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1.
Mol Immunol ; 33(3): 269-78, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8649448

RESUMO

Two-dimensional electrophoretic analysis (2D-PAGE) of cell surface human DP and DR class II antigens identified a glycoprotein, designated pX, that is associated at the cell surface with DP but not DR class II antigen in activated T, B and NK lymphocytes but not in resting B lymphocytes, Raji B lymphoma cells, activated thymic epithelial cells or activated monocytes. pX is a heavily glycosylated protein with an apparent molecular mass spanning between 38 kDa and 22 kDa, that is reduced, after deglycosylation with Endo-F, to 22 kDa. The pX structure appears nonpolymorphic and independent of DP polymorphism, as suggested by 2D-PAGE migrational pattern of 125I-labelled Endo-F deglycosylated DP immunoprecipitates from T cells blasts derived from four donors with different DP allotypes. The apparent absence of polymorphism of pX is further suggested by two-dimensional peptide mapping of a single spot derived from 2D-PAGE of 125I-labelled DP deglycosylated immunoprecipitates from two donors.


Assuntos
Antígenos HLA-DP/metabolismo , Ativação Linfocitária , Subpopulações de Linfócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Linfócitos B/química , Linfócitos B/imunologia , Células Cultivadas , Glicosilação , Antígenos HLA-DP/biossíntese , Antígenos HLA-DP/isolamento & purificação , Humanos , Interfase/imunologia , Subpopulações de Linfócitos/química , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/isolamento & purificação , Peso Molecular , Ligação Proteica/imunologia , Linfócitos T/química , Linfócitos T/imunologia
2.
FEBS Lett ; 386(2-3): 239-42, 1996 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-8647290

RESUMO

Granulocyte colony-stimulating factor (G-CSF) has been recently shown to induce the high-affinity Fc receptor for IgG (Fc(gammaRI)/CD64) in human polymorphonuclear neutrophils (PMN). To elucidate the molecular mechanisms whereby G-CSF exerts this effect, we examined whether the cytokine induces the binding of transcription factors to the IFNgamma response region (GRR), a well characterized regulatory element in the Fc(gammaRI) promoter that is responsible for the transcriptional induction of this gene. Using electrophoretic mobility shift assays, we show that in human PMN, G-CSF activates a GRR-binding complex which contains members of the signal transducer and activator of transcription (STAT) family of proteins, namely STAT1 and STAT3. In keeping with this result, treatment of neutrophils with G-CSF led to tyrosine phosphorylation of STAT3, as determined by immunoprecipitation followed by immunoblotting with antiphosphotyrosine antibodies. This is the first demonstration that in human neutrophils, the induction by G-CSF of Fc(gammaRI) gene expression may be mediated by the binding of STAT1 and STAT3 to the GRR sequence.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neutrófilos/metabolismo , Regiões Promotoras Genéticas , Receptores de IgG/genética , Transativadores/metabolismo , Sítios de Ligação , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Humanos , Interferon gama , Ligação Proteica , Fator de Transcrição STAT1 , Fator de Transcrição STAT3
3.
Virus Res ; 19(2-3): 199-208, 1991 May.
Artigo em Inglês | MEDLINE | ID: mdl-1654017

RESUMO

We have constructed a fibroblast cell line (EL alpha 4-2) which constitutively expresses the alpha 4 gene of herpes simplex virus type 1. We studied the induction of the alpha 4 gene, in the absence of the viral activator VP16, by stimulating EL alpha 4-2 cells with different growth factors. Here we report that a rapid, transient induction of the alpha 4 gene occurs only when EL alpha 4-2 fibroblasts are stimulated with purified epidermal growth factor (EGF). Such an induction does not require de novo protein synthesis. The role of cellular factors on the EGF-mediated induction of the alpha 4 gene has been analyzed by gel mobility shift assays using nuclear extracts from EL alpha 4-2 cells stimulated or not with EGF. The results obtained show that two factors bind to TAATGARAT (R = purine) regardless of EGF-stimulation. We conclude that a mechanism, different from the one involving VP16, is responsible for alpha 4 gene activation in EL alpha 4-2 cells and that the DNA-protein architecture is maintained at the TAATGARAT regulatory site regardless of changes in the transcriptional state induced by EGF.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Genes Virais/efeitos dos fármacos , Simplexvirus/efeitos dos fármacos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA Viral/genética , DNA Viral/metabolismo , Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Simplexvirus/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional , Proteínas Virais/genética , Proteínas Virais/metabolismo
4.
Drug News Perspect ; 11(10): 620-4, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15616631

