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ABSTRACT: Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally, their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigentransfected target cellCTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigengreen fluorescent protein (GFP) fusion proteins. After coculture at various effector : target (E : T) cell ratios, viable and dead GFP-positive cells were quantified by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with human immunodeficiency virus (HIV) and influenza virusspecific CTL clones and revealed cytotoxicity at lower E : T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay provides a versatile tool that will advance our understanding of cell-mediated immunity.
The Journal of Infectious Diseases 11/2005; 192(7):1183-90. · 6.41 Impact Factor
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ABSTRACT: Late-stage CCR5 tropic human immunodeficiency virus type 1 (HIV-1) isolates (R5 HIV-1) can deplete nearly all CD4+ thymocytes from human thymus/liver grafts, despite the fact that fewer than 5% of these cells express CCR5. To resolve this paradox, we studied the replication and cytopathic effects (CPE) of late-stage R5 HIV-1 biological clones from two progressors and two long-term nonprogressors (LTNP) in fetal thymic organ culture (FTOC) with and without added cytokines. We found that R5 HIV-1 clones from progressors but not LTNP were cytopathic in untreated FTOC. Moreover, R5 HIV-1 clones from progressors replicated to higher levels than LTNP-derived R5 HIV-1 clones in this system. In contrast, when FTOC was maintained in the presence of interleukin 2 (IL-2), IL-4, and IL-7, both progressor and LTNP clones exhibited similar replication and CPE, which were equal to or greater than the levels achieved by progressor-derived R5 HIV-1 clones in untreated FTOC. This finding was likely due to IL-2-induced CCR5 expression on CD4+ thymocytes in FTOC. R5 HIV-1 clones showed greater pathogenesis for CCR5+ cells but also showed evidence of CPE on CCR5- cells. Furthermore, infection of FTOC by R5 HIV-1 induced IL-10 and transforming growth factor beta (TGF-beta) expression. Both IL-10 and TGF-beta in turn induced CCR5 expression in FTOC. Induction of CCR5 expression via cytokine induction by R5 HIV-1 infection of CCR5+ thymocytes likely permitted further viral replication in newly CCR5+ thymocytes. CCR5 expression, therefore, is a key determinant of pathogenesis of R5 HIV-1 in FTOC.
Journal of Virology 02/2005; 79(1):458-71. · 5.40 Impact Factor
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Mark J Geels,
Marion Cornelissen,
Hanneke Schuitemaker,
Kiersten Anderson, David Kwa,
Jolanda Maas,
John T Dekker,
Elly Baan,
Fokla Zorgdrager,
Remco van den Burg,
Martijn van Beelen,
Vladimir V Lukashov,
Tong-Ming Fu,
William A Paxton,
Lia van der Hoek,
Sheri A Dubey,
John W Shiver,
Jaap Goudsmit
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ABSTRACT: Control of viremia in natural human immunodeficiency virus type 1 (HIV-1) infection in humans is associated with a virus-specific T-cell response. However, still much is unknown with regard to the extent of CD8(+) cytotoxic T-lymphocyte (CTL) responses required to successfully control HIV-1 infection and to what extent CTL epitope escape can account for rises in viral load and ultimate progression to disease. In this study, we chose to monitor through full-length genome sequence of replication-competent biological clones the modifications that occurred within predicted CTL epitopes and to identify whether the alterations resulted in epitope escape from CTL recognition. From an extensive analysis of 59 biological HIV-1 clones generated over a period of 4 years from a single individual in whom the viral load was observed to rise, we identified the locations in the genome of five CD8(+) CTL epitopes. Fixed mutations were identified within the p17, gp120, gp41, Nef, and reverse transcriptase genes. Using a gamma interferon ELIspot assay, we identified for four of the five epitopes with fixed mutations a complete loss of T-cell reactivity against the wild-type epitope and a partial loss of reactivity against the mutant epitope. These results demonstrate the sequential accumulation of CTL escape in a patient during disease progression, indicating that multiple combinations of T-cell epitopes are required to control viremia.
Journal of Virology 01/2004; 77(23):12430-40. · 5.40 Impact Factor
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ABSTRACT: In approximately half of human immunodeficiency virus (HIV) type 1-infected individuals, the development of CXC chemokine receptor 4-using, syncytium-inducing (SI) virus variants precedes a rapid progression to acquired immunodeficiency syndrome (AIDS). In other individuals, only CC chemokine receptor 5-using (R5), non-SI (NSI) virus variants are present throughout infection. These individuals may be either long-term survivors (LTSs) or rapid progressors. The basis for this variable disease progression in individuals with only R5 virus variants is not yet fully understood. In this study, the beta-chemokine sensitivity of biological HIV-1 clones isolated from 13 individuals who harbored only R5, NSI virus variants (7 LTSs and 6 progressors) was investigated. We found a statistically significant decrease in sensitivity of virus variants to RANTES (regulated on activation, normally T cell-expressed and -secreted) neutralization during the course of progressive infection, but not during follow-up of LTSs. Our data suggest that a role exists for RANTES neutralization sensitivity of HIV-1 in AIDS pathogenesis.
The Journal of Infectious Diseases 10/2003; 188(6):864-72. · 6.41 Impact Factor
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ABSTRACT: A polymorphism at position -589 in the interleukin 4 (IL-4) promoter region was recently described as being associated with the presence of syncytium-inducing CXCR4 using (X4) HIV-1 variants.
To study the IL-4 promoter polymorphism -589T in relation to HIV-1 disease progression and acquisition of X4 HIV-1 variants.
Retrospective longitudinal study among 342 HIV-1-infected homosexual men who participated in the Amsterdam Cohort study. Polymerase chain reaction was used in combination with restriction analysis to identify IL-4 promoter genotypes.
Carriers of the -589T allele (either -589 C/T heterozygotes or -589 T/T homozygotes), showed comparable progression to AIDS [relative hazard (RH), 0.94; P = 0.71], and survival (RH IL-4 -589 C/T or T/T, 0.94; P = 0.69) as carriers of the -589 C/C genotype (the reference group). In contrast to a previous study, we found that the -589T polymorphism was associated with a delayed acquisition of X4 HIV-1 variants (RH, 0.56; P = 0.02 for IL-4 -589 C/T or T/T) and a reduced number of CCR5 expressing memory CD4 T cells.
In the Amsterdam Cohort of homosexual men with HIV infection, the IL-4 -589T promoter polymorphism was associated with a delayed acquisition of X4 variants but did not affect overall disease progression.
AIDS 06/2003; 17(7):981-5. · 6.24 Impact Factor
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ABSTRACT: The presence of only non-syncytium-inducing beta-chemokine receptor 5-restricted (R5/NSI) human immunodeficiency virus type 1 (HIV-1) in an infected individual has been associated with long-term asymptomatic survival. However, the majority of R5/NSI HIV-1-infected individuals do progress to AIDS. Here, we compared the replicative capacity and cytopathicity of R5/NSI HIV-1 variants that were isolated early and late in the clinical course from 7 long-term asymptomatic individuals and 7 individuals with progressive HIV-1 infection. R5/NSI HIV-1 cytopathicity in vitro directly correlated with in vitro replication. HIV-1 variants obtained early and late during long-term asymptomatic HIV infection from the same individual were equally cytopathic. In contrast, HIV-1 variants obtained during late-stage progressive HIV infection were more cytopathic than viruses obtained early in infection from the same individuals. Our data indicate that the cytopathicity of HIV-1 variants may increase with progression to disease.
The Journal of Infectious Diseases 06/2003; 187(9):1397-403. · 6.41 Impact Factor
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AIDS 04/2003; 17(5):759-61. · 6.24 Impact Factor
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ABSTRACT: The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by n-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.
Journal of Virology 07/2002; 76(11):5612-26. · 5.40 Impact Factor
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ABSTRACT: It has been hypothesized that human immunodeficiency virus type 1 (HIV-1) evolves toward increased cytopathicity in conjunction with disease progression in infected patients. A viral property known to evolve in some but not all patients is coreceptor utilization, and it has been shown that a switch in coreceptor utilization is sufficient for the development of increased cytopathicity. To test the hypothesis that the evolution of other viral properties also contributes to accelerating cytopathicity in vivo, we used human lymphoid tissue explants to assay the cytopathicity of a panel of primary HIV-1 isolates derived from various stages of disease characterized by the presence or absence of changes in coreceptor preference. We found no evidence of coreceptor-independent increases in cytopathicity in isolates obtained either before coreceptor preference changes or from patients who progressed to AIDS despite an absence of coreceptor evolution. Instead, the cytopathicity of all HIV-1 isolates was determined solely by their coreceptor utilization. These results argue that HIV-1 does not evolve toward increased cytopathicity independently of changes in coreceptor utilization.
