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Astroviruses are highly divergent and infect a wide variety of animal hosts. In 2009, a genetically divergent human astrovirus (HAstV) strain VA1 was first identified in an outbreak of acute gastroenteritis. This strain has also been associated with fatal central nervous system disease. In this work, we report the isolation of three high-affinity neutralizing monoclonal antibodies (Nt-MAbs) targeting the capsid spike domain of HAstV-VA1. These antibodies (7C8, 2A2, 3D8) were used to select individual HAstV-VA1 mutants resistant to their neutralizing activity and a HAstV-VA1 triple mutant that escapes neutralization from all three Nt-MAbs. Sequencing of the virus genome capsid region revealed escape mutations that map to the surface of the capsid spike domain, define three potentially independent neutralization epitopes, and help delineate four antigenic sites in human astroviruses. Notably, two of the escape mutations were found to be present in the spike sequence of the HAstV-VA1-PS strain isolated from an immunodeficient patient with encephalitis, suggesting that those mutations arose as a result of the immune pressure generated by the patient's immunotherapy. In agreement with this observation, human serum samples exhibiting strong neutralization activity against wild-type HAstV-VA1 had a 2.6-fold reduction in neutralization titer when evaluated against the triple-escape HAstV-VA1 mutant, suggesting that both mouse and human antibody responses target shared neutralization epitopes. The isolated Nt-MAbs reported in this work will help to characterize the functional domains of the virus during cell entry and have the potential for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1. IMPORTANCE: Human astroviruses (HAstVs) have been historically associated with acute gastroenteritis. However, the genetically divergent HAstV-VA1 strain has been associated with central nervous system disease. In this work high-affinity neutralizing monoclonal antibodies directed to HAstV-VA1 were isolated and characterized. The proposed binding sites for these antibodies and for neutralizing antibodies against classical HAstVs suggest that there are at least four neutralization sites on the capsid spike of astroviruses. Our data show that natural infection with human astrovirus VA1 elicits a robust humoral immune response that targets the same antigenic sites recognized by the mouse monoclonal antibodies and strongly suggests the emergence of a variant HAstV-VA1 virus in an immunodeficient patient with prolonged astrovirus infection. The isolated Nt-MAb reported in this work will help to define the functional sites of the virus involved in cell entry and hold promise for developing a specific antibody therapy for the neurological disease associated with HAstV-VA1.
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Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , Humanos , Animais , Anticorpos Neutralizantes/imunologia , Camundongos , Epitopos/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Mamastrovirus/imunologia , Mamastrovirus/genética , Mutação , Infecções por Astroviridae/imunologia , Infecções por Astroviridae/virologia , Testes de NeutralizaçãoRESUMO
Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.
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Infecções por Astroviridae , Proteínas do Capsídeo , Mamastrovirus , Precursores de Proteínas , Infecções por Astroviridae/virologia , Células CACO-2 , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Humanos , Espaço Intracelular/virologia , Mamastrovirus/fisiologia , Precursores de Proteínas/metabolismo , Tripsina/metabolismoRESUMO
Astroviruses are common pathogens of the human gastrointestinal tract, but they have been recently identified from cases of fatal meningoencephalitis. Astrovirus VA1 is the most frequently detected astrovirus genotype from cases of human encephalitis, but the prevalence of neutralizing antibodies to VA1 in human sera is unknown. We developed a focus reduction neutralization assay (FRNT) for VA1 and measured the seroprevalence of neutralizing antibodies from two cohorts of adult and pediatric serum samples: (i) an age-stratified cohort from St. Louis, MO, collected from 2007 to 2008 and (ii) a cohort from the Peruvian Amazonian River Basin collected in the late 1990s. In the St. Louis cohort, the lowest seropositivity rate was in children 1 year of age (6.9%), rising to 63.3% by ages 9 to 12, and 76.3% of adults ≥20 years were positive. The Peruvian Amazon cohort showed similar seropositivity rates across all ages, with individuals under age 20 having a rate of 75%, while 78.2% of adults ≥20 years were seropositive. In addition, we also identified the presence neutralizing antibodies to VA1 from commercial lots of intravenous immunoglobulin (IVIG). Our results demonstrate that a majority of humans are exposed to VA1 