Miwa Morita

A common polymorphism in the interleukin 8 gene promoter is associated with Clostridium difficile diarrhea.

In mouse liver transplantation, tolerance is readily inducible. Recent studies have revealed that CD25+CD4+ regulatory T cells play an important role in regulating various immune responses, including transplant tolerance. However, the contribution of these cells to tolerance in mouse liver transplantation has not been elucidated. We showed here that depletion of CD25+CD4+ T cells increased proliferative response of CD4+ T cells and cytotoxic T lymphocyte induction of CD8+ T cells. Depletion of these cells in the recipient but not in the donor before liver transplantation caused rejection. Furthermore, the number of CD25+CD4+ population and forkhead/winged helix transcription factor expression in liver mononuclear lymphocytes derived from tolerant mice were higher than those from grafts undergoing rejection. In conclusion, these results indicate that CD25+CD4+ regulatory T cells in the recipient but not in the donor of liver transplantation are important for the tolerance induction. Liver Transpl 12:1112–1118, 2006.

Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene.

Background Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing α‐satellite DNA with CENP‐B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co‐factor of aromatic amino acid hydroxylases and nitric oxide synthase.

PD-1/B7-H1 interaction contribute to the spontaneous acceptance of mouse liver allograft.

The programmed death‐1 (PD‐1)/B7‐H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD‐1/B7‐H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7‐H1 is highly expressed on the donor‐derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD‐1/B7‐H1 pathway via anti‐B7‐H1mAb or using B7‐H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody‐treated group in comparison to the controls. Taken together, these data revealed that the B7‐H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.

Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG1 Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG1 revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.

PPAR-γ agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-β-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation

Better ways to prevent the cold ischemia-warm reperfusion (CI/WR) injury associated with liver transplantation are needed, and many investigations have focused on the molecular mechanisms of this injury. However, the mechanisms reported to date are controversial and no improvement in therapy has resulted. Here, using prolonged CI and orthotopic transplantation of rat liver grafts, we found that the CI/WR injury was closely associated with autophagy. By 15 min after the start of WR, small masses of hepatocytes that possessed abundant autophagosomes and autolysosomes frequently dissociated from the hepatic cords and obstructed the sinusoid, causing massive necrosis of hepatocytes within 2 hours. The cell masses included TUNEL-positive nuclei without caspase-3 and -7 activation. Autophagy suppression with the phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin or LY294002, reduced both liver damage and the mortality rate of recipient rats. To elucidate the downstream mechanisms of this autophagic pathway, liver grafts were treated with aspartic and cysteine proteinase inhibitors, pepstatin and leupeptin. This treatment also significantly improved the survival rate of recipient rats. These data suggest that autophagy-associated hepatocyte death triggers liver graft dysfunction. The protective effects of suppressing autophagy may suggest new ways to prevent CI/WR injury of the liver.

Identification of a novel biomarker gene set with sensitivity and specificity for distinguishing between allograft rejection and tolerance

Here we examined whether the expression of a novel immunoregulatory gene set could be used to predict outcomes in murine models of rapamycin‐induced cardiac tolerance, spontaneous hepatic tolerance, and cardiac rejection. The expression of the immunoregulatory gene set was assessed with the GeXP multiplex reverse‐transcription polymerase chain reaction (RT‐PCR) analysis system, and it was correlated to the pathological and biochemical parameters of the allografts. In rejecting cardiac grafts, the increased expression of an inflammatory set of genes, which included CD45, CD4, CD25, suppressor of cytokine signaling 2, cytotoxic T lymphocyte–associated protein 4 (CTLA4), selectin lymphocyte, interferon‐γ (IFN‐γ), programmed cell death 1 (Pdcd1), and granzyme B (Gzmb), was seen 8 days after transplantation along with histological evidence of severe allograft rejection. In tolerant cardiac allografts, the expression of fibrinogen‐like protein 2 (Fgl2), Pdcd1, killer cell lectin‐like receptor G1 (Klrg1), CTLA4, and lymphocyte‐activation gene 3 was associated with tolerance. In a model of liver allograft tolerance, the increased expression of lectin galactose‐binding soluble 1, Fgl2, CD39, phosphodiesterase 3B, Klrg1, forkhead box P3 (Foxp3), and transforming growth factor β as well as the inflammatory set of genes was observed 8 to 14 days after transplantation (ie, when there was severe inflammatory injury). At a later time when the liver allografts had been fully accepted and were histologically normal, the expression of the inflammatory set of genes returned to the baseline, but the expression of the tolerogenic set of genes was still increased. Genes that were expressed in tolerant cardiac and liver allografts included Fgl2, Klrg1, and Foxp3, whereas genes associated with rejection included CD25, Gzmb, and IFN‐γ. Our data indicate that monitoring the graft expression of a novel biomarker gene set with the GeXP multiplex RT‐PCR analysis system may allow differentiation between rejection and tolerance. Liver Transpl 18:444–454, 2012.

