Determinantes associados à variabilidade do índice de fragmentação do ácido desoxirribonucleico espermático em homens saudáveis: um estudo de seguimento

The aim of this work was to study the variability of the sperm deoxyribonucleic acid (DNA) fragmentation index in a cohort of healthy men and to analyze the determinants and associated factors related to that variability, including sexual habits, lifestyle and environmental exposures. This is a prospective study which was carried out for 1 year evaluating multiple semen samples (obtained approximately every 4-6 weeks) from 19 healthy male volunteers. The subjects completed epidemiological questionnaires on lifestyle and environmental exposures in each of the interviews. Individuals were divided into two groups depending on their responses to several lifestyle and environmental exposure questions (“yes” vs. “no”). The coefficient of variation (CV) and the intra-subject coefficient of variation (CVi) of the sperm DNA fragmentation index were calculated and their statistical differences examined with regard to their responses to the studied factors. The CVs of the the sperm DNA fragmentation index were significantly different for all the studied variables, with the exception of the exposure to environmental toxicants (similar CV) and light physical exercise (similar CVi). That index was also positively correlated to the number of hours spent doing sedentary activities (p-value = 0.05). As with the analysis of conventional semen parameters, a single analysis of the sperm DNA fragmentation might be scarcely realiable to determine that parameter in men. Our study shows that certain male factors or characteristics may be related to a higher or lower variability of the sperm DNA fragmentation index.El objetivo de este trabajo fue estudiar la variabilidad del índice de fragmentación del ácido desoxirribonucleico (ADN) espermático en una cohorte de varones sanos y analizar los factores asociados a dicha variabilidad, incluyendo hábitos de vida y exposiciones ambientales. Se trata de un estudio prospectivo llevado a cabo durante 1 año evaluando múltiples muestras seminales (obtenidas aproximadamente cada 4-6 semanas) procedentes de 19 varones voluntarios sanos. Los sujetos cumplimentaron encuestas epidemiológicas sobre hábitos de vida y exposiciones ambientales en cada una de las entrevistas. Los individuos se clasificaron en dos grupos según sus respuestas acerca de determinados hábitos de vida o exposiciones ambientales (“sí” vs. “no”). Se calculó el coeficiente de variación (CV) y el coeficiente de variación intraindividual (CVi) del índice de fragmentación del ADN espermático y se examinaron sus diferencias estadísticas con respecto a sus respuestas a los factores estudiados. Los CV del índice de fragmentación del ADN espermático fueron significativamente distintos para todas las variables estudiadas, a excepción de la exposición a tóxicos ambientales (similar CV) y el ejercicio físico ligero (similar CVi). Dicho índice también se relacionó positivamente con el número de horas empleadas en actividades sedentarias (p-valor = 0,05). Como ocurre con el análisis de los parámetros seminales convencionales, un único análisis de fragmentación del ADN espermático podría ser poco fiable para determinar dicho parámetro en el varón. Este estudio muestra que determinados factores o características del varón podrían estar relacionadas con una mayor o menor variabilidad de su índice de fragmentación del ADN espermático.O objetivo deste trabalho foi estudar a variabilidade do índice de fragmentação do ácido desoxirribonucleico (ADN) espermático numa coorte de homens saudáveis e analisar os fatores associados a essa variabilidade, incluindo estilos de vida e exposições ambientais. Este é um estudo prospetivo, realizado por 1 ano, avaliando várias amostras de sémen (obtidas aproximadamente a cada 4-6 semanas) a partir de 19 voluntários saudáveis do sexo masculino. Os sujeitos preencheram questionários epidemiológicos sobre estilos de vida e exposição a fatores ambientais em cada uma das entrevistas. Os indivíduos foram classificados em dois grupos de acordo com suas respostas sobre determinados estilos de vida ou exposições ambientais (“sim” vs. “não”). Calculou-se o coe ciente de variação (CV) e o coe ciente de variação intra-individual (CVI) do índice de fragmentação do ADN do esperma e analisaram-se as diferenças estatísticas relativamente às respostas aos fatores estudados. Os VC do índice de fragmentação do ADN do esperma foram significativamente diferentes para todas as variáveis estudadas, com exceção da exposição a tóxicos ambientais (CV similar) e do exercício físico ligeiro (VCi similar). O índice também se relacionou positivamente com o número de horas gastas em atividades sedentárias (valor-p = 0,05). Tal como acontece com a análise dos parâmetros seminais convencionais, uma única análise de fragmentação de ADN espermático pode ser pouco fiável para determinar este parâmetro no homem. Este estudo mostra que determinados fatores ou características do indivíduo podem estar relacionados com uma maior ou menor variabilidade do seu índice de fragmentação do ADN espermático

