Detection of bile salt-dependent lipase, a 110 kDa pancreatic protein, in urines of healthy subjects.

Bile salt-dependent lipase (BSDL), a 110 kDa glycoprotein secreted by the pancreatic acinar cells, participates in the duodenal hydrolysis of dietary lipid esters. Recent in vitro and in vivo studies demonstrated that the BSDL reaches the blood via a transcytosis motion through enterocytes, suggesting that this enzyme may play a role in vascular biology. Once in the blood, BSDL should be eliminated. We address the hypothesis that BSDL may be filtered by the glomerulus and eliminated in urines. Immunological methods and proteomic were used to detect and to characterize BSDL in urine. The immunoreactive form of BSDL was detected in urines of 36 male subjects devoid of renal failure. Proteomic demonstrated that the immunoreactive protein is BSDL. Experiments using a monoclonal antibody to the oncofetal glycoform of pancreatic BSDL suggested that the protein is not expressed by renal cells but originates from the pancreas via circulation. We demonstrate that under normal physiological conditions, BSDL, a high-molecular weight blood glycoprotein, can be filtered by the renal glomerulus to be eliminated in urines.

Immunohistochemical detection of C-100 hepatitis C virus antigen in formaldehyde-fixed paraffin-embedded liver tissue. Correlation with serum, tissue and in situ RT-PCR results.

We localized HCV C-100 protein in liver biopsies of 15 patients with chronic hepatitis C using immunohistochemistry. The results were compared to serum, tissue extract analysis of HCV RNA and in situ RT-PCR described in a previous study. HCV was detected in 80% of the sera tested, in 40% of the tissue extracts and in 80% and 60% of the tissue sections tested by immunohistochemistry and in situ RT-PCR respectively. Compared to the serum positive cases, 83% and 67% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR and 41% were positive with tissue extract detection. Compared to the tissue extract positive cases, 25% and 50% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR. Finally, 75% of the cases positive by immunohistochemistry were also positive by in situ RT-PCR. These results underline the complementarity of the different methods for the precise diagnosis of hepatitis C.

Evaluation of glycogen loss in human liver transplants. Histochemical zonation of glycogen loss in cold ischemia and reperfusion.

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.

Effect of ischemia-reperfusion on bile canalicular F-actin microfilaments in hepatocytes of human liver allograft: image analysis by confocal laser scanning microscopy.

We studied and quantified the effect of ischemia-reperfusion on hepatic F-actin on bile canalicular and basolateral membranes in human liver allografts by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was normal in liver hepatocytes before reperfusion but decreased significantly after reperfusion (by 25% of controls). These results indicate that hepatic F-actin alteration is produced during the reperfusion phase. This modification, probably induced by reactive oxygen species, could impair bile canalicular contraction and tight junction permeability and consequently bile secretion in the postoperative period.

Hepatitis C virus RNA detection by in situ RT-PCR in formalin-fixed paraffin-embedded liver tissue. Comparison with serum and tissue results.

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.

Quantitative analysis of glycogen content in hepatocytes of human liver allograft after ischemia and reperfusion.

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).

Hepatotoxic effect of metallic pollutants on enzyme histochemical activities of yellow-legged gull Larus cachinnans michahellis liver.

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.

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.

A quantitative immunocytochemical study of Na+,K+-ATPase in rat hepatocytes after STZ-induced diabetes and dietary fish oil supplementation.

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+,K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+,K+-ATPase catalytic alpha1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic alpha1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the alpha1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+,K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.

Effect of ischemia-reperfusion on the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity in human liver allograft.

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.

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.

Time-related changes of cold-stored rat liver in University of Wisconsin solution. Effect of ursodeoxycholate.

Livers of Wistar rats were stored between 0 and 36 hrs. in the University of Wisconsin preservation liquid in order to determine time-related biochemical and morphological hepatic changes. Ursodeoxycholate (100 microM) was also added in the medium to test the hepatoprotective properties of the bile salt. Biochemical assays were performed on hepatic microsomes, plasma and biliary canalicular membranes. Protein and lipid composition of the microsomal and baso-lateral plasma membranes remained stable. Protein and cholesterol content of the biliary canalicular membranes decreased, phospholipid/cholesterol ratio increased between 0 and 36 hrs.; it resulted in a leak of 5'-nucleotidase and leucine amino peptidase activity of these biliary canalicular membranes, especially up to 12 hrs. Between 0 and 36 hrs., the lipid and protein content remained stable in the plasma membranes, as well as both tested enzymatic activities. Observations under electron microscopy showed alterations and underlined fragility of the bile canaliculi, particularly after 24 hrs. preservation. Ultrastructure of sinusoidal membranes showed damaged microvilli. Endoplasmic reticulum remained unchanged, in relation to the stability of the microsomal lipidic, proteic content and hydroxymethylglutaryl-coenzyme A reductase activity, except the decreased protein content after preservation for 36 hrs without ursodeoxycholate. Ursodeoxycholate by itself did not protect against the described disturbances.

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.

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.