Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
PLoS Pathog ; 16(9): e1008879, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32997728

RESUMO

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.


Assuntos
Moléculas de Adesão Celular , Adesões Focais , Produtos do Gene gag , Vírus Linfotrópico T Tipo 1 Humano , Proteínas dos Microfilamentos , Fosfoproteínas , Linfócitos T , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Adesões Focais/química , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/virologia , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos , Linfócitos T/química , Linfócitos T/metabolismo , Linfócitos T/virologia
2.
Front Microbiol ; 10: 2439, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708905

RESUMO

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 infects CD4+ T-cells via cell-to-cell transmission requiring reorganization of the cytoskeleton and expression of the viral transactivator and oncoprotein Tax. Viruses spread at the virological synapse (VS), a virus-induced specialized cell-cell contact, by polarized budding into synaptic clefts, and by cell surface transfer of viral biofilms (VBs). Since little is known about Tax's role in formation of the VB, we asked which component of the VB is regulated by Tax and important for HTLV-1 transmission. Collagens are not only structural proteins of the extracellular matrix and basal membrane but also represent an important component of the VB. Here, we report that among the collagens known to be present in VBs, COL4 is specifically upregulated in the presence of HTLV-1 infection. Further, we found that transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts in Jurkat and CCRF-CEM T-cells, while robust induction of COL4 protein requires continuous Tax expression as shown in Tax-transformed T-cell lines. Repression of Tax led to a significant reduction of COL4A1/A2 transcripts and COL4 protein. Mechanistically, luciferase-based promoter studies indicate that Tax activates the COL4A2 and, to a less extent, the COL4A1 promoter. Imaging showing partial co-localization of COL4 with the viral Gag protein in VBs at the VS and transfer of COL4 and Gag to target cells suggests a role of COL4 in VB formation. Strikingly, in chronically infected C91-PL cells, knockout of COL4A2 impaired Gag transfer between infected T-cells and acceptor T-cells, while release of virus-like particles was unaffected. Taken together, we identified COL4 (COL4A1, COL4A2) as a component of the VB and a novel cellular target of Tax with COL4A2 appearing to impact virus transmission. Thus, this study is the first to provide a link between Tax's activity and VB formation by hijacking COL4 protein functions.

3.
Front Microbiol ; 9: 400, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29563906

RESUMO

The Human T-cell leukemia virus type 1 (HTLV-1)-encoded accessory protein p8 is cleaved from the precursor protein p12 encoded by the HTLV-1 open reading frame I. Both p12 and p8 are thought to contribute to efficient viral persistence. Mechanistically, p8 induces T-cell conjugates and cellular conduits. The latter are considered to facilitate transfer of p8 to target cells and virus transmission. Transfer of p8 between p8-expressing T-cells and recipient cells has been analyzed by immunofluorescence and live imaging. However, automatic quantitation of p8-transfer between cells has not been studied yet. Here we developed a novel method allowing time saving quantitation of p8 transfer between cells by flow cytometry. After establishing a protocol for the detection of intracellular p8 by flow cytometry and validation of p8 protein expression by western blot and immunofluorescence, we set up a co-culture assay between p8-expressing donor Jurkat T-cells and recipient Jurkat T-cells that had been prestained with a well-retained live cell dye. Upon quantitating the amount of p8 positive recipient cells with regard to the percentage of p8 expressing donor cells, time course experiments confirmed that p8 is rapidly transferred between Jurkat T-cells. We found that p8 enters approximately 5% of recipient T-cells immediately upon co-culture for 5 min. Prolonged co-culture for up to 24 h revealed an increase of relative p8 transfer to approximately 23% of the recipient cells. Immunofluorescence analysis of co-culture experiments and manual quantitation of p8 expression in fluorescence images confirmed the validity of the flow cytometry based assay. Application of the new assay revealed that manipulation of actin polymerization significantly decreased p8 transfer between Jurkat T-cells suggesting an important role of actin dynamics contributing to p8 transfer. Further, transfer of p8 to co-cultured T-cells varies between different donor cell types since p8 transfer could hardly been detected in co-cultures of 293T donor cells with Jurkat acceptor cells. In summary, our novel assay allows automatic and rapid quantitation of p8 transfer to target cells and might thus contribute to a better understanding of cellular processes and dynamics regulating p8 transfer and HTLV-1 transmission.

