RESUMEN
Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 µg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium bovis/inmunología , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Bovina/prevención & control , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Bovinos , Clonación Molecular , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Inmunogenicidad Vacunal , Interferón gamma/biosíntesis , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Bovina/inmunología , Tuberculosis Bovina/microbiología , Vacunación/métodosRESUMEN
Bovine tuberculosis (bTB) is usually diagnosed in vivo and ex vivo on the basis of delayed hypersensitivity reactions with a complex pool of antigens named bovine tuberculin (PPDB). The IFN-γ release assay (IGRA) for bTB is a blood-based assay that improves detection of infected cattle at early stages that escape skin testing. Improvements to IFN-γ testing with specific proteins have been performed to increase sensitivity. DosR regulon-related antigens are well known mycobacterial proteins expressed during the non-replicative phases of infection, this has been useful to improve the diagnosis of subclinical forms of TB in suspected individuals. Transcripts of DosR genes mb2054c, mb2057c, and mb2660c have been identified by our group in lymph nodes of IFN-γ test negative cattle. This led us to hypothesize that DosR-related proteins may potentiate the IFN-γ response to PPDB in animals with a false negative IFN-γ test, making evident subclinical infection. Three hundred animals were evaluated by means of IGRA and post-mortem microbiological analysis of tissue samples to validate M. bovis infection. We found that 176 out of 300 animals showed an overall increased OD in complemented IGRA with two purified protein cocktails in comparison to PPDB alone, and were scrutinized for a subclinical infection; thirty percent when PPDB was supplemented with a cocktail of four DosR antigens, and 70% when PPDB was supplemented with a cocktail of six antigens (four DosR and two RD1 antigens). Forty five animals showed a substantial IFN-γ overproduction but remained negative, and 40 animals changed the result to a positive test. Only 18 out of 176 IFN-γ high producing animals were also positive to M. bovis isolation. Fifty seven animals with no visible lesions at slaughter and with a negative IGRA test result contained M. bovis DNA in tissue samples. In conclusion, Mb1762c, Mb2054c, Mb2057c, and Mb2660c have the potential to increase sensitivity of the IFN-γ in vitro test for bTB diagnosis when supplemented to PPDB.
Asunto(s)
Infecciones Asintomáticas , Ensayos de Liberación de Interferón gamma/métodos , Ensayos de Liberación de Interferón gamma/veterinaria , Mycobacterium bovis/inmunología , Tuberculina/inmunología , Tuberculosis Bovina/diagnóstico , Tuberculosis Bovina/inmunología , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Bovinos , ADN Bacteriano , Diagnóstico Diferencial , Interferón gamma/sangre , Interferón gamma/metabolismo , Ganglios Linfáticos/inmunología , México , Mycobacterium bovis/genética , Mycobacterium bovis/aislamiento & purificación , Proteínas Recombinantes , Regulón , Sensibilidad y Especificidad , Tuberculosis Bovina/microbiologíaRESUMEN
Several studies have demonstrated that the serum of patients with cancer contains antibodies that react with a group of autoantigens denominated tumor-associated antigens (TAA). TAA can be detected prior to clinical diagnosis; thus, they would be ideal biomarkers for early detection of cancer, using only a few microliters of a patient's serum. In the current study, we used an immune proteomic approach, combining two-dimensional (2D) electrophoresis, Western blot, and matrix-associated laser desorption/ionization-mass spectrometry (MALDI-MS) methods to identify TAA in the sera of patients diagnosed with breast cancer. Sera were obtained from 36 newly diagnosed patients with stage II breast cancer and those from 36 healthy volunteers were evaluated for the presence of the TAA. Alpha 2HS-glycoprotein (AHSG) antibodies were detected in 33 of 36 patients with breast cancer (91.7%) and in only 3 of 36 healthy patients (controls, 8.3%). Sensitivity of detection of autoantibodies against AHSG in patients with breast cancer was 91.7%. AHSG was detected in cancer tissue by immunohistochemistry. Our results strongly suggest that the presence of serum autoantibodies against AHSG protein may be useful as serum biomarkers for early-stage breast cancer screening and minimally invasive diagnosis in Mexican populations. BIOLOGICAL SIGNIFICANCE: In the present study, 2D immunoblot analysis was used to make a screening in samples of sera from patients with a diagnosis of early-stage breast cancer, in order to identify some autoantibodies that react against TAA. Proteins identified in the present study, particularly alpha 2HS-glycoprotein (AHSG), might be useful as potential biomarkers for breast cancer in early stages for Mexican populations.
