Cell-density-dependent expression of Cyp1a2 gene in monolayer-cultured adult mouse hepatocytes

The effects of PAF antagonists, of substances which influence the arachidonic acid metabolism, and of dexamethasone and ketotifen were evaluated in an acute PAF-induced mortality model in female NMRI mice. We established a dependence of sensitivity to PAF on strain (AB mice showed no dose dependence) and on sex of the animals as well as on the PAF charges used in our experiments. PAF produced resistance in surviving animals against the PAF-induced death on repeated application. The PAF antagonists, WEB 2170 and WEB 2086, provided the best dose-dependent protection against PAF toxicity, followed by dexamethasone, by the COX/LOX synthetase inhibitor X 86 (a BW 755 C-analogue) and by the PAF receptor antagonist BN 52021. Particularly remarkable was the excellent prevention by aspirin. Aspirin may not only inhibit the cyclooxygenase pathway but also endogenous PAF synthesis. Other drugs, i.e. indomethacin, the thromboxane receptor antagonist, BM 13177, the thromboxane synthetase inhibitor, HOE 944, as well as the lipoxygenase inhibitors (NDGA, esculetin, SHAM and phenidone) exerted a dose-dependent protection only at high doses.     

Effect of triamcinolone on the ultrastructure of mouse hepatocytes

Premature female mice were injected daily with 1.5 mg/kg body weight of triamcinolone diacetate for 10 and 21 consecutive days. Electron-microscopic study of hepatocytes indicated the formation of 'giant' mitochondria concomitant with a marked increase in the number and size of lipid droplets. Hypertrophied mitochondria and enlarged lipid droplets were found to be in a close topographical association. The mechanism of mitochondrial enlargement is discussed as well as the latters' role in the hepatocytes' lipid metabolism.     

Hepatic stimulator substance administration enhances regenerative capacity of hepatocytes in cadmium-pretreated partially hepatectomized rats

The liver is of central importance in the metabolism of essential and toxic metals such as cadmium (Cd). Cd pretreatment suppressed the regenerative capacity of hepatocytes, which normally occurs 24 hr after partial hepatectomy, due to the inhibition of the activity of the enzyme thymidine kinase. The effect of hepatic stimulator substance (HSS) administration (10, 20, and 40 mg protein/kg body weight) on hepatocyte proliferation was investigated in Cd-pretreated partially hepatectomized rats. HSS administration partly restored the suppressed hepatocyte DNA biosynthesis in Cd-pretreated partially hepatectomized rats. The hepatocyte mitotic activity and the percentage of proliferating cell nuclear antigen-positive nuclei were in accordance with the liver proliferative status. The administration of HSS did not affect in a statistically significant manner the activity of the enzyme thymidine kinase in Cd-pretreated partially hepatectomized rats. It is suggested that the administration of HSS ameliorates the diminished hepatocyte regenerative response to partial hepatectomy in this model of acute liver injury, due to Cd intoxication.     

The regenerative potential of oxytalan fibers. An experimental study in the monkey

Although a number of studies have described the oxytalan fibers as being a natural component of the periodontal ligament, little information exists about the regenerative potential of these connective tissue fibers. The aim of the present study was to examine whether oxytalan fibers have the capacity to reform after regenerative periodontal therapy. Intrabony defects were produced surgically at the mesial aspects of teeth 37, 35, 45, 47 and at the distal aspects of teeth 11, 21, 31, 41 in one monkey (Macaca fascicularis). After 3 months, the defects were exposed using a full-thickness flap procedure. The root surfaces were debrided and subsequently PDGF-growth factors were placed in the defects. 4 of the 8 sites were covered with a bioresorbable membrane before closure of the wound. Post-surgically, antibiotics were given systemically for 1 week, and tooth cleaning was carried out 1x a week during the entire experimental period. After 5 months, the animal was sacrificed and the oral tissues were fixed by perfusion with 10% buffered formalin. Specimens containing the defects and surrounding tissues were dissected free and histological sections were cut in the mesio-distal direction, parallel to the long axes of the teeth. The sections were stained with hematoxylin and eosin or with the oxone-aldehyde-fuchsin-Halmi staining method and subsequently examined in the light and in the electron microscope. The results revealed that new oxytalan fibers oriented mainly in an apico-occlusal direction had developed in the regenerated periodontal ligament. Many of the newly-formed fibers were inserted into the new cementum, thus suggesting a strong relationship between this tissue and the oxytalan fibers. It is concluded that the regenerated periodontal ligament connective tissue formed after surgery contains oxytalan fibers similar to those present in the original tissue. These results demonstrate that oxytalan fibers develop de novo in the newly-formed periodontal ligament.     