4.
Chembiochem ; 16(3): 446-54, 2015 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-25639621

RESUMO

Contact between the human immunodeficiency virus (HIV-1) and its target cell is initiated by the interaction of viral gp120 with cellular CD4. An assembled peptide (CD4bs-M) that presents the CD4 binding site of gp120 was previously shown to inhibit the gp120-CD4 interaction. Here, we demonstrate that CD4bs-M selectively enhances infection of cells with HIV-1, whereas infection with herpes simplex virus remains largely unaffected. The effects of CD4bs-M variants containing D-amino acids, or prolines at selected positions, point to the importance of side chain orientation and spatial orientation of this fragment. Furthermore, CD4bs-M was shown to assemble into amyloid-like fibrils that capture HIV-1 particles, which likely contributes to the infection-enhancing effect. Beyond infection enhancement, CD4bs-M enabled HIV-1 infection of CD4-negative cells, suggesting that binding of the peptide to gp120 facilitates interaction of gp120 with coreceptors, which might in turn enhance HIV-1 entry.


Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , HIV-1/patogenicidade , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Células HeLa/virologia , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Células Vero/virologia
5.
Front Immunol ; 4: 257, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24027570

RESUMO

We have recently designed a soluble synthetic peptide that functionally mimics the HIV-1 coreceptor CXCR4, which is a chemokine receptor that belongs to the family of seven-transmembrane GPCRs. This CXCR4 mimetic peptide, termed CX4-M1, presents the three extracellular loops (ECLs) of the receptor. In binding assays involving recombinant proteins, as well as in cellular infection assays, CX4-M1 was found to selectively recognize gp120 from HIV-1 strains that use CXCR4 for cell entry (X4 tropic HIV-1). Furthermore, anti-HIV-1 antibodies modulate this interaction in a molecular mechanism related to that of their impact on the gp120-CXCR4 interaction. We could now show that the selectivity of CX4-M1 pertains not only to gp120 from X4 tropic HIV-1, but also to synthetic peptides presenting the V3 loops of these gp120 proteins. The V3 loop is thought to be an essential part of the coreceptor binding site of gp120 that contacts the second ECL of the coreceptor. We were able to experimentally confirm this notion in binding assays using substitution analogs of CX4-M1 and the V3 loop peptides, respectively, as well as in cellular infection assays. These results indicate that interactions of the HIV-1 Env with coreceptors can be mimicked by synthetic peptides, which may be useful to explore these interactions at the molecular level in more detail.

6.
J Leukoc Biol ; 94(1): 123-35, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23610148

RESUMO

This study tested the hypothesis that mtDNA fragments carry immunostimulatory motifs that naturally induce immune activation by PDC. Genomic and mtDNA induced similar IFN-α production after transfection into PBMCs using the liposomal transfection reagent DOTAP. Shortening of mtDNA to CpG islands enhanced the immunostimulatory activity, based on the presence of unmethylated CpG DNA. Further fragmentation into mtODN, which exhibited similarities to published CpG ODN, resulted in a strong immunostimulatory activity in addition to PDC maturation and migration. The addition of the human cathelicidin LL-37 to CpG islands induced spontaneous PDC IFN-α production. Notably, one phosphodiester mtODN with a double-palindromic structure induced PDC IFN-α production in the absence of DOTAP. Flow cytometry, life-cell, and confocal imaging revealed attachment and spontaneous uptake into PDC, colocalizing, in part, with TLR9 in early endosomal vesicles. This process was accompanied by a moderate but significant PDC maturation in addition to B cell and NK cell activation (P<0.05). Altogether, our data indicate that fragmented mtDNA, which may be released as a consequence of apoptotic, necrotic, and necroptotic cell death, can act as a DAMP. For the first time, our study provides a mechanism how longer and shorter mtDNA fragments can be taken up naturally by the PDC and thus, may contribute to acute and chronic immune activation.


Assuntos
Linfócitos B/imunologia , Ilhas de CpG/imunologia , DNA Mitocondrial/imunologia , Células Dendríticas/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Oligodesoxirribonucleotídeos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células Cultivadas , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , DNA Mitocondrial/genética , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunização , Interferon-alfa/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Oligodesoxirribonucleotídeos/genética , Catelicidinas
7.
PLoS One ; 7(3): e33925, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22470494

RESUMO

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Infecções por HIV/metabolismo , HIV-1/imunologia , Adulto , Idoso , Animais , Fármacos Anti-HIV/uso terapêutico , Ligante de CD40/farmacologia , Linhagem Celular , Doença Crônica , Cricetinae , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Interferon-alfa/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos/farmacologia , Receptores Toll-Like/metabolismo
8.
BMC Infect Dis ; 12: 14, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22264238

RESUMO

BACKGROUND: HIV-associated general immune activation is a strong predictor for HIV disease progression, suggesting that chronic immune activation may drive HIV pathogenesis. Consequently, immunomodulating agents may decelerate HIV disease progression. METHODS: In an observational study, we determined immune activation in HIV patients receiving low-dose (5 mg/day) prednisolone with or without highly-active antiretroviral therapy (HAART) compared to patients without prednisolone treatment. Lymphocyte activation was determined by flow cytometry detecting expression of CD38 on CD8(+) T cells. The monocyte activation markers sCD14 and LPS binding protein (LBP) as well as inflammation markers soluble urokinase plasminogen activated receptor (suPAR) and sCD40L were determined from plasma by ELISA. RESULTS: CD38-expression on CD8+ T lymphocytes was significantly lower in prednisolone-treated patients compared to untreated patients (median 55.40% [percentile range 48.76-67.70] versus 73.34% [65.21-78.92], p = 0.0011, Mann-Whitney test). Similarly, we detected lower levels of sCD14 (3.6 µg/ml [2.78-5.12] vs. 6.11 µg/ml [4.58-7.70]; p = 0.0048), LBP (2.18 ng/ml [1.59-2.87] vs. 3.45 ng/ml [1.84-5.03]; p = 0.0386), suPAR antigen (2.17 µg/ml [1.65-2.81] vs. 2.56 µg/ml [2.24-4.26]; p = 0.0351) and a trend towards lower levels of sCD40L (2.70 pg/ml [1.90-4.00] vs. 3.60 pg/ml [2.95-5.30]; p = 0.0782). Viral load in both groups was similar (0.8 × 105 ng/ml [0.2-42.4 × 105] vs. 1.1 × 105 [0.5-12.2 × 105]; p = 0.3806). No effects attributable to prednisolone were observed when patients receiving HAART in combination with prednisolone were compared to patients who received HAART alone. CONCLUSIONS: Patients treated with low-dose prednisolone display significantly lower general immune activation than untreated patients. Further longitudinal studies are required to assess whether treatment with low-dose prednisolone translates into differences in HIV disease progression.


Assuntos
Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Fatores Imunológicos/administração & dosagem , Ativação Linfocitária/efeitos dos fármacos , Prednisolona/administração & dosagem , ADP-Ribosil Ciclase 1/análise , Proteínas de Fase Aguda/análise , Adulto , Fármacos Anti-HIV/administração & dosagem , Ligante de CD40/sangue , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/análise , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/análise , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Monócitos/química , Monócitos/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue
9.
Virology ; 423(2): 152-64, 2012 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-22209232

RESUMO

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p<0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.


Assuntos
Antígenos CD4/imunologia , Células Dendríticas/virologia , Dinaminas/imunologia , Endocitose , Infecções por HIV/imunologia , HIV-1/fisiologia , Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Dinaminas/genética , Endossomos/imunologia , Endossomos/virologia , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Interferon-alfa/imunologia , Microscopia Imunoeletrônica
10.
AIDS Res Hum Retroviruses ; 26(2): 161-71, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20156099

