Search PubMed⌕ Search

Biomedical subjects

D D Richman

Publications and source records attributed to D D Richman.

At least 145 records · Page 8Linked to original sources
PubMed1995

Cytomegalovirus infection induces high levels of cyclins, phosphorylated Rb, and p53, leading to cell cycle arrest.

Human cytomegalovirus (HCMV) infection stimulates cellular DNA synthesis and causes chromosomal damage. Because such events likely affect cellular proliferation, we investigated the impact of HCMV infection on key components of the cell cycle. Early after infection, HCMV induced elevated levels of cyclin E, cyclin E-associated kinase activity, and two tumor suppressor proteins, p53 and the retinoblastoma gene product (Rb). The steady-state concentration of Rb continued to rise throughout the infection, with most of the protein remaining in the highly phosphorylated form. At early times, HCMV infection also induced cyclin B accumulation, which was associated with a significant increase in mitosis-promoting factor activity as the infection progresses. In contrast, the levels of cyclin A and cyclin A-associated kinase activity increased only at late times in the infection, and the kinetics were delayed relative to those for cyclins E and B. Analysis of the cellular DNA content in the infected cells by flow cytometry showed a progressive shift of the cells from the G1 to the S and G2/M phases of the cell cycle, leading to an accumulation of aneuploid cells at late times. We propose that these HCMV-mediated perturbations result in cell cycle arrest in G2/M.

Cell Cycle↗
PubMed1995

Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro.

Human immunodeficiency virus type 1 (HIV-1) possesses the ability to establish a complete infection in nondividing host cells. The capacity of HIV-1 to infect nondividing cells probably contributes significantly to its pathology in vivo, as reflected by infection of peripheral T lymphocytes, tissue macrophages, and microglial cells. However, the in vitro demonstration of the establishment of stable HIV-1 infection in quiescent T cells remains controversial. We have developed a primary T-cell model of acute HIV-1 infection of quiescent CD4 lymphocytes that demonstrates the development of a complete, reverse-transcribed form of virus that is stable for over 10 days in culture. To ensure that our primary cell culture was representative of a quiescent population, the CD4 lymphocyte targets were monitored for membrane expression of activation antigens and for shifts in cell cycle from G0/G1 to S/G2 phase. The presence of viral DNA fragments reflecting progressive reverse transcription was determined by PCR analysis. HIV entered primary CD4 cells rapidly, but viral DNA accumulated slowly in the resting cell cultures. DNA species containing regions of full-length reverse transcription were not detected until 3 to 5 days after infection. In parallel with the appearance of complete viral DNA, spliced RNA transcripts, predominantly of the nef species, were detected by reverse transcriptase PCR amplification. When infected CD4 cells were sorted on the basis of cell cycle analysis of DNA content, the accumulation of a complete viral DNA form was found to occur in both the purified G0/G1-phase cell subset and the cell fraction enriched for the minor S-phase subset. In contrast, spliced viral RNA products could be detected only in the enriched S-phase cell fraction. These results demonstrate that HIV-1 can infect and establish a complete, stable form of viral DNA in primary CD4 lymphocytes in vitro but is blocked from transcription in the absence of cell activation. The findings are consistent with in vivo data from HIV-infected individuals that show the existence of viral DNA predominantly as a stable, extrachromosomal form in T cells of the peripheral circulation.

Base Sequence↗
PubMed1995

Early versus delayed treatment of HIV infection. Zidovudine should be given before symptoms develop.

An understanding of the virology and pathogenesis of HIV infection provides a rationale for initiating early intervention with antiretroviral drugs. Even at the earliest stages of infection when HIV-infected patients are asymptomatic, viral replication is ongoing, particularly in lymphoid tissues. Initiation of antiretroviral therapy can reduce viral replication and delay disease progression. A possible objection to early intervention therapy with zidovudine is the risk of selecting out resistant isolates of HIV, which would be difficult to treat. In practice, zidovudine-resistant isolates occur significantly less frequently in patients with early-stage disease compared with those with late-stage HIV infection, thus supporting the early use of zidovudine; in addition, alternative therapies, active against zidovudine-resistant isolates, are available. Clinical trials with zidovudine in asymptomatic patients have differed in terms of length of follow-up, patient inclusion criteria, dosages and end-points. However, a number of conclusions are possible based on the results obtained: early intervention delays the progression of AIDS, delays the onset of symptomatic disease, has a favourable effect on surrogate markers of HIV infection and is well tolerated; it does not, however, seem to produce any benefit in terms of survival. It is this last point that has given rise to much of the controversy regarding early intervention with zidovudine in asymptomatic patients. Since the disease is progressive in nature with persistent and high levels of viral replication and as prolonging the period of relative health and quality of life when the patient is asymptomatic is desirable, the choice to treat before symptoms develop would appear to be the optimal therapeutic strategy.