RESUMO

The identification of CD4 as the HIV receptor immediately triggered a search for the development of novel therapeutic agents aimed at blocking receptor binding. Initial experimental approaches to this problem failed, but led to the observation that one or more other receptors for HIV, or co-receptors, must be involved in the entry of the virus in cells. In 1996 evidence was reported of a second viral receptor, already known under several names and renamed "fusin." Shortly thereafter the CCR5 molecule was identified as a co-receptor for the second type of HIV strain. This second discovery left no doubts: the second receptor for the virus encompassed at least two members of the chemokine receptor family. The identification of these co-receptors has led to several important new observations about HIV, including the fact that chemokines are potent in vitro inhibitors of viral replication, at least in T lymphocytes; however, there is still little information on their role in vivo. Nevertheless, unlike chemokines, the role of chemokine receptors in vivo has already emerged as being of substantial importance.

5.
J Biol Regul Homeost Agents ; 14(1): 41-4, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10763892

RESUMO

Interleukin-2 (IL-2), one of the most potent immunoregulatory and inflammatory cytokines, is being tested in phase III clinical trials in order to demonstrate its efficacy in combination with current antiviral agents in preventing the occurrence of opportunistic infections and death in individuals infected by the human immunodeficiency virus (HIV). In the meantime, its capacity to boost the number of CD4+ T cells in peripheral blood has been confirmed by a number of individual phase I/II trials conducted in different countries by independent investigators. In the face of this remarkable result, little is known of the effects exerted by this cytokine once administered to infected individuals in terms of its impact on different immunologic functions. The recent acquisitions on the important role played by latently infected cells in in vivo infection in reinitiating HIV replication and cytopathicity once antiviral therapy is suspended or becomes suboptimal, has shed new light on the possibility of utilizing immunologic strategies, including IL-2, for eradicating the virus from latent reservoirs. Results from a clinical trial conducted at our Institute indicate a decrease in lymphocyte-associated HIV DNA after IL-2 administration, supporting this hypothesis.


Assuntos
Infecções por HIV/tratamento farmacológico , Interleucina-2/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ensaios Clínicos como Assunto , DNA Viral/análise , Infecções por HIV/virologia , Humanos , Interleucina-2/farmacologia , Replicação Viral/efeitos dos fármacos
7.
Biochemistry ; 37(38): 13165-73, 1998 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-9748323

