Upregulation of the apoptosis-associated protein Grb3-3 in HIV-1-infected human CD4(+) lymphocytes.

The mechanism(s) by which HIV-1 infection contributes to depletion of CD4(+) T cell is not well understood. In this report, we investigated whether a recently identified isoform of growth factor receptor bound protein (Grb2), named Grb3-3, a signaling molecule that is associated with the MAP kinase pathway and with apoptosis could be involved. We find that Grb3-3 is markedly up-regulated following HIV-1 infection of CD4(+) peripheral blood mononuclear cells undergoing apoptosis. Although IL-2 deprived CD4(+) cells also undergo apoptosis to a similar extent, Grb3-3 upregulation is not detected under these experimental conditions. Transient overexpression of Grb3-3 in Jurkat T-cells also causes apoptosis. Upon staurosporine stimulation, Grb3-3 predisposes Sup-T1 cell to apoptosis. Finally, analysis of the HIV-1 genes responsible for Grb3-3 expression demonstrates that Tat and Nef can independently induces its expression, suggesting these two earliest viral gene products of HIV-1 may share some common pathway(s) in up-regulating Grb3-3 expression.

Grb3-3 is up-regulated in HIV-1-infected T-cells and can potentiate cell activation through NFATc.

The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factor receptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4(+) peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.

Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies.

We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.

Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS.

The early successes of highly active anti-retroviral therapies (HAART) for the treatment of HIV-1-infection and AIDS have raised the question as to whether there is a legitimate role for gene therapy in the treatment of this chronic infectious disease. However, in many patients the profound suppression of viral replication is short lived, particularly if patients have been treated with sequential monotherapies in the past, have been infected with a highly drug resistant isolate of HIV-1, or have temporarily discontinued therapy as a "holiday" or because of drug intolerance. In addition, life-long adherence to maintenance HAART will probably be required even in responding patients with undetectable viremia because of the reservoirs of latently infected cells that can persist for years. Gene therapy through the introduction of anti-retroviral "resistance" genes into CD4(+) T cells is one approach that could give long term protection to these HIV-1 susceptible cells in vivo. We have explored this approach by developing intrabodies to the critical HIV-1 transactivator protein, Tat that is absolutely required for HIV-1 replication. This provocative treatment approach, that will be tested in a clinical gene therapy trial, sets the groundwork for determining if anti-Tat intrabody gene therapy together with HAART can provide a treatment strategy for the immune reconstitution of HIV-1-infected patients with advanced disease.

Inhibition of human immunodeficiency virus type 1 replication in vitro by a novel combination of anti-Tat single-chain intrabodies and NF-kappa B antagonists.

Human immunodeficiency virus type 1 (HIV-1) Tat, an early regulatory protein that is critical for viral gene expression and replication, transactivates the HIV-1 long terminal repeat (LTR) via its binding to the transactivation response element (TAR) and, along with other cellular factors, increases viral transcription initiation and elongation. Tat also superactivates the HIV-1 promoter through a TAR-independent mechanism, including tumor necrosis factor alpha-induced and protein kinase C (PKC)-dependent activation of NF-kappa B, and inhibitors of Tat and NF-kappa B cooperatively down-regulate this Tat-mediated LTR superactivation. In this study, a combined pharmacologic and genetic strategy using two PKC (NF-kappa B) inhibitors, pentoxifylline (PTX) and Gö-6976, and a stably expressed anti-Tat single-chain intracellular antibody (sFv intrabody) was employed to obtain cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. Treatment of cells with PTX and Gö-6976 resulted in cooperative inhibition of both HIV-1 LTR-driven gene expression and HIV-1 replication. In addition, the combined use of anti-Tat sFv intrabodies and the two NF-kappa B inhibitors retained the virus in the latent state for as long as 45 days. The combined treatment resulted in more durable inhibition of HIV-1 replication than was seen with the NF-kappa B inhibitors alone or the anti-Tat sFv intrabodies alone. Together, these results suggest that in future clinical gene therapy trials, a combined pharmacologic and genetic strategy like the one reported here may improve the survival of transduced cells and prolong clinical benefit.

Enzymatic removal of alpha-galactosyl epitopes from porcine endothelial cells diminishes the cytotoxic effect of natural antibodies.

