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1.
Eur J Immunol ; 40(4): 949-54, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20201043

RESUMEN

The hallmark of chronic viral infections is a progressive exhaustion of antigen-specific CD8(+) T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8(+) T cells that makes them dysfunctional. Here, we show that during human chronic HIV-1 infection, a CD8(+) T-cell positive costimulatory pathway mediated by DNAX-activating molecule-1 is also disrupted. Thus, DNAX-activating molecule-1 downregulation on CD8(+) T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection.


Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/patología , Infecciones por VIH/inmunología , VIH-1/fisiología , Animales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos Virales/inmunología , Proteínas Reguladoras de la Apoptosis/fisiología , Linfocitos T CD8-positivos/inmunología , Regulación hacia Abajo , Glicoproteínas/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Activación de Linfocitos , Recuento de Linfocitos , Coriomeningitis Linfocítica/inmunología , Ratones , Fragmentos de Péptidos/inmunología , Receptor de Muerte Celular Programada 1 , Receptores Virales/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Carga Viral , Proteínas Virales/inmunología , Replicación Viral
2.
Am J Pathol ; 174(4): 1415-25, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19246649

RESUMEN

Ubiquitinated endosomal proteins that are deposited into the lumens of multivesicular bodies are either sorted for lysosomal-mediated degradation or secreted as exosomes into the extracellular milieu. The mechanisms that underlie the sorting of cellular cargo proteins are currently unknown. In this study, we show that the COP9 signalosome (CSN)-associated protein CSN5 quantitatively regulated proteins that were sorted into exosomes. Western blot analysis of exosomal proteins indicated that small interfering (si)RNA knockdown of CSN5 results in increased levels of both ubiquitinated and non-ubiquitinated exosomal proteins, including heat shock protein 70, in comparison with exosomes isolated from the supernatants of 293 cells transfected with scrambled siRNA. Furthermore, 293 cells transfected with JAB1/MPN/Mov34 metalloenzyme domain-deleted CSN5 produced exosomes with higher levels of ubiquitinated heat shock protein 70, which did not affect non-ubiquitinated heat shock protein 70 levels. The loss of COP9-associated deubiquitin activity of CSN5 also led to the enhancement of HIV Gag that was sorted into exosomes as well as the promotion of HIV-1 release, suggesting that COP9-associated CSN5 regulates the sorting of a number of exosomal proteins in both a CSN5 JAB1/MPN/Mov34 metalloenzyme domain-dependent and -independent manner. We propose that COP9-associated CSN5 regulates exosomal protein sorting in both a deubiquitinating activity-dependent and -independent manner, which is contrary to the current idea of ubiquitin-dependent sorting of proteins to exosomes.


Asunto(s)
Exosomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , Ubiquitinación/fisiología , Western Blotting , Complejo del Señalosoma COP9 , Proteína p24 del Núcleo del VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1 , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunoprecipitación , Fragmentos de Péptidos/metabolismo , Transporte de Proteínas/fisiología , ARN Interferente Pequeño , Transfección
3.
Biotechniques ; 40(1): 91-100, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16454046

RESUMEN

The rapid increase of viral strains that are resistant to the currently available antiretroviral drugs is a threat to the success of current human immunodeficiency virus type 1 (HIV-1) treatment and emphasizes the importance of developing novel anti-HIV-1 compounds. To improve the current abilities to screen for novel HIV-1 inhibitors, here we introduce a T-cell-based reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCRS, and provides the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. In this format, JLTRG-R5 cell-based infectivity assays have a Z'-factor of 0.78, which defines the assay as extremely robust and clearly amenable to high-throughput screening. The functional similarity of the JLTRG-R5 cell line and peripheral blood mononuclear cells (PBMCs) was demonstrated through the identity of the inhibitory concentrations, 50% (IC50s) for four antiretroviral compounds or neutralizing antibodies. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. In addition, the EGFP marker allows for data acquisition at an optimal time point by prescreening selected positive control wells using fluorescent microscopy. These characteristics make the system extremely flexible, rapid, and inexpensive. Due to its intrinsic flexibility, the JLTRG-R5 cell-based reporter system provides a powerful tool to greatly facilitate future screening for HIV-1 inhibitors.


