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Biomedical subjects

O Schwartz

Publications and source records attributed to O Schwartz.

At least 37 records · Page 2Linked to original sources
PubMed2000

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation.

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.

Clathrin↗
PubMed1999

Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos.

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.

Cell Membrane↗
PubMed1999

The downregulation of CD4 and MHC-I by primate lentiviruses: a paradigm for the modulation of cell surface receptors.

The human and simian immunodeficiency viruses (HIV and SIV) downregulate the cell surface expression of CD4, their primary receptor, and of class I histocompatibility complex (MHC-I), a critical mediator of immune recognition. While the first of these effects seems important to preserve viral infectivity, the second likely promotes immune evasion. Three HIV-1 proteins, Nef, Env and Vpu, contribute to downregulate CD4, Env forms a complex with CD4 in the endoplasmic reticulum, thereby retaining the receptor in this compartment. Nef and Vpu, on the other hand, act as connectors between CD4 and specific intracellular trafficking pathways, targeting the receptor for degradation in the lysosome and the proteasome, respectively. Some of the downstream partners of the viral proteins in these events have been identified, and include the adaptor complex of clathrin-coated pits, the beta subunit of COP-I coatomer, and the ubiquitin pathway-related h-beta TrCP protein. HIV-induced MHC-I downregulation, mostly the effect of Nef, also reflects a redistribution of this receptor, with its accumulation in the Golgi. The modalities of this process, however, are as yet imperfectly understood. New evidence indicates that the mechanisms employed by primate lentiviruses to downmodulate CD4 and MHC-I are also exploited by a number of cellular regulatory processes.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

Traumatic injuries of the teeth in connection with general anaesthesia and the effect of use of mouthguards.

The aim of the present study was to describe the frequency of dental injuries over a 10-year period, where a mouthguard was used in approximately 10% of intubation cases or endoscopying. The types of injury are presented and the use of different types of mouthguard is discussed. Among 120,086 procedures involving anaesthesia carried out in the period 1983-1992 at Bispebjerg Hospital, Copenhagen, 75 cases of dental trauma occurred. The incidence of dental traumatic injuries was 0.06%. Although eight patients had used mouthguards they were registered with traumatic injuries to the teeth in connection with general anaesthesia. Of the patients, 58% were women and 42% men, with a mean age of 61 years. Seventy-eight percent of the injuries were located in the maxilla and 91% of these involved the incisor area of the maxilla. Forty-one percent were classified as lateral luxations or mobile teeth, 47% were classified as fractured teeth, 10% tooth avulsions and the remaining 2% were classified as concussions. Because of the low frequency of traumatic dental injuries in connection with general anaesthesia and the non-significant effect of the use of mouthguards, routine use of mouthguards is not recommended. Only when a patient is to have an endoscopy done under general anaesthesia, and the surgeon or anaesthesiologist foresees problems (restricted mouth opening, extensive fixed prosthetics, extensive loss of marginal bone) is use of an individually made mouthguard indicated.

Adult↗
PubMed1999

Opposite effects of SDF-1 on human immunodeficiency virus type 1 replication.

The alpha-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.

Anti-HIV Agents↗
PubMed1999

Oligomerization within virions and subcellular localization of human immunodeficiency virus type 1 integrase.

Previous biochemical and genetic evidence indicated that the functional form of retroviral integrase protein (IN) is a multimer. A direct demonstration of IN oligomerization during the infectious cycle was, however, missing, due to the absence of a sensitive detection method. We describe here the generation of infectious human immunodeficiency virus type 1 (HIV-1) viral clones carrying IN protein tagged with highly antigenic epitopes. In this setting, we could readily visualize IN both in producer cells and in viral particles. More interestingly, we detected IN oligomers, the formation of which was dependent on disulfide bridges and took place inside virions. Additionally, expression of a tagged HIV-1 IN in the absence of other viral components resulted in almost exclusive nuclear accumulation of the protein. Mutation of a conserved cysteine in the proposed dimer interface determined the loss of viral infectivity, associated with a reduction of IN oligomer formation and the redistribution of the mutated protein in the nucleus and cytoplasm. Epitope tagging of HIV-1 IN expressed alone or in the context of a replication-competent viral clone provides powerful tools to validate debated issues on the implication of this enzyme in different steps of the viral cycle.

Cell Nucleus↗
PubMed1998

Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules.

The surface expression of MHC I is reduced in HIV-infected cells. We show that the Nef protein affects the intracellular sorting of HLA-A and -B molecules. In the presence of Nef, these proteins accumulate in the Golgi and colocalize with clathrin-coated vesicles. MHC I modulation relies on a tyrosine-based sorting signal located in the cytoplasmic domain of HLA-A and -B heavy chains. This cryptic sorting signal becomes operative only in the presence of Nef. Nef interacts with the medium (mu) subunit of AP adaptor complexes involved in the recognition of tyrosine-based sorting signals, likely facilitating the connection between MHC I and the clathrin-dependent sorting machinery.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1.

We have investigated the cellular uptake of Gag p24 shortly after exposure of cells to human immunodeficiency virus (HIV) particles. In the absence of envelope glycoprotein on virions or of viral receptors or coreceptors at the cell surface, p24 was incorporated in intracellular vesicles but not detected in the cytosolic subcellular fraction. When appropriate envelope-receptor interactions could occur, the nonspecific vesicular uptake was still intense and cytosolic p24 represented 10 to 40% of total intracellular p24. The measurement of cytosolic p24 early after exposure to HIV type 1 is a reliable assay for investigating virus entry and early events leading to authentic cell infection.

CD4 Antigens↗
PubMed1998

Antiviral activity of the proteasome on incoming human immunodeficiency virus type 1.

Following cell surface receptor binding and membrane fusion, human immunodeficiency virus (HIV) virion cores are released in the cytoplasm. Incoming viral proteins represent potential targets for cytosolic proteases. We show that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the efficiency of HIV infection. Proteasome inhibitors were active at the early steps of the viral cycle. Incoming p24Gag proteins accumulated in the cytosol, and larger amounts of proviral DNA were synthesized. In vitro, purified 20S proteasome degraded HIV virion components. Thus, degradation of incoming viral proteins by the proteasome represents an early intracellular defense against infection.

Acetylcysteine↗
PubMed1997

HIV coreceptor downregulation as antiviral principle: SDF-1alpha-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication.

Ligation of CCR5 by the CC chemokines RANTES, MIP-1alpha or MIP-1beta, and of CXCR4 by the CXC chemokine SDF-1alpha, profoundly inhibits the replication of HIV strains that use these coreceptors for entry into CD4(+) T lymphocytes. The mechanism of entry inhibition is not known. We found a rapid and extensive downregulation of CXCR4 by SDF-1alpha and of CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via Gi-proteins. Removal of SDF-1alpha led to rapid, but incomplete surface reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1alpha-induced receptor downregulation and decreased the potency of SDF-1alpha as inhibitor of HIV replication. Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.

Animals↗
PubMed1997

Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation.

Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses. In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects. Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity. We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function. A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds. This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II. Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef. Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase. An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression. These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.

Alleles↗
PubMed1997

Human immunodeficiency virus type I Nef independently affects virion incorporation of major histocompatibility complex class I molecules and virus infectivity.

We have recently reported that HIV-1 Net down-regulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules. MHC-I molecules are one of the predominant cellular proteins associated with HIV-1 virions. Wild-type or nef-mutated HIV-1 virions were analyzed by immunoelectronic microscopy and Western blot for particle-associated MHC-I molecules. The number of MHC-I molecules was significantly higher in HIV-1 virions produced in the absence of Nef than in wild-type virions, indicating that Nef affects the incorporation of MHC-I molecules into virions. Wild-type HIV particles have been shown to be more infectious than Nef- viruses. This difference was maintained when Nef+ and Nef virions devoid of MHC-I molecules were produced in Daudi-CD4 cells. Therefore, the enhancement of virion infectivity and the down-regulation of MHC-I represent independent biological properties of Nef.

Down-Regulation↗
PubMed1997

Positive association of the beta fibrinogen H1/H2 gene variation to basal fibrinogen levels and to the increase in fibrinogen concentration during acute phase reaction but not to coronary artery disease and myocardial infarction.

BACKGROUND: Fibrinogen has been demonstrated to be an independent risk factor of cardiovascular disease. The absence of the HaeIII cutting site (H2 allele) of an H1/H2 gene variation in the promoter region of the beta fibrinogen gene was associated with increased levels of fibrinogen. METHODS AND RESULTS: In the present study, the effects of the H1/H2 gene variation not only on plasma fibrinogen concentrations but also on coronary artery disease (CAD) and myocardial infarction (MI) were investigated in 923 individuals who underwent coronary angiography for diagnostic purposes. Relation of the H1/H2 genotype to fibrinogen plasma levels: A strong association was observed between the H1/H2 gene variation and fibrinogen levels. The differences in fibrinogen plasma levels between H2H2 and H1H1 homozygotes were almost threefold more pronounced within subjects with clinical chemical signs of an acute phase reaction (CRP > or = 7.5 mg/l) than within a subgroup of subjects without these signs (CRP < 7.5 mg/l) (median of CRP distribution: 7.5 mg/l). In 207 patients who underwent aortocoronary bypass surgery plasma fibrinogen levels were almost identical directly after surgery. Two days after operation fibrinogen increased to clearly higher levels in H2H2 homozygotes than in H1H2 and H1H1 genotypes, whereas almost the same maximal increases in fibrinogen concentrations were reached 3-4 days after surgery in all individuals. Relation of the H1/H2 genotype to CAD and MI. Whereas in the total population the plasma fibrinogen concentrations were strongly associated with smoking, CAD and MI, an association of the H1/H2 gene variation to CAD and MI was not detected. However, mean age at first MI of H2H2 individuals (62.9 years) was clearly higher than of H1H2 genotypes (56.9 years) and of H1H1 subjects (56.4 years). In addition, in a subgroup of individuals with a higher risk of MI by either high apoB and/or low apoA1 plasma levels the portion of MI patients was clearly smaller within H2H2 homozygotes than within H1H2 or H1H1 genotypes, although-also in these high risk groups-mean age at first MI of H2H2 individuals were higher than of the other two genotypes. CONCLUSIONS: Obviously, the H2 allele of the fibrinogen H1/H2 genotype does not only influence basal fibrinogen concentrations, but particularly also the extent of fibrinogen level increase during acute phase reaction. Whereas the fibrinogen plasma level is positively associated with coronary artery disease and myocardial infarction, the H2 allele-although exhibiting an association with elevated fibrinogen levels-was not positively associated with CAD and MI.

Acute-Phase Reaction↗
PubMed1996

The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.

A putative chemokine receptor that we previously cloned and termed LESTR has recently been shown to function as a co-receptor (termed fusin) for lymphocyte-tropic HIV-1 strains. Cells expressing CD4 became permissive to infection with T-cell-line-adapted HIV-1 strains of the syncytium-inducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the indentification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the natural ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new chemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 cDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1 alpha and MIP-1 beta, which were shown previously to prevent infection with primary, monocyte-tropic viruses, are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS.

Animals↗
PubMed1996

A cell surface marker gene transferred with a retroviral vector into CD34+ cord blood cells is expressed by their T-cell progeny in the SCID-hu thymus.

Gene transduction into immature hematopoietic cells collected at birth from the umbilical cord could be useful for the treatment of genetic or acquired disorders of the hematopoietic system diagnosed during pregnancy. The SCID-hu mouse is a convenient model to investigate T-cell lineage gene therapy, since it allows replication of human intrathymic T-cell development. CD34+ cells isolated from cord blood were cocultured with CRIP MFG-murine CD2 (mCD2) cells that produce recombinant retroviruses encoding the mCD2 antigen, a cell surface marker easily detectable by flow cytometry. After 3 and 4 days in coculture, a mean of 19% and 39% human hematopoietic cells, respectively, expressed the mCD2 antigen. CD34+ cells cocultured for 4 days were used to reconstitute human fetal thymus implanted in SCID mice. Five to 10 weeks later, the mCD2 antigen was detected on approximately 10% of human thymocytes repopulating the thymic grafts in four of nine SCID mouse chimeras. Vector genomes were detected in graft cell DNA by Southern blot. Analysis of vector integration indicated that positive cells were of polyclonal origin in three animals and predominantly monoclonal in the other one. Our data show that foreign genes can be transduced into CD34+ cord blood cells endowed with T-cell differentiation potential, and suggest strategies for T-cell lineage gene therapy in the neonate.

Animals↗
PubMed1996

Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein.

Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.

Cell Line↗
PubMed1995

Gene polymorphism but not catalytic activity of angiotensin I-converting enzyme is associated with coronary artery disease and myocardial infarction in low-risk patients.

BACKGROUND: An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene has been postulated to be associated with an increased risk of coronary artery disease (CAD) and myocardial infarction (MI). METHODS AND RESULTS: In the present study, the effects of I/D gene polymorphism and of ACE activity on CAD and MI were investigated in 920 individuals who underwent coronary angiography for diagnostic purposes. In the total population and in all CAD and MI groups, a strong association was observed between the gene polymorphism and ACE activities; DD genotypes had approximately twofold higher ACE activities than II genotypes. Although classic risk and protective factors of CAD and MI were identified, associations of ACE genotype and of ACE activity to CAD and MI were not detected in the total population. Among subjects defined to be at lower risk of MI by low body mass index and low cigarette consumption, however, an association of the DD genotype with MI was found. Exclusion of individuals with triglyceride levels > 140 mg/dL and cholesterol levels > 180 mg/dL revealed an association of the DD genotype with CAD. An association of the ACE activity with CAD or MI could not be demonstrated in any of the low-risk populations. CONCLUSIONS: Increased ACE activity obviously is not a risk factor of CAD or MI. The importance of the deletion polymorphism for the development of CAD and MI may be restricted to individuals without classic risk factors.

Case-Control Studies↗