B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE+ B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE+ clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE+ cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE+ and non-IgE–expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE+ B lineage cells in food allergy.

BackgroundReciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases.ResultsWe have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage.ConclusionsDevelopmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.

Knowledge about the modulation of gut microbiota improves our understanding of the underlying mechanism by which probiotic treatment benefits the chickens. This study examined the effects of Bacillus subtilis DSM 32315 on intestinal structure and microbial composition in broilers. Broiler chicks were fed basal diets without or with B. subtilis supplementation (1.0 × 109 spores/kg of diet). Supplemental B. subtilis increased average body weight and average daily gain, as well as elevated villus height and villus height to crypt depth ratio of ileum in broilers. Multi-dimension analysis showed a certain degree of separation between the cecal microbiota from treatment and control groups. Increased Firmicutes abundance and reduced Bacteroidetes abundance in cecum were observed responded to B. subtilis addition, which also increased the abundances of Christensenellaceae and Caulobacteraceae, and simultaneously decreased the abundances of potentially harmful bacteria such as Vampirovibrio, Escherichia/Shigella and Parabacteroides. Network analysis signified that B. subtilis addition improved the interaction pattern within cecal microbiota of broilers, however, it exerted little influence on the metabolic pathways of cecal microbiota by comparison of the functional prediction of metagenomes. In conclusion, supplemental B. subtilis DSM 32315 improved growth performance and intestinal structure of broilers, which could be at least partially responsible by the manipulation of cecal microbial composition.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-β) receptor II knockout mice indicating a role for TGF-β signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-β1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-β signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-β) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-β–induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heatinactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1-or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1-or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heatsensitive cell wall extension of native stipes. These results indicate a chitinasehydrolyzing mechanism for stipe cell wall extension. IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ␤-1,6-branched ␤-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ␤-1,3-glucans under turgor pressure in vivo.O ne of the remarkable characteristics of the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation (1-5). The ...

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Using a combined chromatography method, we simultaneously purified three protein fractions (II-2, II-3 and II-4) with 1,3-b-glucanase activity from extraction of pilei of Coprinopsis cinerea fruiting bodies. MALDI-TOF/TOF amino acid sequencing showed that these three fractions matched a putative exo-1,3-b-glucanase, a putative glucan 1,3-b-glucosidase and a putative glycosyl hydrolase family 16 protein annotated in the C. cinerea genome, respectively; however, they were characterized as a 1,3-b-glucosidase, an exo-1,3-b-glucanase and an endo-1, 3-b-glucanase, respectively, by analysis of their substrate specificities and modes of action. This study explored how these three 1,3-b-glucoside hydrolases synergistically acted on laminarin: the endo-1,3-b-glucanase hydrolysed internal glycosidic bonds of laminarin to generate 1,3-b-oligosaccharides of various lengths, the exo-1,3-b-glucanase cleaved the longer-chain laminarioligosaccharides into short-chain disaccharides, laminaribiose and gentiobiose, and the 1,3-b-glucosidase further hydrolysed laminaribiose to glucose. The remaining gentiobiose must be hydrolysed by other 1,6-b-glucosidases. Therefore, the endo-1,3-b-glucanase, exo-1,3-b-glucanase and 1,3-b-glucosidase may act synergistically to completely degrade the 1,3-b-glucan backbone of the C. cinerea cell wall during fruiting body autolysis. These three 1,3-b-glucoside hydrolases share a similar optimum pH and optimum temperature, supporting the speculation that these enzymes work together under the same conditions to degrade 1,3-b-glucan in the C. cinerea cell wall during fruiting body autolysis.

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