Infection of primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but cytokine production is evident.

We investigated the ability of dengue virus to invade human primary Kupffer cells and to complete its life cycle. The virus effectively penetrated Kupffer cells, but the infection did not result in any viral progeny. Dengue virus-replicating Kupffer cells underwent apoptosis and were cleared by phagocytosis. Infected Kupffer cells produced soluble mediators that could intervene in dengue virus pathogenesis.

Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability.

PURPOSE: Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. METHODS: Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. RESULTS: In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50-100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time. from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. CONCLUSIONS: Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.

Differing infection patterns of dengue and yellow fever viruses in a human hepatoma cell line.

Dengue (DEN) and yellow fever (YF) viruses are responsible for human diseases with symptoms ranging from mild fever to hepatitis and/or hemorrhages. Whereas DEN virus typically induces only limited foci of necrosis in the liver, YF virus infection is characterized by devastating lesions. In a human hepatoma cell line (HepG2), the kinetics of DEN and YF virus replication and release from the cells and the nature of host cell response to viral infection were compared. DEN virus infection was characterized by the early appearance of intracellular viral antigens, major ultrastructural cytopathic changes as early as 32 h after infection, extensive apoptotic cell death, and a low production of infectious particles. In contrast, YF virus grew exponentially to high titers and induced cytopathic changes only 72 h after infection. Differences between the infection processes of the two viruses observed in the hepatoma cell line may explain the different liver pathologies.

[Hepatic macrophages and virus]. / Macrophages hépatiques et virus.

Kupffer cells are located at strategic positions in the liver sinusoids. They are involved in the clearance of about 90% of foreign particles namely viruses and play an important role in the pathogenesis since their interaction with viruses represents a major determinant of viremia. On the one hand, they may protect the host from the infection by taking up and degrading the viral particles as well as by presenting antigen to lymphocytes, on the other hand, they may enhance the infection by producing and disseminating viruses when they are permissive. When they are activated or when they produce interferon alpha, Kupffer cells exert an antiviral effect on adjacent cells. Moreover they participate to the inflammatory response by synthesizing numerous mediators. Their destruction as well as the alteration of their functional properties may have serious physiopathological consequences.

An electron microscopy study into the mechanism of gene transfer with lipopolyamines.

Cationic amphiphiles have been shown to mediate gene transfer to eukaryotic cells, although the nature and fate of the lipid-DNA complexes is still a matter of debate. Negative staining transmission electron microscopy (TEM) of the complexes in physiological medium, as well as thin-section TEM of transfected cells has been used to visualize the particles and the possible pathways leading to transgene expression. Lipopolyamines form a network of tubular micelles into which plasmid DNA is intertwined and condensed; the cationic particles contain hundreds of plasmid molecules and are heterogeneous with respect to size (0.1-0.5 microgram) and shape. Adherent cells (293M, 3T3, MRC5, primary leptomeningeal cells) take them up readily within minutes by spontaneous endocytosis. Among suspension cells, lymphocytes only incidentally show cytoplasmic inclusions and monocytes degrade the particles by phagocytosis. The marked decrease in transfection efficiency generally observed between adherent and nonadherent cells is thus due to reduce cell binding. This suggests that cationic particles bind to membrane components responsible for Ca2+-mediated cell anchoring to the extracellular matrix. Cation/anion-mediated endocytosis leads to endosomes that are entirely filled with the particles. Consequently, two escape mechanisms may operate: disruption of the lamellar envelope in close contact with tubular micelles, and endosome buffering by the lipopolyamine in response to proton entry, leading to osmotic swelling and endosome rupture. Even for moderately transfected MRC5 cells, 10(2)-10(3) particles are found either free or in cytoplasmic vacuoles 24 h after transfection, highlighting a very inefficient nuclear translocation process. Such high numbers are also the clue to the small concentration window between transfection and cytotoxicity that is often observed with nonviral vectors. Nuclear particle inclusions are sometimes seen, yet it is unclear whether plasmid uncoating (before expression) takes place by anion exchange in the cytoplasm or in the nucleus. The still lower efficiency of free plasmid translocation to the nucleus suggests an active role for the cationic lipid during this step. Although the last stages of the transfection mechanism remain unclear, the present work shows that the major barrier which hampers in vitro gene delivery with cationic vectors is nuclear translocation (and cell entry for nonadherent cells), providing precise targets for the design of improved nonviral vectors.

