In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.

Noticias sobre el virus del papiloma humano y su vacuna en la prensa valenciana (2006-2011)

La introducción de la vacuna contra el virus del papiloma humano (VPH) dirigida a mujeres adolescentes ha tenido en España un desarrollo no exento de cierta controversia. Asociada inicialmente al mensaje de «vacuna contra el cáncer de útero» que ofrecía una nueva posibilidad de lucha contra esa enfermedad, obtuvo una réplica que moderaba la euforia con un mensaje dirigido a probar evidencias. Mientras se administraba la segunda dosis de vacuna (febrero de 2009) ocurrió un suceso inesperado en Valencia relacionado con la aparición de acontecimientos adversos tras la administración de la vacuna en dos adolescentes, que tuvo un explosivo tratamiento mediático. Este estudio analiza el alcance y el contenido de las noticias aparecidas en dos periódicos regionales valencianos de gran tirada durante el sexenio 2006-2011 que mencionan al VPH, su vacuna y el cáncer de útero. Se discute la influencia que los mensajes emitidos hayan podido tener en la aceptabilidad de la vacuna.

Notificación de reacciones adversas a la vacuna frente al virus del papiloma humano en la Comunidad Valenciana (2007-2011)

Introducción: En 2009, 2 casos de convulsiones en adolescentes tras la administración de la vacuna tetravalente frente al virus del papiloma humano (VPH) generaron impacto mediático y afectaron negativamente la confianza del público en esta vacuna. Nuestros objetivos fueron describir las sospechas de reacciones adversas (SRA) notificadas al Centro Autonómico de Farmacovigilancia de la Comunidad Valenciana (CAFCV) tras la administración de la vacuna frente al VPH y comparar la tasa de notificación de síncope y convulsiones de esta vacuna con la de otras vacunas administradas en adolescentes. Material y métodos: Estudio descriptivo de las notificaciones de SRA relacionadas con esta vacuna recibidas por el CAFCV entre 2007 y 2011. Resultados: Las manifestaciones clínicas más comunicadas fueron mareos, cefalea y síncope. Las tasas de notificación de síncope o pérdida de conciencia y convulsiones con la vacuna frente al VPH fueron de 17 y 3,2 por 100.000 dosis administradas, respectivamente, y de 15 y 1,6 para síncope o pérdida de conciencia y convulsiones sincopales ocurridas el día de la vacunación. Las tasas de notificación de síncope o pérdida de conciencia y convulsiones fueron de 6,4 y 0,4 para otras vacunas. Conclusiones: Las tasas de notificación de síncope o pérdida de conciencia y convulsiones fueron mayores para la vacuna frente al VPH que para otras vacunas administradas en adolescentes; esto es consistente con la atención mediática originada por la vacuna y con hallazgos de estudios previos. No obstante, la información obtenida sobre las SRA a la vacuna sugiere un buen perfil de seguridad.

Aceptabilidad de la vacuna contra el VPH en estudiantes universitarios españoles durante la etapa pre-vacunal: un estudio transversal

Introducción. El cáncer de cuello de útero (CCU), segunda causa de mortalidad por cáncer en mujeres, está asociado a la infección por virus de papiloma humano (VPH), cuya máxima prevalencia se sitúa entre los 20 y 24 años de edad. Desde 2006 se dispone de una vacuna contra el VPH. El objetivo de este estudio es evaluar los conocimientos sobre CCU, la infección por VPH y su vacuna, valorando su aceptabilidad en población universitaria. Métodos. Estudio transversal sobre 1.750 estudiantes de la Universidad de Alicante (2008) seleccionados al azar, proporcional por sexo y estudios, mediante un cuestionario ad-hoc validado. Se calcularon porcentajes, intervalos de confianza, tablas de contingencia según sexo, edad y tipo de estudios, calculando odds ratios ajustadas (OR). Resultados. Muestra con 58,6% mujeres y 6,6% de estudiantes biosanitarios. Un 87,3% dispuestos a vacunarse frente al VPH, el 94,3% vacunaría a sus hijas, un 48,0% había oído hablar de la vacuna. El 90,6% tiene bajos conocimiento sobre la infección por VPH y un 82,2% sobre la vacuna. Un 22,4% manifiesta conocer la asociación entre VPH y CCU. Las mujeres registran OR mayores en conocimientos y predisposición a vacunarse. La aceptabilidad de la vacuna contra VPH se asocia con el sexo y la confianza en las vacunas como método preventivo, la influencia de los conocimientos previos es escasa sobre la predisposición vacunal. Conclusiones. Alta aceptabilidad de la vacuna en el periodo estudiado. Aumentar la confianza hacia las vacunas puede influir en una mejor predisposición a vacunarse.

