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>

Reprogramação de células mesenquimais de tecido adiposo em células-tronco pluripotentes por meio de proteína de fusão TAT / Nuclear reprogramming of adipose-tissue mesenchymal stem cells into pluripotent stem cells using TAT fusion protein

Os vírus são eficazes na transferência de genes em células devido aos seus mecanismos especializados. No entanto, vírus como veículos de entrega de genes podem acarretar em problemas, particularmente quando proposto para reprogramar células somáticas em células-tronco pluripotentes induzidas (iPS) visando utilização terapêutica. No presente estudo, procurou-se desenvolver um sistema alternativo para entregar diretamente proteínas nucleares (Oct4, Sox2, KLF4, e c-Myc) fusionadas com o domínio de transdução de proteína TAT, para promover a reprogramação de fibroblastos embrionários de camundongos (MEF) ou células mesenquimais derivadas de tecido adiposo humano (hASC) em células iPS. Primeiramente o PTD TAT ou TAT- foi fundido a proteína verde fluorescente (GFP) como modelo para prova de princípio e padronização detalhada. Inesperadamente, TAT-GFP produzido e secretado pelas células NIH-3T3 produtora não foi capaz de ser detectado no meio de cultura por verificação quantitativa fluorimétrica, nem foi capaz de ser detectada em células-alvo, por citometria de fluxo, depois de co-cultura em transwells. Essa observação pode ser explicada por: (1) ineficiência desse tipo de célula em secretar proteínas e (2) falta de resistência à clivagem por endoproteases furinas. Para contornar esses fatores limitantes usou-se citometria de fluxo para avaliar as melhores condições para a transfecção por seis diferentes tipos de células (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t e COS-7) com TAT (modificada para ser resistente à furinas) fundido a GFP. Células 293t-TAT-GFP exibiram a maior eficiência de transfecção e também de secreção. O mesmo pôde ser observado para as seis linhagens celulares expressando fatores de transcrição nucleares TAT, determinados por ELISA. Em seguida, diferentes estratégias de entrega foram testadas. A primeira foi baseada na co-cultura de uma mistura de células produtoras com MEF ou hASC. No entanto, não foi possível observar a reprogramação...

Viruses are effective at transferring genes into cells by its specialized mechanisms. However, viruses as gene delivery vehicles entail problems, particularly when proposed to reprogram somatic cells into induced pluripotent stem cells (iPS) for therapeutic uses. In the present study, we aimed to develop an alternative system for directly delivering nuclear proteins (Oct4, Sox2, Klf4, and c-Myc) fused with TAT protein transduction domain to promote reprogramming of mouse embryonic fibroblasts (MEF) or human adipose tissue derived mesenchymal cells (hASC) into iPS cells. First TAT- or TAT- PTD was fused to green fluorescent protein (GFP) as a proof of principle model and for detailed standardization. Unexpectedly, TAT-GFP produced and secreted by NIH-3T3 producer cells was not detected in the culture medium by quantitative fluorimetric verification, nor detected on target cells, by flow cytometry, after being co-cultured using transwells. This observation maybe explained by: (1) inefficiency of this cell type to be transfected and to secrete proteins and (2) lack of resistance to furin endoproteases cleavage on Golgi of TAT sequence. To circumvent these limiting factors we used flow cytometer to assess the best conditions for transfection in six different cell types (CHO, NIH-3T3, HT1080, HEK-293A, HEK-293t and COS-7) with TAT- (a modified PTD to be resistant to furin endoproteases) fused to GFP. 293t-TAT-GFP cells displayed the highest transfection efficiency and secretion levels. The same could be observed for the six cell lineages expressing TAT- nuclear transcription factors, determined by ELISA.Next, different delivery strategies were tested for TAT- nuclear transcription factor system. Co-culturing a mix of producer cells with MEF or hASC resulted in not reprogramming and this was associated with cell death. The second was based on the use of microconcentrated conditioned cell culture medium, changed every 24h, in four cycles. However, despite the...

Study on the relationship between the polymorphisms and secondary structure of tat exon-1 gene and HIV/ AIDS progress in subtype B' and B'/C / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the polymorphisms and secondary structure of human immunodeficiency virus (HIV-1) tat exon 1 among subtype B' and B'/C HIV-1 infected people in China and to explore the relationship between the polymorphism of tat exon 1 and the disease progression.</p><p><b>METHODS</b>8 subtype B' and 5 B'/C HIV-1 infected patients with slow disease progression were selected from Liaoning, Jilin and Yunnan province. 26 subtype B' and 9 B'/C HIV-1 infected patients with similar sex, age but with typical disease progression were selected. Provirus was extracted from the whole blood. The gene sequences of the Tat exon 1 were amplified by nest-polymerase chain reaction (nest-PCR). Products were purified and sequenced directly. The sequences were aligned, translated, amino acid substitution were analyzed and secondary structures were predicted.</p><p><b>RESULTS</b>Many amino acid substitution could be found in the exon 1 of Tat in HIV-1 subtype B' and B'/C recombinant strain infected persons with different disease progression except A58T,none of them showed definitely relationship with HIV viral load and disease progression. 23N, 31S, 32Y and 46F were subtype-specific substitutions. No characteristic secondary structure of exon 1 of Tat was found.</p><p><b>CONCLUSION</b>Some of the mutations of tat exon 1 might be related to HIV viral load and disease progression. However, there was no relationship found between the secondary structure of Tat protein and the disease progression.</p>

The role of the Tat gene in the pathogenesis of HIV infection.