Studies on fenestral contraction in rat liver endothelial cells in culture.

Liver endothelial cells possess fenestrae, which are pores supported by a cytoskeleton ring composed of actin and myosin. Fenestrae are dynamic structures that can contract or dilate, although the mechanism for this phenomenon remains to be elucidated. Staining of actin and/or of myosin permitted measurement of fenestral diameter and area in cultured rat liver endothelial cells using digitized video-intensified fluorescence microscopy with image analysis. Within 1 minute of incubation with 0.1 micromol/L serotonin, fenestral diameter and area decreased by 24 +/- 5% and 56 +/- 7%, respectively. Contraction of fenestrae by serotonin was inhibited by chelation of extracellular Ca2+ with EGTA and by addition of Ca2+ channel blockers, such as dilthiazem and verapamil. The response of fenestrae to serotonin was mimicked by addition of a Ca2+ ionophore, A23187. Serotonin inhibited cAMP production, had no effect on inositol phosphate production, and activated phospholipase A2, causing release of arachidonic acid. These results suggest that contraction of fenestrae is associated with Ca2+ influx. In response to 0.1 micromol/serotonin, intracellular Ca2+ levels increased within 3 to 5 seconds from 150 nmol/L to >400 nmol/l followed by rapid phosphorylation of the 20-kd subunit of myosin light chain; both events dependent on extracellular Ca2+.

Productive infection of primary cultures of endothelial cells from the cat liver sinusoid with the feline immunodeficiency virus.

Given the similarities between the two viruses, the feline immunodeficiency virus (FIV) is becoming an interesting animal model for human immunodeficiency virus (HIV) studies. To explore the still controversial role of the liver in the development of HIV infection, sinusoidal endothelial cells (SEC) were isolated, and primary cultures were infected with the FIV Villefranche IFFA strain. The isolated cells were characterized by their typical fenestrations, the presence of von Willebrand factor (vWf), and their ability to take up acetylated low-density lipoproteins and denatured collagen. Two weeks after infection, significant amounts of FIV p24 antigen were detected by immunofluorescence in both multinucleated giant and single cells and by enzyme-linked immunosorbent assay in the culture medium. High amounts of viral particles were observed together with different steps of budding at the plasma membrane or at the membrane of intracytoplasmic vacuoles. The released viral particles were shown to be infectious for a permissive cell line. During the first 3 weeks of infection, the only cytopathic effect of FIV was syncytia formation. No noticeable impairment of the pattern of fenestrations and the modulation of their number by a cytoskeleton-mediated process occurred. The productive infection of SEC may contribute to the progression of the infection.

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.

Morphological changes in lymph nodes and expression of VCAM1 and cytokines at the late stages of SIV-induced disease in rhesus monkeys.

Four patterns of structural alterations were found in lymph nodes (LNs) from rhesus monkeys 17 to 34 months after infection with simian immunodeficiency virus (SIV-mac251). SIV p27gag antigen and viral particles were localized either between the processes of follicular dendritic cells (FDCs) or in the cytoplasm of macrophages. In hyperplastic follicles, enlarged germinal centres contained numerous Ki67+ proliferating centroblasts which were rather rare in light zones occupied by the CD23+ FDC network. Involuted follicles contained a small number of Ki67+ centroblasts and the CD23 labelling was limited to a very small apical zone. A correlation was found between the morphological characteristics of the follicles (hyperplasia-involution) and the level of expression of the vascular cell adhesion molecule 1 (VCAM1) on FDCs. A gradient in VCAM1 intensity with no expression in the subcapsular-intermediary sinuses, low membrane labelling in the mantle and strong expression in the FDC network was observed. IL1 alpha+ and IL6+ (interleukin) cells (lymphocytes and macrophages) were detected in the mantle, the interfollicular area and the medulla of LNs. Expression of the tumour necrosis factor alpha and ultrastructural markers of interferon alpha production were found in a few FDC and macrophages. Our findings indicate a close relationship between the morphofunctional properties of FDC and the LN structure in SIV infection.