Aceptabilidad de la vacuna contra el virus del papiloma humano en madres de la provincia de Valencia (España)

Introducción: La Comunidad Valenciana inició en octubre del 2008 el programa de vacunación contra el virus del papiloma humano (VPH) en niñas de 14 años. El objetivo de este estudio es evaluar los conocimientos sobre la infección por VPH y su vacuna en madres de adolescentes e identificar los factores asociados a la predisposición de vacunar a sus hijas. Material y métodos: Estudio observacional transversal mediante cuestionario dirigido a madres de alumnas nacidas en 1995 matriculadas en centros de secundaria de la provincia de Valencia durante 2010-2011. Muestra aleatoria estratificada por conglomerados (n = 1.279). Análisis estadístico: porcentajes, intervalos de confianza, OR, contrastes chi al cuadrado y regresión logística multivariante. Resultados: Ochocientos treinta y tres cuestionarios completados (65,1%). El 76,6% de las madres habían vacunado a sus hijas contra el VPH. El 93,8% conocía la vacuna, sobre todo a través de la televisión (71,5%). El 78,5% recibió consejo favorable de un profesional sanitario, lo que mejoró la vacunación de sus hijas (OR: 2,4). Los conocimientos globales sobre la infección por VPH y la vacuna fueron bajos. La confianza de las madres en las vacunas como método preventivo mejora la vacunación contra VPH (OR: 3,8). El miedo a los efectos adversos (45,6%) fue el primer motivo de rechazo. Conclusiones: No parece que los medios de comunicación influyan en la decisión de vacunar. Sería conveniente minimizar la percepción de riesgo ante esta vacuna. El consejo del profesional sanitario actúa a favor de la vacunación si este interviene activamente en sentido positivo. Existe una brecha entre nivel de conocimientos y toma de decisión para vacunar.

Papilomatose oral : considerações a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Cervical Cancer Control, Priorities and New Directions

Cervical cancer is caused by infection with a range of high risk oncogenic human papillomavirus (HPV) types, and it is now accepted that >99% of cervical cancer is initiated by HPV infection. The estimated lifetime risk of cervical cancer is nevertheless relatively low (less than I in 20 for most community based studies). Although sensitivity and specificity of the available diagnostic techniques are suboptimal, Screening for persistent HPV infection is effective in reducing the incidence of cervical cancer. Infection can be detected by molecular techniques or by cytological examination of exfoliated cervical cells. Persistent infection is the single best predictor of risk of cervical cancer.(1) The latest findings of HPV and cervical cancer research need to be widely disseminated to the scientific and medical societies that are updating screening and management protocols, public health professionals, and to women and clinicians. This report reviews current evidence, clinical implications and directions for further research in the prevention, control and management of cervical cancer. We report the conclusions of the Experts' Meeting at the EUROGIN 2003 conference. (C) 2003 Wiley-Liss, Inc.

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.

RNA interference for the treatment of cancer

RNA interference (RNAi) is the latest new technology in the field of genetic medicine in which specific genes can be turned off, or silenced, so as to affect a therapeutic outcome. It can be highly specific, works in the nanomolar range and is far more effective than the antisense approaches popular 10-15 years ago. Here we review the field and explore the potential role of RNAi in cancer therapy, highlighting recent progress and examining the hurdles that must be overcome before this promising technology is ready for clinical use. (C) 2006 Prous Science. All rights reserved.

Prophylactic HPV vaccines: Underlying mechanisms

Human papillomavirus virus-like particles (HPV VLP) can be generated by the synthesis and self-assembly in vitro of the major virus capsid protein L1. HPV L1 VLPs are morphologically and antigenically almost identical to native virions, and this technology has been exploited to produce HPV L1 VLP subunit vaccines. The vaccines elicit high titres of anti-L I VLP antibodies that persist at levels 10 times that of natural infections for at least 48 months. At present the assumption is that the protection achieved by these vaccines against incident HPV infection and HPV-associated ano-genital pathology is mediated via serum neutralising Immunoglobulin G (IgG). However, since there have been very few vaccine failures thus far, immune correlates of protection have not been established. The available evidence is that the immunodominant neutralising antibodies generated by L1 VLPs are type-specific and are not cross-neutralising, although highly homologous HPV pairs share minor cross-neutralisation epitopes. Important issues remaining to be addressed include the duration of protection and genotype replacement. (c) 2006 Elsevier Ltd. All rights reserved.

Maternal mind-mindedness during infancy, general parenting sensitivity and observed child feeding behavior:a longitudinal study

Maternal mind-mindedness, or the tendency to view the child as a mental agent, has been shown to predict sensitive and responsive parenting behavior. As yet the role of mind-mindedness has not been explored in the context of feeding interactions. This study evaluates the relations between maternal mind-mindedness at 6 months of infant age and subsequently observed maternal sensitivity and feeding behaviors with children at age 1 year. Maternal mind-mindedness was greater in mothers who had breast-fed compared to formula-fed. Controlling for breast-feeding, mind-mindedness at 6 months was correlated with observations of more sensitive and positive feeding behaviors at 1 year of age. Mind-mindedness was also associated with greater general maternal sensitivity in play and this general parenting sensitivity mediated the effect of mind-mindedness on more sensitive and positive feeding behaviors. Interventions to promote maternal tendency to consider their child's mental states may encourage more adaptive parental feeding behaviors. © 2014 Taylor & Francis.

Recombinant protein subunit vaccine synthesis in microbes:a role for yeast?

Objectives Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host. Key findings The yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles. Summary Advances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development.