Asunto(s)
Anticuerpos Antineoplásicos/sangre , Antígenos de Neoplasias/sangre , Autoanticuerpos/sangre , Neoplasias de la Mama/sangre , alfa-2-Glicoproteína-HS/metabolismo , Biomarcadores de Tumor/sangre , Femenino , Humanos , México , Estadificación de Neoplasias , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Host control of mycobacterial infection, in both human and mouse models, has been shown to be associated with the production of interferon (IFN)-gamma by CD4(+) T cells. Interleukin (IL)-12 is known to be a crucial cytokine in the differentiation of IFN-gamma-producing T helper 1 (Th1) cells. To determine whether continuous administration of IL-12 expressed in transgenic tomato (TT-IL-12) has therapeutic efficacy in a murine model of pulmonary tuberculosis, BALB/c mice were infected with either Mycobacterium tuberculosis H37Rv strain or a multi-drug-resistant clinical isolate (MDR) and treated with a daily oral dose of TT-IL12 crude fruit extracts. For the early H37Rv infection, TT-IL-12 administration was started 1 day before infection and continued for 60 days. In the H37Rv or MDR late infection, treatment was started 60 days after infection and continued for another 60 days. In both phases of infection, TT-IL-12 administration resulted in a reduction of bacterial loads and tissue damage compared with wild-type tomato (non-TT). The Th1 response was increased and the Th2 response was reduced. In the late infection, a long-term treatment with TT-IL-12 was necessary. We demonstrate that TT-IL-12 increases resistance to infection and reduces lung tissue damage during early and late drug-sensitive and drug-resistant mycobacterial infection.
Asunto(s)
Inmunoterapia/métodos , Interleucina-12/biosíntesis , Extractos Vegetales/administración & dosificación , Solanum lycopersicum/genética , Tuberculosis Resistente a Múltiples Medicamentos/terapia , Tuberculosis Pulmonar/terapia , Animales , Humanos , Interleucina-12/genética , Pulmón/inmunología , Solanum lycopersicum/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente , Células TH1/inmunología , Tiempo , Tuberculosis Resistente a Múltiples Medicamentos/inmunología , Tuberculosis Pulmonar/inmunologíaRESUMEN
Objetivo: Desarrollar un nuevo índice topográfico que cuantifique microirregularidades en la superficie corneal. Métodos: Se incluyen 155 ojos de 128 pacientes con una edad media de 29,2 años, a los cuales se les realiza una topografía corneal (TC). Se diferencian tres grupos en función de la regularidad corneal: grupo I formado por 50 ojos de pacientes con córneas regulares, grupo II 45 ojos córneas con macroirregularidad, y grupo III 60 ojos córneas con microirregularidad. A partir de los valores de los radios de curvatura aportados por el mapa de datos numéricos de la TC, se elabora un algoritmo que expresa las diferencias en poder dióptrico entre puntos muy próximos. Se realizó una estadística descriptiva en cada grupo y para la comparación entre grupos se utilizó la comparación múltiple de medias de Scheffé. Resultados: El índice de irregularidad medio en el grupo I fue 7.Conclusiones: El nuevo índice de irregularidad es capaz de cuantificar microirregularidades en la superficie corneal. Este índice es tanto mayor de cero cuanto mayores sean las microirregularidades de la córnea (AU)
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Asunto(s)
Persona de Mediana Edad , Adulto , Adolescente , Masculino , Femenino , Humanos , Topografía de la Córnea , CórneaRESUMEN
Esthesioneuroblastoma is a rare tumor that originates in the olfactory epithelium and can invade the orbit. We report a case in which the first symptom was a post-traumatic paralysis of the IV cranial nerve. Whether the tumor itself or the trauma or a combination of the two factors caused the paralysis is discussed.