RESUMO

Abstract Human plasmacytoid dendritic cells (PDC) are the major producers of type I interferons (IFN) after stimulation with CpG oligodeoxynucleotides (ODN). HIV-1-infected patients show a deficit in PDC numbers and function with progression of disease. CpG ODN appear to be attractive therapeutics to support the impaired innate immunity in HIV-1 infection. PDC counts, phenotype, and function were analyzed in 23 HIV-infected untreated individuals and 16 controls. Markers for migration (CCR7), activation (CD80), maturation (CD83), and endocytosis (BDCA2) were evaluated at baseline and 20 h after in vitro stimulation with class A, B, C, and P ODN. PDC counts and the expression of BDCA2 on these cells were significantly lower in HIV-1-infected subjects compared to controls (both p < 0.001). After stimulation with CpG ODN, CD80 and CD83 were upregulated to a similar extent in patients and controls, whereas CCR7 was upregulated more efficiently by CpG-P and CpG-C than CpG-A in HIV-1-infected individuals compared to controls. The IFN-alpha induction significantly differed for the CpG ODN classes (A > P > C > B) in patients and controls (p < 0.05). Functional PDC deficits in IFN-alpha and TNF-alpha induction were particularly evident in subjects with less than 500 CD4(+) cells/mul. CpG-P ODNs not only induced remarkable IFN-alpha production in patient PBMCs, but also significantly upregulated the antibacterial and antiviral CXC chemokine IP-10. In conclusion, PDC counts, phenotype, and function are significantly impaired in HIV-1-infected subjects. Optimized P-class ODN may be effective in reversing this innate immune defect, which should be further evaluated in vivo.


Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunidade Inata , Fatores Imunológicos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Adulto , Antígenos CD/análise , Antígeno B7-1/análise , Células Dendríticas/química , Células Dendríticas/imunologia , Humanos , Imunoglobulinas/análise , Interferon-alfa/biossíntese , Lectinas Tipo C/análise , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Receptores CCR7/análise , Receptores Imunológicos/análise , Antígeno CD83
11.
Immunology ; 129(2): 234-47, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19824924

RESUMO

Human plasmacytoid dendritic cells (PDC) are crucial for innate and adaptive immune responses against viral infections, mainly through production of type I interferons. Evidence is accumulating that PDC surface receptors play an important role in this process. To investigate the PDC phenotype in more detail, a chip-based expression analysis of surface receptors was combined with respective flow cytometry data obtained from fresh PDC, PDC exposed to interleukin-3 (IL-3) and/or herpes simplex virus type 1 (HSV-1). CD156b, CD229, CD305 and CD319 were newly identified on the surface of PDC, and CD180 was identified as a new intracellular antigen. After correction for multiple comparisons, a total of 33 receptors were found to be significantly regulated upon exposure to IL-3, HSV-1 or IL-3 and HSV-1. These were receptors involved in chemotaxis, antigen uptake, activation and maturation, migration, apoptosis, cytotoxicity and costimulation. Infectious and ultraviolet-inactivated HSV-1 did not differentially affect surface receptor regulation, consistent with the lack of productive virus infection in PDC, which was confirmed by HSV-1 real-time polymerase chain reaction and experiments involving autofluorescing HSV-1 particles. Viral entry was mediated at least in part by endocytosis. Time-course experiments provided evidence of a co-ordinated regulation of PDC surface markers, which play a specific role in different aspects of PDC function such as attraction to inflamed tissue, antigen recognition and subsequent migration to secondary lymphatic tissue. This knowledge can be used to investigate PDC surface receptor functions in interactions with other cells of the innate and adaptive immune system, particularly natural killer cells and cytotoxic T lymphocytes.


Assuntos
Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Imunidade Adaptativa , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Separação Celular , Chlorocebus aethiops , Células Dendríticas/imunologia , Células Dendríticas/patologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/efeitos da radiação , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Interleucina-3/imunologia , Interleucina-3/metabolismo , Análise em Microsséries , Células Vero , Internalização do Vírus/efeitos da radiação
12.
J Virol ; 82(17): 8900-5, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18579609

RESUMO

Plasmacytoid dendritic cells (PDC) are major producers of type I interferons (IFN) in response to human immunodeficiency virus type 1 (HIV-1) infection. To better define the underlying mechanisms, we studied the magnitude of alpha IFN (IFN-alpha) induction by recombinant viruses containing changes in the Env protein that impair or disrupt CD4 binding or expressing primary env alleles with differential coreceptor tropism. We found that the CD4 binding affinity but not the viral coreceptor usage is critical for the attachment of autofluorescing HIV-1 to PDC and for subsequent IFN-alpha induction. Our results illustrate the importance of the gp120-CD4 interaction in determining HIV-1-induced immune stimulation via IFN-alpha production.


Assuntos
Antígenos CD4/imunologia , Células Dendríticas/imunologia , HIV-1/imunologia , Interferon-alfa/biossíntese , Adolescente , Adulto , Antígenos CD4/metabolismo , Linhagem Celular , Células Cultivadas , Criança , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Rim/citologia , Pessoa de Meia-Idade , Transfecção , Replicação Viral
13.
J Immunol ; 179(6): 4219-30, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17785862

RESUMO

Plasmacytoid dendritic cells (PDC), the main producers of type I IFNs in the blood, are important for the recognition and control of viral and bacterial infections. Because several viruses induce IFN-alpha production, severe courses of herpes virus infections in nonimmunocompromised patients may be related to numerical or functional PDC deficits. To evaluate this hypothesis, PBMC and PDC were repeatedly isolated from nine patients with acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus. The patients experienced meningitis/encephalitis and frequent infections in childhood (n = 2), recurrent herpes virus infections at unusual localizations (n = 2), ocular surgery (n = 1), infections (n = 4), and stress around ARN (n = 6). The median percentage of isolated PDC was significantly lower in patients compared with 18 age-matched healthy controls (p < 0.001), confirmed by FACS analysis using peripheral blood, and was extremely low during acute disease. PDC counts dropped in five controls suffering from respiratory infections or diarrhea. IFN-alpha production in PDC and PBMC exposed to different stimuli was significantly lower in patients than in controls (p < 0.05). Anergy to these stimuli was observed on four occasions, in particular during acute disease. PDC of patients showed up-regulated IFN regulatory factor-7 mRNA levels and evidence of in vivo activation (CD80) and maturation (CD83) (p < 0.05). CD8+ cell responses were significantly lower in patients vs controls (p = 0.04). These data support a risk factor model in which numerical and functional deficits in PDC-mediated innate immune responses contribute to an impaired control of latent herpes virus infections and subsequent development of ARN.


Assuntos
Células Dendríticas/imunologia , Herpes Simples/imunologia , Síndrome de Necrose Retiniana Aguda/imunologia , Síndrome de Necrose Retiniana Aguda/virologia , Simplexvirus/imunologia , Adolescente , Adulto , Idoso , Contagem de Células Sanguíneas , Anergia Clonal , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Regulação para Baixo/imunologia , Herpes Simples/patologia , Herpesvirus Humano 3/imunologia , Humanos , Imunidade Inata , Fator Regulador 7 de Interferon/biossíntese , Fator Regulador 7 de Interferon/genética , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/metabolismo , Pessoa de Meia-Idade , Síndrome de Necrose Retiniana Aguda/patologia , Regulação para Cima/imunologia
14.
AIDS ; 21(5): 645-7, 2007 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-17314528

RESUMO

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas do Envelope Viral/fisiologia , Replicação Viral/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Vírus GB C/fisiologia , Humanos , Proteínas Recombinantes de Fusão/farmacologia , Transfecção , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/farmacologia , Replicação Viral/efeitos dos fármacos
15.
Microbes Infect ; 8(2): 354-62, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16239118

RESUMO

Whereas the acquired T cell-mediated protection against intracellular pathogens such as Leishmania major has been well studied in the past, the cells and mechanisms involved in their innate control are still poorly understood. Here, we investigated the role of natural killer T (NKT) cells in a high dose L. major mouse infection model. In vitro, L. major only weakly stimulated NKT cells and antagonized their response to the prototypic NKT cell ligand alpha-galactosylceramide, indicating that L. major partially escapes the activation of NKT cells. NKT cell deficiency as analyzed by subcutaneous infection of Jalpha281-/- mice (lacking invariant CD1d-restricted NKT cells) and CD1-/- mice (lacking all CD1d-restricted NKT cells) led to a transient increase in skin lesions, but did not impair the clinical cure of the infection, NK cell cytotoxicity, the production of IFN-gamma, the expression of inducible nitric oxide synthase, and the control of the parasites in the lymph node. In the spleen, however, NKT cells were required for NK cell cytotoxicity and early IFN-gamma production, they lowered the parasite burden, and exerted bystander effects on Leishmania antigen-specific T cell responses, most notably after systemic infection. Thus, NKT cells fulfill organ-specific protective functions during infection with L. major, but are not essential for parasite control.