Internalin B promotes the replication of Listeria monocytogenes in mouse hepatocytes

The uptake of Listeria monocytogenes by a variety of cell types in vitro is facilitated by the protein products of the inlAB (internalin) operon expressed by the organism. In the case of mouse hepatocytes, the extent to which inlAB expression influenced the uptake of Listeria in vitro was markedly dependent upon the ratio of bacteria to cells. At a ratio of 100:1, greater than 40-fold fewer transposon-induced inl4B mutant listeriae entered hepatocytes compared to the isogenic wild-type control; the difference was only fourfold, however, in cultures inoculated at a 1:1 ratio. Similarly, the uptake of in-frame inlB or inlAB deletion mutants differed only fourfold from the uptake of wild-type or inlA mutant Listeria at a 1:1 multiplicity of infection. Mutations affecting inlB or inlAB, on the other hand, resulted in a marked decrease in the capacity of Listeria to proliferate within mouse hepatocytes in vivo and in vitro. Electron micrographs of Listeria-infected hepatocytes demonstrated the impaired capacity of inlB mutants to escape from endocytic vacuoles and to enter the cytoplasm where proliferation occurs. These findings indicate that the protein product of inlB exerts a significant effect on the intracellular replication of Listeria.     

Effects of clofibrate withdrawal on peroxisomes in mouse hepatocytes

Adult male mice (NMRI strain) were initially treated with 0.5% clofibrate in the diet during four days to obtain a high level of peroxisomes in the hepatocytes. Groups of animals were then given control diet for one, two, three, or four days. Liver samples were cytochemically stained for catalase prior to examination by electron microscopy and subsequent morphometric analysis. Various enzyme activities were assayed in liver homogenates. A sixfold increase in peroxisomal area was found after four days of clofibrate feeding compared with untreated animals. The peroxisomal fractional area was unchanged after one day of refeeding control diet but was reduced with over 50% after two days on control diet and was similar to that in the untreated animals after four days. The size distribution of peroxisomes shifted to smaller values during the breakdown of peroxisomes and was similar to the untreated animals after four days of clofibrate withdrawal. During this process a more heterogeneous staining of peroxisomes was observed. The mitochondrial fractional area was not affected by the treatments, but a shift in size distribution to smaller values was observed in the most induced animals. Protein content was not affected by the treatments. The activity of catalase (twofold induction) decreased progressively to control values during recovery but enoyl-CoA hydratase activity (fourty-fold induction) decreased rapidly. NADPH-cytochrome c reductase activity (twofold induction) decreased rapidly to control levels. Citrate synthase activity was not affected by clofibrate treatment but decreased during recovery. Acid phosphatase activity (repressed) increased to control levels. Cathepsin D activity increased somewhat during recovery while proteolytic activity (towards casein) decreased transiently on control diet.     

Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.     

Intraperitoneal transplantation of isolated hepatocytes of the pig: the implantable bioartificial liver]

To elucidate the histopathological features of pancreatic ischemia, we examined postmortem pancreases in which cholesterol emboli were present. Cholesterol emboli were detected in 17 pancreases (6 of 36 cases of aortic aneurysm and 11 of 223 control cases). Two of the 17 pancreases had well-demarcated patchy lesions composed of degenerating acinar cells showing deeply eosinophilic cytoplasm and pyknotic nuclei, indicating fresh ischemia. In the marginal zone of the larger lesions and in the small lesions, the intralobular ductules had avoided the ischemic changes. Five of the 17 pancreases had patchy fibrotic foci containing small ductules with slightly retraction features. These ductules are considered to be the remnant intralobular ductules that have avoided the previous ischemic damage. We conclude that these patchy fibrotic foci are the healed ischemic lesions. The current findings suggest that the healed ischemic lesions can be differentiated from common pancreatic fibrosis. The existence of remnant intralobular ductules and the patchy retraction features may be useful histological markers for the determination of healed ischemic lesions.     

Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.     

Altered differentiation of hepatocytes in a transgenic mouse model of hepatocarcinogenesis

Transgenic mice carrying the SV40 T antigen (TAg) gene, which develop hepatocellular and biliary cell tumors by 4 mo of age, show ductular structures in the neonatal liver. Coexpression of c-myc with TAg increases the extent and persistence of ductular lesions and also accelerates tumor development. To analyze possible links between altered gene expression and cell differentiation and to determine the relationship between the ductular structures and tumor development in these mice, ductular cells in single (TAg) and bitransgenic (TAg x c-myc) mice were characterized for biliary and hepatocellular differentiation, transgene expression, and proliferation activity. The results show that the ductular cells in these transgenic mice have characteristics of biliary cells, including basement membrane formation, positive laminin staining, and bile duct-specific lectin (Dolichos biflorus agglutinin and peanut agglutinin) binding, and characteristics of hepatocytes, including albumin expression and ultrastructural features such as round nuclei with 1 or 2 nucleoli and well-developed cytoplasmic organelles. However, differences in transgene expression and cell proliferation between the ductular cells and nonductular hepatocytes were not apparent. Thus, the ductular cells could not be defined as tumor progenitor cells in these mouse livers. However, this model suggests that manipulation of gene expression can alter differentiation of hepatic parenchymal cells.     

A technique for liver transplantation in the inbred mouse

Action potentials of isolated Purkinje fibres and ventricular muscle fibres of canine hearts treated with ouabain and 16-epi-gitoxin were recorded by microelectrodes. Under the influence of both glycosides, Purkinje system fibres became inexcitable earlier than ventricular muscle fibres. Being exposed to 0.125 muM ouabain and 2.5 muM 16-epi-gitoxin, both Purkinje and ventricular muscle fibres became poisoned in the same time as ventricular muscle fibres exposed to 0.1 muM ouabain and 1.5 muM 16-epi-gitoxin. At the lower 16-epi-gitoxin concentration Purkinje fibres had 2.5 times the survival time of that exposed to the lower ouabain concentration. Compared to 16-epi-gitoxin the inexcitability of Purkinje fibres after ouabain remained irreversible. The semisynthetic glycoside 16-epi-gitoxin exerts a weaker effect on the specialized conducting system.     

Isolation and long-term maintenance of differentiated adult chicken hepatocytes in primary culture

Adult chicken hepatocytes were obtained by an adaptation of the two step in situ collagenase perfusion. Usually 0.5 to 1 x 10(9) cells were obtained, with 75 to 95% viability. Hepatocytes attached within 2 h when plated on plastic cell culture dishes and spread in 4 h, surviving for several months in a specific serum-free medium. These cells retained a typical parenchymal cell morphology and the ability to produce a specific protein (albumin) throughout the culture period. We hereby provide a suitable model for studying hepatic metabolism in birds.     

Treatment of IFN-gamma transgenic mice with anti-IFN-gamma reveals the remodeling capacity of the adult pancreas

Pancreatic expression of gamma-interferon (IFN-gamma) initiates a cascade of pathogenic changes that include pancreatic inflammation, islet cell destruction, hyperglycemia, and islet regeneration. In this study, we explore the developmental plasticity of the adult pancreas and particularly its ability to return to normoglycemia and to remodel itself from an advanced pathogenic state. This was approached by treating adult transgenic mice with a pulse of anti-IFN-gamma antibody and determining the functional and morphological status of the pancreas. We demonstrated that anti-IFN-gamma antibody administration led to the reduction of hyperglycemic blood glucose levels in transgenic mice. We also observed that the pancreas returned from a profoundly perturbed state toward normality. Analysis of the mitotic index indicated that cell proliferation previously associated with islet cell regeneration was greatly reduced after anti-IFN-gamma administration. Our results highlight the ability of the adult pancreas to remodel itself and return from a complex pathological state to normalcy once the trophic signal inducing this pathology is removed. These data also suggest that anti-IFN-gamma administration may have important clinical implications for treatment of chronic pancreatitis in humans.     

