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[Abstract] Aims. After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA. Methods. Using telomerase reverse transcriptase (hTERT) and SV40 large T antigen (SV40LT), we transduced primary OA articular chondrocytes. Proliferative capacity, degree of senescence, and chondrocyte surface antigen expression in transduced chondrocytes were evaluated. In addition, the capacity of transduced chondrocytes to synthesize a tissue similar to cartilage and to respond to interleukin (IL)-1β was assessed. Results. Coexpression of both transgenes (SV40 and hTERT) were observed in the nuclei of transduced chondrocytes. Generated chondrocyte cell lines showed a high proliferation capacity and less than 2% of senescent cells. These cell lines were able to form 3D aggregates analogous to those generated by primary articular chondrocytes, but were unsuccessful in synthesizing cartilage-like tissue when seeded on type I collagen sponges. However, generated chondrocyte cell lines maintained the potential to respond to IL-1β stimulation. Conclusion. Through SV40LT and hTERT transduction, we successfully immortalized chondrocytes. These immortalized chondrocytes were able to overcome senescence in vitro, but were incapable of synthesizing cartilage-like tissue under the experimental conditions. Nonetheless, these chondrocyte cell lines could be advantageous for OA investigation since, similarly to primary articular chondrocytes, they showed capacity to upregulate inflammatory mediators in response to the IL-1β cytokine. Xunta de Galicia; ED431B 2020/55 Xunta de Galicia; IN607A2021/07 Instituto de Salud Carlos III; PI20/00933 Instituto de Salud Carlos III; PI17/02197 Instituto de Salud Carlos III; PI19/01206

Background. Betamethasone, a glucocorticoid used to induce lung maturation when there is a risk of preterm delivery, can affect the immune system maturation and type 1 diabetes (T1D) incidence in the progeny. It has been described that prenatal betamethasone protects offspring from experimental T1D development. The main aim of this study was to evaluate the possible association between betamethasone prenatal exposure and T1D in humans. Research Design and Methods. A retrospective case-control study with a total of 945 children, including 471 patients with T1D and 474 healthy siblings, was performed. Participants were volunteers from the Germans Trias i Pujol Hospital and DiabetesCero Foundation. Parents of children enrolled in the study completed a questionnaire that included questions about weeks of gestation, preterm delivery risk, weight at birth, and prenatal betamethasone exposure of their children. Multiple logistic regression was used to detect the association between betamethasone exposure and T1D. Results. We compared T1D prevalence between subjects prenatally exposed or unexposed to betamethasone. The percent of children with T1D in the exposed group was 37.5% (21 of 56), and in the unexposed group was 49.52% (410 of 828) ( p = 0.139 ). The percentage of betamethasone-treated subjects with T1D in the preterm group (18.05%, 13 of 72) was significantly higher than that found in the control group (12.5%, 9 of 72) ( p = 0.003 ). The odds ratio for T1D associated with betamethasone in the univariate logistic regression was 0.59 (95% confidence interval, 0.33; 1.03 [ p = 0.062 ]) and in the multivariate logistic regression was 0.83 (95% confidence interval, 0.45; 1.52 [ p = 0.389 ]). Conclusions. The results demonstrate that the prenatal exposure to betamethasone does not increase T1D susceptibility, and may even be associated with a trend towards decreased risk of developing the disease. These preliminary findings require further prospective studies with clinical data to confirm betamethasone exposure effect on T1D risk.

