In vivo infection of ramified microglia from adult cat central nervous system by feline immunodeficiency virus.

Infection of microglial cells by the human immunodeficiency virus (HIV) is supposed to play an important role in the pathogenesis of AIDS-related central nervous system (CNS) complications. So far, however, experimental data about interactions between HIV and ramified microglia from the adult CNS were only occasionally reported, making it difficult to understand the exact nature of pathogenic events contributing to HIV-encephalopathy. Therefore, we used the animal model of feline immunodeficiency virus (FIV) infection of domestic cats to establish an experimental system which is suitable for studying the relationships between an immunodeficiency virus and the mature ramified microglia of the central nervous system. By means of density gradient centrifugation approximately 95% pure microglial cells could be isolated from adult feline brain that were characterized by their CD45(low) phenotype. Resident microglia extracted from the CNS of experimentally infected cats harbored FIV-specific DNA and cocultivation with mitogen-activated, but uninfected peripheral blood mononuclear cells (PBMC) resulted in recovery of high-titered infectious virus. Double labeling of brain cell monocultures explanted from persistently infected animals for both microglia and FIV markers disclosed less than 1% of viral antigen expressing microglial cells. This suggests that during the subclinical phase of the infection only a small number of brain-resident macrophages are productively infected. However, interaction of FIV-infected microglia and inflammatory lymphocytes may promote viral replication, thus supporting viral spread in brain tissue.

Aspirin and heparin prevent hepatic blood stasis and thrombosis induced by the toxic glycoprotein Bolesatine in mice.

Bolesatine is a toxic glycoprotein isolated from Boletus satanas Lenz, which inhibits protein synthesis in vivo and in vitro. The LD50 (24 h) is 1 mg /kg bw (i.p.), in mice and rats. When given i.p. to mice (0.1 - 1.0 mg/kg bw) bolesatine induced thrombi and blood stasis in the liver, 5 - 21 h after injection, and modifications of the number of blood corpuscles in peripheral blood. These effects were efficiently reversed by aspirin, ticlopidin and heparin (as attested by histology and electron microscopy) which however failed to prevent death in animals given lethal doses. Together, these results showed that the death of bolesatine poisoned animals given high doses, was rather due to a combination of thrombosis and other toxic effects. In addition, they suggest that these antithrombotic drugs may overcome cases of human poisoning, with low exposures of this boletus, showing a hypertension probably due to mechanical obstruction which resists normal therapy.

In vitro infection of peripheral blood mononuclear cells by hepatitis C virus.

To study the in vitro susceptibility of peripheral blood mononuclear cells (PBMC) to hepatitis C virus (HCV), we incubated cells from healthy donors with HCV-positive sera. Using RT-PCR and in situ hybridization, the genomic viral RNA was detected in PBMC and in their supernatants until 25 days post-incubation. The PBMC of the different donors were not all permissive to HCV, but results were more constantly positive when cells from four donors were pooled. Quantification of the genomic viral RNA by the branched-DNA assay showed a decrease in the HCV RNA concentration during the first week of culture followed by a peak during the second or third week, and also an increase in the total amount of viral RNA in the inoculated cells. Although HCV RNA could be detected in the supernatants by RT-PCR, the concentration was very low. Using a sense-specific RT-PCR method, the HCV negative-strand was also detected in the cells but not in the supernatants. In two experiments PBMC were successfully infected using HCV-positive culture supernatants, therefore suggesting that infectious particles can be produced in this system. Our findings demonstrate that PBMC are permissive for HCV replication in vitro but the replication level is very low. The HCV RNA concentration measured in PBMC of 10 chronically infected patients was not significantly higher than the maximal concentration obtained in PBMC infected in vitro.

Mouse hepatitis virus type 3 infection provokes a decrease in the number of sinusoidal endothelial cell fenestrae both in vivo and in vitro.

Fenestrations of hepatic endothelial cells play an active role as a sieving barrier allowing extensive exchange between the blood and liver parenchyma. Alteration of these structures may be induced in the course of various pathological events and provoke important perturbations of liver function. We demonstrate here that sinusoidal endothelial cells are permissive for mouse hepatitis virus 3 (MHV3) in vivo and in vitro and that this infection leads to a striking decrease in the number of fenestrae. The disappearance of these structures observed under scanning electron microscopy or in cryofracture preparations in vivo and in vitro cannot be reversed by the action of cytochalasin B on the microfilament network. The decrease in the porosity seems to be related directly to the productive infection of the endothelial cells, because it was not observed in A/J mice resistant to the virus and in susceptible BALB/c mice immunized with a thermosensitive mutant in which no viral replication occurs. In conclusion, a viral infection of liver endothelial cells may cause extensive loss of the fenestrations and thus lead to important functional pertubations.

Evidence of feline immunodeficiency virus replication in cultured Kupffer cells.

