Shunsuke Chikuma

Antibodies to different peptides in osteopontin reveal complexities in the various secreted forms.

We have generated synthetic peptides corresponding to various portions of human osteopontin (OPN) and have immunized rabbits and mice with these peptides to generate polyclonal and monoclonal antibodies specific to human OPN. We then generated six distinct sandwich enzyme‐linked immunoabsorbent assay (ELISA) systems by using different pairs of polyclonal and monoclonal antibodies against human OPN. These systems allowed us to detect not only various isoforms and truncated forms of recombinant OPN, but also the glycosylated form of native urinary OPN. Most importantly, tumor‐derived OPN was differentially detected by the six ELISA systems. The ELISA systems that we have developed will be useful for clarifying the functional roles for OPN in vivo in various physiologic and pathologic conditions. J. Cell. Biochem. 77:487–498, 200.

Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis

We previously constructed an adenovirus vector carrying a gene encoding a soluble form of fusion protein, consisting of the extracellular portion of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 (Adex1CACTLA4IgG). Murine type II collagen-induced arthritis (CIA) was treated with Adex1CACTLA4IgG. A single intraarticular injection of 1 x 10(5) PFU was able to support serum CTLA4IgG at more than 10 microg/ml for at least 12 weeks and was able to inhibit the CIA clinically and histologically. In contrast, intravenous, intramuscular, or subcutaneous injection of 1 x 10(5) PFU was unable to support a significant level of serum CTLA4IgG and thus was unable to inhibit the development of arthritis. Thus, we demonstrated that (1) a low-dose intraarticular injection of Adex1CACTLA4IgG was effective in delaying the onset of CIA and reducing the severity of arthritis; (2) an intraarticular (knee joint) injection of Adex1CACTLA4IgG effectively blocked the development of arthritis in distal paws; (3) the inhibitory effect of Adex1CACTLA4IgG lasted at least up to 20 weeks; (4) although serum CTLA4IgG at more than 10 microg/ml persisted for at least 12 weeks, mice treated by intraarticular injection of Adex1CACTLA4IgG were not anergic to adenovirus and were able to mount antibody responses against various antigens.

Long-Term Acceptance of Allografts by in Vivo Gene Transfer of Regulatable Adenovirus Vector Containing CTLA4IgG and loxP

CTLA4IgG was shown to inhibit the costimulatory signal for T cell activation by interfering with the ligation of CD28 and B7-1 or B7-2. To inhibit various immune responses including acute cellular rejection of allografts, a certain level of serum CTLA4IgG should be maintained for an appropriate period. We previously reported on an adenovirus vector containing CTLA4IgG, which we designated Adex1CACTLA4IgG. Adex1CACTLA4IgG was able to maintain a significant level of serum CTLA4IgG for a long period on intravenous injection, which in turn inhibited various immune responses including protective immunity against infectious agents. To overcome the inhibitory effect, we constructed a new adenovirus vector, Adex1CALoxCTLA4IgGLox, by cloning CTLA4IgG cDNA between two loxP sequences under the control of the CAG promoter. We demonstrated that the administration of adenovirus vector containing Cre recombinase gene (Adex1CACre) at the desired time induced Cre-mediated recombination within a gene derived from Adex1CALoxCTLA4IgGLox vector, and the cDNA of CTLA4IgG was excised from the transduced gene and terminated the expression of CTLA4IgG in vitro and in vivo. More importantly, we also demonstrated that the long-term acceptance of allografts was achieved after the termination of CTLA4IgG expression, while the immune response against adenovirus was restored.

Janus kinase 2 is associated with a box 1‐like motif and phosphorylates a critical tyrosine residue in the cytoplasmic region of cytotoxic T lymphocyte associated molecule‐4

It is a consensus that a cytotoxic T lymphocyte associated molecule‐4 (CTLA‐4) transduces inhibitory signal for T cell activation under physiological condition, indicating that this molecule is an important regulator of T cell homeostasis in vivo. It has been reported that phosphorylation and dephosphorylation of tyrosine residue Y‐165 in the cytoplasmic region of CTLA‐4 play an important role in its negative signaling and cell surface expression. Some signaling molecules such as Src homology 2 protein tyrosine phosphatase 2 (SHP‐2) and the p85 subunit of phosphatidylinositol 3 kinase (PI3 kinase) associate with phosphorylated tyrosine residue Y‐165, through Src homology 2 (SH2) domains. On the other hand, the adapter complex proteins, AP‐2 and AP‐50 interact with the same tyrosine residue when unphosphorylated, resulting in clathrin‐mediated endocytosis of CTLA‐4 molecules. The objective of this study is to identify a tyrosine kinase that can directly bind and phosphorylate the critical tyrosine residue, Y‐165 in the cytoplasmic domain of CTLA‐4. Here, we demonstrated that 1) Janus Kinase 2 (Jak2) was directly associated with a box 1‐like motif in the cytoplasmic tail of CTLA‐4 molecule, 2) Jak2 phosphorylated Y‐165 residue in the cytoplasmic region of CTLA‐4 molecule, and 3) Jak2 was associated with CTLA‐4 in HUT 78 T cell lines. J. Cell. Biochem. 78:241–250, 2000.