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Biomedical subjects

Shinichi Miyagawa

Publications and source records attributed to Shinichi Miyagawa.

At least 19 recordsLinked to original sources

Molecular analysis of coordinated bladder and urogenital organ formation by Hedgehog signaling.

The urogenital and reproductive organs, including the external genitalia, bladder and urethra, develop as anatomically aligned organs. Descriptive and experimental embryology suggest that the cloaca, and its derivative, the urogenital sinus, contribute to the formation of these organs. However, it is unknown how the primary tissue lineages in, and adjacent to, the cloaca give rise to the above organs, nor is bladder formation understood. While it is known that sonic hedgehog (Shh) is expressed by the cloacal epithelia, the developmental programs that regulate and coordinate the formation of the urogenital and reproductive organs have not been elucidated. Here we report that Shh mutant embryos display hypoplasia of external genitalia, internal urethra (pelvic urethra) and bladder. The importance of Shh signaling in the development of bladder and external genitalia was confirmed by analyzing a variety of mutant mouse lines with defective hedgehog signaling. By genetically labeling hedgehog-responding tissue lineages adjacent to the cloaca and urogenital sinus, we defined the contribution of these tissues to the bladder and external genitalia. We discovered that development of smooth muscle myosin-positive embryonic bladder mesenchyme requires Shh signaling, and that the bladder mesenchyme and dorsal (upper) external genitalia derive from Shh-responsive peri-cloacal mesenchyme. Thus, the mesenchymal precursors for multiple urogenital structures derive from peri-cloacal mesenchyme and the coordination of urogenital organ formation from these precursors is orchestrated by Shh signals.

Animals↗

Impact of tumor spread to the cystic duct on the prognosis of patients with gallbladder carcinoma.

INTRODUCTION: The importance of gallbladder carcinoma spread to the cystic duct has not yet been described. Although the cystic duct is contiguous with the gallbladder, it is located in the hepatoduodenal ligament and differs in structure from the gallbladder. The incidence and prognostic significance of cancer spread to the cystic duct in patients with gallbladder cancer is unclear. METHODS: Surgical specimens from 42 patients who underwent resection for advanced gallbladder carcinoma were examined retrospectively. RESULTS: Altogether, 13 (31%) of the patients had cancer spread to the cystic duct. The incidences of perineural invasion, lymph node metastasis, and venous invasion were significantly higher in these patients than in the other 29 patients without cancer spread to the cystic duct (P = 0.027, 0.034, and 0.034, respectively). The 3- and 5-year survival rates of these 13 patients were significantly lower than those of the other 29 patients (15.4% vs. 51.0% and 7.7% vs. 46.6%, respectively, P < 0.0001 each). Multivariate analysis using the Cox proportional hazard model identified positive cancer spread to the cystic duct and depth of invasion (beyond serosa) as significant independent indicators of a poor prognosis. CONCLUSIONS: Cancer spread to the cystic duct is an indicator of poor prognosis in patients with gallbladder carcinoma. This may be due to the high incidence of concomitant perineural invasion and lymph node metastasis associated with cancer spread.

Adenocarcinoma↗

Efficacy of S-1 for patients with peritoneal metastasis of gastric cancer.

BACKGROUND: This study was designed to examine the efficacy and compliance of S-1 for the patients with peritoneal metastasis of gastric cancer. METHODS: Sixteen consecutive patients with peritoneal metastasis of gastric cancer were treated with S-1. Their survival was compared with that of the historical control group (25 patients). Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase and orotate phosphoribosyl transferase mRNA expression in the tumor were evaluated. RESULTS: The median survival time of S-1-treated patients was 550 days, which was significantly longer than that of the historical control group (215 days). We elucidated some factors to prolong the survival of the patients treated with S-1 for peritoneal metastasis: peritoneal metastasis without other distant metastases, the combination of S-1 treatment and gastrectomy, and low expression of thymidine phosphorylase mRNA in primary tumors. CONCLUSIONS: S-1 showed a surprisingly long-term survival with minimum toxicity in patients with peritoneal metastasis of gastric cancer.

