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1.
J Virol ; 91(18)2017 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-28659489

RESUMEN

There are no approved therapeutics for the treatment of dengue disease despite the global prevalence of dengue virus (DENV) and its mosquito vectors. DENV infections can lead to vascular complications, hemorrhage, and shock due to the ability of DENV to infect a variety of immune and nonimmune cell populations. Increasingly, studies have implicated the host response as a major contributor to severe disease. Inflammatory products of various cell types, including responding T cells, mast cells (MCs), and infected monocytes, can contribute to immune pathology. In this study, we show that the host response to DENV infection in immunocompetent mice recapitulates transcriptional changes that have been described in human studies. We found that DENV infection strongly induced metabolic dysregulation, complement signaling, and inflammation. DENV also affected the immune cell content of the spleen and liver, enhancing NK, NKT, and CD8+ T cell activation. The MC-stabilizing drug ketotifen reversed many of these responses without suppressing memory T cell formation and induced additional changes in the transcriptome and immune cell composition of the spleen, consistent with reduced inflammation. This study provides a global transcriptional map of immune activation in DENV target organs of an immunocompetent host and supports the further development of targeted immunomodulatory strategies to treat DENV disease.IMPORTANCE Dengue virus (DENV), which causes febrile illness, is transmitted by mosquito vectors throughout tropical and subtropical regions of the world. Symptoms of DENV infection involve damage to blood vessels and, in rare cases, hemorrhage and shock. Currently, there are no targeted therapies to treat DENV infection, but it is thought that drugs that target the host immune response may be effective in limiting symptoms that result from excessive inflammation. In this study, we measured the host transcriptional response to infection in multiple DENV target organs using a mouse model of disease. We found that DENV infection induced metabolic dysregulation and inflammatory responses and affected the immune cell content of the spleen and liver. The use of the mast cell stabilization drug ketotifen reversed many of these responses and induced additional changes in the transcriptome and immune cell repertoire that contribute to decreased dengue disease.


Asunto(s)
Antialérgicos/administración & dosificación , Virus del Dengue/inmunología , Dengue/tratamiento farmacológico , Dengue/patología , Perfilación de la Expresión Génica , Cetotifen/administración & dosificación , Mastocitos/inmunología , Animales , Dengue/inmunología , Modelos Animales de Enfermedad , Ratones
2.
Am J Pathol ; 184(1): 92-100, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24434277

RESUMEN

Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 µmol/L. However, at concentrations >100 µmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis.


Asunto(s)
Adrenérgicos/administración & dosificación , Linfocitos T CD4-Positivos/virología , Regulación de la Expresión Génica , VIH-1/efectos de los fármacos , Metanfetamina/administración & dosificación , MicroARNs/genética , Replicación Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , VIH-1/fisiología , Humanos , MicroARNs/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/efectos de los fármacos
3.
Am J Pathol ; 184(4): 927-936, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24486327

RESUMEN

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 µmol/L concentrations) did not induce apoptosis, but 200 to 1000 µmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.


Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Trastornos Relacionados con Cocaína/complicaciones , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Infecciones por VIH/complicaciones , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Separación Celular , Trastornos Relacionados con Cocaína/inmunología , Trastornos Relacionados con Cocaína/patología , Progresión de la Enfermedad , Citometría de Flujo , Infecciones por VIH/inmunología , Infecciones por VIH/patología , VIH-1 , Humanos , Virión/efectos de los fármacos
4.
Infect Immun ; 82(7): 2728-35, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24733094

RESUMEN

Porphyromonas gingivalis is a Gram-negative obligate anaerobic bacterium and is considered a keystone pathogen in the initiation of periodontitis, one of the most widespread infectious diseases. Bacterial bis-(3'-5') cyclic GMP (cyclic di-GMP [c-di-GMP]) serves as a second messenger and is involved in modulating virulence factors in numerous bacteria. However, the role of this second messenger has not been investigated in P. gingivalis, mainly due to a lack of an annotation regarding diguanylate cyclases (DGCs) in this bacterium. Using bioinformatics tools, we found a protein, PGN_1932, containing a GGDEF domain. A deletion mutation in the pgn_1932 gene had a significant effect on the intracellular c-di-GMP level in P. gingivalis. Genetic analysis showed that expression of the fimA and rgpA genes, encoding the major protein subunit of fimbriae and an arginine-specific proteinase, respectively, was downregulated in the pgn_1932 mutant. Correspondingly, FimA protein production and the fimbrial display on the mutant were significantly reduced. Mutations in the pgn_1932 gene also had a significant impact on the adhesive and invasive capabilities of P. gingivalis, which are required for its pathogenicity. These findings provide evidence that the PGN_1932 protein is both responsible for synthesizing c-di-GMP and involved in biofilm formation and host cell invasion by P. gingivalis by controlling the expression and biosynthesis of FimA.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/patogenicidad , Adhesión Bacteriana/fisiología , Línea Celular , Biología Computacional , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Mutación , Liasas de Fósforo-Oxígeno/genética , Virulencia
5.
Prostate ; 72(8): 886-97, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21932423

RESUMEN

BACKGROUND: Xenotropic murine leukemia virus-related retrovirus (XMRV) is a recently discovered gammaretrovirus that was originally detected in prostate tumors. However, a causal relationship between XMRV and prostate cancer remains controversial due to conflicting reports on its etiologic occurrence. Even though gammaretroviruses are known to induce cancer in animals, a mechanism for XMRV-induced carcinogenesis remains unknown. Several mechanisms including insertional mutagenesis, proinflammatory effects, oncogenic viral proteins, immune suppression, and altered epithelial/stromal interactions have been proposed for a role of XMRV in prostate cancer. However, biochemical data supporting any of these mechanisms are lacking. Therefore, our aim was to evaluate a potential role of XMRV in prostate carcinogenesis. METHODS: Growth kinetics of prostate cancer cells are conducted by MTT assay. In vitro transformation and invasion was carried out by soft agar colony formation, and Matrigel cell invasion assay, respectively. p27(Kip1) expression was determined by Western blot and MMP activation was evaluated by gelatin-zymography. Up-regulation of miR221 and miR222 expression was examined by real-time PCR. RESULTS: We demonstrate that XMRV infection can accelerate cellular proliferation, enhance transformation, and increase invasiveness of slow growing prostate cancer cells. The molecular basis of these viral induced activities is mediated by the downregulation of cyclin/cyclin dependent kinase inhibitor p27(Kip1) . Downstream analyses illustrated that XMRV infection upregulates miR221 and miR222 expression that target p27(Kip1) mRNA. CONCLUSIONS: We propose that downregulation of p27(Kip1) by XMRV infection facilitates transition of G1 to S, thereby accelerates growth of prostate cancer cells. Our findings implicate that if XMRV is present in humans, then under appropriate cellular microenvironment it may serve as a cofactor to promote cancer progression in the prostate.


Asunto(s)
Adenocarcinoma/patología , Proliferación Celular , Transformación Celular Neoplásica/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/fisiología , Neoplasias de la Próstata/patología , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/fisiología , Adenocarcinoma/fisiopatología , Adenocarcinoma/virología , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Masculino , Metaloproteasas/metabolismo , MicroARNs/metabolismo , Invasividad Neoplásica , Neoplasias de la Próstata/fisiopatología , Neoplasias de la Próstata/virología , ARN Mensajero/metabolismo , Regulación hacia Arriba
6.
Front Immunol ; 12: 681950, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34168651

RESUMEN

Dengue virus (DENV), a Flavivirus, causes a broad spectrum of disease in humans with key clinical signs including thrombocytopenia, vascular leakage and hemorrhaging. A major obstacle to understanding DENV immunity has been the lack of a validated immune-competent mouse model. Here, we report the infection profiles of human clinical isolates of DENV serotypes 1-4 in an immune-competent mouse model. We detected replicating DENV in the peritoneal cells, liver and the spleen that was generally resolved within 2 weeks. The DENV target cell types for infection were monocytes/macrophages, dendritic cells, endothelial cells, and we identified a novel DENV cellular target, fibroblast reticular cells of the spleen. We observed gross pathologies in the spleen and liver that are consistent with dengue disease, including hemorrhaging as well as transcriptional patterns suggesting that antiviral responses and tissue damage were induced. Key clinical blood parameters that define human DENV disease such as hemoconcentration, leukopenia and reduced number of platelets were also observed. Thus, immune-competent mice sustain replicating infection and experience signs, such as hemorrhaging, that define DENV disease in humans. This study thoroughly characterizes DENV1-4 infection in immune-competent mice and confirms the wild-type mouse model as a valid and reproducible system for investigating the mechanisms of DENV pathogenesis.


