Chemokines, especially CXC family, play a key role in neutrophil-mediated esophageal inflammatory disease by attracting neutrophils to the site of inflammation. Recent in vitro and in vivo reports suggest that esophageal squamous epithelial cells can produce these chemokines by the stimulation with gastric acid, bile acids, and pancreatic protease. Transcriptome analysis has confirmed the signaling pathway and several transcriptional factors associated with esophageal inflammation. Detailed studies of the interaction between esophageal epithelium and gastric/duodenal refluxates should make it possible to identify a key therapeutic target molecule that regulates esophageal inflammation.

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Modification of cell culture substrates and scaffolds with bioactive molecules is frequently used to enhance and/or regulate cellular functions and metabolism during culture. These biomaterials have contributed to an advanced understanding of cell biology and materials designs in biomedical applications. In contrast, we have proposed a new technology -- cell sheet engineering -- for realization of tissue/organ reconstruction with structural and functional regeneration. Using cell sheet engineering, confluently cultured cells are harvested as viable, contiguous cell monolayers applied for fabrication of three-dimensional biomimetic tissues or cell sheet-utilized therapies. We have recently introduced bio-functionalization of thermoresponsive surfaces with cell adhesive peptides, e.g., RGDS, and/or cell growth factors, e.g., insulin (INS), for rapid fabrication of cell sheets. Surface-immobilized RGDS peptides promote initial cell adhesion, while INS immobilization accelerates proliferation of adherent cells. More pronounced and synergistic influences on cell growth are observed on RGDS-INS co-immobilized thermoresponsive surfaces even under the serum-free culture conditions. Here, we briefly review the current findings of our bio-functionalized thermoresponsive surfaces for rapid, effective cell sheet fabrication and non-invasive harvest as tissue monolayers for further applications in regenerative medicine.

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Glucosamine, a naturally occurring amino monosaccharide, has been used to treat or prevent osteoarthritis in humans. Recently, we have revealed that glucosamine inhibits platelet activation in vitro. However, the effect of in vivo administration of glucosamine has not yet been clear. Recently, we have evaluated the effect of oral glucosamine administration on platelet activation in guinea pigs. Guinea pigs were orally adminis-tered with glucosamine (an average of 400 mg glucosamine/animal/day) for 22 days and thereafter platelet functions were examined.
Glucosamine-administration suppressed platelet aggregation in response to ADP, but not platelet aggregation induced by collagen. Furthermore, glucosamine-administration inhibited the ADP-induced extracellular release of ATP and production of thromboxane A₂. In contrast, glucosamine did not affect the body-weights, platelet counts and bleeding time in guinea pigs after the administration.
These observations suggest that glucosamine is likely to exert an inhibitory action on platelets in vivo by suppressing platelet aggregation, ATP release, and thromboxane A₂ production. Thus, glucosamine could be expected as a novel and safe anti-platelet agent.

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Anti-cytokine therapies have been used for rheumatoid arthritis (RA) and their efficacy has been previously reported. Serum amyloid A (SAA) is an acute phase protein and known as a precursor of amyloid fibril in AA amyloidosis. IL-6 blocking therapy normalized the serum levels of SAA, in RA patients, while anti-TNFα or IL-1 therapy did not. From the clinical point of view, the exact identification of the induction mechanism of SAA is important. We found that IL-6 plays a critical role in the synergistic induction of the SAA gene by combining with proinflammatory cytokines. Moreover, we discovered that STAT3 plays an essential role in cytokine-driven SAA gene expression even though no typical STAT3 response element (RE) is located in its promoters. STAT3 and NF-κB p65 first form a complex following IL-1 and IL-6 stimulation, after which STAT3 interacts with non-consensus sequences at a 3'-site of NF-κB RE on the SAA gene promoter. STAT3 then recruits p300, which coordinates the interaction of NF-κB p65, STAT3 and C/EBPβ in the synergistic induction of the SAA gene. Our findings have a direct bearing on a better understanding of inflammatory status and are expected to contribute to the development of a therapeutic strategy for AA amyloidosis.

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In neutrophils, an intracellular calcium ion plays an important role in regulating cellular functions induced by chemoattractants. In this study, we have investigated the role of intracellular calcium and its effecter molecule, calmodulin, in superoxide anion production and chemotaxis of rat neutrophils. N-formyl-methionylleucyl-phenylalanine (FMLP), one of the chemoattractants, is known to activate phosphlipase C, phosphatidyl inositol 3-kinase, and Akt via activation of its G protein-coupled membrane receptors. Inositol trisphosphate produced by both enzymes increases a release of calcium ion from the store. By inhibition of calcium release, the production of superoxide anion in neutrophils was abrogated, whereas the chemotaxis was unaffected. In contrast, both the superoxide anion production and the chemotaxis of neutrophils were suppressed by inhibition of influx of extracellular calcium ion. Inhibition of calmodulin using a calmodulin antagonist, W-7, attenuated FMLP-induced superoxide anion production and chemotaxis of neutrophils. W-7 also inhibited FMLP-induced Akt phosphorylation. Taken together, our data indicate that a critical role of calcium ion and calmodulin in superoxide anion production and chemotaxis of neutrophils, and suggested that FMLP-induced Akt activation may be meditated by calcium-calmodulin pathway in rat neutrophils.

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