Toshio Nikaido

High Serum IgG4 Concentrations in Patients with Sclerosing Pancreatitis

BACKGROUND Sclerosing pancreatitis is a unique form of pancreatitis that is characterized by irregular narrowing of the main pancreatic duct, lymphoplasmacytic inflammation of the pancreas, and hypergammaglobulinemia and that responds to glucocorticoid treatment. Preliminary studies suggested that serum IgG4 concentrations are elevated in this disease but not in other diseases of the pancreas or biliary tract. METHODS We measured serum IgG4 concentrations using single radial immunodiffusion and an enzyme-linked immunosorbent assay in 20 patients with sclerosing pancreatitis, 20 age- and sex-matched normal subjects, and 154 patients with pancreatic cancer, ordinary chronic pancreatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or Sjögrens syndrome. Serum concentrations of immune complexes and the IgG4 subclass of immune complexes were determined by means of an enzyme-linked immunosorbent assay with monoclonal rheumatoid factor. RESULTS The median serum IgG4 concentration in the patients with sclerosing pancreatitis was 663 mg per deciliter (5th and 95th percentiles, 136 and 1150), as compared with 51 mg per deciliter (5th and 95th percentiles, 15 and 128) in normal subjects (P<0.001). The serum IgG4 concentrations in the other groups of patients were similar to those in the normal subjects. In patients with sclerosing pancreatitis, serum concentrations of immune complexes and the IgG4 subclass of immune complexes were significantly higher before glucocorticoid therapy than after four weeks of such therapy. Glucocorticoid therapy induced clinical remissions and significantly decreased serum concentrations of IgG4, immune complexes, and the IgG4 subclass of immune complexes. CONCLUSIONS Patients with sclerosing pancreatitis have high serum IgG4 concentrations, providing a useful means of distinguishing this disorder from other diseases of the pancreas or biliary tract.

Thioredoxin-dependent Redox Regulation of p53-mediated p21 Activation

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-κB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1.cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.

Human amniotic mesenchymal cells have some characteristics of cardiomyocytes

Background. Cellular cardiomyoplasty (CCM) is a major method for the treatment of heart failure because adult cardiomyocytes do not regenerate after ischemic injury, which results in heart failure. There is a great deal of interest in finding suitable new cell sources for use in CCM. Here, we report that human amniotic mesenchymal cells (hAMC), which are multipotent cells derived from fetal mesoderm, may be a suitable cell source for CCM. Methods. Freshly isolated hAMC were examined to detect the expression of cardiac-specific genes by reverse-transcription polymerase chain reaction and immunocytochemistry. hAMC were cocultivated with neonatal rat heart explants and transplanted into myocardial infarcts in the rat heart. Results. hAMC expressed cardiac-specific transcription factor GATA4, cardiac-specific genes, such as myosin light chain (MLC)-2a, MLC-2v, cTnI, and cTnT, and the &agr;-subunits of the cardiac-specific L-type calcium channel (&agr;1c) and the transient outward potassium channel (Kv4.3). After stimulation with basic fibroblast growth factor (bFGF) or activin A, hAMC expressed Nkx2.5, a specific transcription factor for the cardiomyocyte and cardiac-specific marker atrial natriuretic peptide. In addition, the cardiac-specific gene &agr;-myosin heavy chain was detected after treatment with activin A. Coculture experiments confirmed that hAMC were able to both integrate into cardiac tissues and differentiate into cardiomyocyte-like cells. After transplantation into the myocardial infarcts in rat hearts, hAMC survived in the scar tissue for at least 2 months and differentiated into cardiomyocyte-like cells. Conclusion. The results of the present study suggest that hAMC possess some characteristics of cardiomyocytes.

Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma.

To clarify the role of small GTPases Rho in the biologic behavior of ovarian carcinoma, we first examined the mRNA expression of RhoA, RhoB, and RhoC in benign, borderline, and malignant ovarian tumors using RT-PCR and real-time RT-PCR. The expression and localization of RhoA protein were also analyzed by Western blotting and immunohistochemistry. Finally, we examined whether up-regulation of Rho enhances the invasiveness of ovarian cancer cells in vitro. Analysis of mRNA levels of the Rho family genes revealed that levels of both RhoA and RhoC were significantly higher in carcinomas than in benign tumors (RhoA, p = 0.0035; RhoC, p = 0.0006). According to histologic subtype, both RhoA and RhoC mRNA levels in serous carcinomas were significantly higher than those in other histologic types. With regard to the International Federation of Gynecological and Obstetrics stage classification, both of RhoA and RhoC mRNA levels were significantly higher in tumors of Stages III+IV than in those of Stages I+II (RhoA, p = 0.0200; RhoC, p = 0.0057). In addition, analysis of matched pairs of primary and disseminated lesions demonstrated that expression of both RhoA and RhoC mRNA was significantly higher in metastatic than in primary tumors. Examination of the protein level showed that expression of RhoA was also increased in advanced ovarian carcinomas, especially those of serous histology. Accordingly, we hypothesized that up-regulation of Rho GTPases plays an important role in the progression of ovarian carcinoma. Matrigel invasion assay using the ovarian cancer cell line, SKOV3, showed that up-regulation and activation after treatment with lysophosphatidic acid was associated with enhanced invasion of the cancer cells. This increase in invasiveness was suppressed by the addition of C3, a specific inhibitor of Rho. These findings suggest that up-regulation of Rho GTPases is important in the tumor progression of ovarian carcinoma and that Rho family proteins could be a molecular target in cancer therapy.

Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice.

Whole pancreas or β-cell transplantation has opened the way for the treatment of advanced stage of diabetes mellitus. However, it is always limited by the scarcity of transplantation materials. The amniotic membrane is part of the fetal membrane and is composed of amniotic epithelium (HAE) and mesenchymal (HAM) cells that are derived from the inner cell mass in the blastocyst. Thus, HAE and HAM cells may have the potential to differentiate into various organs. The aim of our study was to assess the possibility of HAE cells differentiating into insulin-producing cells. In vitro, HAE cells stimulated with nicotinamide induced insulin mRNA in the culture cells. In vivo, HAE cells were capable of normalizing the blood glucose level of diabetic mice after several weeks of implantation into streptozotocin-induced diabetic mice. The distribution of human cells and human insulin secretion in mouse tissue studied by immunohistochemistry for anti-human-specific β-2-microglobulin and anti-human-specific insulin shows the same location in mouse tissue. These studies suggest that HAE cells have the potential to differentiate into β-cells in vivo, and hence that HAE cells have therapeutic potential for the treatment of type I diabetes mellitus.

Expression of steroid receptors, Ki-67, and p53 in uterine leiomyosarcomas

The expression of estrogen receptor (ER), progesterone receptor (PR), tumor suppressor oncogene p53, and Ki-67 was compared in uterine smooth muscle tumors, including leiomyosarcoma (LMS), tumor of uncertain malignant potential (UMP), cellular leiomyoma (CL), bizarre leiomyoma (BL), and usual leiomyoma (UL). ER and PR were expressed in all ULs. PR was expressed in UL irrespective of the phase of the menstrual cycle; this staining was also observed in CL, UMP, and BL, although BL showed variable staining for ER. Compared to these tumors, the expression of both ER and PR was markedly reduced in LMS. The results of ER and PR transcripts by reverse transcription-polymerase chain reaction were compatible with those of immunohistochemistry. The number of Ki-67 positive cells in LMS was significantly higher than in UMP, BL, CL, and UL. p53 immunoreactivity was seen in 10 of 14 LMSs, and missense mutation in the p53 gene was found in 4 of 10 LMSs. These results suggest that abnormal expression of ovarian steroid receptors, p53, and Ki-67 is frequently associated with LMS of the uterus.

Clear cell carcinoma has an expression pattern of cell cycle regulatory molecules that is unique among ovarian adenocarcinomas.

The purpose of this study was to identify biologic differences between ovarian clear cell carcinoma and other ovarian adenocarcinomas by comparing the expression of cell cycle regulatory molecules and by analyzing the survival of the patients.

Growth Inhibition and Differentiation of Pancreatic Cancer Cell Lines by Pparγ Ligand Troglitazone

INTRODUCTION Ligand activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) results in growth inhibition and differentiation of various cancer cells. AIMS We determined whether the PPAR gamma ligand, troglitazone, inhibits the growth of pancreatic cancer cells and clarified the underlying mechanisms with a special focus on restriction point control of the late G1 phase of the cell cycle. METHODOLOGY Nine pancreatic cancer cell lines were used to study a variety of troglitazone effects on cell growth by MTT assay, on cell cycle by flow cytometry, on cell cycle regulating factors of late G1 phase by Western and Northern blotting and CDK2 kinase assay, and on morphology by collagen gel culture and electron-microscopy. RESULTS Troglitazone showed a potent dose-response effect on the growth inhibition of six pancreatic cancer cell lines, which were suppressed to less than 50% of control at the concentration of 10 microM. The growth inhibition was linked to the G1 phase cell cycle arrest through the upregulation of p21 mRNA and protein expression simultaneously with the inhibition of CDK2 kinase activity and the hypophosphorylation of Rb protein. The upregulation of expression of p21 mRNA was mainly due to stabilization of mRNA. Troglitazone induced significant morphologic changes of duct structure with apoptotic cells in the lumen. CONCLUSION Troglitazone had growth inhibitory and differentiation induction effects on the pancreatic cancer cell lines through the upregulation of p21 expression, suggesting that ligand activation of PPAR gamma is a new molecular target for effective therapy against pancreatic cancer.

Stepwise abnormality of sex steroid hormone receptors, tumor suppressor gene products (p53 and Rb), and cyclin E in uterine endometrioid carcinoma.

In the normal cell cycle, the appropriate interaction of factors such as tumor suppressor gene products (retinoblastoma susceptibility [Rb], p53) and cyclins is essential. Abnormalities in the interaction of these factors may result in malignant transformation of the cell. Malignant transformation of the endometrium, which is believed to be a sex steroid‐dependent tumor, probably involves a process of uncoupling of these factors and sex steroid hormone receptors. This study is designed to test this hypothesis.

Coabnormal expression of cyclin D1 and p53 protein in human uterine endometrial carcinomas.

In the normal cell cycle, tumor suppressor gene products (p53) and cyclin (cyclin D1) cooperate. Abnormalities in the cooperation of these factors may result in malignant transformation of the cell. Mutant p53 protein overexpression is defined in many human cancers, including endometrial carcinoma. This study investigated the role of cyclin D1 in the development of human uterine endometrial carcinoma.