Asunto(s)
Virus del Dengue/inmunología , Dengue/inmunología , Dengue/virología , Interacciones Huésped-Patógeno/inmunología , Animales , Biopsia , Dengue/patología , Virus del Dengue/clasificación , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Regulación de la Expresión Génica , Huésped Inmunocomprometido , Ratones , Especificidad de Órganos , Serogrupo
7.
Res Microbiol ; 160(1): 57-62, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19028569

RESUMEN

Sequencing of three housekeeping genes, mdh, dnaE and recA, and ribotyping for seven non-toxigenic Vibrio cholerae O1 strains isolated from different geographic sources indicate a phylogenetic relationship among the strains. Results of MLST and ribotyping indicate a clear difference between three toxigenic strains (N16961, O395, and 569B) and three non-toxigenic strains from India (GS1, GS2, and GW87) and one Guam strain (X392), the latter of which were similar in both MLST and ribotyping, while two other non-toxigenic strains from the USA and India (2740-80 and OR69) appeared to be more closely related to toxigenic strains than to non-toxigenic strains, although this was not supported by ribotyping. These results provide clues to the emergence of toxigenic strains from a non-toxigenic progenitor by acquisition of virulence gene clusters. Results of split decomposition analysis suggest that widespread recombination occurs among the three housekeeping genes and that recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1.


Asunto(s)
Filogenia , Ribotipificación , Vibrio cholerae O1/genética , Secuencia de Bases , ADN Polimerasa III/genética , ADN Bacteriano/genética , Genes Bacterianos , Variación Genética , Malato Deshidrogenasa/genética , Datos de Secuencia Molecular , Familia de Multigenes , Rec A Recombinasas/genética , Recombinación Genética , Análisis de Secuencia de ADN , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/patogenicidad , Virulencia
8.
Environ Microbiol ; 10(4): 866-73, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18201198

RESUMEN

In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1, aadA2, aadA5 and dfrA15, in the Class I integron and SXT, an integrative conjugative element containing dfr18, sulII and strAB, in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of intSXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18, sulII, strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.


Asunto(s)
Antibacterianos/farmacología , Vibriosis/microbiología , Vibrio cholerae no O1/efectos de los fármacos , Vibrio cholerae no O1/genética , Ampicilina/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana Múltiple/genética , Antagonistas del Ácido Fólico/farmacología , Furazolidona/farmacología , Humanos , India , Integrasas/genética , Integrones/genética , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Familia de Multigenes , Estreptomicina/farmacología , Trimetoprim/farmacología , Combinación Trimetoprim y Sulfametoxazol/farmacología , Vibrio cholerae no O1/aislamiento & purificación , Microbiología del Agua
9.
PLoS One ; 13(6): e0199338, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29897982

RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0051387.].

10.
J Med Microbiol ; 56(Pt 2): 260-265, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17244810

RESUMEN

Isolates of Vibrio cholerae O1 biotype El Tor serotype Inaba associated with an outbreak of cholera in Trivandrum, southern India, were characterized. PCR testing revealed that all five isolates examined carried the TCP pathogenicity island, the CTX genetic element and the RTX toxin, and produced cholera toxin (CT). RFLP analysis revealed that these Inaba isolates possessed a single copy of the CTX element flanked by two tandemly arranged copies of the RS element upstream of the core region. The isolates were resistant to ampicillin, nalidixic acid, trimethoprim, sulfamethoxazole, streptomycin and the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Ribotyping of these Inaba isolates revealed a hybridization profile similar to a strain of serotype Ogawa prevalent in southern India.


Asunto(s)
Antibacterianos/farmacología , Cólera/microbiología , Farmacorresistencia Bacteriana , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/genética , Cólera/epidemiología , Toxina del Cólera/genética , ADN Bacteriano/genética , Brotes de Enfermedades , Dosificación de Gen , Genes Bacterianos , Islas Genómicas/genética , Humanos , India/epidemiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Ribotipificación , Serotipificación , Vibrio cholerae O1/efectos de los fármacos , Vibrio cholerae O1/aislamiento & purificación
11.
FEMS Microbiol Lett ; 265(2): 208-14, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17081197

RESUMEN

In this study, we describe a septaplex PCR assay for rapid identification of Vibrio cholerae including detection of the virulence and intsxt genes. Conditions were optimized to amplify fragments of ISRrRNA (encoding for 16S-23S rRNA gene, Intergenic spacer regions), O1rfb (O1 serogroup specific rfb), O139rfb (O139 serogroup specific rfb), ctxA (cholera toxin subunit A), tcpA (toxin coregulated pilus), and intsxt (sxt integron) simultaneously in a single PCR. The septaplex PCR was evaluated using 211 strains of V. cholerae and six water samples for in situ testing. PCR results were correlated with genotype data obtained by individual PCR and slot-blot assays. The one-step PCR described here can be used to identify V. cholerae accurately and rapidly. Also, the virulence and intsxt genes can be simultaneously detected, providing a useful method for monitoring pathogenic, intsxt-positive and nonpathogenic, intsxt-negative V. cholerae serogroups both in the environment and clinical settings.


