Forever vulnerable : pledge for a former child soldier status

Child soldiers, as a prevalent phenomenon widely condemned by the international community, have been slowly becoming a policy priority in the humanitarian field. Regrettably, once this condition becomes part of their past, there is almost a loss of interest or even forgetfulness of their vulnerable condition. Driven by disbelief due to ICC’s disregard for the “former child soldier condition” of the defendant Dominic Ongwen, and the insufficient—and sometimes non-existent— international legal framework for former child soldiers, we propose a protective status for individuals who fall within the definition of former child soldiers. Eligibility for this status is based on the very own definition of former child soldiers, which, in turn, should highlight the heterogeneity of this group, whose vulnerability stems from the past gross violation of their human rights. Accordingly, the proposal of a protective status implies addressing different experiences that are directly linked to different protection needs. Yet, due to the number of issues that this status can raise, we selected the type of participation in hostilities as our guiding element. Nevertheless, the ultimate protection of a vulnerable group can only be obtained through a legal instrument, which is why we aim to set this status in a binding article. Thus, we ask ourselves: why are child soldiers deserving of international law’s protection, but not individuals whose past is tainted by the same violations of their human rights and whose future is conditioned by their past experiences?As crianças-soldado, enquanto fenómeno prevalecente e veementemente repudiado pela comunidade internacional, têm vindo a aferir-se como uma prioridade na ação política internacional em matéria de direito humanitário. Lamentavelmente, assim que esta condição passa a fazer parte do seu passado, verifica-se uma súbita indolência e até negligência perante a vulnerabilidade inerente a estes indivíduos. Motivados pelo desapreço evidenciado pelo Tribunal Penal Internacional quanto à condição de ex-criança-soldado inerente a Dominic Ongwen, bem como pela insuficiência - e por vezes, inexistência - do regime jurídico referente esta mesma condição, propomos-mos, com esta dissertação, a apresentar um estatuto protetor para os indivíduos que se aferem como ex-crianças-soldado. A elegibilidade para a obtenção deste estatuto é baseada na própria definição do conceito de ex-criança-soldado que, por sua vez, deverá evidenciar a heterogeneidade e vulnerabilidade patentes a este grupo. Destarte, a proposta do referido estatuto, implica que nos debrucemos sobre as diversas experiências destes indivíduos, estando estas diretamente ligadas a necessidades distintas de proteção. Contudo, pela multiplicidade e complexidade das questões que este estatuto pode suscitar, selecionámos a forma de participação nas hostilidades enquanto o elemento condutor do nosso estudo. Não obstante, a proteção efetiva de um grupo de pessoas vulneráveis é apenas possível de ser alcançada através de garantia jurídica. É precisamente neste sentido que nos propomos a desenvolver uma disposição jurídica vinculativa do estatuto de ex criança-soldado. Enfim, questionamo-nos: porque é que as crianças-soldado são dignas da proteção do direito internacional, mas o mesmo não se verifica quanto aos indivíduos cujos passados estão manchados pelas mesmas violações de direitos humanos e cujo futuro é condicionado pelo trauma de experiências passadas

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.Fil: Pereyra Gerber, Federico Pehuén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Cabrini, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Jancic, Carolina Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Paoletti, Luciana Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Banchio, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Von Bilderling, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Sigaut, Lorena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pietrasanta, Lia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Duette, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Freed, Eric O.. National Cancer Institute at Frederick; Estados UnidosFil: Basile, Genevieve de Saint. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Moita, Catarina Ferreira. Instituto Gulbenkian de Ciencia; PortugalFil: Moita, Luis Ferreira. Instituto Gulbenkian de Ciencia; PortugalFil: Amigorena, Sebastian. Institute Curie; FranciaFil: Benaroch, Philippe. Institute Curie; FranciaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Ostrowski, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies

Genetic dissection of antigen presentation pathways

Tese de doutoramento, Ciências Biomédicas (Ciências Morfológicas), Universidade de Lisboa, Faculdade de Medicina, 2010.Effective immune responses against tumor antigens that are not endogenously expressed by dendritic cells (DCs) and against virus that do not infect antigen presenting cells (APCs) require extracellular antigens to stimulate CD8+ T cells via the MHC I pathway through a process known as cross-presentation. The molecular mechanisms that direct endocytosed antigens from the classical MHC II-restricted presentation pathway to the MHC I pathway are for the most part not understood. We have completed a screen for genes with a role in this process using a subset of an shRNA lentiviral library enriched for mouse kinases and phosphatases. In this screen we measured the proliferation of CD8+ T (OT-I) cells in response to the stimulation of bone marrow derived DCs (BMDCs) by OVA-expressing S. cerevisiae. After testing over 1000 genes in duplicate and two rounds of phenotypic validation, we have chosen ~80 genes that reproducibly caused increased (more than 1.5 SDEV) or reduced (less than 1.2 SDEV) rates of CD8+ T proliferation. We also tested these 80 genes in a similar assay but using B3Z as the responder cell line. We have found that 15 genes caused a significant change in the levels of IL-2 production. Because IL-2 production by this cell line requires presentation of the SIINFEKL peptide by MHC I but it is co-stimulator independent, it is likely that this group of genes regulates presentation while the other group regulates co-stimulator induction. We performed qPCR validation of the knockdowns and additional secondary screens on this group of 80 genes so that we could identify those that are specifically required for cross-presentation. Among these we validated 1 novel gene that seems to be predominatly involved in cross-presentation, which is a good candidate for follow-up mechanistic studies. Interestingly, it is neither a kinase nor a phosphatase. Therefore, my results suggest that the overwhelming majority of kinases and phosphatases involved in antigen presentation play a role in both canonical and antigen cross-presntation pathways. In the hope to find genes that specifically regulate antigen cross-presentation, I decided to expand our search to include most known genes with a role in vesicle traffic within the cell. In this second phase of our screen we measured the proliferation of CD8+ T (OT-I) cells in response to the stimulation of BMDCs after incubation with soluble OVA. After testing 155 genes, we have chosen 44 genes that reproducibly caused increased (more than 1.0 SDEV) or reduced (less than -0.7 SDEV) rates of CD8+ T proliferation to be tested in the MHC class II pathway. We found 12 genes that caused a significant change in the levels of OT-I proliferation, but had moderate or no effect on Class II antigen presentation. We performed qPCR validation of the knockdowns on this group of 12 genes and identified 8 novel genes with good correlation profiles. These are excellent candidates for future mechanistic studies and the exploration of their in vivo role in antigen cross-presentation in different biologically relevant contexts.Uma resposta imunitária eficaz contra antigénios tumorais, que não são endogenamente expressos por células dendríticas, e contra vírus que não infectem células apresentadoras de antigénios, requer a estimulação de células T CD8+ por parte de antigénios extracelulares via moléculas MHC I, num processo denominado de “apresentação cruzada de antigénios”. Os mecanismos moleculares que direccionam antigénios endocitados do processo clássico de apresentação restringido a moléculas MHC II, para a via de classe I são largamente desconhecidos. Realizámos um screen com o objectivo de identificar genes envolvidos neste processo, usando uma parcela de uma biblioteca lentiviral de shRNA enriquecida em cinases e fosfatases de ratinho. Neste screen, foi medida a proliferação de células T CD8+ (OT-I) em resposta à estimulação de células dendríticas derivadas da medula óssea após incubação com S. cerevisiae expressando ovalbumina. Após testar mais de 1000 genes em duplicado e realizando duas fases de validação fenotípica, foram escolhidos cerca de 80 genes que repetidamente causaram um aumento (mais de 1.5 SDEV) ou diminuição (menos de -1.2 SDEV) na taxa de proliferação de células T CD8+. Os referidos 80 genes foram igualmente testados num ensaio idêntico utilizando a linha celular B3Z. Foram identificados 15 genes cuja secreção de interleucina-2 (IL-2) foi significativamente diferente dos controlos. Dado que a secreção de IL-2 por esta linha celular requer a apresentação do péptido SIINFEKL associado à molécula de MHC I, mas esta é independente da expressão de moléculas co-estimuladoras, é plausível que este grupo de genes possa regular a apresentação, enquanto que os restantes tenham um papel na indução de co-estimuladores. Realizaram-se validações por qPCR dos níveis de knockdown, bem como screens secundários adicionais no grupo de 80 genes para que pudessem ser identificados os especificamente envolvidos na apresentação cruzada de antigénios. Neste grupo identificámos 1 gene que parece estar predominantemente envolvido na apresentação cruzada em antigénios, o qual é um bom candidato para estudos mecanísticos posteriores. Os resultados descritos sugerem que a maioria das cinases e fosfatases envolvidas na apresentação de antigénios desempenham um papel quer na via canónica, quer na de apresentação cruzada dos mesmos. Como tal, decidimos expandir a nossa pesquisa por forma a incluir todos os genes descritos com um papel no tráfego vesicular da célula. Nesta segunda fase, medimos a proliferação de células T CD8+ (OT-I) em resposta à estimulação de células dendríticas derivadas da medula óssea incubadas com ovalbumina solúvel. Após testar 155 genes no contexto da apresentação cruzada de antigénios, foram escolhidos 44 genes que reprodutivelmente causaram um aumento (mais de 1.0 SDEV) ou diminuição (menos de -0.7 SDEV) na taxa de proliferação de células T CD8+ para serem testados no contexto de apresentação por moléculas MHC II. Foi observado que 12 genes causam uma alteração específica nos níveis de proliferação de células OT-I e com um efeito moderado ou mesmo inexistente ao nível da apresentação em classe II. Realizaram-se validações por qPCR dos níveis de knockdown destes 12 genes e foram identificados 8 genes com bons perfis de correlação. Estes constituem excelentes candidatos para futuros estudos mecanísticos e para a investigação do seu papel in vivo na apresentação cruzada de antigénios em diferentes contextos biológicos.Fundação para a Ciência e Tecnologia (FCT); Fundação Luso-Americana para o Desenvolvimento; Fundação Calouste Gulbenkia

