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OBJECTIVE Vaccines and biologics containing CD4 molecules or HIV-1 gp120 might induce antibodies targeting CD4. We evaluated temporal variability of CD4 levels in healthy volunteers to quantify declines that could indicate true adverse events. DESIGN Prospective observational cohort study of 100 healthy adults without HIV-1 infection from the Baltimore region. METHODS Participants enrolled and consented to blood draws for immunologic laboratory panels performed once every 8 weeks for 48 weeks. The primary CD4 measurements were CD4 absolute count (cells/mm) and CD4 percentage (CD4%, total CD4 cells/total lymphocyte cells). CD4 changes over time were modeled using fold changes for CD4 absolute counts and differences for CD4 percentages. RESULTS Variation of average CD4 cell counts and percentages were highly participant-specific (P < 0.001 for both). However, changes in both CD4 measurements over time were stable in the population. We proposed thresholds to flag unusual drops using 1.5 SD estimates, calculated as 1.5-fold declines for CD4 count and 6.4% declines for CD4 percentage. In this healthy cohort, flagging simultaneous declines in both measurements corresponded to a low false-positive rate (5.26%). CONCLUSIONS Normal biological variation in large lymphocytes should be taken into account to establish thresholds for adverse changes in clinical trials. The inherent subject-specific variability in CD4 levels makes establishing absolute cutoffs difficult. However, this study proposes that thresholds for declines using 1.5 SDs from these data (50% in absolute count and 6.4% for CD4 percentage) allow a small false-positive rate (∼5%) that could maintain sensitivity for true adverse events in a clinical trial.

BACKGROUND The role of plasmacytoid dendritic cells (pDC) and interferon alpha (IFN alpha) in HIV-1 infection is still unclear. On one hand, HIV-1 disease is associated with a progressive decline of pDC, which displays reduced ability to produce IFN alpha after in vitro challenge. On the other hand, high IFN alpha serum levels in HIV-1-infected individuals have been proposed to promote immune hyper-activation and disease progression. METHODS We sought to determine whether disappearance of pDC in HIV-1 disease is due to homing in lymphoid tissues. We also studied IFN alpha and myxovirus resistance protein A (MxA) expression in unstimulated pDC and correlated these results with selected clinical and laboratory parameters. RESULTS We found that pDC decline markedly in peripheral blood of patients progressing to disease but at the same time express much higher levels of IFN alpha and MxA compared to control individuals. On the other hand, we observed steady pDC counts in lymph nodes of HIV-1 patients. The frequency of circulating pDC correlated directly with CD4 cell counts and inversely with viral load. However, we found no correlation between IFN alpha and MxA expression levels, CD4 counts, and viral load. CONCLUSIONS Circulating pDC decline sharply in the course of HIV-1 disease, but express high levels of IFN alpha, which may represent a hallmark of systemic immune dysfunction.

Objective and design Studies in cell lines have demonstrated that CCR5 coreceptor levels influence the replication efficiency and Enfuvirtide (T-20) susceptibility of R5 HIV-1 strains. At present, however, the role that CCR5 levels on primary CD4 T cells--which are markedly lower than in cell lines and vary only approximately fivefold among most donors--may play in virus replication levels or susceptibility to T-20 is not known. In the present study we evaluated the impact of differences in CCR5 levels among donor CD4 T cells on the infection efficiency and T-20 susceptibility of R5 HIV-1. Methods CD4 and CCR5 density levels were determined by Quantitative FACS analysis. Virus infectivity assays were conducted in cell lines and primary cells. Associations between coreceptor density, virus replication and T-20 sensitivity were tested using the Spearman's correlation test. Results We found a positive correlation (r, 0.55; P = 0.011) between CCR5 density levels on primary CD4 T cells and replication of R5 HIV-1. In cell lines expressing physiologically relevant levels of CD4 and CCR5, T-20 was significantly more potent in cells with low CCR5 levels. In addition, T20 50% inhibitory concentrations for R5 HIV-1 replication varied approximately 100-fold among primary cells from different donors and they were positively correlated with CCR5 density values (r, 0.84; P = 0.00004). Conclusions These results suggest that CCR5 density levels in HIV-1 patients may impact the activity of T-20 against R5 strains and that therapeutic approaches to alter CCR5 density may potentiate T-20.

OBJECTIVE To evaluate the effects of the cyclin dependent kinase (CDK) inhibitor Indirubin-3'-monoxime (IM) on Tat-mediated transactivation function, a step of the HIV-1 cycle that is not currently targeted in antiviral therapy. METHODS The effects of IM on CDK implicated in HIV-1 Tat transactivation function were evaluated by kinase assays, transfection experiments, RNase protection assay and RT-PCR analysis of viral transcripts. The antiviral effect of IM was investigated in cells from HIV-1 infected individuals as well as in cell lines, primary lymphocytes and monocyte-derived macrophages. The antiviral activity of IM was also tested against drug-resistant HIV-1. RESULTS IM inhibits the kinase activity of CDK9 [50% inhibitory concentration (IC50) of 0.05 microM], the catalytic subunit of Positive transcription elongation factor b (P-TEFb). Inhibition of CDK9 activity by IM results in abrogation of Tat-induced expression of HIV-1 RNA in cell lines. In addition, IM inhibits the replication of HIV-1 in both peripheral blood mononuclear cells (IC50 of 1 microM) and macrophages (IC50 of 0.5 microM). IM is effective against primary and drug-resistant strains of HIV-1. Importantly, the antiviral effects of the drug were seen at concentrations that did not affect cell proliferation. CONCLUSIONS Non-toxic concentrations of IM inhibit HIV-1 by blocking viral gene expression mediated by the cellular factor P-TEFb. The drug is effective against wild-type and drug-resistant strains of HIV-1. IM may help control replication of HIV-1 in patients by disrupting a step of the HIV-1 cycle that is not being targeted in current antiretroviral treatments.

Summary: The recent detection of herpesvirus-like DNA sequences in Kaposi's sarcoma (KS) lesions has led to numerous speculations regarding the role of this new agent in KS pathogenesis. However, recent studies indicate a far wider distribution of such viral sequences, shadowing the potential etiologic role of this agent in KS. In this report we show that malignant KS cell lines do not harbor such viral sequences while B cells, CD14+ and CD34+ cells do, suggesting that if a KS malignancy originates from infection with HHV-8, the virus can be lost and is not necessary for maintenance of the neoplastic state. Alternatively, HHV-8 may be a “passenger” in KS.

The immunophenotype of spindle cells in epidemic, endemic, and classic (sporadic) Kaposi's sarcoma (KS) lesions was defined by the demonstration of various cell markers and compared with that of KS-derived cell lines. No significant histological or immunophenotypic differences were observed between the three clinical types of KS at comparable stages. The spindle-cell compartment of the different KS types was composed predominantly of a mixture of proliferating CD45+/CD68+ bone-marrow-derived monocytes and TE7+/collagen+ fibroblastic cells with varying expression of EN4/PAL-E/CD31/CD34/CD36 endothelial-associated antigens and/or smooth-muscle-specific alpha-actin (alpha-actin). The latter cells appeared to represent transitional forms of fibroendothelial and fibromyocytic cells. The in vitro cultured KS-derived cell lines (KS-3, KS-6, and KS-8) expressed the fibroblastic antigen TE7 and smooth-muscle-specific alpha-actin but not leukocytic or endothelial-associated antigens consistent with the phenotype of fibromyoid spindle cells of primary lesions. Neither HIV antigen nor provirus DNA was demonstrable in the epidemic KS lesions. The observed heterogeneity of the spindle-cell compartment further substantiates the view that Kaposi's sarcoma, irrespective of clinical setting, expresses salient features more compatible with reactive, tumor-like lesion than clonal sarcoma.

Six children with the acquired immunodeficiency syndrome (AIDS) and 12 of their household contacts were investigated serologically for evidence of infection with human T-lymphotropic virus/lymphadenopathy-associated virus (HTLV-III/LAV), the presumed etiologic agent of AIDS. All six children had antibody against HTLV-III/LAV, as measured by enzyme-linked immunosorbent assay, in each specimen tested. Of the two mothers studied both were seropositive; one was diagnosed with and died from AIDS. Four of the remaining 10 household members were seropositive, including three adults in groups at high risk for the development of AIDS and one sibling who was younger than the child with AIDS. Among the seronegative household contacts were four foster mothers or grandmothers of the children with AIDS, three of whom had cared for the children since infancy. Household contact with children with AIDS may include persons in groups at high risk for AIDS who have been infected with HTLV-III/LAV. However, the negative findings in household contacts without risk factors for AIDS suggest that horizontal transmission of the virus within households by means other than sexual contact must be infrequent.

Three human T-lymphotropic viruses have been isolated and characterized in the past 5 years. The ability to culture target cells with T-cell growth factor and sensitive detection systems for the virally encoded polymerase reverse transcriptase permitted isolation of HTLV-I, which is strongly linked to the cause of adult T-cell leukemia and associated with other lymphoid malignancies in endemic areas. The same techniques, using a permissive human tumor cell line, allowed the isolation and characterization of HTLV-III/lymphadenopathy-associated virus, which is implicated as the primary cause of the acquired immunodeficiency syndrome (AIDS). This virus shares some features with HTLV-I and HTLV-II, such as additional genes not found in most retroviruses. One gene codes for a transcriptional activator protein and may be a feature of a larger group of related retroviruses. The clear identification of the primary cause of AIDS has resulted in the development of specific immunologic reagents, preventive and therapeutic proposals, and comprehensive identification of the clinical diseases associated with this virus.