RESUMEN
Clopidogrel and aspirin are the most commonly used medications worldwide for dual antiplatelet therapy after percutaneous coronary intervention. However, clopidogrel hyporesponsiveness related to gene polymorphisms is a concern. Populations with higher degrees of genetic admixture may have increased prevalence of clopidogrel hyporesponsiveness. To assess this, we genotyped CYP2C19, ABCB1, and PON1 in 187 patients who underwent percutaneous coronary intervention. Race was self-defined by patients. We also performed light transmission aggregometry with adenosine diphosphate (ADP) and arachidonic acid during dual antiplatelet therapy. We found a significant difference for presence of the CYP2C19*2 polymorphism between white and non-white patients. Although 7% of patients had platelet resistance to clopidogrel, this did not correlate with any of the tested genetic polymorphisms. We did not find platelet resistance to aspirin in this cohort. Multivariate analysis showed that patients with PON1 and CYP2C19 polymorphisms had higher light transmission after ADP aggregometry than patients with native alleles. There was no preponderance of any race in patients with higher light transmission aggregometry. In brief, PON1 and CYP2C19 polymorphisms were associated with lower clopidogrel responsiveness in this sample. Despite differences in CYP2C19 polymorphisms across white and non-white patients, genetic admixture by itself was not able to identify clopidogrel hyporesponsiveness.
Asunto(s)
Aspirina/farmacología , Plaquetas/efectos de los fármacos , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/farmacología , Ticlopidina/análogos & derivados , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Alelos , Arildialquilfosfatasa/genética , Clopidogrel , Enfermedad de la Arteria Coronaria/genética , Citocromo P-450 CYP2C19/genética , Quimioterapia Combinada , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea , Polimorfismo Genético , Estudios Prospectivos , Ticlopidina/farmacologíaRESUMEN
In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
Asunto(s)
Animales , Masculino , Ratones , Linfocitos B/inmunología , Proteínas de Choque Térmico/inmunología , Inmunomodulación/genética , /genética , ARN Mensajero/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos B/metabolismo , Citometría de Flujo , Expresión Génica/genética , Proteínas de Choque Térmico/uso terapéutico , Memoria Inmunológica/fisiología , Inmunofenotipificación/clasificación , Mediadores de Inflamación/análisis , Interferón gamma/análisis , /inmunología , /análisis , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/genética , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/clasificación , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéuticoRESUMEN
In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
Asunto(s)
Linfocitos B/inmunología , Proteínas de Choque Térmico/inmunología , Inmunomodulación/genética , Interleucina-10/genética , ARN Mensajero/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Linfocitos B/metabolismo , Citometría de Flujo , Expresión Génica/genética , Proteínas de Choque Térmico/uso terapéutico , Memoria Inmunológica/fisiología , Inmunofenotipificación/clasificación , Mediadores de Inflamación/análisis , Interferón gamma/análisis , Interleucina-10/inmunología , Interleucina-12/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/citología , Bazo/inmunología , Subgrupos de Linfocitos T/clasificación , Vacunas de ADN/inmunología , Vacunas de ADN/uso terapéuticoRESUMEN
Autologous hematopoietic SCT (AHSCT) has been investigated in the past as a therapeutic alternative for multiple sclerosis (MS). Despite advances in clinical management, knowledge about mechanisms involved with clinical remission post transplantation is still limited. Abnormal microRNA and gene expression patterns were described in MS and have been suggested as disease biomarkers and potential therapeutic targets. Here we assessed T- and B-cell reconstitution, microRNAs and immunoregulatory gene expression after AHSCT. Early immune reconstitution was mainly driven by peripheral homeostatic proliferation. AHSCT increased CD4(+)CD25(hi)FoxP3(+) regulatory T-cell counts and expression of CTLA-4 and GITR (glucocorticoid-induced TNFR) on CD4(+)CD25(hi) T cells. We found transient increase in exhausted PD-1(+) T cells and of suppressive CD8(+)CD28(-)CD57(+) T cells. At baseline, CD4(+) and CD8(+) T cells from MS patients presented upregulated miR-16, miR-155 and miR-142-3p and downregulated FOXP3, FOXO1, PDCD1 and IRF2BP2. After transplantation, the expression of FOXP3, FOXO1, PDCD1 and IRF2BP2 increased, reaching control levels at 2 years. Expression of miR-16, miR-155 and miR-142-3p decreased towards normal levels at 6 months post therapy, remaining downregulated until the end of follow-up. These data strongly suggest that AHSCT normalizes microRNA and gene expression, thereby improving the immunoregulatory network. These mechanisms may be important for disease control in the early periods after AHSCT.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , MicroARNs/biosíntesis , Esclerosis Múltiple/genética , Esclerosis Múltiple/terapia , Acondicionamiento Pretrasplante/métodos , Adulto , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Esclerosis Múltiple/metabolismo , Trasplante Autólogo , Adulto JovenRESUMEN
The low number of hematopoietic stem cells (HSC) in umbilical cord blood (UCB) is directly related to increased risk of transplant failure. Effective ex vivo expansion of HSC has been tried for many years, with conflicting results because of the inability to reproduce in vitro HSC proliferation in the same way it occurs in vivo. We compared freshly isolated HSC with their expanded counterparts by microarray analysis and detected activation of the noncanonical Wnt (wingless-type MMTV integration site family) pathway. Study of early alterations during ex vivo UCB-HSC expansion could contribute to improvement of ex vivo expansion systems.
Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Vía de Señalización Wnt/genética , ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD34/metabolismo , Calibración , Recuento de Células , Proliferación Celular , Humanos , Reproducibilidad de los ResultadosRESUMEN
In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.
Asunto(s)
Animales , Masculino , Ratones , Células Presentadoras de Antígenos/inmunología , Proteínas Bacterianas/administración & dosificación , /administración & dosificación , Mycobacterium tuberculosis/inmunología , ARN Mensajero/inmunología , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis/inmunología , Proteínas Bacterianas/efectos adversos , Proteínas Bacterianas/inmunología , /efectos adversos , /inmunología , Ratones Endogámicos BALB C , ARN Mensajero/efectos adversos , Bazo/inmunología , Transfección , Vacunas contra la Tuberculosis/efectos adversos , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & controlRESUMEN
In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.