The human immunodeficiency virus Tat regulatory protein is essential for virus replication and for the efficient transcription of HIV-1 provirus, and in the pathogenesis of AIDS. The role of the tat gene was investigated in 300 samples. It was found that 71.7% were subtype CRF_01AE, 9.3% were subtype B, while 11.7 and 7.3% of them were cross-reactive and non-typeable, respectively. Moreover the results from peptide ELISA also showed that a low CD4 cell count was related to a low anti-Tat antibody (p < 0.05), which may be due to the progression of HIV-1, which can be found predominantly in AIDS patients. The results of nested PCR showed that the second Tat exon might also play a role in T-cell activation. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure HIV-1 mRNA expression in PBMC. RT-PCR negative results were found mostly in the asymptomatic HIV-seropositive group (88%). HIV-1 mRNA expression was found to correlate with current immunologic status. The differences in Tat protein sequences from DNA sequencing between the patients who had anti-Tat antibody positive and anti-Tat antibody negative, were not significant (p > 0.05). These results suggested that the Tat amino acid sequences were conserved among each group of samples and did not change significantly compared with the consensus sequence in previous studies. Several factors make Tat an attractive target for vaccine design.

Construction and analysis of activity of an HIV-1/bovine immunodeficiency virus chimeric clone cDNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Chimeric human/bovine immunodeficiency virus (HBIV) cDNA was constructed by replacing HIV tat and LTR with bovine immunodeficiency virus (BIV) tat and LTR to study the activity of BIV tat and LTR in the chimerae.</p><p><b>METHODS</b>The target fragments of BIV tat, LTR and HIV gag, pol, env were respectively amplified by using PCR and sequentially inserted into pBluescript SK(+) vector. The chimeric clone was transfected into human MT4 cells. The transcript and gene expression of the HBIV chimeric virus were detected by using RT-PCR and a reverse transcriptase assay, respectively.</p><p><b>RESULTS</b>BIV tat mRNA and HIV gag mRNA were detected. The reverse transcriptase activity of the chimeric virus was analyzed in the fluctuation curve.</p><p><b>CONCLUSIONS</b>In chimeric HBIV cDNA transfected MT?4 cells, BIV tat and HIV gag were transcripted. The reverse transcriptase of the chimeric virus had biological activity. These data suggest that in MT4 cells, BIV LTR had promoter activity and BIV tat had the function of transactivation in the chimeric virus. The study of the chimeric virus with infectivity is in progress.</p>

Enhanced Induction of CEA Specific Tumor Immunity by TatCEA Fusion Protein

PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.

A simple human immunodeficiency virus vector system for selective infection of CD4(+) cells and inducible expression of foreign genes

The alteration of T lymphocyte functions as a consequence of human immunodeficiency virus (HIV) infection is a potential target for the genetic treatment of the acquired immunodeficiency syndrome (AIDS). One approach to the gene therapy for AIDS is to block the replication of HIV-1. Tat-dependent expression of forein gene and selective infection of CD4(+) cells by retroviral vector might be useful for abrogating the production of HIV-1 from cells. As part of studies to examine the feasibility of this concept, I constructed tat(+) and tat(-) HIV-1 proviral vectors that express all HIV-1 genes except for env and/or tat gene. When tat(+) or tat(-) HIV-1 particles were used for infection of HeLa T4 cells containing the endogenous beta-galactosidase (lacZ) gene under the control of the HIV-1 promoter and transactivation response element sequences, only the tat(+) HIV-1 particles transactivated the lacZ gene expression. This activation of lacZ expression following HIV infection of Tat(-) cells that stably contained but did not express the lacZ construct was determined to be an efficient process. I also constructed simple HIV-1 vectors that express the lacZ gene in a Tat-dependent manner or the hygromycin B phosphostransferase gene (Hyg(r)) under the control of the SV40 early promoter. The Tat-dependent vector conferring the lacZ(+) phenotype was assayed by beta-gal staining after infection of Tat(+) or Tat(-) cells. The activation of lacZ expression was observed only in tat(+) cells. Another simple HIV-1 vector containing the Hyg(r) gene was used for retroviral production from HeLa cells expressing the HIV-1 env gene and infection of CD4(+) or CD4(-) cells, but Hyg(r) colony was observed only from CD4(+) cells. These results provide a rationale for the use of HIV-1 retroviral vector system in the design of gene therapy of HIV infection.