Isolation of antigen/antibody complexes through organic solvent (ICOS) method.

We developed a method termed ICOS (isolation of antigen-antibody complexes through organic solvent) for comprehensive isolation of monoclonal antibodies (mAbs) bound to molecules on the cell surface. By mixing a large number of phage particles of an antibody (Ab) library with living cells, antigen (Ag)-Ab complexes were formed on the cell surface. The mixture was overlaid on organic solution in a tube and subjected to centrifugation. Phages bound to cells were recovered from the precipitate. The phage fraction isolated turned out to contain mAbs that bind to very heterogeneous epitopes and show strong binding activity to Ags. The ICOS method was applied to isolation of human mAbs that may be therapeutic against cancers. Sixty percent of clones isolated by the screening of a phage Ab library against cancer cells turned out to bind to various kinds of tumor-associated Ags. The precise protocol of ICOS method and the rationale of efficient screening were described.

Fulminant hepatitis by Fas-ligand expression in MRL-lpr/lpr mice grafted with Fas-positive livers and wild-type mice with Fas-mutant livers.

Background. Fulminant hepatitis in mice could be induced by gene-transfection of Fas ligand (FasL). However, the mechanisms of this event still remain controversial as to whether it is mediated by direct Fas/FasL interaction and/or neutrophil migration. To investigate the role of exogenous FasL-expression, we established a simple but clear mouse model on which we performed liver transplantation between Fas-mutant mice (MRL-lpr/lpr) and wild-type mice (MRL+/+). Methods. The controls were nontransplanted wild-type (group 1) and MRL-lpr/lpr (group 2) mice. We obtained recipients with a Fas defect only in the liver (group 3; MRL-lpr/lpr liver graft in wild-type mice) and Fas-defected recipients with Fas-positive livers (group 4; wild-type graft in MRL-lpr/lpr). We successfully expressed FasL in the liver by cotransfection of two types of adenoviral vectors, AxCALNFasL and AxCANCre, with a Cre-loxP switching system. Results. FasL-expression in the livers in groups 3 and 4 resulted in animal death due to fulminant hepatitis within 48 hr after administration of the vectors. We obtained similar findings in group 1, whereas the mice in group 2 survived without any evidence of hepatitis. Immune staining revealed a marked infiltration of CD11b-positive cells in group 1 and group 3. Despite the number of apoptotic cells, a few infiltration of CD11b-positive cells were seen in group 4. We observed no remarkable findings in the FasL-expressed livers in group 2. Conclusion. The results indicated that exogenous FasL-expression induces hepatocyte apoptosis both by direct interaction with Fas and by recruiting Fas-positive inflammatory cells. These findings are important for generating a new strategy to prevent hepatitis as well as for understanding the role of the Fas/FasL interaction in the pathophysiology of hepatitis.

Polycystic kidney disease in Han:SPRD Cy rats is associated with elevated expression and mislocalization of SamCystin

Polycystic kidney disease (PKD) in Han:SPRD Cy rats is caused by a missense mutation in Anks6 (also called Pkdr1), leading to an R823W substitution in SamCystin, a protein that contains ankyrin repeats and a sterile alpha motif (SAM). The cellular function of SamCystin and the role of the Cy (R823W) mutation in cyst formation are unknown. In normal SPRD rats, SamCystin was found to be expressed in proximal tubules and glomeruli; protein expression was highest at 7 days of age and declined by ∼50-60% at 45-84 days of age. In Cy/+ and Cy/Cy kidneys, expression of SamCystin was lower than in +/+ kidneys at 3 and 7 days but became elevated at 21 days. Immunohistochemical analysis revealed that SamCystin was distributed on the brush border of proximal tubules in normal rat kidneys. In Cy/+ kidneys, there were robust SamCystin staining in cyst-lining epithelial cells and loss of apical localization, and increased number of PCNA-positive cells in cyst-lining epithelia. Verapamil, an L-type Ca(2+) channel blocker, accelerated PKD progression in this model and caused a further increase in the expression and abnormal distribution of SamCystin. We conclude that aberrant expression and mislocalization of R823W SamCystin lead to increased cell proliferation and renal cyst formation.