Identification de composé sensibilisant préférentiellement les cellules exprimant les protéines E6 et E7 du VPH à l'irradiation

Le cancer du col utérin (CCU) est dans plus de 99% des cas provoqué par une infection avec le virus du papillome humain (VPH), dont le potentiel oncogénique réside dans l'expression des proto-oncogènes viraux E6/E7. Le potentiel carcinogénique de ces protéines virales réside essentiellement dans leurs actions sur les produits des gènes suppresseurs de tumeur p53 et RB. Les produits de ces gènes, p53 et Rb, font parti des voies de signalisation de réponse aux dommages de l'ADN cellulaire (RDA) et leur perte entraine une perte de fonctionnalité qui mène à une instabilité génomique. À long terme et en présence de d'autres facteurs ceux-ci mèneront au développement d'un cancer. Les protéines E6 et E7 sont constitutivement exprimées dans les cellules du CCU ainsi que dans les cellules de tout autre cancer induit par le VPH et seulement dans ces dernières. La prise en charge des cas avancés de ces cancers se fait principalement par radiothérapie et chimiothérapie concomitante. La chimio-radiothérapie utilisée en traitement est efficace mais résulte en un taux élevé de morbidité et un nombre important de patientes récidiveront. Nous proposons que l'exploitation de l'expression spécifique d’E6 et d’E7 dans les cellules du CCU permette d’envisager une stratégie de létalité synthétique afin d'amplifier l'effet létal de l'irradiation sur les cellules CCU. Ceci permettrait potentiellement d'augmenter l'efficacité du traitement et de diminuer les récidives, ainsi que la morbidité liée au traitement. En s'appuyant sur cette hypothèse, notre objectif est d’identifier des composés dont l'action seule ou couplée à l'irradiation provoquerait préférentiellement la mort des cellules exprimant les protéines E6 et E7 du VPH. Les cellules testées comprennent des cellules isogéniques humaines issues de kératinocytes normaux que nous avons modifiées séquentiellement pour obtenir les modifications associées aux cellules CCU (hTERT, E6 et E7), ainsi que les lignées de cellules de CCU HeLa et CaSki .Nous avons procédé à la mise au point et à la validation du protocole de criblage et des méthodes d’évaluation de la sensibilisation, qui se définit comme une perte de viabilité, un arrêt ou ralentissement de la croissance, par détection d’ATP ainsi que par coloration d’ADN génomique au DRAQ5. Suite à un criblage ciblé impliquant des inhibiteurs connus de la voie de réparation des dommages à l’ADN, nous avons identifié l’inhibiteur de mdm2, Nutlin-3, comme étant un composé sensibilisant et radio-sensibilisant préférentiellement les cellules exprimant E6 et E7 du VPH. La Nutlin-3 a été testée sur des cellules HEKn-hTERT-E6-E7, des cellules CaSki et HeLa. L’effet de sensibilisation et de radio-sensibilisation a été confirmé dans ces trois lignées. Tel que suggéré par son action sur mdmd2, la Nutlin-3 permet la stabilisation de p53 dans les cellules HEKn-hTERT-E6-E7 et CaSki et sa réactivation dans les lignées cellulaires HeLa et CaSki. Malgré cette stabilisation de p53, de façon surprenante, l’effet de la Nutlin-3 sur la sensibilisation et la radio-sensibilisation des cellules HeLa et CaSki semble indépendant de p53, tel qu’observé en utilisant des cellules HeLa-GSE et CaSki-GSE dont le p53 est déficient. In vivo la Nutlin-3a montre dans un essai préliminaire l’inhibition de la croissance tumorale des xénogreffes HeLa chez des souris RAG2γc. Ce résultat reste à confirmer avec un essai impliquant un nombre d’échantillons plus grand. À plus long terme, nous comptons étudier l’implication de mdm2 dans l’effet de sensibilisant de la Nutlin-3 dans les cellules CCUs, ainsi que les autres cibles pouvant être impliquées dans la création de cet effet sensibilisant observé.

Uso de lisados bacterianos en la prevención de la infección respiratoria recurrente en pediatría. Revisión sistemática de la literatura

Las infecciones respiratorias altas y bajas son una causa común de morbimortalidad infantil. Se ha propuesto el uso de los lisados bacterianos para prevenir las infecciones recurrentes sin embargo su uso aún se considera controversial. Metodología: Se realizó una revisión sistemática de la literatura. La búsqueda se realizó a través de las bases de datos PUBMED, Embase, Ovid, LiLaCS y Cochrane library plus. Se incluyeron metanálisis publicados en idiomas inglés y español, entre los años 1998 y 2012. Se realizó una evaluación de calidad siguiendo la estrategia Quorum y un análisis cualitativo y cuantitativo de los resultados. Resultados: Se incluyeron 4 revisiones sistemáticas de la literatura con metanálisis. Fue apreciable la disminución de las recurrencias de las infecciones respiratorias relacionadas con el uso de los lisados bacterianos. Los lisados bacterianos disminuyen la necesidad de uso de antibióticos. No se encontró evidencia sobre el uso de los lisados sobre desenlaces como la necesidad de intervenciones adicionales, tiempo de hospitalización, costo relacionado con la atención en salud. No se reportaron eventos adversos de importancia. Conclusión: Los lisados bacterianos son eficaces en disminuir la recurrencia de las infecciones respiratorias en pacientes en edad pediátrica.

Genotipificación de HLA-B en pacientes colombianos afectados por el síndrome Stevens-Johnson y la Necrólisis Epidérmica Tóxica

Las reacciones alérgicas a medicamentos cutáneas severas (RAM) como el Síndrome Stevens Johnson (SJS) y la Necrólisis Epidérmica Tóxica (NET),caracterizadas por exantema, erosión de la piel y las membranas mucosas, flictenas, desprendimiento de la piel secundario a la muerte de queratinocitos y compromiso ocular. Son infrecuentes en la población pero con elevada morbi-mortalidad, se presentan luego de la administración de diferentes fármacos. En Asia se ha asociado el alelo HLA-B*15:02 como marcador genético para SJS. En Colombia no hay datos de la incidencia de estas RAM, ni de la relación con medicamentos específicos o potenciales y tampoco estudios de aproximación genómica de genes de susceptibilidad.

Rodents as carriers of disease: preliminary studies in the United Kingdom

The University of Reading has conducted some preliminary work on the prevalence of Campylobacter spp., Salmonella spp. and Arenavirus in Norway rats trapped from farms and semi-urban areas in central southern England. Campylobacter is the cause of a notificable disease in the UK, with 57,772 cases reported for England and Wales in 2009. Transmission to humans is believed to be primarily through undercooked meat, from contaminated water, and through contact with pets; and symptoms include a high temperature, severe diarrhoea, vomiting and abdominal pain. Ninety-seven per-cent of sporadic cases have been attributed to farm animals, and in particular the meat and poultry industry. There are eighteen species of Campylobacter, eleven of which can be pathogenic to humans; although the principal species that cause gastrointestinal disease in humans are C. jejuni and C. coli; although C. lari, C. helveticus and C. upsaliensis are also involved. Salmonella species also causes a gastrointestinal disease, and in the UK, is common in chicken and has been linked to egg production. Species are typed using antigen specific agglutination tests, or by their susceptibility to specific bacteriophage. Some strains are known to be linked with human disease (eg. S. enteritidis PT4).

Isolation and characterization of an AINTEGUMENTA-like gene in different coconut (Cocos nucifera L.) varieties from Sri Lanka