Adult↗

Immunohistochemical demonstration of proliferating lymphatic vessels in colorectal carcinoma and its clinicopathological significance.

Lymphatic metastasis to the regional lymph nodes through the lymphatic vessels is an important indicator of poor prognosis in many types of malignant tumors. Recently, much attention has been paid to lymphangiogenesis for its possible role on tumor progression in various carcinomas. However, morphological evidence that lymphatic vessels actively proliferate in colorectal carcinoma has not been reported. Here, we first devised a triple immunostaining method to detect proliferating lymphatic vessels utilizing antibody to Ki-67 antigen as a marker of cell proliferation, antibody to cytokeratin as an epithelial cell marker, and antibody to podoplanin as a lymphatic vessel-specific marker. Ki-67/podoplanin-immunoreactivity enabled us to identify proliferating lymphatic vessels, while cytokeratin immunoreactivity allowed us to distinguish proliferating lymphatic vessels from Ki-67/cytokeratin-positive carcinoma cells in lymphatic lumens. Analyzing 64 colorectal carcinoma patients' samples using this technique, we showed that both lymphatic vessel density and proliferating activity of lymphatic vessels were significantly increased in colorectal carcinoma tissues compared with their normal counterparts. We then examined the correlation between the degree of lymphangiogenesis and patients' prognosis or clinicopathological variables, but no statistically significant differences were obtained in these analyses. Thus, these results combined together indicate that extensive lymphangiogenesis occurs in colorectal carcinoma, but that the degree of lymphangiogenesis alone is not an independent prognostic factor for this disease.

Adult↗

Characterization of cytochrome P450 expression in murine embryonic stem cell-derived hepatic tissue system.

An in vitro system for liver organogenesis from murine embryonic stem (ES) cells has been recently established. This system is expected to be applied to the development of a new drug metabolism assay system that uses ES cells as a substitute for animal experiments. The objective of this study was to elucidate the drug metabolism profiles of the murine ES cell-derived hepatic tissue system compared with those of primary cultures of murine adult and fetal hepatocytes. The expression of the genes of the cytochrome P450 (P450) family, such as Cyp2a5, Cyp2b10, Cyp2c29, Cyp2d9, Cyp3a11, and Cyp7a1, was observed in the murine ES cell-derived hepatic tissue system at 16 days and 18 days after plating (A16 and A18). To investigate the activities of these P450 family enzymes in the murine ES cell-derived hepatic tissue system at A16 and A18, testosterone metabolism in this system was analyzed. Testosterone was hydroxylated to 6beta-hydroxytestosterone (6beta-OHT), 16alpha-OHT, 2alpha-OHT, and 2beta-OHT in this system, and was not hydroxylated to 15alpha-OHT, 7alpha-OHT, and 16beta-OHT. This metabolism profile was similar to that of fetal hepatocytes and different from that of adult hepatocytes. Furthermore, pretreatment with phenobarbital resulted in a 2.5- and 2.6-fold increase in the production of 6beta-OHT and 16beta-OHT. Thus, evidence for drug metabolic activities in relation to P450s has been demonstrated in this system. These results in this system would be a stepping stone of the research on the development and differentiation to adult liver.

Animals↗

Predictive factors for intrahepatic cholangiocarcinoma recurrence in the liver following surgery.

BACKGROUND: We performed hepatectomy without lymph node (LN) dissection for intrahepatic cholangiocarcinoma (ICC) limited to the peripheral region of the liver, and hepatectomy with extrahepatic bile duct resection and regional LN dissection for any types of ICC extending to the hepatic hilum. Surgical outcomes were evaluated to elucidate the prognostic factors that influence patient survival with respect to intrahepatic recurrence. METHODS: Forty-one patients underwent resection of ICC with no macroscopic evidence of residual cancer. RESULTS: Significant risk factors for poorer survival included preoperative jaundice (P = 0.0115), serum CA19-9 levels >37 U/ml (P = 0.0089), tumor diameter >4.5 cm (P = 0.017), ICC extending to the hepatic hilum (P = 0.0065), mass-forming with periductal-infiltrating type (P = 0.003), poorly differentiated adenocarcinoma, portal vein involvement (P = 0.0785), LN metastasis at initial hepatectomy (P < 0.0001), and positive surgical margin (P = 0.023). Intrahepatic recurrence, which was the predominant manner of recurrence, was detected in 20 patients (74.1%). Patients with intrahepatic recurrence had a significantly high incidence of high serum CA19-9 levels (>37 U/ml; P = 0.0006), preoperative jaundice (P = 0.0262), ICC extended to the hepatic hilum (P = 0.0349), large tumors (>4.5 cm; P = 0.0351), portal vein involvement (P = 0.0423), and LN metastasis at initial hepatectomy (P = 0.009) compared with disease-free patients. The multiple logistic regression analysis revealed that preoperative CA19-9 elevation and obstructive jaundice influenced intrahepatic recurrence of ICC. CONCLUSIONS: Although LN metastasis is a significant prognostic factor, the most obvious recurrence pattern after surgery was intrahepatic recurrence, which could be predicted preoperatively by a combination of elevated serum CA19-9 levels and manifestation of obstructive jaundice.

Aged↗

Trefoil factor 2 in gland mucous cell mucin in the mucous gel covering normal or damaged gastric mucosa using the Mongolian gerbil model.

OBJECTIVE: Trefoil factor 2 (TFF2) is localized in gastric gland mucous cells. The purpose of the study was to determine whether TFF2 and gastric mucin are localized in mucous cells and in the surface mucous gel layer (SMGL) of the normal gastric mucosa or in the mucoid cap adherent to gastric mucosal lesions in Mongolian gerbils. MATERIAL AND METHODS: Gastric mucosal lesions were induced in Mongolian gerbils using oral administration of Helicobacter pylori (H. pylori), subcutaneous administration of indomethacin, or oral administration of 30% ethanol. Tissue samples were fixed in Carnoy's solution for preservation of the SMGL, dehydrated, and embedded in paraffin. Histochemical staining for gastric mucins and immunostaining for TFF2 were performed. RESULTS: It was found that surface mucous cell mucin and gland mucous cell mucin were segregated in the SMGL covering the normal gastric mucosa, and the mucin of the mucoid cap covering the mucosal lesions was primarily gland mucous cell mucin. There was a co-localization of TFF2 in gland mucous cell mucin in gland mucous cells, the SMGL, and the mucoid cap. CONCLUSIONS: The co-localization of TFF2 in gland mucous cells and in the adherent mucus suggests a physical interaction between TFF2 and gland mucous cell mucin, and the participation of TFF2 trapped in the adherent mucus functions in mucosal defense, healing, and repair.

Animals↗

Clinical utility of quantitative RT-PCR targeted to alpha1,4-N-acetylglucosaminyltransferase mRNA for detection of pancreatic cancer.

alpha1,4-N-Acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase responsible for the biosynthesis of alpha1,4-GlcNAc-capped O-glycans, and is frequently expressed in pancreatic cancer cells but not peripheral blood cells. In the present study, we tested the clinical utility of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood as a biomarker of pancreatic cancer. Total RNA isolated from the peripheral blood mononuclear cells from 55 pancreatic cancer patients, 10 chronic pancreatitis patients, and 70 cancer-free volunteers was analyzed quantitatively by reverse transcription-polymerase chain reaction with primers specific for alpha4GnT, and the expression level of alpha4GnT mRNA relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured. When the ratio of alpha4GnT to GAPDH transcripts exceeded a defined cut-off value, patients were considered to have pancreatic cancer. By these standards, 76.4% of the pancreatic cancer patients were detected by this assay. A strong correlation was obtained between positivity in this assay and the expression of alpha4GnT protein detected immunohistochemically in pancreatic cancer tissues resected subsequently, suggesting that alpha4GnT mRNA detected in the peripheral blood is derived from circulating pancreatic cancer cells. Although increased levels of alpha4GnT mRNA was detected in 40.0% of chronic pancreatitis patients and 17.1% of cancer-free volunteers, the expression levels were significantly lower than those seen in pancreatic cancer patients. These results suggest that quantitative analysis of alpha4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood will contribute to the detection of pancreatic cancer.