Asunto(s)
Fármacos Anti-VIH/farmacología , Línea Celular , Proteínas Fluorescentes Verdes/análisis , VIH-1/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Linfocitos T/citología , Amidas/farmacología , Biomarcadores , Antagonistas de los Receptores CCR5 , Técnicas de Cultivo de Célula , Genes Reporteros , Anticuerpos Anti-VIH/farmacología , Inhibidores de Fusión de VIH/farmacología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Indinavir/farmacología , Lamivudine/farmacología , Pruebas de Sensibilidad Microbiana/instrumentación , Microscopía Fluorescente , Compuestos de Amonio Cuaternario/farmacología , ARN Viral/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Linfocitos T/virología , Replicación Viral/efectos de los fármacos
4.
J Exp Med ; 207(4): 763-76, 2010 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-20368576

RESUMEN

Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.


Asunto(s)
Anticuerpos Antifosfolípidos/farmacología , Anticuerpos Monoclonales/farmacología , Quimiocinas CC/metabolismo , VIH-1/fisiología , Receptores CCR5/fisiología , Tropismo Viral/fisiología , Internalización del Virus/efectos de los fármacos , Anticuerpos Antifosfolípidos/genética , Anticuerpos Antifosfolípidos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Cardiolipinas/inmunología , Fusión Celular , Quimiocina CCL3/inmunología , Quimiocina CCL3/metabolismo , Quimiocina CCL4/inmunología , Quimiocina CCL4/metabolismo , Quimiocinas/metabolismo , Regiones Determinantes de Complementariedad/genética , Medios de Cultivo Condicionados/farmacología , Endotoxinas/farmacología , Células Epiteliales/virología , Células Gigantes/citología , VIH-1/clasificación , Humanos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Cinética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Mutación/genética , Mutación/inmunología , Fosfatidiletanolaminas/inmunología , Fosfatidilserinas/inmunología , beta 2 Glicoproteína I/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
5.
AIDS ; 23(16): 2069-77, 2009 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-19710597

RESUMEN

OBJECTIVES: There is a need to develop HIV-1 vaccine formulations that incorporate inexpensive antigens and clinically acceptable potent adjuvants for inducing neutralizing antibodies. The purpose of this initial vaccine study was to produce peptide- and lipid-induced murine mAbs that replicate the characteristics of the 2F5 and/or 4E10 human antibodies in binding both to the membrane proximal external region (MPER) of glycoprotein 41 and the adjacent lipid bilayer for neutralizing HIV-1 infection of CD4 lymphocytes. RESEARCH DESIGNS AND METHODS: Liposomes containing a synthetic MPER peptide as a peptide antigen, phosphatidylinositol-4-phosphate (PIP) as a lipid antigen, and monophosphoryl lipid A as a potent adjuvant were used as a formulation to immunize mice. mAbs were then produced and tested for binding to MPER, glycoprotein 41, and PIP and for the ability to neutralize HIV-1 infection of CD4 cells in a human peripheral blood mononuclear cell assay. RESULTS: Polyclonal antisera contained antibodies that bound both to MPER and PIP. Immunoglobulin M mAbs were produced that bound both to the core MPER site of 2F5, or that overlapped with the 4E10 site, and that simultaneously bound PIP. High concentrations of these mAbs neutralized infection of peripheral blood lymphocytes by a primary infectious molecular clone of HIV-1. CONCLUSION: Liposomes containing MPER peptide as an antigen, PIP as a lipid antigen, and lipid A as an adjuvant induce anti-MPER-specific multispecific antibodies that simultaneously bind glycoprotein 41 MPER and adjacent lipid and neutralize HIV-1 infection in a human peripheral blood mononuclear cell assay.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Vacunas contra el SIDA/economía , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Liposomas/inmunología , Ratones , Fosfatos de Fosfatidilinositol/inmunología
6.
J Exp Med ; 206(6): 1273-89, 2009 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-19487424

RESUMEN

Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling the direct analysis of those viruses actually responsible for productive clinical infection. We show in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for the molecular cloning and biological analysis of transmitted/founder viruses and a comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses.


