RESUMO
Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4(+) T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy-naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-alpha, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/farmacologia , HIV-1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Divisão Celular/efeitos dos fármacos , Antígenos HLA-DR/imunologia , Humanos , Infecções/virologia , Receptores de Interleucina-2/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4(+) cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Linfócitos B/virologia , Complemento C3/fisiologia , HIV-1/fisiologia , Ativação Linfocitária , Receptores de Complemento 3d/fisiologia , Linfócitos T/virologia , Vírion/fisiologia , Síndrome da Imunodeficiência Adquirida/virologia , Anticorpos Monoclonais/imunologia , Doença Crônica , Humanos , RNA Viral/análiseRESUMO
Macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell expressed and secreted), which are the natural ligands of the CC-chemokine receptor CCR5, inhibit replication of MT-2- negative strains of HIV-1 by interfering with the ability of these strains to utilize CCR5 as a coreceptor for entry in CD4(+) cells. The present study investigates the capacity of natural killer (NK) cells isolated from HIV-infected individuals to produce CC-chemokines and to suppress HIV replication in autologous, endogenously infected cells as well as to block entry of MT-2-negative HIV into the CD4(+) T cell line PM-1. NK cells freshly isolated from HIV-infected individuals had a high number of mRNA copies for MIP-1alpha and RANTES. NK cells produced significant amounts of RANTES, MIP-1alpha, and MIP-1beta constitutively, in response to stimulation with IL-2 alone and when they were performing their characteristic lytic activity (K562 killing). After CD16 cross-linking and stimulation with IL-2 or IL-15 NK cells produced CC-chemokines to levels comparable to those produced by anti-CD3-stimulated CD8(+) T cells. Furthermore, CD16 cross-linked NK cells suppressed (49-97%) viral replication in cocultures of autologous CD8/NK-depleted PBMC to a degree similar to that of PHA or anti-CD3-stimulated CD8(+) T cells. In 50% of patients tested, NK-mediated HIV suppression could be abrogated by neutralizing antibodies to MIP-1alpha, MIP-1beta and RANTES; in contrast, CD8(+) T cell-mediated suppression was not significantly overcome upon neutralization of CC-chemokines. Supernatants derived from cultures of CD16 cross-linked NK cells stimulated with IL-2 or IL-15 dramatically inhibited entry of a MT-2-negative strain of HIV, BaL, in the CD4(+)CCR5(+) PM-1 T cell line. These data suggest that activated NK cells may be an important source of CC-chemokines in vivo and may suppress HIV replication by CC-chemokine-mediated mechanisms in addition to classic NK-mediated lytic mechanisms.
Assuntos
Quimiocinas CC/biossíntese , Infecções por HIV/imunologia , HIV-1/fisiologia , Células Matadoras Naturais/fisiologia , Replicação Viral , Complexo CD3/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Quimiocinas CC/genética , Quimiocinas CC/farmacologia , Infecções por HIV/virologia , Humanos , Interleucina-12/farmacologia , Interleucina-15/farmacologia , Interleucina-2/farmacologia , RNA Mensageiro/análise , Receptores de IgG/fisiologiaRESUMO
The bld2-1 mutation in the green alga Chlamydomonas reinhardtii is the only known mutation that results in the loss of centrioles/basal bodies and the loss of coordination between spindle position and cleavage furrow position during cell division. Based on several different assays, bld2-1 cells lack basal bodies in > 99% of cells. The stereotypical cytoskeletal morphology and precise positioning of the cleavage furrow observed in wild-type cells is disrupted in bld2-1 cells. The positions of the mitotic spindle and of the cleavage furrow are not correlated with respect to each other or with a specific cellular landmark during cell division in bld2-1 cells. Actin has a variable distribution during mitosis in bld2-1 cells, but this aberrant distribution is not correlated with the spindle positioning defect. In both wild-type and bld2-1 cells, the position of the cleavage furrow is coincident with a specialized set of microtubules found in green algae known as the rootlet microtubules. We propose that the rootlet microtubules perform the functions of astral microtubules and that functional centrioles are necessary for the organization of the cytoskeletal superstructure critical for correct spindle and cleavage furrow placement in Chlamydomonas.
