Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver. However, the recipients died when the replacement index (RI) of the human hepatocytes exceeded 50%. The hosts (chimeric mice) survived at RI >50% when treated with a drug that has anti-human complement factor activity, and these mice developed livers with RI values as high as 96%. In total, 36 chimeric mice were generated, and the rate of successful engraftment was as high as 92%. The yield of chimeric mice with RI >70% was 32%. The human hepatocytes in the murine host liver expressed mRNAs for a variety of human cytochrome P450 (hCYP) subtypes, in a manner that was similar to the donor liver. The mRNAs for hCYP3A4 and hCYP1A1/2 were induced in the liver in a CYP type-specific manner when the mice were treated with rifampicin and 3-methylcholanthrene, respectively. These results indicate that human hepatocytes that propagate in mice retain their normal pharmacological responses. We conclude that the chimeric mouse developed in the present study is a useful model for assessing the functions and pharmacological responses of human hepatocytes.

Wnt-5a is a representative ligand that activates a B-cateninindependent pathway in the Wnt signaling. Although abnormal activation of B-catenin-dependent pathway is often observed in human cancer, the relationship between Bcatenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.

We have previously proven that human macrophages can phagocytose porcine cells even in the absence of Ab or complement opsonization, indicating that macrophages present a pivotal immunological obstacle to xenotransplantation. A recent report indicates that the signal regulatory protein (SIRP)␣ is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRP␣, prevents autologous phagocytosis. Considering the limited compatibility (73%) in amino acid sequences between pig and human CD47, we hypothesized that the interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. In the present study, we have demonstrated that porcine CD47 does not induce SIRP␣ tyrosine phosphorylation in human macrophage-like cell line, and soluble human CD47-Fc fusion protein inhibits the phagocytic activity of human macrophages toward porcine cells. In addition, we have verified that manipulation of porcine cells for expression of human CD47 radically reduces the susceptibility of the cells to phagocytosis by human macrophages. These results indicate that the interspecies incompatibility of CD47 significantly contributes to the rejection of xenogeneic cells by macrophages. Genetic induction of human CD47 on porcine cells could provide inhibitory signaling to SIRP␣ on human macrophages, providing a novel approach to preventing macrophage-mediated xenograft rejection.xenotransplantation ͉ phagocytosis ͉ reticuloendothelial system

The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase 1 and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.

No abstract