Search PubMed⌕ Search

Biomedical subjects

R Kurth

Publications and source records attributed to R Kurth.

At least 127 records · Page 7Linked to original sources
PubMed1991

Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey.

The in vitro fidelity of highly purified recombinant reverse transcriptase from simian immunodeficiency virus of African green monkeys (SIVagm) was determined. By using the phi X174am16 reversion assay an overall error rate of 1/19,000 was determined. This is 2.4-fold higher than the overall accuracy of purified recombinant HIV-1 reverse transcriptase, measured in parallel. The evaluation of error frequencies from nucleotide pool bias studies suggest an even higher accuracy for the SIVagm-derived reverse transcriptase. T:dGMP mismatches were formed most frequently with an error rate of 1/155,000, followed by G:dGMP (1/230,000), A:dGMP (1/315,000), G:dAMP (1/340,000), T:dCMP (1/540,000), T:dTMP (1/790,000), and A:dCMP (1/1,050,000) mispairs. Thus, according to pool bias effects and depending on the mismatch under consideration SIVagm reverse transcriptase appears to be 2 to 20-fold more accurate than the homologous enzyme from the human immunodeficiency virus type 1. This higher accuracy is not due to a co-purifying exonuclaease activity. Like the enzyme from HIV-1, the simian monkey-derived enzyme was found to be devoid of a proofreading 3' to 5' exonuclease.

Animals↗
PubMed1991

Neutralizing antibodies and antigens in AIDS.

Until recently, much of the effort put into development of an AIDS vaccine has focussed on the elicitation of a neutralizing antibody response. The viral target of neutralization, HIV envelope glycoprotein, has been produced in bulk through recombinant techniques, but has had little success as a vaccine. The specific epitopes to which neutralizing antibodies bind have been mapped, and although the major epitope is hypervariable, others are conserved. This allows the design of second generation vaccines. Meanwhile, vaccine studies in the SIV animal model simply using inactivated virus as immunogen have demonstrated that an effective vaccine is at least possible. A variety of HIV vaccine preparations are now under investigation and the outlook for the future is promising.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The quest for an AIDS vaccine: the state of the art and current challenges.

Despite intense efforts worldwide, using state-of-the-art methods and techniques and despite ever-increasing knowledge about the molecular and structural make-up of HIV, a practical vaccine against acquired immunodeficiency syndrome (AIDS) has yet to be developed. The increasing use of recombinant DNA techniques and synthetic peptide technology has allowed many groups to identify at the epitope level the regions of HIV proteins which act as targets for (and stimulate) the immune response. Epitopes which stimulate and bind neutralizing antibodies have been examined in detail and an ever-increasing number of antibody-dependent cellular cytotoxicity (ADCC) and cytotoxic T lymphocytes (CTL) epitopes are being defined, as are potentially harmful (immunosuppressive or enhancing) domains. It still is not clear which of the different immune responses (or combinations thereof) it will be necessary to stimulate in order to protect from infection. Infected humans develop neutralizing antibodies, ADCC-inducing antibodies and CTL responses against a variety of viral proteins but it is not known which of these can control or prevent infection in vivo. The extensive knowledge of HIV and the immune response it elicits is being used to design and produce a wide variety of putative vaccines, ranging from whole inactivated virus, through recombinant organisms/proteins, to synthetic peptides although each has its inherent advantages and disadvantages. The very nature of HIV makes vaccine development difficult at best. However, recent successes using whole inactivated virus or virus-infected cells in the macaque simian immunodeficiency virus (SIVmac) model system at least show that protection against lethal lentivirus infection can be achieved.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1991

Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives.

HIV-1 proviral DNA contains two binding sites for the transcription factor NF-kappa B. HIV-1-infected individuals have, on average, abnormally high levels of tumour necrosis factor alpha (TNF alpha) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and NF-kappa B expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit NF-kappa B activity as determined by electrophoretic mobility shift assays and chloramphenicol acetyl-transferase (CAT) gene expression under control of NF-kappa B binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of NF-kappa B-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.