Permissiveness of Kupffer cells for simian immunodeficiency virus (SIV) and morphological changes in the liver of rhesus monkeys at different periods of SIV infection.

The pathogenesis of liver injury, which remains unclear in the course of human immunodeficiency virus infection, can be investigated in simian immunodeficiency virus-infected macaques, which develop an immunodeficiency disease resembling human acquired immune deficiency syndrome (AIDS). We studied the livers of 21 monkeys infected with simian immunodeficiency virus (SIVmac251) for 4 days to 39 months and detected viral antigens in Kupffer cells, macrophages, and lymphocytes in 65% of the livers tested. Virus-containing cells were present in 5 out of 9 livers tested as early as 4 days postinoculation. The number of positive cells as well as their content in viral proteins substantially increased in sinusoidal cells with the progression of the disease. Morphological features and double immunolabeling indicated that Kupffer cells constituted the predominant cell type containing viral antigens. The presence of multinucleated giant cells displaying the ultrastructural features of resident liver macrophages was another sign of the productive infection of Kupffer cells in vivo, which was attested by the observation of budding, immature, and mature SIV particles. Kupffer cell hyperplasia and hypertrophy were evident and appeared to be related to the development of SIV infection, because a close correlation was found between antigenemia and the surface area occupied by these cells. The Kupffer cells contained apoptotic lymphocytes, indicating that resident liver macrophages could play a role in the uptake of such cells from the blood. The production of tumor necrosis factor alpha (TNF alpha) and, possibly, interferon-alpha by Kupffer cells, the expression of vascular adhesion molecule-1, (VCAM-1), intralobular and periportal inflammation, and the proliferation and expansion of bile duct cells were other signs of liver involvement in SIV infection.

Feline immunodeficiency virus can productively infect cultured endothelial cells from cat brain microvessels.

Feline immunodeficiency virus (FIV) provokes a disease in cats characterized by histopathological lesions similar to those observed in AIDS patients. In order to determine whether endothelial cells from brain microvessels are involved in the central nervous system disease to the same extent as macrophages and microglia, cells were isolated from healthy cat brains, cultured and infected in vitro with the FIV Villefranche IFFA 1/88 strain. The isolated cells displayed typical endothelial cell ultrastructural features and were characterized further by von Willebrand factor-labelling and the binding of specific lectins such as Ulex europaeus lectin on their membrane. They were also able to take up acetylated low density lipoproteins. Two weeks after infection, significant amounts of FIV p24 antigen were detected by indirect immunofluorescence in syncytia and single cells. Concomitantly, the same antigen could be detected in the culture medium of the infected cells by an ELISA technique. Numerous viral particles as well as different steps in the process of viral budding were observed under transmission electron microscopy. The synthesis of FIV p24 antigens still occurred in cells in which replication was blocked in the G2 phase with taxol. Our results suggest the possibility of a productive infection of brain microvascular endothelial cells by FIV in vivo, which could lead to important perturbations in the functions of the blood-brain barrier.

HIV-1 infection induces functional alterations in human liver endothelial cells in primary culture.