Adult↗

Identification of oligopeptides binding to peritoneal tumors of gastric cancer.

This is a report of in vivo intraperitoneal biopanning, and we successfully identified a novel peptide to target the multiple peritoneal tumors of gastric cancer. A phage display library was injected directly into the abdominal cavity of mice bearing peritoneal tumors of human gastric cancer, and phages associated with the tumors were subsequently reclaimed from isolated samples. The tumor-associated phages were amplified and the biopanning cycle was repeated five times to enrich for high affinity tumor-selective binding peptides. Finally, a tri-peptide motif, KLP, which showed homology with laminin 5 (a ligand for alpha3beta1 integrin), was identified as a binding peptide for peritoneal tumors of gastric cancer. Phage clones displaying the sequence KLP showed 64-fold higher binding to peritoneal tumors than control phage and were preferentially distributed in tumors rather than in normal organs after intraperitoneal injection into mice. In addition, the KLP phages were more likely to bind to cancer cells in malignant ascites derived from a patient with recurrent gastric cancer. Synthesized peptide containing the motif KLP (SWKLPPS) also showed a strong binding activity to peritoneal tumors without cancer growth effect. Liposomes conjugated with SWKLPPS peptide appeared significantly more often in tumors than control liposomes after intraperitoneal injection into mice. Furthermore, modification of liposomes with SWKLPPS peptide enhanced the antitumor activity of adriamycin on gastric cancer cells. The peptide motif KLP seems a potential targeting ligand for the treatment of peritoneal metastasis of gastric cancer.

Amino Acid Motifs↗

Cloning and differential expression of estrogen receptor and aromatase genes in the self-fertilizing hermaphrodite and male mangrove rivulus, Kryptolebias marmoratus.

The mechanisms underlying sex determination and differentiation in fishes are labile in response to environmental parameters. Sex-specific phenotypes are largely regulated by sex steroids, and the inhibition or the stimulation of aromatase can reverse sex as well as alter secondary sexual characteristics in fishes. Among vertebrates, the mangrove rivulus is the only known self-fertilizing hermaphrodite. Throughout most of its range, rivulus appear to exist as clonally reproducing hermaphrodites. However, outcrossing has been documented in Belize, where up to 25% of rivulus collected are males. The direct development of (primary) males occurs when embryos are incubated at 18 degrees C and hermaphrodites develop into secondary males when held at 28 degrees C. Given the importance of sex steroids, their receptors, and aromatase in sex determination and differentiation of fishes, we cloned, sequenced, and quantified the expression of estrogen receptors (ERalpha, ERbeta) and ovarian (AroA) and brain (AroB) aromatase genes. Hermaphrodites had increased ERalpha, ERbeta, AroA, and AroB gene expression in the liver, gonad, gonad, and brain respectively, compared to males. These data are consistent with the gene expression data reported for other species and are reflective of the presence of ovarian tissue in the hermaphrodites. Interestingly, we show the elevated expression of brain aromatase in the hermaphrodite brain. The role of the dimorphic expression of brain aromatase in the regulation of sex-specific characteristics is intriguing and requires further research. Because of the uniqueness of its reproductive biology, rivulus is an excellent model for elucidating the mechanisms regulating vertebrate sex determination and sexual differentiation.