Development of an efficient tissue culture protocol in coconut is hampered by numerous technical constraints. Thus a greater understanding of the fundamental aspects of embryogenesis is essential. The role of AINTEGUMENTA-like genes in embryogenesis has been elucidated not only in model plants but also in economically important crops. A coconut gene, CnANT, that encodes two APETALA2 (AP2) domains and a conserved linker region similar to those of the BABY BOOM transcription factor was cloned, characterized, and its tissue specific expression was examined. The full-length cDNA of 1,780 bp contains a 1,425-bp open reading frame that encodes a putative peptide of 474 amino acids. The genomic DNA sequence includes 2,317 bp and consists of nine exons interrupted by eight introns. The exon/intron organization of CnANT is similar to that of homologous genes in other plant species. Analysis of differential tissue expression by real-time polymerase chain reaction indicated that CnANT is expressed more highly in in vitro grown tissues than in other vegetative tissues. Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one single nucleotide polymorphism and one indel in the first exon and first intron, respectively, which differentiate the Tall group of trees from Dwarfs. The indel sequence, which can be considered a simple sequence repeats marker, was successfully used to distinguish the Tall and Dwarf groups as well as to develop a marker system, which may be of value in the identification of parental varieties that are used in coconut breeding programs in Sri Lanka.

Local Corticosterone Infusion Enhances Nocturnal Pineal Melatonin Production In Vivo

Melatonin, an important marker of the endogenous rhythmicity in mammals, also plays a role in the body defence against pathogens and injuries. In vitro experiments have shown that either pro- or anti-inflammatory agents, acting directly in the organ, are able to change noradrenaline-induced pineal indoleamine production. Whereas corticosterone potentiates melatonin production, incubation of the gland with tumour necrosis factor-alpha decreases pineal hormonal production. In the present study, we show that nocturnal melatonin production measured by intra-pineal microdialysis is enhanced in pineals perfused with corticosterone at concentrations similar to those measured in inflamed animals. In vitro experiments suggest that this enhancement may be due to an increase in the activity of the two enzymes that convert serotonin to N-acetylserotonin (NAS) and NAS to melatonin. The present results support the hypothesis that the pineal gland is a sensor of inflammation mediators and that it plays a central role in the control of the inflammatory response.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Influence of age on the development of immunological lung response in intrauterine undernourishment

Objective: We investigated the effect of intrauterine undernourishment on some features of asthma using a model of allergic lung inflammation in rats. The effects of age at which the rats were challenged (5 and 9 wk) were also evaluated. Methods: Intrauterine undernourished offspring were obtained from dams that were fed 50% of the nourished diet of counterparts and were immunized at 5 and 9 wk of age. They were tested for immunoglobulin E anti-ova titers (by passive cutaneous anaphylaxis), cell count in the bronchoal-veolar fluid, leukotriene concentration, airway reactivity, mucus production, and blood corticosterone and leptin concentrations 21 d, after immunologic challenge. Results: Intrauterine undernourishment significantly reduced the antigen-specific immunoglobulin E production, inflammatory cell infiltration into airways, mucus secretion, and production of leukotrienes B-4/C-4 in the lungs in both age groups compared with respective nourished rats. The increased reactivity to methacholine that follows antigen challenge was not affected by intrauterine undernourishment. Corticosterone levels increased with age in the undernourished rats` offspring, but not in the nourished rats` offspring. Undernourished offspring already presented high levels of corticosterone before inflammatory stimulus and were not modified by antigen challenge. Leptin levels increased with challenge in the nourished rats but not in the undernourished rats and could not be related to corticosterone levels in the. undernourished rats. Conclusion: Intrauterine undernourishment has a striking and age-dependent effect on the off spring, reducing lung allergic inflammation. (C) 2008 Elsevier Inc. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

Determinantes envolvidos na resposta imune celular humana à infecção por Leishmania infantum chagasi

Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients

O memorial autobiográfico: uma tradição acadêmica do ensino superior no Brasil

The thesis has as object of study the autobiographical memmorials. The general objective is to describe the history of the memmorial as an academic tradition of higher education in Brazil. Considered a hybrid genre, memmorials are known for focusing on life stories from a scientific perspective. The investigation revolves around three intertwined branches: History of Education, educational practices and language usages, which allow us to conduct a dialogue with multiple theoretical-methodological references with a view to supporting our analyses. The corpus used for the analysis was made up of 40 autobiographical memmorials, distributed as follows: 16 academic memmorials, dated from 1935 to 1970; 07 academic memmorials, dated from 1980 to 2007; and 17 formation memmorials, dated from 1995 to 2000. In this corpus, we also included official documents, which relate to legislation contained in edicts, resolutions, ordinances, regulations, which we used with a view to: 1) getting to know and understanding the big picture of higher education regulation in Brazil and the aspects related to the higher education teaching career; 2) investigating the text of memmorials in the light of the injunctive discourse characteristic of the edicts and resolutions in which they were based. The analysis of the memmorial supported by the legislation which regulates it allowed us to reconstitute the image of the professor throughout 80 years in the Brazilian public university. For this purpose, the study was conducted in the theoretical-methodological perspective of the (auto)biographical research in Education and of the sociolinguistic studies on discourse genres and discursive traditions. The investigations reveal the memmorial as an academic genre in which the professor's academic-professional history and the history of the higher education teaching career in Brazil intertwine. Anchored in the Bakhtinian perspective on discourse genres, according to which the memmorials evolve and become more complex as their contexts of usage also evolve and become more complex themselves, the results of our analyses allowed us to correlate genre changes to the sociohistorical context and to its usage as an educational practice in the university, in the decades under study. Therefore, the analyses showed that these self-writings: go from latent subjectivity to pure objectivity from the 1930s to 1960s; they show total annulment of the subject from the 1960s to the 1970s; they reappear in the 1980s, having Professor Magda Soares' memmorial as perspective; they expand and diversify from the 1990s onwards, taking on a formative role and a perspective of future as well. So far as language usages are concerned, we investigated the relationship of the subject with the language, especifically the manifestation of alterity on the discursive tissue of the memmorials. In this branch, the analyses pointed to the influence of the authoritative discourse on the formation of the professor and of the injunction and reinventing discourses on the authorship process. Therefore, the autobiographical memmorial reveals itself as a specific expression of the Brazilian academy's cultural sphere and allows us to confirm the hypothesis that each memorial tackles a singular-plural situation, by presenting a dialectical articulation between private and public, according to the institutional structures, in which and with which the professor has already formed him/herself and with which he/she dialogues

Violência doméstica contra adolescentes: o olhar dos educadores sociais

Domestic violence as a specific expression of violence against adolescents, is historically constructed from phenomenon of power relations that permeate the gender, ethnicity and social class, requiring differentiated attention. Sorting in A mass grave of what is understood by a child and teenager under the doctrines of the law of the minor and of irregular situation resulted, in General, in the preparation for the teenage audience, of policies and actions, fragile and inefficient with regard to domestic violence. Despite the status of children and adolescents clearly define what is meant by child and teenager, break with the menoristas doctrines and embrace the doctrine of integral protection, even if there is little guidance for the public actions and policies geared to the theme. Such deficiency contributes to characterize the current practice of social educators about domestic violence, marked by the absence of specific training; by inadequate working conditions; lack of preparation of the network of care. Thus, with the objective to start the discussion regarding the elements that make up the practice focused on this issue, the present study proposes to problematizing the design held by social educators working in public Social assistance of the city of Natal/RN, regarding the issue of domestic violence against adolescents, by paying attention to such things as their vocational integration, their training and their working conditions. Adopts norteadoras references as having its roots in the Pedagogia Problematizadora, Paulo Freire, and socio-historical perspective. The methodological procedure of nature quali-quantitative was lifting censitário and psychosocial characterization of educators together to State and local Departments of assistance; the application of questionnaire to educators, composed of open and closed issues; and observation and field journaling activities of their work. Of network professionals, 111 64 replied to the questionnaire, which represents more than 50% of the total. Psychosocial characterization exposes data as the predominance of low family income and female professionals; the note identified that do not have a professional technical parameter, and the activities carried out in accordance with the representations of each professional the respect of labour, problematic and adolescents involved, revealing an individual perspective of action. And a preliminary analysis of responses to the questionnaire pointed out that such professionals are subjected to precarious working conditions, as well as one realizes a relative ignorance about the network of care for adolescents at risk and ACE, all this resulting in restricted marginalizantes conceptions, and misleading information regarding domestic violence against teenagers.

Chicken skeletal muscle-associated macroarray for gene discovery

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effects of propolis on antibody production by laying hens

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)