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Biomedical subjects

A Kirn

Publications and source records attributed to A Kirn.

At least 19 recordsLinked to original sources
PubMed1996

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.

Animals↗
PubMed1996

HIV-1 envelope glycoprotein gp120 down-regulates CD4 expression in primary human macrophages through induction of endogenous tumour necrosis factor-alpha.

Among immunological abnormalities present in human immunodeficiency virus type 1 (HIV-1)-infected individuals are dysregulation of cytokine production and CD4 down-regulation in both T-helper cells and monocytes/macrophages. The HIV-1 envelope glycoprotein 120 (gp120) has the ability to induce different cytokines in peripheral blood mononuclear cells and in monocytes/macrophages in vitro which in some instances have been reported to down-regulate macrophage CD4 expression. This study provides evidence that HIV-1 recombinant gp120 (rgp120) down-regulates both surface and total CD4 expression in primary tissue culture-differentiated macrophages (TCDM) at the level of transcription. The CD4 down-regulation observed in TCDM occurred between 6 and 12 hr after rgp120 treatment preceded by a peak of endogenous tumour necrosis factor-alpha (TNF-alpha) observed at 3-6 hr post-treatment. We demonstrate that the TCDM CD4 down-regulation observed after rgp120 treatment was inhibited by the use of an anti-huTNF-alpha monoclonal antibody (mAb), but not by mAb directed against other cytokines induced by rgp120, such as interleukin-1 beta (IL-1 beta) and interferon-alpha (IFN-alpha). The present findings roughly parallel those observed both in the sera of patients and in the monocytes/macrophages isolated from HIV-positive individuals, suggesting that gp120 by stimulating endogenous TNF-alpha production could be a good candidate for the CD4 down-regulation observed in the monocytes/macrophages of HIV-1-infected individuals. In contrast to CD4 down-regulation in HIV-infected lymphocytes, which results from a direct effect of viral genes on CD4 expression, soluble factors such as cytokines induced during HIV infection might explain the monocyte/macrophage CD4 dysregulation observed in acquired immune deficiency syndrome.

Antibodies, Monoclonal↗
PubMed1995

A new series of pyridinone derivatives as potent non-nucleoside human immunodeficiency virus type 1 specific reverse transcriptase inhibitors.

4-(Arylthio)-pyridin-2(1H)-ones variously substituted in their 3-, 5-, and 6-positions have been synthesized as a new series of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)-pyridinone hybrid molecules. Biological studies revealed that some of them show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 16 and 7c, the most active ones, inhibit the replication of HIV-1 at 3 and 6 nM, respectively.

Antiviral Agents↗
PubMed1995

Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphate-protecting groups: intracellular delivery of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate.

The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.

Antiviral Agents↗
PubMed1995

Symptomatic mumps virus reinfections.

Although natural mumps virus infection is believed to induce lifelong immunity, our laboratory was confronted with 82 patients who developed mumps-evoking lesions but exhibited serological evidence of a booster immune response, namely a rise or a high titer of virus-specific IgG, without IgM. In order to provide arguments favoring the existence of recurrent mumps attacks, the age, symptomatology, and humoral response of these patients (group 1) were compared to that of 82 randomly selected true primary infected patients (group 2), 10 parainfluenza virus-infected patients (group 3), and 20 noninfected mumps-immune subjects (group 4). Enzyme-linked immunosorbent assay (ELISA) procedures with different viral antigenic preparations were used for determination of specific IgM, IgA, IgG, IgG subclasses, and IgG avidity. The patients of group 1, older than those of group 2 (28 vs. 10 years, P < 0.0001), presented a significantly less severe and less typical symptomatology. Against the whole virus they exhibited IgG of higher avidity (P < 0.001), a lower prevalence and titer of IgA (10 vs. 68%, P < 0.0001 and 278 vs. 5,009, P < 0.001, respectively). Values obtained for IgG 1, 2, and 3 were significantly different between the two groups. Prevalence and absorbance of nucleocapsid-directed IgG 3 were significantly lower in group 1 (27 vs. 46%, P < 0.01 and 0.444 vs. 0.869, P < 0.01, respectively). A significant discrepancy also allowed patients from group 1 to be distinguished from those of groups 3 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

High hepatitis C viraemia and impaired antibody response in patients coinfected with HIV.

