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Background/Aim: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. Methods: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. Results: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1β increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. Conclusion: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K D ] ‫؍‬ 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K i ‫؍‬ 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

We previously reported that treatment with a JNK inhibitory peptide (11R-JNKI) prevents islet apoptosis and enhances the islet function in vivo. In the present study, we explored more efficient JNK inhibitors. The inhibition of the JNK activity by five types of deletion peptides in 11R-JNKI was investigated. One of the peptides, 8R-sJNKI(-9), significantly prevented JNK activation. At a concentration of 1 µM, 8R-sJNKI(-9) inhibited JNK activity similarly to 10 µM 11R-JNKI and the inhibition of the JNK activity by 10 µM 8R-sJNKI(-9) was significantly greater than that by 10 µM 11R-JNK. To evaluate the effects of 8R-sJNKI(-9), porcine islets were cultured with 1 µM of 8R-sJNKI(-9) or 8R-mutant sJNKI(-9) (8R-mJNKI(-9)). After 1 day of culture, the numbers of islets in the 8R-sJNKI(-9)-treated group was significantly higher than that in the 8R-mJNKI(-9)-treated group. After islet transplantation, the blood glucose levels reached the normoglycemic range in 58.3% of streptozotocin-induced diabetic mice in the 8R-sJNKI(-9) group and 0% of the mice in the 8R-mJNKI(-9)-treated group. These data suggest that 8R-sJNKI(-9) inhibits islet apoptosis and improves islet function.

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.