Miradas y voces de la investigación educativa I

Fil: Ferreyra, Horacio Ademar. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Calneggia, María Isabel. Universidad Católica de Córdoba. Facultad de Educación; ArgentinaFil: Di Francesco, Adriana Carlota. Universidad Católica de Córdoba. Facultad de Educación; Argentin

Espacio y territorios: razón, pasión e imaginarios

En este caleidoscopio de acercamientos hacia lo espacial y territorial, las visiones se mueven desde aquellas románticas y existencialistas, pasando por aquellas objetivistas y positivistas, hasta las estructuralistas y postestructuralistas. Por el espacio y el territorio se interesan con enfoques diversos numerosas disciplinas, desde la psicología, la etología o la literatura, y las ciencias naturales como la biología o la ecología, hasta las ciencias sociales y políticas, como la geografía, la antropología, la economía y la sociología. Este interés multidisciplinario demuestra la importancia y la complejidad del tema espacial y territorial, y reclama la necesidad de su estudio y comprensión interdisciplinarios, como se intenta con esta publicación

Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.

Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314-6025 pg/ml), TNF-α (39-359 pg/ml), and IL-10 (2-96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria

Biochemical analysis of flagella.

<p>(A) Depolymerization of purified flagella in 12% SDS-PAGE gel. (B) Immunoblot assays with anti-flagella antibody recognizing the flagellin of ∼28-kDa. (C) Analysis of the protein band of ∼28-kDa by mass spectrometry. Molecular weight (MW).</p

Transfection assay in HEK293-hTLR5 cells enables IL-8 secretion in response to <i>C. sakazakii</i> flagella and flagellin.

<p>293-hTLR5 cells (HEK293 cells transfected with TLR5) secreted 197 pg/ml and 133 pg/ml of IL-8 after treatment with <i>C. sakazakii</i> (ST1) flagella and flagellin, respectively; and 196 pg/ml of IL-8 were released with FliC-<i>Salmonella enterica</i> serovar Typhimurium. 293-hTLR5 cells pre-incubated with anti-hTLR5-IgA antibodies at a concentration of 10 µg/ml showed a reduction of 76% in IL-8 release.</p

Flagella identification by transmission electron microscopy.

<p>(A) <i>C. sakazakii</i> ATCC BAA-894 strain shows flagellar structures protruding from the bacteria after growth on TSA agar at 37°C. (B) Immunogold-labeling of flagella produced by <i>C. sakazakii</i> on TSA agar using anti-flagella antibody. The inset shows the micrograph of the whole bacteria using immunogold-labeling obtained by TEM. (C) Purified flagella. (D) Immunogold-labeling of purified flagella. Samples were negatively stained and electron micrographs were taken at a magnification of 19,000x.</p

Flagella from <i>C. sakazakii</i> activate cytokine’s release.

<p>Macrophage cells were exposed to flagella (1, 10, 25, 50 and 100 ng/ml) and flagellin (100 ng/ml Tx) for 24 h. (A) High values were observed in the IL-8 release due to the presence of different concentrations of purified flagella from <i>Cronobacter</i> species. (B) Induction of TNF-α in macrophage cells in response to different concentrations of purified flagella from <i>Cronobacter</i> species. (C) A non-significant release of IL-10 was observed when two different concentrations of purified flagella from <i>Cronobacter</i> species were incubated. In addition, cytokines release was also observed when the purified flagella of each <i>Cronobacter</i> species were dissociated in monomers by heat. Lipopolysaccharide 100 ng/ml (LPS), RPMI-1640 medium. Flagellin of <i>Cronobacter</i> (Tx). Flagella of EHEC strain O157:H7 and Flagella from <i>S. enterica</i> serovar Typhimurium (100 ng/ml). * and ** <i>p<</i>0.05.</p