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Type II interferon (IFNγ) is a representative Th1 cytokine and it possesses a variety of functions, including immune regulation, antiviral and antitumor activity. Because of its multifunctional nature, IFNγ is expected to be applied to the treatment of autoimmune diseases, cancer and viral infection. Although IFNγ has therapeutic potential for such diseases, the clinical use of IFNγ has been limited due to its short in vivo half-life and serious adverse effects. In contrast, gene delivery of IFNγ is an alternative approach to increasing the retention time of IFNγ. To extend transgene expression after plasmid DNA (pDNA) gene transfer, we designed and developed pDNA with varying numbers of CpG motifs. CpG-reduced pDNA resulted in more durable transgene expression than its CpG replete counterpart in mice. Comparison of the effect of promoter/enhancer elements on transgene expression showed that ROSA26 promoter-mediated IFNγ expression was safe because of the lack of an initial surge after hydrodynamic gene transfer. We also designed an IFNγ-mouse serum albumin (MSA) fusion protein, IFNγ-MSA. Gene transfer of this fusion protein resulted in a sustained concentration of IFNγ fusion protein in mouse serum, and inhibited tumor metastasis in mice. These results provide experimental evidence that IFNγ gene therapy can be a useful treatment for a variety of diseases.

The 7H6 antigen was recently found in our laboratory, which localized at tight junctions of various tissues. Using the 7H6 monoclonal antibody, we screened several rat liver cDNA libraries and isolated a 5.3 Kb cDNA termed RL5.3. The nucleotide sequence of the RL5.3 cDNA revealed one open reading frame encoding the 130 Kd polypeptide （p 130） homologous to a putative mechanochemical motor protein belonging to the SMC family. RT-PCR was set using a combination of primers corresponding to the nucleotide sequneces of RL5.3 cDNA. This analysis revealed the expression of specific 263 by PCR products in the thymus, liver, kidney and lung, in which epithelial cells have been known to possess tight junctions. Several independent antibodies raised against synthetic oligopeptides and recombinant fusion proteins based on the amino acid sequneces of the p 130 were employed for the detection of p 130 in the rat tissues. The immunofluorescent localization of antigens reacting with these antibodies was found to be characteristic of tight junctions and indistinguishable from that of the 7H6 antigen. Collectively, the p 130 polypeptide encoded by the RL5.3 cDNA was shown to be identical to the 7H6 antigen.

Employing in vitro cultured and virus-infected monocytes as target cells, the response of antibody dependent cell-mediated cytotoxicity （ADCC） to respiratory syncytial virus （RSV） infection was studied in humans. Human peripheral blood monocytes were successfully cultured in vitro and the cells were infected with RSV. These RSV-infected monocytes were lysed by fresh autologous lymphocytes in the presence of RSV-immune human serum. The major viral antigen involved in the ADCC reaction seemed to be fusion （F） protein since the lysis was inhibited strongly by monoclonal antibody specific for RSV F proteins. Reciprocal analysis demonstrated that RSV ADCC reaction was highly cross-reactive to subtype A and B RSV strains. Although the ADCC activity was often dissociated from neutralizing antibody titers to RSV, the highest ADCC response was observed with the serum of subjects of 6 months or older examined at the convalescence of the illness. In view of the importance of anti-F immunity in RSV infection, our results seem to indicate that cellular immune mechanisms, mediated by specific antibodies, play some role in the prevention of and recovery from RSV illness.

2014