Assuntos
Centríolos/ultraestrutura , Chlamydomonas reinhardtii/genética , Genes de Plantas , Genes de Protozoários , Microtúbulos/ultraestrutura , Proteínas de Plantas/fisiologia , Proteínas de Protozoários/fisiologia , Fuso Acromático/ultraestrutura , Actinas/análise , Animais , Divisão Celular , Centríolos/fisiologia , Chlamydomonas reinhardtii/ultraestrutura , Flagelos/ultraestrutura , Meiose , Mitose , Mutação , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Protozoários/análise , Proteínas de Protozoários/genéticaRESUMO
Guanosine triphosphate and formycin triphosphate (FTP) in the presence of excess Mg2+ can bind to empty non-catalytic sites of spinach chloroplast ATPase (CF1). This results in a greatly reduced capacity for ATP hydrolysis compared to the enzyme with non-catalytic sites filled with ATP. With two GTP bound at non-catalytic sites the inhibition is about 90%; with two FTP bound about 80% inhibition is obtained. Binding and release of the nucleotides from the non-catalytic sites are relatively slow processes. Exposure of CF1 with one or two empty non-catalytic sites to 5-10 microM FTP or GTP for 15 min suffices for about 50% of the maximum inhibition. Reactivation of CF1 after exposure to higher FTP or GTP concentrations requires long exposure to 2 microM EDTA. The findings show that, contrary to previous assumptions, GTP can bind tightly to non-catalytic sites of CF1. They suggest that the presence of adenine nucleotides at non-catalytic sites might be essential for high catalytic capacity of the F1 ATPases.
Assuntos
Cloroplastos/enzimologia , Formicinas/metabolismo , Guanosina Trifosfato/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , Ribonucleotídeos/metabolismo , Difosfato de Adenosina/fisiologia , Trifosfato de Adenosina/metabolismo , Bicarbonatos , Sítios de Ligação , Cálcio/fisiologia , Temperatura Alta , Cinética , Magnésio/fisiologia , Plantas/enzimologiaRESUMO
Despite exposure to multiple strains of both macrophage (M)-tropic and T cell (T)-tropic HIV, primary infection is largely restricted to relatively homogeneous M-tropic virus. Since dendritic cells (DCs) play a pivotal role in the early events of HIV infection, several studies have focused on the role of DCs in this restriction. It has been proposed that DCs are more efficiently infected with M-tropic versus T-tropic viruses; however, the infectability of DCs and the relevance of their infectability for inducing productive infection is controversial. It has also been suggested that variability in DC expression of coreceptors for M-tropic versus T-tropic virus could explain the restriction in the transmitting virus. Using HIV-pulsed DCs from individuals with a homozygous deletion in the CCR5 gene as a human "knockout" model, we demonstrate that infection of DCs per se is not necessary to promulgate infection in CD4+ T cells. The data also suggest that transmission of HIV to CD4+ T cells is not dependent on DC coreceptor expression.
Assuntos
Linfócitos T CD4-Positivos/virologia , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV/fisiologia , Receptores CCR5/genética , Células Dendríticas/metabolismo , Infecções por HIV/virologia , Homozigoto , Humanos , Macrófagos/virologia , Receptores CCR5/biossíntese , Replicação ViralRESUMO
The purpose of this study was to determine the effects of 6 wk of essential amino acid (EAA) supplementation on body composition and exercise performance in untrained women (n = 21). Subjects were randomly assigned to a placebo (cellulose) or an EAA (average daily dose of 18.3 g of EAAs in pill form) group. Each subject participated in aerobic and heavy-resistance training three times per week. Body composition was assessed via dual x-ray absorptiometry analysis. Muscular endurance was determined via treadmill time to exhaustion, and strength was assessed by the total amount of weight lifted for one set to exhaustion at an estimated 12 repetitions maximum. No changes occurred in either group for body weight, lean body mass, fat mass, or bone mineral content. Treadmill time to exhaustion (TTE) improved significantly (P < 0.05) in the EAA group (mean +/- SD; pre-TTE = 13.15 +/- 3.67 min, post-TTE = 14. 73 +/- 4.26 min), whereas the placebo group did not change significantly. The total weight lifted at the subject's maximum 12 repetitions did not significantly change in either group. In previously untrained individuals, the ingestion of EAAs combined with aerobic and heavy-resistance training for 6 wk did not have a significant effect on body composition or muscular strength; however, aerobic muscular endurance increased significantly.
