Effects of HIV-1 tat on secretion of TNF-α and IL-1β by U87 cells in AIDS patients with or without AIDS dementia complex / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.</p><p><b>METHODS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.</p><p><b>RESULTS</b>HIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.</p><p><b>CONCLUSION</b>Tat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.</p>

Cloning and eukaryotic expression of HIV-1B gp120 genes from a patient with AIDS dementia complex / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.</p><p><b>METHODS</b>Using the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.</p><p><b>RESULTS</b>HIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.</p><p><b>CONCLUSION</b>All the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.</p>

Analysis of genetic diversity and amino acid sequence of HIV-1tat in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the diversity of HIV-1 tat gene in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC, so as to research HIV evolution, the mechanism of CNS invasion and the pathogenesis of ADC.</p><p><b>METHODS</b>The tat gene was amplified with nested PCR from genomic DNA which was extracted from spleen and basal ganglia of one non-ADC patient with a wide range of cerebral artery atherosclerosis and one ADC patient. PCR products were cloned into the PGEM-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid motifs were all done, so as to research the diversity of HIV-1 tat gene in CNS and peripheral tissue.</p><p><b>RESULTS</b>Gene mutation of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between CNS and peripheral tissue in ADC patient were greater than the non-ADC patient. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.</p><p><b>CONCLUSIONS</b>The compartmentation of tat gene in CNS and peripheral tissue of the two patients was different, the reason may be related to the pathway of HIV into the CNS, the relationship between HIV gene mutation in CNS and ADC still need more investigation.</p>

HIV-1B gp120 genes from one patient with AIDS dementia complex can affect the secretion of tumor necrosis factor and interleukin 1β in glial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>HIV-1 infected and immune-activated macrophages and microglia secrete neurotoxins, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which play major role in the neuronal death. It has been shown that different HIV-1 variants have varying abilities to elicit secretion of TNF-α by peripheral blood mononuclear cell (PBMC); however, whether the difference of gp120 gene could affect the secretion of TNF-α and IL-1β by glial cells is unknown. The aim of this study was to explore the association between gene diversity and induction of neurotoxic cytokines.</p><p><b>METHODS</b>In this study, we constructed retroviral vectors MSCV-IRES-GFP/gp120 using HIV-1 gp120 genes isolated from four different tissues of one patient who died of AIDS dementia complex (ADC). Recombinant retroviruses produced by cotransfection of MSCV-IRES-GFP/gp120, pCMV-VSV-G and pUMVC into 293T cells were collected and added into U87 glial cells. Concentrations of TNF-α and IL-1β secreted by transduced U87 cells were assayed with ELISA separately.</p><p><b>RESULTS</b>The four HIV-1 gp120 were in the different branch of the neighbor-joining tree. Compared to the pMIG retrovirus (gp120-negative) or U87 cells, all the gp120-positive recombinant retroviruses induced more TNF-α (P < 0.01) and IL-1β (P < 0.01). In addition, compared with the L/MIG retrovirus, all the three brain gp120-positive recombinant retroviruses induced less TNF-α (P < 0.01) and IL-1β (P < 0.01).</p><p><b>CONCLUSIONS</b>HIV-1 gp120 could induce U87 cells secret more TNF-α and IL-1β again. The more important is that difference of HIV-1 gp120, especially cell-tropism may account for the different ability in eliciting secretion of TNF-α and IL-1β, which might supply a novel idea helping understand the pathogenesis of ADC.</p>