Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responsesFil: Hiriart, Yanina. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Rossi, Andrés Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Errea, Agustina Juliana. Universidad Nacional de La Plata; Argentina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Moreno, Griselda Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Cayet, D.. Universidad Nacional de La Plata; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blancá, Bruno Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Sirard, J.C.. Centre National de la Recherche Scientifique; FranciaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.Fil: Crispo, Martina. Unidad de Animales Transgénicos y de Experimentación – Institut Pasteur de Montevideo; UruguayFil: Van Maele, Laurye. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Tabareau, Julien. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Cayet, Delphine. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Errea, Agustina Juliana. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Ferreira, Ana María. Cátedra de Inmunologíaa. Facultad de Ciencias/Facultad de Química. Universidad de la República; UruguayFil: Rumbo, Martin. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Sirard, Jean Claude. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; Franci

Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes

BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff ≤ 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p ≤ 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression

Design and validation of an instrument about quality of the advance care planning for professionals

[ES] Objetivo: Diseñar y validar un cuestionario dirigido a profesionales para la medición de la calidad del proceso de planificación anticipada de decisiones y determinar sus propiedades psicométricas. Método: Estudio instrumental de validación de cuestionario. Emplazamiento: centros de atención pri maria y consultas externas de hospitales de Ferrol, Ourense, Monforte-Lugo y el Área Sanitaria V del Servicio de Salud de Asturias. Participantes: médico/as y enfermera/os. Fases: 1) diseño de la primera versión del cuestionario mediante técnica Delphi; 2) construcción de la segunda versión de 21 ítems, tras acuerdo de jueces y prueba W de Kendal; 3) pilotaje, análisis de consistencia interna mediante alfa de Cronbach y coeficiente omega,test-retest mediante coeficiente de correlación de Pearson; y 4) validación, test de esfericidad de Barlett y medida de Kaiser-Meyer-Olkin, análisis factorial exploratorio con rotación varimax y estudio de las dimensiones del cuestionario (número, coeficiente de correlación intraclase y correlación), consistencia interna mediante alfa de Cronbach. Resultados: Estudio piloto con 28 profesionales. Fiabilidad ɷ = 0.917, α = 0, 841 y coeficiente de correlación test-retest 0,785 (intervalo de confianza del 95%: 0,587-0,894; p < 0,001). Validación del cuestionario (21 ítems) en 204 profesionales. El análisis de los ítems y el factorial exploratorio (test de Barlett [χ2 = 1298,789] y KMO = 0,808; p < 0,001) arrojaron un modelo de cinco factores que explican el 64,377% de la varianza total, con 18 ítems agrupados en 5 dimensiones (información, preferencias, intención de conducta, capacitación y comunicación). El alfa de Cronbach del cuestionario global fue de 0,841. Conclusiones: El cuestionario ProPAD-pro ha demostrado ser un instrumento válido y fiable para evaluar la calidad del proceso de planificación anticipada de decisiones. [EN] Objective: To develop a questionnaire aimed to measure the quality of the advance care planning process in healthcare professionals and to assess its psychometric quality. Method: Instrumental questionnaire validation study. Scope: primary care centers and outpatients clinics from general hospitals in Ferrol, Ourense, Monforte-Lugo and Health Area V of the Health Service of Asturias. Participants: physicians and nurses. Phases: (1) design of the first version of the questionnaire using the Delphi technique; (2) construction of the second version of 21 items, after judges' agreement and Kendall's W test; (3) pilot study: internal consistency using Cronbach's alpha and omega coefficient, retest-test by Pearson's correlation coefficient; (4) validation: Barlett's spherity test and Kaiser-Meyer-Olkin measurement, exploratory factor analysis with varimax rotation and study of the dimensions of the questionnaire (number, CCI and correlation), internal consistency using Cronbach's alpha. Results: Pilot study with 28 professionals. Reliability ɷ=0.917, α=0.841, and test-retest correlation coefficient of 0.785 (95% confidence interval: 0.587-0.894; p<0.001). Validation of the questionnaire (21 items) in 204 professionals. Ítem analysis and exploratory factor analysis (Barlett's test [χ2=1298,789] and KMO=0.808; p<0.001) yielded a five-factor model explaining 64.377% of the total variance, with 18 items grouped into 5 dimensions (information, preferences, behavioral intention, training and communication). Cronbach's alpha of the global questionnaire was 0.841. Conclusions: The ProPAD-pro questionnaire has showed to be a valid and reliable instrument to assess the quality of the advance care planning process.Proyecto financiado por los Proyectos de Investigación en Salud (Modalidad Proyectos de Investigación) de la convocatoria 2016 de la Acción Estratégica en Salud 2013-2016, y cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER), Núm. exp. PI16/01686.S

Defining new criteria for selection of cell-based intestinal models using publicly available databases

Peer reviewe

Research Priorities for Neglected Infectious Diseases in Latin America and the Caribbean Region

Dujardin, J. C. et al. 5 p.-1 tab.Global priorities for research in neglected infectious diseases (NIDs) can be assessed in different ways, but it is important to realize that regional priorities may significantly differ one from another. The region of Latin America and the Caribbean (LAC) is—along with Africa and Asia—more affected by NIDs than other regions of the world. Some of the Latin American NIDs are common to other continents, while others are very specific or disproportionately affect the Latin American region [1– 3] (Table 1). Because of its huge ecological diversity, ongoing environmental changes, and massive migrations, LAC is also a catalyst for the (re-)emergence and spreading of NIDs, both inside and outside the subcontinent. Following a colloquium on NIDs in LAC held in Lima, Peru, between 12 and 14 November 2009, a thematic workshop was organized with the support of the European Commission (EC). It involved 29 scientists (16 from the Americas, two from the Democratic Republic of Congo and India, respectively, and nine from Europe) working on different NIDs and representing several research areas from basic to applied. This report summarizes the consensus comments of the expert group after oral and written consultation. It is envisaged that this document should stimulate a debate within the scientific community and serve as a recommendation for future actions by international or regional funding agencies in the area of NIDs in LACThis work was supported by the Directorate-General for Development Cooperation of the Belgian Government (framework agreement 03, project 95502) and the European CommissionPeer reviewe

Management of multidrug resistant Gram-negative bacilli infections in solid organ transplant recipients: SET/GESITRA-SEIMC/REIPI recommendations

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant (MDR) Gram-negative bacilli (GNB), as they are frequently exposed to antibiotics and the healthcare setting, and are regulary subject to invasive procedures. Nevertheless, no recommendations concerning prevention and treatment are available. A panel of experts revised the available evidence; this document summarizes their recommendations: (1) it is important to characterize the isolate´s phenotypic and genotypic resistance profile; (2) overall, donor colonization should not constitute a contraindication to transplantation, although active infected kidney and lung grafts should be avoided; (3) recipient colonization is associated with an increased risk of infection, but is not a contraindication to transplantation; (4) different surgical prophylaxis regimens are not recommended for patients colonized with carbapenem-resistant GNB; (5) timely detection of carriers, contact isolation precautions, hand hygiene compliance and antibiotic control policies are important preventive measures; (6) there is not sufficient data to recommend intestinal decolonization; (7) colonized lung transplant recipients could benefit from prophylactic inhaled antibiotics, specially for Pseudomonas aeruginosa; (8) colonized SOT recipients should receive an empirical treatment which includes active antibiotics, and directed therapy should be adjusted according to susceptibility study results and the severity of the infection.J.T.S. holds a research contract from the Fundación para la Formación e Investigación de los Profesionales de la Salud de Extremadura (FundeSalud), Instituto de Salud Carlos III. M.F.R. holds a clinical research contract “Juan Rodés” (JR14/00036) from the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III

Review article: Direct-acting antivirals for the treatment of HCV during pregnancy and lactation: implications for maternal dosing, foetal exposure, and safety for mother and child

BACKGROUND: With the global efforts to eradicate hepatitis C virus (HCV), treatment during pregnancy is becoming a priority for research as this, and maternal cure should reduce vertical transmission. However, as information on the efficacy and safety of direct-acting antivirals (DAAs) in pregnancy is generally lacking, treatment of HCV infection during pregnancy is not currently recommended. AIM: To provide an overview of current knowledge regarding maternal exposure, placental handling and safety of DAAs during pregnancy and lactation METHODS: A literature search was performed focusing on the effect of pregnancy on maternal exposure to DAAs, the placental handling of DAAs, the safety of DAAs for mother and child during pregnancy and the safety of DAAs during lactation. RESULTS: Exposure to all DAAs studied is likely to be altered during pregnancy, mostly related to pregnancy-induced effects on drug absorption and metabolism. Although animal studies show that most DAAs are reported to cross the placenta and transfer into breast milk, most DAA combinations show a favourable safety profile. Because of the rapid viral decline after treatment initiation, and to avoid the critical period of organogenesis, treatment may be started at the end of the second trimester or early third trimester. CONCLUSIONS: Treatment of HCV infection during pregnancy is realistic, as DAAs are highly effective and treatment duration is relatively short. There is an urgent need to study DAAs during pregnancy and lactation to contribute to the goal of HCV elimination

Studying young recipients of alcohol marketing : Two research paradigms and their possible consolidation

Peer reviewe

Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa.

Flagella contribute to the virulence of pathogenic bacteria through chemotaxis, adhesion to and invasion of host surfaces. Flagellin is the structural protein that forms the major portion of flagellar filaments. Thus, flagellin consists of a conserved domain that is widespread in bacterial species and is dedicated to filament polymerization. Conversely, mammalian hosts detect the conserved domain on flagellin monomers through Toll-like receptor (TLR) 5, which triggers proinflammatory and adaptive immune responses. This review describes the relationships among flagellin molecular structure, bacterial virulence and host defenses, with special emphasis on mucosal tissues