Owen Wood

Identification of a Potential HIV-Induced Source of Bystander-Mediated Apoptosis in T Cells: Upregulation of TRAIL in Primary Human Macrophages by HIV-1 Tat

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.

Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion

BACKGROUND:  West Nile virus (WNV) transmission by transfusion was documented in 2002. Approximately 80 percent of WNV infections are asymptomatic and 1 percent develop severe neurological illness. In animals, Langerhans‐dendritic cells support initial viral replication, followed by replication in lymphoid tissues and dissemination to organs and possibly to the CNS. The cellular tropism of WNV infection after transfusion and the particular human blood cells that sustain viral replication remain largely unknown. Whether primary monocyte‐derived macrophages (MDMs) support WNV infection‐replication and produce infectious virions, with an in vitro system, was investigated.

Bcl-2 Upregulation by HIV-1 Tat during Infection of Primary Human Macrophages in Culture

The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1(BAL )infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.

Absence of detectable xenotropic murine leukemia virus–related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus Type 1–infected blood donors or individuals in Africa

BACKGROUND: Since the identification of xenotropic murine leukemia virus–related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV‐1)‐infected individuals to gain evidence of XMRV infection in this geographic region.

Identification of new, emerging HIV-1 unique recombinant forms and drug resistant viruses circulating in Cameroon

BackgroundThe HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients.MethodologyBlood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database.ResultsPhylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies.ConclusionsOur results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.

Characterization of Immune Responses to Capsid Protein p24 of Human Immunodeficiency Virus Type 1 and Implications for Detection

ABSTRACT To further refine our current nanoparticle-based HIV-1 p24 antigen assay, we investigated immune responses to p24 to identify diagnostically significant immune dominant epitopes (IDEs) in HIV-infected human sera, to address cross-reactivity of anti-p24 antibodies to different subtypes, and to identify new biomarkers that distinguish acute from chronic HIV infection for more accurate incidence estimation. We identified two major linear epitope regions, located in the CypA binding loop and adjacent helices and at the end of the C-terminal domain. Most sera (86%) from acutely HIV-1-infected individuals reacted with multiple peptides, while 60% and 30% of AIDS patient samples reacted with multiple and single peptides, respectively. In contrast, 46% and 43% of chronically HIV-1-infected individuals reacted with one and none of the peptides, respectively, and only 11% reacted with multiple p24 peptides, indicating a progression of immune responses from polyclone-like during acute infection to monoclone-like or a nonresponse to linear epitopes during chronic infection. Anti-p24 antibodies (subtype B) show broad cross-reactivity to different HIV-1 subtypes, and the synergistic action of different combinations of anti-HIV antibodies improves capture and detection of divergent HIV-1 subtypes. Our results indicate that the modified peptide immunoassay is sensitive and specific for the rapid identification of HIV-1 p24 IDEs and for investigation of immune responses to p24 during natural HIV-1 infection. The data provide the foundation for development and refinement of new assays for improved p24 antigen testing as future tools for rapid and accurate diagnosis as part of early intervention strategies and estimations of incidence.

beta-estradiol attenuates the anti-HIV-1 efficacy of Stavudine (D4T) in primary PBL

BackgroundFemale hormones are known to play an important role in predisposition for many infectious diseases. Recent work suggests there are gender effects in HIV/AIDS progression. Here we ask whether the sex steroid hormone β-estradiol affects the replication of HIV-1 or the efficacy of a common anti-retroviral drug, Stavudine (D4T).ResultsHuman PBL were infected with HIV-1 in the presence or absence of combinations of sex steroid hormones and the anti-retroviral drug, D4T. After seven days in culture, viral supernatants were assayed for HIV-1 p24 protein. β-estradiol resulted in a modest inhibition of HIV-1 replication of ~26%. However, 2 nM β-estradiol increased the amount of HIV-1 replication in the presence of 50 nM D4T from a baseline of 33% (+/- SE = 5.4) to 74% (+/- SE = 5.4) of control virus levels in the absence of drug. Both results were statistically highly significant (p < 0.001). β-estradiol did not increase the replication of a D4T-resistant strain of HIV in the presence of D4T. The effects were unlikely to be due to general cell inhibition or toxicity because these concentrations of drug and hormone cause no cytotoxicity in PBL as measured by trypan blue exclusion.Conclusionβ-estradiol inhibited both HIV-1 replication in primary human PBL and the antiretroviral efficacy of D4T in PBL cultures. To optimize antiretroviral drug therapy, it may be necessary to monitor patient hormonal status.

HIV type 2 primary isolates induce a lower degree of apoptosis "in vitro" compared with HIV type 1 primary isolates.

To determine whether subtypes of HIV-1 and HIV-2 vary in their ability to induce T cell apoptosis in vitro, human peripheral blood mononuclear cells (PBMC) from healthy donors and CEM.NKR-CCR5 cells were infected with a variety of HIV-1 and HIV-2 isolates in vitro. Apoptotic cell levels and chemokine and cytokine production were analyzed. Significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes were observed in PBMCs. The percent of apoptotic cells from each individual ranged from 2 to 78% after HIV-1 infection and from 0 to 28% after HIV-2 infection (p < 0.01). We did not observe significant differences in the degree of apoptosis induced among cells infected with different HIV-1 group M subtypes or group O virus, nor among cells infected with different HIV-2 isolates. However, HIV-2 induced significantly lower degree of apoptosis overall in PBMC and CEM.NKR-CC5 cells when compared with HIV-1 subtypes (p < 0.0001). No significant differences were observed in the production of chemokines, such as RANTES, MIP-1alpha, and MIP-1beta, and cytokines, such as TNF-alpha and TNF-beta when PBMC cultures were infected with different HIV-1 subtype viruses, or HIV-2 isolates. In conclusion, HIV-2 isolates induced significantly lower levels of T cell apoptosis in both PBMC and CEM.NKR-CCR5 cells than HIV-1 isolates. No differences in T cell apoptosis levels were seen between different subtypes of HIV-1 group M or group O isolates. This is consistent with the mild clinical course of infection with HIV-2 that has been reported relative to that observed with HIV-1.

Effect of sex steroid hormones on replication and transmission of major HIV subtypes

BACKGROUND The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. METHODS Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. RESULTS Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. CONCLUSIONS Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV.

In Vitro Evaluation of the Protective Role of Human Antibodies to West Nile Virus (WNV) Produced during Natural WNV Infection

BACKGROUND West Nile virus (WNV) is endemic in the United States and transmissible by transfusion. Since 2003, the US blood supply has been screened by nucleic-acid tests (NAT) for WNV in minipools (MP-NAT) of 6 or 16 specimens. WNV infection begins with low-level viremia detectable only by individual testing (ID-NAT) and no detectable WNV antibodies. Viremia then increases to levels detectable by MP-NAT, and antibodies become detectable; later, viremia decays to levels detectable only by ID-NAT before becoming undetectable. All but 1 documented WNV transmission by transfusion involved blood components negative for WNV antibodies, raising the question whether WNV antibody-positive blood components with low levels of WNV RNA are infectious. METHODS Specimens from 102 viremic donors with and without WNV antibodies were used to investigate infectivity in cultures of Vero cells and human monocyte-derived macrophages (MDMs). RESULTS In Vero cell culture, 54 (74%) of 73 WNV antibody-negative specimens and 10 (36%) of 28 WNV antibody-positive specimens were infectious. In a random subset of 20 specimens tested in MDM culture, 7 (88%) of 8 WNV antibody-positive specimens and 12 (100%) of 12 WNV antibody-negative specimens were infectious. CONCLUSION WNV antibodies do not always protect susceptible cells from WNV infection in vitro. RNA positivity in the presence of antibody cannot be ignored as a theoretical risk for blood recipients and needs further investigation.