Shigella-Induced Emergency Granulopoiesis Protects Zebrafish Larvae from Secondary Infection

Emergency granulopoiesis is a hematopoietic program of stem cell-driven neutrophil production used to counteract immune cell exhaustion following infection. Shigella flexneri is a Gram-negative enteroinvasive pathogen controlled by neutrophils. In this study, we use a Shigella-zebrafish (Danio rerio) infection model to investigate emergency granulopoiesis in vivo. We show that stem cell-driven neutrophil production occurs in response to Shigella infection and requires macrophage-independent signaling by granulocyte colony-stimulating factor (Gcsf). To test whether emergency granulopoiesis can function beyond homoeostasis to enhance innate immunity, we developed a reinfection assay using zebrafish larvae that have not yet developed an adaptive immune system. Strikingly, larvae primed with a sublethal dose of Shigella are protected against a secondary lethal dose of Shigella in a type III secretion system (T3SS)-dependent manner. Collectively, these results highlight a new role for emergency granulopoiesis in boosting host defense and demonstrate that zebrafish larvae can be a valuable in vivo model to investigate innate immune memory

TNFα Impairs Rhabdoviral Clearance by Inhibiting the Host Autophagic Antiviral Response.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections

Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish

The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age

A molecular roadmap of the AGM region reveals BMP ER as a novel regulator of HSC maturation

In the developing embryo, hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros (AGM) region, but the molecular regulation of this process is poorly understood. Recently, the progression from E9.5 to E10.5 and polarity along the dorso-ventral axis have been identified as clear demarcations of the supportive HSC niche. To identify novel secreted regulators of HSC maturation, we performed RNA sequencing over these spatiotemporal transitions in the AGM region and supportive OP9 cell line. Screening several proteins through an ex vivo reaggregate culture system, we identify BMP ER as a novel positive regulator of HSC development. We demonstrate that BMP ER is associated with BMP signaling inhibition, but is transcriptionally induced by BMP4, suggesting that BMP ER contributes to the precise control of BMP activity within the AGM region, enabling the maturation of HSCs within a BMP-negative environment. These findings and the availability of our transcriptional data through an accessible interface should provide insight into the maintenance and potential derivation of HSCs in culture.Peer reviewe

Mycobacterium abscessus-Induced Granuloma Formation Is Strictly Dependent on TNF Signaling and Neutrophil Trafficking

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections

Human haematopoietic stem cell development: From the embryo to the dish:From the embryo to the dish

Publisher Copyright: © 2017. Published by The Company of Biologists Ltd.Haematopoietic stem cells (HSCs) emerge during embryogenesis and give rise to the adult haematopoietic system. Understanding how early haematopoietic development occurs is of fundamental importance for basic biology and medical sciences, but our knowledge is still limited compared with what we know of adult HSCs and their microenvironment. This is particularly true for human haematopoiesis, and is reflected in our current inability to recapitulate the development of HSCs from pluripotent stem cells in vitro. In this Review, we discuss what is known of human haematopoietic development: the anatomical sites at which it occurs, the different temporal waves of haematopoiesis, the emergence of the first HSCs and the signalling landscape of the haematopoietic niche. We also discuss the extent to which in vitro differentiation of human pluripotent stem cells recapitulates bona fide human developmental haematopoiesis, and outline some future directions in the field.publishersversionPeer reviewe

Caracterización funcional de los receptores de TNF de pez cebra : Functional characterization of TNF receptors in cebrafish.

