RESUMEN

The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.

Asunto(s)

Antígenos CD40 , Leishmania major , Leishmaniasis Cutánea , Macrófagos , Ratones Endogámicos BALB C , Transducción de Señal , Animales , Leishmania major/inmunología , Leishmania major/fisiología , Antígenos CD40/metabolismo , Ratones , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Macrófagos/inmunología , Macrófagos/parasitología , Humanos , Femenino , Fosforilación , Interacciones Huésped-Parásitos/inmunología , Sistema de Señalización de MAP Quinasas/inmunología

RESUMEN

CD40 plays dual immunoregulatory roles in Leishmania major infection and tumor regression. The functional duality emerges from CD40-induced reciprocal p38MAPK and ERK-1/2 phosphorylations. Because phosphotyrosine-based signaling in hematopoietic cells is regulated by the phosphotyrosine phosphatase SHP-1, which is not implied in CD40 signaling, we examined whether SHP-1 played any roles in CD40-induced reciprocal signaling and anti-leishmanial function. We observed that a weaker CD40 stimulation increased SHP-1 activation. ERK-1/2 inhibition or p38MAPK overexpression inhibited CD40-induced SHP-1 activation. An ultra-low-dose, CD40-induced p38MAPK phosphorylation was enhanced by SHP-1 inhibition but reduced by SHP-1 overexpression. A reverse profile was observed with ERK-1/2 phosphorylation. SHP-1 inhibition reduced syk phosphorylation but increased lyn phosphorylation; syk inhibition reduced but lyn inhibition enhanced CD40-induced SHP-1 phosphorylation. Corroborating these findings, in L. major-infected macrophages, CD40-induced SHP-1 phosphorylation increased and SHP-1 inhibition enhanced CD40-induced p38MAPK activation and inducible NO synthase expression. IL-10 enhanced SHP-1 phosphorylation and CD40-induced ERK-1/2 phosphorylation but reduced the CD40-induced p38MAPK phosphorylation, whereas anti-IL-10 Ab exhibited reverse effects on these CD40-induced functions, identifying IL-10 as a crucial element in the SHP-1-MAPK feedback system. Lentivirally overexpressed SHP-1 rendered resistant C57BL/6 mice susceptible to the infection. Lentivirally expressed SHP-1 short hairpin RNA enhanced the CD40-induced L. major parasite killing in susceptible BALB/c mice. Thus, we establish an SHP-1-centered feedback system wherein SHP-1 modulates CD40-induced p38MAPK activation threshold and reciprocal ERK-1/2 activation, establishing itself as a critical regulator of CD40 signaling reciprocity and mechanistically re-emphasizing its role as a potential target against the diseases where CD40 is involved.

Asunto(s)

Antígenos CD40/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Animales , Activación Enzimática/inmunología , Interleucina-10/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Leishmaniasis Cutánea/patología , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Óxido Nítrico Sintasa de Tipo II , Fosforilación/inmunología , Proteínas Tirosina Quinasas/inmunología , Quinasa Syk , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología

RESUMEN

Human immunodeficiency virus regulatory protein Rev (regulator of viral expression) is translated from a monocistronic transcript produced early in the viral replication cycle. Rev binds to the cis-acting, highly structured viral RNA sequence Rev response element (RRE) and the Rev-RRE complex primarily controls nucleocytoplasmic transport of viral RNAs. Inhibition of Rev-RRE interaction therefore is an attractive target to block viral transport. We have developed a stable cell line carrying a lentiviral vector harboring a rev gene and a co-linear Rev-dependent GFP/luciferase reporter gene cassette and thus constitutively expressing the reporter proteins. Dose-dependent luciferase activity inhibition in the indicator cell line by known small molecule inhibitors Proflavin and K37 established the specificity of the assay. This novel single step assay, that involves use of very small amount of reagents/cells and addition of test material as the only manipulation, can therefore be useful for screening therapeutically potential Rev-RRE interaction inhibitors.