Morphological investigation on phenylhydrazine-induced erythropoiesis in the adult mouse liver

In adult mice suffering from a phenylhydrazine (PHZ)-induced hemolytic anemia, erythropoietic islands were observed in the liver. These islands were studied with the light and electron microscope. Within two days after the beginning of four daily injections of PHZ, erythoid elements appeared in the sinusoids and central veins. A maximum number of erythroblasts was found on day 7. Light and electron microscopic observations revealed that the erythropoietic islands consisted of centrally located macrophages(CM) with a Kupffer cell-like morphology, surrounded by erythroblasts, which were often of the same maturation stage. CM in central veins (CM-V) and in sinusoids (CM-S) were found to have a different morphology. The CM-V phagocytized less circulating red blood cells and were in contact with a smaller number of erythroblasts. Furthermore, the contact areas between erythroblasts and CM-S extended for a much longer distance than those between erythroblasts and CM-V. The progenitor cell for the CM-V is most likely a monocyte, since cells which were morphologically determined as monocytes were found to appear on the first day of the PHZ treatment and differentiated into macrophages within about 2 days. The origin of the CM-S population was less clear, but could be monocytic as well. These data are tentatively explained as a migration of a progenitor of a cellular component of the erythroid micro-environment into the liver after appropriate stimuli. In contrast to fetal liver erythropoiesis, erythroblasts in the adult liver occurred only incidentally extrasinusoidally. Furthermore, specialized membrane contacts between erythroblasts and CM or hepatocytes could not be observed in adult liver. Ferritin could not be detected on the erythroid cell membrane or located in coated vesicles. Also, no ferritin could be observed within or attached to the finger-like processes of CM. The observations suggest that the coated vesicles in erythoid elements are partly exocytotic vesicles and are not specific for ferritin transport. The morphological aspects of PHZ-induced extramedullary erythropoiesis is discussed in relation to the hemopoietic microenvironment.     

Sex difference in immunostaining of RET in the adult mouse kidney

1. Primary and secondary resistance to the widely used antimetabolite 5-fluorouracil (5-FU) are common phenomena in cancer chemotherapy. Because 5-FU still remains the agent of choice in the treatment of, for example, colorectal cancer, circumvention of resistance is of vital importance. 2. Resistance to fluoropyrimidines is a multifactorial event, which includes transport mechanisms, metabolism, molecular mechanisms, protection from apoptosis, and resistance via cell cycle kinetics. To date, the prediction of primary resistance to 5-FU in the clinic is limited to few studies focusing mainly on the key enzyme thymidylate synthase. To gain a deeper insight into the key events responsible for 5-FU resistance in vivo, the evaluation of additional parameters such as other (fluoro)pyrimidine converting enzymes, the mutational status of regulators of apoptosis, and tumour angiogenesis is currently under investigation. 3. Most studies on the circumvention of fluoropyrimidine resistance refer to preclinical investigations and were rarely confirmed in clinical trials. Although our understanding of resistance to 5-FU leaves many open questions, the fundamental insights accomplished during the last years provide a rational understanding to exceed the bounds of the actual therapeutic schedules.     

Intestinal transplantation. The first Swedish transplantation of the small intestine in an adult patient with pseudoobstruction

Recent advances, first and foremost the development of new immunosuppressive agents, have markedly improved the outcome of intestinal transplantation, which is a treatment option for patients with serious intestinal diseases who have become dependent on total parenteral nutrition. The first small bowel transplantation in Sweden was performed at Huddinge Hospital in 1997, in the adult patient with intestinal pseudo-obstruction. The article reports the course of this patient and an update of international progress in intestinal transplantation.