Kiminori Toyooka

Generation and Characterization of Monoclonal Antibodies That Specifically Recognize p65/L-Plastin Isoform but Not T-Plastin Isoform

The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca2+-, calmodulin-, and β-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis

OK‐432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK‐432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pre‐treatment of OK‐432 reduced the lethality of lipopolysaccharide (LPS)‐induced endotoxic shock in d‐(+)‐galactosamine‐sensitized C3H/HeN mice. OK‐432 did not affect the TNFα production in blood, but it did decrease the susceptibility to TNFα. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK‐432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK‐432 against the endotoxemia and bacteremia may depend on an up‐regulation of clearance of LPS and bacteria and the augmented resistance to TNFα.