RNAi screen for kinases and phosphatases that play a role in antigen presentation by dendritic cells

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimEffective CD8(+) T-cell responses against tumor or microbial antigens that are not directly expressed in antigen-presenting cells (APCs) depend on the cross-presentation of these antigens on MHC class I in APCs. To identify signaling molecules that regulate cross-presentation, we used lentiviral-based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross-presentation. Depletion of Acvr1c, a type I receptor for TGF-β family of signaling molecules, led to an increase in CD80 and CD86 co-stimulator surface expression and secreted IL-12 in mouse bone marrow-derived DCs, as well as antigen-specific T-cell proliferation.This work was supported by Fundação para a Ciência e Tecnologia (PIC/IC/82991/2007, PTDC/SAU-MII/100780/2008, and PTDC/SAU-IMU/110303/2009), Fundação Luso-Americana para o Desenvolvimento, and the Human Frontier Science Program.info:eu-repo/semantics/publishedVersio

Adherence to European guidelines for the use of aspirin in primary health care

Introduction and objectives: Cardiovascular disease remains a leading cause of global morbidity and mortality. The administration of low doses of aspirin in secondary prevention of atherosclerotic cardiovascular disease (ASCVD) has been clearly established. However, the most recent guidelines do not recommend aspirin in primary prevention, reserving it for high-risk patients and after a risk/benefit assessment. The aim of this study was to assess adherence to European guidelines for the use of aspirin in primary and secondary prevention of ASCVD in primary health care. Methods: The study population consisted of individuals aged >50 years registered at two primary health care units without (primary prevention) and with previous ASCVD events (secondary prevention). Results: We studied a total of 1262 individuals, 720 in primary prevention and 542 in secondary prevention. A total of 61 individuals (8.5%) were under aspirin therapy in primary prevention, most of them taking 150 mg/day (57%). In secondary prevention, 195 patients (27%) were receiving aspirin only, most taking 150 mg/day (52%), and 166 patients (31%) were not under any antithrombotic or anticoagulant therapy. The 100 mg dosage was predominant in patients with ischemic heart disease with (64%) and without (64%) angina, as well as those with myocardial infarction (61.5%) and peripheral vascular disease (62%). Conclusions: In this study, the prevalence of aspirin use in primary prevention was 8.5%. We found that 30% of patients were not taking either antithrombotic or anticoagulation therapy in secondary prevention. In both primary and secondary prevention, the 150 mg dosage was predominant. Resumo: Introdução e objetivos: As doenças cardiovasculares permanecem uma das principais causas de morbilidade e mortalidade em nível mundial. A administração de baixas doses de ácido acetilsalicílico (AAS) na prevenção secundária de doença cardiovascular aterosclerótica (DCVA) foi claramente estabelecida. Concomitantemente, as recomendações mais recentes não o aconselham na prevenção primária, propondo ser reservado para doentes de alto risco e após uma avaliação risco/benefício. O objetivo deste estudo foi avaliar a adesão às recomendações europeias para o uso de AAS na prevenção primária e secundária de DCVA nos cuidados de saúde primários. Métodos: A população em estudo correspondeu a indivíduos >50 anos registados em duas unidades de cuidados de saúde primários sem (prevenção primária) e com eventos de DCVA anteriores (prevenção secundária). Resultados: Foram estudados 1262 indivíduos, 720 em prevenção primária e 542 em prevenção secundária. Verificámos que 61 dos indivíduos (8,5%) estavam sob terapia com AAS em prevenção primária, com uma predominância da dose de 150 mg (57%). Em prevenção secundária, 195 dos doentes (27%) estavam a realizar ASA exclusivamente, com uma predominância da dose de 150 mg (52%) e 166 dos doentes (31%) não estavam sob qualquer agente antitrombótico ou anticoagulante. A dosagem de 100 mg foi predominante em doentes com doença cardíaca isquémica com (64%) e sem (64%) angina, enfarte agudo do miocárdio (61,5%) e doença vascular periférica (62%). Conclusões: Neste estudo, a prevalência do uso de AAS na prevenção primária foi de 8,5%. Identificámos 30% de doentes que não cumpriam terapia antitrombótica ou anticoagulante em prevenção secundária. Quer em prevenção primária como secundária, o uso da dosagem de 150 mg foi predominante