[EN] The partial remission (PR) phase, a period experienced by most patients with type 1 diabetes (T1D) soon after diagnosis, is characterized by low insulin requirements and improved glycemic control. Given the great potential of this phase as a therapeutic window for immunotherapies because of its association with immunoregulatory mechanisms and beta-cell protection, our objective was to find peripheral immunological biomarkers for its better characterization, monitoring, and prediction. The longitudinal follow-up of 17 pediatric patients with new-onset T1D over one year revealed that, during the PR phase, remitter patients show increased percentages of effector memory (EM) T lymphocytes, terminally differentiated EM T lymphocytes, and neutrophils in comparison to non-remitter patients. On the contrary, remitter patients showed lower percentages of naive T lymphocytes, regulatory T cells (T-REG), and dendritic cells (DCs). After a year of follow-up, these patients also presented increased levels of regulatory B cells and transitional T1 B lymphocytes. On the other hand, although none of the analyzed cytokines (IL-2, IL-6, TGF-beta 1, IL-17A, and IL-10) could distinguish or predict remission, IL-17A was increased at T1D diagnosis in comparison to control subjects, and remitter patients tended to maintain lower levels of this cytokine than non-remitters. Therefore, these potential monitoring immunological biomarkers of PR support that this stage is governed by both metabolic and immunological factors and suggest immunoregulatory attempts during this phase. Furthermore, since the percentage of T-REG, monocytes, and DCs, and the total daily insulin dose at diagnosis were found to be predictors of the PR phase, we next created an index-based predictive model comprising those immune cell percentages that could potentially predict remission at T1D onset. Although our preliminary study needs further validation, these candidate biomarkers could be useful for the immunological characterization of the PR phase, the stratification of patients with better disease prognosis, and a more personalized therapeutic management. Funding for this study was provided by the Spanish Government (FIS PI18/00436) co-financed with the European Regional Development funds (FEDER), and by DiabetesCero Foundation. LGM is supported by the Generalitat de Catalunya (PERIS PIF-Salut Grant No. SLT017/20/000049). This work has been supported by positive discussion through Consolidated Research Group #2017 SGR 103, AGAUR, Generalitat de Catalunya.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of insulin-producing cells. Due to the ability of apoptotic cells clearance to induce tolerance, we previously generated liposomes rich in phophatidylserine (PS) –a feature of apoptotic cells– loaded with insulin peptides to mimic apoptotic beta-cells. PS-liposomes arrested autoimmunity in experimental T1D through the induction of tolerance. The aim of this study was to investigate the potential of several peptides from different T1D autoantigens encapsulated in (PS)-liposomes for T1D prevention and to assess its safety. T1D autoantigens (Insulin, C-peptide, GAD65 and IA2) were encapsulated in PS-liposomes. Liposomes were administered to the ’gold-standard’ model for the study of autoimmune T1D, the Non-Obese Diabetic mouse, that spontaneously develop the disease. Safety and toxicity of liposomes were also determined. Only PS-liposomes encapsulating insulin peptides decrease T1D incidence in the Non-Obese Diabetic mouse model. Disease prevention correlates with a decrease in the severity of the autoimmune islet destruction driven by leukocytes. PS-liposomes neither showed toxic effect nor secondary complications. Among the here referred autoantigens, insulin peptides are the best candidates to be encapsulated in liposomes, like an artificial apoptotic cell, for the arrest of autoimmunity in T1D in a safe manner., This work was suported by the Foundation La Marato de TV3 under Grant 201632_10. CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM) is an initiative from Instituto de Salud Carlos III (Spain). SRF is supported by the Agency for Management of University and Research Grants (AGAUR) of the Generalitat de Catalunya.

Resumen Xunta de Galicia; R2016/036 Xunta de Galicia; R2014/050 Xunta de Galicia; CN2012/142 Xunta de Gaicia; GPC2014/048 Instituto de salud Carlos III; PI17/02197

Les malalties autoimmunitàries estan causades per defectes en la tolerància immunològica, i afecten a gairebé un 10% de la població. Darrerament, diverses intervencions mèdiques han convertit aquestes malalties en cròniques, però el seu diagnòstic encara comporta morbiditat i mortalitat elevades. Així, un repte biomèdic urgent és el desenvolupament de teràpies que puguin restablir selectivament la tolerància, aturin l’atac autoimmunitari i permetin la regeneració del teixit danyat. En condicions fisiològiques, la fagocitosi de cèl·lules apoptòtiques per part de fagòcits com les cèl·lules dendrítiques (CDs) —procés que rep el nom d’eferocitosi— els indueix propietats tolerogèniques i l’habilitat de restaurar la tolerància. Una demostració d’això és que una immunoteràpia cel·lular consistent en CDs tolerogèniques (CDtols) degut a l’eferocitosi