RESUMEN

Intramacrophage pathogen Leishmania donovani escapes host immune response by subverting Toll-like receptor (TLR) signaling, which is critically regulated by protein ubiquitination. In the present study, we identified tumor necrosis factor receptor-associated factor (TRAF) 3, degradative ubiquitination of which is essential for TLR4 activation, as a target for Leishmania to deactivate LPS-mediated TLR4 signaling. We used LPS-treated RAW 264.7 cells and compared the TLR4-mediated immune response in these cells with L. donovani and L. donovani + LPS costimulated macrophages. TRAF3, which was ubiquitinated (2.1-fold over control) at lys 48 position and subsequently degraded following LPS treatment, persisted in L. donovani and L. donovani + LPS costimulated cells due to defective lys 48 ubiquitination. Lys 63-linked ubiquitination of upstream proteins in the cascade (cIAP1/2 and TRAF6), mandatory for TRAF3 degradation, was also reduced postinfection. This may be attributed to reduced association between ubiquitin-conjugating enzyme Ubc13 and TRAF6 during infection. Inhibition of TRAF3 before infection by shRNA in Balb/c mice showed enhanced IL-12 and TNF-α (10.8- and 8.1-fold over infected control) and decreased spleen parasite burden (61.3% suppression, P<0.001), thereby marking reduction in disease progression. Our findings identified TRAF3 as a novel molecular regulator exploited by Leishmania for successful infection.

Asunto(s)

Leishmania donovani/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/inmunología , Receptor Toll-Like 4/inmunología , Animales , Línea Celular , Células Cultivadas , Expresión Génica/inmunología , Interacciones Huésped-Parásitos/inmunología , Immunoblotting , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-12/metabolismo , Leishmania donovani/fisiología , Leishmaniasis Visceral/genética , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Lisina/inmunología , Lisina/metabolismo , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Modelos Inmunológicos , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor 3 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/inmunología

RESUMEN

Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.

Asunto(s)

Eritropoyetina/biosíntesis , Vectores Genéticos , Lentivirus/genética , Elastasa Pancreática/metabolismo , ARN Interferente Pequeño/metabolismo , Clonación Molecular , Regulación hacia Abajo , Epoetina alfa , Eritropoyetina/genética , Células HEK293 , Humanos , Lentivirus/aislamiento & purificación , Elastasa Pancreática/genética , Plásmidos/genética , Plásmidos/metabolismo , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transfección , Transgenes

RESUMEN

Human herpesvirus 6B (HHV-6B) primary infections occur in early childhood and establish a life-long latency in the most healthy adults. HHV-6B was detectable in the peripheral blood mononuclear cells (PBMC) and granulocytes by serial genomic DNA dilution PCR till 10 pg of template DNA, in a healthy adult. Epstein Barr virus (EBV) mediated transformation of the PBMC resulted in establishment of a B-cell line. Southern hybridization with the PBMC as well as the cell line DNA showed distinct signals for high copy viral genomes and Gardella gel analysis indicated chromosomal integration of the HHV-6B. Integration site analysis in the PBMC and the cell line indicated an atypical viral integration in non-telomeric region of chromosome 12. Cell free culture medium of the cell line could infect different mononuclear cell lines, naïve or mitogen stimulated PBMC and was found to impart productive infection in a recipient T cell line. An HIV-1 LTR driven luciferase based reporter cell line was made and a single step assay was developed for estimating HHV-6B relative concentration in the culture supernatants. This study thus reports a new infectious HHV-6B isolate with uncommon integration site, spontaneous production from a cell line and also development of a simple relative HHV-6B titer assay.

