Immunopathological properties of the Campylobacter jejuni flagellins and the adhesin CadF as assessed in a clinical murine infection model

Background: Campylobacter jejuni infections constitute serious threats to human health with increasing prevalences worldwide. Our knowledge regarding the molecular mechanisms underlying host-pathogen interactions is still limited. Our group has established a clinical C. jejuni infection model based on abiotic IL-10-/- mice mimicking key features of human campylobacteriosis. In order to further validate this model for unraveling pathogen-host interactions mounting in acute disease, we here surveyed the immunopathological features of the important C. jejuni virulence factors FlaA and FlaB and the major adhesin CadF (Campylobacter adhesin to fibronectin), which play a role in bacterial motility, protein secretion and adhesion, respectively. Methods and results: Therefore, abiotic IL-10-/- mice were perorally infected with C. jejuni strain 81-176 (WT) or with its isogenic flaA/B (ΔflaA/B) or cadF (ΔcadF) deletion mutants. Cultural analyses revealed that WT and ΔcadF but not ΔflaA/B bacteria stably colonized the stomach, duodenum and ileum, whereas all three strains were present in the colon at comparably high loads on day 6 post-infection. Remarkably, despite high colonic colonization densities, murine infection with the ΔflaA/B strain did not result in overt campylobacteriosis, whereas mice infected with ΔcadF or WT were suffering from acute enterocolitis at day 6 post-infection. These symptoms coincided with pronounced pro-inflammatory immune responses, not only in the intestinal tract, but also in other organs such as the liver and kidneys and were accompanied with systemic inflammatory responses as indicated by increased serum MCP-1 concentrations following C. jejuni ΔcadF or WT, but not ΔflaA/B strain infection. Conclusion: For the first time, our observations revealed that the C. jejuni flagellins A/B, but not adhesion mediated by CadF, are essential for inducing murine campylobacteriosis. Furthermore, the secondary abiotic IL-10-/- infection model has been proven suitable not only for detailed investigations of immunological aspects of campylobacteriosis, but also for differential analyses of the roles of distinct C. jejuni virulence factors in induction and progression of disease

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

<p>Abstract</p> <p>Background</p> <p>Host cell invasion by the foodborne pathogen <it>Campylobacter jejuni </it>is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin^-/-, integrin-beta1^-/-, focal adhesion kinase (FAK)^-/-and Src/Yes/Fyn^-/-deficient mice, and wild-type control cells, to investigate <it>C. jejuni</it>-induced mechanisms leading to Cdc42 activation and bacterial uptake.</p> <p>Results</p> <p>Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and <it>C. jejuni </it>invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2^-/-knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using <it>C. jejuni </it>mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry.</p> <p>Conclusion</p> <p>Collectively, our findings led us propose that <it>C. jejuni </it>infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.</p

Approach to Estimate the Phase Formation and the Mechanical Properties of Alloys Processed by Laser Powder Bed Fusion via Casting

A high-performance tool steel with the nominal composition Fe85Cr4Mo8V2C1 (wt%) was processed by three different manufacturing techniques with rising cooling rates: conventional gravity casting, centrifugal casting and an additive manufacturing process, using laser powder bed fusion (LPBF). The resulting material of all processing routes reveals a microstructure, which is composed of martensite, austenite and carbides. However, comparing the size, the morphology and the weight fraction of the present phases, a significant difference of the gravity cast samples is evident, whereas the centrifugal cast material and the LPBF samples show certain commonalities leading finally to similar mechanical properties. This provides the opportunity to roughly estimate the mechanical properties of the material fabricated by LPBF. The major benefit arises from the required small material quantity and the low resources for the preparation of samples by centrifugal casting in comparison to the additive manufacturing process. Concluding, the present findings demonstrate the high attractiveness of centrifugal casting for the effective material screening and hence development of novel alloys adapted to LPBF-processing

Stringent Constraints on Cosmological Neutrino-Antineutrino Asymmetries from Synchronized Flavor Transformation

We assess a mechanism which can transform neutrino-antineutrino asymmetries between flavors in the early universe, and confirm that such transformation is unavoidable in the near bi-maximal framework emerging for the neutrino mixing matrix. We show that the process is a standard Mikheyev-Smirnov-Wolfenstein flavor transformation dictated by a synchronization of momentum states. We also show that flavor ``equilibration'' is a special feature of maximal mixing, and carefully examine new constraints placed on neutrino asymmetries. In particular, the big bang nucleosynthesis limit on electron neutrino degeneracy xi_e < 0.04 does not apply directly to all flavors, yet confirmation of the large-mixing-angle solution to the solar neutrino problem will eliminate the possibility of degenerate big bang nucleosynthesis.Comment: 11 pages, 6 figures; minor changes to match PRD versio

Distribution of Alarin Immunoreactivity in the Mouse Brain

Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic–pituitary–gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism

Prion Protein Is a Key Determinant of Alcohol Sensitivity through the Modulation of N-Methyl-D-Aspartate Receptor (NMDAR) Activity

The prion protein (PrP) is absolutely required for the development of prion diseases; nevertheless, its physiological functions in the central nervous system remain elusive. Using a combination of behavioral, electrophysiological and biochemical approaches in transgenic mouse models, we provide strong evidence for a crucial role of PrP in alcohol sensitivity. Indeed, PrP knock out (PrP−/−) mice presented a greater sensitivity to the sedative effects of EtOH compared to wild-type (wt) control mice. Conversely, compared to wt mice, those over-expressing mouse, human or hamster PrP genes presented a relative insensitivity to ethanol-induced sedation. An acute tolerance (i.e. reversion) to ethanol inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated excitatory post-synaptic potentials in hippocampal slices developed slower in PrP−/− mice than in wt mice. We show that PrP is required to induce acute tolerance to ethanol by activating a Src-protein tyrosine kinase-dependent intracellular signaling pathway. In an attempt to decipher the molecular mechanisms underlying PrP-dependent ethanol effect, we looked for changes in lipid raft features in hippocampus of ethanol-treated wt mice compared to PrP−/− mice. Ethanol induced rapid and transient changes of buoyancy of lipid raft-associated proteins in hippocampus of wt but not PrP−/− mice suggesting a possible mechanistic link for PrP-dependent signal transduction. Together, our results reveal a hitherto unknown physiological role of PrP on the regulation of NMDAR activity and highlight its crucial role in synaptic functions