RESUMO
OBJECTIVE: To investigate the usefulness of polymerase chain reaction (PCR) from cerebrospinal fluid (CSF) for rapid diagnosis and assessing treatment response of tuberculous meningitis (TBM) in AIDS patients. PATIENTS: Forty-four CSF samples from 10 patients with TBM confirmed by autopsy or by a culture of CSF (41 samples) and from two patients with highly probable TBM (three samples) were analysed. CSF specimens were collected before and during standard antituberculous treatment. CSF samples from 24 AIDS patients with autopsy evidence of other neurologic diseases were studied as controls. METHODS: A nested PCR amplifying a 123 base-pair fragment of the IS6110 sequence was developed. Heating to 95 degrees C for 15 min was used for pre-PCR treatment of samples. RESULTS: Detection limit was 10(2) colony-forming units per ml or 10 fg purified Mycobacterium tuberculosis DNA. M.tuberculosis DNA was detected in CSF from all the 12 confirmed or highly probable TBM cases. CSF was positive by nested PCR in 17 of 17 (100%) and 18 of 27 (67%) samples collected before and during therapy, respectively. Clinical and microbiological follow-up > or = 2 weeks was available for seven patients. PCR-positive CSF converted to M. tuberculosis DNA negative in four patients that showed improvement during treatment, but it remained positive in three patients who died of disseminated tuberculosis. All the CSF samples from the non-TBM controls were negative by nested PCR. CONCLUSIONS: Nested PCR for detection of M. tuberculosis DNA is specific for diagnosis of TBM and more sensitive than conventional bacteriology. Moreover, nested PCR could be a useful method for assessing treatment response in AIDS patients with TBM.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Reação em Cadeia da Polimerase/métodos , Tuberculose Meníngea/complicações , Tuberculose Meníngea/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/líquido cefalorraquidiano , DNA Bacteriano/genética , Estudos de Avaliação como Assunto , Humanos , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Tuberculose Meníngea/microbiologiaRESUMO
RANTES and sCD30 were measured in ex vivo culture supernatants of unstimulated or stimulated PBMC in order to investigate their potential role as markers of acute immune activation. Patients in an advanced stage of HIV infection (AIDS A) were compared to AIDS patients who were evaluated for pneumonia at the time of blood withdrawal (AIDS B); HIV(+) individuals with nonprogressive infection (LTNP) and healthy donors (N) served as controls. Constitutive levels of RANTES were significantly elevated in AIDS B patients (P 0.0001), whereas spontaneous release of sCD30 was strongly correlated with the presence of both pneumonia (P 0.002) and HIV infection (P 0.004). LPS was a strong inducer of RANTES in all four categories; however, in AIDS B patients a negative and positive correlation between constitutive and induced levels was observed with LPS (P 0.0004) and IFN-gamma (P 0.006), respectively. We clearly showed that IFN-gamma reached a fourfold superinduction of sCD30 release in both HIV-positive and -negative individuals, whereas IL-6-driven production of both sCD30 and RANTES occurred only in healthy donors. Ex vivo RANTES levels may also be monitored as an index of acute immune activation under conditions of chronic activation of the immune system, whereas sCD30 release may be equally indicative of both acute and chronic processes of T cell activation. Proinflammatory stimuli differentially affected RANTES and sCD30 secretion in ex vivo PBMC cultures, suggesting complex pathways in the in vivo regulation of these two molecules.
Assuntos
Quimiocina CCL5/sangue , Soronegatividade para HIV/imunologia , Soropositividade para HIV/sangue , Antígeno Ki-1/sangue , Leucócitos Mononucleares/citologia , Adulto , Feminino , Humanos , Masculino , SolubilidadeRESUMO
U937 cell clones which sustain efficient or poor replication of human immunodeficiency virus type 1 (HIV-1) (referred to herein as plus clones and minus clones, respectively) have been previously described. 1,25-Dihydroxyvitamin D3 (vitamin D3) potently induced HIV-1 replication and proviral DNA accumulation in minus clones but not in plus clones. Vitamin D3 did not induce NF-kappaB activation but selectively upregulated CXCR4 expression in minus clones. The CXCR4 ligand stromal-cell derived factor-1 induced Ca2+ fluxes and inhibited both constitutive and vitamin D3-enhanced HIV replication in minus clones.
Assuntos
Calcitriol/farmacologia , HIV-1/metabolismo , NF-kappa B/metabolismo , Receptores CXCR4/metabolismo , Replicação Viral , Sequência de Bases , Linhagem Celular , Células Clonais , Primers do DNA , DNA Viral , HIV-1/fisiologia , HumanosRESUMO
U937 cell clones in which efficient (plus) vs poor (minus) replication of HIV-1 occurs have been described. We evaluated the role of host factors in their differential ability to support HIV-1 replication. Plus clones constitutively produced TNF-alpha and viral replication was inhibited by neutralization of endogenous TNF-alpha. However, HIV-1 replication was strongly upregulated in minus clones by exogenous TNF-alpha, which also further accelerated the kinetics of infection in plus clones. We observed an increased accumulation of proviral DNA within one round of HIV-1 replication following TNF-a treatment of plus cells. This effect was associated with increased surface density of CXCR4 in both plus and minus clones. Our results identify TNF-alpha as one correlate that contributes to the higher ability of U937-plus clones to sustain HIV-1 replication. Furthermore, we suggest that TNF-alpha may affect steps of the viral life cycle that occur earlier than transcription and also enhance HIV-1 replication by increasing the surface density of CXCR4.
Assuntos
HIV-1/fisiologia , Receptores CXCR4/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Replicação Viral/fisiologia , Sequência de Bases , Quimiocina CXCL12 , Quimiocinas CXC/genética , Primers do DNA/genética , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Humanos , NF-kappa B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Células U937 , Regulação para Cima/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.
Assuntos
HIV-1/imunologia , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Naturais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Técnicas de Cocultura , Citotoxicidade Imunológica , Transcriptase Reversa do HIV/biossíntese , HIV-1/crescimento & desenvolvimento , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-2/farmacologia , Leucócitos Mononucleares/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Células U937RESUMO
Induction of chemokine gene expression from peripheral blood mononuclear cells (PBMCs) stimulated by proinflammatory cytokines plays an important role in both wound repair and response to infectious agents. In the present study, we show that the proinflammatory cytokine interleukin-6 (IL-6) potently induced mRNA expression and secretion of the CC chemokine monocyte chemotactic protein 1 (MCP-1) in PBMCs. In addition, because human immunodeficiency virus (HIV) infection in vivo and in vitro has been shown to dysregulate the production of and/or the response to cytokines, PBMCs from both healthy uninfected and HIV-infected individuals were studied for their constitutive and IL-6-induced expression of MCP-1. No substantial differences were observed between the two groups of individuals. In addition, IL-6 upregulated MCP-1 expression in the promonocytic cell line U937 and in its chronically HIV-infected counterpart, U1. In these cell lines, IL-6 selectively induced MCP-1 and not other chemokines, including regulated upon activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and IL-8. IL-6 induction of MCP-1 was partially inhibited by hydrocortisone in U1 cells. Thus, IL-6 activates PBMCs to secrete MCP-1, a CC chemokine pivotal for monocyte recruitment in tissue and organs in which important inflammatory events occur.