OBJECTIVE: To determine if cultured feline Kupffer cells (KC) are as permissive for feline immunodeficiency virus (FIV) as cultured human liver macrophages are for HIV. Two types of infection likely to be relevant to the in vivo situation were used. KC were infected with either free virus or autologous infected peripheral blood mononuclear cells (PBMC). METHODS: Feline KC were isolated by centrifugal elutriation from collagenase-perfused liver; cultured cells were characterized by their morphological appearance and their erythrophagocytotic properties. After infection, viral replication was measured by enzyme-linked immunosorbent assay, reverse transcriptase activity, immunofluorescence assay, in situ hybridization and electron microscopic observations. RESULTS: Three days after isolation, 85% of cultured KC were able to internalize red blood cells; 45% were CD4-positive and 65% expressed a 24 kD protein thought to be a receptor for FIV (CD9). After the addition of autologous infected PBMC or cell-free supernatant of chronically infected IRC4 cells to KC cultures, a peak of viral replication was detected at day 28. Antigen revealed by immunofluorescence assay was present in only 0.4%, and viral RNA was detected by in situ hybridization in 2% of the infected cells. CONCLUSIONS: FIV can replicate in cultured feline KC without inducing any cytopathic effect, which suggests that these cells may play a role in the physiopathology of FIV infection.

Modification of the amount of cholesterol in hepatic steatosis induced in susceptible and resistant mice infected with MHV3: a biochemical and ultrastructural study.

A mouse hepatitis virus-3 strain subcultured in our laboratory is a unique experimental model in which to study virus-induced liver steatosis. This strain produces massive lipid deposition not only in sensitive adult BALB/c mice but also (though less extensive) in virus-resistant adult A/J mice. Biochemical determinations have shown that this steatosis is characterized by an increased amount of neutral lipids (sterols and triglycerides) in infected livers of BALB/c mice and by a smaller increase in those of A/J mice. However, the relative percentage of cholesterol and triglycerides is similar in both strains. Liver phospholipid content was significantly decreased in both strains of mice. To discriminate between cytoplasmic and membrane cholesterol content in different types of liver cells, an ultrastructural study was performed with filipin, a specific cholesterol marker. This study shows on one hand an important increase in the cholesterol in the hepatocytes of BALB/c mice and a smaller increase in those of A/J mice, in agreement with biochemical data. However, marked cholesterol decrease and abnormal cholesterol distribution were observed in the endothelial liver cells of infected BALB/c mice. This decreased cholesterol content probably led to higher fluidity of these membranes, which could be related to the important drop in the number of endothelial cell fenestrae observed after mouse hepatitis virus-3 infection. Because in A/J infected mice neither a decrease in the amount and distribution of cholesterol nor decreased fenestration were observed in endothelial liver cells, these findings could be correlated with the resistance of these mice to the infection.

Altered pathogenicity in the liver induced by a mouse hepatitis virus type 3 thermosensitive mutant.

Intraperitoneal inoculation into sensitive BALB/c mice of D85, a thermosensitive (ts) mutant, provokes acute hepatitis followed by recovery of the mice. The ts mutant was able to replicate in the liver. However, the maximal viral titre was obtained 2 days later than was the case with the wild-type (wt) MHV 3 infection; the viral antigens remained localized within small foci and no invasion of the entire liver was observed. The hepatocytes infected with D85 showed strong steatosis similar to that induced by wt virus, but the other lesions induced by MHV 3 (closing of endothelial cell fenestrae and hepatocytolysis) were not seen. An important feature noticed with the D85 mutant concerned the establishment, in the surviving animals, of persistent infection: this phenomenon was demonstrated by the decrease of viral titre in the liver, viral RNA detection, and the fact that viral antigens gradually decreased until the 3rd month post-infection.

Alterations of hepatocellular peroxisomes in viral hepatitis in the mouse.

In addition to being found in peroxisomal diseases, peroxisomal alterations are also seen in viral hepatitis, though quantitative data are lacking. Experiments were performed on BALB/c mice. These mice were infected with Mouse Hepatitis Virus type 3 or were starved. The peroxisomes were cytochemically stained for catalase. Light microscopic, ultrastructural and morphometric analysis were performed. Several peroxisomal changes were observed 24 h after infection, and these changes became more pronounced after 40 h. There was a decrease in catalase activity, which was more pronounced in some regions, in some cells and in individual organelles; and there was also the onset of a progressive decrease in the number of organelles. It is believed that peroxisomes disappear by lysis. Proliferation probably occurs simultaneously up to 40 h after infection. At 48 h, necrotic foci are found to have swollen peroxisomes, and thus destruction is enhanced. Although peroxisomes seem to be sensitive markers of hepatic injury, they show a heterogeneous reaction pattern. Our results are discussed in relation to human viral hepatitis.

