Study of the cross-talk between Fasciola hepatica juveniles and the intestinal epithelial cells of the host by transcriptomics in an in vitro model.

Fasciolosis is a globally widespread trematodiasis with a major economic and veterinary impact. Therefore, this disease is responsible for millions of dollars in losses to the livestock industry, and also constitutes an emerging human health problem in endemic areas. The ubiquitous nature of Fasciola hepatica, the main causative agent, is one of the key factors for the success of fasciolosis. Accordingly, this parasite is able to subsist in a wide variety of ecosystems and hosts, thanks to the development of a plethora of strategies for adaption and immune evasion. Fasciolosis comprises a growing concern due to its high prevalence rates, together with the emergence of strains of the parasite resistant to the treatment of choice (triclabendazole). These facts highlight the importance of developing novel control measures which allow for an effective protection against the disease before F. hepatica settles in a niche inaccessible to the immune system. However, knowledge about the initial phases of the infection, including the migration mechanisms of the parasite and the early innate host response, is still scarce. Recently, our group developed an in vitro host-parasite interaction model that allowed the early events to be unveiled after the first contact between the both actors. This occurs shortly upon ingestion of F. hepatica metacercariae and the emergence of the newly excysted juveniles (FhNEJ) in the host duodenum. Here, we present a transcriptomic analysis of such model using an approach based on RNA sequencing (RNA-Seq), which reveals changes in gene expression related to proteolysis and uptake of metabolites in FhNEJ. Additionally, contact with the parasite triggered changes in host intestinal cells related to pseudogenes expression and host defence mechanisms, including immune response, among others. In sum, these results provide a better understanding of the early stages of fasciolosis at molecular level, and a pool of targets that could be used in future therapeutic strategies against the disease.

Molecular Characterization of the Interplay between Fasciola hepatica Juveniles and Laminin as a Mechanism to Adhere to and Break through the Host Intestinal Wall.

Fasciola hepatica is the main causative agent of fasciolosis, a zoonotic parasitic disease of growing public health concern. F. hepatica metacercariae are ingested by the host and excyst in the intestine, thereby releasing the newly excysted juveniles (FhNEJ), which traverse the gut wall and migrate towards the biliary ducts. Since blocking F. hepatica development is challenging after crossing of the intestinal wall, targeting this first step of migration might result in increased therapeutic success. The intestinal extracellular matrix (ECM) is constituted by a network of structural proteins, including laminin (LM) and fibronectin (FN), that provide mechanical support while acting as physical barrier against intestinal pathogens. Here, we employed ELISA and immunofluorescent assays to test for the presence of LM- and FN-binding proteins on a tegument-enriched antigenic fraction of FhNEJ, and further determined their identity by two-dimensional electrophoresis coupled to mass spectrometry. Additionally, we performed enzymatic assays that revealed for the first time the capability of the juvenile-specific cathepsin L3 to degrade LM, and that LM degradation by FhNEJ proteins is further potentiated in the presence of host plasminogen. Finally, a proteomic analysis showed that the interaction with LM triggers protein changes in FhNEJ that may be relevant for parasite growth and adaptation inside the mammalian host. Altogether, our study provides valuable insights into the molecular interplay between FhNEJ and the intestinal ECM, which may lead to the identification of targetable candidates for the development of more effective control strategies against fasciolosis.

Fasciola hepatica juveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism.

BACKGROUND: The trematode Fasciola hepatica is the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. During F. hepatica infection, newly excysted juveniles (FhNEJ) emerge in the duodenum of the mammalian host and migrate towards their definitive location, the intra-hepatic biliary ducts. Understanding how F. hepatica traverses the intestinal wall and migrates towards the liver is pivotal for the development of more successful strategies against fasciolosis. The central enzyme of the mammalian fibrinolytic system is plasmin, a serine protease whose functions are exploited by a number of parasite species owing to its broad spectrum of substrates, including components of tissue extracellular matrices. The aim of the present work is to understand whether FhNEJ co-opt the functions of their host fibrinolytic system as a mechanism to facilitate trans-intestinal migration. METHODOLOGY/PRINCIPAL FINDINGS: A tegument-enriched antigenic extract of FhNEJ (FhNEJ-Teg) was obtained in vitro, and its capability to bind the zymogen plasminogen (PLG) and enhance its conversion to the active protease, plasmin, were analyzed by a combination of enzyme-linked immunosorbent, chromogenic and immunofluorescence assays. Additionally, PLG-binding proteins in FhNEJ-Teg were identified by bidimensional electrophoresis coupled to mass spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins. CONCLUSIONS/SIGNIFICANCE: Our results show that FhNEJ-Teg contains proteins that bind PLG and stimulate its activation to plasmin, which could facilitate the traversal of the intestinal wall by FhNEJ and contribute to the successful establishment of the parasite within its mammalian host. Altogether, our findings contribute to a better understanding of host-parasite relationships during early fasciolosis and may be exploited from a pharmacological and/or immunological perspective for the development of treatment and control strategies against this global disease.

Regulation of BDNF Release by ARMS/Kidins220 through Modulation of Synaptotagmin-IV Levels.

BDNF is a growth factor with important roles in the nervous system in both physiological and pathological conditions, but the mechanisms controlling its secretion are not completely understood. Here, we show that ARMS/Kidins220 negatively regulates BDNF secretion in neurons from the CNS and PNS. Downregulation of the ARMS/Kidins220 protein in the adult mouse brain increases regulated BDNF secretion, leading to its accumulation in the striatum. Interestingly, two mouse models of Huntington's disease (HD) showed increased levels of ARMS/Kidins220 in the hippocampus and regulated BDNF secretion deficits. Importantly, reduction of ARMS/Kidins220 in hippocampal slices from HD mice reversed the impaired regulated BDNF release. Moreover, there are increased levels of ARMS/Kidins220 in the hippocampus and PFC of patients with HD. ARMS/Kidins220 regulates Synaptotagmin-IV levels, which has been previously observed to modulate BDNF secretion. These data indicate that ARMS/Kidins220 controls the regulated secretion of BDNF and might play a crucial role in the pathogenesis of HD.SIGNIFICANCE STATEMENT BDNF is an important growth factor that plays a fundamental role in the correct functioning of the CNS. The secretion of BDNF must be properly controlled to exert its functions, but the proteins regulating its release are not completely known. Using neuronal cultures and a new conditional mouse to modulate ARMS/Kidins220 protein, we report that ARMS/Kidins220 negatively regulates BDNF secretion. Moreover, ARMS/Kidins220 is overexpressed in two mouse models of Huntington's disease (HD), causing an impaired regulation of BDNF secretion. Furthermore, ARMS/Kidins220 levels are increased in brain samples from HD patients. Future studies should address whether ARMS/Kidins220 has any function on the pathophysiology of HD.

Generation and Characterization of Antibodies against Opioid Receptors from Zebrafish.

The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.

Impacto de un protocolo de crisis hipertensiva en el Servicio de Urgencia en la Clínica Geriátrica del Ejército; Marzo 2009 - Marzo 2011 / Impact of a protocol for hypertensive crisis in the Emergency Service at the Geriatric Clinic of the Army; March 2009-March 2011

OBJETIVO: Determinar el impacto de un protocolo de crisis hipertensiva en el Servicio de Urgencia, en cuanto al diagnóstico y tratamiento de la crisis hipertensiva, mediante un protocolo de manejo en la Clínica Geriátrica del Ejército, Marzo 2009 - Marzo 2011. METODOS: El estudio realizado fue de tipo prospectivo-longitudinal, con una muestra aleatoria de 60 pacientes. La recolección de datos fue a través del llenado de fichas clínicas, mediante la revisión de las historias clínicas de urgencia de pacientes con diagnóstico y manejo de crisis hipertensiva, entre marzo a diciembre del 2009 sin uso de ningún protocolo; luego se dictó una charla de capacitación sobre aspectos relacionados al diagnóstico y manejo de la Crisis Hipertensiva según protocolo, dirigida a médicos generales y especialista del Servicio de Urgencia. Luego se revisaron las historias clínicas de urgencias con diagnóstico de crisis hipertensiva, entre enero del 2010 a marzo del 2011, con capacitación y uso de protocolo. Para el análisis de resultados se emplearon porcentajes, medidas de tendencia central, de dispersión, además de las pruebas estadísticas: Comparación de Medias, U de Mann-Whitney y Chi Cuadrado, con una significancia de p=0.05. RESULTADOS: El 63.3 por ciento de los pacientes con crisis hipertensiva fueron diagnosticados y tratados mediante el uso del protocolo de crisis hipertensiva, el 48.3 por ciento correspondía a edades entre 60 a 70 años, el 50 por ciento correspondió a ambos sexo, el 30 por ciento tenían secundaria completa, el 71.7 por ciento eran amas de casa. Las características clínicas fueron: el 85 por ciento clasificó como urgencia hipertensiva, el 56.7 por ciento tuvo como antecedente patológico personal a la hipertensión arterial; así mismo, la hipertensión arterial constituyó el 20 por ciento como antecedente patológico familiar, el 73.3 por ciento recibió tratamiento médico adecuado, el 8.3 por ciento presentó enfermedad cerebro vascular isquémica aguda en la...

OBJECTIVE: Determine the impact of a hypertensive crisis protocol in the emergency department, in terms of diagnosis and treatment of hypertensive crisis, through a management protocol Geriatric Clinic of the Army, March 2009 - March 2011. METHODS: The study was a prospective, longitudinal, with a random sample of 60 patients. The data were collected through medical records filling, by reviewing the medical records of patients with emergency diagnosis and management of hypertensive crisis, from March to December 2009 without using any protocol, then gave a talk on training on issues related to the diagnosis and management of hypertensive crisis according to protocol, aimed at general practitioners and specialist Emergency Room. Then we reviewed the medical records of emergency with acute hypertensive, between January 2010 to March 2011, with training and protocol use. For the analysis results were used percentages, measures of central tendency, dispersion, and statistical tests: comparison of means, Mann-Whitney and Chi Square, with a significance of p=0.05. RESULTS: The 63.3 per cent of patients with hypertensive crisis were diagnosed and treated using the protocol of hypertensive crisis, 48.3 per cent were aged 60-70 years, 50 per cent were both sex, 30 per cent had secondary complete, 71.7 per cent were housewives. Clinical characteristics were: 85 per cent classified as hypertensive urgency, 56.7 per cent had a pathological personal as hypertension, likewise, hypertension accounted for 20 per cent as pathological family history, 73.3 per cent received appropriate medical treatment, and the 8.3 per cent presented acute ischemic cerebrovascular disease in hypertensive emergency. As for the characteristics of the intervention were: 78.3 per cent received training in the handling and use of hypertensive crisis protocol, to 83.3 per cent of patients was discharged from the emergency department and 46.7 per cent had a hospital stay of 2 hours using the protocol...