OBJECTIVES: Since human liver endothelial cells allow HIV-1 multiplication in vitro, we investigated whether HIV induced functional alterations in these cells in primary culture. DESIGN: Direct evidence of the replication of HIV in endothelial cells is sparse, but clotting abnormalities and thrombi, which suggest the existence of an endothelial dysfunction, have been observed in HIV-infected patients. We therefore studied the storage and release of endothelial-specific factors in primary cultures of liver endothelial cells infected with HIV, as well as their cytoskeleton, pinocytic and phagocytic properties. METHODS: Intracellular storage of von Willebrand's factor (vWF) was determined by immunofluorescence and computer image analysis. Excretion of vWF, protein S and endothelin-1 was measured using an enzyme-linked immunosorbent assay and radioimmunoassay. Cytoskeletal constituents were studied by light microscopy. The pinocytosis of acetylated low-density lipoproteins and the phagocytosis of latex beads were analysed under light and electron microscopy. RESULTS: The synthesis of vWF is markedly decreased in HIV-infected liver endothelial cells, as is the excretion of endothelin-1. In contrast, the excretion of protein S remains unaffected and the cytoskeletal network appears to be unaltered. Pinocytosis and phagocytosis are preserved. CONCLUSIONS: HIV infection triggers non-lethal functional alterations in cultured human liver sinusoidal endothelial cells, with a selective impairment in the storage and/or the excretion of endothelial-specific factors such as vWF. This functional modulation could play a role in the pathophysiology of HIV-induced disease.

Phagocytosis and degradation of human serum albumin microspheres and nanoparticles in human macrophages.

Nanoparticles and microspheres made from human serum albumin are biodegradable and, as a physiological material, less cytocidal than cyanoacrylates. Therefore, they should be a suitable carrier system for targeting drugs into cells of the mononuclear phagocyte system. Nevertheless, the process of phagocytic uptake and degradation of albumin particles by macrophages has so far not been documented in detail. For this reason the presented electron microscopical investigation was performed. To study both cellular particle uptake and intracellular degradation, human monocytes were isolated from the peripheral blood of healthy donors and cultivated in plastic plates. After maturation to macrophages, the cells were incubated with the particles for 2h, then washed with buffer and further cultivated for 1-7 days. After fixing with glutaraldehyde, the cells were prepared for electron microscopy. The process of incorporation was demonstrated to be phagocytosis, by scanning and transmission electron microscopy. The degradation of the microspheres was followed by transmission electron microscopy. The metabolism started some hours after particle uptake. After 3 days the process was almost terminated. After 7 days of cultivation only small numbers of intact microspheres were found in the cytoplasm.

Inhibition of HIV in vitro by antiviral drug-targeting using nanoparticles.

Nanoparticles are known to accumulate in the phagocytic cells of the mononuclear phagocyte system. Therefore, the use of this carrier system for the targeting of antiviral drugs to monocytes/macrophages (MO/MAC) is an attractive concept in the treatment of diseases involving MO/MAC, e.g. infection with HIV. In this study, the ability of macrophages isolated from peripheral blood of healthy blood donors to phagocytose and metabolize human serum albumin microspheres was investigated by transmission electron microscopy. Furthermore, nanoparticles manufactured using human serum albumin or polyhexylcyanoacrylate were loaded with nucleoside analogues (AZT and ddC) and tested for their ability to prevent HIV infection in MO/MAC cultures. Our results demonstrate the effectiveness of this drug-targeting system to one of the major target cells for HIV.

Signs of Kupffer cell involvement in productive simian immunodeficiency virus infection in monkey liver.

The livers of 21 rhesus monkeys inoculated with SIVmac251 were examined at 4 days to 39 months after infection. SIV antigens were detected in the cytoplasm of Kupffer cells (KC), macrophages and lymphocytes in two-thirds of the livers tested. The number of cells containing viral proteins substantially increased during the development of the disease, and KC were the main cell type displaying SIV proteins at an advanced stage of infection. Mature and immature lentiviral particles were found in cytoplasmic vacuoles or associated with worm-like structures in KC, indicating that SIV replication could occur within resident liver macrophages. Another sign of the permissiveness of KC was the formation of multinucleated giant cells within the hepatic sinusoids. Some of these cells containing 3-6 nuclei still retained ultrastructural features of KC. Most of them contained a high quantity of viral particles. Numerous lymphocytes displaying signs of apoptosis were taken up by KC, especially at the beginning of infection. Hyperplasia and hypertrophy of KC were noted in the course of SIV disease in the liver. The present data indicate that KC can be infected in vivo and may serve as a reservoir for SIV during the progression of the disease.

Human endothelial cells isolated from the hepatic sinusoids and the umbilical vein display a different permissiveness for HIV1.

Endothelial cells are now considered as potential targets for HIV infection, together with other non-lymphoid cells. We previously demonstrated that endothelial cells isolated from the human liver sinusoids (SEC) are permissive for HIV1 in vitro (Steffan et al., 1992), whereas human umbilical vein endothelial cells (HUVEC) do not allow replication of seven HIV1 and HIV2 strains under the same infection conditions (Lafon et al., 1992). The aim of our work was to compare the permissiveness of SEC and HUVEC and to determine whether the lack of HIV replication in HUVEC, which do not possess CD4 receptors (Lafon et al., 1992), could be related to the absence of virus penetration. The bypass of the early events in the virus life cycle, including the stage of fusion, by transfection of HUVEC with the HTLV-IIIB provirus led to one cycle of viral replication. Moreover, a low level of viral replication was observed in HUVEC infected with the NDK HIV strain, known to penetrate its target cell in a CD4-independent fashion. These results indicate that the block of infection in HUVEC takes place during the early stages of the viral replicative cycle. However, given the low level of NDK replication in HUVEC (0.02% infected cells) in comparison with NDK-infected SEC (30 to 50% infected cells), the absence of the CD4 receptor on HUVEC may not be the only limiting factor for viral growth. Although these two types of endothelial cells display a very different permissiveness for HIV, the present data suggest that they could both serve as a viral reservoir.

Phagocytosis of nanoparticles by human immunodeficiency virus (HIV)-infected macrophages: a possibility for antiviral drug targeting.

Human monocytes/macrophages (MO/MAC) were isolated from peripheral blood and cultivated on hydrophobic Teflon membranes. This culture system is suitable for HIV infection of MO/MAC in vitro. After transfer into 24-well plates the mature macrophages (infected or uninfected) were used for measurements of phagocytosis. The uptake of different, radioactively labeled nanoparticles (NP) made of polyalkylcyanoacrylate, polymethylmethacrylate (PMMA), and human serum albumin (HSA) by the macrophages was determined. In addition, the influence on phagocytosis of size and composition, concentration, and surface of the NP was studied. Further, macrophages of different state of activation were tested. NP made of polyhexylcyanoacrylate (PHCA) or human serum albumin with a diameter of about 200 nm were found most useful for targeting antiviral substances such as azidotymidine to macrophages. Cells infected in vitro with HIV-1D117/III, a monocytotropic HIV isolate from a perinatally infected child, possessed an even higher phagocytotic activity than noninfected cells. Macrophages isolated from HIV-infected patients also showed good incorporation of NP. Thus, the concept of a specific targeting of antiviral substances to macrophages in HIV-infected individuals appears quite promising.

Primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1.

Human endothelial cells isolated from hepatic sinusoids were infected in vitro with human immunodeficiency virus type 1 (HIV-1). An early sign of infection occurring in the culture was the formation of multinucleated cells. By double-labeling immunofluorescence, 5-15% of the cells recognized as endothelial cells owing to the presence of von Willebrand factor were found to contain HIV p24 and gp120 antigens after 2 weeks. Reverse transcriptase activity was released into the medium, and different steps in the process of viral budding were observed by electron microscopy. The virus produced by the endothelial cells was found to be infectious for CEM cells, a human T-cell line. CD4 molecules are present at the surface of the endothelial cells, as demonstrated by immunogold-silver staining and backscattered electron imaging. Treatment with an anti-CD4 antibody abolished productive infection of the sinusoidal endothelial cells. The possibility that endothelial cells of the liver sinusoid are infected in vivo with HIV remains to be clearly shown.