The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27Kip1

AbstractE5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27Kip1 expression at protein level

Production and purification of immunologically active core protein p24 from HIV-1 fused to ricin toxin B subunit in E. coli

<p>Abstract</p> <p>Background</p> <p>Gag protein from HIV-1 is a polyprotein of 55 kDa, which, during viral maturation, is cleaved to release matrix p17, core p24 and nucleocapsid proteins. The p24 antigen contains epitopes that prime helper CD4 T-cells, which have been demonstrated to be protective and it can elicit lymphocyte proliferation. Thus, p24 is likely to be an integral part of any multicomponent HIV vaccine. The availability of an optimal adjuvant and carrier to enhance antiviral responses may accelerate the development of a vaccine candidate against HIV. The aim of this study was to investigate the adjuvant-carrier properties of the B ricin subunit (RTB) when fused to p24.</p> <p>Results</p> <p>A fusion between ricin toxin B subunit and p24 HIV (RTB/p24) was expressed in <it>E. coli</it>. Affinity chromatography was used for purification of p24 alone and RTB/p24 from cytosolic fractions. Biological activity of RTB/p24 was determined by ELISA and affinity chromatography using the artificial receptor glycoprotein asialofetuin. Both assays have demonstrated that RTB/p24 is able to interact with complex sugars, suggesting that the chimeric protein retains lectin activity. Also, RTB/p24 was demonstrated to be immunologically active in mice. Two weeks after intraperitoneal inoculation with RTB/p24 without an adjuvant, a strong anti-p24 immune response was detected. The levels of the antibodies were comparable to those found in mice immunized with p24 alone in the presence of Freund adjuvant. RTB/p24 inoculated intranasally in mice, also elicited significant immune responses to p24, although the response was not as strong as that obtained in mice immunized with p24 in the presence of the mucosal adjuvant cholera toxin.</p> <p>Conclusion</p> <p>In this work, we report the expression in <it>E. coli </it>of HIV-1 p24 fused to the subunit B of ricin toxin. The high levels of antibodies obtained after intranasal and intraperitoneal immunization of mice demonstrate the adjuvant-carrier properties of RTB when conjugated to an HIV structural protein. This is the first report in which a eukaryotic toxin produced in <it>E. coli </it>is employed as an adjuvant to elicit immune responses to p24 HIV core antigen.</p

CD43 signals induce Type One lineage commitment of human CD4+ T cells

<p>Abstract</p> <p>Background</p> <p>The activation and effector phenotype of T cells depend on the strength of the interaction of the TcR with its cognate antigen and additional signals provided by cytokines and by co-receptors. Lymphocytes sense both the presence of an antigen and also clues from antigen-presenting cells, which dictate the requisite response. CD43 is one of the most abundant molecules on the surface of T cells; it mediates its own signalling events and cooperates with those mediated by the T cell receptor in T cell priming. We have examined the role of CD43 signals on the effector phenotype of adult CD4⁺and CD8⁺human T cells, both alone and in the presence of signals from the TcR.</p> <p>Results</p> <p>CD43 signals direct the expression of IFNγ in human T cells. In freshly isolated CD4⁺T cells, CD43 signals potentiated expression of the IFNγ gene induced by TcR activation; this was not seen in CD8⁺T cells. In effector cells, CD43 signals alone induced the expression of the IFNγ gene in CD4⁺T cells and to a lesser extent in CD8⁺cells. The combined signals from CD43 and the TcR increased the transcription of the T-bet gene in CD4⁺T cells and inhibited the transcription of the GATA-3 gene in both populations of T cells, thus predisposing CD4⁺T cells to commitment to the T1 lineage. In support of this, CD43 signals induced a transient membrane expression of the high-affinity chains of the receptors for IL-12 and IFNγ in CD4⁺T cells. CD43 and TcR signals also cooperated with those of IL-12 in the induction of IFNγ expression. Moreover, CD43 signals induced the co-clustering of IFNγR and the TcR and cooperated with TcR and IL-12 signals, triggering a co-capping of both receptors in CD4⁺populations, a phenomenon that has been associated with a T1 commitment.</p> <p>Conclusion</p> <p>Our results suggest a key role for CD43 signals in the differentiation of human CD4⁺T cells into a T1 pattern.</p

The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination

CD4+ T cells are major players in the immune response against several diseases; including AIDS, leishmaniasis, tuberculosis, influenza and cancer. Their activation has been successfully achieved by administering antigen coupled with antibodies, against DC-specific receptors in combination with adjuvants. Unfortunately, most of the adjuvants used so far in experimental models are unsuitable for human use. Therefore, human DC-targeted vaccination awaits the description of potent, yet nontoxic adjuvants. The nontoxic cholera B subunit (CTB) can be safely used in humans and it has the potential to activate CD4+ T cell responses. However, it remains unclear whether CTB can promote DC activation and can act as an adjuvant for DC-targeted antigens. Here, we evaluated the CTB's capacity to activate DCs and CD4+ T cell responses, and to generate long-lasting protective immunity. Intradermal (i.d.) administration of CTB promoted late and prolonged activation and accumulation of skin and lymphoid-resident DCs. When CTB was co-administered with anti-DEC205-OVA, it promoted CD4+ T cell expansion, differentiation, and infiltration to peripheral nonlymphoid tissues, i.e., the skin, lungs and intestine. Indeed, CTB promoted a polyfunctional CD4+ T cell response, including the priming of Th1 and Th17 cells, as well as resident memory T (RM) cell differentiation in peripheral nonlymphoid tissues. It is worth noting that CTB together with a DC-targeted antigen promoted local and systemic protection against experimental melanoma and murine rotavirus. We conclude that CTB administered i.d. can be used as an adjuvant to DC-targeted antigens for the induction of broad CD4+ T cell responses as well as for promoting long-lasting protective immunity