de cèl·lules β apoptòtiques va aturar l’atac autoimmunitari contra les cèl·lules β en un model experimental de diabetis tipus 1 (DT1). Donades les dificultats en obtenir i estandarditzar cèl·lules β apoptòtiques autòlogues humanes per la seva implementació clínica, es va dissenyar una nanoteràpia basada en liposomes que simulen cèl·lules apoptòtiques. Les principals característiques d’aquestes vesícules sintètiques són: elevat percentatge de fosfatidilserina (FS) —fosfolípid característic de la membrana de les cèl·lules apoptòtiques—, diàmetre major de 500 nm, càrrega negativa i eficient encapsulació de pèptids d’insulina. Aquesta estratègia és tant efectiva en generar CDtols i aturar l’autoimmunitat contra les cèl·lules β com la immunoteràpia basada en cèl·lules apoptòtiques. La hipòtesi d’aquest treball és que els FS-liposomes poden restablir la tolerància en diverses malalties autoimmunitàries antigen-específiques mitjançant la generació de CDtols i l’expansió de limfòcits T reguladors, i que tenen el potencial translacional per abordar patologies autoimmunitàries humanes. L’objectiu principal d’aquest estudi ha estat caracteritzar globalment el potencial tolerogènic dels FS-liposomes. Amb aquesta finalitat, diferents pèptids autoantigènics rellevants en malalties autoimmunitàries s’han encapsulat en FS-liposomes de manera eficient i sense dificultats en mantenir el diàmetre i la càrrega, demostrant la versatilitat de l’estratègia a diferents patologies autoimmunitàries. En el model experimental de DT1, l’administració de FS-liposomes ha expandit clons de cèl·lules T CD4+ reguladores i T CD8+ que contribueixen a l’efecte tolerogènic de la teràpia a llarg termini. En el mateix model, s’ha demostrat la biocompatibilitat i seguretat del producte final donada la seva òptima tolerabilitat. D’altra banda, els FS-liposomes s’han adaptat al model d’esclerosi múltiple experimental simplement reemplaçant el pèptid encapsulat. En aquest model, els FS-liposomes han induït CDtols i han reduït la incidència i severitat de la malaltia correlacionant amb un increment en la freqüència de cèl·lules T reguladores, fet que valida el potencial dels FS-liposomes a constituir una plataforma per a la recuperació de tolerància en diferents malalties autoimmunitàries. Finalment, i de cara a una futura implementació clínica, s’han determinat els efectes de la teràpia en CDs humanes obtingudes de pacients amb DT1. En CDs de pacients adults, els FS-liposomes han estat fagocitats eficientment per les CDs amb una ràpida cinètica dependent de la FS, i això ha causat l’adquisició d’un transcriptoma, fenotip i funcionalitat tolerogènics similars als observats en els models experimentals. En CDs de pacients pediàtrics, però, les CDs han presentat defectes en la seva capacitat fagocítica correlacionant amb el temps de progressió de la malaltia; tanmateix, el seu fenotip i expressió de gens immunoreguladors després de la fagocitosi dels FS-liposomes són indicatius d’una habilitat tolerogènica òptima. En conclusió, la immunoteràpia liposomal descrita, que es basa en l’eferocitosi com a mecanisme inductor de tolerància, assoleix el mimetisme apoptòtic de manera simple, segura i eficient. A més, els liposomes ofereixen avantatges quant a producció i estandardització. Per tant, els FS-liposomes tenen potencial translacional i constitueixen una estratègia prometedora per recuperar la tolerància immunològica en malalties autoimmunitàries antigen-específiques. Autoimmune diseases are caused by defective immunological tolerance, and reportedly affect up to 10% of the global population. In the last years, current medical interventions have transformed these disorders into chronic and manageable, but they still entail high rates of morbidity and mortality. Hence, there is an urgent need to develop therapies capable of restoring the breach of tolerance selectively, which halt the autoimmune aggression and allow the regeneration of the targeted tissue. In physiological conditions, the phagocytosis of apoptotic cells performed by phagocytes such as dendritic cells (DCs) —a process termed efferocytosis— prompts the acquisition of tolerogenic features and the ability to restore tolerance. Indeed, a cell immunotherapy consisting of DCs rendered tolerogenic (tolDCs) by apoptotic β-cell efferocytosis arrested the autoimmune attack against β-cells in an experimental model of type 1 diabetes (T1D). However, in light of the hurdles in obtaining and standardising human autologous apoptotic β-cells for its implementation in the clinics, a nanotherapeutic strategy based on liposomes mimicking apoptotic cells was designed. The fundamental characteristics of these synthetic vesicles are: a high percentage of phosphatidylserine (PS) —phospholipid unique to the apoptotic cell membrane—, diameter superior to 500 nm, negative charge and efficient encapsulation of insulin peptides. Importantly, this strategy was equally effective in inducing tolDCs and blunting β-cell autoimmunity as the immunotherapy based on apoptotic cells. The hypothesis of this work is that autoantigen-loaded PS-liposomes can re-establish tolerance in several antigen-specific autoimmune diseases through the induction of tolDCs and the expansion of regulatory T lymphocytes, and