RESUMO

Human neutrophils can be induced to actively transcribe a number of early-response genes, in particular those encoding cytokines, chemokines, and the high-affinity surface receptor for IgG, FcgammaRI. Although little is known to date about the regulation of gene transcription in neutrophils, several indications point to a role for distinct transcription factors, such as members of the NF-kappaB and STAT families. In this study, we investigated whether these transcription factors become activated under stimulatory conditions which are known to induce gene transcription in neutrophils. Unexpectedly, we found that conventional procedures employed to prepare cellular extracts cause the release of proteolytic activities that are normally stored in intracellular granules, resulting in the degradation of various NF-kappaB/Rel and STAT proteins. To circumvent this problem, we developed an alternative procedure which allowed us to show that in neutrophils, LPS and TNFalpha induce a NF-kappaB DNA-binding activity which essentially consists of p50/RelA dimers, and that IFNgamma promotes the binding of STAT1 homodimers to the IFNgamma response region of the FcgammaRI promoter. Moreover, we report that neutrophil stimulation with GM-CSF results in the formation of a STAT5-containing DNA-binding activity. Collectively, the current findings open new perspectives about mechanisms that are likely to regulate gene transcription in neutrophils. In addition, the procedure described herein could prove useful in other cell types that express high levels of endogenous proteases.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Inibidores de Proteases/farmacologia , Fatores de Transcrição/metabolismo , Fracionamento Celular , Proteínas de Ligação a DNA/metabolismo , Detergentes , Endopeptidases/metabolismo , Humanos , Hidrólise , NF-kappa B/química , NF-kappa B/isolamento & purificação , NF-kappa B/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/genética , Neutrófilos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-rel , Fator de Transcrição STAT1 , Transativadores/metabolismo
8.
J Biol Chem ; 273(33): 20779-84, 1998 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-9694822

RESUMO

The STAT5 activation has important roles in cell differentiation, cell cycle control, and development. However, the potential implications of STAT5 in the control of apoptosis remain unexplored. To evaluate any possible link between the erythropoietin receptor (EpoR) JAK2/STAT5 transduction pathway and apoptosis, we have investigated apoptosis-resistant cells (ApoR) that arose from positive selection of the erythroid-committed Ba/F3EpoR cells triggered to apoptosis by ectopic expression of the HOX-B8 homeotic gene. We show that JAK2 is normally activated by Epo in both Ba/F3EpoR and ApoR cells. In contrast, both STAT5a and STAT5b isoforms are uniquely activated in a C-truncated form (86 kDa) only in ApoR cells. Analysis of ApoR and Ba/F3EpoR subclones confirmed that the switch to the truncated STAT5 isoform coincides with apoptosis survival and that ApoR do not derive from preexisting cells with a shortened STAT5. In addition, ApoR cells die in the absence of Epo. This indicates that resistance to apoptosis is not because of a general defect in the apoptotic pathway of ApoR cells. Furthermore, we show that the 86-kDa STAT5 protein presents a dominant-negative (DN) character. We hypothesize that the switch to a DN STAT5 may be part of a mechanism that allows ApoR cells to be selectively advantaged during apoptosis. In conclusion, we provide evidence for a functional correlation between a naturally occurring DN STAT5 and a biological response.


Assuntos
Apoptose , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas do Leite , Transativadores/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Proteínas de Ligação a DNA/genética , Eritropoetina/farmacologia , Genes Dominantes , Camundongos , Fosforilação , Fator de Transcrição STAT5 , Transdução de Sinais , Células-Tronco/metabolismo , Transativadores/genética
9.
J Immunol ; 160(2): 911-9, 1998 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9551929

RESUMO

Since IL-10 has been shown to up-regulate the expression of the high affinity receptor for IgG (FcgammaRI/CD64) in human monocytes, we examined whether the cytokine exerts a similar action toward polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in neutrophils, IL-10 failed to induce either the mRNA accumulation or the surface expression of FcgammaRI. Consistent with these findings, stimulation of PMN with IFN-gamma, but not with IL-10, resulted in the induction of specific DNA-binding activities to the IFN-gamma response region (GRR), a regulatory element located in the FcgammaRI gene promoter, required for transcriptional activation. In electrophoretic mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces the binding to the GRR of both STAT1 and STAT3, two members of the STAT family. In neutrophils, however, these activators did not bind to the GRR in response to IL-10, despite the fact that both STAT1 and STAT3 are expressed in these cells. On the other hand, IFN-gamma was an efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The lack of inducible GRR-binding activity in IL-10-treated PMN could not be ascribed to a lack of IL-10R, and did not appear to reflect an inhibitory effect of the cytokine. Taken together, our data suggest that IL-10 is unable to induce FcgammaRI gene expression in neutrophils because the intracellular signaling pathway triggered by the cytokine is impaired at the level of, or upstream of, STAT1 and/or STAT3 activation.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon gama/genética , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptores de IgG/genética , Sequências Reguladoras de Ácido Nucleico/imunologia , Transativadores/metabolismo , Genes de Imunoglobulinas , Humanos , Interferon gama/farmacologia , Interleucina-10/farmacologia , Leucócitos Mononucleares/metabolismo , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/biossíntese , Receptores de IgG/efeitos dos fármacos , Receptores de IgG/metabolismo , Receptores de Interleucina/biossíntese , Receptores de Interleucina-10 , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Transdução de Sinais/imunologia
10.
J Immunol ; 150(1): 139-50, 1993 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8417120

