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We studied and quantified the effect of cyclosporine A on hepatic F-actin on bile canalicular and basolateral membranes in rats fed either soybean lecithin, triacylglycerol-enriched diet, or low-fat diet by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was quite normal in the lecithin-cyclosporine A group but decreased significantly in the other cyclosporine A-treated groups (by 40% and 25% of control in triacylglycerol-cyclosporine A and cyclosporine A groups, respectively). The alteration of F-actin by cyclosporine A, related to cholestasis evidenced by a decrease in bile salt secretion, was prevented by dietary soybean lecithin and amplified by dietary soybean triacylglycerol. [ABSTRACT FROM AUTHOR]

ABSTRACT- The ultrastructure of the lobular patterns of hepatic glycogen was studied in four human liver surgical biopsies, obtained 14 h after the last meal. In centrilobular hepatocytes, glycogen particles were regularly dispersed with elements of SER between the particles. Periportal hepatocytes showed dense glycogen deposits with SER restricted to the periphery of the glycogen masses. The ultrastructural heterogeneity of the lobular distribution of glycogen in our human cases is similar to that found in control-fed rats and mice. The metabolic signification of these findings is as yet unclear. [ABSTRACT FROM AUTHOR]

Ductal adenocarcinoma of the pancreas is ranking 4 for patient' death from malignant disease in Western countries, with no satisfactory treatment. We re-examined more precisely the histone deacetylases (HDAC) and Sirtuin (SIRT) gene expression patterns in pancreatic cancer with more pancreatic tumors and normal tissues. We also examined the possible relationship between HDAC gene expression levels and long term disease outcome. Moreover, we have evaluated by using an in vitro model system of human pancreatic tumor cell line whether HDAC7 knockdown may affect the cell behavior. We analyzed 29 pancreatic adenocarcinoma (PA), 9 chronic pancreatitis (CP), 8 benign pancreatic (BP) and 11 normal pancreatic tissues. Concerning pancreatic adenocarcinoma, we were able to collect biopsies at the tumor periphery. To assess the possible involvement of HDAC7 in cell proliferation capacity, we have generated recombinant human Panc-1 tumor which underexpressed or overexpressed HDAC7. The expression of HDAC1,2,3,4,7 and Nur77 increased in PA samples at levels significantly higher than those observed in the CP group (p = 0.0160; 0.0114; 0.0227; 0.0440; 0.0136; 0.0004, respectively). The expression of HDAC7, was significantly greater in the PA compared with BP tissue samples (p = 0.05). Mean mRNA transcription levels of PA for HDAC7 and HDAC2 were higher when compared to their counterpart biopsies taken at the tumor periphery (p = 0.0346, 0.0053, respectively). Moreover, the data obtained using confocal microscopy and a quantitative method of immunofluorescence staining strongly support the HDAC7 overexpression in PA surgical specimens. The number of deaths and recurrences at the end of follow up were significantly greater in patients with overexpression of HDAC7. Interestingly, the rate of growth was significantly reduced in the case of cell carrying shRNA construct targeting HDAC7 encoding gene when compared to the parental Panc-1 tumor cells (p = 0.0015) at 48 h and 96 h (p = 0.0021). This study strongly support the notion that HDAC7play a role in pancreatic adenocarcinoma progression.

Aberrant glycosylation or overexpression of cell-surface glycosylated tumor-associated Ags (TAA) distinguish neoplastic from normal cells. Interactions of TAA MUC1 and HER2/neu with dendritic cells (DC) preclude efficient processing, which impairs immune responses. It is thus important to define the mechanisms of interactions between DC and glycosylated TAA and their trafficking and processing for further T cell activation. In this work, we study interactions between DC and the oncofetal fucose-rich glycovariants of bile salt-dependent lipase (BSDL), expressed in pancreatic cancer tissues and referred to as pathological BSDL carrying the fucosylated J28 glycotope (pBSDL-J28) because it is characterized by the mAb J28. The expression of pBSDL-J28 was assessed by immunohistochemistry and quantified by confocal microscopy. Nontumoral pancreatic tissues and cells do not express pBSDL-J28. Using multidisciplinary approaches and functional studies, we provide the first evidence, to our knowledge, that this tumoral glycoprotein is rapidly internalized by human DC through macropinocytosis and endocytosis via mannose receptors and then transported to late endosomes for processing. Interestingly, pBSDL-J28 per se induced DC maturation with increased expression of costimulatory and CD83 molecules associated with cytokine secretion (IL-8 and IL-6). Surprisingly, DC retained their full ability to internalize Ags, making this maturation atypical. Finally, the allogeneic pBSDL-J28-treated DC stimulated lymphocyte proliferation. Besides, pulsing DC with pBSDL-J28 C-terminal glycopolypeptide and maturation with CD40L triggered CD4(+) and CD8(+) T cell proliferation. Therefore, interactions of pBSDL-J28, expressed on tumoral pancreatic tissue, with DC may lead to adequate Ag trafficking and processing and result in T cell activation.

The alphaGal epitope is a carbohydrate structure, Galalpha1,3Galbeta1,4GlcNAc-R, synthesized on glycoconjugates in many mammals by alpha1,3galactosyltransferase. Humans do not express this epitope and present in serum large amounts of naturally occuring antibodies, which recognize the alphaGal epitopes and participate in the hyperacute rejection of xenograft. Studies indicated that the fundamental mechanism of hyperacute rejection involving the alphaGal epitope expression can be used in cancer therapy. We have previously suggested that the alphaGal epitope expression by human pancreatic tumoral cells could decrease the tumorigenic behavior of these cells. To determine whether the expression of the alphaGal epitope can modify the tumorigenicity of pancreatic cancer cells, we used a Syrian golden hamster pancreatic adenocarcinoma experimental model. The expression of alphaGal epitopes in the Syrian golden hamster pancreatic cancer cell line HaP-T1 was obtained by selecting stable cell clones transfected with murine alpha1,3galactosyltransferase gene. The alphaGal epitope expression resulted in a delay in the tumoral development of HaP-T1 cells in vivo after allograft transplantation of Syrian golden hamsters (2.5-fold, P < 0.05) and of nude mice. This result is associated with an 100% increase in survival time of nude mice bearing tumors expressing the alphaGal epitope. Our results confirm that the cell surface expression of alphaGal epitope decreases the tumorigenic behavior of pancreatic cancer cells. This novel property may be useful for the development of cancer gene immunotherapy strategy., ((c) 2005 Wiley-Liss, Inc.)

We have analyzed the effect of ischemia-reperfusion on expression of hepatic Na+,K+-ATPase on bile canalicular (BCM) and basolateral membranes (BLM) in human liver allografts using confocal laser scanning microscopy imaging. Na+, K+-ATPase, an integral membrane enzyme, plays a key role in the physiology and structure of hepatocytes, where it maintains the electrochemical gradients for Na+ and K+ across the cell membrane. The concentrations of these ions as well as their gradients regulate the active transport across the plasma membrane for bile acid and water from sinusoidal to canalicular membranes. In addition, Na+,K+-ATPase is also involved in cellular structure because of its close relationship with submembrane microfilaments and its implication in tight junction assembly. Therefore, Na+,K+-ATPase appears as an indicator of tissue viability and hepatic functionality during liver transplantation. Its localization and its function in BCM are still controversial. As in previous studies, we found an enzyme expression in both BLM and BCM. We show that ischemia induced a decrease in Na+,K+-ATPase expression only in BCM. This result could be explained by the differences in biochemical membrane environment between basolateral and bile canalicular Na+,K+-ATPase. Membrane lipid fluidity, which is more elevated in BLM than in BCM, could protect the enzyme during ischemia. After reperfusion, Na+,K+-ATPase expression was strongly decreased in both BCM and BLM. This alteration following reperfusion is probably due to multiple factors: direct alteration of the enzyme catalytic subunit and modification of its environment and membrane lipid fluidity by free radicals and changes in ATP levels and ionic distribution. This important decrease in Na+,K+-ATPase expression of both BLM and BCM could disturb not only hepatic secretory function but also cellular volume and structure during the postoperative period.

We studied and quantified the effect of ischemia-reperfusion on expression of intercellular adhesion molecule-1 (ICAM-1) on sinusoidal endothelial cells and hepatocyte plasma membranes by means of confocal laser scanning microscopy imaging. We found that ischemia induced an increase in ICAM-1 expression on sinusoidal endothelial cells and hepatocytes. After reperfusion, ICAM-1 expression was increased on sinusoidal endothelial cells, whereas it was unmodified on hepatocytes. On the other hand, ICAM-1 expression was not correlated to ischemia-reperfusion liver injury. Therefore, hepatocellular ischemia-reperfusion injury could be induced by other causes than immune-mediated damages.

To find a prognosis model of human liver transplant, we evaluate 62 surgical biopsies for the loss of glycogen and its variations in relation to cold ischemia, reperfusion, lobular zonation and donor's ages. We applied univariate, multivariate and discriminant analysis and logistic regression. There was a clear lobular zonation of glycogen during cold ischemia and at reperfusion. During cold ischemia, the mean loss was 48% in periportal zones and 74% in pericentrilobular zones. At reperfusion, it was in the range of 60% in periportal zones and 95% in pericentrilobular zones. It was observed in 64% of the grafts for an ischemia time less than 10 hr and in 82% of the grafts for an ischemia time of 10 hr or more. It was increased by 90% at reperfusion with pericentral predominance. Donors' age was an aggravating factor of glycogen loss beyond 28 years of age. In conclusion, in periportal zones, mean global glycogen depletion was about 54% during cold ischemia and reperfusion. It decreased by 90% at reperfusion with pericentral predominance. Logistic regression has allowed modelization of cold ischemia and reperfusion.

The purpose of this study was to localize HCV RNA in formalin-fixed paraffin-embedded liver biopsies of 15 patients with chronic hepatitis C using in situ RT-PCR method. The results were compared to serum and tissue extract analysis of HCV RNA. HCV RNA was detected in 80% of the sera tested, in 40% of the corresponding hepatic tissue extract and in 60% of the tissue sections tested by in situ RT-PCR. Compared to the serum positive cases, 67% of the cases were positive with in situ RT-PCR and 41% were positive with tissue extract detection. 50% of the cases in situ RT-PCR positive were also positive with tissue extract detection. These results underlined the complementarity of the different methods of viral detection for the precise diagnosis of hepatitis C.

We studied the hepatotoxic effect of heavy metals (cadmium, mercury, copper) on Mg2+ -ATPase, NADH diaphorase, succinic dehydrogenase and acid phosphatase of yellow-legged gull liver, using enzyme histochemical methods. The lysosomal enzyme activity of acid phosphatase was increased in all cases. However, the other enzyme activities appeared to be insensitive to the different metallic pollutants and to their respective levels, in contrast with literature experimental data showing plasma membrane and mitochondrial alterations. This controversy could be explained by the differences in dietary conditions and metal overloads. The molecular basis of the toxicities of metallic pollutants is discussed.

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content was studied using histochemical quantitative analysis. In most of the cases, this heterogeneous pattern of glycogen was observed after preservation and reperfusion. However, a 42% reduction of glycogen content, expressed as the ratio between stained surface and total surface of liver biopsies, was observed in biopsies after reperfusion. Moreover, both periportal and centrilobular hepatocytes showed a significant decrease in mean optical density after reperfusion (18% and 25%, respectively). The comparison of our results to early postoperative liver function tests and cold ischemia times showed no significant correlation (p<0.05).

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity was studied using histochemical methods. The characteristic heterogeneous lobular distribution pattern of glycogen and glucose-6-phosphatase was maintained after preservation and reperfusion. However, it appeared that glycogen content decreased in both periportal and centrilobular hepatocytes after reperfusion. The glycogen decrease was higher in periportal hepatocytes. Glucose-6-phosphatase activity was maintained after reperfusion in most of the cases in periportal hepatocytes. In centrilobular hepatocytes, more cases showed a decrease in enzyme activity. It is suggested that ischemia-reperfusion mainly affects the glycogen content in both periportal and centrilobular hepatocytes and that centrilobular glucose-6-phosphatase activity is more sensitive to ischemia-reperfusion injury than periportal hepatocytes.

We studied the effect of cyclosporine A on hepatic Ca2+, Mg2+-ATPase and F-actin on bile canalicular and basolateral membranes in rats fed either soyabean lecithin, or triacylglycerol enriched diet, or low fat diet. Ca2+, Mg2+-ATPase histochemical activity was not modified in lecithin-cyclosporine A group, whereas the activity was decreased in the other groups. The triacylglycerol-cyclosporine A group had the lower activity. The histochemical staining of F-actin was quite normal in lecithin-cyclosporine group but decreased in the other cyclosporine A treated groups. The lower staining was observed in the triacylglycerol-cyclosporine group. The alteration of Ca2+, Mg2+-ATPase and F-actin by cyclosporine A, related to cholestasis evidenced by a decrease in bile salt secretion, were prevented by dietary soyabean lecithin and amplified by dietary soyabean triacylglycerol.