OBJECTIVE: To compare hepatitis C virus (HCV) load in patients infected with HCV alone and those coinfected with HIV, and to evaluate the antibody response to HCV in the case of HIV infection. DESIGN: Patients coinfected with both HCV and HIV have been shown to develop hepatic changes more rapidly, which may be due to an interaction between HCV and HIV. In a prospective study, serum samples were taken from 150 patients. METHODS: Using reverse transcription followed by polymerase chain reaction and the branched DNA assay, we detected HCV RNA in 75 patients coinfected with HIV and HCV and in 75 patients infected with HCV alone. The HIV RNA was also quantified by the branched DNA assay and the p24 antigenaemia was determined by enzyme-linked immunosorbent assay. The immune response to HCV was studied in the 150 patients by the use of third generation recombinant immunoblot assay (RIBA). RESULTS: Although a comparable number of patients had detectable HCV viraemia in both groups, HCV RNA was quantifiable in 79% of HIV-positive patients and in only 43% of HIV-negative patients (P < 10(-5)), and the mean HCV RNA level was much higher in the HIV-positive group than in the HIV-negative group (P < 10(-7)). The quantity of HCV RNA did not correlate with the CD4 count, p24 antigenaemia or HIV RNA level. The analysis of RIBA showed 14.7% indeterminate or negative results in the HIV-positive group and only 4% indeterminate results in the HIV-negative group. HIV-positive patients had reactivity to less antigen bands than HIV-negative patients (P < 10(-3)), and they had a weaker reactivity to c100, c33c and NS5 antigen bands than HIV-negative patients. CONCLUSION: Our results show that in the case of HIV infection, the HCV RNA levels are strongly increased, but HCV load is not linked to the immunosuppression induced by HIV; therefore, the present data do not support the hypothesis of a direct interaction between HIV and HCV.

Adolescent↗
PubMed1995

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.

Base Sequence↗
PubMed1995

Human immunodeficiency virus type 1 infection of human CD4-transgenic rabbits.

Investigations of human immunodeficiency virus (HIV) infection of man have benefited from the study of relevant animal models of the infection and disease. However, the ultimate models use primate species which are either endangered, not generally available, or expensive to maintain. A transgenic rabbit specifically and stably expressing human CD4 protein on T lymphocytes was assessed as a new laboratory animal model for HIV-1 infection. In vitro studies demonstrate that lymphocytes derived from the transgenic rabbits are more susceptible to HIV-1IIIB infection than those from normal rabbits. In vivo infection of huCD4-transgenic rabbits using HIV-1IIIB-infected autologous lymphocytes was demonstrated by virus isolation, detection of HIV-1-specific DNA in peripheral blood lymphocytes and seroconversion to various HIV-1 proteins. Viral DNA was detected in the tissues of one rabbit sacrificed 7 weeks post-infection and virus was isolated from lymph node. Although these transgenic rabbits are less sensitive to HIV-1 infection than man, such a small and inexpensive animal model may be a useful tool.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Presence of virion protein x (Vpx) of simian immunodeficiency virus SIVmac 251 in target cells in vivo.

Localization of virion-associated protein x (Vpx) of SIVmac 251 was studied in lymph nodes and liver of six SIVmac-infected monkeys. Vpx was found associated with the network of follicular dendritic cells and macrophages in lymph nodes and/or livers from five out of six animals by immunohistochemistry. Although the humoral response to Vpx occurs in only 50% of the animals, the presence of Vpx in target cell or antibodies to Vpx in all the monkeys studied, suggests that Vpx may be necessary for viral replication in vivo.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

Contrary results on mouse hepatitis virus type 3 susceptibility in A/J mouse hepatocytes of phosphatidylserine treatment and of a hypercholesterolaemic diet: no correlation with membrane fluidity levels.