Asunto(s)
Evolución Molecular , Genoma Viral , Infecciones por VIH , VIH-1/genética , VIH-1/inmunología , Fenotipo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Femenino , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/clasificación , Humanos , Funciones de Verosimilitud , Macrófagos/inmunología , Macrófagos/virología , Masculino , Modelos Teóricos , Mutación , Filogenia , Receptores CCR5/inmunología , Virión/genética , Replicación Viral/genética , Replicación Viral/inmunología
7.
Immunol Rev ; 206: 149-59, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16048547

RESUMEN

The gastrointestinal mucosa is the largest reservoir of macrophages in the body. These important effector cells are derived from blood monocytes that are recruited to the lamina propria by endogenous chemoattractants in the non-inflamed mucosa and by inflammatory chemokines and bacterial products during inflammation. In the non-inflamed mucosa, newly recruited pro-inflammatory monocytes are exposed to lamina propria stromal (extracellular matrix) factors that induce phenotypic and functional differentiation into non-inflammatory macrophages. As a consequence of this differentiation, resident lamina propria macrophages are strikingly downregulated for the expression of innate response receptors, such as the receptors for lipopolysaccharide, immunoglobulin G (IgG), and IgA, and the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-alpha. Despite downregulated pro-inflammatory function, strong phagocytic and bactericidal activities remain intact. Thus, in the non-inflamed intestinal mucosa, lamina propria macrophages are non-inflammatory but retain avid scavenger and host defense functions, a unique but ideal phenotype and functional profile for effector cells in close proximity to immunostimulatory microorganisms and products.


Asunto(s)
Inmunidad Innata , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Animales , Humanos , Inmunidad Mucosa , Mucosa Intestinal/citología , Macrófagos/citología , Macrófagos/metabolismo
8.
Pigment Cell Res ; 18(3): 167-80, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15892713

RESUMEN

Summary WNT1 and WNT3a have been described as having redundant roles in promoting the development of neural crest-derived melanocytes (NC-Ms). We used cell lineage restricted retroviral infections to examine the effects of WNT signaling on defined cell types in neural crest cultures. RCAS retroviral infections were targeted to melanoblasts (NC-M precursor cells) derived from transgenic mice that express the virus receptor, TVA, under the control of a melanoblast promoter (DCT). As expected, over 90% of DCT-TVA+ cells expressed early melanoblast markers MITF and KIT. However, by following the fate of infected cells in standard culture conditions, we find that only 5% of descendents were NC-Ms. The majority of the descendents were not NC-Ms, but expressed smooth muscle cell markers, demonstrating that mammalian melanoblasts are not committed to the NC-M lineage. RCAS infection of DCT-TVA+ cells demonstrated that overexpression of canonical WNT signaling genes (betaCAT, WNT3a or WNT1) can increase NC-M numbers in an endothelin dependent manner. However, WNT1 and WNT3a have different modes of action with respect to melanoblast fate. Intrinsic over-expression of betaCAT or WNT3a can increase NC-M numbers by biasing the fate of DCT-TVA+ cells to NC-Ms. In contrast, the DCT-TVA+ melanoblasts cannot respond to WNT1 signaling and do not alter their fate towards NC-M. Instead, WNT1 only increases NC-M numbers through paracrine signaling on melanoblast precursors to increase the numbers of neural crest cells that become NC-Ms.


Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Melanocitos/fisiología , Cresta Neural/citología , Proteínas/fisiología , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Melanocitos/citología , Ratones , Ratones Mutantes , Ratones Transgénicos , Músculo Liso/citología , Músculo Liso/fisiología , Cresta Neural/embriología , Regiones Promotoras Genéticas/genética , Proteínas/genética , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt1 , Proteína Wnt3 , Proteína Wnt3A
9.
J Virol ; 76(15): 7518-27, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12097564

RESUMEN

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Virus de la Leucosis Aviar/metabolismo , Virus del Sarcoma Aviar/metabolismo , Receptores Virales/química , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Proteínas Aviares , Sitios de Unión , Western Blotting , Línea Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos BALB C , Receptores Virales/inmunología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/química
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