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

Emotional Speech Analysis using Artificial Neural Networks

Abstract—In the present text, we deal with the problem of classification of speech emotion. Problems of speech processing are addressed through the use of artificial neural networks (ANN). The results can be use for two research projects- for prosody modelling and for analysis of disordered speech. The first ANN topology discussed is the multilayer neural network (MLNN) with the BPG learning algorithm, while the supervised SOM (SSOM) are the second ANN topology. Our aim is to verify the various of knowledge from phonetics and ANN but also to try to classify speech signals which are described by musical theory. Finally, one solution is given for this problem which is supplemented with a proof. I

Two sisters with cardiac‐urogenital syndrome secondary to pathogenic splicing variant in the MYRF gene with unaffected parents: A case of gonadal mosaicism?

Abstract Background Cardiac‐urogenital syndrome [MIM # 618280] is a newly described very rare syndrome associated with pathogenic variants in the myelin regulatory factor (MYRF) gene that leads to loss of protein function. MYRF is a transcription factor previously associated only with the control of myelin‐related gene expression. However, it is also highly expressed in other tissues and associated with various organ anomalies. The clinical picture is primarily dominated by complex congenital cardiac developmental defects, pulmonary hypoplasia, congenital diaphragmatic hernia, and urogenital malformations. Case Presentation We present case reports of two siblings of unrelated parents in whom whole‐exome sequencing was indicated due to familial occurrence of extensive developmental defects. A new, previously undescribed splicing pathogenic variant c.1388+2T>G in the MYRF gene has been identified in both patients. Both parents are unaffected, tested negative, and have another healthy daughter. The identical de novo event in siblings suggests gonadal mosaicism, which can mimic recessive inheritance. Conclusions To our knowledge, this is the first published case of familial cardiac‐urogenital syndrome indicating gonadal mosaicism

Cytokine array analysis of rat intestinal loops washes.

<p>Layout of the arrays (A), cytokine profiles from loops treated with PBS (control) (B), gliadin (C), gliadin+IFN-γ (D), <i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ (E), <i>E. coli</i> CBL2+gliadin+IFN-γ (F), and <i>E. coli</i> CBL2+<i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ (G). The data are expressed as relative levels of selected cytokines (percentage of positive controls). Cytokine-induced neutrophil chemoattractant (CINC)-2 and -3, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-3α, nerve growth factor β-(NGF), tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF). The signal intensity was measured using the LAS-1000 luminescence detector (Fujifilm, Tokyo, Japan).</p

Adhesion of different bacterial strains to IEC-6 cells.

<p>The highest percentage of adhered bacteria was observed for <i>E. coli</i> CBL2 and <i>Shigella</i> CBD8. The differences between tested bacterial strains, as well as the effect of simultaneously added gliadin fragments were non-significant as established by applying the Mann-Whitney U-test. Data are expressed as medians and interquartile ranges (25% to 75%) of adhesion of four independent experiments. None of the differences was found to be statistically significant (P<0.05). The separate dot indicates an outlier.</p

Effects of bacterial strains and gliadin on goblet cells.

<p>Histological staining of PAS-positive goblet cells in rat intestinal loops exposed to: <i>B. bifidum</i> IATA-ES2+gliadin (200 µg) (A), IFN-γ (225 U) (B), <i>Shigella</i> CBD8+gliadin (C), <i>Shigella</i> CBD8+gliadin +IFN-γ (D), <i>E. coli</i> CBL2+gliadin (E) and <i>E. coli</i> CBL2+gliadin+IFN-γ (F). Bacteria were applied at 10⁶/loop. Changes in goblet cells are expressed as medians and interquartile ranges (25% to 75%) of the number of PAS-positive goblet cells/100 epithelial cells (G). These values were for <i>B. bifidum</i> IATA-ES2 (39, 35–41), <i>E. coli</i> CBL2 (38, 35–41) and <i>Shigella</i> CBD8 (25, 20–27) when applied alone to the loops. Different letters (a–e) indicate statistically significant differences between medians as calculated by Mann-Whitney U test (P<0.05). Identical letters correspond to non-significant differences. The separate dots or asterisks indicate outliers. The pictures were obtained for the specimens viewed under an Olympus BX 40. Scale bar, 50 µm.</p

Changes in intestinal permeability induced by gliadin and various bacterial strains.

<p>Intestinal loops were exposed to: gliadin fragments with IFN-γ and <i>B. bifidum</i> IATA-ES2 (A,D), gliadin+IFN-γ+<i>E. coli</i> CBL2 (B,E) and <i>Shigella</i> CBD8+gliadin+IFN-γ (C,F); The white arrows indicate gliadin fragments found by immunofluorescence (A–C) using mouse peroxidase-labeled monoclonal anti-gliadin antibody and TSA™ Plus Fluorescence systems and black arrows indicate goblet cells (D, E). Bacteria were applied at 10⁶/loop. The specimens were viewed under confocal microscope Olympus FV 1000 SIM using differential interference contrast (D–F). Scale bar, 20 µm.</p

Distribution of claudin-1 and ZO-1 in rat intestinal loops.

<p>Exposure of intestinal tissue to gliadin digest alone (A) or with IFN-γ (C) led to reduced ZO-1 expression at the periphery of the villi. The combination of gliadin+IFN-γ+<i>B. bifidum</i> IATA-ES2 (E) maintained the original level of ZO-1 as in PBS-treated loops (I). When gliadin+IFN-γ were applied with <i>E. coli</i> CBL2 (G) ZO-1 fluorescence was weaker. No changes in claudin-1 localization (B, D, F, H) were detected after any stimulus in comparison with PBS control (J). Representative pictures of three experiments are shown. The specimens were viewed under an Olympus BX 40 microscope. Scale bar, 20 µm. Western blot of tissue lysates (K) from intestinal loops stimulated with: 1. gliadin, 2. gliadin+IFN-γ, 3. <i>B. bifidum</i> IATA-ES2+gliadin, 4. <i>E. coli</i> CBL2+gliadin +IFN-γ, 5. <i>E. coli</i> CBL2+<i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ, 6. <i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ, and 7. PBS. The separated proteins on membranes were stained with anti ZO-1 or claudin-1 antibodies and re-probed with antibodies against β-actin to document the same protein concentration in all samples.</p

<i>Bordetella</i> Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation, Migration and T Cell Stimulatory Capacity of Dendritic Cells

<div><p>Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent <i>Bordetella pertussis</i>. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 <i>in vitro</i>. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells <i>in vitro</i>. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during <i>B. pertussis</i> infection.</p></div