by adulthood, with the majority of infections occurring between 2 and 9 years of age. In addition, our results indicate that VA1 has been circulating in two geographically and socioeconomically divergent study cohorts over the past 20 years. Nonetheless, a significant proportion of the human population lacks neutralizing immunity and remains at risk for acute infection. IMPORTANCE Astroviruses are human pathogens with emerging disease associations, including the recent recognition of their capacity to cause meningoencephalitis. Astrovirus VA1 is the most commonly identified astrovirus genotype from cases of human encephalitis, but it is unknown what percentage of the human population has neutralizing antibodies to VA1. We found that 76.3 to 78.2% of adult humans ≥20 years of age in two geographically and socioeconomically distinct cohorts are seropositive for VA1, with the majority of infections occurring between 2 and 9 years of age. These results demonstrate that VA1 has been circulating in human populations over the past 2 decades and that most humans develop neutralizing antibodies against this virus by adulthood. However, a subset of humans lack evidence of neutralizing antibodies and are at risk for diseases caused by VA1, including encephalitis.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Astroviridae/epidemiologia , Mamastrovirus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Modelos Logísticos , Masculino , Mamastrovirus/genética , Pessoa de Meia-Idade , Missouri/epidemiologia , Peru/epidemiologia , RNA Viral/genética , Estudos Soroepidemiológicos , Adulto JovemRESUMO
RESUMEN Introducción: las parafilias, reconocidas como trastornos de la preferencia sexual, conducen a una relación morbosa donde el deseo o el comportamiento sexual son aberrantes. Ocasionan intenso malestar, pueden extenderse por seis meses o más, determinando conductas violatorias de buenas costumbres y normas sociales que ocasionan molestias constituidas delitos, y que necesitan tratamiento especializado para su rehabilitación y reivindicación social. Objetivo: destacar las peculiaridades clínicas de las parafilias, la importancia de su diagnóstico, trascendencia social en su repercusión en las tendencias modernas facilitadas por las nuevas técnicas de la información y la comunicación, peculiaridades del tratamiento médico legal especializado que incluyan rehabilitación y reivindicación de las relaciones con el medio social. Métodos: lógico dialéctico, reflexivo e histórico a partir de experiencias y búsqueda de información científica de actualidad. Resultados: se destacan particularidades del diagnóstico clínico, sus pautas específicas y el interés que en la actualidad despiertan en los medios modernos de información. Se plantean las medidas que la ley autoriza y recomiendan las mejores formas de aplicación ante estos casos para garantía de su rehabilitación y reivindicación cuando perturben el orden o violen normas de convivencia social. Conclusiones: se plantea la necesidad de un diagnóstico clínico especializado y basado en pautas reconocidas que sirvan al tratamiento médico legal encaminado a imponer medidas que propicien el tratamiento continuo, especializado y riguroso enfocado en la rehabilitación y la reeducación del paciente.
ABSTRACT Introduction: paraphilias, recognized as disorders of sexual preference, lead to a morbid relationship, sexual desire or behavior is aberrant, causing intense discomfort, extending over time for six months or more, determining behaviors that contravene good customs and social norms, causing discomfort that sometimes constitute crimes, which can be punished, needing specialized treatment for rehabilitation and enable social demands. Objective: to underline the clinical peculiarities of paraphilias, the importance of their diagnosis, social significance in their repercussion on modern trends facilitated by the new information and communication technologies, peculiarities of specialized medical-legal treatment that includes rehabilitation and vindication of relations with the social environment. Methods: logical-dialectical, reflexive and historical methods based on experiences and application of searching methods for up-to-date scientific information. Results: the particularities of the clinical diagnosis, its specific guidelines and the interest these disorders arouse on the modern means of information, raising the measures that the law authorizes and recommending the best forms of application in these cases guaranteeing their rehabilitation and vindication when they disturb the order or contravene the norms of social coexistence. Conclusions: the need for a specialized clinical diagnosis based on recognized guidelines is raised, which will provide the medical-legal treatment aimed at imposing measures that will promote continuous, specialized and rigorous treatment aimed at the rehabilitation and re-education of the patient.