CD4 Lymphocyte Count↗
PubMed1994

Phospholipid prodrug inhibitors of the HIV protease. Antiviral activity and pharmacokinetics in rats.

The aspartyl protease of the human immunodeficiency virus (HIV) is an important target for chemotherapeutic intervention because of its key role in cleaving the HIV gag-pol polyprotein during viral assembly and budding. Short peptides and peptidomimetics, which bind to the active site of the HIV aspartyl protease and inhibit processing of the polyprotein, have been synthesized. These compounds are active against HIV in vitro, but many face substantial development problems because of their rapid elimination from the body in bile and urine. Refinement of these agents appears to be necessary if they are to become useful clinically. Recently, we developed a novel chemical strategy for increasing plasma levels of HIV protease inhibitory peptides, which involves the attachment of a biodegradable phospholipid group to the C-terminus of a pentapeptide, iBOC-[L-Phe]-[D-beta-Nal]-Pip-[alpha-(OH)-Leu]-Val (7194). We coupled phosphatidylethanolamine to the C-terminal valine of 7194 to make a phospholipid prodrug (7196). In vitro assays in HT4-6C cells infected with HIV-1 showed that the antiviral activity of the C-terminal phospholipid prodrug, 7196, was equal to that of the free peptide, 7194. Similar results were obtained in vitro when a related pentapeptide (7140) was derivatized at the N-terminal with dipalmitoylphosphatidylethanolamine-succinic acid (7172). Tritium-labeled 7194 and 7196 were prepared and injected intravenously into rats at 3 mumol/kg; then the plasma was assayed for native compound and metabolites by HPLC radioactivity flow detection. The peak plasma level of the tritium-labeled lipid prodrug (7196) was 36 microM versus 1.6 microM for the free protease inhibitor pentapeptide (7194). The area under the curve of the phospholipid prodrug (7196) was 48-fold greater and its mean residence time was increased 43-fold versus the free peptide (7194). Phospholipid prodrugs appear to offer an alternative approach to optimizing in vivo performance of HIV protease inhibitors and other small peptides.

Amino Acid Sequence↗
PubMed1994

Antiproliferative effects of enediynes on AIDS-derived Kaposi's sarcoma cells.

We have investigated the antiproliferative and cytotoxic effects of selected enediynes against three Kaposi's sarcoma (KS) cell lines. The enediynes tested were found to be very potent in inhibiting the growth of KS cells. Treatment with concentrations of 10(-10) M or less were capable of producing 50% inhibition of growth. Furthermore, treatment of KS cells with enediynes induced apoptosis in up to 80% of the cells. This unique class of antitumor agents has therapeutic potential for the treatment of KS.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Synthesis and antiviral activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT.

Phospholipid conjugates of 3'-azido-3'-deoxythymidine (AZT) show activity against the human immunodeficiency virus (HIV) in vitro. In a previous report (K.Y. Hostetler, L.M. Stuhmiller, B.H.M. Lenting, H. van den Bosch and D.D. Richman (1991), J. Biol. Chem. 265, 6112-6117) the syntheses and anti-HIV activities of AZT mono- and diphosphate diglyceride have been described. We now report on the synthesis, characterization and biological activity of 3'-azido-3'-deoxythymidine triphosphate distearoylglycerol (AZTTP-DSG). The compound was prepared by the condensation of AZT diphosphate with distearoylphosphatidic acid morpholidate in anhydrous pyridine at room temperature and purified by means of high-performance liquid chromatography using a silica column. Characterization was performed with 31P-NMR and IR analyses and determination of the fatty acid, phosphorus and nucleoside content of the product. AZTTP-DSG inhibited HIV-1 replication in both CEM and HT4-6C cells at a level intermediate in potency between its mono- and diphosphate analogs. The IC50 values of AZTTP-DSG were 0.33 and 0.79 microM in these two cell lines, respectively. In addition, AZTTP-DSG was less toxic to CEM cells in vitro than the other AZT liponucleotides and reduced viable cell numbers in this cell type by 50% at 1000 microM. Initial studies on the metabolism of AZTTP-DSG revealed that both AZT and AZT monophosphate were liberated from the lipid pro-drug by a rat liver mitochondrial enzyme preparation. These phospholipid derivatives of AZT nucleotides represent pro-drugs for the intracellular delivery of phosphorylated antiviral nucleoside analogs.

