Gliadin induces neutrophil migration via engagement of the formyl peptide receptor, FPR1

Background Gliadin, the immunogenic component within gluten and trigger of celiac disease, is known to induce the production of Interleukin-8, a potent neutrophil-Activating and chemoattractant chemokine.We sought to study the involvement of neutrophils in the early immunological changes following gliadin exposure. Methods Utilizing immunofluorescence microscopy and flow cytometry, the redistribution of major tight junction protein, Zonula occludens (ZO)-1, and neutrophil recruitment were assessed in duodenal tissues of gliadin-gavaged C57BL/6 wild-Type and Lys-GFP reporter mice, respectively. Intravital microscopy with Lys-GFP mice allowed monitoring of neutrophil recruitment in response to luminal gliadin exposure in real time. In vitro chemotaxis assays were used to study murine and human neutrophil chemotaxis to gliadin, synthetic alpha-gliadin peptides and the neutrophil chemoattractant, fMet-Leu-Phe, in the presence or absence of a specific inhibitor of the fMet-Leu-Phe receptor-1 (FPR1), cyclosporine H. An irrelevant protein, zein, served as a control. Results Redistribution of ZO-1 and an influx of CD11b+Lys6G+ cells in the lamina propria of the small intestine were observed upon oral gavage of gliadin. In vivo intravital microscopy revealed a slowing down of GFP+ cells within the vessels and influx in the mucosal tissue within 2 hours after challenge. In vitro chemotaxis assays showed that gliadin strongly induced neutrophil migration, similar to fMet-Leu-Phe.We identified thirteen synthetic gliadin peptide motifs that induced cell migration. Blocking of FPR1 completely abrogated the fMet-Leu-Phe-, gliadin- and synthetic peptide-induced migration. Conclusions Gliadin possesses neutrophil chemoattractant properties similar to the classical neutrophil chemoattractant, fMet-Leu-Phe, and likewise uses FPR1 in the process. Copyright

GEF-H1 controls microtubule-dependent sensing of nucleic acids for antiviral host defenses

Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I and Mda5-dependent phosphorylation of IRF3 and induction of interferon-β expression in macrophages. Generation of Arhgef2−/− mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways

VESGEN Mapping of Bioactive Protection against Intestinal Inflammation: Application to Human Spaceflight and ISS Experiments

Challenges to successful space exploration and colonization include adverse physiological reactions to micro gravity and space radiation factors. Constant remodeling of the microvasculature is critical for tissue preservation, wound healing, and recovery after ischemia. Regulation of the vascular system in the intestine is particularly important to enable nutrient absorption while maintaining barrier function and mucosal defense against micro biota. Although tremendous progress has been made in understanding the molecular circuits regulating neovascularization, our knowledge of the adaptations of the vascular system to environmental challenges in the intestine remains incomplete. This is in part because of the lack of methods to observe and quantify the complex processes associated with vascular responses in vivo. Developed by GRC as a mature beta version, pre-release research software, VESsel GENeration Analysis (VESGEN) maps and quantifies the fractal-based complexity of vascular branching for novel insights into the cytokine, transgenic and therapeutic regulation of angiogenesis, lymphangiogenesis and microvascular remodeling. Here we demonstrate that VESGEN can be used to characterize the dynamic vascular responses to acute intestinal inflammation and mucosal recovery from in vivo confocal microscopic 3D image series. We induced transient intestinal inflammation in mice by DSS treatment and investigated whether the ability of the pro biotic yeast Saccharomyces boulardii (Sb) to protect against intestinal inflammation was due to regulation of vascular remodeling. A primary characteristic of inflammation is excessive neovascularization (angiogenesis) resulting in fragile vessels prone to bleeding. Morphological parameters for triplicate specimens revealed that Sb treatment greatly reduced the inflammatory response of vascular networks by an average of 78%. This resulted from Sb inhibition of vascular endothelial growth factor receptor signaling, a major angiogenesis signaling pathway. It needs to be determined whether pro biotic yeast represents a promising approach to GI protection in space. GRC performed only the VESGEN post-testing analysis

Catching-up and falling behind knowledge spillover from American to German machine tool makers

