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Biomedical subjects

R Kurth

Publications and source records attributed to R Kurth.

At least 109 records · Page 6Linked to original sources
PubMed1994

The immunosuppressive peptide of HIV-1: functional domains and immune response in AIDS patients.

OBJECTIVES: To study the biological properties of the immunosuppressive peptide (ISU-peptide) of HIV-1, a 17-mer corresponding to the amino-acid domain 583-599 of the transmembrane glycoprotein gp41 of HIV-1. This peptide exhibits sequence homology to the highly conserved ISU-peptide of type C and D retroviruses. Also, to study the immune response against the corresponding gp41 epitope in AIDS patients. DESIGN: The ISU-peptide and control peptides were synthesized and tested for immunosuppressive activity in different in vitro lymphocyte proliferation assays. Antibody responses were tested using a peptide enzyme-linked immunosorbent assay. A new property of the ISU-peptide, inhibition of HIV-1 replication, was investigated using a cytopathogenicity assay. RESULTS: The ISU-peptide of HIV-1 and the immunosuppressive peptides of type C and type D retroviruses possess similar functional properties. They inhibit mitogen-induced and lymphokine-dependent T-lymphocyte proliferation, they are interspecies-reactive, they must be conjugated to a carrier protein in order to be immunosuppressive, and their N-terminal octamers represent the minimal immunosuppressive domain. HIV-infected individuals develop antibodies against an epitope located at the C-terminal end of the ISU-peptide and the number of responders and antibody titres decrease during progression to AIDS. In addition to its immunosuppressive activity, the ISU-peptide of HIV-1 inhibits the cytopathic effect of HIV-1 on human MT4 cells, suggesting interference with virus replication. CONCLUSIONS: The immunosuppressive property of the ISU-peptide suggests that gp41 might contribute to the development of AIDS. The evolutionary conservation of the immunosuppressive domain and the ability of the corresponding ISU-peptide to inhibit HIV replication suggest that this domain plays an important role in the life cycle of HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

Base Sequence↗
PubMed1993

Insufficient inactivation of HIV-1 in human cryo poor plasma by beta-propiolactone: results from a highly accurate virus detection method.

Human cryo poor plasma was spiked with high titre HIV-1 and subjected to a beta-propiolactone (beta-PL) inactivation procedure. The residual titre of HIV-1 was measured using a sensitive and accurate titration technique based upon the killing of MT-4 cells by HIV and the subsequent inability to incorporate 3H-thymidine. It was found that under these conditions a reduction of only 1 log virus titre was achieved after one hour exposure to beta-PL and of 1.8 logs after 5 h. The presence of viable HIV was confirmed using immunohistochemical techniques, by transfer of live virus to fresh cells, and by production of reverse transcriptase. These results have obvious implications for products where human plasma is treated with beta-PL as the only virus inactivation step.

Blood↗
PubMed1993

A general method for the identification of transcribed retrovirus sequences (R-U5 PCR) reveals the expression of the human endogenous retrovirus loci HERV-H and HERV-K in teratocarcinoma cells.

During the past decade, different types of endogenous retroviral sequences have been defined in the human genome usually by low stringency hybridization employing DNA probes of evolutionary conserved animal retrovirus genes. Although all human genomic loci sequenced were found to be defective or interspersed with stop codons, indirect evidence is accumulating that human endogenous retroviral loci are expressed at least in some instances. One example is the synthesis of retroviral particles in human teratocarcinoma cell lines observed by electron microscopy. To establish a link between virus expression and genomic loci we searched for retroviral RNA in human cellular mRNA populations using a generally applicable method. A tRNA-derived primer complementary to a putative retroviral primer binding site was extended by reverse transcription and this product was elongated with a homopolymeric stretch and amplified by PCR (R-U5 PCR). Cloning and sequencing of such products revealed that the endogenous retroviral loci HERV-H and HERV-K are expressed in those human teratocarcinoma cell lines which produce retroviral particles. The size distribution of four HERV-K mRNAs detected in Northern blots is reminiscent of the complex expression pattern seen with a number of exogenous retroviruses.

Animals↗
PubMed1993

Self-limiting infection by int/nef-double mutants of simian immunodeficiency virus.

Simian immunodeficiency virus (SIVmac) infectious for rhesus monkeys was altered by site-directed mutagenesis of genes influencing in vivo replication and persistence with the long-term goal to develop attenuated lentiviruses with limited replication capacity in vivo. Double mutants of SIVmac (termed delta-int 1 to 3) were generated by introducing frameshift and deletion mutations into the nef gene and into the pol gene region coding for the integrase protein. Delta-int/delta-nef viruses formed after transfection of CD(4+)-lymphocyte cell lines were unable to establish sustained replication. In contrast, both wild-type SIVmac and mutant SIVmac delta-nef (coding for a truncated NEF protein and a wild-type INT protein) replicated continuously and at a comparable rate. However, a transient and self-limiting infection of the C8166 T-cell line was observed subsequent to transfection of double mutant proviruses into HeLa-tat-III cells. Viruses attenuated by int/nef-double mutation were able to enter the T-cells, initiate synthesis of viral DNA as shown by PCR amplification of closed circular episomes, and express viral antigens in infected cells as demonstrated by immunocytochemical staining. Integration of the int mutant viruses into the chromosome was completely inhibited. Episomal viral DNA was detectable in the infected cells for up to 2 weeks, after which it disappeared. Thus, SIVmac attenuated by int and nef mutation established a transient infection of permissive cells resulting in the expression of viral antigen from episomal viral DNA over a limited period of time.

Antigens, Viral↗
PubMed1993

Evidence that HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma-derived retrovirus HTDV.

Human teratocarcinoma-derived viruses (HTDV) are retrovirus-like particles that are regularly observed by electron microscopy at low frequency in cell lines established from human teratocarcinomas. Over the last years, one of our teratocarcinoma cell lines spontaneously began to produce high amounts of HTDV. This cell line is stained in immunofluorescence tests by an antiserum raised against recombinant gag protein of HERV-K, an expressed human endogenous retrovirus sequence. In immunoelectron microscopy of ultrathin frozen sections, this anti HERV-Kgag-specific antiserum reacts specifically with HTDV particles. In Western blots, the antiserum recognizes predominantly a protein with an apparent molecular weight of 30 kDa, presumably the major core protein of HTDV particles. Taken together, these results provide evidence that HERV-K codes for HTDV.

Fluorescent Antibody Technique↗
PubMed1993

Inactivation of HIV infectivity by the chlorite-oxygen reaction product tetrachlorodecaoxygen.

OBJECTIVE: Since the chlorite-oxygen reaction product tetrachlorodecaoxygen (TCDO) anion complex promotes efficaciously tissue repair and has antibacterial activity, our aim was to determine the effects of TCDO on the replication of HIV and on the infectivity of free HIV particles. DESIGN: The effects of TCDO on cellular HIV replication machinery and the consequences of TCDO for infectivity of HIV virions were evaluated. METHODS: Virus yields in supernatants of TCDO-supplemented cultures of HIV-infected cells or virus infectivity in TCDO-treated virus stocks were quantified by titration assays and then calculating the 50% tissue culture infectious dose. RESULTS: First, TCDO did not affect the replication of HIV in persistently infected lymphocytic and monocytic cell lines or in peripheral blood mononuclear cells. Second, supplementation of HIV stocks with TCDO markedly decreased the infectivity of HIV particles in a concentration dependent manner. Third, the binding of gp120 envelope glycoprotein of HIV-1 to cells is blocked by pre-incubation with TCDO. Fourth, the inhibition of HIV replication by the reverse transcriptase inhibitor 3'-azido-3'-deoxythymidine (zidovudine) in de novo infected cell cultures was not affected by the simultaneous addition of TCDO. However, the delayed virus spread of HIV in cultures in the presence of suboptimal concentrations of zidovudine could significantly be blocked by the simultaneous addition of TCDO. Fifth, TCDO failed to induce the chromosomally integrated HIV-1 provirus in the T-lymphoma cell line ACH2. CONCLUSIONS: TCDO appears to inactivate HIV particles directly, but has no influence on the intracellular replicative machinery of HIV. Our results suggest that a clinical evaluation of the TCDO complex as chemotherapy for HIV infection and full-blown AIDS should be considered, particularly in patients concomitantly receiving zidovudine.