Asunto(s)

Herpesvirus Humano 6/genética , Herpesvirus Humano 6/aislamiento & purificación , Provirus/genética , Provirus/aislamiento & purificación , Infecciones por Roseolovirus/virología , Adulto , Enfermedades Asintomáticas , Cromosomas/virología , ADN Viral/química , ADN Viral/genética , Granulocitos/virología , Humanos , Leucocitos Mononucleares/virología , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Carga Viral/métodos

RESUMEN

Using the Indian Human immunodeficiency virus type 2 (HIV-2) isolate derived lentiviral vector (LV) system reported earlier, we have derived multiple differently configured transfer vectors. Among the features imparted, the novel ones include a blue/white colony screening platform, a shorter vector backbone candidate and availability of default dual tags. Simultaneously, panels with different utilities were also made using this LV. These include neomycin or puromycin or hygromycin selection markers, with options of default promoter, dual multiple cloning site (MCS) availability and drug inducible transgene expression. All the transfer vectors contain the main MCS with the option of single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from cohesive and blunt end cloning sites, as described for the original parent vector. Each transfer vector format was tested by appropriate transgene expression function by transduction of target cells. This is the most comprehensive HIV-2 based lentiviral vector system developed so far and it will significantly aid in preferential applications and thus increase its utility as a versatile system for gene transfer technology.

Asunto(s)

Vectores Genéticos/genética , VIH-2/genética , Lentivirus/genética , Animales , Biomarcadores/metabolismo , Clonación Molecular , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción Genética , Transfección , Transgenes

RESUMEN

There is a growing need for developing a simple, rapid, reliable and cost effective method for detection of HIV-1 for early diagnosis of the infection especially in developing countries and in resource limited settings. A method for simultaneous detection of the HIV-1 p17 gene and the house keeping human ß-actin gene from dried blood spots (DBS) by a monoplex polymerase chain reaction (PCR) is described. Genomic DNA was extracted from 40 HIV-1 positive and 40 HIV-1 negative DBS and used as templates to amplify both the HIV-1 p17 and ß-actin genes simultaneously under the same cycling condition by a single round PCR. This method of detection of HIV-1 may provide a simple, rapid and cost effective alternative in resource limited settings; however, it would require testing a larger number of samples before widespread use.

Asunto(s)

Actinas/sangre , ADN Viral/sangre , Antígenos VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/sangre , Actinas/genética , ADN Viral/genética , Pruebas con Sangre Seca , Antígenos VIH/genética , Infecciones por VIH/sangre , Infecciones por VIH/virología , Humanos , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Provirus/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética

RESUMEN

Depending on the strength of signal dose, CD40 receptor (CD40) controls ERK-1/2 and p38MAPK activation. At low signal dose, ERK-1/2 is maximally phosphorylated but p38MAPK is minimally phosphorylated; as the signal dose increases, ERK-1/2 phosphorylation is reduced whereas p38MAPK phosphorylation is reciprocally enhanced. The mechanism of reciprocal activation of these two MAPKs remains un-elucidated. Here, our computational model, coupled to experimental perturbations, shows that the observed reciprocity is a system-level behavior of an assembly of kinases arranged in two modules. Experimental perturbations with kinase inhibitors suggest that a minimum of two trans-modular negative feedback loops are required to reproduce the experimentally observed reciprocity. The bi-modular architecture of the signaling pathways endows the system with an inherent plasticity which is further expressed in the skewing of the CD40-induced productions of IL-10 and IL-12, the respective anti-inflammatory and pro-inflammatory cytokines. Targeting the plasticity of CD40 signaling significantly reduces Leishmania major infection in a susceptible mouse strain. Thus, for the first time, using CD40 signaling as a model, we show how a bi-modular assembly of kinases imposes reciprocity to a receptor signaling. The findings unravel that the signalling plasticity is inherent to a reciprocal system and that the principle can be used for designing a therapy.

Asunto(s)

Antígenos CD40/metabolismo , Modelos Biológicos , Proteínas Quinasas/metabolismo , Transducción de Señal , Animales , Línea Celular , Retroalimentación Fisiológica , Leishmania major/fisiología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Fosforilación

RESUMEN

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-ß synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.

