RESUMO
This study was undertaken to establish an assay system to detect the survival advantage of anti-HIV ribozyme expressing cells under the selective pressure of HIV infection. In a mixture with wild type cells the proportion of ribozyme expressing cells was increased up to 12-fold. As a mechanism of the selective advantage an inhibition of HIV induced apoptotic cell death could be demonstrated. Furthermore, a dose dependency of the antiviral ribozyme effects was observed.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , HIV-1/genética , RNA Catalítico/genética , Adulto , Apoptose/fisiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Citometria de Fluxo , Terapia Genética , Vetores Genéticos , Infecções por HIV/terapia , Repetição Terminal Longa de HIV/genética , Humanos , RNA Catalítico/biossíntese , Receptor de Fator de Crescimento Neural/biossíntese , Receptor de Fator de Crescimento Neural/genética , Retroviridae/genética , Transdução GenéticaRESUMO
Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine. Its ability to induce interferon-g suggests a potential virustatic effect. On the other hand, it stimulates NFkB - an activator of HIV replication. Recently, stimulation of HIV-1 in monocytic cells has been demonstrated. In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated. Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E. coli or eucaryotically by baculovirus in Sf9 cells. HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells. The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells. By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75%. Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells. Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.
Assuntos
HIV-1/fisiologia , Interleucina-18/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , Escherichia coli , Proteína do Núcleo p24 do HIV/biossíntese , HIV-1/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/farmacologia , Spodoptera , Linfócitos T , TransfecçãoRESUMO
To date, a selective advantage of cells expressing anti-HIV ribozymes has not been shown. This study was undertaken to determine whether such a selective advantage can be demonstrated in vitro. A retroviral vector coding for a hairpin ribozyme targeting the HIV 5'LTR and for the low affinity nerve growth factor receptor (LNGF-RDelta) was designed. Since we demonstrated by RT-PCR that the amount of ribozyme transcripts was highly correlated with the level of surface LNGF-RDelta expression, the vector was utilized to assess ribozyme expression by flow cytometry. Transduced Hut78 and primary CD4+ T cells were purified and subsequently mixed with unmodified cells. After HIV challenge the percentage of ribozyme expressing cells in the cell mixture was monitored by flow cytometry. Twenty-one days after HIV infection the proportion of ribozyme expressing CD4+ T cells was 2.6 times higher in comparison to cells with the control vector. CD4+ T cells with a strong ribozyme expression conferred a 7.4-fold selective advantage at day 21 and a 11.7-fold at day 28. For Hut78 cells a selective advantage was detected exclusively for strongly ribozyme expressing cells. As a mechanism underlying the selective advantage an inhibition of HIV induced apoptosis was shown. These results demonstrate that anti-HIV ribozymes are able to confer a selective survival advantage and indicate that the protective effect is dependent on the amount of ribozyme expression. Gene Therapy (2000) 7, 408-416.