Acetylcysteine↗
PubMed1991

Lack of HIV-1 V3 region sequence diversity in two haemophiliac patients infected with a putative biologic clone of HIV-1.

Peripheral blood mononuclear cells (PBMC) from two haemophilia B patients, who presumably became infected with a putative HIV-1 biological clone following treatment with the same suspected batch of commercial factor, were used to clone and sequence the hypervariable V3 region of the HIV-1 envelope protein. In 10 of 12 clones the V3 region was identical and two (one from each patient) had a single non-synonymous point mutation. Viable reisolates (shown to be authentic by sequencing of V3) currently appear to be monocyte tropic. These results strongly indicate that the patients were infected from a common source with a very low number of infectious particles and indicate that variability under these conditions is limited.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of the simian immunodeficiency virus SIVmac nef gene.

We are using site-directed mutagenesis of single viral genes to identify and analyze the genetic determinants of human and simian immunodeficiency virus pathogenicity. In a first approach, we have constructed a series of simian immunodeficiency virus SIVmac nef mutants by partial deletion and insertions in the nef gene, as this gene is a candidate gene for the establishment and maintenance of latency. nef insertion mutants replicated faster than wild-type SIVmac, suggesting that the nef gene product acts as a negative factor for replication. Surface phenotyping revealed that cultures permanently infected with nef mutants exhibit an enhanced expression of viral proteins on the outer cell surface. We have analyzed the properties of the mutant viruses in cell culture and intend to use rapidly replicating mutants (putatively unable to undergo latency) as model vaccine viruses in the rhesus monkey.

Base Sequence↗
PubMed1991

Epidemiology and pathogenicity of human retroviruses.

The human retroviruses can be divided into oncovirus (HTLV-I and HTLV-II) and lentivirus strains (HIV-1 and -2). The HTLVs are endemic in Central Africa, the Caribbean Islands and in southwest Japan, but now tend to spread through the i.v.-drug user population in the USA and in some countries in Western Europe. HTLV infection is associated with a malignant form of adult T-cell leukemia, tropical spastic paraparesis (TSP) and an associated myelopathy (HAM). The pathogenic mechanisms of HTLV are as yet poorly understood. HIV infection is spreading rapidly almost world-wide and has reached epidemic proportions in Central Africa, parts of South America and in certain populations in industrialized countries that have risk behaviour for contracting venereal or blood-borne infections. The mechanisms of HIV-induced immune suppression are still not entirely clear, as direct T-lymphocyte destruction after viral infection cannot account for the almost complete loss of CD4 T-cells in the final stages of disease. Various indirect mechanisms of HIV-induced immune cell destruction are outlined below.

Biological Products↗
PubMed1990

Demonstration of cross-reactive antibodies able to elicit lysis of both HIV-1- and HIV-2-infected cells.

A total of 100% of sera from a large number of HIV-1-infected patients contained antibodies able to elicit Antibody-dependent cellular cytotoxicity lysis of cells infected with the HIV-1 isolates IIIB or RF. Levels of activity could not be correlated with activities in ELISA or neutralizing antibody assays nor with the clinical status of the patients. Surprisingly, 8 of 156 patients sera could additionally elicit lysis of HIV-2-infected cells, and cold target competition assays demonstrated that the cross-reactivity was apparently mediated via recognition of common epitope(s) expressed on the surface of cells infected with either group of HIV. The ADCC mechanism was shown to be mediated by a CD16+ lymphocyte. This demonstration of an effector mechanism able to attack and eliminate cells infected with a wide range of HIV strains has obvious implications for development of putative vaccines.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization.

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.

Animals↗
PubMed1990

Decreased accessory cell function of macrophages after infection with human immunodeficiency virus type 1 in vitro.

Peripheral blood monocytes from human immunodeficiency virus (HIV)-infected individuals or AIDS-related complex/AIDS patients ex vivo exhibit distinct alterations in some but not all immune functions. In studies presented here, monocytes from healthy donors were infected with HIV 1 in vitro and co-cultures with autologous uninfected T lymphocytes were set up. The monocyte/macrophage (M phi)-dependent T cell function was determined by measurement of proliferative and secretory [interleukin (IL)2, interferon-gamma] responses to lectin (phytohemagglutinin), mitogen (anti-CD3 monoclonal antibody), or recall antigen (tetanus toxoid, tuberculin). Accessory function of M phi was normal after HIV infection when optimal amounts (10%-20%) were added to the T lymphocytes. However, HIV infection of M phi significantly decreased T cell proliferative responses and secretion of IL2 when supplemented at limited dilution (0.5%-5%), although interferon-gamma production was not affected. Whereas the lipopolysaccharide-triggered M phi production of IL1 was not impaired by HIV 1 infection, there was a significant decrease in this response when anti-CD3 monoclonal antibody or tetanus toxoid were used to trigger the peripheral blood mononuclear cells. The impairment of proliferation of T lymphocytes in the presence of HIV 1-infected M phi could be overcome by addition of exogenous IL 1. Taken together, these data clearly show that the mononuclear phagocyte-dependent enhancement of stimulated T cell proliferation and lymphokine secretion is decreased when the restricted numbers of monocytes/M phi are HIV 1 infected. There are, therefore, two possible roles of M phi in HIV infection and progression to disease. First, as a reservoir and vehicle for dissemination of the virus, and second, as an immune cell whose essential functions are impaired by infection.

Antigen-Presenting Cells↗
PubMed1990

Complete nucleotide sequence of a simian immunodeficiency virus from African green monkeys: a novel type of intragroup divergence.

We have determined the entire nucleotide sequence of a full-length molecular clone, termed SIVagm3, which is infectious in vitro and in vivo. The genomic organization was found to be similar to other immunodeficiency viruses of human and simian origin. Comparison of SIVagm3 with SIVagmTYO-1, the only other completely sequenced molecular SIVagm clone, revealed a novel type of intragroup divergence, which is characterized by (1) an unusually high degree of variability in pol in relation to gag and env and (2) a high degree of divergence in the rev and tat genes. Thus, since SIVagm3 and SIVagmTYO-1 evolved from their common ancestor, they diverged in a different manner than human immunodeficiency viruses. Hypervariable regions in env were defined and shown to be relatively restricted in comparison to HIV-1 and HIV-2.

Amino Acid Sequence↗
PubMed1990

Immunological studies of the basis for the apathogenicity of simian immunodeficiency virus from African green monkeys.

Potential reasons for the lack of pathogenicity of the simian immunodeficiency virus SIVagm in its natural host, the African green monkey (AGM, Cercopithecus aethiops), were investigated with respect to immunological mechanisms. The functional immune response of monkeys to infection was similar (though not identical) to that of humans to infection with human immunodeficiency virus type 1 (HIV-1). In the sera of infected animals, neutralizing antibodies were found to be low or absent, and in particular there was no neutralization of the various isolates by homologous sera. There was no detectable antibody/complement cytotoxicity, though AGM sera were able to initiate antibody-dependent cellular cytolysis of infected cells in the presence of healthy effector peripheral blood lymphocytes. As in the human/HIV system, macrophages from AGMs are readily infected by SIVagm. Two possibly important differences between the AGM/SIVagm system and the human/HIV system are (i) the low immune response of the AGMs to the core protein of SIVagm and (ii) the significantly lower inhibitory effect of SIVagm proteins on the proliferation of AGM lymphocytes.

Animals↗
PubMed1990

Productive infection of both CD4+ and CD4- human cell lines with HIV-1, HIV-2 and SIVagm.

Human monolayer cells of various origins were shown to be susceptible to infection by HIV-1, HIV-2 and simian immunodeficiency virus obtained from African green monkeys (SIVagm). Immunoperoxidase staining revealed infection of 2-7% of the monolayer cells, although in order to achieve infection approximately 50-fold more virus was necessary than with CD4(+)-permissive lymphoma cells. No CD4-receptor antigen expression by fibroblastoid cells was detectable by immunofluorescence using several monoclonal antibodies (MAbs), although a low level of CD4-specific messenger RNA expression was revealed by Northern analysis (with the exception of Tera-1 and RD cells). Attempts to block viral infection by anti-CD4 MAbs indicated a CD4 receptor-mediated mechanism for all lines tested except RD cells. We conclude that a low level of CD4-receptor expression is sufficient to allow infection of fibroblastoid cells. The infectability of a CD4-negative cell line indicates a second pathway of cellular infection, possibly mediated by a cellular receptor distinct from the CD4 molecule.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Soluble CD4 enhances simian immunodeficiency virus SIVagm infection.

The CD4 molecule is expressed on T-helper cells and serves as the cellular receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for the simian immunodeficiency viruses SIVmac and SIVagm. HIV-1, HIV-2, and SIVmac infectivity can be blocked by monoclonal antibodies (MAbs) directed against the CD4 molecule and by soluble CD4 proteins (sCD4). In the present study, we demonstrated not only lack of inhibition, but 10- to 100-fold sCD4-dependent enhancement of SIVagm infectivity of human T-cell lymphoma lines, although SIVagm infection was blocked by MAbs OKT4a and Leu3a. SIVagm enhancement with sCD4 was suppressed by MAbs OKT4a and Leu3a to levels observed without addition of sCD4. The infectivity of all four tested SIVagm variants was enhanced by sCD4 on all tested lymphoma cell lines. These results suggest a second step (second or secondary receptor) required for enhancing virus entry into the cell and may have serious implications for approaches to the treatment of acquired immunodeficiency syndrome on the basis of modified sCD4 molecules.

Animals↗
PubMed1990

Does HIV cause AIDS? An updated response to Duesberg's theories.

AIDS is an acquired immunodeficiency syndrome caused by the lentivirus HIV and characterized by a successive depletion of CD4 helper/inductor lymphocytes. The humoral and cellular immune deficiency is the basis for the development of opportunistic infections and tumors. Because of a multitude of different bacterial, viral, and parasitic opportunistic agents, AIDS is typified by a number of divergent clinical symptoms. As with many lentiviruses, HIV is difficult to demonstrate in the organism, especially in asymptomatic carriers. Although the rate of infection in peripheral lymphocytes appears to be low, there is an increasing amount of virological and immunological data which help to explain the slow but irreversible failure of the immune defense. We still know relatively little about the pathogenic mechanisms of HIV, although a number of the presently available experimental results provide useful starting points for subsequent investigations. Peter H. Duesberg recently published that HIV and AIDS may well be correlated, but stated that HIV is not the cause of AIDS. Duesberg bases his hypothesis on the fact that HIV fulfills neither Koch's classic postulates nor several more of his own postulates for viral pathogenesis. Following the summary of individual pathogenic mechanisms of HIV infection, the separate points of Duesberg's hypothesis are discussed in detail. It is made very clear that the magnitude of epidemiologic, clinical, and experimental observations and results argue for a causal role of HIV and AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Cloning and expression of the complete SIVagm pol region in E. coli. Purification and partial characterization of the reverse transcriptase.

The complete pol region of the simian immunodeficiency virus from African green monkeys was cloned and expressed in E. coli. The reverse transcriptase was purified to high specific activity and could be shown to contain both reverse transcriptase activity as well as an associated RNase H activity. As is observed with other reverse transcriptases the enzyme is composed of two subunits which cannot be separated by conventional techniques. When comparing the recombinant enzyme with the authentic enzyme isolated from virus no differences were found by biochemical, enzymological, or immunological criteria. Moreover, the action of inhibitors against this enzyme did not show significant differences when compared to reverse transcriptases from HIV-1 and HIV-2.

Bacterial Proteins↗