Peyer's patch-deficient mice demonstrate that Mycobacterium avium subsp. paratuberculosis translocates across the mucosal barrier via both M cells and enterocytes but has inefficient dissemination.

Mycobacterium avium subsp. paratuberculosis, the agent of Johne's disease, infects ruminant hosts by translocation through the intestinal mucosa. A number of studies have suggested that M. avium subsp. paratuberculosis interacts with M cells in the Peyer's patches of the small intestine. The invasion of the intestinal mucosa by M. avium subsp. paratuberculosis and Mycobacterium avium subsp. hominissuis, a pathogen known to interact with intestinal cells, was compared. M. avium subsp. paratuberculosis was capable of invading the mucosa, but it was significantly less efficient at dissemination than M. avium subsp. hominissuis. B-cell knockout (KO) mice, which lack Peyer's patches, were used to demonstrate that M. avium subsp. paratuberculosis enters the intestinal mucosa through enterocytes in the absence of M cells. In addition, the results indicated that M. avium subsp. paratuberculosis had equal abilities to cross the mucosa in both Peyer's patch and non-Peyer's patch segments of normal mice. M. avium subsp. paratuberculosis was also shown to interact with epithelial cells by an alpha(5)beta(1) integrin-independent pathway. Upon translocation, dendritic cells ingest M. avium subsp. paratuberculosis, but this process does not lead to efficient dissemination of the infection. In summary, M. avium subsp. paratuberculosis interacts with the intestinal mucosa by crossing both Peyer's patches and non-Peyer's patch areas but does not translocate or disseminate efficiently.

A Mycobacterium avium subsp. paratuberculosis LuxR regulates cell envelope and virulence.

Mycobacterium avium subsp. paratuberculosis adapts to the environment via the regulation of genes affecting its envelope's composition. Bacteria grown in milk (in vivo conditions) presented differences in the cell wall-associated lipids and in the expression of genes involved in lipid metabolism (FadE8, FadE6 and MAP1420) and host cell invasion (MAP1203, LprL). A different lipid profile was also observed in the envelope of intracellular bacteria after 1 h of infection. Intracellular bacteria showed up-regulation of a LuxR regulator which controls the envelope's composition by up-regulation of FadE8, MAP1420, MAP1203 and LprL and by down-regulation of pks12, mmpL2 and MAP2594. A LuxR-overexpressing strain with a lipid-deficient envelope phenotype, infected epithelial cells more efficiently than the wild-type bacteria; however, it was not more resistant than the wild-type strain to the action of bactericidal proteins. Here we show that LuxR regulates virulence determinants and is involved in mycobacteria adaptation to the host.

Mycobacterium avium subspecies paratuberculosis suppresses expression of IL-12p40 and iNOS genes induced by signalling through CD40 in bovine monocyte-derived macrophages.

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular organism that resides in host macrophages. MAP causes a fatal wasting syndrome in ruminants, typified by granulomatous enteritis in the small intestine. MAP has also been suspected as a causative or exacerbating factor in some cases of human Crohn's disease. In MAP infections, a cytotoxic and proinflammatory Th1-like response is essential to control disease. While such a response may initially develop, this typically gives way to a Th2-like response later in infection. Interaction between CD40 receptors on macrophages and CD154 (CD40L) on activated T cells is crucial for maintaining a Th1 response and activation of macrophages. In this report, we investigated the hypothesis that CD40 signalling is impaired in MAP-infected macrophages. Uninfected bovine monocyte-derived macrophages (MDM) responded to CD40L by up-regulating expression of genes encoding IL-6, TNFalpha, IL-8, iNOS, IL-10, and IL-12p40. In contrast, MDM cells infected with MAP failed to up-regulate expression of iNOS and IL-12p40 genes in response to CD40L. CD40L stimulation caused a transient activation of the mitogen-activated protein kinase (MAPK) family member extracellular signal-regulated kinases (ERK) 1/2, stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) and p38 in MDM cells. In uninfected cells, inhibition of MAPK revealed that CD40L-mediated increase in IL-6 gene expression was dependent on activation of ERK1/2, while increases in IL-12p40, iNOS, and IL-10 gene expression were dependent on activation of p38. Because early activation of p38 was unimpaired in MAP-infected macrophages, we propose that MAP interferes with gene expression of iNOS and IL-12p40 genes downstream of p38.

Evaluación del uso de video juego en escolares de la Región Metropolitana / Evaluation of the use of video game in school children of Metropolitan Region

Introducción: Los video juegos (VJ) producen alteraciones fisiológicas y conductuales a corto plazo. Un 10-15 por ciento de los jugadores desarrollan conductas adictivas. Objetivo: describir el uso de los VJ en población escolar de la región metropolitana, analizar variables individuales y familiares, determinando asociación entre el uso de los VJ y factores de riesgo para adicción. Diseño: Estudio prospectivo descriptivo. Autoaplicación de encuesta con criterios de Tejeiro para adicción a VJ y Apgar familiar. Se utilizó stata 8.1. Resultados: 363 niños encuestados entre 9 y 16 años. Los varones cumplen más criterios para adicción a VJ (p<0.05). Se diferenciaron 2 grupos, bajo riesgo (52.03 por ciento) y alto riesgo de adicción a VJ (47.97 por ciento). Mayor riesgo de adicción en niños insertos en familias disfuncionales con mala supervisión parental y que juegan más de una hora de lunes a viernes. Conclusiones: Se observa asociación entre mayores puntajes en la escala de adicción a VJ con sexo masculino, disfunción familiar, menor supervisión parenteral, menor rendimiento escolar y mayor número de horas dedicadas al uso de video juegos. Es fundamental ampliar la muestra para determinar si los criterios pueden ser aplicados ala población general.

Introduction: video games produce physiologic and conductual abnormalities in short terms. 10 to 15 percent of the video game players develop addictive conducts. Objetive: To describe the use of the video games in the school age population in the metropolitan region and analyze individual and family variables, to determine an association between tha use of video games and the presence of risk factors for addiction. Design: Prospective descriptive study consistent in the selfaplication questionnaire based on Tejeiros addiction to video game criteria and family Apgar. Stata 8.1 was used. Results: The questionnaire was aplicated to 363 children between 9 and 16 years old. Men fulfill more video game addiction criteria. (p < 0.05). Two groups were determined, low risk (52,03 percent) and high risk for videogame addiction was found in children with family malfunction and poor parental supervision that played video games for more than one four from monday to friday. Conclusions: There is an association between high score in the video game addiction scale, male gender, family malfunction, low parental supervision, low scholastic yield and greater number of hours dedicated to video games. It is fundamental to extend the sample, to determine whether the criteria can be applied to the general population.

Relationship between Mycobacterium avium subspecies paratuberculosis, IL-1alpha, and TRAF1 in primary bovine monocyte-derived macrophages.

Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular pathogen that resides in host macrophage cells. Presently, little is known about how MAP is able to subvert the normal bacteriocidal functions of infected macrophages. Previously, we reported that ileal tissues from MAP infected cattle contained high levels of interleukin-1 alpha (IL-1alpha) and tumor necrosis factor receptor-associated factor 1 (TRAF1), relative to ileal tissues from uninfected cattle. High-level expression of these two proteins could have profound effects on macrophage function, intracellular signaling, and apoptosis. We now demonstrate that high levels of TRAF1 protein are located primarily within macrophages infiltrating areas of MAP infection. We have also utilized cultured bovine monocyte-derived macrophage cells (MDM) either infected with live MAP or stimulated with recombinant IL-1alpha (rIL-1alpha) to determine if there is a relationship between IL-1alpha and TRAF1 expression. These studies have identified a dose dependent increase in TRAF1 protein levels in bovine MDM in response to infection with live MAP or following treatment with rIL-1alpha. Sustained TRAF1 protein expression was dependent upon interaction of rIL-1alpha with it's receptor and rIL-1beta was also able to enhance TRAF1 gene expression. Our results suggest that MAP may use the IL-1-TRAF1 system to enhance TRAF1 protein expression in infected bovine MDM. These novel results provide evidence for a new avenue of research on the effect of MAP and other intracellular pathogens on macrophage signaling and apoptosis.

Gene expression profiling of monocyte-derived macrophages following infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) and Mycobacterium avium subspecies avium (MAA) represent two closely related intracellular bacteria with vastly different associated pathologies. MAA can cause severe respiratory infections in immune compromised humans but is nonpathogenic in ruminants and is more readily controlled by the bovine immune system than MAP. MAP causes a fatal wasting syndrome in ruminants, typified by granulomatous enteritis localized in the small intestine. MAP has also been cited as a potential cause of human Crohn's disease. We used a bovine immune-specific microarray (BOTL-5) to compare the response of mature bovine monocyte-derived macrophages (MDM cells) to MAP and MAA. Statistical analysis of microarray data revealed 21 genes not appreciably expressed in resting MDM cells that were activated following infection with either MAA or MAP. Further analysis revealed 144 genes differentially expressed in MDM cells following infection with MAA and 99 genes differentially expressed following infection with MAP. Of these genes, 37 were affected by both types of mycobacteria, with three being affected in opposite directions. Over 41% of the differentially expressed genes in MAA and MAP infected MDM cells were members of, regulated by, or regulators of the MAPK pathways. Expression of selected genes was validated by quantitative real-time reverse transcriptase PCR and in several key genes (i.e., IL-2 receptor, tissue inhibitor of matrix metalloproteinases-1, and Fas-ligand) MAA was found to be a stronger activating factor than MAP. These gene expression patterns were correlated with prolonged activation of p38 MAPK and ERK1/2 by MAA, relative to MAP.

Correlates of public health workforce acceptance of smallpox immunization in Virginia.

OBJECTIVE: By October 24, 2003, 38,577 of 500,000 targeted civilians received smallpox vaccine in the Pre-Event Smallpox Vaccination Campaign, Phase I. We investigated reasons for the low vaccination uptake. DESIGN: Cross-sectional survey, conducted in May 2004. SAMPLE: We surveyed 225 health care personnel, potential members of smallpox response teams in Virginia, who were offered vaccination. We assessed respondents' acceptance of vaccination and its association with factors potentially influencing vaccination: perceptions of vaccine safety, contraindications, concerns about bioterrorism, and workplace influences. RESULTS: Among nonvaccinees (n=44), 70% had a contraindication to the vaccine compared with 8% among vaccinees (n=132). The desire to prepare America for potential bioterrorist attack was associated with acceptance of smallpox vaccination (odds ratio [OR]: 17.7, 95% confidence interval [CI]: 3.6-85.9). Among respondents with contraindications, vaccinees reported more often than nonvaccinees having been asked by their supervisors to be vaccinated (OR: 5; 95% CI: 1.1-22.1) and to have been concerned that their vaccination choice would affect positively their job evaluation (OR: 11; 95% CI: 1.6-81.1). CONCLUSION: Concerns about bioterrorism and willingness to help in the preparedness effort were motivations for vaccination. Continued vigilance to avoid vaccination of those with contraindications is needed.