Long-term continuous positive airway pressure treatment ameliorates biological clock disruptions in obstructive sleep apnea

Background Obstructive Sleep Apnea (OSA) is a highly prevalent and underdiagnosed sleep disorder. Recent studies suggest that OSA might disrupt the biological clock, potentially causing or worsening OSA-associated comorbidities. However, the effect of OSA treatment on clock disruption is not fully understood. Methods The impact of OSA and short- (four months) and long-term (two years) OSA treatment, with Continuous Positive Airway Pressure (CPAP), on the biological clock was investigated at four time points within 24 h, in OSA patients relative to controls subjects (no OSA) of the same sex and age group, in a case-control study. Plasma melatonin and cortisol, body temperature and the expression levels and rhythmicity of eleven clock genes in peripheral blood mononuclear cells (PBMCs) were assessed. Additional computational tools were used for a detailed data analysis. Findings OSA impacts on clock outputs and on the expression of several clock genes in PBMCs. Neither short- nor long-term treatment fully reverted OSA-induced alterations in the expression of clock genes. However, long-term treatment was able to re-establish levels of plasma melatonin and cortisol and body temperature. Machine learning methods could discriminate controls from untreated OSA patients. Following long-term treatment, the distinction between controls and patients disappeared, suggesting a closer similarity of the phenotypes. Interpretation OSA alters biological clock-related characteristics that differentially respond to short- and long-term CPAP treatment. Long-term CPAP was more efficient in counteracting OSA impact on the clock, but the obtained results suggest that it is not fully effective. A better understanding of the impact of OSA and OSA treatment on the clock may open new avenues to OSA diagnosis, monitoring and treatment

Anthracyclines induce DNA damage response-mediated protection against severe sepsis

Copyright © 2013 Elsevier Inc. All rights reserved.Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.L.F.M. receives support from FLAD and FCT (grants PTDC/SAU-IMU/110303/2009, PTDC/SAU-MII/100780/2008, and PTDC/SAU-IMU/110303/2009), A.C. receives support from FCT (PTDC/SAU-IMU/110303/2009), J.A.F. receives support from a Gulbenkian grant (96526/2009), and P.P. is an FCT fellow (SFRH/BD/45502/2008).info:eu-repo/semantics/publishedVersio

The MHC class Ib protein ULBP1 is a nonredundant determinant of leukemia/lymphoma susceptibility to γδ T-cell cytotoxicity

© 2010 by The American Society of HematologyOn the path to successful immunotherapy of hematopoietic tumors, γδ T cells offer great promise because of their human leukocyte antigen (HLA)–unrestricted targeting of a wide variety of leukemias/lymphomas. However, the molecular mechanisms underlying lymphoma recognition by γδ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to γδ T cell–mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all Vγ9+ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to γδ T cell–based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials.This work was distinguished with the Pfizer Award for ClinicalResearch 2009. This work was further supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-MII/71662/2006) and Fundação Calouste Gulbenkian (SDH Oncologia 2008; Projecto 99293). L.F.M. is a Young Investigator from the Human Frontier Science Program and was supported by Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e Tecnologia (FCT; PTDC/SAU-MII/69280/2006 and PTDC/SAU-MII/78333/2006). T.L. and D.V.C. received PhD fellowships (47342/2008 and 37898/2007) from FCT