Assuntos
Aminoácidos Essenciais/farmacologia , Composição Corporal/efeitos dos fármacos , Suplementos Nutricionais , Exercício Físico/fisiologia , Resistência Física , Absorciometria de Fóton , Adolescente , Adulto , Aminoácidos Essenciais/administração & dosagem , Método Duplo-Cego , Feminino , Humanos , Músculo Esquelético/efeitos dos fármacos , Esforço Físico/efeitos dos fármacos , Fatores de Tempo , Levantamento de PesoRESUMO
In Chlamydomonas reinhardtii, specialized cytoskeletal structures known as rootlet microtubules are present throughout interphase and mitosis. During cytokinesis, an array of microtubules termed the phycoplast is nucleated from rootlet microtubules and forms coincidentally with the cleavage furrow [Johnson and Porter, 1968: J. Cell Biol. 38:403-425; Holmes and Dutcher, 1989: J. Cell Sci. 94:273-285; Gaffel and el-Gammel, 1990: Protoplasma 156:139-148; Schibler and Huang, 1991: J. Cell Biol. 113:605-614]. We have obtained two independent lines of evidence that support the hypothesis that the rootlet and phycoplast microtubules play a direct role in cleavage furrow placement and assembly. First, the destabilization of spindle and phycoplast microtubules by pharmacological agents was accompanied by the aberrant distribution of actin and a failure of cytokinesis. Second, we characterized mutant strains that failed to complete cytokinesis properly. Actin and myosin were mislocalized to additional rootlet microtubules in the cyt2-1 strain, and this mislocalization was correlated with the presence of additional cleavage furrows. This evidence suggests that microtubules are necessary for the correct positioning and assembly of functional cleavage furrows in C. reinhardtii.
Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Sulfanilamidas , Animais , Divisão Celular/efeitos dos fármacos , Chlamydomonas reinhardtii/genética , Dinitrobenzenos/farmacologia , Microtúbulos/ultraestrutura , Mutação , Fenótipo , Fuso Acromático/efeitos dos fármacos , Vimblastina/farmacologiaRESUMO
The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.
Assuntos
Trifosfato de Adenosina/metabolismo , Cloroplastos/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Sítios de Ligação , Hidrólise , Cinética , Substâncias Macromoleculares , Magnésio/farmacologia , Plantas/enzimologiaRESUMO
The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.
Assuntos
Cloroplastos/enzimologia , Nucleotídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Catálise , Cloretos/química , Guanosina Trifosfato/metabolismo , Hidrólise , Ácidos Sulfúricos/químicaRESUMO
Wheat storage proteins are deposited in the vacuole of maturing endosperm cells by a novel pathway that is the result of protein body formation by the endoplasmic reticulum followed by autophagy into the central vacuole, bypassing the Golgi apparatus. This model predicts a reduced role of the Golgi in storage protein accumulation, which has been supported by electron microscopy observations. To study this issue further, wheat cDNAs encoding three distinct proteins of the endomembrane system were cloned and characterized. The proteins encoded were homologues (i) of the ER translocon component Sec61 alpha, (ii) the vacuolar sorting receptor BP-80 which is located in the Golgi and clathrin-coated prevacuole vesicles (CCV), and (iii) the Golgi COPI coatomer component COP alpha. During endosperm development, the levels of all three mRNAs were highest in young stages, before the onset of storage protein synthesis, and declined with seed maturation. However, the relative mRNA levels of BP-80/Sec61 alpha and the COP alpha/Sec61 alpha were lower during the onset of storage protein synthesis than at earlier stages of endosperm development. These results support previous studies, suggesting a reduced function of the Golgi apparatus in wheat storage protein transport and deposition.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/fisiologia , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Triticum/fisiologia , Clonagem Molecular , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico , RNA Mensageiro , RNA de Plantas/metabolismo , Sementes/genética , Sementes/metabolismo , Triticum/genética , Triticum/metabolismo , Vacúolos/genética , Vacúolos/metabolismoRESUMO
In higher plants, meristem organization and cell division regulation are two fundamentally important and intimately related biological processes. Identifying and isolating regulatory genes in these processes is essential for understanding higher plant growth and development. We describe the molecular isolation and analyses of an Arabidopsis gene, TSO1, which regulates both of these processes. We previously showed that tso1 mutants displayed defects in cell division of floral meristem cells including partially formed cell walls, increased DNA content, and multinucleated cells (Liu, Z., Running, M. P. and Meyerowitz, E. M. (1997). Development 124, 665-672). Here, we characterize a second defect of tso1 in influorescence meristem development and show that the enlarged influorescence in tso1 mutants results from repeated division of one inflorescence meristem into two or more influorescence meristems. Using a map-based approach, we isolated the TSO1 gene and found that TSO1 encodes a protein with cysteine-rich repeats bearing similarity to Drosophila Enhancer of zeste and its plant homologs. In situ TSO1 mRNA expression pattern and the nuclear localization of TSO1-GFP are consistent with a regulatory role of TSO1 in floral meristem cell division and in influorescence meristem organization.