Assuntos
Células Matadoras Naturais/imunologia , Leishmania major/patogenicidade , Leishmaniose Cutânea/imunologia , Linfonodos/imunologia , Baço/imunologia , Animais , Antígenos CD1/metabolismo , Antígenos CD1d , Humanos , Imunidade Inata , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Pele/imunologia , Pele/parasitologia , Baço/citologia , Baço/parasitologia
16.
Microbes Infect ; 7(15): 1461-8, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16269265

RESUMO

The pro-inflammatory cytokine TNF is essential for a protective immune response to some but not all strains of Leishmania major. TNF-deficient mice of a resistant genetic background succumbed rapidly to an infection with L. major BNI. Another member of the TNF superfamily, Fas ligand (FasL), has also been reported to be critical for the immune response to L. major. To test the relative importance of TNF versus FasL for the control of L. major BNI, we infected wildtype C57BL/6 (B6.WT), B6.TNF(-/-), B6.gld and C57BL/6.gld x TNF(-/-) (B6.gld.TNF(-/-)) double-negative mice. Visceral, fatal disease was only observed in B6.TNF(-/-) mice, but not in B6 gld mice. The course of infection and the immune response of B6.gld.TNF(-/-) mice were similar to those of B6.TNF(-/-) mice. B6.gld.TNF(-/-) mice had a high tissue parasite burden and expressed prominent amounts of inducible nitric oxide synthase (iNOS) in the skin, the lymph nodes (LN) and the spleen as previously reported for B6.TNF(-/-) mice, whereas the tissue parasite load and the iNOS expression of B6.gld mice resembled that of B6.WT controls. Neither the TNF- nor the FasL-deficiency exerted a detectable intrinsic effect on the proliferation of T cells. Thus, TNF, but not FasL is essential for the control of L. major BNI. The discrepancy between these and other published data are most likely due to the use of different strains of the pathogen.


Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Glicoproteínas de Membrana/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas , Imuno-Histoquímica , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Linfonodos/parasitologia , Linfonodos/patologia , Ativação Linfocitária , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/análise , Pele/parasitologia , Pele/patologia , Baço/parasitologia , Baço/patologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genética , Fatores de Necrose Tumoral/genética
17.
AIDS ; 19(12): 1267-72, 2005 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-16052081

RESUMO

OBJECTIVE: A number of studies concerning the pathogenesis of GB virus C (GBV-C) in HIV-infected people suggest a beneficial effect and improved survival for dually infected individuals. However there has remained controversy regarding the clinical relevance of these findings, as some studies have not confirmed these observations. To address the possibility of direct inhibitory mechanisms, we studied the impact of GBV-C on HIV-1 replication in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with sera from GBV-C positive individuals or transfected with GBV-C specific RNA and superinfected with HIV. Replication kinetics of HIV were studied by quantification of HIV-p24 release. Induction of soluble antiretroviral factors were monitored with an HIV infection assay and by quantification of chemokine secretion. Changes in chemokine receptor expression were analysed by flow cytometry. RESULTS: We demonstrate that GBV-C infection of PBMC leads to significant replication inhibition of R5- and X4-HIV isolates representing eight HIV clades. The inhibitory effect is mediated by GBV-C infection and also by expression of GBV-C structural glycoproteins and/or of non-structural proteins NS2/NS3. Upon GBV-C infection CD4 and CD8 T lymphocytes produce soluble HIV-suppression factors. Induction of stromal cell-derived factor (SDF)-1 and subsequent internalization of CXCR4 was not observed. CONCLUSIONS: CD4 and CD8 T lymphocytes are stimulated by GBV-C to secrete antiretroviral factors, inhibiting R5- and X4-HIV strains. As no induction of SDF-1 and no down-regulation of the respective receptor CXCR4 could be observed, it is likely that additional unidentified factors causing inhibition of X4-HIV strains are induced by GBV-C.


Assuntos
Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Vírus GB C/fisiologia , HIV/fisiologia , Leucócitos Mononucleares/virologia , Replicação Viral/fisiologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Humanos , Transfecção
18.
Infect Immun ; 70(8): 4638-42, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12117977

RESUMO

Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo.


Assuntos
Leishmania mexicana/imunologia , Leishmaniose Cutânea/enzimologia , Leishmaniose Tegumentar Difusa/enzimologia , Óxido Nítrico Sintase/biossíntese , Animais , Humanos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leishmaniose Tegumentar Difusa/imunologia , Leishmaniose Tegumentar Difusa/parasitologia , Leishmaniose Tegumentar Difusa/patologia , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase Tipo II , Pele/enzimologia , Pele/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...