RESUMO

To gain insight into the developmentally regulated expression of the mouse TCR V delta-gene segments, we have investigated the role of the 5' promoter region of the V delta 1-gene. Transient transfection assays showed that a construct encompassing 267 nucleotides upstream from the mapped transcriptional start site was capable of driving promoter activity when transfected into V delta 1+ T cells. The inclusion of an additional 459-bp 5' segment to this construct did not affect promoter activity. However, a deletion of 222 5' nucleotides from the same construct dramatically decreased promoter activity. In vivo genomic footprinting localized several protein-DNA interactions to the stretch of DNA shown to have transcriptional activity. A computer analysis revealed that the segments of DNA participating in these protein-DNA interactions were identical to the previously described cyclic AMP response element (CRE), E box, and leukemia virus E26 cis-acting elements. Transient transfection assays performed with -267 bp constructs containing mutations at each of the localized cis-acting elements revealed that the CRE, E box, and Ets elements work together in driving promoter activity and that the CRE and Ets elements are the most important for driving transcription. Gel mobility shift analyses showed that each of these cis-acting elements is capable of binding specific nuclear factors present in V delta 1-expressing cells. These data indicate that multiple transcription factors acting in concert are responsible for V delta 1 gene expression.


Assuntos
Genes Reguladores , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transcrição Gênica , Animais , Sequência de Bases , Mapeamento Cromossômico , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação
11.
Pathobiology ; 61(5-6): 288-92, 1993.
Artigo em Inglês | MEDLINE | ID: mdl-8297495

RESUMO

To study the effects of the regulatory phosphoprotein ICP4 of the Herpes simplex virus, (HSV), a DNA tumor virus, on the induction of gene expression by the epidermal growth factor (EGF), we have constructed a cell line, ELa4-2, which constitutively expresses the a-4 gene product. The ELa4-2 cells are derived from the rat fibroblast EL2, in which EGF induces a marked c-fos and c-myc proto-oncogene transcription. Here we report that in ELa4-2 cells, the gene expression induced by EGF was negatively affected in respect to that obtained stimulating the parental EL2 cells. In particular, we studied the c-fos and c-myc proto-oncogene transcription induced by EGF. We found that in ELa4-2 cells the c-fos induction was dramatically reduced in comparison with the c-fos induction obtained in the parental EL2 cells. On the contrary, the c-myc induction by EGF was not affected by the presence of ICP4. Finally, we compared the HSV infectivity in ELa4-2 versus the EL2 cells. We showed that the virus growth capability was reduced, in the cells expressing ICP4.


Assuntos
Fibroblastos/citologia , Genes fos/genética , Transcrição Gênica/genética , Proteínas Virais , Animais , Linhagem Celular Transformada , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Genes myc/genética , Herpesvirus Humano 1/fisiologia , Proteínas Nucleares/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos/análise , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Transativadores/farmacologia
12.
Clin Immunol ; 99(1): 75-81, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11286543

RESUMO

Intermittent administration of recombinant interleukin-2 (rIL-2) to individuals infected with human immunodeficiency virus (HIV) has been shown to raise and maintain the absolute number of circulating CD4(+) T cells to normal or near normal levels. One of the signaling pathways triggered by IL-2 is the Janus kinase-signal transducer and activator of transcription (JAK-STAT). In particular, IL-2 activates the tyrosine kinases JAK1 and JAK3 and the transcription factors STAT3 and STAT5. We have previously observed that most HIV(+) individuals, unlike healthy seronegative controls, show a constitutive activation of STAT1 and a C-terminal truncated isoform of STAT5 (STAT5 Delta). In the present study, we have analyzed the protein level and activation state of STAT5 isoforms expressed in peripheral blood mononuclear cells of two HIV-infected individuals who showed a good or a poor response to intermittent IL-2 administration, respectively, and of a single individual before and after initiation of Zidovudine monotherapy. We provide evidence that both therapeutic interventions enhanced the expression and activation of the C-terminal truncated isoform of STAT5 (STAT5 Delta) in vivo.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Proteínas de Ligação a DNA/metabolismo , Infecções por HIV/tratamento farmacológico , Interleucina-2/uso terapêutico , Proteínas do Leite , Transativadores/metabolismo , Zidovudina/uso terapêutico , Adulto , DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Fator de Transcrição STAT5
13.
J Virol ; 73(9): 7515-23, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10438841

RESUMO

The human immunodeficiency virus (HIV) replicates in activated CD4(+) T lymphocytes. However, only CD4(+) Th2 and Th0, but not Th1, CD4(+) T-cell clones have been reported to efficiently support HIV-1 replication. This dichotomous pattern was further investigated in the present study in Th1, Th2, or Th0 cell lines derived from umbilical human cord blood and in T-cell clones obtained from the peripheral blood mononuclear cells (PBMC) of healthy adults. Both primary and laboratory-adapted HIV-1 strains with CCR5 as the exclusive entry coreceptor (R5 viruses) efficiently replicated in Th1, Th2, and Th0 cells. In sharp contrast, CXCR4-dependent (X4) viruses poorly replicated in both polarized and unpolarized CD4(+) T cells, including adults' PBMC infected several days after mitogenic stimulation. Unlike the X4 HIV-1(NL4-3), a chimera in which the env gene had been replaced with that of the R5 HIV-1(NL(AD8)), efficiently replicated in both Th1 and Th2 cells. This X4-dependent restriction of HIV replication was not explained by either the absence of functional CXCR4 on the cell surface or by the inefficient viral entry and reverse transcription. T-cell receptor stimulation by anti-CD3 monoclonal antibodies fully rescued X4 HIV-1 replication in both Th1 and Th2 cells, whereas it did not alter the extent and kinetics of R5 HIV-1 spreading. Thus, R5 HIVs show a replicative advantage in comparison to X4 viruses in their ability to efficiently propagate among suboptimally activated T lymphocytes, regardless of their polarized or unpolarized functional profiles. This observation may help to explain the absolute predominance of R5 HIVs over X4 viruses observed after viral transmission and during early-stage disease.


Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Células Th1/virologia , Células Th2/virologia , Replicação Viral , Adulto , Anticorpos Monoclonais/metabolismo , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Proteína gp120 do Envelope de HIV/genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Fatores de Tempo , Transcrição Gênica
14.
J Immunol ; 162(1): 323-30, 1999 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-9886402

RESUMO

IFN-gamma induces transcription of several IFN-stimulated genes (ISGs). Recently, the IFN-gamma-dependent Janus kinase (JAK)/STAT pathway has been shown to mediate the activation of some ISGs, by the sequential phosphorylation of two JAK kinases (JAK1 and JAK2) and of STAT1. Given that the JAK/STAT is the major, but not the only pathway linked to the IFN-gammaR, aim of our work was to investigate the signal-transduction pathway(s) by which IFN-gamma exerts its effects on acute replication of HIV in monocytic cells. To this end, we utilized clones previously derived from the U937 promonocytic cell line, differing for their efficient (plus clones) or inefficient (minus clones) abilities of supporting HIV replication. Unlike IFN-alpha, IFN-gamma did not inhibit HIV replication in plus clones, whereas virus production in minus cells was efficiently inhibited by both types of IFN. Plus clones generated a JAK/STAT signal-transduction pathway in response to IFN-alpha, but not IFN-gamma. In contrast, minus clones responded to either cytokines. The functional defect of plus clones in response to IFN-gamma was correlated to a selective defect of IFN-gammaR2, but not IFN-gammaR1, membrane expression. Surprisingly enough, IFN-gamma stimulation of plus clones induced IFN-stimulated gene factor 3 (ISGF3gamma). These results strongly support the hypothesis that the JAK/STAT pathway is responsible for the antiretroviral effect of IFN-gamma, and further provide evidence for a potential second pathway triggered by IFN-gamma in the absence of IFN-gammaR2 chain cell surface expression and involving ISGF3gamma.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/imunologia , Interferon-alfa/farmacologia , Interferon gama/antagonistas & inibidores , Interferon gama/fisiologia , Proteínas Tirosina Quinases/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/virologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/imunologia , HIV-1/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Imunidade Inata , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Interferon gama/deficiência , Fosfoproteínas/biossíntese , Proteínas Tirosina Quinases/deficiência , RNA Mensageiro/biossíntese , Receptores de Interferon/biossíntese , Fator de Transcrição STAT1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Transativadores/metabolismo , Fatores de Transcrição/biossíntese , Células U937 , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia , Receptor de Interferon gama
15.
Blood ; 94(12): 4202-9, 1999 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10590065

RESUMO

Infection by the human immunodeficiency virus (HIV) either upregulates or downregulates the expression of several cytokines and interferons (IFNs) that use the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway for signal transduction. However, very little is known on the state of activation of the JAK/STAT pathway after HIV infection either in vivo or in vitro. In this regard, we report here that a constitutive activation of a C-terminal truncated STAT5 (STAT5triangle up) and of STAT1alpha occurs in the majority ( approximately 75%) of individuals with progressive HIV disease. We have further demonstrated that, among peripheral blood mononuclear cells (PBMCs), STAT5triangle up is activated preferentially in CD4(+) T cells. In contrast to a published report, expression of STATs from PBMCs of infected individuals was comparable with that of seronegative donors. In addition, in vitro infection of mitogen-activated PBMCs with a panel of laboratory-adapted and primary HIV strains characterized by differential usage of chemokine coreceptors did not affect STAT protein levels. However, enhanced activation of STAT was observed after in vitro infection of resting PBMCs and nonadherent PBMCs by different viral strains. Thus, constitutive STAT activation in CD4(+) T lymphocytes represents a novel finding of interest also as a potential new marker of immunological reconstitution of HIV-infected individuals.


Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Proteínas de Ligação a DNA/imunologia , HIV-1 , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Proteínas do Leite , Transdução de Sinais/imunologia , Transativadores/imunologia , Síndrome da Imunodeficiência Adquirida/sangue , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/imunologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT5
16.
J Immunol ; 163(12): 6892-7, 1999 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10586091

RESUMO

Leader binding protein-1 (LBP-1)/late SV40 factor (LSF) and ying yang-1 (YY1) transcription factors are involved in the regulation of HIV expression. In particular, YY1 and LBP-1 have been shown to cooperate in repressing HIV-1-long terminal repeat reporter gene expression by in vitro cotransfection experiments. However, no information is available on the levels of expression and activation of these transcription factors in PBMC of HIV-infected individuals. Therefore, we have evaluated the expression and DNA binding activity of YY1 and LBP-1 (LSF) in PBMC of HIV-infected individuals before, during, and after administration of IL-2 in association with antiretroviral therapy (ART), a regimen under consideration for broad clinical use in this disease based on its ability to stably raise the absolute number of circulating CD4+ T lymphocytes. Both YY1- and LBP-1 (LSF)-DNA binding were profoundly down-modulated during administration of IL-2/ART, and a proteolytic activity probably responsible for the reduced expression of the two cellular transcription factors was found activated in PBMC of individuals receiving the immunotherapeutic regimen. This study is the first evidence of modulation of cellular transcription factors following IL-2/ART administration and provides a potential correlate of the transient raises in plasma viremia early reported in patients receiving IL-2 in the absence of ART, thus underscoring the importance of always administering this cytokine to HIV-infected individuals together with potent antiretrovirals.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Regulação para Baixo/imunologia , Soropositividade para HIV/imunologia , Interleucina-2/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Vírus do Sarcoma do Macaco-Barrigudo/imunologia , Fatores de Transcrição/antagonistas & inibidores , Adulto , Especificidade de Anticorpos , Western Blotting , DNA/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endopeptidases/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Feminino , Soropositividade para HIV/enzimologia , Soropositividade para HIV/metabolismo , Humanos , Hidrólise , Injeções Subcutâneas , Interleucina-2/genética , Interleucina-2/farmacologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Proteínas de Ligação a RNA , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1
17.
J Immunol ; 166(3): 1863-70, 2001 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11160233

RESUMO

We have recently shown that the binding subunit of pertussis toxin (PTX-B) inhibits the entry and replication of macrophage-tropic (R5) HIV-1 strains in activated primary T lymphocytes. Furthermore, PTX-B suppressed the replication of T cell-tropic (X4) viruses at a postentry level in the same cells. In this study we demonstrate that PTX-B profoundly impairs entry and replication of the HIV-1(ADA) (R5), as well as of HIV pseudotyped with either murine leukemia virus or vesicular stomatitis virus envelopes, in primary monocyte-derived macrophages. In addition, PTX-B strongly inhibited X4 HIV-1 replication in U937 promonocytic cells and virus expression in the U937-derived chronically infected U1 cell line stimulated with cytokines such as TNF-alpha and IL-6. Of interest, TNF-alpha-mediated activation of the cellular transcription factor NF-kappaB was unaffected by PTX-B. Therefore, PTX-B may represent a novel and potent inhibitor of HIV-1 replication to be tested for efficacy in infected individuals. In support of this proposition, a genetically modified mutant of PTX (PT-9K/129G), which is safely administered for prevention of Bordetella pertussis infection, showed an in vitro anti-HIV profile superimposable to that of PTX-B.


Assuntos
Antivirais/imunologia , HIV-1/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana , Monócitos/imunologia , Fragmentos de Peptídeos/imunologia , Toxina Pertussis , Fatores de Virulência de Bordetella/imunologia , Replicação Viral/imunologia , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Regulação Viral da Expressão Gênica/imunologia , Genes Reporter/imunologia , HIV-1/genética , HIV-1/fisiologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/virologia , Humanos , Interleucina-6/farmacologia , Vírus da Leucemia Murina/genética , Luciferases/genética , Macrófagos/virologia , Monócitos/virologia , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/fisiologia , Células U937 , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/metabolismo , Replicação Viral/genética
18.
J Virol ; 69(7): 4173-81, 1995 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7539506

RESUMO

Vesicular stomatitis virus (VSV) has a broad host range. It replicates in the cytoplasm and causes rapid cytopathic effects. We show that following VSV infection, a nuclear factor that binds to a select set of interferon-stimulated responsive elements (ISRE) is induced in many cell types. This factor, tentatively called VSV-induced binding protein (VIBP), was estimated to have an approximate molecular mass of 50 kDa and was distinct from known members of the interferon regulatory factor family, that are known to bind to the ISRE. Induction of VIBP required tyrosine kinase activity but did not require cellular transcription. Treatment of cells with cycloheximide, which inhibits translation, only partially inhibited induction of VIBP. However, type I interferons and staurosporine, both of which inhibit VSV transcription, inhibited VIBP induction. Moreover, a double-stranded RNA analog, poly(I)-poly(C) also induced a DNA-binding activity very similar to that of VIBP. These results indicate that a preexisting cellular protein is activated upon VSV infection and that this activation requires primary viral transcripts. The functional activity of VIBP was analyzed in cells stably transfected with a herpesvirus thymidine kinase-luciferase reporter gene that is under control of the ISRE. While activity of the control promoter without ISRE was strongly inhibited following VSV infection (as a result of virus-mediated transcriptional shutdown of the host cell), the inhibition was reversed by the ISRE-containing promoter, albeit partially, which suggests that VSV infection differentially affects transcription of host genes. Although VIBP was induced in all other cells tested, it was not induced in embryonal carcinoma cells after VSV infection, suggesting developmental regulation of VIBP inducibility.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Interferons/farmacologia , Proteínas Nucleares/biossíntese , Vírus da Estomatite Vesicular Indiana/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/fisiologia , Dactinomicina/farmacologia , Células-Tronco de Carcinoma Embrionário , Humanos , Fator Regulador 1 de Interferon , Camundongos , Dados de Sequência Molecular , Peso Molecular , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/fisiologia , Poli I-C/farmacologia
19.
Artigo em Inglês | MEDLINE | ID: mdl-2825402

RESUMO

The Southern blot hybridization technique has been applied to study the configuration of immunoglobulin and T-cell receptor genes in 6 cases of the so called mediastinal large cell lymphoma with sclerosis. This lymphoma has been recently recognized as a separate entity among non-Hodgkin lymphomas mainly affecting young adult patients. The B-cell origin of this neoplasm was suggested by means of immunohistochemical analysis. However, the immunophenotypical B-cell related markers used do not always exhibit lineage fidelity. The Southern blot analysis demonstrated the presence of unique heavy and k-light chain immunoglobulin gene rearrangements, establishing genotypically their B-cell origin.


Assuntos
Linfoma/genética , Neoplasias do Mediastino/genética , Linfócitos B/imunologia , DNA de Neoplasias/análise , Genes de Imunoglobulinas , Genótipo , Humanos , Imunoensaio , Imunoglobulinas/análise , Imunoglobulinas/genética , Imuno-Histoquímica , Linfoma/imunologia , Linfoma/patologia , Neoplasias do Mediastino/imunologia , Neoplasias do Mediastino/patologia , Hibridização de Ácido Nucleico , Receptores de Superfície Celular/genética , Esclerose , Linfócitos T
20.
Blood ; 94(8): 2880-9, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10515892

RESUMO

We have recently shown that, in human neutrophils, interleukin-10 (IL-10) fails to induce specific DNA-binding activities to the gamma-interferon response region (GRR), a regulatory element located in the FcgammaRI gene promoter, which is required for transcriptional activation by IL-10 and interferon gamma (IFNgamma) in monocytic cells. In this study, we report that IL-10 is also unable to induce the binding of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite the fact that both proteins are expressed in neutrophils. Whereas IFNgamma and granulocyte colony-stimulating factor (G-CSF) are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in polymorphonuclear neutrophils (PMN), IL-10 fails to trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcgammaRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible SH2-containing protein (CIS) gene family (which also includes CIS1, CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part, under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth arrest, macrophage-like differentiation, and nitric oxide synthesis, but not IL-6 mRNA expression. Collectively, our data suggest that, in neutrophils, the activation of STAT1 and STAT3 phosphorylation is neither required for CIS3/SOCS3 induction by IL-10 nor involved in the regulatory effects of IL-10 on cytokine production.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-10/farmacologia , Neutrófilos/efeitos dos fármacos , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Proteínas Repressoras , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Família Multigênica , Neutrófilos/metabolismo , Óxido Nítrico/biossíntese , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/metabolismo , Transfecção , Domínios de Homologia de src
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