that they have translational potential to tackle human autoimmune disorders. The main aim of the present work has been to characterise the tolerogenic potential of PS-liposomes globally. To this end, different autoantigenic peptides relevant in autoimmune diseases have been efficiently encapsulated into PS-liposomes, without difficulties in preserving their appropriate diameter and charge, thus demonstrating the versatility of the therapy to different autoimmune pathologies. In the experimental model of T1D, the administration of PS-liposomes causes the expansion of clonal CD4+ regulatory T cells and CD8+ T cells, which contribute to the long-term re-establishment of tolerance. Moreover, in the same model, the biocompatibility and safety of the final product have been confirmed given its optimal tolerability. Furthermore, PS-liposomes have been adapted to the experimental multiple sclerosis model by merely replacing the encapsulated autoantigen. In this model, PS-liposomes elicit the generation of tolDCs and decrease the incidence and severity of the disease correlating with an increase in the frequency of regulatory T cells, a fact that validates the potential of PS-liposomes to serve as a platform for tolerance re-establishment in different autoimmune diseases. Finally, considering its future clinical implementation, the effect of the PS-liposomes therapy has been determined in human DCs obtained from patients with T1D. In DCs from adult patients, PS-liposomes are efficiently phagocyted by DCs with rapid kinetics dependent on the presence of PS, and this induces a tolerogenic transcriptome, phenotype and functionality that are similar to those observed in experimental models. However, DCs from paediatric patients display defects in their phagocytic capacity correlating with the time of disease progression, albeit their phenotype and immunoregulatory gene expression after PS-liposomes phagocytosis point to an optimal tolerogenic ability. In conclusion, the liposomal immunotherapy herein described, which is based on efferocytosis as a powerful tolerance-inducing mechanism, achieves apoptotic mimicry in a simple, safe and efficient manner. Additionally, liposomes offer advantages in terms of production and standardisation. Therefore, PS-liposomes possess translational potential and constitute an encouraging strategy to restore immunological tolerance in antigen-specific autoimmune diseases.

Altres ajuts: ISCIII/PI15/00198, AGAUR, Fundación DiabetesCero Autoimmunity against β-cells in type 1 diabetes (T1D) is prompted by defective immunological tolerance, an event in which dendritic cells (DCs) play a crucial role as orchestrators of the immune response. Relying on the inherent ability of a apoptotic cell clearance to induce tolerance, we designed a liposomal nanotherapy rich in phophatidylserine (PS) -a characteristic signal of the apoptotic cell membrane- and loaded with insulin to mimic apoptotic β-cells. PS-liposomes administration blunted autoimmunity in experimental T1D through the generation of tolerogenic DCs. Moreover, human DCs from adult patients with T1D were also rendered tolerogenic after PS-liposomes phagocytosis. However, since T1D in children is often more complicated to manage and severe dysglycaemia could impair DCs functionality, our aim was to explore the therapeutic value of PS-liposomes in DCs from paediatric patients (PP) with T1D.

Altres ajuts: This work has been co-financed with the European Regional Development funds (FEDER), and supported by the Deutsche Forschungsgemeinschaft (KFO296). CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM) is an initiative from Instituto de Salud Carlos III. SRF is supported by the Agency for Management of University and Research Grants (AGAUR) of the Generalitat de Catalunya. Non-genetic factors are crucial in the pathogenesis of type 1 diabetes (T1D), a disease caused by autoimmunity against insulin-producing β-cells. Exposure to medications in the prenatal period may influence the immune system maturation, thus altering self-tolerance. Prenatal administration of betamethasone -a synthetic glucocorticoid given to women at risk of preterm delivery- may affect the development of T1D. It has been previously demonstrated that prenatal betamethasone administration protects offspring from T1D development in nonobese diabetic (NOD) mice. The direct effect of betamethasone on the immature and mature immune system of NOD mice and on target β-cells is analysed in this paper. In vitro, betamethasone decreased lymphocyte viability and induced maturation-resistant dendritic cells, which in turn impaired γδ T cell proliferation and decreased IL-17 production. Prenatal betamethasone exposure caused thymus hypotrophy in newborn mice as well as alterations in immune cells subsets. Furthermore, betamethasone decreased β-cell growth, reduced C-peptide secretion and altered the expression of genes related to autoimmunity, metabolism and islet mass in T1D target tissue. These results support the protection against T1D in the betamethasone-treated offspring and demonstrate that this drug alters the developing immune system and β-cells. Understanding how betamethasone generates self-tolerance could have potential clinical relevance in T1D.

Resumen del póster publicado en el II Annual Meeting CINBIO abstracts book [Internet], p. 65 [Abstract] Introduction: Endothelium is the inner layer of the cornea, which must be viable for transplanting. The limited availability of corneas makes necessary the developing of preservation techniques that allow a long storage without losing endothelial viability.Objectives: Optimization of a cell viability assay in preserved corneas.Methods: One half of an endothelium from a cornea that was storage in hypothermic conditions and an endothelium of a cryopreserved cornea were stained with LIVE/DEAD imaging kit and Hoechst. The other half of endothelium was the negative control. Corneal endothelia were imaged using a fluorescence microscope.Results: Four sort of cells were visualized on both endothelia: viable cells with high esterase activity, intermediate cells with low esterase activity, non-viable cells without esterase activity, and cells only stained by Hoechst. Conclusions: Triple stain is effectiveto detect different sort of cells in endothelium of preserved corneas, included viable cells, depending on their esterase enzymatic activity and on cell and nuclear membrane damage.

[Resumen] El objetivo de este trabajo ha sido evaluar la eficacia de cuatro protocolos de criopreservación diferentes atendiendo a la viabilidad celular endotelial y a la estructura de las córneas tras su descongelación. Un total de n=3 córneas fueron criopreservadas con cada uno de los protocolos siguientes: criopreservación lenta con albúmina (Grupo 1), criopreservación lenta sin albúmina (Grupo 2), criopreservación rápida mediante vitrificación con VS55 (Grupo 3) y criopreservación rápida mediante vitrificación con DP6 (Grupo 4). Tras la descongelación, se realizó sobre dos córneas de cada grupo un ensayo de viabilidad para evaluar la celularidad y la viabilidad de las células endoteliales. Paralelamente, se realizó la tinción de tricrómico de Masson en una córnea descongelada de cada grupo para analizar la estructura histomorfológica. Las córneas del Grupo 4 mostraron, cualitativamente, más celularidad que las córneas del Grupo 3, seguidas de las córneas del Grupo 1 y Grupo 2. Respecto a la viabilidad, el Grupo 1 mostró un 66,79% de células viables, el Grupo 2 presentó muy pocas células viables, y los grupos 3 y 4, ninguna. Los daños de la criopreservación se observaron en los desprendimientos del endotelio en los Grupos 2, 3 y 4, en las oquedades amplias del estroma en los Grupos 1, 3 y 4, y en el estrechamiento y pérdida de células y de volumen celular en el epitelio de todos los grupos. La criopreservación con cualquiera de los cuatro protocolos utilizados aportan celularidades endoteliales altas tras la descongelación, mostrando la mayor celularidad la criopreservación rápida. Sin embargo, la viabilidad celular endotelial se ve más afectada en la criopreservación rápida que en la criopreservación lenta, que sí retiene cierta viabilidad celular. El endotelio, el epitelio y el estroma de la córnea son capas sensibles a los daños de la criopreservación, en especial durante la criopreservación rápida. [Resumo] O obxectivo deste traballo foi a avaliación da eficacia de catro protocolos de criopreservación diferentes, atendendo á viabilidade celular endotelial e á estrutura das córneas tras a súa desconxelación. Un total de n=3 córneas foron criopreservadas con cada un dos protocolos seguintes: criopreservación lenta con albumina (Grupo 1), criopreservación lenta sen albumina (Grupo 2), criopreservación rápida mediante vitrificación con VS55 (Grupo 3) e criopreservación rápida mediante vitrificación con DP6 (Grupo 4). Tras a desconxelación, realizouse sobre dúas córneas de cada grupo un ensaio de viabilidade para avaliar a celularidade e a viabilidade das células endoteliais. Paralelamente, realizouse a tinguidura de tricrómico de Masson nunha córnea desconxelada de cada grupo para analizar a estrutura histomorfolóxica. As córneas do Grupo 4 mostraron, cualitativamente, máis celularidade que as córneas do Grupo 3, seguidas das córneas do Grupo 1 e Grupo 2. Respecto á viabilidade, o Grupo 1 mostrou un 66,79% de células viables, o Grupo 2 presentou moi poucas células viables, e os grupos 3 e 4, ningunha. Os danos da criopreservación observáronse nos desprendementos do endotelio nos Grupos 2, 3 y 4, nos ocos amplos do estroma nos Grupos 1, 3 y 4, e no estreitamento e perda de células e de volume celular do epitelio de todos os grupos. A criopreservación con calquera dos catro protocolos utilizados aportan celularidades endoteliais altas tras a desconxelación, mostrando a maior celularidade a criopreservación rápida. Sen embargo, a viabilidade celular endotelial vese máis afectada na criopreservación rápida que na criopreservación lenta, que si retén certa viabilidade celular. O endotelio, o epitelio e o estroma da córnea son capas sensibles aos danos da criopreservación, en especial durante a criopreservación rápida. [Abstract] The aim of this study was the evaluation of four protocols of cryopreservation, attending to the cell endothelial viability and to the cornea structure after thawing. A total of n=3 corneas were cryopreserved using the following: slow cryopreservation with albumin (Group 1), slow cryopreservation without albumin (Group 2), fast cryopreservation with vitrification using VS55 solution (Group 3), fast cryopreservation with vitrification using DP6 solution media (Group 4). After thawing, a viability assay was performed over two corneas to evaluate the cellularity and viability of endothelial cells. A Masson staining was performed in one thawed cornea of each group. Corneas in Group 4 showed the huge cellularity, followed by corneas of Group 3, Group 1 and Group 2. Corneas of Group 1 showed 66,79% of viable cells, while in Group 2 there were very few viable cells, and no viable cells in Groups 3 and 4. Cryoinjuries were observed in endothelium detachment in Groups 2, 3 and 4 and in the holes among collagen fibres of strome in Groups 1, 3 and 4. Also, the thinner epithelial layer and its cell losing in all the groups are cryoinjuries. Cryopreservation with any of the four protocols allow a high endothelial cellularity after thawing. Fast cryopreservation offers the best result in cellularity. Despite of all, endothelial cell viability is affected with fast cryopreservation. Slow cryopreservation retains some cell viability. Endothelium, epithelium and strome are three sensitive layers of cornea that suffer cryoinjuries, in special, when the fast cryopreservation is carries out. Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en Fundamentos de investigación biomédica. Curso 2017/2018.

[Resumen] Identificar los procesos que rigen el ensamblado y desensamblado de las comunidades animales resulta fundamental para contribuir a su conservación. Por ello, exploramos arversos aspectos de la estructura de las comunidades de anfibios del noroeste ibérico durante una secuencia temporal de once años (2003-2013), a lo largo de un gradiente altitudinal (1-2.036 m.s.n.m) en 3.627 puntos de muestreo, con énfasis en el papel de las perturbaciones antrópicas. Los análisis revelaron que aunque los anfibios gallegos utlnrnron todo tipo de medios acuáticos y terrestres, la riqueza de especies fue mayor en los puntos de agua con elevada cobertura de macrófitos sumergidos, ubicados a menor altitud, y de gran superficie. Detectamos anidamiento en la comunidad, de magnitud moderada y temporalmente estable, lo que indica cierta plasticidad, resistencia y resiliencia de estas especies ante las perturbaciones humanas y los cambios ambientales. Además, los anfibios se vieron más influenciados por los impactos indirectos de la actividad humana que por perturbaciones directas. Se destaca la necesidad de conservar la metacomunidad en su conjunto, la idoneidad de generar una red de microrreservas para anfibios en Galicia, y la necesidad de mantener al menos, dentro de los espacios naturales protegidos, el tradicional paisaje en mosaico de las comunidades agrosilvopastorales gallegas., [Resumo] Identilicar os procesos que rexen o ensamblado e desensamblado das comunidades animais resulta fundamental para contribuir á súa conservación. Por iso, exploramos diversos aspectos da estrutura das comunidades de anfibios do noroeste ibérico durante un~a secuencia temporal de once anos (2003-2013), ao langa dun gradiente altitudinal (1-2.036 m.s.n.m) en 3.627 puntos de mostraxe, con énfase no papel das pert~rbacións antrópicas. As análises revelaron que ainda que os anfibios galegos utilizaron todo tipo de medios acuáticos e terrestres, a riqueza de especies foi maior nos puntos de auga con elevada cobertura de macrófitos mergullados, situados a menor altitude, e de gran superficie. Detectamos aniñamento na comunidade, de magnitude moderada e temporalmente estable, o que indica certa plasticidade, resistencia e resiliencia destas especies ante as perturbacións humanas e os cambios ambientais. Ademais, os anfibios vrronse máis influenciados polos impactos indirectos da actividade humana que por perturbacións directas. Destácase a necesldade de conservar a metacomunldade no seu conxunto, a idoneidade de xerar unha red e de microrreservas para anfibios en Galicia, e a necesidade de manter polo menos, dentro dos espazos naturais protexidos, a tradicional paisaxe en mosaico das comunidades agrosilvopastorales galegas., [Abstract] Identifying the processes governing the assembly and disassembly of animal communities is essential to contribute to their conservation. Thus, we explored different features of the structure of amphibian communities in the NW of the Iberian 'Peninsula during a time sequence of eleven years (2003-2013), along an altitudinal gradient (1-2.036 m.a.s.l.) in 3.627 sample points, with emphasis on the role of human disturbance on community arquitecture. Qur analyses revealed that although Galician amphibians used all kinds of aquatic and terrestrial habitats, species richness was higher in wetlands with high coverage of submerged macrophytes, located at a lower allitude, and with a larger area. We detected nestedness of moderate magnitude and temporally stable in the metacommunity, which shows certain plasticity, resistance and resilience of these species both to human disturbance and environmental changes. IR addition, the presence/absence of amphibians was more influenced by indirect impacts of human activity than by direct perturbance. These results highlight the need to preserve the metacommunity as a whole, the suitability of creating a network of micro-reserves for amphibians in Galicia, and the need to maintain, at least within protected areas, the mosaic landscape of traditional human rural Galician communities.

[Resumo] A diabetes MODY é una enfermidade monoxénica pouco frecuente dentro de todos os casos de diabetes, que se caracteriza por una progresiva disfunción nas células β- pancreáticas, por ser independente de insulina e comezar a desenvolverse en idades moi tempranas. Un diagnóstico molecular pode chegar a supoñer un cambio de tratamento nun paciente, un diagnóstico precoz ou un seguimento efectivo da progresión da enfermidade. Existen unha serie de xenes implicados na diabetes MODY e diversas institucións están identificando variantes relacionadas con esta patoloxía a través da secuenciación dun panel de xenes mediante NGS (Next Generation Sequencing). A NGS está afianzándose na xenética clínica pois permite a realización de diagnóstico molecular a varios pacientes simultaneamente e en moi pouco tempo. Para este traballo foron seleccionados once pacientes baixo os criterios do Complexo Hospitalario Universitario de A Coruña para a realización dun diagnóstico molecular para diabetes MODY. Este consistiu na secuenciación dun panel de xenes relacionados coa mesma (ABCC8, GCK, HNF1A, HNF1B, HNF4A, KCJ11, INS) mediante tecnoloxía NGS. Deste xeito foi realizada unha posta a punto da técnica para a elaboración dun Protocolo Normalizado de Traballo (PNT) que se utilizará rutinariamente no laboratorio e unha análise preliminar das variantes obtidas. Atopáronse variantes de interese clínico en cinco dos once pacientes (unha patolóxica, dúas probablemente patolóxicas e catro de significado incerto), as cales serán estudadas posteriormente e confirmadas mediante secuenciación Sanger. A posterior integración desta información será clave para complementar o diagnóstico clínico de cada paciente. [Abstract] Maturity-onset diabetes of the young (MODY) is a monogenic disorder with a low frequency amongst all diabetes cases. Some of the characteristics features of MODY are the progressive pancreatic β-cell dysfunction, as well as non-insulin dependence, and early-onset diabetes. A molecular diagnosis may imply a change in the treatment, an early diagnosis or a better management of the clinical course of this disease. Several genes are associated with MODY. Some institutions are currently performing variant detection using a gene panel, one of the applications of the NGS (Next Generation Sequencing) technology. NGS technologies have enabled a dramatic transformation in clinical genetics, as they allow molecular diagnoses for many patients at once and in a short time. Following the criteria established by the Complexo Hospitalario Universitario de A Coruña, eleven patients were selected to participate in a molecular diagnosis of MODY. For this, we sequenced a gene panel (ABCC8, GCK, HNF1A, HNF1B, HNF4A, KCJ11, INS) using NGS technology. As a result of this pilot study, we set up a Standard Operating Procedure (SOP), which will be used as a routine in this laboratory. Additionally, we proceeded with a preliminary variant analysis. Clinically relevant variants were found in five out of eleven patients: one pathogenic, two likely pathogenic and four variants of uncertain significance. All of them will be studied and validated by Sanger sequencing. The subsequent integration of this information will be key to complement the clinic diagnosis of each patient. Traballo fin de grao (UDC.CIE). Bioloxía. Curso 2015/2016

Resumen del trabajo presentado al X Congrés de la Societat Catalana d'Inmunologia: "Immunoteràpia contra el Càncer", session III: "From Innate to Adaptative Immunity"; celebrado en Barcelona (España) del 17 al 18 de noviembre de 2016.-- et al., Autoimmune diseases are caused by the destruction of the host’s own cells by autoreactive lymphocytes. There are nearly 100 autoimmune diseases, with increasing incidence and affecting around 5% of the population. Genetic and environmental factors contribute to autoimmunity but the triggering factors remain unknown, and current therapies are poorly effective and cause side effects. Therefore, new and safe approaches are required to arrest autoimmunity and allow target tissue regeneration. Based on apoptotic cells’ ability to induce self-tolerance after phagocytosis, liposomes that possess their inherent tolerogenic features have been designed. Liposomes are phospholipid bilayer vesicles that constitute a versatile system for modulation of immune responses. The herein reported liposomes are made of phosphatidylcholine, cholesterol, phosphatidylserine (PS) —the main 'eat me' signal exposed in the membrane of apoptotic cells—, and display a diameter greater than 500 nm to promote phagocytosis. Liposome’s ability to restore self-tolerance was tested in experimental models of autoimmune diseases: the non-obese diabetic (NOD) mouse for type 1 diabetes (T1D), and the experimental autoimmune encephalomyelitis (EAE)-induced mouse for multiple sclerosis (MS). Liposomes were generated encapsulating disease-specific autoantigens: insulin peptides for T1D and myelin-oligodendrocyte glycoprotein peptide for MS. Liposomes were efficiently engulfed by dendritic cells, inducing a tolerogenic phenotype and function. Liposomes were successful at arresting autoimmunity in vivo: T1D and EAE’s incidence was decreased and their onset delayed. Moreover, EAE’s severity was reduced. PS-Liposomes induced an antigen-specific CD4+ FoxP3+ and FoxP3– T cell response in the T1D model, and FoxP3– T lymphocytes correlated with minor clinical signs and hinted at belonging to Type 1 regulatory T cells in the MS model. This work validates PS-liposomes as a powerful tool for the re-establishment of tolerance. Working synergistically, autoantigen-loaded PS-liposomes co-deliver a double signal of tolerance and specificity to arrest autoimmune reactions in an effective and safe manner.

El dolor crónico continúa siendo un reto para las ciencias de la salud y para la enfermería debido a la repercusión psicológica en el individuo, así como, a la elevada prevalencia y gasto en nuestra sociedad. Comprender el dolor desde una perspectiva multidisciplinar, conseguir un mejor control de este y de las variables psicológicas, insomnio y ansiedad, son los objetivos expuestos. La revisión bibliográfica en numerosos libros, revistas científicas, páginas web y la utilización de la herramienta online NNNConsult han sido imprescindibles para el desarrollo de este trabajo. La personalidad condiciona la forma de vivenciar el dolor. El afrontamiento activo, permite controlar el dolor y disminuir las consecuencias psicológicas. Por el contrario, el afrontamiento pasivo y los mecanismos de defensa se asocian a una peor adaptación. El desarrollo de actitudes como la empatía, la aceptación incondicional o la autenticidad están relacionadas con resultados positivos en los pacientes. Se debe abandonar el tratamiento farmacológico exclusivo sustituyéndolo o combinándolo con terapias cognitivo-conductuales y con métodos físicos. En el plan de cuidados, las actividades de enfermería propuestas van a estar dirigidas a un mejor manejo del dolor y de las respuestas psicológicas adversas. La implicación familiar y una correcta educación sanitaria son necesarias para la mejora en el afrontamiento de los problemas desencadenados. Las múltiples opciones terapéuticas y su accesibilidad para toda la población son algunos de los aspectos positivos que se contraponen a los problemas económicos y al uso exclusivo de tratamientos farmacológicos. Nuestra presencia y paciencia son, y serán siempre, el mejor analgésico., Grado en Enfermería

Type 1 diabetes (T1D) is a metabolic disease that results from the autoimmune attack against insulin-producing β-cells in the pancreatic islets of Langerhans. Currently, there is no treatment to restore endogenous insulin secretion in patients with autoimmune diabetes. In the last years, the development of new therapies to induce long-term tolerance has been an important medical health challenge. Apoptosis is a physiological mechanism that contributes to the maintenance of immune tolerance. Apoptotic cells are a source of autoantigens that induce tolerance after their removal by antigen presenting cells (APCs) through a process called efferocytosis. Efferocytosis will not cause maturation in dendritic cells, one of the most powerful APCs, and this process could induce tolerance rather than autoimmunity. However, failure of this mechanism due to an increase in the rate of β-cells apoptosis and/or defects in efferocytosis results in activation of APCs, contributing to inflammation and to the loss of tolerance to self. In fact, T1D and other autoimmune diseases are associated to enhanced apoptosis of target cells and defective apoptotic cell clearance. Although further research is needed, the clinical relevance of immunotherapies based on apoptosis could prove to be very important, as it has translational potential in situations that require the reestablishment of immunological tolerance, such as autoimmune diseases. This review summarizes the effects of apoptosis of β-cells towards autoimmunity or tolerance and its application in the field of emerging immunotherapies.