The Effects of Interleukin‐6 Signal Blockade on Immune System,Reproductive and Skeletal Development in Juvenile Mice

Interleukin‐6 (IL‐6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL‐6 participates in a broad spectrum of physiological events, and the IL‐6 receptor (IL‐6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL‐6 signaling called for evaluations of anti–IL‐6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL‐6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti‐mouse IL‐6R antibody, MR16‐1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading‐dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL‐6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T‐cell counts were observed, normal levels of antigen‐specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL‐6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.     

Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.     

Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction

The establishment of methods for directive differentiation from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is important for regenerative medicine. Although Sry-related HMG box 17 (SOX17) overexpression in ESCs leads to differentiation of either extraembryonic or definitive endoderm cells, respectively, the mechanism of these distinct results remains unknown. Therefore, we utilized a transient adenovirus vector-mediated overexpression system to mimic the SOX17 expression pattern of embryogenesis. The number of alpha-fetoprotein-positive extraembryonic endoderm (ExEn) cells was increased by transient SOX17 transduction in human ESC- and iPSC-derived primitive endoderm cells. In contrast, the number of hematopoietically expressed homeobox (HEX)-positive definitive endoderm (DE) cells, which correspond to the anterior DE in vivo, was increased by transient adenovirus vector-mediated SOX17 expression in human ESC- and iPSC-derived mesendoderm cells. Moreover, hepatocyte-like cells were efficiently generated by sequential transduction of SOX17 and HEX. Our findings show that a stage-specific transduction of SOX17 in the primitive endoderm or mesendoderm promotes directive ExEn or DE differentiation by SOX17 transduction, respectively.     

Phosphatidic Acid-dependent Recruitment and Function of the Rac Activator DOCK1 during Dorsal Ruffle Formation

Activation of receptor tyrosine kinases leads to the formation of two different types of plasma membrane structures: peripheral ruffles and dorsal ruffles. Although the formation of both ruffle types requires activation of the small GTPase Rac, the difference in kinetics suggests that a distinct regulatory mechanism operates for their ruffle formation. DOCK1 and DOCK5 are atypical Rac activators and are both expressed in mouse embryonic fibroblasts (MEFs). We found that although PDGF-induced Rac activation and peripheral ruffle formation were coordinately regulated by DOCK1 and DOCK5 in MEFs, DOCK1 deficiency alone impaired dorsal ruffle formation in MEFs. Unlike DOCK5, DOCK1 bound to phosphatidic acid (PA) through the C-terminal polybasic amino acid cluster and was localized to dorsal ruffles. When this interaction was blocked, PDGF-induced dorsal ruffle formation was severely impaired. In addition, we show that phospholipase D, an enzyme that catalyzes PA synthesis, is required for PDGF-induced dorsal, but not peripheral, ruffle formation. These results indicate that the phospholipase D-PA axis selectively controls dorsal ruffle formation by regulating DOCK1 localization.     

Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4

Temperate phages can integrate their genomes into a specific region of a host chromosome to produce lysogens (prophage). During genome insertion, prophages may interrupt the gene coding sequence. In Bacillus subtilis, the sigma factor gene sigK is interrupted by a 48 kb prophage‐like element. sigK is a composite coding sequence from two partial genes during sporulation. For over two decades, however, no further examples of DNA element‐mediated gene reconstitution other than sigK have been identified in spore formers. Here we report that the gene for dipicolinic acid (DPA) synthetase β subunit spoVFB in B. weihenstephanensis KBAB4 is interrupted by a prophage‐like element named vfbin. DPA is synthesized in the mother cell and required for maintaining spore dormancy. We found that spoVFB was a composite coding sequence generated in the mother cell via chromosomal rearrangement that excised vfbin. Furthermore, vfbin caused excision after phage‐inducer treatment, but vfbin appeared to be defective as a prophage. We also found various spore‐forming bacteria in which sporulation‐related genes were disrupted by prophage‐like DNA elements. These results demonstrate the first example of a similar mechanism that affects a sporulation gene other than sigK and suggest that this prophage‐mediated DNA rearrangement is a common phenomenon in spore‐forming bacteria.