Xenotransplantation of tissues between discordant species such as pig into human is not yet feasible due to the problem of hyperacute rejection. This rapid response to xenogeneic tissue is mediated by natural antibodies that react with antigens on the xenograft. A number of xenoantigens consist of carbohydrate residues, and a terminal galactose in alpha linkage has been shown to be involved in hyperacute rejection of pig-to-human xenografts. We show that alpha-linked galactose on porcine endothelial cells is a major epitope recognized by IgG and IgM antibodies present in monkey and human sera. Endothelial cells that had been treated with alpha-galactosidase did not react with fluorescein-labeled Griffonia simplicifolia I B4 (GS-IB4), a lectin that detects the alpha-galactosyl epitope on intact cells. The reactivity of both human and cynomolgus monkey serum with endothelial cells was decreased by 59% to 90% after treatment with coffee bean alpha-galactosidase. Using a colorimetric assay for cell viability, we show that natural antibodies present in the sera of cynomolgus monkey and humans are cytotoxic to porcine endothelial cells in the presence of exogenously added complement. When the terminal alpha-galactosy residues were removed by enzymatic digestion, the cytotoxic effect of natural antibodies on porcine endothelial cells was diminished by > 80%. Evaluation of the time course of reappearance of the alpha-galactosyl epitope at the cell surface revealed that 48 hr after alpha-galactosidase treatment, binding of GS-IB4 was diminished by 60%. These results suggest that glycosidase treatment of cells to be transplanted could prevent hyperacute rejection mediated by natural antibodies.

Production and characterization of monoclonal antibodies to Ha-ras and N-ras p21.

The mammalian ras family consists of the Ha, Ki and N-ras genes that encode a series of 21,000 dalton proteins (p21). The three ras proteins participate in normal cell physiology and have been implicated in cellular transformation by either overexpression of the normal p21 or by mutation at positions 12, 13, or 61. To help understand the biological roles of the different ras proteins, we have generated monoclonal antibodies (Mabs) to the Ha-ras and N-ras p21. Mab Ha-770, raised to a Ha-specific synthetic peptide, reacts with Ha-ras recombinant p21 (r-p21) as well as cellular Ha-ras p21 by immunoprecipitation. Western blot and sandwich ELISA assays. Mab N-838, raised to an N-ras specific synthetic peptide, reacts with the N-ras recombinant p21 by immunoprecipitation, Western blot and sandwich ELISA assays. Mabs to the Ha-ras and N-ras p21 should be valuable reagents in assessing the individual roles of ras proteins in normal and neoplastic cells.

Generation of monoclonal antibodies specific for ras p21 Glu-12 oncoproteins: detection in carcinogen-induced mammary carcinomas.

ras genes have been shown to become oncogenes by single point mutations which result in amino acid substitutions that affect either their GTPase activity (positions 12, 13, 59, 61) or their affinity for GTP and GDP. Ras oncogenes and their corresponding proteins have been described in a variety of human cancers as well as in animal tumors induced by physical and chemical carcinogens. One of these animal tumor systems involves the induction of mammary carcinomas in rats by a single dose of N-nitroso-N-methylurea (NMU), a methylating carcinogen. These NMU-induced mammary carcinomas contain transforming H-ras genes activated by G----A transitions in the second nucleotide of their 12th codon, presumably a consequence of the pre-mutagenic lesions induced by NMU. These G----A mutations result in the replacement of the normal glycine in the 12th position of the ras p21 protein by a glutamic acid residue. In this study, we report the generation of monoclonal antibodies (Mab) reactive with oncogenic ras p21 proteins containing glutamic acid at position 12 (p21 Glu-12). Mab designated E184 specifically recognized activated ras p21 Glu-12 proteins but not normal p21 (Gly-12) or p21 proteins activated by other position 12 substitutions including arginine, aspartic acid, cysteine, valine or serine residues. Western blot analysis of NMU-induced mammary carcinomas demonstrated that Mab E184 recognized p21 proteins expressed in these rat tumors but not p21 present in normal tissues nor in other carcinogen-induced tumors known to carry H-ras oncogenes activated by mutations at position 61.(ABSTRACT TRUNCATED AT 250 WORDS)

Production and characterization of anti-RAS p21 monoclonal antibodies.

Monoclonal antibodies (MAb) Ras 10 and Ras 11 were raised to an activated human Harvey-ras p21 and shown to react with recombinant p21 as well as p21 derived from human and rodent cells. Characterization studies by ELISA, immunoprecipitation and Western blot procedures demonstrated that MAb Ras 10 (IgG2a) and Ras 11 (IgG2b) react with normal p21, activated p21, and p21 from each of the Harvey, Kirsten and N-ras families. Studies illustrated that MAb Ras 10 and Ras 11 can also be used in flow cytometry and immunohistochemistry to specifically detect cellular p21. ELISA, immunoprecipitation and Western blot studies comparing rat anti-p21 MAb Y13-259 with Ras 10 and Ras 11 demonstrated that Ras 10 and Ras 11 had a greater sensitivity for ras protein detection than Y13-259. Collectively, these studies illustrate that MAb Ras 10 and Ras 11 can be applied to a variety of assay formats to detect ras proteins and, therefore, may be valuable tools in detecting and measuring of ras protein expression in normal, neoplastic and pre-neoplastic cells.