Amino Acid Sequence↗

Immunoglobulin G4-related lymphoplasmacytic sclerosing cholangitis that mimics infiltrating hilar cholangiocarcinoma: part of a spectrum of autoimmune pancreatitis?

BACKGROUND: Autoimmune pancreatitis has been designated as sclerosing pancreatocholangitis, because this disease shows a high prevalence of bile-duct lesions. We present herein the clinical characteristics of unusual cases that show dominant bile-duct lesions and mimicking infiltrating hilar cholangiocarcinomas. METHODS: Clinical and pathologic findings of 3 patients with immunoglobulin (Ig) G4 related sclerosing cholangitis who had no apparent pancreatic lesions comparable with autoimmune pancreatitis were analyzed. OBSERVATIONS: All patients were middle-aged or elderly individuals with slightly elevated serum IgG4 concentrations and showed long-segment narrowing of the bile-duct system, mimicking infiltrating hilar cholangiocarcinoma without significant pancreatic change. The first patient was treated with a corticosteroid, resulting in amelioration of the narrowing of the bile duct. The second patient underwent surgery based on a diagnosis of cholangiocarcinoma. In the third patient, the bile-duct stricture reversed spontaneously 1 month after the drainage procedure. Pathologic findings of the bile ducts for all patients disclosed significant lymphoplasmacytic infiltration, including abundant IgG4-bearing plasma cells. CONCLUSIONS: The use of IgG4 immunostaining in biopsy specimens of the bile duct may identify the presence of corticosteroid-responsive lymphoplasmacytic sclerosing cholangitis.

Aged↗

Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liver organogenesis system.

Recent studies in the field of regenerative medicine have exploited the pluripotency of embryonic stem (ES) cells to generate a variety of cell lineages. However, the target has always been only a single lineage, which was isolated from other differentiated cell populations. In the present study, we selected sublines with a high capability for differentiation to contracting cardiomyocytes and also produced germ-line chimeric mice from a parent ES line. We also succeed in establishing embryoid bodies prepared from the ES cells that differentiated into not only hepatocytes but also at least two mesodermal lineages: cardiomyocytes that supported liver development and endothelial cells corresponding to sinusoids. This allowed the development of an in vitro system using murine ES cells that approximated the events of liver development in vivo. The expression of albumin was significantly higher in cardiomyocytes that had arisen in differentiated ES cells than in those that had not. Our in vitro system for liver organogenesis consists of a blood/sinusoid vascular-like network and hepatocyte layers and shows higher levels of hepatic function, such as albumin production and ammonia degradation, than hepatic cell lines and primary cultures of murine adult hepatocytes. This innovative system will lead to the development of second-generation regenerative medicine techniques using ES cells and is expected to be useful for the development of bioartificial liver systems and drug-metabolism assays.

Ammonia↗

[Survey of gemcitabine treatment for advanced pancreatic cancer in 20 hospitals of Nagano Prefecture].

To establish an effective therapy for pancreatic cancer, we made a retrospective survey of gemcitabine treatment performed at 20 hospitals in Nagano Prefecture. We analyzed data of 106 patients (64 men and 42 women, median age 66 years (33-83 years old)), half of whom had stage IV disease. Gemcitabine was administered for 3 consecutive weeks with one week rest in 57 patients, biweekly in 30 patients, initially for 3 weeks with 1 week rest and switched to biweekly schedule to 15 patients, and with another regimen to 4 patients. Analysis of the results of gemcitabine treatment between the 3 weeks with 1-week-rest regimen and the biweekly regimen revealed no differences between regimens in growth inhibition and symptom alleviation. However, we found less occurrence of blood toxicity in the biweekly regimen (40%) than in the 3 weeks with 1 week-rest regimen (59%). Median survival time for the biweekly regime was 9.7 months, only slightly longer than that of the 3 weeks with 1-week-rest regimen (8.5 months). The present study showed that a biweekly regimen for gemcitabine administration may be equivalent to the standard regime of 3 weeks with 1-week-rest regimen. Moreover, the biweekly regimen has advantages over the 3 weeks with 1-week rest regimen both economically and in terms of convenience for outpatient treatment. Therefore, the present results should be confirmed in future prospective studies, with the hope of developing a new standard treatment regimen for pancreatic cancer.