The aim of this study was to examine whether or not membrane fluidity directly influences infection by enveloped viruses, and, more precisely here, the susceptibility of A/J mouse hepatocytes to Mouse Hepatitis Virus type 3 (MHV3). We therefore studied, in parallel, the effects on hepatocyte membrane fluidity and on intracellular viral titre of two treatments, i) a hypercholesterolaemic diet to increase the hepatocyte membrane cholesterol content, ii) direct phosphatidylserine incorporation into hepatocyte membrane. Membrane fluidity was monitored on isolated hepatocytes by fluorescence anisotropy with TMA-DPH, and the viral titre was determined by plaque assay. The results clearly demonstrate that membrane fluidity is not directly involved in viral infection mechanisms.

Animals↗
PubMed1994

Equal inhibition of the replication of human immunodeficiency virus in human T-cell culture by ddA bis(SATE)phosphotriester and 3'-azido-2',3'-dideoxythymidine.

It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway.

Antiviral Agents↗
PubMed1994

Isolation and identification of two CD34+ cell subpopulations from normal human peripheral blood.

Circulating CD34+ progenitors were separated from normal human peripheral blood on the basis of size and density by counterflow centrifugal elutriation (CCE). The CD34+ cells, 0.15% of peripheral blood mononuclear cells, were heterogeneous with respect to their elutriation characteristics, mainly size and density. The least mature CD34+ cells, characterized by lack of CD38 antigen, were predominantly found in the small lymphoid cell fraction. In fractions containing larger and denser cells (large lymphocytes, monocytes, and granulocytes), CD38 was increasingly expressed on the CD34+ cells, as were lineage commitment markers CD10 (B lymphoid), CD33 (myeloid), CD13 (myelomonocytic) and CD71 (erythroid) antigens. The smaller and less dense CD34+ cells expressed CD34 antigen brightly while the larger and denser CD34+ cells expressed it dimly. The smaller and less dense CD34+ high cells failed to establish colony growth in short-term culture while the larger and denser CD34+low cells gave rise to high counts of colony forming units-granulocyte macrophage (CFU-GM). Physical separation on the basis of size and density by CCE differentiates between two main classes of steady-state CD34+ cells from normal human peripheral blood. The smaller and less dense CD34+high cells correspond to the earliest progenitors that express differentiation markers poorly but CD34 antigen brightly, do not give rise to short-term colony growth in vitro, and thus represent indirect evidence for pluripotent hematopoietic stem cells (PHSC). The larger and denser CD34+low cells are the more mature progenitor cells, already committed to myeloid, lymphoid or erythroid differentiation but only dimly expressing CD34 antigen, and these cells were responsible for short-term colony growth in vitro.

Adult↗
PubMed1994

Bicyclic imidazo derivatives, a new class of highly selective inhibitors for the human immunodeficiency virus type 1.

In the search for new antiviral agents against human immunodeficiency virus, different members of two imidazoheterocycle families (imidazothiazoles, imidazopyridines) have been found to display potent inhibitory effects on the replication of HIV-1. Three of these derivatives, which show significant anti-HIV-1 activity, have been chosen for further studies. The analysis of these compounds and their comparison to AZT and TIBO revealed that these bicyclic imidazo derivatives represent a class of highly specific inhibitors of HIV-1, but not of HIV-2 or simian immunodeficiency virus (SIV). Their inhibition of HIV-1 is mediated through interaction with the reverse transcriptase (RT). The mechanism of action of these bicyclic imidazo derivatives may be similar to that of the other non-nucleoside RT inhibitors (NNRTIs).

Antiviral Agents↗
PubMed1994

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.

Cells, Cultured↗