Michiaki Unno

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model.

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.

A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells

PurposeIn the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems.MethodsWe have produced an anti-epidermal growth-factor receptor (EGFR) × anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E. coli) expression system with refolding method. The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro. This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen.ResultsWhen T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-γ, GM-CSF, and TNF-α than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37°C for 48xa0h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice. When Ex3 diabody (20xa0μg/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth.ConclusionThe Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity.

Abstract. 4–1BB ligand (4–1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4–1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4–1BB in immune function, but relatively few investigations of human 4–1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4–1BBL (Ad4–1BBL) and its stimulation of antitumor immunity. Ad4–1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4–1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1×anti-CD3). Cocultivation of Ad4–1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-γ), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4–1BBL and T-LAK cells were administered when tumor size exceeded 5xa0mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4–1BBL for genetic immunotherapy of cancer.

Inhibition of heme oxygenase ameliorates sepsis-induced liver dysfunction in rats.

Abstract.Purpose: The disintegration of heme produces carbon monoxide (CO), a known vasodilator, which is catalyzed by heme oxygenase (HO). This study aimed to clarify the effect of HO inhibition on septicrat livers using two types of HO inhibitors; Sn-protoporphyrin (Sn-PP) and Zn-protoporphyrin (Zn-PP).nMethods: Sepsis was induced in male Sprague-Dawley rats by cecal ligation and puncture (CLP). Either NaOH or HO inhibitors were injected intraperitoneally; first 18u2009h prior to CLP, then immediately after CLP. The animals were killed 12 and 24u2009h after CLP and the liver tissue and plasma were harvested.nResults: Using Northern blotting, we found that mRNA of the stress-inducible isozyme, HO-1, was dramatically induced 12u2009h after CLP. Administering the HO inhibitors, Sn-PP and Zn-PP (5u2009μmol/kg), induced a significant inhibition of the elevation of aspartate aminotransferase plasma levels, the elevation of cyclic guanosine monophosphete (cGMP) in the liver tissue, and the increase in the sinusoidal space ratio, 24u2009h after CLP. Both Sn-PP and Zn-PP decreased the mortality rate 24u2009h after CLP compared with normal saline.nConclusions: CO produced by excessively induced HO-1 after CLP promotes an immoderate dilation of the sinusoidal space through the up-regulation of cGMP, resulting in liver dysfunction. Therefore, administering HO inhibitors at appropriate doses could be beneficial for the amelioration of sepsis-induced liver dysfunction.