Mis casos clínicos de especialidades odontológicas

Libro que muestra la atención de casos clínicos particulares referente a las diferentes especialidades odontológicasLibro que muestra la atención de casos clínicos particulares referente a las diferentes especialidades odontológicasUniversidad Autónoma de Campeche Universidad Autónoma del Estado de Hidalgo Universidad Autónoma del Estado de Méxic

Vacunas, estrategia biológica contra patógeno

En la actualidad se cuenta con un gran número de vacunas disponibles para ser aplicadas en la población humana cuya producción masiva se basa en el crecimiento de los organismos patógenos in vitro, los cuales son tratados posteriormente para atenuarlos y reducir la posibilidad de que se reviertan a su forma virulenta y produzcan alguna enfermedad. Por otra parte, existen vacunas constituidas solo por algunos componentes de los organismos patógenos o por subunidades de algunas toxinas, desarrolladas a partir de bacterias principalmente. Estas vacunas se conciben mediante técnicas avanzadas de biología celular y molecular que permiten optimizar su producción. Si bien las vacunas tradicionales han disminuido drásticamente la mortalidad y morbilidad causada por muchas infecciones, hay muchos mecanismos por esclarecer implicados en el desarrollo de respuestas protectoras. En general, una vacuna ideal debe ser segura, inductora de protección de larga duración y contra variantes del mismo agente patógeno, de rápida producción, que no involucre el uso de patógenos vivos o activos, que sea de bajo costo, y que su aplicación implique una sola dosis, con mínimos efectos secundarios. El desarrollo de tecnologías para mejorar las vacunas existentes y la creación de nuevas para otras enfermedades, así como el conocimiento acerca de su funcionamiento, permitirán que la vacunación sea un recurso más accesible para la prevención de enfermedades graves actuales y emergentes en la población en general, pero sobre todo en los grupos vulnerables

Expresión del ARNm de la IL-2 en bazos de pollos vacunados contra el virus de la influenza aviar

The aim of this study was to standardize a Reverse transcriptase polymerase chain reaction (RT/PCR) assay to detect IL-2 mRNA in lymphocytes from chickens inoculated with an experimental avian influenza vaccine, an experimental avian influenza vaccine added with rabies virus N recombinant protein, and a commercial influenza vaccine. For standardization, chicken lymphocytes were obtained in different quantities (1 x 106, 1 x 107 cells/ml), and activated by means of Concanavalin A (Con A) at 5, 10 and 30 mg/ml for 5, 24 and 48 h. RNA was extracted from lymphocytes and cDNA using oligo (dT) was synthesized. Primers to amplify avian IL-2 were designed from consensus sequences obtained from the GeneBank database. It was determined that 10 mg of Con-A added to 1 x 107 cells for 24 h showed the best results to differentiate activated lymphocytes from non-activated cells (control). b-actin was used as constitutive gene. A kinetic expression analysis was performed using lymphocytes obtained at 0, 1, 4 and 10 d post-vaccination. Cells coming from non-vaccinated chickens were used as control. It was established that avian influenza vaccines increased the ratio expression of avian IL-2 when compared to the control group. It can be concluded that RT/PCR may be used to analyze avian IL-2 expression.El objetivo de este estudio fue estandarizar un ensayo de Transcripción reversa en cadena de la polimerasa (RT/PCR) para detectar la presencia de ácido ribonucleico mensajero (ARNm) de la Interleucina 2 (IL-2), con la finalidad de determinar su expresión en linfocitos de pollos inmunizados con una vacuna inactivada experimental contra influenza aviar (IA), la vacuna inactivada experimental contra IA adicionada con proteína N recombinante del virus de la rabia (N-Bac) y una vacuna comercial. Para la estandarización, se obtuvieron linfocitos de pollos que fueron cultivados a 1 x 106 y 1 x 107 células/ml en presencia de Concanavalina A (Con-A) a una concentración de 5, 10 y 30 mg/ml, durante 5, 24 y 48 h. Se extrajo el ARN de los linfocitos y se sintetizó el ácido desoxirribonucleico complementario (ADNc) utilizando oligo(dT). Para llevar a cabo el PCR se diseñaron cebadores específicos dentro de la secuencia del gen de la IL-2 de pollo. Se determinó que 10 mg de Con-A durante 24 h y una concentración celular de 1 x 107 mostraron los mejores resultados con respecto a los niveles de ARNm para IL-2 entre los linfocitos activados y los controles (linfocitos sin activar). Como gen constitutivo se empleó la b-actina. La cinética de expresión se realizó en linfocitos obtenidos a los días 0, 1, 4 y 10 posvacunación. El grupo control fueron linfocitos de pollos no vacunados. Se estableció que las vacunas contra IA incrementaron la expresión de la IL-2 en pollos, en comparación de los controles. Se concluye que el RT/PCR puede ser empleado para analizar la expresión de la IL-2 de pollo

Protection against live rotavirus challenge in mice induced by parenteral and mucosal delivery of VP6 subunit rotavirus vaccine

Live oral rotavirus (RV) vaccines are part of routine childhood immunization but are associated with adverse effects, particularly intussusception. We have developed a non-live combined RV – norovirus (NoV) vaccine candidate consisting of human RV inner-capsid rVP6 protein and NoV virus-like particles. To determine the effect of delivery route on induction of VP6-specific protective immunity, BALB/c mice were administered a vaccine containing RV rVP6 intramuscularly, intranasally or a combination of both, and challenged with murine RV. At least 65 % protection against RV shedding was observed regardless of delivery route. The levels of post-challenge serum VP6-specific IgA titers correlated with protection