Antígeno heterólogo no diagnóstico imunológico da wuchereriose: extrato de Setaria equina utilizado na reação imunoenzimática - ELISA

A reação imunoenzimática - ELISA foi empregada na sorologia da Wuchereriose, utilizando como antígeno extrato bruto de verme adulto de Setaria equina. Foram estudados soros de 139 indivíduos em três grupos: grupo 1 - de pacientes com diagnóstico parasitológico de Wuchereriose; grupo 2 - de pacientes com diversas patologias; grupo 3 - de pessoas clinicamente normais, de área endêmica e não endêmica. O antígeno utilizado mostrou alta comunidade antigênica coma W. bancrofti. As reações cruzadas, obtidas em particular com soros de pacientes com parasitoses intestinais (áscaris, ancilóstoma) recomendam a investigação de frações antigênicas de Setaria equina que possam fornecer testes de maior especificidade.

Diagnóstico histopatológico de la leishmaniasis cutánea y cutáneo-mucosa en Bolivia

En 98 casos de Leishmaniasis cutánea y cutáneo-mucosa, se encontró relación importante entre infiltrado plasmocitario y la presencia del parásito; en cuanto el infiltrado linfocitario y/o granulomatoso, aparecia en los casos donde el parásito estaba ausente. Por otra parte no se encontró diferencia entre los tipos histopatológicos predominantes en relación con el tiempo de evolución de las lesiones. Estos aspectos son una contribución para la interpretación de material de biopsias en las áreas endémicas de Leishmianiasis.

Anticorpos anti-HBs obtidos em coelhos e sua utilização como reagentes para testes sorológicos no diagnóstico da hepatite B

Pela técnica de cromatografia de afinidade, utilizando-se a resina de Sepharose 4B ligada ao anti-HBs, obteve-se na passagem de plasma de portador assintomático de antígeno HBs, um antígeno parcialmente purificado. Este antígeno foi utilizado para a inoculação em coelhos, num esquema de cinco doses, sendo a primeira dose de 1mg e as quatro subseqüentes de 0,5 mg, com intervalos aproximadamente de quinze dias. Observando-se que os títulos não mais variaram após a quinta inoculação, os animais foram sangrados no 62° dia e os anticorpos anti-HBs obtidos foram padronizados através dos seguintes métodos para detecção de antígeno HBs: a) Hemaglutinação passiva reversa (HAPR) – utilizando-se a gamaglobulina específica obtida de soro imune dos coelhos através de cromatografia de afinidade, alcançando uma concentração ótima de apenas 10µg/ml para a sensibilização de hemácias de carneiro a 5%, fixadas com glutaraldeído. B) Contraimunoeletroforese (CIEF) – utilizando-se o soro imune diluído até 1/20 como reagente para a detecção do antígeno HBs. O soro imune anti-HBs foi também utilizado para a conjugação com uma nova resina de Sepharose 4b tendo uma captação aproximada de 0,5 a 1,0mg antígeno HBs por ml de resina após completa saturação.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.

Diagnóstico rápido de citomegalovirus (CMV) en pacientes inmunocomprometidos mediante anticuerpos monoclonales que reconocen proteinas precoces virales

Se aplicó la técnica de detección de antigenos precoces fluorescentes (DAPF) usando el anticuerpo monoclonal E-13 McAb, mediante el cual se lograron detectar 15 casos positivos a CMV de 75 muestras de orina o sangre ("buffy coat") tomadas de 52 pacientes inmunocomprometidos ingresados en el Instituto de Nefrología de ciudad Habana. Aplicando las técnicas clásicas de aislamiento en fibroblastos humanos diploides (MRC-5), se lograron aislar 12 cepas de CMV de casos previamente positivos por DAPF; lográndose además un aislamiento en una muestra reportada negativa por fluorescencia. Se observó una coincidencia de un 80% entre ambas técnicas. Se detectó la presencia de anticuerpos IgG contra CMV en todos los casos estudiados, utilizando para ello la técnica ELISA.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Summary : Internal ribosome entry sites (IRES) are used by viruses as a strategy to bypass inhibition of cap-dependent translation that commonly results from viral infection. IRES are also used in eukaryotic cells to control mRNA translation under conditions of cellular stress (apoptosis, heat shock) or during the G2 phase of the cell cycle when general protein synthesis is inhibited. Variation in cellular expression levels has been shown to be inherited. Expression is controlled, among others, by transcriptional factors and by the efficiency of cap-mediated translation and ribosome activity. We aimed at identifying genomic determinants of variability in IRES-mediated translation of two representative IRES [Encephalomyocarditis virus (EMCV) and X-linked Inhibitor-of-Apoptosis (XIAP) IRES]. We used bicistronic lentiviral constructions expressing two fluorescent reporter transgenes. Lentiviruses were used to transduce seven different laboratory cell lines and B lymphoblastoid cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209); representing an in vitro approach to family structure allowing genome scan analyses. The relative expression of the two markers was assessed by FACS. IRES efficiency varies according to cellular background, but also varies, for a same cell type, among individuals. The control of IRES activity presents an inherited component (h2) of 0.47 and 0.36 for EMCV and XIAP IRES, respectively. A genome scan identified a suggestive Quantitative Trait Loci (LOD 2.35) involved in the control of XIAP IRES activity. Résumé : Les sites internes d'entrée des ribosomes (IRES = internal ribosome entry sites) sont utilisés par les virus comme une stratégie afin d'outrepasser l'inhibition de traduction qui résulte communément d'une infection virale. Les IRES sont également utilisés par les cellules eucaryotes pour contrôler la traduction de l'ARN messager dans des conditions de stress cellulaire (apoptose, choc thermique) ou durant la phase G2 du cycle cellulaire, situations durant lesquelles la synthèse générale des protéines est inhibée. La variation des niveaux d'expression cellulaire de transcription est un caractère héréditaire. L'expression des gènes est contrôlée entre autre par les facteurs de transcription et par l'efficacité de la traduction initiée par la coiffe ainsi que par l'activité des ribosomes. Durant cette étude nous avons eu pour but d'identifier les déterminants génomiques responsables de la variabilité de la traduction contrôlée par l'IRES. Ceci a été effectué en étudiant deux IRES représentatifs : l'IRES du virus de l'encéphalomyocardite (EMCV) et l'IRES de l'inhibiteur de l'apoptose XIAP (X-linked Inhibitor-of-Apoptosis). Nous avons utilisés des lentivirus délivrant un transgène bicistronique codant pour deux gènes rapporteurs fluorescents. Ces lentivirus ont été utilisés pour transduire sept différentes lignées cellulaires de laboratoire et des lignées cellulaires lymphoblastoïdes B du Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209) qui représentent une approche in vitro de la structure familiale et qui permettent des analyses par balayage du génome. L'expression relative des deux marqueurs fluorescents a été analysée par FACS. Nos résultats montrent que l'efficacité des IRES varie en fonction du type de cellules. Il varie aussi, pour le même type de cellules, selon les individus. Le contrôle de l'activité de l'IRES est un caractère héritable (héritabilité h2) de 0.47 et 0.36 pour les IRES de EMCV et XIAP respectivement. Le balayage du génome a permis l'identification d'un locus à effets quantitatifs [QTL Quantitative Trait Loci (LOD 2.35)] impliqué dans le contôle de l'activité de l'IRES de XIAP.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Diagnóstico de la cuenca de río Frío, Arenal - Huetar Norte, Costa Rica

El diagnóstico de la cuenca de río Frío forma parte del proyecto Araucaria XXI "Unidos por el agua", el objetivo principal de este estudio es conseguir una visión general en 4 ámbitos: biofísico, social, económico y administrativo para conocer el estado actual de la zona y poder conocer las principales problemáticas para posteriormente realizar el Plan de Manejo. La cuenca del río Frío se localiza en el Área de Conservación Huetar- Norte y esta constituida principalmente por los cantones de Guatuso y Los Chiles. Se trata de una zona con una importante variedad de ecosistemas y con una importante diversidad biológica, principalmente la avifauna. Esta zona ha sufrido a lo largo de los años una importante transformación paisajística, las grandes masas boscosas han sido modificadas muy rápidamente en pastos y cultivos, que son las principales actividades económicas. Se trata de una zona aislada de Costa Rica y fronteriza con Nicaragua, la situación de la zona tanto económica como social es mala, presenta índices de analfabetismo elevados, falta de fuentes de empleo, falta de recursos económicos y de presencia institucional. Las infraestructuras viales presentan un estado muy deteriorado que dificulta el desarrollo de actividades turísticas. A todo este conjunto de problemática observadas gracias al diagnóstico se les han planteado toda una serie de estrategias orientativas para mejorar la gestión de la cuenca del río Frío que buscan un desarrollo más sostenible de la zona.

Diagnóstico del Refugio Nacional de Vida Silvestre Caño Negro, Arenal - Huetar Norte, Costa Rica

Se ha elaborado un diagnóstico a partir de un exhaustivo análisis del Refugio Nacional de Vida Silvestre Caño Negro. Esta zona pertenece al Área de Conservación Arenal Huetar Norte, situado al norte de Costa Rica. El refugio forma parte de la cuenca de río Frío y consiste en un sistema de humedales continentales. El diagnóstico contiene la descripción de los aspectos biofísicos, socioeconómicos y administrativo-institucionales del área. La metodología utilizada se basa en un estudio de la bibliografía sobre la zona y posteriormente un análisis detallado de este para observar las problemáticas y oportunidades. Se ha observado que el humedal consta de una gran riqueza ecológica tanto de flora como de fauna; presenta una situación socioeconómica con cifras preocupantes de empleo, salud y educación, por tratarse de una zona aislada. Administrativamente hablando tiene fuertes déficits tanto de presupuesto como de personal. También se trata de un ecosistema con grandes oportunidades como la gran biodiversidad que presenta (por lo que incluso recibió un reconocimiento RAMSAR), su gran potencial eco-turístico, su gran importancia migratoria para la avifauna e incluso su riqueza paisajística. A partir de esta realidad se han planteado unas estrategias de mejora como son la incorporación de nuevas actividades, una mejor gestión de los recursos o incluso un mayor control del Refugio para así mejorar la situación actual.

Viral envelope glycoprotein processing by proprotein convertases.

The proprotein convertases (PCs) are a family of nine mammalian enzymes that play key roles in the maintenance of cell homeostasis by activating or inactivating proteins via limited proteolysis under temporal and spatial control. A wide range of pathogens, including major human pathogenic viruses can hijack cellular PCs for their own purposes. In particular, productive infection with many enveloped viruses critically depends on the processing of their fusion-active viral envelope glycoproteins by cellular PCs. Based on their crucial role in virus-host interaction, PCs can be important determinants for viral pathogenesis and represent promising targets of therapeutic antiviral intervention. In the present review we will cover basic aspects and recent developments of PC-mediated maturation of viral envelope glycoproteins of selected medically important viruses. The molecular mechanisms underlying the recognition of PCs by viral glycoproteins will be described, including recent findings demonstrating differential PC-recognition of viral and cellular substrates. We will further discuss a possible scenario how viruses during co-evolution with their hosts adapted their glycoproteins to modulate the activity of cellular PCs for their own benefit and discuss the consequences for virus-host interaction and pathogenesis. Particular attention will be given to past and current efforts to evaluate cellular PCs as targets for antiviral therapeutic intervention, with emphasis on emerging highly pathogenic viruses for which no efficacious drugs or vaccines are currently available.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

IL28B polymorphism is significantly correlated with ifn anti-viral effectiveness already on first day of pegylated interferon-alpha and ribavirin therapy of chronic HCV infection

Background and Aims: Recently, single nucleotide polymorphisms (SNPs) in IL28B were shown to correlate with response to pegylated interferon-a (IFN) and ribavirin therapy of chronic HCV infection. However, the cause for the SNPs effect on therapy response and its application for direct anti-viral (DAV) treatment are not clear. Here, we analyze early HCV kinetics as function of IL28B SNPs to determine its specific effect on viral dynamics. Methods: IL28B SNPs rs8099917, rs12979860 and rs12980275 were genotyped in 252 chronically HCV infected Caucasian naïve patients (67% HCV genotype 1, 28% genotype 2-3) receiving peginterferonalfa- 2a (180 mg/qw) plus ribavirin (1000-1200 mg/qd) in the DITTO study. HCV-RNA was measured (LD = 50 IU/ml) frequently during first 28 days. Results: RVR was achieved in 33% of genotype 1 patients with genotype CC at rs12979860 versus 12-16% for genotypes TT and CT (P < 0.03). Significant (P < 0.001) difference in viral decline was observed already at day 1 (see Figure). First phase decline was significantly (P < 0.001) larger in patients with genotype CC (2.0 log) than for TT and CT genotypes (0.6 and 0.8), indicating IFN anti-viral effectiveness in blocking virion production of 99% versus 75-84%. There was no significant association between second phase slope and rs12979860 genotype in patients with a first phase decline larger than 1 log. HCV kinetics as function of IL28b SNP. The same trend (not shown) was observed for HCV genotype 2-3 patients with different SNP genotype distribution that may indicate differential selection pressure as function of HCV genotype. Similar results were observed for SNPs rs8099917 and rs12980275, with a strong linkage disequilibrium among the 3 loci allowing to define the composite haplotype best associated with IFN effectiveness. Conclusions: IFN effectiveness in blocking virion production/ release is strongly affected by IL28B SNPs, but not other viral dynamic properties such as infected cell loss rate. Thus, IFN based therapy, as standard-of-care or in combination with DAV, should consider IL28B SNPs for prediction and personalized treatment, while response to pure DAV treatment may be less affected by IL28B SNPs. Additional analyses are undergoing to pinpoint the SNP effect on IFN anti-viral effectiveness.