Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue

BACKGROUND: Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. METHODS: Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). RESULTS: Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P<.001). Histologic analysis identified M. catarrhalis in crypts, intraepithelially, subepithelially, and (using CLSM) intracellularly. M. catarrhalis colocalized with macrophages and B cells in lymphoid follicles. CONCLUSIONS: Colonization by M. catarrhalis is more frequent than is determined by surface culture, because the organism resides both within and beneath the epithelium and invades host cells.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

A BAYESIAN APPROACH TO DOSE-RESPONSE ASSESSMENT AND DRUG-DRUG INTERACTION ANALYSIS: APPLICATION TO IN VITRO STUDIES

The considerable search for synergistic agents in cancer research is motivated by the therapeutic benefits achieved by combining anti-cancer agents. Synergistic agents make it possible to reduce dosage while maintaining or enhancing a desired effect. Other favorable outcomes of synergistic agents include reduction in toxicity and minimizing or delaying drug resistance. Dose-response assessment and drug-drug interaction analysis play an important part in the drug discovery process, however analysis are often poorly done. This dissertation is an effort to notably improve dose-response assessment and drug-drug interaction analysis. The most commonly used method in published analysis is the Median-Effect Principle/Combination Index method (Chou and Talalay, 1984). The Median-Effect Principle/Combination Index method leads to inefficiency by ignoring important sources of variation inherent in dose-response data and discarding data points that do not fit the Median-Effect Principle. Previous work has shown that the conventional method yields a high rate of false positives (Boik, Boik, Newman, 2008; Hennessey, Rosner, Bast, Chen, 2010) and, in some cases, low power to detect synergy. There is a great need for improving the current methodology. We developed a Bayesian framework for dose-response modeling and drug-drug interaction analysis. First, we developed a hierarchical meta-regression dose-response model that accounts for various sources of variation and uncertainty and allows one to incorporate knowledge from prior studies into the current analysis, thus offering a more efficient and reliable inference. Second, in the case that parametric dose-response models do not fit the data, we developed a practical and flexible nonparametric regression method for meta-analysis of independently repeated dose-response experiments. Third, and lastly, we developed a method, based on Loewe additivity that allows one to quantitatively assess interaction between two agents combined at a fixed dose ratio. The proposed method makes a comprehensive and honest account of uncertainty within drug interaction assessment. Extensive simulation studies show that the novel methodology improves the screening process of effective/synergistic agents and reduces the incidence of type I error. We consider an ovarian cancer cell line study that investigates the combined effect of DNA methylation inhibitors and histone deacetylation inhibitors in human ovarian cancer cell lines. The hypothesis is that the combination of DNA methylation inhibitors and histone deacetylation inhibitors will enhance antiproliferative activity in human ovarian cancer cell lines compared to treatment with each inhibitor alone. By applying the proposed Bayesian methodology, in vitro synergy was declared for DNA methylation inhibitor, 5-AZA-2'-deoxycytidine combined with one histone deacetylation inhibitor, suberoylanilide hydroxamic acid or trichostatin A in the cell lines HEY and SKOV3. This suggests potential new epigenetic therapies in cell growth inhibition of ovarian cancer cells.

Moraxella catarrhalis AcrAB-OprM efflux pump contributes to antimicrobial resistance and is enhanced during cold shock response

Moraxella catarrhalis is a common pathogen of the human respiratory tract. Multidrug efflux pumps play a major role in antibiotic resistance and virulence in many Gram-negative organisms. In the present study, the role of the AcrAB-OprM efflux pump in antibiotic resistance was investigated by constructing mutants that lack the acrA, acrB, and oprM genes in M. catarrhalis strain O35E. We observed a moderate (1.5-fold) decrease in the MICs of amoxicillin and cefotaxime and a marked (4.7-fold) decrease in the MICs of clarithromycin for acrA, acrB, and oprM mutants in comparison with the wild-type O35E strain. Exposure of the M. catarrhalis strains O35E and 300 to amoxicillin triggered an increased transcription of all AcrAB-OprM pump genes, and exposure of strains O35E, 300, and 415 to clarithromycin enhanced the expression of acrA and oprM mRNA. Inactivation of the AcrAB-OprM efflux pump genes demonstrated a decreased ability to invade epithelial cells compared to the parental strain, suggesting that acrA, acrB, and oprM are required for efficient invasion of human pharyngeal epithelial cells. Cold shock increases the expression of AcrAB-OprM efflux pump genes in all three M. catarrhalis strains tested. Increased expression of AcrAB-OprM pump genes after cold shock leads to a lower accumulation of Hoechst 33342 (H33342), a substrate of AcrAB-OprM efflux pumps, indicating that cold shock results in increased efflux activity. In conclusion, the AcrAB-OprM efflux pump appears to play a role in the antibiotic resistance and virulence of M. catarrhalis and is involved in the cold shock response.

Manifestation of depression symptoms among Mexican American adolescents

Background. Previous findings reported more depression among Mexican American adolescents than among adolescents from other groups. There has been minimal research published on distribution of depression symptoms among Mexican American adolescents and practically no data has been published from community samples. ^ Objective. To examine the phenomenology of DSM-IV major depression symptoms across groups defined by ethnic status, by gender and language use focusing on the Mexican American group. ^ Methods. Secondary data from 2624 adolescents (ages 10-17) among three ethnic subgroups, Mexican (26.7%), African (45%) and Anglo Americans (28.3%), was analyzed. Data come from the Teen Life Changes (TLC) Survey conducted in 1994 by Roberts et al. (1997). A self-report questionnaire, which includes the DSD scale to measure depression, was used. ^ Results. Analysis of data showed significant differences among youth in the phenomenology of depression symptoms by ethnicity, by gender and by language use at home. ^ Conclusion. This study adds knowledge to the psychopathology and mental health literature from the identification of depression symptoms profile as well as permits the design of more appropriate policy for prevention and intervention programs among culturally diverse youth. ^

A dual oxidase generates a protective oxidative burst during infection in C. elegans

Caenorhabditis elegans has recently been developed as a model system to study both pathogen virulence mechanisms and host defense responses. We have shown that C. elegans produces reactive oxygen species (ROS) in response to exposure to the important Gram-positive, noscomial pathogen, Enterococcus faecalis. We have also shown evidence of oxidative stress and upregulation of stress response after exposure to the pathogen. As in mammalian systems, this work shows that production of ROS for innate immune functions occurs via an NADPH oxidase. Specifically, reducing expression of a dual oxidase, Ce-duox1/BLI-3 causes a decrease in ROS production in response to E. faecalis. We also present evidence that reduction of expression of Ce-duox1/BLI-3 increases susceptibility to this pathogen, specifically when expression is reduced in the intestine and the hypodermis. This dual oxidase has previously been localized to the hypodermis, but we show that it is additionally localized to the intestine of C. elegans. To further demonstrate the protective effects of the pathogen-induced ROS production, we demonstrate that antioxidants that scavenge ROS, increase the sensitivity of the nematode to the infection, in stark contrast to their longevity-promoting effects under non-pathogenic conditions. In conclusion, we postulate that the generation of ROS by NADPH oxidases in the barrier epithelium is an ancient, highly conserved innate immune defense mechanism.^

Producción de materiales didácticos en portugués: enfoque interdisciplinario. 16H368

El presente proyecto es una continuación del proyecto de investigación: Producción de materiales teóricodidácticos en portugués: enfoque interdisciplinario (Cód. 16h286). El contexto de investigación es la Carrera de Profesorado en Portugués de la Facultad de humanidades y Ciencias Sociales – UNaM. El equipo de investigadores y auxiliares involucrados son docentes de las cátedras y graduados adscriptos, que planificarán y llevarán a cabo el trabajo de campo en disciplinas del Profesorado. Como lo indica el título uno de los propósito de la investigación es el de desarrollar materiales didácticos adecuados a las necesidades de las cátedras y estratégicos en la resolución de problemas de aprendizaje. Desde el paradigma interpretativo, se realizará una investigación aplicada e interdisciplinaria, en la que se utilizará el método etnográfico a fin de estudiar el contexto socio-cultural del estudiante y el análisis del discurso para analizar sus producciones, en procura de plantear resoluciones.

Producción de materiales teórico-didácticos en portugués: enfoque interdisciplinario.16H286

El proyecto: Producción de materiales teórico-didácticos en portugués: enfoque interdisciplinario, tiene como propósito el desarrollo de propuestas interdisciplinarias para la elaboración de materiales didácticos. Inicialmente con producciones que apuntan a la proficiencia en portugués en respuesta a la demanda de tres disciplinas de primer año: Lengua Portuguesa I, Comprensión y Producción Discursiva I, Introducción a los Estudios Literarios, así como la articulación de contenidos entre las mismas y de otras cátedras como: Cultura Brasileña, Cultura Portuguesa y Literatura Portuguesa. El contexto de investigación es el Profesorado de Portugués de la Facultad de Humanidades y Ciencias Sociales de la UNAM, en interacción con sectores donde se enseña portugués. Se pretende realizar este trabajo en el lapso de tres años, durante el cual los docentes investigadores planificarán y desarrollarán el trabajo de campo optimizando sus propias actividades en las disciplinas a cargo, tanto en el Profesorado en Portugués como en otras Carreras o contextos de enseñanza del idioma.

Escuelas bilingues de frontera: discursos (des) encontrados. 16H225

El proyecto tiene como propósito analizar los discursos de docentes y funcionarios argentinos y brasileños que integran el Programa bilateral de Enseñanza Común para Escuelas de Frontera Español-portugués. El interés por la temática reside en desvendar problemas existentes en el programa. Problemas que creemos que en parte se fundan en las diferencias conceptuales e ideológicas que se vislumbran al comparar los discursos de los participantes argentinos y brasileños. Para realizar la investigación se seleccionó un corpus de textos basados en diferentes trabajos de campo, tales como: entrevistas, observaciones de clases, notas, textos de circulación interna del programa (acuerdos, informes, relatorías, grabaciones de reuniones, planificaciones conjuntas, etc.). El análisis del mismo fue abordado desde la perspectiva del análisis del discurso.) Objetivos del Proyecto -Identificar cuáles son los conceptos que subyacen en los discursos políticos pedagógicos de equipos técnicos y docentes de ambos países. -Reconocer los factores discursivo-culturales que provocan confusiones y malos entendidos entre argentinos y brasileños. -Interpretar los efectos de sentidos producidos en la interdiscursividad del programa. -Estudiar la problemática para contribuir a mejorar las re

Producción de materiales teórico-didácticos en portugués: enfoque interdisciplinario. 16H286

Actividades desarrolladas durante el período: fueron realizadas aproximadamente veinte reuniones, con el propósito de dar a conocer testimonios sobre las lecturas, las situaciones que se presentan en el aula referidas a nuestra problemática y la elaboración de materiales con fines didácticos presentados en este informe: A) Avance de los materiales elaborados por la cátedra de Cultura Brasileña en articulación con la cátedra de Introducción a los Estudios Literarios: ARTES VISUAIS: Roteiro para a análise, B) Avance de los materiales para Lengua Portuguesa I. La elaboración de los mismos se articuló con contenidos de Comprensión y Producción Discursiva I – materia también de primer año, dando el carácter interdisciplinario que se considera de suma importancia en la adquisición de conocimiento y manejo de competencias lingüísticodiscursivas en portugués. C) Avance de materiales elaborados entre las cátedras de Literatura Portuguesa y Comprensión y Producción Discursiva I: Modelo de análise semiótica segundo Greimas.

Dinámica poblacional de planococcus ficus sign. (Hemiptera-Pseudcoccidae) en viñedos : Mendoza (Argentina)

Planococcus ficus Signoret es un insecto perteneciente al orden Hemiptera, familia Pseudococcidae, cuya población ha aumentado en los últimos años en Mendoza. Ataca gran variedad de hospederos y está distribuido en las principales zonas vitícolas del mundo. Su acción disminuye el vigor general de las plantas, perjudica la calidad de los frutos y las características organolépticas de los vinos elaborados con racimos infestados. El objetivo de este trabajo fue realizar estudios bioecológicos durante la temporada 2004-2005, analizando la forma de pasaje invernal y el número de generaciones anuales. Se marcaron y muestrearon plantas con los signos característicos del ataque. Se recolectó quincenalmente material vegetal infestado. En invierno: trozos de corteza. En primavera-verano: brotes, hojas y racimos. Los resultados indicaron que pasa el invierno principalmente como huevos agrupados en masas algodonosas, bajo la corteza. Se distinguieron seis generaciones, movilizándose conforme al aumento de las temperaturas hacia los brotes y hojas y, a partir de la tercera generación, a los racimos, permaneciendo en todas las partes aéreas de la planta el resto de la temporada hasta la caída de hojas. Con la disminución de las temperaturas retornó hacia la corteza del tronco para invernar.

Trips (Insecta: Thysanoptera) en montes de cerezo en Mendoza, Argentina

En las últimas temporadas se ha relacionado la presencia de trips con daños observados en cerezas en Mendoza, Argentina. Los daños serían producidos por la oviposición de estos insectos sobre frutos pequeños. El objetivo del trabajo fue comparar la evolución de poblaciones de las especies de trips en dos montes frutales con diferente manejo de malezas. Se tomaron muestras de malezas y de flores de cerezo en los dos montes y se analizó la evolución de las poblaciones de trips. Tres especies de trips fueron frecuentes en flores de cerezo en ambos montes: Frankliniella australis, F. occidentalis y Thrips tabaci. F. australis es la de mayor reproducción de las tres. No se observaron diferencias consistentes en cuanto al número de trips por flor de cerezo en ambas fincas y esto puede atribuirse a que la abundancia de dichos insectos en las flores de cerezo depende de otras variables tales como densidad de plantación, aporte por migración de áreas vecinas además de los niveles de infestación de malezas en el cultivo.