Two different methods, fluorescent phallotoxins and immunohistochemical amplification systems were used to visualize actin in normal human hepatocytes. With fluorescent phallotoxins (NBD-phallacidin or rhodamine phalloidin), F-actin was distributed along the plasma membranes and at the bile canaliculi. With immunohistochemical methods (biotin-avidin, biotin-streptavidin, silver enhancement), actin was found at the same level, however a cytoplasmic staining was observed and discussed as G-actin localization.

Histochemical and cytochemical methods induce a loss of endoplasmic reticulum (ER) membrane integrity in hepatocytes. In order to evaluate the degree of ER membrane integrity, glucose-6-phosphatase (G6P-A) was localized in light and electron microscopy using glucose-6-phosphate (G6P) and mannose-6-phosphate (M6P) as substrates. In case of ER membrane alteration, M6P diffuses inside the ER and is hydrolysed by a non-specific phosphohydrolase. G6P and M6P hydrolysis was quantified with image analysis methods. In light microscopy, the ratio of reaction of M6P hydrolysis/G6P hydrolysis gave 75% of non specific reaction. In electron microscopic study this ratio was about 30%. These results showed that enzyme localization methods in electron microscopy produced less ER membrane alteration than light microscopic methods.

The glucose-6-phosphatase activity of periportal and perivenous human hepatocytes was studied with a quantitative method. The results obtained with histogram of light intensity distributions indicate that the enzyme reaction was 1.3 to 2.5 fold higher in periportal zone than in perivenous zone. The profiles of light intensity along portal----hepatic venous distances show a progressive decrease of enzyme activity with highest values in periportal hepatocytes.

A new case of primary biliary cirrhosis associated with sarcoidosis is reported in a 38 year-old woman. The diagnosis of sarcoidosis was based on the occurrence of cutaneous nodules, mediastinal lymph node enlargement, pulmonary reticular infiltrate, parotitis and diffuse arthritic pain, all disappearing after steroid therapy. Hepatic biopsy showed epithelioid-cell granuloma without necrosis. The diagnosis of primary biliary cirrhosis was made 6 years later as based on intrahepatic cholestasis associated with high titer of antimitochondrial antibodies and chronic destructive cholangitis. This association (5 other cases have already been reported) raises the problem of a possible link between the two diseases which could represent different patterns of a single immunological disorder.

With respect to a particularly demonstrative case of Leydig cell tumour with gynaecomastia in a 40 year old man, the authors review the current data in the literature concerning the clinical and biological features and treatment of this tumour. They pay particular attention to the classical anatomo-pathological features, the ultra-structure and the ultrastructural and histo-enzymatic appearances.

The authors compare the fusion of sheep fibroblasts induced by low multiplicities of infection using visna virus and by high concentrations of polyethylene-glycol. In the case of Visna virus cell fusion is of the endogenous type, while fusion induced by polyethylene-glycol is of the exogenous type. The ultrastructural features are discussed for each type of cell fusion. The main differences between the two systems involve the intracellular microfilaments and Golgi apparatus.

Visna virus induced cell fusion of sheep choroid plexus cells was explored in vitro. Fusion is early rapid, and of exogenous origin for multiplicities of infection equal to or greater than 2 UFP per cell; whereas fusion is slow, late-occurring and of endogenous origin for multiplicities of infection less than or equal to 0.75 UFP per cell.

Sizes for ovarian stroma cells in culture are according to a log normal distribution. Cell stimulation by H.C.G. promotes a significative decrease of these sizes.

During the first moments of the in vitro infection of Sheep choroid plexus cells with high multiplicities of infection of Visna virus, the authors observed: a strong development of GERL, some signs of an intensive protein synthesis and numerous filament bundles. This infection leads to an exogenous cell fusion.

To determine the cytochemical localization of glucose-6-phosphatase in the human hepatocyte, lead - based and cerium - based media were used. By studying the effects of systematic variation of the incubation medium components, the optimal experimental conditions were determined. The exclusive localization of the cytochemical reaction in the endoplasmic reticulum and nuclear envelope, together with the results of control experiments ensured that these findings could be correlated with the phosphohydrolase activity of the multicomponent glucose-6-phosphatase system.

Sheep choroid plexus cells infected with low multiplicities of infection of Visna Virus were stellate and had long and thin processes containing filaments and forming cytoplasmic bridges between adjacent cells. Enlargement of the bridges resulted in the formation of multinucleated cells. Some glycoproteins were clustered on filaments outside the cell. The cytoplasmic changes showed : an intensive protein synthesis; numerous mitochondria closely associated with filaments and some lysosomes and numerous vesicules near the plasma membrane.