Assesment of Hypoderma infestation in a wild population of Cervus elaphus from mountains Atlantic ecosystems in southwestern Europe (Spain)

[EN] Hypodermosis in Cervus elaphus was studied in the Riaño Regional Hunting Reserve, Province of León, north-western Spain. One hundred and ten red deer were examined for the presence of warble fly larvae. They were analyzed by PCR analysis of the COI region of mt-DNA and identified as Hypoderma actaeon. The prevalence of larvae was 42.7% with a mean intensity of 12.5 ± 18 (range 1–80) warbles/deer infested. The distribution of larvae in the infested animals showed an aggregated/overdispersed pattern (aggregation index = 25.84), where the larvae are not randomly or uniformly distributed, but strongly aggregated among their hosts. Larvae were found in all three states. First and second-instars were observed mainly in the autumn until the end of winter (November-March) and third-instars in late winter until mid-spring (March–May). The adult animals and the males had a higher prevalence than the young and the females, finding statistically significant differences only according to the sex of the animals. Seasonal variations were observed in the prevalence with the highest number of infested animals in winter and autumn, but not in terms of the mean intensity of parasites. Additionally, we assessed the presence of anti-Hypoderma antibodies in serum by means of indirect ELISA tests, using a crude larval extract (CLE) and a purified fraction the hypodermin C (HyC) obtained from first instars of Spanish isolates of Hypoderma lineatum (cattle). These findings confirm that H. actaeon is widely distributed in northern Spain, and provide new information about its chronobiology in mountainous Atlantic ecosystems from southwestern Europe.SIPublicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCL

Identification of Hypoderma actaeon (Diptera: Oestridae) in red deer ( Cervus elaphus ) from northern Spain: Microscopy study and molecular analysis

[EN] Hypoderma spp. larvae were observed subcutaneously in the dorsal and lumbar regions of red deer (Cervus elaphus) hunted in the province of León (northwestern Spain) causing a myiasis. They were removed and initially classified by their size, shape, color, and location under the skin into the three larval stages that parasitize these animals. The morphological characteristics of the first and second-instar are described and from the features of the third-instar the species was identified as Hypoderma actaeon. To accurately identify this species, five isolates of genomic DNA from the third-instar, two from the second-instar and two from first-instar of H. actaeon were analyzed by PCR analysis of the COI region of mt-DNA. The results confirmed that the examined samples exactly matched with H. actaeon. This study has shown the morphological identification of the three larval stages of H. actaeon and, for the first time, the first and second-instar larvae have been molecularly characterized. Finally, identification of only H. actaeon suggests that this species is the only affecting red deer in the Iberian Peninsula.S

Manual de disección del ratón para la localización y el aislamiento de los órganos linfoides

[ES] En los órganos linfoides primarios o centrales tiene lugar el origen y diferenciación de diversas células competentes del sistema inmunitario. Forman parte de este grupo la médula ósea y el timo. El timo es un órgano linfoide primario que ocupa un lugar fundamental en la biología de las células T, que está localizado cranealmente al corazón. Aparte de ser la fuente primordial de células T, es el lugar en el que las células T se diferencian y maduran para generar un amplio repertorio de células T, que posteriormente serán exportadas a la periferia como células T naïve maduras capaces de reconocer péptidos en el contexto del complejo mayor de histocompatibilidad (MHC), tras sufrir un proceso de selección positiva (restricción MHC) y negativa (tolerancia a lo propio) que tiene lugar en la cortical y medular, respectivamente. La médula ósea, como órgano linfoide primario, es el sitio donde se produce la he-matopoyesis y en el que se originan y maduran las células B en los mamíferos. Las células B proliferan y se diferencian dentro de la médula ósea y, de manera similar a lo que ocurre en el timo, mediante un proceso de selección se eliminarán aquellas células B que porten receptores de alta afinidad para antígenos propios. En los órganos linfoides secundarios o periféricos (bazo y nódulos linfáticos) es donde se produce el encuentro entre el antígeno y las células del sistema inmunita-rio. Existen varios tipos de organización de tejido linfoide que se localiza a lo largo de los vasos del sistema linfático. En algunos casos, este tejido se dispone de manera difusa en el pulmón y en la lámina propia, mientras que en otros lugares el tejido linfoide está organizado en forma de folículos linfoides, que consisten en agregados tanto de células linfoides, como no linfoides rodeados por una red de capilares de drenaje linfático. El bazo es un órgano linfoide secundario localizado en el hipocondrio izquierdo y ejerce un papel primordial en la respuesta inmunitaria frente a microorganismos procedentes del torrente sanguíneo. Los nódulos linfáticos están distribuidos por todo el organismo y están conectados entre sí a través del sistema linfático y su función se centra en servir de sitio de encuentro entre los antígenos que son transportados por la linfa o capturados por las células fagocíticas en los tejidos para ser presentados a las células efectoras del sistema inmunitario. Tejidos linfoides terciarios asociados a las mucosas. A pesar de que el bazo y los nódulos linfáticos son los órganos linfoides secundarios más organizados, existe otro tejido linfoide, menos organizados, que se denomina tejido linfoide asociado a mucosas (MALT). Las placas de Peyer, por ejemplo, son acúmulos linfoides macroscópicos que están presentes en la submucosa del intestino delgado murino y humano. Estas estructuras constituyen los sitios donde se produce la respuesta inmunitaria frente a antígenos de la dieta, bacterias comensales o microorganismos patógenos y, a diferencia, de otros órganos linfoides periféricos, como son los nódulos linfáticos mesentéricos, que recogen el drenaje linfático de las vellosidades intestinales y de las placas de PeyerLa identificación y obtención de los órganos linfoides primarios (timo y médula ósea), secundarios (bazo y nódulos linfoides) y terciarios (placas de Peyer) en el ratón.Contenido docenteActiv

PD-1/PD-L1, PD-1/PD-L2, and other co-inhibitory signaling pathways in transplantation

[EN] Transplantation of cells, tissues and vascularized solid organs is a successful therapeutic intervention for many end-stage chronic diseases. The combination of co-stimulatory blockade with the delivery of negative signals to T cells through co-inhibitory receptors would provide a robust approach to modulating T-cell receptor signaling and improving alloantigen-specific control of transplant rejection. This approach based on fundamental knowledge of APC/T-cell interactions may complement conventional therapies in the near future to reinforce long-term allograft survival, and permit minimal immunosuppression. The focus of this review was primarily on two major co-inhibitory signaling pathways, namely PD-1/PD-L1/PD-L2 and BTLA/CD160/HVEM/LIGHT that have been thoroughly characterized in murine models of transplantation using genetically modified mice, specific monoclonal antibodies and fusion proteinsSIThis work has been supported by grants FIS 01-3026 and FIS PI-050021 of Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Spanish Government, Spain) to JIR

Critical role of PD-L1 expression on non-tumor cells rather than on tumor cells for efective anti-PD-L1 immunotherapy in a transplantable mouse hematopoietic tumor model

[EN] The expression of PD-L1 on tumor cells or within the tumor microenvironment has been associated with good prognosis and sustained clinical responses in immunotherapeutic regimens based on PD-L1/PD-1/CD80 immune checkpoint blockade. To look into the current controversy in cancer immunotherapy of the relative importance of PD-L1 expression on tumor cells versus non-tumor cells of the tumor microenvironment, a hematological mouse tumor model was chosen. By combining a genetic CRISPR/Cas9 and immunotherapeutic approach and using a syngeneic hematopoietic transplantable tumor model (E.G7-cOVA tumor cells), we demonstrated that dual blockade of PD-L1 interaction with PD-1 and CD80 enhanced anti-tumor immune responses that either delayed tumor growth or led to its complete eradication. PD-L1 expression on non-tumor cells of the tumor microenvironment was required for the promotion of tumor immune escape and its blockade elicited potent anti-tumor responses to PD-L1 WT and to PD-L1-defcient tumor cells. PD-L1+ tumors implanted in PDL1-defcient mice exhibited delayed tumor growth independently of PD-L1 blockade. These fndings emphasize that PD-L1 expression on non-tumor cells plays a major role in this tumor model. These observations should turn our attention to the tumor microenvironment in hematological malignancies because of its unappreciated contribution to create a conditioned niche for the tumor to grow and evade the anti-tumor immune response.SIThis work has been supported by Grant FIS PI# 1300029 (Fondo de Investigaciones Sanitarias, Ministry of Health, Spanish Government, and co-funded by European Union ERDF/ESF, “Investing in your future”), LE093U13 and Unit of Excellence Research UIC #012 (Department of Education of the Regional Government, Junta de Castilla y Leon) and Gerencia Regional de Salud (BIO/01/15) to JIRB. It was also funded by Miguel Servet National Grant (Health National Organization Research) CP12/03063, CPII17/00002 and FIS PI16/00002 (Instituto de Salud Carlos III and co-funded by European Union ERDF/ESF, “Investing in your future”), and Gerencia Regional de Salud GRS963/A/2014, GRS1142/A/2015 and GRS 1505/A/2017 to M.L.R.G. This work has been partially funded by the National Network CIBER-ONC (oncology research) CB16/12/00480

Characteristcs of the firm and export performance

Este traballo trata de demostra-la influencia de tres características do perfil das empresas (tamaño, participación do capital estranxeiro e sector de actividade) no seu rendemento exportador, medido a través das variables probabilidade de exportar e propensión exportadora. A utilización da análise de varianza, de regresión (lineal e loxística), log-lineal xerárquico e do algoritmo "CHAlD" permite mostra-la influencia daminante do tamaño da empresa na probabilidade de exportar -o sector determina a probabilidad e de exportar só nas empresas de tamaño máis pequeno-. O considera-la propensión exportadora verificase o impacto prevalecente do sector, con electos diferenciados do tamaño segundo o sector industrial examinado. A participación do capital estranxeiro en ningún caso presenta efectos significativosSome characteristics of the firm such as size, rata of foreign capital or industry could affect export. Linear and logistic regression, variance and log-linear analysis, and CHAlD algorithm have been used to test those relationships. Results have shown that export probability is related to the size of the company. lndustry has only some effects on small and medium-sized enterprises. Export intensity of the firm depends on industry and the relationship between size and export intensity is different in different industries. The influence of foreign trade capital rate cannot be testedS

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma

[EN] Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints.We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunitySIThe authors thank Praveen Mathoor and Margarete Mijatovic for excellent technical assistance. The authors are supported by Deutsche Forschungsgemeinschaft (SFB 1039 TP B04 and B06; FOR 2438), Deutsche Krebshilfe (70112451), and Else Kröner Fresenius-Foundation (Graduate school Translational Research Innovation—Pharma (TRIP); and Else Kröner Fresenius Graduate School

LIGHT/HVEM/LTβR Interaction as a Target for the Modulation of the Allogeneic Immune Response in Transplantation

[EN] The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC-cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell-mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR) engagement. LIGHT-deficient mice, or WT mice treated with LIGHT-targeting decoy receptors HVEM-Ig, LTβR-Ig or sDcR3-Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTβR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor-specific tolerance. This review discusses how targeting the interaction of LIGHT with HVEM and/or LTbR using recombinant soluble decoy receptors or monoclonal antibodies may represent an innovative therapeutic intervention for the prevention and treatment of allograft rejection and promotion of donor-specific tolerance. © 2013 The American Society of Transplantation and the American Society of Transplant SurgeonsSIThis work has been supported by grants FIS reference # PI10/01039 from Ministry of Health and Department of Education from Junta of Castilla and Leon reference # LE007A10-2 (to JIRB), and by the Swiss National Science Foundation (to PS

Therapeutic implications of NK cell regulation of allogeneic CD8 T cell-mediated immune responses stimulated through the direct pathway of antigen presentation in transplantation

[EN]Natural killer (NK) cells are a population of innate type I lymphoid cells essential for early anti-viral responses and are known to modulate the course of humoral and cellular-mediated T cell responses. We assessed the role of NK cells in allogeneic CD8 T cell-mediated responses in an immunocompetent mouse model across an MHC class I histocompatibility barrier to determine its impact in therapeutic clinical interventions with polyclonal or monoclonal antibodies (mAbs) targeting lymphoid cells in transplantation. The administration of an NK cell depleting antibody to either CD8 T cell replete or CD8 T cell-depleted naïve C57BL/6 immunocompetent mice accelerated graft rejection. This accelerated rejection response was associated with an in vivo increased cytotoxic activity of CD8 T cells against bm1 allogeneic hematopoietic cells and bm1 skin allografts. These findings show that NK cells were impli-cated in the control host anti-donor cytotoxic responses, likely by competing for common cell growth factors in both CD8 T cell replete and CD8 T cell-depleted mice, the latter reconstituting in response to lymphopenia. Our data calls for precaution in solid organ transplantation under tolerogenic protocols involving extensive depletion of lymphocytes. These pharmacological biologics with depleting proper-ties over NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic allore-sponses refractory to current immunosuppression.SIThis work has been supported by grant FIS PI# 1300029 (Fondo de Investigaciones Sanitarias, Ministry of Health, Spanish Government and co-funded by European Union ERDF/ESF, “Investing in your future”), LE093U13 and Unit of Excellence Research UIC #012 (Department of Education of the Regional Government, Junta de Castilla y Leon) and Gerencia Regional de Salud (BIO/01/15) to JIRB, and by Miguel Servet National Grant (Health National Organization Research) CP12/03063, CPII17/00002 and FIS PI16/00002 (Instituto de Salud Carlos III and co-funded by European Union ERDF/ESF, “Investing in your future”), and Gerencia Regional de Salud GRS963/A/2014, GRS1142/A/2015 and GRS 1505/A/2017 to M.L.R.G.This work has been partially funded by the National Network CIBER-ONC (oncology research) CB16/12/00480. P.S. is funded by grant 31003A-17-6256 of the Swiss National Science Foundation. We thank University of Leon for providing funding to cover publication expenses

The role of TNFR2 and DR3 in the in vivo expansion of tregs in T cell depleting transplantation regimens

[EN] Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNF