Effects of HIV-1 Tat derived from central nervous system of a HIV-associated dementia patient on the activity of human umbilical vein endothelial cells / 中华实验和临床病毒学杂志

Objective@#BG-derived HIV-1 Tat protein from an HIV-associated dementia (HAD) patient was expressed in E. coli BL21(DE3) and purified in order to research the effects on human umbilical vein endothelial cells (HUVECs) activity.@*Methods@#The recombinant plasmid pGEX-KG-tat with HIV-1 tat stored in our laboratory was amplified by PCR. The PCR product was cloned into pET-32a-tat. The recombinant plasmid pET-32a-tat was transfected into E. coli, and Tat protein was expressed in BL21(DE3), which was induced by IPTG. Then it was purified by Ni-chelating chromatography column and gel filtration preloaded column, and identified by SDS-PAGE and Western blot(WB). The concentration was determined by BCA Kit. Different concentrations of Tat were added into HUVECs to detect their effects on cell activity by cck-8.@*Results@#The Tat with high purity was efficiently expressed in BL21 (DE3) and obtained by using the Ni-chelating chromatography column and gel filtration preloaded column. The concentration was 0.47 mg/ml by using BCA Kit. As the concentration of Tat increased, HUVECs activity decreased. There was no significant difference in cells viability between negative control with 100 ng/ml and 200 ng/ml group (P>0.05). There was significant difference in cells viability between negative control with 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group (P<0.05). But the difference between 300 ng/ml, 400 ng/ml, 500 ng/ml and 1000 ng/ml group was not statistically significant (P>0.05).@*Conclusions@#The HIV-1 Tat with biological activity was efficiently expressed in BL21 (DE3), and the activity of HUVECs was significantly decreased when the concentration reached 300 ng/ml.

HIV-1 Tat Protein Promotes Amyloid beta Generation and Tau Phosphorylation in Rat Hippocampal Slices

HIV-1 Tat protein has been implicated as a causative agent in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND) and Alzheimer's disease (AD)-like pathology in HIV-1 infected patients. Here, we provide insights into the potential roles of extracellular HIV-1 Tat protein in amyloid beta (Abeta) generation and Tau phosphorylation, two major neuropathological features of AD. Exposure of the rat hippocampal slices to the full-length HIV-1 Tat protein (Tat1-86) for 3 days led to the increased levels of Abeta precursor protein (APP) accumulation, which accompanied by Abeta generation in the hippocampus, the brain region most commonly damaged in HIV-1-associated dementia (HAD). Moreover, extracellular HIV-1 Tat significantly stimulated the level of phosphrylated Tau (pTau) identified using immunoblotting with AT8 antibody, which recognizes abnormally hyperphosphorylated Tau. Collectively, our data suggest that HIV-1 Tat plays important roles in increasing the levels of APP accumulation, Abeta generation and Tau phosphorylation in the hippocampus, and thereby might contribute to the development of AD-like pathology in HIV-1-infected patients.

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>

HIV-1 Tat Protein-dependent Cytotoxicity is Attenated by 15-deoxy-Delta12,14-Prostaglandin J2 in Rat Hippocampal Slices: Involvement of the ERK1/2 Signaling Pathway

15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) may hold promise in treatment of the pathologies associated with human immunodeficiency virus (HIV) infection of the central nervous system. However, its precise role and neuroprotective mechanism in the hippocampus remain poorly understood. In the present study, rat hippocampal slices were stimulated with HIV-1 Tat protein to investigate the protective role of 15d-PGJ2 on the hippocampal cytotoxicity. Full-length HIV-1 Tat protein (Tat1-86), but neither its Tat32-62 nor Tat30-86 fragment, significantly induced cytotoxicity in the hippocampus, the brain region most commonly damaged in HIV-associated dementia. This Tat-induced cytotoxicity was associated with inactivation of MEK/extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, Tat1-86 did not alter Wnt signaling pathway necessary for cell survival. Pretreatment of slices with 15d-PGJ2 markedly reduced Tat-driven cytotxicity. Interestingly, this reduction was accompanied by suppression of ERK inactivation in response to Tat. Moreover, the inhibition of the MEK/ERK pathway with SL327 enhanced the Tat-induced cytotoxicity, confirming the ERK-dependent mechanism of Tat-driven cytotoxicity. Collectively, these data demonstrate that the protective action of 15d-PGJ2 against the hippocampal cytotoxicity upon Tat stimulation is exerted through suppression of Tat-mediated ERK1/2 inactivation.

Analysis of genetic diversity and amino acid sequence of HIV-1 tat from a patient with AIDS dementia complex / 中华微生物学和免疫学杂志

Objective To study the variation and characteristics of HIV-1 tat exon 1 gene from a patient with AIDS dementia complex( ADC), so as to research the pathogenesis of ADC. Methods The tat gene was amplified with nested PCR from genomic DNA which was extracted from lymph node, spleen and different brain tissues( meninges, grey matter from frontal cortex, white matter from frontal cortex, temporal cortex and basal ganglia) of a patient who died of ADC. 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. Results The samples were all identified as HIV-1 B and genetic variation exists in HIV-1 tat isolated from different tissue;Compared with HXB2, sixteen sites of the amino acid seque nce coded by the HIV-1 tat gene which was isolated from the patient changed. In addition, part of the changes were different between periphery and brain,especially, the five Q54R changes from basal ganglia and one Q54R change from temporal cortex are deserve to follow with interest. Conclusion Variations exist in the HIV-1 tat genes extracted from the ADC patient and the variations from peripheral and central nerve tissues were different, whether the variations concerned with the pathogenesis of ADC need more research.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

BACKGROUND: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. METHODS: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining. RESULTS: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. CONCLUSION: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Enhancement of Adenoviral Transduction and Immunogenecity of Transgenes by Soluble Coxsackie and Adenovirus Receptor-TAT Fusion Protein on Dendritic Cells

BACKGROUND: Investigating strategy to enhance efficiency of gene transfer via adenovirus is critical to sustain gene expression in targeted cells or tissues to regulate immune responses. However, the use of adenovirus as a gene delivery method has been limited by the native tropism of the virus. In this study, the critical parameter is to improve the efficient binding of viral particles to the plasma membrane prior to cellular uptake. METHODS: Human immunodeficiency virus (HIV-1) trans-acting activator of transcription (TAT), a protein transduction domain, was fused to the ectodomain of the coxsackie-adenovirus receptor (CAR). The CAR-TAT protein was produced from a Drosophila Schneider 2 cells (S2) transfected with CAR-TAT genes. The function of CAR- TAT was analyzed the efficiency of adenoviral gene transfer by flow cytometry, and then immunizing AdVGFP with CAR-TAT was transduced on dendritic cells (DCs). RESULTS: S2 transfectants secreting CAR-TAT fusion protein has been stable over a period of 6 months and its expression was verified by western blot. Addition of CAR-TAT induced higher transduction efficiency for AdVGFP at every MOI tested. When mice were vaccinated with DC of which adenoviral transduction was mediated by CAR-TAT, the number of IFN-gamma secreting T-cells was increased as compared with those DCs transduced without CAR-TAT. CONCLUSION: Our data provide evidence that CAR-TAT fusion protein enhances adenoviral transduction and immunogenecity of transgenes on DCs and may influence on the development of adenoviral- mediated anti-tumor immunotherapy.

p53-mediated HIV-1 Tat Suppression is Likely to be Assaciated with duble-stranded RNA-dependent Protein Kinase, PKR

No abstract available.