Assuntos
Proteínas de Arabidopsis , Proteínas de Plantas/fisiologia , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Sequência de Bases , Divisão Celular , Clonagem Molecular , Cisteína , DNA de Plantas , Genes de Plantas , Meristema , Dados de Sequência Molecular , Proteínas de Plantas/genética , RNA Mensageiro , Distribuição TecidualRESUMO
Microbial coinfections have been associated with transient bursts of human immunodeficiency virus (HIV) viremia in patients. In this study, we have investigated whether microbial coinfections can induce replication of HIV-1 in latently infected CD4(+) T cells derived from HIV-infected patients who are receiving highly active antiretroviral therapy and in whom plasma viremia is undetectable by sensitive assays. We demonstrate that supernatants from macrophages exposed to the bacterial product lipopolysaccharide can induce in vitro activation of HIV-1 from latently infected, resting CD4(+) T cells obtained from HIV-infected individuals. Depletion of proinflammatory cytokines from the supernatant markedly reduced-whereas depletion of ss chemokines increased--the ability of the supernatant to induce replication of HIV-1. Our results suggest that coinfection with microbial pathogens such as bacteria may induce viral replication in the latent viral reservoirs in vivo.
Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Lipopolissacarídeos/toxicidade , Ativação Viral , Latência Viral , Fármacos Anti-HIV/uso terapêutico , Citocinas/fisiologia , Humanos , Replicação ViralRESUMO
Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral , Linfócitos T CD4-Positivos/virologia , DNA Viral , Células Gigantes , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Replicação ViralRESUMO
This study demonstrates that the beta-chemokines macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta) and, RANTES (regulated on activation, normally T-cell expressed and secreted) inhibit human immunodeficiency virus (HIV) replication in anti-CD3 or recall antigen-stimulated peripheral blood mononuclear cells (PBMCs) of asymptomatic HIV-infected subjects. Significant levels of beta-chemokines were produced by both CD4+ and CD8+ PBMC subsets from HIV-infected individuals. Neutralization of endogenous MIP-1 alpha, MIP-1 beta, and RANTES did not rescue HIV replication in cultures to which greater than 10% CD8+ T cells had been added, indicating that the HIV suppressor activity of CD8+ T cells cannot be explained entirely by the beta-chemokines. However, significant enhancement of viral replication was observed upon neutralization of endogenous beta-chemokines in CD8-depleted or CD4+ PBMCs from most donors, particularly in cultures with low inducible levels of HIV production. In contrast, certain endogenous proinflammatory cytokines induced HIV replication in these same cells. These data suggest that the levels of HIV replication in CD4+ PBMC reflect the balance of the opposing effects of endogenous suppressive factors, such as the beta-chemokines, and HIV-inducing cytokines, such as tumor necrosis factor alpha and interleukin 1 beta.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Quimiocina CCL5/biossíntese , Quimiocina CCL5/farmacologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/fisiologia , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas Inflamatórias de Macrófagos/farmacologia , Replicação Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , HIV/efeitos dos fármacos , Humanos , Cinética , Depleção Linfocítica , Proteínas Recombinantes/farmacologia , Toxoide Tetânico/farmacologia , Fatores de Tempo , Replicação Viral/efeitos dos fármacosRESUMO
Virus replication in a human immunodeficiency virus (HIV)-infected individual, as determined by the steady-state level of plasma viremia, reflects a complex balance of viral and host factors. We have previously demonstrated that immunization of HIV-infected individuals with the common recall antigen, tetanus toxoid, disrupts this steady state, resulting in transient bursts of plasma viremia after immunization. The present study defines the viral genetic basis for the transient bursts in viremia after immune activation. Tetanus immunization was associated with dramatic and generally reversible shifts in the composition of plasma viral quasispecies. The viral bursts in most cases reflected a nonspecific increase in viral replication secondary to an expanded pool of susceptible CD4(+) T cells. An exception to this was in a patient who harbored viruses of differing tropisms (syncytium inducing and non-syncytium inducing [NSI]). In this situation, immunization appeared to select for the replication of NSI viruses. In one of three patients, the data suggested that immune activation resulted in the appearance in plasma of virus induced from latently infected cells. These findings illustrate certain mechanisms whereby antigenic stimulation may influence the dynamics of HIV replication, including the relative expression of different viral variants.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/fisiologia , Replicação Viral/imunologia , Sequência de Aminoácidos , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Primers do DNA , Humanos , Memória Imunológica , Dados de Sequência Molecular , Filogenia , Especificidade da EspécieRESUMO
CD8+ T cell-mediated antiviral activity against HIV has been described consistently in infected individuals; however, the role of this activity in controlling replication of HIV in the latently infected, resting CD4+ T cell reservoir is unclear. By using an ex vivo system, we show that replication of HIV in this viral reservoir is effectively suppressed in coculture by autologous CD8+ T cells in long-term nonprogressors (LTNPs) and in patients whose viremia was controlled by highly active antiretroviral therapy (HAART), but not in therapy-naive patients who had substantial levels of plasma viremia. This antiviral activity was largely independent of cytotoxic CD8+ T lymphocytes (CTL). When the role of soluble CD8+ T cell-derived factors was examined, we found that CC-chemokines played a major role in inhibition of viral replication in the latent viral reservoir in some LTNPs and patients receiving HAART, but not in chronically infected patients who were not receiving antiretroviral therapy. Potent antiviral activity, independent of CC-chemokines, was found mainly in patients in whom HAART was initiated shortly after the acute phase of HIV infection. These results indicate that CD8(+) T cells provide potent suppressive activity against HIV replication in the latent viral reservoir via direct cellular contact in patients who are naturally LTNPs or in those who are treated with HAART. Furthermore, the profound antiviral activity exerted by non-CC-chemokine soluble factors in infected patients who began HAART early in HIV infection suggests that preservation of this HIV-suppressive mechanism by early initiation of therapy may play an important role in the containment of viral replication in infected patients following interruption of therapy.
Assuntos
Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/terapia , HIV-1/imunologia , HIV-1/fisiologia , Replicação Viral , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Quimiocina CCL4 , Quimiocina CCL5/análise , Doença Crônica , Técnicas de Cocultura , Progressão da Doença , Citometria de Fluxo , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Proteínas Inflamatórias de Macrófagos/análise , Fenótipo , Latência ViralRESUMO
It has been suggested that CD4+ T cell proliferative responses to HIV p24 Ag may be important in the control of HIV infection. However, these responses are minimal or absent in many HIV-infected individuals. Furthermore, while in vitro and in vivo responses to non-HIV recall Ags improve upon administration of highly active antiretroviral therapy, there does not appear to be a commensurate enhancement of HIV-specific immune responses. It is possible that CD4+ p24-specific T cells are deleted early in the course of infection. However, it is also possible that a discrete unresponsiveness, or anergy, contributes to the lack of proliferation to p24. To evaluate the possible contribution of unresponsiveness to the lack of CD4+ T cell proliferation to p24 in HIV-infected individuals, we attempted to overcome unresponsiveness. CD40 ligand trimer (CD40LT) and IL-12 significantly increased PBMC and CD4+ T cell proliferative responses to p24 Ag in HIV-infected, but not uninfected, individuals. No increase in proliferative response to CMV Ag was observed. CD40LT exerted its effect through B7-CD28-dependent and IL-12- and IL-15-independent mechanisms. Finally, the increase in proliferation with CD40LT and IL-12 was associated with an augmented production of IFN-gamma in most, but not all, individuals. These data suggest the possible contribution of HIV-specific unresponsiveness to the lack of CD4+ T cell proliferation to p24 Ag in HIV-infected individuals and that clonal deletion alone does not explain this phenomenon. They also indicate the potential for CD40LT and IL-12 as immune-based therapies for HIV infection.
