Effect of dietary lipid (soybean lecithin and triacylglycerol) on hepatic F-actin microfilaments in cyclosporine A-treated rats: image analysis by confocal laser scanning microscopy.

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.

Effect of dietary lipids on hepatic Na+,K(+)-ATPase in cyclosporine A-treated rats: immunocytochemical analysis of alpha1 subunit by confocal laser scanning microscopy imaging.

We studied the effect of dietary soybean lecithin or triacylglycerol on hepatic Na+,K(+)-ATPase in cyclosporine A-treated rats by means of quantitative immunocytochemistry. Cyclosporine A-treated rats were fed lecithin or a triacylglycerol-enriched diet or a low-fat diet. As a control, one group was only fed the low-fat diet; the three other groups were treated with cyclosporine A solvent and received the low fat, lecithin, or triacylglycerol diet. Bile canalicular staining significantly decreased in all cyclosporine A-treated groups with the higher values in lecithin-fed rats. In basolateral membranes, no decrease was observed in the lecithin-cyclosporine group, in contrast to the other groups. The triacylglycerol-cyclosporine group had lower values in both membrane domains. The alteration of Na+,K(+)-ATPase by cyclosporine A was related to cholestasis evidenced by a decrease in bile salt secretion. These modifications were prevented by dietary soybean lecithin and amplified by dietary soybean triacylglycerol.

Modification of Ca2+, Mg2+-ATPase and F-actin distribution in hepatocytes of cyclosporine A treated rats. Effect of soyabean lecithin and triacylglycerol.

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.

Analysis by confocal laser scanning microscopy imaging of undilated bile canaliculi F-actin staining in the hepatocytes of human extrahepatic cholestatic liver.

Many studies have demonstrated the role of bile canalicular microfilaments in bile secretion and bile flow. It is now admitted that modification of bile canalicular network of microfilaments play a role in dysfunction of bile secretion observed in many cases of cholestasis. This work intends to study F-actin, a major component of microfilaments, in human hepatocytes in extrahepatic cholestasis. Normal and extrahepatic cholestatic liver were studied. F-actin was stained with fluorescent phallotoxin and quantified by using confocal laser scanning microscopy and an image analysis method. Mean specific fluorescence (MSF) of bile canaliculi was measured. Since dilated and bile plugged canaliculi were rarely observed in cholestatic liver sections, only undilated bile canaliculi were analysed. Bile canalicular MSF was significantly increased (p < 0.05) in cholestatic hepatocytes (1.3 to 1.7 fold higher than in controls). These data demonstrate a pericanalicular thickening of F-actin microfilaments in human extrahepatic cholestatis, similar to that described in literature in many cases of human intrahepatic and extrahepatic cholestasis cases as well as in experimentally induced cholestasis. However, further studies are needed to understand this increase in F-actin pericanalicular microfilaments in human extrahepatic cholestasis.

Altered compartmentalization of transforming growth factor-beta in asthmatic airways.

BACKGROUND: Asthma is characterized by alterations of the bronchial epithelium associated with inflammatory cell infiltrates and sub-epithelial fibrosis. Transforming Growth Factor-beta (TGF-beta) is an anti-inflammatory and fibrosing cytokine normally present in bronchial epithelial cells and also potentially produced by inflammatory cells. Thus, TGF-beta could play a role in the asthmatic process, and its expression could be modified in asthmatic airways. OBJECTIVE: To test this latter hypothesis, we studied the bronchial distribution of TGF-beta in asthmatic patients. METHODS: TGF-beta 1, 2, 3 distribution was studied by immunohistochemistry in bronchial biopsies from 12 asthmatic patients and 10 non-asthmatic subjects. RESULTS: Bronchial epithelial cells from asthmatics were negative or faintly positive while a bright staining was detected in these from non-asthmatics (P < 0.01). In both groups, when inflammatory cells were present beneath the basement membrane, they were stained by the anti-TGF-beta antibody. CONCLUSION: This study shows an altered compartimentalization of TGF-beta in asthma. (a) TGF-beta is scarse in asthmatic bronchial epithelial cells, which could favour the perennization of the bronchial inflammation, and (b) TGF-beta is present in inflammatory cells beneath the basement membrane, where it could be involved in the frequent sub-epithelial fibrosis.