Antiviral Agents↗
PubMed1994

Risk factors for Kaposi's sarcoma in patients with advanced human immunodeficiency virus disease treated with zidovudine. Zidovudine Epidemiology Study Group.

BACKGROUND: Although the cause of Kaposi's sarcoma (KS) is unknown, its unique epidemiology suggests that an infectious, sexually transmitted agent or agents may contribute to its pathogenesis. METHODS: To assess the natural history of KS associated with the acquired immunodeficiency syndrome and to identify factors associated with its development, data were analyzed from a multicenter, observational cohort study of 1044 persons with the acquired immunodeficiency syndrome or the acquired immunodeficiency syndrome-related complex and a total CD4 cell count of less than 0.25 x 10(9)/L who were treated with zidovudine between April 1987 and April 1988. Records were reviewed bi-monthly. Follow-up continued for 2 years or until death. RESULTS: One hundred thirty-one patients (13%) had KS a study enrollment, and 143 developed KS (14%) during follow-up, with a 2-year actuarial risk of 21%. The probability of KS at 2 years for patients with initial CD4 cell counts of less than 0.1 x 10(9)/L was 25%, compared with 15% for those with counts of 0.1 x 10(9)/L or more. By logistic regression, a baseline CD4 cell count of less than 0.1 x 10(9)/L (relative odds, 1.43; 95% confidence interval, 1.04 to 1.95), homosexuality (relative odds, 3.71; 95% confidence interval, 1.82 to 7.56), cytomegalovirus disease (relative odds, 1.56; 95% confidence interval, 1.01 to 2.41), and white race (relative odds, 1.64; 95% confidence interval, 1.11 to 2.43) were independently associated with KS. Median survival after KS was 408 days, and KS was an independent predictor of death (relative hazard, 1.78; 95% confidence interval, 1.26 to 2.52). CONCLUSIONS: Kaposi's sarcoma contributes to human immunodeficiency virus-related morbidity and mortality, especially among male homosexuals. This large cohort study provides further evidence for an association between risk for cytomegalovirus infection and KS.

AIDS-Related Complex↗
PubMed1994

The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes.

The viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nef gene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study nef function in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nef on viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nef mutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nef confers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study nef function in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

Base Sequence↗
PubMed1994

Biological importance and cooperativity of HIV-1 regulatory gene splice acceptors.

The replication of HIV-1 mutants containing altered splice acceptor sequences was studied. The splice acceptor sites 5' of the essential tat and rev AUG codons were altered to eliminate specifically spliced species from the viral repertoire of mRNAs. All splice site mutants were attenuated or fully defective. Mutation of the tat splice acceptor (exon 4) caused loss of the mRNA species containing exon 4 and resulted in an attenuated but replication-competent phenotype. Mutation of the rev splice acceptor sites resulted in viral genomes that failed to propagate in vitro. Mutation of the more 5' of the two major rev acceptors (exon 4A) caused loss of the mRNA species containing exon 4A together with a compensatory increase in use of the more 3' of the rev acceptors (exon 4B). Mutation of the splice acceptor for exon 4B caused the unexpected loss of both exon 4A- and 4B-containing mRNAs. In addition to these effects on the rev splice acceptors, mutations at the 4A and 4B sites also resulted in decreased use of the tat splice site (exon 4) located 175 nucleotides upstream. These effects on utilization of the tat splice acceptor site may explain the requirement for tat to efficiently complement these mutants. The 4A mutant was complemented by tat but not by rev. The 4B mutant was complemented by rev but required both tat and rev for maximum complementation. These data suggested a cooperativity among these splice sites necessary for efficient viral replication. They also indicated that while viral replication persisted at low levels in the absence of splicing to the known site 5' of the tat AUG, failure to splice to at least one of the two major sites 5' of the rev AUG resulted in insufficient rev activity for replication competence.

Cell Line↗
PubMed1994

2-LTR circular viral DNA as a marker for human immunodeficiency virus type 1 infection in vivo.

The unintegrated viral DNA found in human immunodeficiency virus type 1 infection includes linear and circular forms. We targeted the circular form containing two copies of the viral long terminal repeat (2-LTR circle) and developed specific assays to detect this molecule in peripheral blood mononuclear cells from HIV-infected patients. In vitro HIV-1 infection of peripheral blood mononuclear cells showed rapid accumulation and rapid decay of 2-LTR circular viral DNA. Examination of 2-LTR circular viral DNA levels provides a view of spreading infection based on a viral DNA form that is structurally distinct and has a known, short half-life in infected cells. In patients not receiving antiviral therapy, the levels of 2-LTR circular viral DNA and total viral DNA were significantly correlated to CD4 cell counts. Similar correlations were not observed in patients receiving zidovudine (AZT), didanosine (ddA), or zalcitabine (ddC).

Base Sequence↗
PubMed1994

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.

CD4 Antigens↗
PubMed1994

Drug resistance in viruses.

The mechanisms of action of antiviral drugs and of drug resistance are known in remarkable detail, partly because X-ray crystallographic structures are known for many target viral proteins. Resistance usually involves amino acid substitutions in the target protein that prevent drug binding or prevent an enzyme from accepting the drug as a substrate. This information allows more effective use of available drugs and aids the development of new drugs.

Alphaherpesvirinae↗
PubMed1994

Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates.

Reverse transcriptase-associated amino acid substitutions related to ddC, d4T, and nevirapine resistance have been found in isolates of human immunodeficiency virus type 1 (HIV-1) from patients treated with AZT only. Sequence analysis of 23 isolates documented the presence of 4 unexpected mutations at amino acid residues related to drug resistance. Two isolates contained an aspartic residue in codon 69 associated with ddC resistance, and another a change in codon 75 associated with resistance to d4T. The Y-to-C alteration in codon 181 associated with nevirapine resistance was observed in another isolate after serial passage in cell culture in the absence of drug. Changes in substitution patterns were also noted after serial passage of four AZT resistant isolates in cell culture without inhibitors. One of the strains showed changes in codons 67 and 70 to wild-type residues. Clonal analysis showed that this alteration occurred by the selection during cell culture passage of the wild-type genotype, which was present as a minority subpopulation in the initially resistant virus stock, rather than to genetic reversion. In summary, we present evidence documenting the presence of mutations associated with drug resistance in the absence of drug treatment and supporting the role played by gentic variability in the emergence of HIV-1 antiviral resistance.

Amino Acid Sequence↗
PubMed1994

Resistance, drug failure, and disease progression.

The clinical significance of the reduced in vitro susceptibility of HIV to antiretroviral agents has been difficult to elucidate for nucleoside analogs such as zidovudine. However, the virological significance of resistance to nevirapine and other HIV-1-specific reverse transcriptase inhibitors has been established. With antiretroviral therapy, disease progression is not equivalent to drug failure, which is not equivalent to drug resistance. Clinical disease progression is only indirectly linked to HIV replication. Drug resistance is complex, and combining drugs does not appear to delay emergence of resistant strains of HIV although it may affect the specific amino acid substitutions. Drug resistance does appear to contribute to drug failure. The clinical trial ACTG 116B/117 found that the duration of prior zidovudine therapy was not related to the relative benefit of switching to didanosine. Preliminary results of analysis of resistant strains of HIV isolated from ACTG 116B/117 patients revealed that the relative hazard of progression was about threefold higher for patients with high-level resistance to zidovudine, syncytium-inducing biological phenotype, and an AIDS diagnosis at baseline. This study showed clearly that acquisition of an HIV strain with high-level resistance to zidovudine was a poor prognostic factor. Although nevirapine resistance emerges rapidly, preliminary data suggest that high dosages may be active against HIV even in the presence of resistant HIV strains. At the present time, viral resistance and biological phenotype are not useful in the management of individual patients.

Antiviral Agents↗
PubMed1994

The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression.

Many patients infected with human immunodeficiency virus (HIV) yield syncytium-inducing (SI) virus isolates that are cytopathic in cell culture. The presence of SI virus was assessed in 325 persons entering 11 antiretroviral therapy trials and correlated with both CD4 cell declines and clinical end points. Adjusted mean rates of CD4 cell count decline were 40 and 102 cells/year in the non-SI (NSI) and SI groups, respectively (P < .0001). Rates of decline in 16 persons converting from NSI to SI virus averaged 31 cells/year before conversion and 142 cells/year afterward (P = .04). In a nested case-control analysis, persons who experienced surrogate marker end points or opportunistic infections were 2.3-3.5 times more likely to have SI virus than were controls (P = .01-.04) but who died were similar to controls with respect to virus phenotype (P = .70). Presence of the SI phenotype of HIV is a strong predictor of decline in CD4 cell count and progression of disease; however, controlling for the CD4 cell count, the SI phenotype did not increase the immediate risk of death.

Adult↗
PubMed1994

Phosphatidylazidothymidine and phosphatidyl-ddC: assessment of uptake in mouse lymphoid tissues and antiviral activities in human immunodeficiency virus-infected cells and in Rauscher leukemia virus-infected mice.

During the early stages of human immunodeficiency virus (HIV) infection, although symptoms are absent and viral replication in peripheral blood mononuclear cells is low, substantial levels of HIV replication can be documented in lymphoid tissue [G. Pantaleo, C. Graziosi, J.F. Demarest, L. Butini, M. Montroni, C.H. Fox, J.M. Orenstein, D.P. Kotler, and A.S. Fauci, Nature (London) 362:355-358, 1993, and J. Embretsen, M. Zupancic, J.L. Ribas, A. Burke, P. Racz, K. Tenner-Tacz, and A.T. Haase, Nature (London) 362:359-362, 1993]. This observation suggests that earlier treatment of HIV infection may be indicated and that strategies for enhancing drug targeting to the lymphoid tissue reservoris of HIV infection may be beneficial. To address this issue, we synthesized dioleoylphosphatidyl-ddC (DOP-ddC) and dipalmitoylphosphatidyl-3'-azido-3'-deoxythymidine (DPP-AZT), phospholipid prodrugs which form lipid bilayers and which are readily incorporated into liposomes. The anti-HIV activity of DOP-ddC was similar to that of ddC in HIV type 1-infected HT4-6C cells, but DPP-AZT was considerably less active than AZT in HT4-6C cells. Liposomes containing DOP-[3H]ddC or DPP-[3H]AZT administered intraperitoneally to mice produced greater levels of total radioactivity over time in plasma, spleen, and lymphoid tissue relative to the results with [3H]ddC and [3H]AZT, respectively. DPP-AZT administered intraperitoneally in liposomes as a single daily dose to mice infected with Rauscher leukemia virus prevented increased spleen weight and reverse transcriptase levels in serum with a dose-response roughly comparable to that of AZT given continuously in the drinking water. DOP-ddC, DPP-AZT, and lipid conjugates of other antiretroviral nucleosides may provide higher levels of drug over time in plasma and in lymph nodes and spleen, important reservoirs of HIV infection, and may represent an interesting alternative approach to antiviral nucleoside treatment of AIDS.

Animals↗
PubMed1994

Influence of host cell type and V3 loop of the surface glycoprotein on susceptibility of human immunodeficiency virus type 1 to polyanion compounds.

Dextran sulfate is a potent inhibitor of human immunodeficiency virus (HIV) binding and replication in lymphocytic cell lines. In this study, we demonstrate that the effect of dextran sulfate and heparin depends on the host cell type and on the V3 loop, the principal neutralizing determinant of HIV gp120. In particular, when dextran sulfate was tested on primary human macrophages infected with macrophage-tropic viruses, enhancement of infection was observed in 6 of 11 independent macrophage preparations and with 5 of 13 primary HIV isolates. Our in vitro observations might explain why enhanced HIV replication was observed in HIV-infected patients treated with dextran sulfate.

Cell Line↗