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Biomedical subjects

C Flexner

Publications and source records attributed to C Flexner.

At least 55 records · Page 3Linked to original sources
PubMed1994

Effect of dipyridamole on zidovudine pharmacokinetics and short-term tolerance in asymptomatic human immunodeficiency virus-infected subjects.

Zidovudine delays the progression of infection and prolongs the survival of human immunodeficiency virus (HIV)-infected patients, but these benefits are limited by dose-related toxicity and the cost of the drug. Dipyridamole, in micromolar concentrations, acts synergistically with zidovudine, reducing the anti-HIV 95% inhibitory concentration of zidovudine 5- to 10-fold in vitro. We sought to establish a well-tolerated dose of dipyridamole for use in combination with zidovudine and to detect clinically significant pharmacokinetic interactions. Both objectives are essential for planning studies of the efficacy of the zidovudine-dipyridamole combination. Eleven asymptomatic HIV-infected subjects (median CD4+ cell count, 311 cells per mm3), 10 of whom had been on zidovudine at 500 mg/day for at least 6 months, were admitted to the study. Zidovudine pharmacokinetics were measured on day 1. Dipyridamole was then begun at 600 mg/day (subjects 1 to 3) or 450 mg/day (subjects 4 to 11), and zidovudine and dipyridamole pharmacokinetics were measured on day 5. All subjects given 600 mg of dipyridamole per day developed headache or nausea, or both. Six of eight subjects given dipyridamole at 450 mg/day developed headache or mild nausea that resolved after a median of 2 days. The area under the zidovudine concentration-time curve was not significantly different on day 1 in comparison with that on day 5 (P = 0.11). Symptoms were significantly correlated with the maximum zidovudine concentrations, which were achieved when dipyridamole was dosed concomitantly (p = 0.03). Total (free and protein-bound) dipyridamole trough concentrations were near those demonstrating synergy with zidovudine against HIV in vitro. Dipyridamole was highly protein bound, with a median free/total dipyridamole ratio of 0.7%; the percent free/total dipyridamole ratio was inversely correlated with alpha 1 acid glycoprotein concentrations (r2 =0.66). Results of the study indicate that adjustment of the zidovudine dose was not required to achieve equivalent zidovudine concentrations when zidovudine was administered in combination with dipyridamole at the doses studied. In the short study described here, the zidovudine-dipyridamole combinations was well tolerated in asymptomatic HIV-infected subjects after the occurrence of mild transient symptoms.

Adult↗
PubMed1993

Clearance of recombinant vaccinia virus expressing IL-2: role of local host immune responses.

Recombinant vaccinia viruses that express the human or mouse IL-2 gene are rapidly eliminated from immunoincompetent nude mice whereas control viruses cause lethal infections. To understand the role that virus-encoded IL-2 plays in attenuation, we investigated the mechanism of virus elimination from nude mice. Survival correlated with accelerated clearance of the virus. Treatment of infected mice with antibodies to eliminate NK cells or to neutralize interferon-gamma suggested that both are involved in the elimination of IL-2-producing virus. However, lytic activity of NK cells was not necessary as shown by studies with beige mice. Coinfection with IL-2-expressing and control virus resulted in lethal infection of nude mice, indicating the absence of significant systemic immunity. Focally acting immunopotentiating and chemotactic activities of virus-encoded IL-2 and host-derived interferon-gamma seem to confer protection in this novel approach to virus attenuation.

Animals↗
PubMed1993

Comparison of the effects of 3'-azidothymidine with those of neutralizing antibodies on simian immunodeficiency virus infection in macrophages.

We previously showed that simian immunodeficiency virus-infected macaque macrophages contacting uninfected CD4+ lymphocytes caused extensive cell fusion and synthesis of phlogistic cytokines. In this study, macaque macrophage cultures inoculated with SIVmac251 and treated simultaneously with 10 microM 3'-azidothymidine (AZT) became infected and produced small amounts of viral antigen (p27) but failed to fuse with CD4+ CEM174 cells. When AZT was added 1 to 3 days after virus inoculation, the infected cells caused fusion and the release of tumor necrosis factor and produced increasing amounts of p27. In contrast, neutralizing antibodies prevented infection when added at the time of virus inoculation, and they were much more effective than AZT in limiting virus replication, fusion cytopathic effect, and cytokine production when added up to 3 days postinoculation. Neutralizing antibodies may be more effective than AZT in reducing the virus load in the macrophage population and in preventing both cell fusion and the production of potentially pathogenic cytokines.

Animals↗
PubMed1991

Non-Hodgkin's lymphoma in patients with advanced HIV infection treated with zidovudine.

We wished to determine the incidence of human immunodeficiency virus-related high-grade non-Hodgkin's lymphoma (NHL) and identify factors associated with the development of NHL in patients receiving zidovudine. Data are from a 2-year prospective, observational, multisite study of 1030 patients with the acquired immunodeficiency syndrome (AIDS) and advanced AIDS-related complex who received zidovudine. Non-Hodgkin's lymphoma developed in 24 (2.3%) of 1030 patients who received zidovudine during 1463 person-years of follow-up (rate, 1.6 per 100 person-years of therapy). The relative hazard for development of NHL was stable throughout 2 years of therapy, with the risk of developing NHL 0.8% for each additional 6 months of therapy. Factors associated with development of NHL were a prior diagnosis of Kaposi's sarcoma, herpes simplex virus infection, or lower mean neutrophil count. Less strongly associated was a prior diagnosis of oral hairy leukoplakia or homosexual transmission of HIV. By Cox proportional hazards analysis, a prior diagnosis of Kaposi's sarcoma, cytomegalovirus disease, or oral hairy leukoplakia was most strongly associated with development of NHL. Our study demonstrates a relatively high incidence of NHL in patients with advanced human immunodeficiency virus disease who are undergoing antiretroviral therapy and suggests possible risk factors for development of NHL.

AIDS-Related Complex↗
PubMed1991

Rhesus monkey macrophages infected with simian immunodeficiency virus cause rapid lysis of CD4-bearing lymphocytes.

Inoculation of simian immunodeficiency virus into cultures of primary rhesus monkey macrophages or CD4-bearing transformed T lymphocytes resulted in persistent infection, with minimal virus replication in the macrophages and extensive replication in the lymphocytes. However, uninfected T cells added to infected macrophages underwent rapid fusion and lysis and were almost completely eliminated without the production of virus particles. Lysis required direct contact between the T cells and the infected macrophages, which enabled binding between CD4 on the former and viral gp120 on the latter to occur. This process was blocked by soluble CD4 and dextran sulphate. Neutralizing antibodies in the serum of an infected macaque prevented cell fusion by preventing infection of the macrophages. However, these antibodies did not prevent fusion when added to previously infected macrophages. Infected macrophages were incorporated into the syncytia of lymphocytes and continued incorporation of new lymphocytes into the syncytia required infected macrophages to be metabolically active. One inference from these studies is that infected macrophages in vivo could help mediate the well known depletion of T4 cells in patients with AIDS.

Animals↗
PubMed1991

Pharmacokinetics, toxicity, and activity of intravenous dextran sulfate in human immunodeficiency virus infection.

Polysulfated polysaccharides are attractive candidates for antiviral drug development because of their potent in vitro activities against human immunodeficiency virus (HIV), herpesviruses, and other enveloped viruses. To determine the potential anti-HIV activity of a prototypical polysulfated polysaccharide, we administered the maximally tolerated dose of dextran sulfate by continuous intravenous infusion to 10 subjects with symptomatic HIV infection for up to 14 days. Since parenteral dextran sulfate is an anticoagulant, the infusion was adjusted to produce the greatest acceptable increase in activated partial thromboplastin time. Drug concentrations in plasma achieved with this protocol were up to 200-fold greater than the 50% inhibitory concentration for free HIV infectivity in vitro. Despite this, circulating HIV antigen (p24) levels increased in all eight subjects who received the drug for more than 3 days (median proportional increase, 73.5%; range, 32 to 130%); this increase was highly significant when it was compared with that in a large cohort of untreated historical controls (Fisher's exact test, P less than 0.001). Frequent decreases in infusion rate were required in all subjects to maintain a constant activated partial thromboplastin time; plasma dextran sulfate levels did not fall as the infusion rate decreased, suggesting a decline in estimated drug clearance over time. Continuous intravenous dextran sulfate was toxic, producing profound but reversible thrombocytopenia in all eight subjects who received drug for more than 3 days and extensive but reversible alopecia in five of these subjects. Because of its toxicity and lack of beneficial effect on surrogate markers, dextran sulfate is unlikely to have a practical role in the treatment of symptomatic HIV infection.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Anchor sequence-dependent endogenous processing of human immunodeficiency virus 1 envelope glycoprotein gp160 for CD4+ T cell recognition.

Human CD4+ T cell clones and cell lines were shown to lyse recombinant vaccinia virus-infected cells that synthesize the HIV-1 envelope glycoprotein gp160. The processing of endogenously synthesized gp160 for recognition by CD4+ T cells required that the protein, after synthesis on the rough endoplasmic reticulum and during subsequent cellular transport, remain attached to the luminal/extracellular membrane face by a hydrophobic anchor sequence.

Amino Acid Sequence↗
PubMed1990

Attenuation and immunogenicity in primates of vaccinia virus recombinants expressing human interleukin-2.

Vector-directed lymphokine expression represents a novel approach to the attenuation of live recombinant viruses which might be used as vaccines. Expression of interleukin-2 (IL-2) by recombinant vaccinia virus has been shown to significantly attenuate virus virulence in rodent species without diminishing immunogenicity. Skin lesion formation and immunogenicity of vaccinia/IL-2 recombinants in three species of primates was examined. IL-2 expression was associated with a 15-fold reduction in the area of induration after intradermal inoculation of recombinant viruses in patas monkeys. Wild type and a control vaccinia recombinant produced large (greater than 5000 mm2) skin ulcers in this species, but the IL-2 expressing recombinant produced no ulceration. Production of antibodies to vaccinia virus and to influenza A virus haemagglutinin expressed by recombinant vectors was examined in rhesus and squirrel monkeys. IL-2 expression accelerated the resolution of skin lesions in rhesus but not squirrel monkeys. Despite this, antibody production was equivalent in the presence or absence of IL-2. IL-2 expression can greatly reduce the skin lesions formed by live recombinant vaccinia vectors in primates, indicating significant attenuation, without reducing the immunogenicity of the vaccine.

Animals↗
PubMed1990

An epitope in human immunodeficiency virus 1 reverse transcriptase recognized by both mouse and human cytotoxic T lymphocytes.

T-cell-mediated cytotoxicity may play an important role in control of infection by the human immunodeficiency virus (HIV). In this study, we have identified and characterized a relatively conserved epitope in the HIV-1 reverse transcriptase recognized by murine and human cytotoxic T cells. This epitope was identified using a murine antigen-specific CD8+ class I major histocompatibility complex-restricted cytotoxic T-cell (CTL) line, a transfected fibroblast cell line expressing the HIV-1 pol gene, recombinant vaccinia viruses containing different truncated versions of the pol gene, and overlapping synthetic peptides. The optimal antigenic site was identified as residues 203-219 by synthesizing extended or truncated peptide analogs of the antigenic fragment. The optimal peptide was then tested for sensitization of autologous Epstein-Barr virus-transformed B-cell targets for killing by fresh human peripheral blood mononuclear cells. It was recognized by CTLs from several HIV-seropositive patients but not from any seronegative donor. Therefore, this peptide is a good candidate for inclusion in an AIDS vaccine. This study demonstrates that the same CTL epitope can be seen by murine and human CD8+ CTLs, as previously demonstrated for epitopes recognized by CD4+ helper T cells, and suggests the utility of screening for immunodominant CTL epitopes in mice prior to carrying out studies in humans.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Dextran sulfate is poorly absorbed after oral administration.

STUDY OBJECTIVE: To determine whether dextran sulfate (molecular weight, 7000 to 8000 daltons; 17% to 20% sulfur), a synthetic heparin analogue with anti-human immunodeficiency virus (HIV) activity in vitro, is absorbed after oral administration. DESIGN: Open-label, single-center study in two parts. The first part was a bioavailability study in which six subjects received a single 1800-mg oral dose and a single 225- or 300-mg intravenous dose. The second part was a study of the dose-response relation between dextran sulfate and total plasma lipolytic activity in which twelve subjects were given a single infusion of either 0.05, 0.5, 5, or 50 mg of dextran sulfate. SUBJECTS: Eighteen healthy volunteers. MEASUREMENTS AND MAIN RESULTS: In the bioavailability study, plasma and urine dextran-sulfate concentrations were measured by a competitive binding assay after each dose. In addition, two bioassays were used to assess plasma concentrations: plasma lipolytic activity and activated partial thromboplastin time (APTT). After the oral dose, plasma concentrations were not measurable with the competitive binding assay (lower limit of sensitivity, 1 microgram/mL); less than 0.5% of the dose was recovered in the urine, the APTT did not increase, and the median increase in the plasma lipolytic activity was only twofold (maximum increase, 11-fold). In contrast, after the intravenous dose of 225 mg, peak plasma concentrations by competitive binding assay were 26 to 35 micrograms/mL (median, 28 micrograms/mL); 25% to 29% (median, 25%) of the dose was recovered in the urine; the APTT increased to 3.5 to 9.2 times the baseline value (median increase, 6.9 times), and the plasma lipolytic activity increased by 185 to 548 times the baseline value (median increase, 438 times). In the dose-response study, intravenous doses as low as 0.5 mg produced significant increases in the plasma lipolytic activity. There was a steep dose-response curve between 0.5 and 50 mg. CONCLUSION: Dextran sulfate is very poorly absorbed after oral administration.

Administration, Oral↗
PubMed1989

Long-term culture and fine specificity of human cytotoxic T-lymphocyte clones reactive with human immunodeficiency virus type 1.

The definition of human immunodeficiency virus type 1 (HIV-1) immunogenic epitopes is central to the rational design of AIDS vaccine strategies. In this study, we have generated seven HIV-1 reverse transcriptase-specific cytotoxic T-lymphocyte (CTL) clones from the peripheral blood of two seropositive subjects. Epitopes recognized by these CTL clones were identified by using target cells infected with recombinant HIV-1-vaccinia virus vectors expressing truncated reverse transcriptase proteins and further defined by using target cells incubated with overlapping 25-amino acid synthetic reverse transcriptase peptides. Five different CTL epitopes were identified, and in each case recognition was restricted by class I human leukocyte antigens (HLA). Clones maintained specific cytolytic function in continuous culture for up to 11 months, requiring only periodic restimulation with a CD3-specific monoclonal antibody. These results indicate that HIV-1-specific, major histocompatibility class I-restricted CTL recognize multiple epitopes of a single viral gene product in conjunction with different host HLA antigens. In addition, they demonstrate that human virus-specific CTL can be grown in long-term culture without the need for reexposure to viral antigen.

Cell Line↗
PubMed1988

Successful vaccination with a polyvalent live vector despite existing immunity to an expressed antigen.

A global vaccination strategy must take into account production and delivery costs as well as efficacy and safety. A heat-stable, polyvalent vaccine that requires only one inoculation and induces a high level of humoral and cellular immunity against several diseases is therefore desirable. A new approach is to use live microorganisms such as mycobacteria, enteric bacteria, adenoviruses, herpesviruses and poxviruses as vaccine vectors. A potential limitation of live polyvalent vaccines, however, is existing immunity within the target population not only to the vector, but to any of the expressed antigens. This could restrict replication of the vector, curtail expression of antigens, and reduce the total immune response to the vaccine. Recently acquired immunity to vaccinia virus can severely limit the efficacy of a live recombinant vaccinia-based vaccine, so a strategy involving closely spaced inoculations with the same vector expressing different antigens may present difficulties. We have constructed a recombinant vaccinia virus that expresses surface proteins from two diverse pathogens, influenza A virus haemagglutinin and herpes simplex virus type 1 (HSV-1) glycoprotein D. Mice that had recently recovered from infection with either HSV-1 or influenza A virus could still be effectively immunized with the double recombinant.

Animals↗
PubMed1988

HIV-1 reverse transcriptase is a target for cytotoxic T lymphocytes in infected individuals.

Characterization of the host immune response to human immunodeficiency virus type 1 (HIV-1) is critical to the rational design of an effective AIDS vaccine. In this study, cytotoxic T lymphocytes (CTL) specific for HIV-1 reverse transcriptase (RNA-dependent DNA polymerase) were found in blood samples from HIV-1-infected individuals. CTL targets were prepared by immortalizing B cells from ten seropositive and six seronegative individuals, and then infecting these cells with recombinant vaccinia viruses containing HIV-1 genes. CTL directed against autologous B lymphoblasts expressing HIV-1 reverse transcriptase were detected in fresh blood samples from eight HIV-1 seropositive subjects, but in no seronegative controls. The effector cells were identified as major histocompatibility complex-restricted CD3+CD8+ lymphocytes. Because the HIV-1 pol gene is highly conserved among different isolates and generates both humoral and cellular immune responses, it bears consideration for inclusion in a candidate AIDS vaccine.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Characterization of human immunodeficiency virus gag/pol gene products expressed by recombinant vaccinia viruses.

Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.

Antigens, Viral↗
PubMed1988

Roles of vaccinia virus in the development of new vaccines.

Vaccinia virus is an efficient expression vector with broad host range infectivity and large DNA capacity. This vector has been particularly useful for identifying target antigens for humoral and cell-mediated immunity. With increased levels of gene expression, obtained either with stronger vaccinia promoters or through incorporation of the bacteriophage T7 RNA polymerase gene into the vaccinia genome, proteins may be synthesized in mammalian cells for use as subunit vaccines. For use as a live recombinant vaccine, efforts are being made to attenuate vaccinia virus further, either by inactivating genes contributing to virulence or by introducing human lymphokine genes into the vaccinia genome.

Genetic Vectors↗