Role of Intestinal Bacteria in Gliadin-Induced Changes in Intestinal Mucosa: Study in Germ-Free Rats

10 pages, 6 figures.[Background and Aims]: Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production.[Methodology/Principal Findings]: Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-γ) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro. Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-γ significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-γ induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-γ and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection.[Conclusions]: Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.This work was supported by grants 310/07/0414, 303/08/0367, P304/10/P406 of the Grant Agency of the Czech Republic; IAA500200801, IAA500200710, KJB50020094 of the Academy of Sciences; AV CR-C.S.I.C. 09/10, Project 2B06155 of the Ministry of Education; and Institutional Research Concept AVOZ50200510. This work was also supported by grants 2006CZ0030 and 2008CZ0023 from Consejo Superior de Investigaciones Científicas (CSIC, Spain) and AGL2008-01440/ALI and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation. The scholarship to G. De Palma from Junta de Ampliación de Estudios - Consejo Superior de Investigaciones Científicas (JAE-CSIC; Spain) is fully acknowledged.Peer reviewe

Preliminary Results of Ocular Artefacts Identification in EEC Series by Neural Network

The human electroencephalogram (EEG), is record of the electrical activity of the brain and contains useful diagnostic information on a variety of neurological disorders. Normal EEG signal are usually registered from electrodes placed on the scalp, and are often very small in amplitude, of 20 µV. The EEG, like all biomedical signals, is very susceptible to a variety of large signal contamination or artefacts (signals of other than brain activity) which reduce its clinical usefulness. For example, blinking or moving eyes produces large electrical potentials around the eyes called the electrooculogram (EOG). The EOG spreads across the scalp to contaminate the EEG, when it is referred to as an ocular artefact (OA). This paper includes method of identification portion of the EEG record where ocular artefact appears and classification its type by neural network

Formación profesional : revista europea

Monográfico sobre la formación profesional en los nuevos países de la Unión EuropeaLos rasgos educativos característicos de la población Checa son el fuerte porcentaje de cualificaciones CINE 3 y la escasa proporción de mano de obra con educación básica (CINE 1 y 2) o sin educación alguna (CINE 0) y de personas con cualificación terciaria (CINE 5, 6). El decenio de los 90 ha sido testigo de un descenso radical en la cifra de alumnos matriculados en escuelas profesionales secundarias y de un incremento en la participación de programas secundarios conducentes al examen de titulación de bachillerato y a la educación terciaria. El desarrollo ulterior de la educación terciaria se encuentra impedido por la insuficiente capacidad de los centros de enseñanza.LU

The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells.

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture

Bordetella Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells

Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent Bordetella pertussis. The adenylate cyclase toxin (CyaA) mediates immune evasion of B. pertussis by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. Bordetella pertussis, the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing B. pertussis bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on in vitro differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated in vitro. The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens

The RT-PCR analysis of hAECs.

<p>The RT-PCR analysis of hAECs after de-epithelialization and each passage (P0-P5). The iPS cells were used as a positive (iPS) and corneal fibroblasts as negative control (CF). Sample without cDNA (NTC) was used as non-template control. One representative experiment of 3 (with identical results) is shown.</p