Asunto(s)

Cisteína Endopeptidasas/fisiología , Regulación hacia Abajo/inmunología , Glicoesfingolípidos/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leishmania donovani/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 2/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Animales , Línea Celular , Femenino , Glicoesfingolípidos/antagonistas & inhibidores , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leishmania donovani/patogenicidad , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 2/fisiología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa

RESUMEN

Human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter-mediated gene expression is regulated by the viral Tat protein that relieves a block to viral transcription elongation after binding with a viral hairpin loop RNA structure called the trans-activation-responsive region (TAR). Tat protein significantly up-regulates viral genome transcription and hence it has long been considered a potential target for antiretrovirals. Here we report the construction of a plasmid containing an HIV-1 LTR-driven reporter cassette with a colinear tat gene under control of a viral promoter and thus conditionally configured for constitutive expression of reporter genes. Inhibition of luciferase reporter expression in a cell line harboring the plasmid in the presence of tat-targeted shRNA confirmed the specificity of the assay and a dose-dependent reporter activity inhibition by the fluoroquinoline derivative K-37, a class of small RNA binding molecule that inhibits Tat and other RNA-dependent transactivations, further validated the method. Subsequently we also made a lentiviral vector (LV) containing the same transcription units and derived a stable cell line using the said LV and similar dose-dependent inhibition was documented using K-37. This quick and sensitive reporter-based method is the simplest screening assay for putative inhibitors of HIV-1 Tat-induced LTR-driven gene expression requiring test material addition as the only manipulation.

Asunto(s)

Duplicado del Terminal Largo de VIH , VIH-1/genética , Activación Transcripcional/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Células Cultivadas , Regulación Viral de la Expresión Génica , VIH-1/metabolismo , Humanos , Luciferasas , ARN Viral/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Sensibilidad y Especificidad , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética

RESUMEN

To reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of reactive oxygen species (ROS), which are a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling proteins are associated with mitochondrial ROS generation, which is the major contributor of total cellular ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation located at the inner membrane of mitochondria. Functional knockdown of macrophage UCP2 by small interfering RNA-mediated silencing was associated with increased mitochondrial ROS generation, lower parasite survival, and induction of marked proinflammatory cytokine response. Induction of proinflammatory cytokine response in UCP2 knocked-down cells was a direct consequence of p38 and ERK1/2 MAPK activation, which resulted from ROS-mediated inhibition of protein tyrosine phosphatases (PTPs). Administration of ROS quencher, N-acetyl-l-cysteine, abrogated PTP inhibition in UCP2 knocked-down infected cells, implying a role of ROS in inactivating PTP. Short hairpin RNA-mediated in vivo silencing of UCP2 resulted in decreased Src homology 2 domain-containing tyrosine phosphatase 1 and PTP-1B activity and host-protective proinflammatory cytokine response resulting in effective parasite clearance. To our knowledge, this study, for the first time, reveals the induction of host UCP2 expression during Leishmania infection to downregulate mitochondrial ROS generation, thereby possibly preventing ROS-mediated PTP inactivation to suppress macrophage defense mechanisms.

Asunto(s)

Regulación hacia Abajo/inmunología , Mediadores de Inflamación/fisiología , Canales Iónicos/fisiología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/prevención & control , Mitocondrias/metabolismo , Proteínas Mitocondriales/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Animales , Línea Celular , Regulación hacia Abajo/genética , Activación Enzimática/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Mediadores de Inflamación/metabolismo , Canales Iónicos/deficiencia , Canales Iónicos/genética , Leishmania donovani/inmunología , Leishmaniasis Visceral/patología , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Mitocondrias/genética , Mitocondrias/inmunología , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas Tirosina Fosfatasas/metabolismo , ARN Interferente Pequeño/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteína Desacopladora 2

RESUMEN

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regiones de Fijación a la Matriz , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Acetilación , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Secuencia de Bases , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , ADN/genética , ADN/metabolismo , Daño del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Histona Desacetilasa 1/metabolismo , Humanos , Ratones , Modelos Biológicos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteína de la Leucemia Promielocítica , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética

RESUMEN

BACKGROUND & OBJECTIVE: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. METHODS: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. RESULTS: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. INTERPRETATION & CONCLUSION: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.

