Characterization of differentiation markers of the BEM epithelial barrier.

<p>Immunofluorescence staining for a) ZO-1 (green, blue is DNA) and b) F-actin (red). c) Immunofluorescence staining of laminin and integrin alpha-6 (ITGA6) (blue is DNA; red is laminin; green is ITGA6). d) IHC staining of laminin. The arrow indicates the presence of a continuous layer of laminin at the interface between the epithelial sheet and the stromal compartment; antibody isotype control is represented in (e). Images are representative of 4 different experiments.</p

Stable iron isotopes and microbial mediation in red pigmentation of the Rosso Ammonitico (mid-late Jurassic, Verona area, Italy).

The iron (Fe) isotopic composition of 17 Jurassic limestones from the Rosso Ammonitico of Verona (Italy) have been analyzed by Multiple-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICP-MS). Such analysis allowed for the recognition of a clear iron isotopic fractionation (mean -0.8 per thousand, ranging between -1.52 to -0.06 per thousand) on a millimeter-centimeter scale between the red and grey facies of the studied formation. After gentle acid leaching, measurements of the Fe isotopic compositions gave delta(56)Fe values that were systematically lower in the red facies residues (median: -0.84 per thousand, range: -1.46 to +0.26 per thousand) compared to the grey facies residues (median: -0.08 per thousand, range: -0.34 to +0.23 per thousand). In addition, the red facies residues were characterized by a lighter delta(56)Fe signal relative to their corresponding leachates. These Fe isotopic fractionations could be a sensitive fingerprint of a biotic process; systematic isotopic differences between the red and grey facies residues, which consist of hematite and X-ray amorphous iron hydroxides, respectively, are hypothesized to have resulted from the oxidizing activity of iron bacteria and fungi in the red facies. The grey Fe isotopic data match the Fe isotopic signature of the terrestrial baseline established for igneous rocks and low-C(org) clastic sedimentary rocks. The Fe isotopic compositions of the grey laminations are consistent with the influx of detrital iron minerals and lack of microbial redox processes at the water-interface during deposition. Total Fe concentration measurements were performed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) (confirmed by concentration estimations obtained by MC-ICP-MS analyses of microdrilled samples) on five samples, and resultant values range between 0.30% (mean) in the grey facies and 1.31% (mean) in the red facies. No correlation was observed between bulk Fe content and pigmentation or between bulk Fe content and Fe isotopic compositions. The rapid transformation of the original iron oxyhydroxides to hematite could have preserved the original isotopic composition if it had occurred at about the same temperature. This paper supports the use of Fe isotopes as sensitive tracers of biological activities recorded in old sedimentary sequences that contain microfossils of iron bacteria and fungi. However, a careful interpretation of the iron isotopic fractionation in terms of biotic versus abiotic processes requires supporting data or direct observations to characterize the biological, (geo)chemical, or physical context in relation to the geologic setting. This will become even more pertinent when Fe isotopic studies are expanded to the interplanetary realm.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Characterization of epithelial markers by confocal microscopy.

<p>a) Immunofluorescence staining of cytokeratin 5 (blue is DNA; green is CK5; white is transmitted light). b) Immunofluorescence staining of nerve growth factor receptor and integrin alpha-6 (blue is DNA; red is NGFR; green is ITGA6; white is transmitted light). c) Immunofluorescence double staining for cytokeratin 5 and nerve growth factor receptor (blue is DNA; red is NGFR; green is CK5). d) Differential expression of basal cells CK14 and p63 markers (blue is DNA; red is CK14; green is p63). e) Enlargement of an area represented in d); the yellow arrow indicates positivity for both p63 and CK14. Images are representative of 4 different experiments.</p

Club and goblet secretory cells are both present in the epithelial space of the BEM.

<p>a) Immunofluorescence staining of ClCs (positive for CCSP, red). b) Immunofluorescence staining of ClCs (positive for CCSP, red) and goblet cells (positive for MUC5AC, green). c) Immunofluorescence staining reveals the presence of cells positive for both Club cells and goblet cells markers (blue is DNA; red is CCSP; green is MUC5AC). White dotted lines separate the epithelium from the scaffold. Images are representative of 4 different experiments.</p

3D Reconstruction of the Human Airway Mucosa <i>In Vitro</i> as an Experimental Model to Study NTHi Infections

<div><p>We have established an <i>in vitro</i> 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular, we reported that the mature model, entirely constituted of primary cells of human origin, develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable <i>Haemophilus influenzae</i> results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary, we propose our <i>in vitro</i> model as a reliable and potentially customizable system to study mid/long term host-pathogen processes.</p></div

Immunofluorescence characterization of NTHi-infected BEM.

<p>a) Bacteria (red), revealed by using polyclonal antibodies against the whole bacterium, are embedded into a thick mucus layer (white). The contour of epithelial cells is delineated by phalloidin staining (green). b) Bacteria (red) associated with and traversing the epithelial layer (phalloidin, green; DNA, blue). c) NTHi bacteria (red) colonizing the stromal compartment (transmitted light, grey; DNA, blue). d) Outer Membrane Vesicles (red) detected within the bottom side of the BEM stromal compartment (transmitted light, grey; DNA, blue) and bacteria (red), highlighted by asterisks.</p

Mucociliary differentiation of NHBE cells grown on a BEM.

<p>Mucus granules are visible by SEM (a) (indicated by arrows), TEM (b) and confocal analysis (c) (blue is DNA, green is MUC5AC, red is F-actin). Cilia are visible by SEM (d), TEM (e) and confocal analysis (f) (blue is DNA, white is B-tubulin, red is F-actin). Images are representative of 3 independent experiments.</p

SslE contributes to the capacity of IHE3034 strain to reach the surface of mucus-producing epithelial cells.

<p>(A) Schematic representation of the experimental procedure. (B), Percentage of mucus trapped (left panel) and cell associated (right panel) <b>I</b>HE3034 (WT), IHE3034Δ<i>sslE</i> (KO), IHE3034Δ<i>sslE</i>::<i>sslE</i>_WT (COMPL) and IHE3034Δ<i>sslE</i>::<i>sslE</i>_mut (MUT) bacteria after 4hrs of infection. <i>n</i> = 4, **P≤0.01; Error bars, SD; percentages were calculated on recovered CFU respect to the starting inoculum. (C) Co-infection experiments: percentage of mucus trapped (left panel) and cell associated (right panel) <b>I</b>HE3034 (WT), IHE3034Δ<i>sslE</i> (KO) or IHE3034Δ<i>sslE</i> plus IHE3034 (KO_COINF) bacteria. Strains were plated on both non-selective and selective plates to differentiate WT and Δ<i>sslE</i> for CFU counts. <i>n</i> = 4, **P≤0.01; Error bars, SD;</p

Schematic representation of the contribution of SslE to <i>E.coli</i> pathogenesis.

<p>Gut mucus forms two layers, an inner firm mucus layer devoid of bacteria, and an outer layer that is not sterile and is a major habitat for commensal bacteria. <i>E. coli</i> can penetrate this barrier through the SslE-mediated enzymatic degradation of the mucus, targeting epithelial cells. This interaction will eventually lead to IL-8 release and neutrophils recruitment.</p

Different morphology of NHBE cells grown on BEM or transwell.

<p>Haematoxylin-eosin-alcian blue staining of NHBE cells differentiated on a BEM (a) or on a transwell system (b). The arrow indicates the basal layer that forms when NHBE cells are grown on a BEM. Images are representative of 4 different experiments.</p