Assuntos
Adjuvantes Imunológicos/fisiologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/metabolismo , Anergia Clonal/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Interferon gama/biossíntese , Interleucina-12/fisiologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/fisiologia , Fármacos Anti-HIV/uso terapêutico , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Ligante de CD40 , Células Cultivadas , Relação Dose-Resposta Imunológica , Sinergismo Farmacológico , Epitopos/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Interleucina-12/uso terapêutico , Interleucina-15/fisiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Ligantes , Glicoproteínas de Membrana/uso terapêuticoRESUMO
CD4(+) T cells have been shown to play a critical role in the maintenance of an effective anti-viral CD8(+) CTL response in murine models. Recent studies have demonstrated that CD4(+) T cells provide help to CTLs through ligation of the CD40 receptor on dendritic cells. The role of CD4(+) T cell help in the expansion of virus-specific CD8(+) memory T cell responses was examined in normal volunteers recently vaccinated to influenza and in HIV-1 infected individuals. In recently vaccinated normal volunteers, CD4(+) T cell help was required for optimal in vitro expansion of influenza-specific CTL responses. Also, CD40 ligand trimer (CD40LT) enhanced CTL responses and was able to completely substitute for CD4(+) T cell help in PBMCs from normal volunteers. In HIV-1 infection, CD4(+) T cell help was required for optimal expansion of HIV-1-specific memory CTL in vitro in 9 of 10 patients. CD40LT could enhance CTL in the absence of CD4(+) T cell help in the majority of patients; however, the degree of enhancement of CTL responses was variable such that, in some patients, CD40LT could not completely substitute for CD4(+) T cell help. In those HIV-1-infected patients who demonstrated poor responses to CD40LT, a dysfunction in circulating CD8(+) memory T cells was demonstrated, which was reversed by the addition of cytokines including IL-2. Finally, it was demonstrated that IL-15 produced by CD40LT-stimulated dendritic cells may be an additional mechanism by which CD40LT induces the expansion of memory CTL in CD4(+) T cell-depleted conditions, where IL-2 is lacking.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/fisiologia , Epitopos de Linfócito T/imunologia , HIV-1/imunologia , Memória Imunológica , Ativação Linfocitária/imunologia , Cooperação Linfocítica , Linfócitos T Citotóxicos/imunologia , Adjuvantes Imunológicos/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Comunicação Celular/imunologia , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Citocinas/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Infecções por HIV/imunologia , Humanos , Vírus da Influenza A/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologiaRESUMO
The chemokine receptors CXCR4 and CCR5 have been identified as major coreceptors for HIV-1 entry into CD4+ T cells. The majority of primary HIV-1 isolates in early disease use CCR5 as a coreceptor, whereas during disease progression with the emergence of syncytium-inducing viruses, CXCR4 is also used. We performed a cross-sectional study in which we evaluated the expression of two HIV-1 coreceptors, CCR5 and CXCR4, in whole blood samples taken from HIV-1-infected and uninfected individuals. We demonstrate that CXCR4 on CD4+ and CD8+ T cells, and CD14+ monocytes is significantly down-regulated, and CCR5 expression on CD4+ T cells is up-regulated in HIV-infected individuals compared with uninfected controls. Coreceptor expression correlated with the level of cellular activation in vivo in both HIV-infected and uninfected individuals, with CXCR4 being expressed predominantly on quiescent (HLA-DR-) T cells and CCR5 being expressed predominantly on activated (HLA-DR+) T cells. Lower expression of CXCR4 and higher expression of CCR5 on CD4+ T cells correlated with advancing disease. In addition, a tendency for greater activation of CXCR4+CD4+ T cells in patients with advanced disease was observed. Patients who harbored syncytium-inducing viruses, however, could not be distinguished from those who harbored nonsyncytium-inducing viruses based on the level of CD4+ T cell activation or chemokine receptor expression.