[Effects of estrogen treatment on the antiviral properties of murine Kupffer cells]. / Répercussions d'un traitement aux oestrogènes sur les propriétés antivirales des cellules de Kupffer murines.

Adult female mice were administered 17 beta-oestradiol at pharmacological dosages by subcutaneous injections. Histopathological examination revealed an increase in cells of the mononuclear lineage infiltrating the sinusoids of the liver. In vitro culture of Kupffer cells demonstrated that the treatment did not alter their antiviral properties but did rather induce an activation of their non-specific immune properties. This activation might be beneficial or deleterious for the infected host and must be discussed in each particular pathological process.

The Kupffer cell is the first target in ricin-induced hepatitis.

The first target of ricin in the liver appears to be the Kupffer cells which are heavily damaged as early as four hours after the intravenous inoculation of 6 LD100 into mice. At that time, the only endothelial cell damage is constituted by more or less extended interruptions of the fenestrated cytoplasm. Hepatocyte injury affects the endoplasmic reticulum, the glycogen, the mitochondria as well as the plasmic membrane; however, it never results in cytolysis or complete necrosis. The intravascular coagulation observed may be induced either by the endotoxins, which are no longer cleared by the Kupffer cells, or as a result of the loss of the sinusoidal lining with the ensuing platelet activation.

Isolation and immunohistochemical characterization of two developmentally regulated highly insoluble antigens from the membrane of sinusoidal rat liver cells.

Using sequential extractions with buffers containing or not containing neutral detergent, two highly insoluble protein components were found in livers of 30-day-old rats. These compounds (molecular weights (MW) 52 900 and 33 200 respectively) were practically absent from livers of young rats (between 5 and 8 postnatal days). After two-third hepatectomy performed on 30-day-old rats followed by a 24 h recovery, the level of these compounds is drastically decreased (about 50%). Monospecific antibodies against these components were obtained. Using immunohistochemical techniques, these antigens were localized in the membranes (essentially plasma membranes and sometimes internal membranes) of sinusoidal cells of adult rat livers. After partial hepatectomy, these antigens are no more present in the sinusoidal cells of the regenerating parts of the liver.

Probable role of endogenous endotoxins in hepatocytolysis during murine hepatitis caused by frog virus 3.

Three new observations bear out the role of endogenous endotoxins in the pathogenesis of murine hepatitis caused by frog virus 3. First, the LD50 of endotoxin is 20 times lower in mice pretreated for 2.5 hr with a sublethal dose of frog virus 3 than in untreated mice. Animals inoculated with one sublethal dose of lipopolysaccharide 2.5 hr after injection of one sublethal dose of virus die, all having developed extensive hepatocellular necrosis. This hypersensitivity varies according to the intensity of virus-induced destruction of Kupffer cells, which are the intrahepatic target of the virus. Second, mortality is significantly lower and the interval between infection and death longer in axenic mice, which are largely protected from portal endotoxemia. Third, the impairment of some biologic activities of endotoxin (through treatment with polymyxin B or indomethacin, for example) protects mice against hepatic damage and death. Likewise, mice rendered tolerant to endotoxins, and C3H/HeJ mice, which are genetically resistant to endotoxins, survive challenge with frog virus 3 and are refractory with regard to hepatocytolysis . These findings suggest that, in hepatitis caused by frog virus 3, endogenous endotoxins are responsible for extensive hepatocytolysis since virus-induced damage to the hepatic reticuloendothelial system prevents their detoxification.

Protective effect of colectomy in frog virus 3 hepatitis of rats: possible role of endotoxin.

Four to six days after colectomy, rats resisted a challenge of frog virus 3 that in sham-operated animals led to lethal hepatitis. Furthermore, the beneficial effect of colectomy was lost after intravenous administration of a dose of bacterial endotoxin as small as 0.01 100% lethal dose. The protection was related to neither a different distribution of the virus in body organs nor a stimulation of the reticuloendothelial system. The virus-induced early events--destruction of liver sinusoidal cells with leakage of cathepsin D into serum and inhibition of liver macromolecular synthesis--evolved similarly in both groups of rats. After an identical consumption of complement at the beginning of infection, a renewal in complement activity in the protected rats contrasted with an increasing deficiency in the control animals. The protective role of colectomy seems to be related to the suppression of the main source of bacterial endotoxin.

Localization by autoradiography of viral proteins in the parenchymal cells of the liver during frog virus 3 induced hepatitis of mice.

Frog virus 3 inoculated into mice induces an acute degenerative hepatitis. This hepatitis is of toxic origin since the virus is unable to multiply at 37 degrees C. The Kupffer cells, which are the target cells for FV3, reveal the presence of viral particles, viral DNA and proteins. Although the hepatocytes present early and drastic nuclear lesions, viral particles were never observed in these cells. Viral proteins however but not DNA, could be found inside parenchymal cells.