OBJETIVOS 1. Caracterización de los receptores de Tnf (Tnfr1 y Tnfr2) en la homeostasis vascular durante el desarrollo en pez cebra. 2. Caracterización de las rutas de señalización de Tnfr1 y Tnfr2 involucradas en el desarrollo de las células endoteliales y su homeostasis. 3. Caracterización del papel que juegan Tnfrs y sus ligandos (Tnfa y Lta) en la ola primitiva de hematopoyesis en el pez cebra. 4. Caracterización de los Tnfrs y sus ligandos (Tnfa y Lta) en la especificación y mantenimiento de las HSCs en el embrión de pez cebra. METODOLOGÍA Para la presente tesis doctoral, se ha utilizado el pez cebra (Danio rerio) como modelo de experimentación animal. Además, para la experimentación in vitro se han usado varias líneas celulares y técnicas de cultivo celulares. En cuanto a las técnicas de biología molecular, podemos destacar la microinyección en estadio de huevo de RNAm, morfolinos y DNA; RT-qPCR e hibridación in situ. También se han usado técnicas de microscopía tales como la microscopía confocal y de fluorescencia; técnicas inmunohistoquímicas y TUNEL para la detección de apoptosis; y técnicas de citometría y aislamiento físico de células mediante fluorescencia (FACS). RESULTADOS Durante la presente tesis doctoral, hemos caracterizado el papel que desempeñan el factor de necrosis tumoral (Tnfa) y sus receptores (receptores del factor de necrosis tumoral, Tnfrs) en el desarrollo de las células endoteliales y del sistema hematopoyético. Para ello hemos usado el pez cebra (Danio rerio), como modelo animal de vertebrados. Por un lado, nuestros resultados nos han llevado a concluir que la deficiencia en el gen de tnfr2 en embriones de pez cebra conlleva la inducción en células endoteliales de un programa apoptótico que depende de caspasa-8, caspasa-2 y P53, pero no caspasa-3. Además, la deficiencia simultánea de Tnfr1 o la activación de NF-ĸB, rescata la apoptosis de las células endoteliales, lo que indica que debe haber un equilibrio entre los dos receptores de Tnf para la correcta homeostasis vascular de este tipo celular. De forma similar, el Tnfa promueve la apoptosis de las células endoteliales humanas a través de Tnfr1, desencadenando la activación de caspasa-2 y P53. Por otro lado, en este trabajo demostramos que la señalización de Tnfa a través de Tnfr2 se necesita de forma intrínseca por las células madre hematopoyéticas (HSCs) para su mantenimiento y expansión. La deficiencia genética de Tnfa o Tnfr2, pero no de linfotoxina (Lta) o Tnfr1, conlleva la apoptosis de las HSCs cuando se especifican en el embrión. CONCLUSIONES 1. El silenciamiento génico dirigido contra Tnfr2 resulta en la inducción de un programa apoptótico dependiente de caspasa-8 en células endoteliales. Esta apoptosis se rescata mediante la eliminación de Tnfr1, lo que indica que se requiere un balance adecuado entre los Tnfrs para la integridad de las células endoteliales y la homeostasis vascular. 2. En las células endoteliales, el Tnfr1 señaliza apoptosis a través de la formación del complejo II y la consecuente activación de caspasa-8 y caspasa-2, mientras que Tnfr2 señaliza supervivencia mediante el comple I y la activación de NF-ĸB. 3. El programa apoptótico inducido por Tnfr1, en el cual participa caspasa-8, caspasa-2 y P53, se encuentra conservado evolutivamente en células endoteliales humanas. 4. La señalización de Tnfrs es dispensable para la hematopoyesis primitiva en el embrión de pez cebra. 5. La inhibición génica de Tnfa o Tnfr2, pero no Tnfr1 o Lta, resulta en la apoptosis de las HSCs cuando éstas emergen de la aorta dorsal. 6. La señalización Tnfa/Tnfr2 se requiere de forma intrínseca para la supervivencia de las HSCs a partir del endotelio hemogénico. OBJECTIVES 1. Characterization of Tnfa receptors (Tnfr1 and Tnfr2) in vascular homeostasis during the zebrafish embryo development. 2. Characterization of the Tnfr1 and Tnfr2 signaling pathways involved in endothelial cell development and homeostasis. 3. Characterization of the role played by Tnfrs and their ligands (Tnfa and Lta) in the primitive wave of hematopoiesis in the zebrafish embryo. 4. Characterization of Tnfrs and their ligands (Tnfa and Lta) in HSCs specification and maintenance in the zebrafish embryo. METHODOLOGY In this Doctoral Thesis, zebrafish (Danio rerio) has been utilized as research animal model. In addition, several cell lines and tissue culture techniques have been used for the in vitro experimentation. Related to the molecular biology techniques utilized, the injection at one-cell stage with mRNA, morpholinos and DNA; RT-qPCR and in situ hybridization can be emphasized. Moreover, other techniques used have been used such as confocal microscopy and fluorescence microscopy; immunohystochemistry techniques and TUNEL for apoptosis detection; as well as cytometry-related assays as fluorescence activated cell sorting (FACS). RESULTS During this Doctoral Thesis, we have characterized the role of Tnfa and Tnf receptors (Tnfrs) during endothelial and hematopoiesis development in the embryo using zebrafish (Danio rerio) as a vertebrate model. Targeted gene knockdown of Tnfr2 in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of Tnfr1 or the activation of NF-ĸB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. Similarly, Tnfa promotes the apoptosis of human endothelial cells through Tnfr1 and triggers caspase-2 and P53 activation. On the other hand, we show that Tnfa signaling through Tnfr2 is intrinsically required for hematopoietic stem cells (HSCs) maintenance and expansion. The genetic inhibition of Tnfa or Tnfr2, but not of lymphotoxin α (Lta) or Tnfr1, results in the apoptosis of HSCs soon after their emergence. CONCLUSIONS 1. Target gene silencing of Tnfr2 results in the induction of a caspase-8-dependent apoptotic program in endothelial cells. This apoptosis can be rescued by depletion of Tnfr1, indicating that an appropriate signaling balance between both Tnfrs is required for endothelial cell integrity and vascular homeostasis. 2. In endothelial cells, Tnfr1 signals apoptosis through complex II formation and caspase-8 and caspase-2 activation, while Tnfr2 signals survival via complex I and NF-ĸB activation. 3. The apoptotic program induced by Tnfr1, which involves caspase-8, caspase-2 and P53, is evolutionary conserved in human endothelial cells. 4. Tnfrs signaling is dispensable for primitive hematopoiesis in the zebrafish embryo. 5. Genetic inhibition of Tnfa or Tnfr2, but not of Tnfr1 or Lta, results in HSC apoptosis soon after their emergence from the floor of the dorsal aorta. 6. Tnfa/Tnfr2 signaling is intrinsically required for HSC survival after their emergence from the hemogenic endothelium

Md1 and Rp105 regulate innate immunity and viral resistance in zebrafish

11 páginas, 7 figuras, 2 tablasTLR4 was the first TLR family member identified in mammals and is responsible for the activation of the immune response by bacterial LPS. Later, MD1 and RP105 were shown to form complexes that directly interact with the MD2-TLR4 complex, acting as physiological negative regulators of LPS signaling. Despite the general conservation of various TLR families from fish to mammals, several differences can be appreciated, such as the high tolerance of fish to LPS, the absence of the crucial accessory molecules Md2 and Cd14 for Tlr4 signaling in fish, the absence of Tlr4 in some fish species, and the confirmation that LPS does not signal through Tlr4 in zebrafish. The present study has identified the Rp105 and Md1 homologs in zebrafish, confirming (i) Rp105 and Tlr4 evolved from a common ancestor before the divergence between fish and tetrapods and (ii) the presence of Md1 in teleost fish and the lack of Md2, suggesting that the divergence of these accessory molecules occurred in the tetrapod lineage. Biochemical and functional studies indicate that Md1 binds both Rp105 and Tlr4 in zebrafish. Genetic inhibition of zebrafish Md1 and Rp105 reveals that Md1 or Rp105 deficiency impairs the expression of genes encoding pro-inflammatory and antiviral molecules, leading to increased susceptibility to viral infection. These results shed light on the evolutionary history of Md1 and Rp105 and uncover a previously unappreciated function of these molecules in the regulation of innate immunityThis work was supported by the Spanish Ministry of Science and Innovation (grants BIO2011-23400 and CSD2007-00002 to VM, and PhD fellowship to SC, all co-funded with Fondos Europeos de Desarrollo Regional/European Regional Development Funds), the Fundación Séneca-Murcia (PhD fellowship to RE-P), the European 7th Framework Initial Training Network FishForPharma (PhD fellowship to SDT, PITG-GA-2011-289209), and Fundação para a Ciência e Tecnologia (PhD fellowship to SdO, SFRH/BD/62674/2009)Peer reviewe