot), its activity (colorimetric assay) and expression of tissue transglutaminase mRNA (RT-PCR).
Moreover, in human bile the presence of FXIII has been investigated (immunologic
test).We found TG2 presence and activity in hepatocytes and cholangiocyets cell lines. The treatment with bile acid at non-toxic concentrations increased the expression and activity of TG2 in all the cell lines (from +20 to +40% vs. untreated cultures). TG activity was present in the medium of Hep-G2 cells but not in the medium of cholangiocytes cell lines. The base line expression is significantly higher
in cholangiocytes compared to hepatocytes. A TG2 activity was demonstrated in
hepatocytes cell culture medium and not in the cholangiocytes one; moreover a transglutaminasic activity was demonstrated in human bile (0.25±0.12 mU/ml) associated
with the presence of the FXIII.
CONCLUSION: Our preliminary in vitro data demonstrates that TG2 is represented
in Hep-G2, SK-CHA-1 and MZ-CHA-1 human hepatic/biliary cell lines and in human
bile. The treatment with bile acid in cell cultures increases its expression, suggesting
a possible homeostatic regulation.
REFERENCE(S): [1] Elli et al. Dig Liv Dis 2009; 41(8): 541−50.
Disclosure of Interest: None Declare
Resveratrol induced inhibition of cholangiocarcinoma cell growth is transglutaminase dependent
No full textINTRODUCTION/OBJECTIVES: Cholangiocarcinoma is a tumour with a poor prognosis. An efficient therapy is unavailable in unoperable patients and new drugs are widely requested. Resveratrol (RES) is a natural molecule with a well known anticancer effect proved on different tumour cell lines. Previous studies demonstrated the cytotoxic effect of RES on cholangiocarcinoma cell cultures [1] and the related increase of transglutaminase type 2 (TG2), which is involved in carcinogenesis and apoptosis.
AIMS & METHODS: Aim of the present study was to evaluate if the cytotoxic effect of RES on cholangiocarcinoma cell lines could be abolished by TG inhibition.
Cholangiocarcinoma cell lines (SK-CHA-1 and MZ-CHA-1), growth in a three dimensional
cell culture systems, have been treated for 72 hours with RES (32, 64 microM) alone or combined with three different TG2 inhibitors: cystamine and the selective inhibitors B003 and T101. At the end of the treatments we investigated:
(1) cells viability (clonigenic test); (2) cell morphology with light microscopy on
histological sections and electron microscopy (TEM); 3) TG2 activity (colorimetric technique).
RESULTS: RES treatment induced a significant inhibition of cell growth (90% and 62% vs 100% controls). The cotreatement RES/TG2 inhibitors prevented these growth
inhibition in both cell lines; the cell growth for MZ-CHA-1 at higher dose of RES was
respectively (cystammine, B003 and T101) 50%, 60% and 90% vs. 100% controls; for SK-CHA-1 (cystammine, B003 and T101) was 55%, 60% and 80% vs. 100% controls. Histological sections and TEM results demonstrated a partial protection with both cystammine and B003/T101 inhibitors. The normalization of cell growth was associated to an inhibition of TG2 activity both in MZ-CHA-1 (60% with cystamine, 80% with B003 and 90% with T101 vs. 100% controls) and SK-CHA-1 (20% with
cystammine, 40% with B003 and 60% with T101 vs. 100% controls).
CONCLUSION: Our data demonstrated that the cytotoxic effect of RES in SK-CHA-1 and MZ-CHA-1 is TG2 mediated.
REFERENCE(S): [1] Roncoroni et al. Liver Int. 2008;10:1426−36.
Disclosure of Interest: None Declared
Cytoskeleton reorganization and ultrastructural damage induced by gliadin in a three-dimensional in vitro model
No full textAIM: To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns. METHODS: We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure (by means of light microscopy and scanning electron microscopy), and the effect of gliadin on actin (phalloidin fluorescence) and the tight-junction protein occludin and zonula occluden-1. RESULTS: The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gliadin decreased the number of intracellular actin fi laments, impaired and disassembled the integrity of the tight-junction system. CONCLUSION: Our data obtained from an "in vivo - like" polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions
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