Antiviral Agents↗
PubMed1993

Interferon-gamma-activated monocytes impair infectivity of HIV particles by an oxygen metabolite-dependent reaction.

Mononuclear phagocytes generate microbicidal oxygen metabolites spontaneously and after phagocytic stimulation by a NADPH-dependent enzymatic reaction called the oxidative burst. The spontaneous release of reactive oxygen radicals and intermediates (ROI) increases five- to eightfold after treatment of monocytes with the lymphokine interferon-gamma (IFN-gamma). The effect of the IFN-gamma-activated release of ROI by human monocytes on the infectivity of free human immunodeficiency virus (HIV) in the supernatant was investigated with the following results. First, IFN-gamma-activated, but neither control monocytes nor lipopolysaccharide-stimulated monocytes effectively decreased the infectivity of cell-free HIV-1 in culture medium supernatant. Second, the mechanism of inactivation was dependent on the enhanced spontaneous release of ROI by IFN-gamma-activated mononuclear phagocytes, since either the enzyme catalase or the free radical scavenger butylated hydroxyanisole (BHA) could block this activity. Third, soluble and solid-phase HIV-1 outer envelope glycoprotein (gp120) failed to trigger the oxidative burst activity after specific gp120-monocytic CD4 receptor interaction. These results indicate an anti-viral effect of IFN-gamma-activated monocytes/macrophages on HIV-1 which may have important implications for our understanding of spread of the virus in the body and the development of full-blown AIDS after a long period of latency.

Butylated Hydroxyanisole↗
PubMed1992

Development of a quasispecies of human immunodeficiency virus type 1 in vivo.

During treatment with one specific batch of blood clotting factor IX, a number of hemophilia B patients in Germany recently became infected with human immunodeficiency virus type 1 (HIV-1). The nucleotide sequences of cloned HIV-1 envelope gene regions including the variable V3 loop and the V4 region derived from short-term virus cultures and directly from peripheral blood cells of these patients were shown to be highly homologous. Based on the assumption that the corresponding consensus sequence (termed HIV-1MBK) was identical to the genotype of the initially infecting virus, we were able to construct phylogenetic trees of the developing quasispecies in two patients studied in detail. True intermediates between input and multiply mutated genotypes were found in individual blood samples. Except for the initially infecting variant HIV-1MBK, variants found at 11 months postinfection had replaced those seen at 5 months postinfection. Variability early after infection was shown to cluster in two small regions located 3' of the V3 loop (i.e., outside the loop) and within the V4 region. This communication therefore describes the evolution of an HIV-1 quasispecies in humans starting from a single genotype.

Amino Acid Sequence↗
PubMed1992

AIDS vaccine: present problems and future perspectives.

Vaccination has proved to be an effective means for the prevention of infectious diseases. Advances in our understanding of the human immunodeficiency virus (HIV) and the immune system of the host may lay the foundation for the development of an AIDS vaccine. Current attempts to develop vaccines focus on the development of substances that will produce a different type of immune response from that which occurs naturally. Progress has been made in understanding the mechanisms by which the AIDS virus stimulates an neutralizing antibody response and triggers specific cytotoxic T lymphocytes in the host. The pressing need for a vaccine has prompted the testing of several candidate vaccines based on the simian immunodeficiency virus (closely related to HIV) in the macaque animal model for AIDS. The lessons learned from these trials will be valuable for developing future vaccines.

AIDS Vaccines↗
PubMed1992

Vaccination against HIV.

The spread of AIDS progresses unrelentingly despite all efforts of public education and the only real hope of epidemiological control lies in the development of an effective vaccine. The very nature of the AIDS virus (HIV) and the manner with which it interacts with the host makes development of a practical vaccine very difficult. Recent successes using whole inactivated virus as immunogen in the SIVmac animal model system now show that it is possible to protect against infection. However, due to supposed limited efficacy in the field of such a vaccine and the fact that large scale production and administration of a multi-shot whole HIV-based product would be technically impossible, it is now important to identify which of the proteins are responsible and which immune response is protective. With this knowledge a recombinant or synthetic vaccine could be produced on a large scale. However, despite recent protection of a few chimpanzees against HIV infection itself there is currently no proven effective and practical vaccine even in the laboratory and it will be many years before such a product is available to the general public.

Animals↗
PubMed1992

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.

Animals↗
PubMed1992

B-cell epitope mapping of the entire SIVagm envelope glycoprotein including fine mapping of immunogenic regions.

To allow the precise definition of the anti-lentiviral immune response in the natural host and to facilitate the development of an alternative animal model for vaccine development, we are identifying the immunogenic domains of SIVagm proteins. First, a total of 173 synthetic 15-mer peptides with an overlap of 10 amino acids were produced spanning the entire envelope glycoprotein of the molecular clone SIVagm3. These peptides were used as antigen in enzyme-linked immunosorbent assays for identifying regions recognized by antibodies from naturally infected African green monkeys and monkeys infected with a molecular clone. Regions corresponding to the HIV-1 V3, the transmembrane protein (TMP) "Gnann peptide", and the C-terminal area of the outer envelope protein were shown to be immunodominant. These regions were re-synthesized as 15-mer peptides with an overlap of 14 amino acids and used to precisely map the epitopes recognized. Sera from 93 captive and 61 wild animals were tested by SIVagm-specific Western blot (WB) and for ELISA reactivity against the immunodominant TMP peptide. One hundred percent (76/76) of the WB-positive captive animals and 98% (41/42) wild WB-positive animals also reacted against the peptide. In contrast, only 62% of the WB-positive sera reacted with the "V3" epitope and 46% with the gp130 C-terminal epitope.

Amino Acid Sequence↗
PubMed1992

Vaccine protection against SIVmac infection by high- but not low-dose whole inactivated virus immunogen.

Eight rhesus macaques were immunized intramuscularly four times (0, 1, 2, and 4 months) over a period of 4 months with a formalin-inactivated whole Simian immunodeficiency virus (SIV) vaccine in the presence of muramyl dipeptide (MDP) as adjuvant. Four animals received 0.5 mg and the other four received 0.1 mg immunogen per injection. Three weeks after the final immunization, the vaccinated monkeys along with two control monkeys were challenged intravenously with 50 MID50 of SIVmac251-32H. At the time of challenge, one monkey from the high dose group had a high titer (1:761) of antibody able to neutralize in vitro the homologous 32H strain of SIV mac. All other animals had lower but measurable titers (1:57-1:453), although there was no correlation between the levels of neutralizing antibody and subsequent protection. Upon challenge, three of four animals from the low dose group (plus the nonvaccinated control animals) became infected as demonstrated by reisolation of virus from peripheral blood mononuclear cells, by SIVmac-specific polymerase chain reaction, and by the development of a strong anamnestic response. In the sera from these animals the titer of neutralizing antibody rose to over 1:5,100. All other animals (one from low dose group and all four of the high dose group) remained negative by all parameters at 4 months post challenge. These data indicate that when used in conjunction with MDP, the amount of immunogen required per immunization for protection against challenge is between 0.1 and 0.5 mg.

Animals↗
PubMed1991

Development of vivo of genetic variability of simian immunodeficiency virus.

Rapid development of genetic variability may contribute to the pathogenicity of lentiviruses as it may allow escape from immune surveillance and/or from suppression of virus replication. Although apathogenic in African green monkeys, simian immunodeficiency virus isolated from African green monkeys is shown to display extensive genetic variability and defectiveness in the V1- and V2-like variable domains of the external envelope protein comparable to that known for human immunodeficiency virus. However, in contrast to the situation in human immunodeficiency virus-infected individuals, a predominant major virus variant was detected neither in a monkey naturally infected for more than 10 years nor in two monkeys infected with a molecular virus clone for 15-20 months. Extensive variability evolves from a single genotype with a maximal rate of 7.7 mutations per 1000 nucleotides per year. A remarkable selection for nonsynonymous mutations that accounts for 92% of all changes indicates continuous selection of variants.

Amino Acid Sequence↗