Asunto(s)
Integrones/genética , Juego de Reactivos para Diagnóstico/microbiología , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Factores de Virulencia/genética , Genes Virales , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Vibrio cholerae/aislamiento & purificación , Virulencia/genética , Factores de Virulencia/aislamiento & purificación
12.
J Leukoc Biol ; 97(4): 779-90, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25691383

RESUMEN

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4(+) T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM-100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4(+) T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4(+) T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4(+) T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients.


Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Cocaína/farmacología , VIH-1/efectos de los fármacos , Integración Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Núcleo Celular/metabolismo , Células Cultivadas , Cocaína/toxicidad , Trastornos Relacionados con Cocaína/complicaciones , ADN Viral/genética , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Células HEK293 , Infecciones por VIH/complicaciones , VIH-1/fisiología , Humanos , Técnicas In Vitro , Activación de Linfocitos/efectos de los fármacos , Fitohemaglutininas/farmacología , Provirus/efectos de los fármacos , Vesiculovirus/ultraestructura , Carga Viral , Virión/fisiología
13.
Microbiologyopen ; 4(1): 53-65, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25524808

RESUMEN

Porphyromonas gingivalis is a keystone periopathogen that plays an essential role in the progress of periodontitis. Like other gram-negative bacteria, the ability of P. gingivalis to produce outer membrane vesicles is a strategy used to interact with, and survive within its biological niches. Here we compared the protein components associated with vesicles derived from a fimbriated strain (33277) and an afimbriated strain (W83) of P. gingivalis using proteomic analyses. Some well-known virulence factors were identified in vesicles from both strains, such as gingipains and hemagglutinin. In contrast, FimC, FimD, and FimE, minor components of long fimbriae were found exclusively in 33277 vesicles, while proteins with a tetratricopeptide repeat (TPR) domain were unique to W83 vesicles. We found that significantly more 33277 than W83 vesicles were internalized into human oral keratinocytes and gingival fibroblasts. Interestingly, FimA, a well-known adhesin responsible for the attachment and invasion of P. gingivalis into host cells, was not essential for the invasive capabilities of P. gingivalis vesicles. Rather minor components of long fimbriae were required for an efficient invasive activity of vesicles. The most striking finding was that P. gingivalis strains lacking or having a reduced FimA expression showed a significant reduction in vesiculation. These results suggest that production and pathogenicity of P. gingivalis vesicles may largely depend on expression of the fim locus, and that the integration of vesicle production and pathogenicity with fimbrial expression may allow P. gingivalis to confer upon itself certain functional advantages.


Asunto(s)
Fimbrias Bacterianas/fisiología , Porphyromonas gingivalis/fisiología , Vesículas Transportadoras/metabolismo , Proteínas Fimbrias/biosíntesis , Proteínas Fimbrias/metabolismo , Proteínas Fimbrias/fisiología , Humanos , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidad , Transporte de Proteínas , Proteómica
14.
PLoS One ; 7(12): e51387, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23251514

RESUMEN

The main objective of this study was to examine effects of cocaine on HIV-1 replication in primary CD4+ T cells. Cocaine a commonly used drug among HIV-1 positive individuals serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS). Accumulating evidence suggest that cocaine increases HIV-1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. Intriguingly, there are no studies on cocaine-induced alterations in HIV-1 replication in primary CD4+ T cells that serve as the main targets for HIV-1 replication in vivo. In this report, we demonstrate cocaine-induced enhancement of HIV-1 replication in primary CD4+ T cells isolated from human PBMCs. To decipher a potential mechanism, we examined whether cocaine targets the innate antiviral immunity of CD4+ T cells mediated by cellular microRNAs (miRNAs). This is because recently a network of anti-HIV miRNAs in CD4+ T cells is highlighted to suppress viral replication. Our genome wide miRNA expression analysis indicated downregulation of several anti-HIV miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) in cocaine treated CD4+ T cells. However, our real-time quantitative PCR analysis revealed significant downregulation of miR-125b only. Our results illustrated that miR-125b knockdown enhances HIV-1 replication, whereas overexpression of miR-125b decreases HIV-1 replication in these cells. Therefore, we believe miR-125b is a key player for the cocaine induced enhancement of HIV-1 replication in CD4+ T cells. Since, miR-125b targets the 3' UTR regions of HIV-1 transcripts and inhibits viral protein translation, our data suggest modulation of post entry steps of HIV-1 by cocaine. Given that a plethora of studies suggest that cocaine regulates HIV entry, our results implicate a potentially novel mechanism by which cocaine can increase viral replication in CD4+ T cells.


Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Cocaína/farmacología , Regulación hacia Abajo/efectos de los fármacos , VIH-1/efectos de los fármacos , MicroARNs/metabolismo , Replicación Viral/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Técnicas de Silenciamiento del Gen , VIH-1/fisiología , Humanos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa
15.
Environ Microbiol Rep ; 3(6): 783-90, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23761370

RESUMEN

In this study, we report the presence of the El Tor CTXΦ and classical CTXΦ in Vibrio cholerae O1 strains isolated from Varanasi, India. Polymerase chain reaction, DNA sequencing and restriction fragment length polymorphism revealed that, although ctx-positive strains isolated after 1990 contain CTXΦ harbouring El Tor type of rstR and classical ctxB, strains isolated before 1990 contain El Tor type of rstR and El Tor ctxB. Two V. cholerae O1 strains (VC104 and VC106) represent an altered/hybrid strain containing the RS1 element followed by CTXΦ prophage harbouring El Tor type rstR and classical ctxB on the chromosome-I and RS2 element followed by second copy of CTXΦ prophage harbouring classical type rstR and classical ctxB on the chromosome-II. This is the first report of occurrence of El Tor CTXΦ harbouring classical ctxB and classical CTXΦ harbouring classical ctxB in chromosome-I and -II, respectively in diarrhoeal isolates of V. cholerae O1 El Tor strains from Varanasi, India, and that had been isolated in 1992.

16.
Infect Genet Evol ; 10(7): 925-30, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20621579

RESUMEN

We examined the effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains. We found that one strain of V. cholerae O139 VO146P showed loss of the complete phage array, and other strain VO170P showed partial loss of the phage array giving rise to altered strains designated as VO146N and VO170N. Results of PCR and RFLP analysis revealed that both strains (VO146P and VO170P) possessed a single copy of pre-CTX(ET)Phi and two copies of CTXPhi comprising CTX(Class)Phi and CTX(Calc)Phi arranged in tandem, and integrated in the large chromosome. The presence of classical ctxB was detected in CTX(Calc)Phi of both V. cholerae O139 strains. Nucleotide sequencing of three housekeeping genes showed no difference between parent and altered strains of V. cholerae O139.


Asunto(s)
Toxina del Cólera/genética , Profagos/genética , Cloruro de Sodio/farmacología , Vibrio cholerae O139/virología , Activación Viral/fisiología , ADN Viral/química , ADN Viral/aislamiento & purificación , Variación Genética , Genoma Bacteriano , Genoma Viral , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Preservación Biológica/métodos , Profagos/efectos de los fármacos , Profagos/fisiología , Factores de Tiempo , Vibrio cholerae O139/clasificación , Vibrio cholerae O139/efectos de los fármacos , Activación Viral/efectos de los fármacos
17.
Environ Microbiol Rep ; 2(1): 67-75, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23766000

RESUMEN

A multiplex PCR was developed to detect pre-CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre-CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre-CTXΦ and CTXΦ. None of the V. cholerae non-O1 and non-O139 that were tested had pre-CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre-CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre-CTX(ET) Φ and CTX(ET) Φ lineage with V. cholerae El Tor and pre-CTX(Class) Φ, pre-CTX(Calc) Φ, and CTX(Calc) Φ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi-locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre-CTXΦ and/or CTXΦ.

18.
Environ Microbiol Rep ; 2(1): 50-7, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23765998

RESUMEN

In this study, we report the presence of SXT in environmental Vibrio cholerae O1 El Tor strains isolated before 1992 from Varanasi, India. All isolates, except one, were resistant to Tm, and/or Sul, Sm, Fr, Na and Am. None contained plasmids. PCR and DNA sequencing revealed the presence of SXT containing dfrA1 and/or sulII, strAB in six isolates and dfr18, sulII and strAB in five isolates. Three clinical V. cholerae O1 isolated during 1992 contained the antibiotic resistance gene cassette aadA1 in the class 1 integron. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence of the transferable nature of SXT and associated antibiotic resistance genes, and its integration into the prfC site. Results of phylogenetic analysis of the intSXT gene of clonally similar V. cholerae showed a clear difference between dfr18(+) and dfrA1(+) V. cholerae O1 isolates. This is the first report of the occurrence of SXT harbouring sulII, strAB, dfr18 and/or dfrA1 genes in environmental V. cholerae O1 isolated prior to 1992 from Varanasi, India, and suggests emergence of SXT(+) antibiotic-resistant V. cholerae O139 and O1 from an environmental V. cholerae progenitor by acquisition of SXT and antibiotic-resistant gene clusters.

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