In our days, German machine tool makers accuse their Chinese competitors of violating patent rights and illegally imitating German technology. A century ago, however, German machine tool makers used exactly the same methods to imitate American technology. To understand the dynamics of this catching-up process we use patent statistics to analyze firms? activities between 1877 and 1932. We show that German machine tool makers successfully deployed imitating and counterfeiting activities in the late 19th century and the 1920s to catchup to their American competitors. The German administration supported this strategy by stipulating a patent law that discriminated against foreign patent holders and probably also by delaying the granting of patents to foreign applicants. Parallel to the growing international competitiveness of German firms, however, the willingness to guarantee intellectual property rights of foreigners was also increasing because German firms had now to fear retaliatory measures in their own export markets when violating foreign property rights within Germany

Complement activation capacity in plasma before and during high-dose prednisolone treatment and tapering in exacerbations of Crohn's disease and ulcerative colitis

BACKGROUND: Ulcerative colitis (UC) and Crohn's disease (CD) are characterized by intestinal inflammation mainly caused by a disturbance in the balance between cytokines and increased complement (C) activation. Our aim was to evaluate possible associations between C activation capacity and prednisolone treatment. METHODS: Plasma from patients with exacerbations of UC (n = 18) or CD (n = 18) were collected before and during high dose prednisolone treatment (1 mg/kg body weight) and tapering. Friedman's two way analysis of variance, Mann-Whitney U test and Wilcoxon signed-rank sum test were used RESULTS: Before treatment, plasma from CD patients showed significant elevations in all C-mediated analyses compared to the values obtained from 38 healthy controls (p < 0.02), and in mannan binding lectin (MBL)-concentration and MBL-C4-activation capacity (AC) values compared to UC patients (p < 0.02). Before treatment, plasma from UC patients showed significant elevations only in the classical pathway-mediated C3-AC compared to values obtained from healthy controls (p < 0.01). After treatment was initiated, significant reductions, which persisted during follow-up, were observed in the classical pathway-mediated C3-AC and MBL-C4-AC in plasma from CD patients (p < 0.05). CONCLUSION: Our findings indicate that C activation capacity is up-regulated significantly in plasma from CD patients. The decreases observed after prednisolone treatment reflect a general down-regulation in immune activation

Identification and Comparative Expression Analysis of Interleukin 2/15 Receptor β Chain in Chickens Infected with E. tenella

BACKGROUND: Interleukin (IL) 2 and IL15 receptor β chain (IL2/15Rβ, CD122) play critical roles in signal transduction for the biological activities of IL2 and IL15. Increased knowledge of non-mammalian IL2/15Rβ will enhance the understanding of IL2 and IL15 functions. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] Chicken IL2/15Rβ (chIL2/15Rβ) cDNA was cloned using 5'/3'-RACE. The predicted protein sequence contained 576 amino acids and typical features of the type-I cytokine receptor family. COS-7 cells transfected with chIL2/15Rβ produced proteins of approximately 75 and 62.5 kDa under normal and tunicamycin-treated conditions, respectively. The genomic structure of chIL2/15Rβ was similar to its mammalian counterparts. chIL2/15Rβ transcripts were detected in the lymphoblast cell line CU205 and in normal lymphoid organs and at moderate levels in bursa samples. Expression profiles of chIL2/15Rβ and its related cytokines and receptors were examined in ConA-stimulated splenic lymphocytes and in ceca-tonsils of Eimeria tenella-infected chickens using quantitative real-time PCR. Expression levels of chIL2/15Rβ, chIL2Rα, and chIL15Rα were generally elevated in ceca-tonsils and ConA-activated splenic lymphocytes. However, chIL2 and chIL15 expression levels were differentially regulated between the samples. chIL2 expression was upregulated in ConA-activated splenic lymphocytes, but not in ceca-tonsils. In constrast, chIL15 expression was upregulated in ceca-tonsils, but not in ConA-activated splenic lymphocytes. CONCLUSIONS/SIGNIFICANCE: We identified an avian form of IL2/15Rβ and compared its gene expression pattern with those of chIL2, chIL15, chIL2Rα, and chIL15Rα. Our observations suggest that chIL15 and its receptors, including chIL2/15Rβ, play important roles in mucosal immunity to intestinal intracellular parasites such as Eimeria