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Dengue is the most prevalent and rapidly transmitted mosquito-borne viral disease of humans. One of the fundamental innate immune responses to viral infections includes the processing and release of pro-inflammatory cytokines such as interleukin (IL-1ß and IL-18) through the activation of inflammasome. Dengue virus stimulates the Nod-like receptor (NLRP3-specific inflammasome), however, the specific mechanism(s) by which dengue virus activates the NLRP3 inflammasome is unknown. In this study, we investigated the activation of the NLRP3 inflammasome in endothelial cells (HMEC-1) following dengue virus infection. Our results showed that dengue infection as well as the NS2A and NS2B protein expression increase the NLRP3 inflammasome activation, and further apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) oligomerization, and IL-1ß secretion through caspase-1 activation. Specifically, we have demonstrated that NS2A and NS2B, two proteins of dengue virus that behave as putative viroporins, were sufficient to stimulate the NLRP3 inflammasome complex in lipopolysaccharide (LPS)-primed endothelial cells. In summary, our observations provide insight into the dengue-induced inflammatory response mechanism and highlight the importance of DENV-2 NS2A and NS2B proteins in activation of the NLRP3 inflammasome during dengue virus infection.
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Vírus da Dengue/imunologia , Dengue/imunologia , Células Endoteliais/fisiologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular Transformada , Dengue/virologia , Vírus da Dengue/patogenicidade , Humanos , Imunidade Inata , Interleucina-1beta/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas Viroporinas/genética , VirulênciaRESUMO
RESUMEN Introducción: el ritmo acelerado de vida, el stress crónico, la aparición de ansiedad, depresión y malestar, con frecuencia se intentan resolver a través de la automedicación con psicofármacos. Estos pueden ser de gran ayuda si se prescriben de manera adecuada y se acompañen de otras acciones. En la mayoría de los casos cuando los problemas son psicológicos y el consumo prolongado, crea hábito y provoca efectos patológicos. Objetivo: determinar la automedicación con psicofármacos y sus efectos provocados por el uso prolongado, en la población de un grupo de consultorios del municipio Pinar del Río. Métodos: investigación descriptiva, transversal, epidemiológica, mediante la visita a 10 636 familias de 37 consultorios de tres áreas de salud de la ciudad de Pinar del Río, con una población de 29 877 personas, entre septiembre 2017 y junio 2018. Resultados: del universo de población a encuestar, el 82,61 % eran mayores de 20 años y entre estos el 8,9 % se auto medicaban. Reconocen habituación un 38,5 % y dependencia el 11 %. Conclusiones: la automedicación con psicofármacos es frecuente en la población estudiada, implicaron riesgos de habituación y dependencia, se determinaron situaciones sociales preocupantes, que aconsejan una política sanitaria de regulación y control, con el reclamo de experiencias comprobadas e integralidad.
ABSTRACT Introduction: the accelerated rhythm of life, chronic stress, onset of anxiety, depression and distress, often lead to self-medication with psychotropic drugs, with the risk of getting into the habit, without being effective in solving the situation, these can be of great help if prescribed correctly and accompanied by other actions, but in many cases is being excessive and causes pathological effects when the problems are psychological and people are exposed to prolonged consumption. Objective: to determine the self-medication with psychoactive drugs in the population of a group of clinics from Pinar del Río municipality, and the effects provoked by prolonged use. Methods: descriptive, cross-sectional, epidemiological research carried out by means of visiting 10,636 families from 37 clinics in three health areas of the city of Pinar del Río, between September 2017 and June 2018, with a population of 29,877 people, to determine self-medication with psychotropic drugs in people over 20 years old, and to diagnose the effects recognized due to their prolonged use, through a survey prepared for this purpose. Results: 82,61 % of the target population to be surveyed were over 20 years old and among these 8,9 % practiced self-medication. They recognized getting into the habit (38,5 %) and dependency 11 %. Conclusions: self-medication with psycho-pharmaceuticals is frequent in the studied population; it implies getting into the habit and suffer from risks of dependence, which lead to social situations and impose regulations in order to control health policy and appealing to proven and integrative experiences.
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In recent decades, the incidence of death and morbidity due to diabetes has increased worldwide, causing a high social and economic impact. Diabetes is a major cause of blindness, kidney failure, heart attack, stroke and lower limb amputation. However, the molecular mechanisms that make the heart and kidneys the main targets of diabetes are not completely understood. To better understand the complex biochemical mechanism of diabetic cardiomyopathy, we investigated the effects of hyperglycemia with concomitant digoxin and ouabain stimulation in H9c2 cells. Total extracted proteins were analyzed by label-free LC-MS/MS, quantified by Scaffold software and validated by parallel reaction monitoring (PRM) methodology. Here, we show that the eukaryotic initiation factors (Eifs) and elongation factors (Eefs) Eif3f, Eef2 and Eif4a1 are overexpressed following cardiotonic steroid (CTS) stimulation. Similarly, the expression of four 14-3-3 proteins that play a key role in cardiac ventricular compaction was altered after CTS stimulation. In total, the expression of nine protein groups was altered in response to the stimulation of H9c2 cells. Here, the biological consequences of these changes are discussed in depth. SIGNIFICANCE: Hyperglycemia is the main physiological condition that provokes tissue and vascular injuries in heart of diabetic patients. However, the changings at large scale in the expression of proteins of cardiomyocytes generated by this condition was not yet studied. Here we report for the first time the altered biosynthesis of nine groups of proteins of H9c2 cells activated by high glucose concentrations and by cardiotonic steroids (CTS). Furthermore, the increased biosynthesis of Eifs, Eefs and 14-3-3 protein groups by CTS, which play a crucial role in cardiomyopathies are original data reported in this work. These findings not only enhance our knowledge concerning to the effects of hyperglycemia and CTS on H9c2 cells but also indicate potential molecular targets to interfere in diabetes cardiomyopathy progression.
Assuntos
Glicosídeos Cardíacos , Cardiotônicos , Cromatografia Líquida , Glucose , Humanos , Miócitos Cardíacos , Proteômica , Espectrometria de Massas em TandemRESUMO
RESUMEN Introducción: se han realizado esfuerzos por lograr la proyección comunitaria de la Psiquiatría en función de los principios reconocidos en la Carta de La Habana, facilitado por la existencia del modelo de medicina familiar. La Atención Primaria de Salud constituye el eslabón fundamental del Sistema Nacional de Salud, donde se desarrollan los programas dirigidos a elevar el nivel de salud, al situar a la Salud Mental Comunitaria al nivel de los países desarrollados. Objetivos: determinar la relación de Enfermedades mentales, somáticas y riesgos en salud mental en las familias de tres Grupos Básicos de Trabajo de diferentes áreas de salud Métodos: se realizó una investigación descriptiva, epidemiológica, se visitó a 12 909 familias, en tres Grupos Básicos de Trabajo urbanos de la ciudad de Pinar del Río, seleccionados de forma aleatoria, con 36 087 integrantes, se entrevistó al representante del núcleo mediante una encuesta aplicada. Resultados: el número de enfermedades somáticas reconocidas alcanza el 57,8 % de la población, las enfermedades mentales y trastornos del comportamiento al 4,9 %, con un 1,5 % de alcohólicos, 3,5 % de bebedores de riesgo y un 0,8 % que reconocen dependencia de la automedicación con psicofármacos. Conclusiones: se analizaron las proporciones de las diferentes enfermedades reconocidas, se destacó la interacción entre ellas y la importancia de la responsabilidad, tanto del sector salud como de los sectores interrelacionados, realzando la importancia y responsabilidad del Grupo Básico de Trabajo y el Equipo de Salud Mental Comunitaria en la aspiración de una mejor Salud Mental Integral.
ABSTRACT Introduction: in our country, efforts have been made to achieve community projection of psychiatry based on the principles recognized in the Havana Agreement, facilitated by the existence of the family medicine model. Primary Health Care is the fundamental link of the National Health System, where programs are designed aimed at increasing the level of health, inserting the Cuban Mental Health System of the community, at the level of developed countries. Objectives: to determine the relationship of mental and somatic disorders along with risks in Mental Health in the families of three Basic Working Teams from the different health areas to contribute to the understanding of the problem of the community mental health disorders in order to improve the conduction of the intra and intersectorial efforts to reach a better program concerning Comprehensive Mental Health System. Methods: a descriptive, epidemiological research was carried out, visiting 12,909 families, in three urban Basic Working Teams from Pinar del Río city, randomly chosen, with 36,087 members, interviewing the representative of the nucleus by means of a survey directly applied. Results: the number of recognized somatic diseases reaches 57,8 % of the population, mental and behavioral disorders at 4,9 %, with 1,5 % of alcoholics, 3,5 % of risk drinkers and the 0,8 % identifying their dependence on self-medication with psychopharmaceutical substances. Conclusions: the proportions of the different diseases listed were analyzed, highlighting the interaction between them and the importance of responsibilities, both the health sector and the interrelated sectors; giving emphasis to the significance and the responsibilities of the Basic Working Teams.
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Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.