David M. Asmuth

`modeling' relationships among Hiv-1 replication, immune activation and Cd4+ T-cell losses using adjusted correlative analyses

ObjectiveTo model the relationships among HIV-1 replication, immune activation and CD4+ T-cell losses in HIV-1 infection. MethodsCross-sectional analysis of baseline data from the Viral Activation by Transfusion Study. Comparisons of unadjusted and adjusted correlative analyses to establish models for mechanisms of cell loss in AIDS. ResultsUsing these analyses, significant correlations were found among plasma levels of tumor necrosis factor alpha (TNFα) and its type two receptor (TNFrII), interleukin-6 (IL-6), β2-microglobulin, expression of CD38 and HLA-DR on CD8+ T lymphocytes and plasma levels of HIV-1 RNA. When correlations among these indices were adjusted for possible intermediary correlations, the relationship between HIV-1 RNA levels and all plasma markers of immune activation could be accounted for by the correlation between plasma HIV-1 RNA and plasma TNFrII levels. In addition, the negative correlations that both HIV-1 RNA levels and TNFrII levels had with CD4+ T-cell counts were partially accounted for by the correlations of HIV-1 RNA and TNFrII with CD38 expression on CD8+ T cells. In persons with advanced disease (CD4+ T cells <u200450u2004×u2004106/l) IL-6 levels were inversely correlated with CD4+ T-cell counts. ConclusionsThis analysis is consistent with a model wherein HIV-1 replication induces TNFα expression that induces multiple other indices of immune activation. In this model, HIV-1 replication and TNFα expression induce CD4+ T-cell losses at least in part through mechanisms reflected in heightened CD38 expression.

Chemokine/CD4 receptor density ratios correlate with HIV replication in lymph node and peripheral blood of HIV-infected individuals

ObjectivesLymphoid tissue is a major reservoir for virus replication in HIV-infected subjects. The relationship of CCR5 and CXCR4 coreceptor density and HIV replication in peripheral blood mononuclear cells (PBMC) and lymph node (LN) mononuclear cells (LNMC) of HIV-infected subjects was examined. MethodsPBMC and cervical LNMC from 12 HIV-infected patients were examined for virological and immunological parameters including chemokine receptor density, HIV plasma and cellular viral load, coreceptor usage and CD38/HLA-DR expression. ResultsThe number of CCR5 and CXCR4 molecules on CD4 lymphocytes in the LN were significantly higher than in PBMC. In contrast the number of CD4 molecules/CD4 T cell was higher in PBMC than in LNMC. The CXCR4/CD4 and CCR5/CD4 ratios in the LN were significantly higher than in the PBMC. This was associated with a cellular viral load in the LN that was ~110-fold higher than in PBMC. The absolute number of coreceptor molecules per cell did not correlate with the viral load. However, the CCR5/CD4 and CXCR4/CD4 ratios in the LN positively correlated with HIV cellular and plasma RNA. Characterization of the viral isolates suggested an association between clinical isolates using a distinct coreceptor and the upregulation of the corresponding chemokine receptor. ConclusionsThe ratios of chemokine receptors to CD4 molecules in CD4 T cells from LN is higher than in PBMC and may account for the relative difference in cellular viral load in these compartments. Additionally, the coreceptor/CD4 ratios, particularly in the lymphoid tissue, were highly related to HIV replication.

Cytomegalovirus Glycoprotein B Groups in Human Immunodeficiency Virus–Infected Patients with Incident Retinitis

Cytomegalovirus (CMV) strains may be categorized into 4 different groups on the basis of glycoprotein B (gB) genotype. gB genotypes in CMV polymerase chain reaction (PCR)-positive samples from case patients who were diagnosed with retinitis during prospective follow-up were compared with genotypes in CMV PCR-positive samples from an equal number of retinitis-free matched control subjects. All patients were infected with human immunodeficiency virus (HIV) and CMV. Control subjects and their plasma samples were matched with case patients according to baseline CD4(+) T cell count, transfusion history, HIV risk factor, and follow-up time. CMV DNA was genotyped by restriction-enzyme digestion. Eighteen patients met our case definition. Approximately one-third of patients had gB genotype 1, and half had gB genotype 2. These rates were similar among case patients and control subjects and were similar by risk group. The CMV gB2 genotype is not a major determinant of retinitis pathogenicity but appears to be highly prevalent among HIV-infected patients.

Entrapment of recent thymic emigrants in lymphoid tissues from HIV-infected patients: Association with HIV cellular viral load

Objective(s): Depletion of thymus derived naive T-cells is a feature of HIV infection. Here the impact of HIV infection on the compartmentalization of recent thymic emigrants of (RTE) and naive T-cells was examined. Methods: Peripheral blood mononuclear cells (PBMC) and lymphoid tissue (LT) from 43 HIV-infected patients and 12 controls were examined for RTE distribution by measuring coding joint T-cell receptor excisional circles (cjTREC) by PCR and naive and memory T-cell subsets and adhesion molecules (L-selection, LFA-1) by flow cytometry. Results: In HIV-infected patients, the RTE as quantified by cjTRECs in CD4 LT cells were significantly higher than in PBMC. Their values, however, were less than in control subjects, in both the LT and PBMC compartments. This was associated with an increase in L-selectin and LFA-1 expression on LT derived T cells. In PBMC, a significant positive relationship between TREC and naive CD4 cells and an inverse relationship between TREC and cellular viral load (CVL) was observed. Whereas in LT, there was a positive relationship between cjTREC and both naive CD4 cell percentage and CVL. Conclusions: Collectively, the data suggests that LT is a significant reservoir for RTE. The RTE appeared to be entrapped in LT from HIV-infected subjects. Such entrapment is probably a response to the high viral load in these tissues. These observations may partially explain the decline in RTE observed in the peripheral blood of HIV-infected patients, and the delay in recovery of naive cells in blood after initiation of HAART.

Homeostasis of Naive and Memory T Cell Subpopulations in Peripheral Blood and Lymphoid Tissues in the Context of Human Immunodeficiency Virus Infection

To understand the nature of naive and memory T cell depletion in human immunodeficiency virus (HIV) immunopathogenesis, their homeostasis in peripheral blood (PB) and lymph node (LN) compartments of HIV-infected patients was examined. Although the percentage of naive CD4+ cells was higher in LN than in PB mononuclear cells (LNMC and PBMC, respectively), the memory cells were higher in PBMC than in LNMC. The ratio of naive:memory CD4+ cells from PB positively correlated with that in LNs and with the absolute CD4+ cell counts and recall antigen responses, and the ratio inversely correlated with the cellular virus load from the corresponding compartment. These findings indicate that although the pattern of naive and memory cells in the LN and PB compartments appear divergent, their relationship is nonrandom and is significant. The naive&rcolon;memory ratio in PB appears to reflect the lymphoid microenvironment and may potentially be useful as a surrogate marker for treatment efficacy and immune reconstitution.

The importance of high-throughput cell separation technologies for genomics/proteomics-based clinical diagnostics

Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. High-throughput cell separation technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNAs) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using microamplification PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to be about 100 times more sensitive than they are now to be able to do many biologically and biomedically meaningful experiments and clinical tests.