Vascular Patterning Analysis by VESGEN 2D/3D with Bioinformatics: Updates for Rodent Tissues

Fractally branching vascular systems are a complex physiological requirement shared by humans with all higher terrestrial life forms, including other vertebrates, insects, and higher land plants. Vascular trees, networks, and tree-network composites are therefore mapped and quantified by the VESsel GENeration Analysis (VESGEN) software according to weighted physiological vascular rules that include vessel connectivity, tapering and bifurcational branching. According to fluid dynamics, successful vascular transport depends upon a complex distributed system of highly regulated laminar flow. VESGEN has elucidated changes in vascular patterning resulting from inflammatory, developmental and other signaling pathways within numerous tissues of major model organisms important for Space Biology, especially for rodents. Important early stage regenerative opportunities have been identified by VESGEN vascular analysis for visual impairments in the human retina, and is currently being used for research into astronaut visual and ocular disorders associated with long duration missions. The VESGEN 2D software is a mature, automated, widely published capability for which beta testing and public release by NASA is planned for the upcoming year. Early-stage capabilities for VESGEN 3D analysis are under development for the rodent retina and intestine as prototype tissues. A prototype VESGEN 2D Bioinformatics software capability has also been developed to associate phenotypic changes in molecular expression with vascular structure and function. By new VESGEN bioinformatic innovations, expression patterns of the genetic, transcriptional, protein and other markers for regulatory molecules such as vascular endothelial growth factor (VEGF) and their receptors, often indicators of tissue oxygenation status, are co-localized with alterations in vascular pattern. Biomarkers are therefore mapped and quantified as information dimensions directly correlated with the spatial dimensions of a vascular pattern. Further important technology innovations by NASA include substantial image segmentation advances for more automated binary extraction of the grayscale vascular patterns, together with informative associated image quality assessments. Vascular mapping and quantification capabilities for the rodent retina and intestine are illustrated for VESGEN 2D, along with technology status reports on VESGEN 3D and Bioinformatic capabilities. Research partially supported by Ames Center Innovation Awards

Wiskott-Aldrich syndrome protein deficiency in innate immune cells leads to mucosal immune dysregulation and colitis in mice

BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich Syndrome protein (WASP), a hematopoietic-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T-cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2 deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared to RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients, and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin (IL)-10. Administration of IL-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of IL-10, and homeostasis of regulatory T cells

Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

The ability of Interleukin-15 (IL-15) to activate many immune antitumor mechanisms renders the cytokine a good candidate for the therapy of solid tumors, particularly renal cell carcinoma. Although IL-15 is being currently used in clinical trials, the function of the cytokine on kidney's components has not been extensively studied; we thus investigated the role of IL-15 on normal and tumor renal epithelial cells. Herein, we analyzed the expression and the biological functions of IL-15 in normal renal proximal tubuli (RPTEC) and in their neoplastic counterparts, the renal clear cell carcinomas (RCC). This study shows that RPTEC express a functional heterotrimeric IL-15Rαβγc complex whose stimulation with physiologic concentrations of rhIL-15 is sufficient to inhibit epithelial mesenchymal transition (EMT) commitment preserving E-cadherin expression. Indeed, IL-15 is not only a survival factor for epithelial cells, but it can also preserve the renal epithelial phenotype through the γc-signaling pathway, demonstrating that the cytokine possess a wide range of action in epithelial homeostasis. In contrast, in RCC in vitro and in vivo studies reveal a defect in the expression of γc-receptor and JAK3 associated kinase, which strongly impacts IL-15 signaling. Indeed, in the absence of the γc/JAK3 couple we demonstrate the assembly of an unprecedented functional high affinity IL-15Rαβ heterodimer, that in response to physiologic concentrations of IL-15, triggers an unbalanced signal causing the down-regulation of the tumor suppressor gene E-cadherin, favoring RCC EMT process. Remarkably, the rescue of IL-15/γc-dependent signaling (STAT5), by co-transfecting γc and JAK3 in RCC, inhibits EMT reversion. In conclusion, these data highlight the central role of IL-15 and γc-receptor signaling in renal homeostasis through the control of E-cadherin expression and preservation of epithelial phenotype both in RPTEC (up-regulation) and RCC (down-regulation)