Person: Pastor Fernández, Iván
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First Name
Iván
Last Name
Pastor Fernández
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Sanidad Animal
Area
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7 results
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Item NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model(Pathogens, 2022) Rico San Román, Laura; Amieva, Rafael; Regidor Cerrillo, Javier; García Sánchez, Marta Eulalia; Collantes Fernández, Esther; Pastor Fernández, Iván; Saeij, Jeroen P. J.; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del PilarThe intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.- Project number: 290
Item Parasitología Interactiva(2016) Gómez Bautista, Mercedes; Ortega Mora, Luis Miguel; Álvarez García, Gema; Ferré Pérez, Ignacio; Moreno Gonzalez, Javier; Gutierrez Expósito, Daniel; Arranz-Solís, David; Pastor Fernández, Iván; Rojo Montejo, Silvia; Sánchez Sánchez, Roberto; García Lunar, Paula; Jiménez Meléndez, Alejandro; Jimenez Pelayo, Laura; García Sánchez, Marta; Diezma Díaz, Carlos; Horcajo Iglesias, María Del Pilar; Regidor Cerrillo, Javier; Pérez Villalobos, Natividad; Collantes Fernández, EstherDesarrollo de una guía interactiva donde se muestran los ciclos biológicos,imágenes y dibujos de los estadios evolutivos de los principales géneros y especies de parásitos protozoos relevantes en al ámbito veterinario. Item Modeling the Ruminant Placenta-Pathogen Interactions in Apicomplexan Parasites: Current and Future Perspectives(Frontiers in Veterinary Science, 2021) Jiménez-Pelayo, Laura; Pastor Fernández, Iván; Collantes Fernández, Esther; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del PilarNeospora caninum and Toxoplasma gondii are one of the main concerns of the livestock sector as they cause important economic losses in ruminants due to the reproductive failure. It is well-known that the interaction of these parasites with the placenta determines the course of infection, leading to fetal death or parasite transmission to the offspring. However, to advance the development of effective vaccines and treatments, there are still important gaps on knowledge on the placental host-parasite interactions that need to be addressed. Ruminant animal models are still an indispensable tool for providing a global view of the pathogenesis, lesions, and immune responses, but their utilization embraces important economic and ethics restrictions. Alternative in vitro systems based on caruncular and trophoblast cells, the key cellular components of placentomes, have emerged in the last years, but their use can only offer a partial view of the processes triggered after infection as they cannot mimic the complex placental architecture and neglect the activity of resident immune cells. These drawbacks could be solved using placental explants, broadly employed in human medicine, and able to preserve its cellular architecture and function. Despite the availability of such materials is constrained by their short shelf-life, the development of adequate cryopreservation protocols could expand their use for research purposes. Herein, we review and discuss existing (and potential) in vivo, in vitro, and ex vivo ruminant placental models that have proven useful to unravel the pathogenic mechanisms and the host immune responses responsible for fetal death (or protection) caused by neosporosis and toxoplasmosis.- Project number: 25
Item Parasit`Xpert, un blog de Parasitología Veterinaria(2023) Ferré Pérez, Ignacio; Ortega Mora, Luis Miguel; Álvarez García, Gema; Collantes Fernández, Esther; Horcajo Iglesias, María Del Pilar; Calero Bernal, Rafael; Pastor Fernández, Iván; Arranz-Solís, David; Sánchez Sánchez, Roberto; Regidor Cerrillo, Javier; Hecker, Yanina PaolaParasit’Xpert es un blog sobre enfermedades parasitarias de los rumiantes domésticos para el veterinario (https://parasitxpert.es/). Su objetivo es ser un punto de encuentro y referencia para los profesionales del sector y estudiantes de veterinaria que quieran conocer y profundizar en el campo de las enfermedades parasitarias. La plataforma se inició en enero de 2021 y se estructura en diversas secciones prácticas: parásito del mes, ¿sabías qué?, diagnóstico, control, zoonosis y avances. El grupo SALUVET y la empresa de transferencia del conocimiento universitario SALUVET-Innova han puesto en marcha la web Parasit’xpert junto a la empresa farmacéutica Boehringer Ingelheim Animal Health España. El objetivo general del presente proyecto de innovación docente fue desarrollar el blog Parasit’Xpert (https://parasitxpert.es/) ya en activo e implementar un plan de difusión promocional para incrementar el número de usuarios, especialmente en países hispanohablantes. Item Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells(Veterinary Parasitology, 2023) Rico San Román, Laura; Amieva, Rafael; Horcajo Iglesias, María Del Pilar; García Sánchez, Marta Eulalia; Pastor Fernández, Iván; Ortega Mora, Luis Miguel; Collantes Fernández, EstherBovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.Item Whole-transcriptome analysis reveals virulence-specific pathogen−host interactions at the placenta in bovine neosporosis(Frontiers in Immunology, 2023) Horcajo Iglesias, María Del Pilar; Coronado, Montserrat; Pastor Fernández, Iván; Collantes Fernández, Esther; Rico San Román, Laura; Reyes Palomares, Armando Adolfo; Ortega Mora, Luis MiguelResearch on bovine neosporosis has achieved relevant milestones, but the mechanisms underlying the occurrence of foetal death or protection against foetal death remain unclear. In a recent study, placentas from heifers challenged with the high-virulence isolate Nc-Spain7 exhibited focal necrosis and inflammatory infiltrates as soon as 10 days post-infection (dpi), although parasite detection was minimal. These lesions were more frequent at 20 dpi, coinciding with higher rates of parasite detection and the occurrence of foetal death in some animals. In contrast, such lesions were not observed in placentas from animals infected with the low-virulence isolate Nc-Spain1H, where the parasite was detected only in placenta from one animal at 20 dpi. This work aimed to study which mechanisms are triggered in the placentas (caruncles and cotyledons) of these pregnant heifers at early stages of infection (10 and 20 dpi) through whole-transcriptome analysis. In caruncles, infection with the high-virulence isolate provoked a strong proinflammatory response at 10 dpi. This effect was not observed in heifers infected with the low-virulence isolate, where IL-6/JAK/STAT3 signalling and TNF-alpha signalling via NF-κB pathways were down-regulated. Interestingly, the expression of E2F target genes, related to restraining the inflammatory response, was higher in these animals. At 20 dpi, more pronounced proinflammatory gene signatures were detectable in heifers infected with the high-virulence isolate, being more intense in heifers carrying dead fetuses. However, the low-virulence isolate continued without activating the proinflammatory response. In cotyledons, the response to infection with the high-virulence isolate was similar to that observed in caruncles; however, the low-virulence isolate induced mild proinflammatory signals at 20 dpi. Finally, a deconvolutional analysis of gene signatures from both placentome tissues revealed a markedly higher fraction of activated natural killers, M1 macrophages and CD8+ T cells for the high-virulence isolate. Therefore, our transcriptomic analysis supports the hypothesis that an intense immune response probably triggered by parasite multiplication could be a key contributor to abortion. Further studies are required to determine the parasite effectors that govern the distinct interactions of high- and low-virulence isolates with the host, which could help elucidate the molecular processes underlying the pathogenesis of neosporosis in cattle.Item Ovine placental explants: a new ex vivo model to study host‒pathogen interactions in reproductive pathogens(Theriogenology, 2023) Horcajo Iglesias, María Del Pilar; Ortega Mora, Luis Miguel; Benavides, Julio; Sánchez Sánchez, Roberto; Ammieva, Rafael; Collantes Fernández, Esther; Pastor Fernández, IvánReproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.