Immunohistochemical detection of hepatitis C virus related C100-3 and core antigens in formalin-fixed liver tissue.

HCV C100-3 non-structural and core proteins have been detected by immunohistochemical methods on paraffin-embedded tissue sections using monoclonal antibodies in 22 cases of chronic hepatitis C. C100-3 protein was detected in cytoplasm and nuclei of hepatocytes whereas core protein was only detected in nuclei. The specificity of the nuclear localization of HCV antigens was discussed in relation to cross-reactivity of the anti core antibody with host-derived GOR antigen.

Balance between alveolar macrophage IL-6 and TGF-beta in lung-transplant recipients. Marseille and Montréal Lung Transplantation Group.

Acute inflammation in the lung is characterized by a phase of tissue injury followed by a phase of tissue repair. When the latter is excessive, fibrosis occurs. Alveolar macrophages (AM) can produce cytokines involved in both phases of acute lung inflammation, notably interleukin-6 (IL-6), involved in injury and transforming growth factor-beta (TGF-beta), mediating repair. We hypothesized that AM were activated in both phases, and studied IL-6 and TGF-beta production by AM during complications of lung transplantation, acute rejection (AR), and cytomegalovirus pneumonitis (CMVP). In addition, we analyzed these cytokines in bronchiolitis obliterans (BO), a fibrotic complication of lung transplantation linked to previous AR and CMVP. At the onset of AR and CMVP, IL-6 secretion increased, whereas AM TGF-beta content was increased, but not its secretion. In contrast, with time, IL-6 reached control value whereas TGF-beta secretion rose significantly. In BO, IL-6 was not oversecreted, but TGF-beta increased, notably before functional abnormalities occurred. These results show that during acute complications of lung transplantation, AM display an early activation with oversecretion of IL-6, which is involved in tissue injury, counterbalanced by a late activation in which TGF-beta predominates, mediating tissue repair. The results provide new insights into the pathogenesis of BO, which is linked to acute complications of lung transplantation through this biphasic AM activation.

Immunocytochemical study of NA+ K(+)-ATPase alpha 1 and beta 1 subunits in human and rat normal hepatocytes using confocal microscopy.

Hepatic Na+ K(+)-ATPase was recently shown to be composed of two alpha 1 and beta 1 subunits similar to that of kidney. Its localization on hepatocyte plasma membranes was not clearly established. We have studied the localization of alpha 1 and beta 1 subunits using immunocytochemical method and confocal microscopy. In accordance with previous cytochemical findings, the catalytic alpha 1 subunit was distributed in basolateral and bile canalicular membranes of hepatocytes. The beta 1 subunit was not demonstrated, due to its very low amount in the liver. The controversy about the bile canalicular localization was discussed.

Histoenzymatic study of human renal tissue preservation: II--Catalase activity in proximal tubular cells is uncorrelated with transplant evolution.

Enzyme histochemical activity of catalase, a peroxisomal enzyme involved in cellular antioxidant systems, was studied in proximal tubular cells of human renal transplants as a marker of ischemia-reperfusion injury in the prediction of the evolution of renal transplants. A low enzymatic activity was observed in all renal biopsies performed at 30 min. reperfusion with no difference between the several evolution types of renal transplants. Reduced catalase activity due to ischemia-reperfusion injury could not be correlated with renal function or used as an index of renal function recovery.

Histoenzymatic study of human renal tissue preservation: I--Proximal tubular glucose-6-phosphatase is correlated with transplant evolution.

Glucose-6-phosphatase was tested histochemically as a gluconeogenesis marker of ischemia-reperfusion injury of proximal tubular cells in human renal transplants. Histochemical enzyme activity, histology and transplantation conditions (preservation solution, cold and warm ischemia time, donor age), were compared to renal transplant evolution. Neither histology nor transplantation conditions were correlated with renal transplant evolution. Only glucose-6-phosphatase activity was significantly correlated with transplant evolution and could be used as a more sensitive marker than histology for the detection of ischemia-reperfusion injury of proximal tubules.

Microscopic localization of mercury-selenium interaction products in liver, kidney, lung and brain of Mediterranean striped dolphins (Stenella Coeruleoalba) by silver enhancement kit.

Microscopic observation, using physical development of silver, was carried out to localize the mercury-selenium interaction products in the organs of Mediterranean striped dolphins. The silver-metal reaction products were located mainly in hepatocytes and macrophages for liver, in proximal tubules for kidney. They were less abundant in lung than in liver and kidney. The result of semi-quantitative histochemistry tests showed that silver staining deposits were more abundant at relatively high metal concentrations than low metal contents, but independent of the metal contents. Comparisons with the most concentrated metal contents suggested that there might be a new complex of mercury and selenium, which could not be stainable by physical silver development.

Localization of myosin in normal human liver hepatocytes using immunohistochemical amplification methods.

Different immunohistochemical amplification systems were used to visualise myosin in normal human hepatocytes. With biotin-streptavidin-peroxidase and immunogold silver staining, myosin was distributed along the plasma membranes and at the bile canaliculi. With biotin-streptavidin-rhodamine, myosin was mainly found at the bile canaliculi level, however with confocal microscopy, the plasma membrane fluorescent staining was more apparent. The staining pattern of myosin appeared to be similar to that of actin in normal human hepatocytes.

Nuclear immunostaining of hepatitis C infected hepatocytes with monoclonal antibodies to C100-3 nonstructural protein. Comparison of immunogold silver staining with other immunohistochemical methods.

Immunohistochemical methods have been used to localize an HCV antigen on paraffin embedded liver tissue sections by means of monoclonal antibodies to C100-3 nonstructural protein. Peroxidase-antiperoxidase, alkaline phosphatase-antialkaline phosphatase, biotin-streptavidin-peroxidase and immunogold silver staining methods showed a nuclear staining of the hepatocytes in cases of chronic hepatitis with positive HCV serology, alcoholic liver disease and hepatocarcinoma. No cross reactions were observed with viral hepatitis B and delta antigens. The strongest reaction without background staining was obtained with immunogold silver staining. Nuclear localization was compared to the cytoplasmic staining described in the literature.

Localization of actin in normal human hepatocytes using fluorescent phallotoxins and immunohistochemical amplification.

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.

Loss of endoplasmic reticulum membrane integrity: an image analysis of the glucose-6-phosphatase system in human hepatocyte.

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.

Quantitative histochemical study of glucose-6-phosphatase in periportal and perivenous human hepatocytes.

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.

Image analysis for histochemical study of glucose-6-phosphatase inactivation by diethyl pyrocarbonate in normal human liver.

Glucose-6-phosphatase (G6Pase) is a multicomponent system that catalyzes G6P hydrolysis. To determine the specificity of the histochemical reaction of G6Pase, we investigated the inhibitory effect of diethyl pyrocarbonate (DEPC), a specific and very effective inhibitor of the phosphohydrolase component of the G6Pase system, in normal human liver. The inactivation of the histochemical enzymatic activity by DEPC was monitored by determining the mean brightness of the microscopic image and the histogram of light intensity distributions. The results obtained indicate that the histogram is more sensitive than the mean brightness to variations of enzymatic activities, and that the percent of pixels brighter than a convenient level is directly proportional to DEPC concentration. This study indicates that DEPC can be used as an efficient inhibitor of the histochemical reaction of G6Pase.

Cytochemical demonstration of glucose-6-phosphatase in human liver.

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.