Asunto(s)

Proteínas de la Cápside/inmunología , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/inmunología , Anticuerpos Antivirales/análisis , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Ensayo de Inmunoadsorción Enzimática , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/ultraestructura , Humanos , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomyces cerevisiae/genética

RESUMEN

CD40, a costimulatory molecule expressed on macrophages, induces expression of interleukin 12 (IL-12) in uninfected macrophages and IL-10 in macrophages infected with Leishmania major. IL-12 suppresses, whereas IL-10 enhances, L. major infection. The mechanisms that regulate this difference in CD40-induced cytokine production remain unclear, but it is known that L. major depletes cholesterol. Here we show that cholesterol influenced the assembly of distinct CD40 signalosomes. Depletion of membrane cholesterol inhibited the assembly of an IL-12-inducing CD40 signalosome containing the adaptors TRAF2, TRAF3 and TRAF5 and the kinase Lyn and promoted the assembly of an IL-10-inducing CD40 signalosome containing the adaptor TRAF6 and the kinase Syk. Thus, cholesterol depletion might represent an immune-evasion strategy used by L. major.

Asunto(s)

Antígenos CD40/inmunología , Colesterol/metabolismo , Leishmania major/inmunología , Macrófagos/inmunología , Animales , Antígenos CD40/metabolismo , Células Cultivadas , Colesterol/inmunología , Leishmania major/metabolismo , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Transducción de Señal/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/inmunología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo

RESUMEN

An infectious Indian human immunodeficiency virus-2 (HIV-2) subtype A isolate was completely sequenced and analyzed and its phylogenetic relatedness was investigated. The unusual limited size of the long terminal repeat (LTR) from the isolate was caused due to a truncation within the nef open reading frame (ORF) located at the U3 region of the LTR. The genetic relatedness and lineage of this HIV-2 strain were investigated. The close relatedness of this isolate to West African HIV-2 isolates confirms a geographical entry route of HIV-2 to this part of the Indian subcontinent. This is the first report of an HIV-2 full genome analysis from the Indian subcontinent as well as from Asia.

Asunto(s)

Genoma Viral , Infecciones por VIH/virología , VIH-2/genética , VIH-2/aislamiento & purificación , ARN Viral/genética , Análisis por Conglomerados , Genotipo , Humanos , India , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN

RESUMEN

Gene delivery has become an important tool for biological research and gene therapy trials. Lentiviral vector (LV) mediated gene transfer is a preferred approach for stable, sustained transgenic expression. We report here step-wise development of an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived third generation lentiviral vector with a novel, versatile multiple cloning site (MCS) that can also facilitate single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from useful cohesive/blunt end cloning. Efficiency of the vector systems was functionally demonstrated by development of a transgenic enhanced green fluorescence protein (GFP) expressing cell line. Further, a GFP down regulated cell line was derived from the said cell line through LV mediated shRNA expression by cloning the GFP-shRNA cassette using the T/A cloning strategy. Subsequently long term expression of GFP transgene in nude mouse spleen/liver was also documented till 30 days. This LV platform with the enhanced user friendly cloning options will be an important advancement in gene transfer technology.

Asunto(s)

Técnicas de Transferencia de Gen , Vectores Genéticos/genética , VIH-2 , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Desnudos , MicroARNs/genética , Datos de Secuencia Molecular , Transgenes

RESUMEN

An infectious Indian human immunodeficiency virus type 2 isolate from Mumbai, propagated in this laboratory, was found to bear an unusually short long terminal repeat (LTR) region. Complete sequencing of the 601 bp LTR indicated a loss of around 250 nucleotide pairs from the unique 3' (U3) region as compared to other well-characterized HIV-2 isolates. Phylogenetic analysis of this LTR shows closest relatedness to the Guinea-Bissau subtype A isolates HIV-2(CAM2) and HIV-2(ALI). The LTR from the biologically active infectious clone with the observed deletion contained all functionally relevant promoter and polyadenylation sequences.

Asunto(s)

Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/genética , VIH-2/patogenicidad , Secuencia de Bases , VIH-2/genética , Humanos , India , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADN