Transcriptomic Profile of Canine DH82 Macrophages Infected by Leishmania infantum Promastigotes with Different Virulence Behavior

Zoonotic visceral leishmaniosis caused by Leishmania infantum is an endemic disease in the Mediterranean Basin affecting mainly humans and dogs, the main reservoir. The leishmaniosis outbreak declared in the Community of Madrid (Spain) led to a significant increase in human disease incidence without enhancing canine leishmaniosis prevalence, suggesting a better adaptation of the outbreak’s isolates by other host species. One of the isolates obtained in the focus, IPER/ES/2012/BOS1FL1 (BOS1FL1), has previously demonstrated a different phenotype than the reference strain MCAN/ES/1996/BCN150 (BCN150), characterized by a lower infectivity when interacting with canine macrophages. Nevertheless, not enough changes in the cell defensive response were found to support their different behavior. Thus, we decided to investigate the molecular mechanisms involved in the interaction of both parasites with DH82 canine macrophages by studying their transcriptomic profiles developed after infection using RNA sequencing. The results showed a common regulation induced by both parasites in the phosphoinositide-3-kinase–protein kinase B/Akt and NOD-like receptor signaling pathways. However, other pathways, such as phagocytosis and signal transduction, including tumor necrosis factor, mitogen-activated kinases and nuclear factor-κB, were only regulated after infection with BOS1FL1. These differences could contribute to the reduced infection ability of the outbreak isolates in canine cells. Our results open a new avenue to investigate the true role of adaptation of L. infantum isolates in their interaction with their different hosts

Immunization with the HisAK70 DNA Vaccine Induces Resistance against Leishmania Amazonensis Infection in BALB/c Mice

Leishmania amazonensis is the aetiological agent of a broad spectrum of leishmaniosis in South America. It can cause not only numerous cases of cutaneous leishmaniosis but also diffuse cutaneous leishmaniosis. Considering the diversity of parasite species causing different forms of the disease that coexist in the same region, it is desirable to develop a vaccine capable of eliciting cross-protection. We have previously described the use of HisAK70 DNA vaccine for immunization of mice to assess the induction of a resistant phenotype against Leishmania major and infantum infections. In this study, we extended its application in the murine model of infection by using L. amazonensis promastigotes. Our data revealed that 14 weeks post-infection, HisAK70-vaccinated mice showed key biomarkers of protection, such as higher iNOS/arginase activity, IFN-γ/IL-10, IFN-γ/IL-4, and GM-CSF/IL-10 ratios, in addition to an IgG2a-type response when compared to the control group. These findings correlated with the presentation of lower footpad swelling and parasite burdens in the immunized compared to the control mice. Overall, this study suggests that immunization with HisAK70 may be considered a suitable tool to combat leishmaniosis as it is able to induce a potent cellular immune response, which allows to control the infection caused by L. amazonensis

Veterinaria es calidad: Evaluación contínua y autoevaluación

El documento recoge los resultados del proyecto de Innova-Gestion UCM desarrollado durante los años 2016/2017 para la evaluación por rúbrica y online, formando, aplicando y analizando sus resultados. También se exponen los datos preliminares de la opinión de docentes y alumnos sobre su satisfacción con el actual Grado en Veterinaria. Estos resultados son, además, el punto de partida, para continuar mejorando la calidad de la docencia del centro

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Epitope Selection for Fighting Visceral Leishmaniosis: Not All Peptides Function the Same Way

Author Contributions Conceptualization, A.M.-R., G.D.-B., and J.C.; methodology, A.M., D.Á.-C., and A.M.-R.; formal analysis, A.M.-R.; resources, G.D.-B. and J.C.; data curation, A.M.-R., G.D.-B., and J.C.; writing—original draft preparation, A.M.-R.; writing—review and editing, A.M., A.M.-R., G.D.-B., J.C., and J.A.O.; supervision, G.D.-B. and J.C.; funding acquisition, A.M.-R., G.D.-B., J.A.O., and J.C. All authors have read and agreed to the published version of the manuscript. Patents The University Complutense of Madrid (Spain) has filed a patent on the HisDTC multiepitope chimera (P202030547).Visceral leishmaniosis (VL) caused by Leishmania infantum is a disease with an increasing prevalence worldwide. Treatments are expensive, toxic, and ineffective. Therefore, vaccination seems to be a promising approach to control VL. Peptide-based vaccination is a useful method due to its stability, absence of local side effects, and ease of scaling up. In this context, bioinformatics seems to facilitate the use of peptides, as this analysis can predict high binding affinity epitopes to MHC class I and II molecules of different species. We have recently reported the use of HisAK70 DNA immunization in mice to induce a resistant phenotype against L. major, L. infantum, and L. amazonensis infections. In the present study, we used bioinformatics tools to select promising multiepitope peptides (HisDTC and AK) from the polyprotein encoded in the HisAK70 DNA to evaluate their immunogenicity in the murine model of VL by L. infantum. Our results revealed that both multiepitope peptides were able to induce the control of VL in mice. Furthermore, HisDTC was able to induce a better cell-mediated immune response in terms of reduced parasite burden, protective cytokine profile, leishmanicidal enzyme modulation, and specific IgG2a isotype production in immunized mice, before and after infectious challenge. Overall, this study indicates that the HisDTC chimera may be considered a satisfactory tool to control VL because it is able to activate a potent CD4+ and CD8+ T-cell protective immune responses.Universidad Complutense de Madrid-SantanderComunidad de MadridDepto. de Sanidad AnimalCAI Ciencias de la Tierra y ArqueometríaTRUEpu

Subtilase cytotoxin-encoding subAB2 variants in verotoxin-producing Escherichia coli strains isolated from goats and sheep

Subtilase cytotoxin (SubAB) is a cytotoxin which might contribute to the virulence of verotoxin-producing Escherichia coli (VTEC) strains in humans. Three variants of SubAB encoding genes have been described (subAB1, subAB2-1, and subAB2-2) and it has been suggested that the strains positive for two variants of subAB may be more pathogenic for humans. In this study, 188 subAB2-positive VTEC strains isolated from goats and sheep were investigated for the presence of the subAB2-1 and subAB2-2 variants by PCR. Eighty-one of the 132 (61.4%) caprine strains and 36 of the 56 (64.3%) ovine strains possessed the subAB2-1 variant and all ovine and caprine strains, except one, were positive for the subAB2-2 variant. The results of this study show for first time that the subAB2-1 and subAB2-2 variants are found in caprine subAB2-positive VTEC strains and confirm that both subAB2 variants are detected in ovine subAB2-positive VTEC strains. Since no significant difference in the presence of both subAB2 variants was found among strains belonging to serotypes associated with severe illness in humans and strains not belonging to these serotypes, the occurrence of two subAB2 variants seems not to be associated with a higher risk of severe disease in humans.Universidad Complutense de Madrid / Banco Santander Central Hispano (INBAVET 920338)Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ±4.40 vs 38.58 ±2.19; P <0.0001) and intensity of infection (3.64 ±0.13 vs 1.83 ±0.12; P <0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ±5.54 vs 26.37 ±2.93; P =0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ±26.29 vs 69.80 ±9.02; P =0.0151) and decreased IL- 10 production (165.06 ±23.87 vs 264.41 ±30.58; P =0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.UCM-SANTANDERCOMUNIDAD DE MADRIDDepto. de Sanidad AnimalFac. de VeterinariaTRUEpu

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells

CRediT authorship contribution statement Alicia Mas: Conceptualization, Methodology, Formal analysis, Writing - original draft. Abel Martínez-Rodrigo: Methodology, Formal analysis, Writing - review & editing. Jose Antonio Orden: Resources, Writing - review & editing. Luis Miguel Vinals: ˜ Resources. Gustavo Domínguez-Bernal: Conceptualization, Writing - original draft, Supervision, Funding acquisition. Javier Carrion: ´ Conceptualization, Supervision, Funding acquisition.Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ± 4.40 vs 38.58 ± 2.19; P < 0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ± 0.12; P < 0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ± 5.54 vs 26.37 ± 2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ± 26.29 vs 69.80 ± 9.02; P = 0.0151) and decreased IL-10 production (165.06 ± 23.87 vs 264.41 ± 30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus syrian hamster model

Authors’ contributions AN performed the histopathological analyses and participated in the design of the study. GDB, JAO, RDF and NME participated in the design and the discussion section of this paper. JC conceived of the study, carried out the experiments and wrote the paper. All authors read and approved the final manuscript.Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.Ministerio de Educación, Cultura y DeporteAGL2007-62207Depto. de Sanidad AnimalFac. de VeterinariaTRUEpu

SYNTHETIC MULTI-EPITOPE CHIMERA AS A VACCINE Al\D TREAT\:IENT FOR LEISHMANIASIS IN MAMMALS

CONTRATO DE LICENCIA DE PATENTE ENTRE FATRO S.P.A Y LA UNIVERSIDAD COMPLUTENSE DE MADRID REUNIDOS De una parte, Universidad Complutense de Madrid (en adelante, la “UCM”) con número de identificación fiscal Q2818014I, y domicilio en Avenida de Séneca, 2, 28040, Madrid, representada en este acto por Doña Margarita San Andrés Moya, representación que deriva de su calidad de Vicerrectora de Investigación y Transferencia de la UCM, nombrada por Decreto Rectoral 22/2019, de 14 de junio, y en virtud de lo que establece el Artículo 66.2 del Decreto 32/2017, de 21 de marzo, del Consejo de Gobierno de la Comunidad de Madrid, por el que se aprueban los Estatutos de la Universidad Complutense de Madrid (BOCM nº 71, de 24 de marzo), y de las competencias que le han sido delegadas por Decreto Rectoral 1/2021, de 14 de enero, de establecimiento de los Vicerrectorados de la Universidad Complutense de Madrid, de delegación de competencias y de diversas cuestiones de índole organizativo. De otra parte, FATRO S.P.A. (en adelante, la “Empresa”), con número de identificación fiscal 01125080372, VAT code no. IT 01665321202, y domicilio en Via Emilia no. 285, 40064 Ozzano dell’Emilia, Bologna, Italia, representada en este acto por Carlo Gazza, con D.N.I. número AT 5842680, representación que deriva de su calidad de Vicepresidente de la Empresa, según se desprende de l’acta del consejo de administración del 24.06.2022.Quimera sintética multiepitópica como vacuna y tratamiento frente a leishmaniosis en mamíferos. La invención se refiere a quimeras sintéticas que incluyen 4 péptidos multiepitópicos frente a Leishmania. Cada uno de los péptidos se ha seleccionado de una proteína de Leishmania infantum. Se trata de las histonas nucleosomales H2A, H2B, H3 y H4. La invención también se refiere a una composición farmacéutica que incluye una de estas quimeras sintéticas. También se refiere a una vacuna profiláctica y/o terapéutica frente a Leishmania spp para su uso en mamíferos y, especialmente, en humana y en perros.Depto. de Sanidad AnimalFac. de Veterinariapu