RESUMEN
Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage-associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane-bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag-specific T-cell responses. Our results demonstrated the ability of membrane-bound and secreted HSP70 to significantly enhance gag-specific T-cell responses and increase the breadth of T-cell responses to include subdominant epitopes. Membrane-bound or secreted HSP70 also significantly improved the multifunctionality of HIV-specific T cells and T-cell proliferation, which is important for maintaining T-cell integrity. Most importantly, the inclusion of membrane-bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.
Asunto(s)
Vacunas contra el SIDA/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Vacunas de ADN/inmunología , Animales , Células Dendríticas/fisiología , Femenino , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Humanos , Interferón gamma/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Linfocitos T/inmunología , Vacunación , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
BACKGROUND: An internal evaluation of the inpatient pharmacy order entry database (WORx) at a university hospital revealed that the nature of the reaction was documented for only 47% of patients with reported drug allergies/intolerance. Insufficient documentation of drug allergy/intolerance may result in administration of drugs that should not be prescribed. Similarly, valuable agents that should be used may not be prescribed due to an unnecessary fear of adverse drug reaction. More complete description of drug allergy/intolerance may result in more correct prescribing of medications. OBJECTIVE: Evaluate the impact of a pharmacist-driven protocol on the quality of drug allergy/intolerance documentation. METHODS: Four pre-intervention evaluations were conducted every 2 weeks documenting the completeness of drug allergy/intolerance information in the pharmacy order entry database. One week following the implementation of a pharmacist-driven protocol intended to improve the completeness of drug allergy/intolerance information, a series of 4 postintervention evaluations was repeated. Proportional analysis of pre- and postinterventional data was performed to evaluate the effectiveness of the intervention. RESULTS: A total of 1,686 allergies from 2,174 patients were reviewed pre and post intervention. The frequency of complete drug allergy/intolerance documentation pre intervention was 52% to 62%. Following implementation of the hospitalwide, pharmacist-driven protocol, this rate increased to 60% to 76%. Pediatric services demonstrated the most substantial improvement, increasing from 53% to 79% to 67% to 93%. Blank reaction fields decreased by 10% in both age groups. CONCLUSION: A pharmacy-driven initiative intended to improve the completeness of drug allergy/intolerance documentation was associated with modest success. Other mechanisms, including electronic health record systems with computerized physician order entry and decision support, are needed to improve the completeness of drug allergy/intolerance information.
RESUMEN
A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (αGalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA(0)) cleavage loop as a vaccine. Peptides designed across the HA(0) of influenza A/H3N2 viruses, delivered to mice via the intranasal route with αGalCer as an adjuvant, provided 100â% protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA(0) of influenza A/H5N1 with αGalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20â% of mice vaccinated with αGalCer-adjuvanted peptides spanning the HA(0) of H5N1 survived homologous viral challenge, possibly because the HA(0) of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA(0) peptides adjuvanted with αGalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Peso Corporal , Protección Cruzada , Femenino , Galactosilceramidas/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Histocitoquímica , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/administración & dosificación , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Microscopía , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/prevención & control , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Carga ViralRESUMEN
Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.
Asunto(s)
Muerte Celular , Virus del Dengue/patogenicidad , FN-kappa B/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Células Cultivadas , Virus del Dengue/inmunología , Células Epiteliales/inmunología , Células Epiteliales/virología , Humanos , Macrófagos/inmunología , Macrófagos/virología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
After fusion of the human immunodeficiency virus type 1 (HIV-1) envelope with the host cell membrane, the HIV-1 core enters the cell cytoplasm. Core components are then restructured to form the reverse transcription complex (RTC); the biochemical details of this process are currently unclear. To investigate early RTC formation, we characterized the endogenous reverse transcription activity of virions, which was less efficient than reverse transcription during cell infection and suggested a requirement for a cell factor. The addition of detergent to virions released reverse transcriptase and capsid, and reverse transcription products became susceptible to the action of exogenous nucleases, indicating virion disruption. Disruption was coincident with the loss of the endogenous reverse transcription activity of virions, particularly late reverse transcription products. Consistent with this observation, the use of a modified "spin thru" method, which uses brief detergent exposure, also disrupted virions. The addition of lysates made from mammalian cell lines (Jurkat, HEK293T, and NIH 3T3 cells) to virions delipidated by detergent stimulated late reverse transcription efficiency. A complex with reverse transcription activity that was slower sedimenting than virions on a velocity gradient was greatly stimulated to generate full-length reverse transcription products and was associated with only relatively small amounts of capsid. These experiments suggest that cell factors are required for efficient reverse transcription of HIV-1.
Asunto(s)
Extractos Celulares , ADN Viral/biosíntesis , VIH-1/fisiología , Transcripción Reversa/fisiología , ADN Viral/análisis , Detergentes , HumanosRESUMEN
Low-level drug resistance is not detected by routine consensus sequence genotype analysis (CSA) but low levels of specific mutations, such as the non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutation K103N, can be quantitated by allele-specific PCR (ASP). This study has applied an ASP to quantitate low-level K103N in patients presenting for clinical HIV genotyping and assess the correlation with antiretroviral treatment history and outcomes. HIV RNA was extracted from patient plasma and subjected to PCR amplification of the reverse transcriptase (RT) region followed by genotyping by CSA and real-time ASP for K103N. When applied to samples from patients presenting for genotyping, the ASP detects K103N, not K103 nor K103R, but cross-reacts with K103S. ASP identified all samples that were K103N by CSA (10.5%) and an additional 14% by ASP only, representing patients who were therapy naïve and with NNRTI treatment history. ASP detected therapy-acquired K103N at low levels up to 6 years after cessation of NNRTI therapy. In three patients with new HIV diagnosis and K103N detected by ASP only, K103N virus declined rapidly from the circulation but persisted in PBMC DNA at >12 months post-diagnosis. Efavirenz (EFV) combination therapy in three patients with low-level K103N suppressed successfully viral load, although one patient developed failure and CSA-detectable K103N after 15 months of therapy. Thus, analysis of K103N by ASP in conjunction with CSA genotyping provides additional information that reflects K103N transmission and persistence but detection of low-level K103N does not preclude successful EFV-containing combination therapy.
Asunto(s)
Alelos , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , VIH/genética , Mutación Missense , Reacción en Cadena de la Polimerasa , Alquinos , Benzoxazinas/uso terapéutico , Ciclopropanos , Farmacorresistencia Viral/genética , Femenino , Variación Genética , VIH/efectos de los fármacos , Infecciones por VIH/sangre , Humanos , Masculino , Técnicas de Diagnóstico Molecular , ARN Viral/sangre , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND: HIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells. RESULTS: Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p66(8.44) and p51(8.31)). The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs), and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p66(8.53) and p51(8.60)), suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p66(8.44) and p51(8.31) differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay. CONCLUSION: The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.
Asunto(s)
VIH-1/química , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/metabolismo , Virión/química , Extractos Celulares/química , Línea Celular , Electroforesis en Gel Bidimensional , Humanos , Punto Isoeléctrico , Fosforilación , Isoformas de Proteínas/análisis , Procesamiento Proteico-Postraduccional , Proteoma/análisisRESUMEN
Water column bulk Pseudo-nitzschia abundance and the dissolved and particulate domoic acid (DA) concentrations were measured in the Santa Barbara Basin (SBB), California from 2009â»2013 and compared to bulk Pseudo-nitzschia cell abundance and DA concentrations and fluxes in sediment traps moored at 147 m and 509 m. Pseudo-nitzschia abundance throughout the study period was spatially and temporally heterogeneous (<200 cells L-1 to 3.8 × 106 cells L-1, avg. 2 × 105 ± 5 × 105 cells L-1) and did not correspond with upwelling conditions or the total DA (tDA) concentration, which was also spatially and temporally diverse (<1.3 ng L-1 to 2.2 × 105 ng L-1, avg. 7.8 × 10³ ± 2.2 × 104 ng L-1). We hypothesize that the toxicity is likely driven in part by specific Pseudo-nitzschia species as well as bloom stage. Dissolved (dDA) and particulate (pDA) DA were significantly and positively correlated (p < 0.01) and both comprised major components of the total DA pool (pDA = 57 ± 35%, and dDA = 42 ± 35%) with substantial water column concentrations (>1000 cells L-1 and tDA = 200 ng L-1) measured as deep as 150 m. Our results highlight that dDA should not be ignored when examining bloom toxicity. Although water column abundance and pDA concentrations were poorly correlated with sediment trap Pseudo-nitzschia abundance and fluxes, DA toxicity is likely associated with senescent blooms that rapidly sink to the seafloor, adding another potential source of DA to benthic organisms.
Asunto(s)
Diatomeas , Ácido Kaínico/análogos & derivados , Toxinas Marinas/análisis , Contaminantes del Agua/análisis , California , Monitoreo del Ambiente , Sedimentos Geológicos , Ácido Kaínico/análisis , Agua de Mar/análisis , Agua de Mar/microbiología , Factores de TiempoRESUMEN
The establishment of reservoirs of latently infected cells is thought to contribute to the persistence of HIV-1 infection in the host. Studies so far have mainly focused on the long-lived reservoir of HIV-infected resting CD4+ T cells. A discrete population of HIV-infected CD4-/CD8- double negative (DN) T cells has recently been shown to exist and may also play a role in HIV-1 persistence. DN T cells are CD3 positive, either TCRalphabeta or TCRgammadelta positive, but lack both CD4 and CD8 surface markers. We developed a novel, magnetic bead column-based cell fractionation procedure for isolating >99% pure DN T cells. CD4+, CD8+, and DN T cells were purified from 23 samples of a cohort of 18 HIV-1-infected patients. Each cell fraction was analyzed for levels of total and integrated HIV-1 DNA. A correlation was observed between the presence of HIV-1 DNA in the DN T cell fraction and plasma viral load (VL). Using a micrococulture technique, we saw an initial release of virus from DN T cells of a patient with high VL. Analysis of env and nef sequence data suggested that the HIV-1 present in CD4+ and DN T cells originated from a common infecting strain. Different from the published literature, we have demonstrated the presence of HIV-1 DNA in DN T cells only in patients who are experiencing HAART failure. While these cells may have a limited role in viral persistence in high VL patients, our results suggest DN T cells are unlikely to be a major reservoir in patients on HAART with clinically undetectable plasma viral RNA.
Asunto(s)
Antígenos CD4/inmunología , Antígenos CD8/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Linfocitos T/virología , Adulto , Fármacos Anti-VIH/farmacología , Terapia Antirretroviral Altamente Activa , Infecciones por VIH/genética , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/análisis , Linfocitos T/metabolismo , Carga ViralRESUMEN
BACKGROUND: Current research suggests that human immunodeficiency virus type-1 (HIV-1) virion infectivity factor (Vif) acts during viral assembly in producer cells to ensure infectivity in target cells but the exact mechanism of action has not been defined. Vif interacts with Gag, viral protease and RNA and these interactions are proposed to be important for correct particle assembly and stability of the reverse transcription complex. OBJECTIVES: The existence of cells that are either permissive or non-permissive for replication of Vif deficient viruses suggests the involvement of host cellular factors in its function. Current research suggests an association of Vif with the intermediate filament protein, vimentin, and the tyrosine kinase, Hck, but the significance of these associations remains to be defined. More recently HP68, a cellular ATP binding protein, has been shown to be important for capsid formation and an interaction between Vif and HP68 has been shown. Our aim was to further identify host cellular factors involved in Vif function. STUDY DESIGN: We have employed the yeast 2-hybrid system to identify cellular proteins which interact with HIV-1 Vif. Sixteen clones were isolated from a high stringency yeast-2-hybrid screen of a human leucocyte cDNA library with Vif derived from the T-cell tropic HIV-1 strain NL4.3. Of these, 8 clones were confirmed as specifically binding Vif, fully sequenced and identified via GenBank homology searches. RESULTS: Thus far 3 of these clones, spermine/spermidine N1-acetyltransferase, Triad 3 and a novel gene which we have termed 'novel Vif binding protein', have been characterised and represent attractive candidates for mediating Vif action during HIV replication. CONCLUSIONS: Through identification and characterisation of cellular factors interacting with HIV-1 Vif we hope to unravel the mechanism of action of Vif which may ultimately aid therapeutic design.
Asunto(s)
Productos del Gen vif/fisiología , Infecciones por VIH/virología , VIH-1/fisiología , Productos del Gen vif/genética , Infecciones por VIH/etiología , VIH-1/genética , Humanos , Mutación , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-hck , ARN Viral/metabolismo , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Vimentina/metabolismo , Proteínas Virales/metabolismo , Ensamble de Virus , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia HumanaRESUMEN
Integration of HIV-1 DNA is essential both for productive viral replication and for viral persistence in patients. Methods to measure specifically proviral HIV DNA are required for investigating the mechanisms of HIV integration, for screening novel integrase inhibitors in cell culture and for monitoring levels of persistent integrated viral DNA in patients. In this report, the linker primer polymerase chain reaction (LP-PCR) and Alu-PCR methods for the quantitation of integrated HIV-1 DNA have been modified and evaluated. Each of the two modified assays allowed the quantitative detection of 4 copies of integrated HIV DNA in presence of 2 x 10(5) cell-equivalents of human chromosomal DNA. The results show that proper DNA isolation procedures and the inclusion of appropriate controls in these assays are important for the accurate quantitation of integrated HIV DNA. With further improvements, it should be possible to use these methods as diagnostic tools to monitor closely the efficacy of antiretroviral therapy.
Asunto(s)
ADN Viral/aislamiento & purificación , VIH-1/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Línea Celular , Cartilla de ADN , ADN Viral/genética , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Células Tumorales Cultivadas , Integración Viral , Replicación ViralRESUMEN
Traditional vaccine strategies that induce antibody responses have failed to protect against HIV infection in clinical trials, and thus cell-mediated immunity is now an additional criterion. Recent clinical trials that aimed to induce strong T cell responses failed to do so. Therefore, to enhance induction of protective T cell responses, it is crucial that the optimum antigen combination is chosen. Limited research has been performed into the number of antigens selected for an HIV vaccine. This study aimed to compare DNA vaccines encoding either a single HIV antigen or a combination of two antigens, using intradermal vaccination of C57BL/6 mice. Immune assays were performed on splenocytes, and in vivo protection was examined by challenge with a chimeric virus, EcoHIV, able to infect mouse but not human leukocytes, at 10 days (short term) and 60 days (long term) post final vaccination. At 60 days there was significantly lower frequency of induced antigen-specific CD8(+) T cells in the spleens of pCMVgag-pol-vaccinated mice compared with mice which received pCMVgag only. Most importantly, short term viral control of EcoHIV was similar for pCMVgag and pCMVgag-pol-vaccinated mice at day 10, but only the pCMVgag-vaccinated significantly controlled EcoHIV at day 60 compared with pCMV-vaccinated mice, showing that control was reduced with the inclusion of the HIV pol gene.
Asunto(s)
Vacunas contra el SIDA/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/prevención & control , Vacunas/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/genética , Animales , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Femenino , Antígenos VIH/genética , Infecciones por VIH/inmunología , Ratones Endogámicos C57BL , Bazo/inmunología , Vacunas/administración & dosificación , Vacunas/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Four IgG(1kappa) monoclonal antibodies (mAbs) against Influenza A/Chicken/Vietnam/8/2004 (H5N1) virus are described. Three of these showed neutralizing activities against H5N1 strains from clades 1, 2 and 3 using a retroviral pseudotype or live virus microneutralization assay. In the pseudotype assay, the IC(90) neutralizing titre range was >1600-51,200, and with the microneutralization was 80> or =10,240. MAb 1C1 showed strong neutralizing activities in both assays. All four mAbs reacted specifically to the immunogen by immunohistochemical staining and to A/Hong Kong/483/1997 (H5N1) and A/Thailand/1(KAN-1)/2004 (H5N1)-infected MDCK cells by immunofluorescence. ELISA titrations of the mAbs showed specificity for H5N1 haemagglutinin (HA) and no cross-reactivity to 15 other Influenza A subtypes. Only mAbs 1C1 and the non-neutralizing 1F7 reacted with HA(1), the cleaved subunit of HA, by Western blot. These results suggest that the mAbs recognize distinct or overlapping epitopes and will be useful reagents for construction of specific rapid point-of-care assays or for therapeutic use.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Pollos , Reacciones Cruzadas , Hemaglutininas Virales/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Concentración 50 Inhibidora , Pruebas de NeutralizaciónRESUMEN
Virion infectivity factor (Vif) facilitates HIV infection by counteracting APOBEC3G late in replication in virus-producer cells. Here, we show that early after infection of new target cells Vif is part of the HIV reverse transcription machinery and acts as an accessory factor for reverse transcription. Vif protein was present in gradient fractions containing reverse transcription complexes (RTCs), and anti-Vif antibody immunoprecipitated HIV reverse transcription products from these gradient fractions. To investigate a role for Vif in RTCs independent of APOBEC3G, we created an intracellular environment that would restrict reverse transcription by pre-treating permissive target cells with 5-Fluoro 2-deoxyuridine, a thymidylate synthetase inhibitor, prior to infection with virus from permissive cells. Infectivity assays and quantitation of reverse transcription products demonstrated that replication of HIV lacking Vif was inhibited to a greater degree than wild type, without concurrent mutation of reverse transcription products, suggesting compromised reverse transcription in the absence of Vif.
Asunto(s)
VIH-1/fisiología , VIH-1/patogenicidad , Transcripción Reversa , Replicación Viral , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Centrifugación por Gradiente de Densidad , Floxuridina/farmacología , VIH-1/genética , Células HeLa , Humanos , Inmunoprecipitación , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
BACKGROUND: The 2005 southern hemisphere formulation of the inactivated split-virion influenza vaccine Vaxigrip unintentionally contained a lower concentration of haemagglutinin (HA) than European Pharmacopoeia (EP) and WHO specifications for one of the three strains. OBJECTIVES: To evaluate the immunogenicity of the 2005 southern hemisphere formulation of an influenza vaccine containing 9 microg/dose of HA for A/Wellington/1/2004(H3N2) strain, and 15 microg/dose for each of the A/New Caledonia/20/99(H1N1) strain and B/Shanghai/361/2002-like strains. PATIENTS/METHODS: In an open, non-controlled multicentre clinical trial, 75 healthy adults (18-59 years) and 65 healthy older adults (> or =60 years) were vaccinated once. Serum samples were obtained on D0 and 21 for haemagglutination inhibition (HAI) antibody titration. RESULTS: A high proportion of adults (64%) and elderly (68%) were already seroprotected (HAI titre of > or =40) against A/Wellington/1/2004(H3N2) before vaccination, probably due to high circulation of an antigenically similar H3N2 strain and a high 2004 vaccination rate. By D21, seroprotection rates against H3N2 attained 93.8% and 96.0% in adults and elderly respectively. The other two immunogenicity criteria for annual licensure of influenza vaccines in Europe were also met in both age groups for the H3N2 strain, and also for the H1N1 and B strains. CONCLUSIONS: These results enabled the 2005 southern hemisphere vaccine to be used in expectation that it would provide satisfactory protection against influenza, despite the reduced H3N2 antigen content.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Aprobación de Drogas , Femenino , Pruebas de Inhibición de Hemaglutinación , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Masculino , Persona de Mediana Edad , Vacunas de Productos Inactivados/inmunología , Vacunas de Subunidad/inmunología , Adulto JovenRESUMEN
Tumor necrosis factor alpha (TNF-alpha) is believed to play a significant role in the pathogenesis of dengue virus (DV) infection, with elevated levels of TNF-alpha in the sera of DV-infected patients paralleling the severity of disease and TNF-alpha release being coincident with the peak of DV production from infected monocyte-derived macrophages (MDM) in vitro. Since macrophages are a primary cell target in vivo for DV infection, we investigated the potential antiviral role of TNF-alpha in regulating DV replication in MDM. While pretreatment of MDM with TNF-alpha had a minor inhibitory effect, addition of TNF-alpha to MDM with established DV infection had no effect on DV replication as measured by DV RNA levels or progeny virus production. Blocking endogenous TNF-alpha using short interfering RNA or inhibitory TNF-alpha antibodies also had no effect on infectious DV production or viral RNA synthesis. Together, these results demonstrate that DV replication in MDM is not affected by TNF-alpha. Additionally, normal cellular TNF-alpha signaling, measured by quantitation of TNF-alpha-induced stimulation of transcription from an NF-kappaB-responsive reporter plasmid or NF-kappaB protein nuclear translocation, was blocked in DV-infected MDM and Huh7 cells. Thus, DV replication in MDM is not affected by TNF-alpha, and infected cells do not respond normally to TNF-alpha stimulation. It is therefore unlikely that the increased production of TNF-alpha seen in DV infection directly effects DV clearance by reducing DV replication, and the ability of DV to alter TNF-alpha responsiveness highlights another example of viral subversion of cellular functions.
Asunto(s)
Núcleo Celular/metabolismo , Virus del Dengue/metabolismo , Dengue/metabolismo , Macrófagos/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/inmunología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Núcleo Celular/inmunología , Núcleo Celular/patología , Núcleo Celular/virología , Chlorocebus aethiops , Dengue/inmunología , Dengue/patología , Virus del Dengue/inmunología , Humanos , Macrófagos/inmunología , Macrófagos/patología , Macrófagos/virología , Ratones , FN-kappa B/inmunología , ARN Interferente Pequeño/farmacología , ARN Viral/biosíntesis , ARN Viral/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
This report describes a 43 yr old man diagnosed with U.K. acquired cyclospora cayetanensis infection resulting in fever and diarrhoea. In course of the febrile illness, he suffered an out of hospital cardiac arrest. Extensive cardiac investigation including a transthoracic echocardiogram, coronary angiogram, and cardiac electrophysiological studies failed to identify the cause. The possible links between cyclospora infection and cardiac arrest are explored. Fever has been reported as a precipitant for idiopathic ventricular fibrillation in patients with the Brugada syndrome but also rarely in individuals with normal hearts. Clinicians should be aware of a possible link between any febrile illness and potentially fatal ventricular dysrhythmia.
Asunto(s)
Cyclospora/aislamiento & purificación , Ciclosporiasis/complicaciones , Fiebre/etiología , Paro Cardíaco/etiología , Adulto , Animales , Diarrea/etiología , Humanos , Masculino , Reino UnidoRESUMEN
Astrocytes persistently infected with HIV-1 can transmit virus to CD4+ cells, suggesting that astrocytes may be a source of viral persistence and dissemination in the brain. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. HIV-1 was observed in vesicle-like structures. Unspliced genomic RNA and extrachromosomal HIV-1 DNA were detected in astrocytes, with levels declining over time. The extrachromosomal viral DNA was not de novo reverse transcribed in astrocytes but most likely the products of intravirion reverse transcription present in the virus inoculum. Integrated HIV-1 DNA was not detected in assays sensitive to detect 2 integrated copies of provirus. However, the majority of astrocyte cultures released infectious virus that could be transmitted to CD4+ cells. Our findings suggest a novel pathway of HIV-1 uptake and release in astrocytes that does not necessarily require virus replication, which may contribute to persistence and spread of HIV-1 in the brain.
Asunto(s)
Astrocitos/virología , VIH-1/fisiología , Astrocitos/química , Linfocitos T CD4-Positivos/virología , Vesículas Citoplasmáticas/virología , ADN Viral/análisis , Proteína p24 del Núcleo del VIH/análisis , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Biológicos , ARN Viral/análisis , Factores de Tiempo , Integración ViralRESUMEN
Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.
Asunto(s)
ADN Circular/análisis , ADN Viral/análisis , Infecciones por Hepadnaviridae/virología , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B del Pato/fisiología , Hepatitis Viral Animal/virología , Glándulas Suprarrenales/virología , Animales , ADN Circular/aislamiento & purificación , ADN Viral/aislamiento & purificación , Patos , Genoma Viral , Corazón/virología , Riñón/virología , Leucocitos Mononucleares/virología , Hígado/virología , Reacción en Cadena de la Polimerasa , Bazo/virologíaRESUMEN
PURPOSE: Thrombin injection is becoming well established for the percutaneous management of iatrogenic pseudoaneurysms. All the published series to date use bovine thrombin, and there have been reports of adverse immunologic effects following its use. Our study aimed to assess the efficacy of human thrombin injection for pseudoaneurysm occlusion. METHODS: Fourteen patients with iatrogenic pseudoaneurysms underwent a color Doppler ultrasound examination to assess their suitability for percutaneous human thrombin injection. Human thrombin 1000 IU was then injected into the pseudoaneurysm sac under sterile conditions and with ultrasound guidance. A further color Doppler ultrasound examination was performed 24 hr later to confirm occlusion. RESULTS: All 14 pseudoaneurysms were successfully occluded by human thrombin injection. In two cases a second injection of thrombin was required, but there were no other complications, and all pseudoaneurysms remained occluded at 24 hr. CONCLUSION: Ultrasound-guided human thrombin injection is simple to perform, effective and safe. We recommend that human thrombin becomes the agent of choice for percutaneous injection into iatrogenic pseudoaneurysms.