Adult↗

Defective development of splenic and epidermal CD4+ dendritic cells in mice deficient for IFN regulatory factor-2.

Dendritic cells (DCs) play important roles in the initiation and regulation of immune responses. Although several subsets of DCs were identified according to their expression of surface molecules such as CD4, CD8, and CD11b, the regulatory mechanism for the development and homeostasis of these DC subsets remains unclear. Here we show that mice lacking IFN regulatory factor-2 (IRF-2(-/-) mice) exhibited a marked and selective defect in splenic CD4(+)CD11b(+)DCs, instead of CD8 alpha(+)CD11b(-)DCs that were reported to be missing in mice lacking the related transcription factor IRF-8. Furthermore, the numbers of epidermal Langerhans cells in IRF-2(-/-) mice were reduced at least in part because of the lack of the CD4(+)CD11b(+) subset. Studies with radiation bone marrow chimeras as well as in vitro retrovirus-mediated gene transduction showed that IRF-2 was required cell-autonomously for the development of myeloid-related DCs. Notably, these abnormalities in DCs diminished in mice lacking both IRF-2 and the IFN-alpha/beta receptor, indicating that IRF-2 acted through negatively regulating IFN-alpha/beta signals. In contrast, natural killer cells still showed developmental arrest in these double mutant mice, indicating that the mode of action of IRF-2 for CD4(+)DC development is distinct from that for natural killer cell development. Our current findings thus pointed to a previously unknown unique cell-type-selective multimode function of IRF-2 in the regulation of lymphohematopoiesis.

Animals↗

Estrogen-independent activation of erbBs signaling and estrogen receptor alpha in the mouse vagina exposed neonatally to diethylstilbestrol.

Growth factors and estrogen receptor (ER) signaling cooperate to play essential roles in cell proliferation, differentiation and tumor progression in mouse reproductive organs. Treatment of neonatal mice with diethylstilbestrol (DES) induces an estrogen-independent persistent proliferation and cornification of the vaginal epithelium, which results in cancerous lesions later in life. However, the mechanisms of the estrogen-dependent and -independent pathways essentially remain unknown. We characterized the expression of epidermal growth factor (EGF)-like growth factors (EGF, transforming growth factor alpha (TGF-alpha), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC), amphiregulin (APR), epiregulin (EPR) and neuregulin (NRG) 1) and erbB receptors (EGF receptor (EGFR), erbB2/neu, erbB3 and erbB4) in the vaginae of mice treated either neonatally (0-4 day) or as adults (55-59 day) with estrogens. EGFR and erbB2 were activated in the vaginal epithelium of mice by estrogen treatment. This activation was also encountered in vaginae from neonatally DES-exposed mice, along with the expression of EGF, TGF-alpha, HB-EGF, BTC, APR, EPR and NRG1. Immunohistochemical analysis indicated that erbB2 was primarily expressed in vaginal epithelium. Finally, we found that serine 118 and 167 located in the AF-1 domain of ERalpha were phosphorylated in these vaginae. AG825, AG1478 or ICI 182,780 administration blocked proliferation of vaginal epithelium induced by neonatal DES exposure. Thus, signal transduction via EGFR and erbB2 could be related to the estrogen-induced vaginal changes and persistent erbBs phosphorylation and sustained expression of EGF-like growth factors, leading to ERalpha activation that may result in cancerous lesions in vaginae from neonatally DES-exposed mice later in life.

Animals↗

Prognostic significance of mature dendritic cells and factors associated with their accumulation in metastatic liver tumors from colorectal cancer.

Although dendritic cells (DCs) play an important role in tumor immunity, their prognostic significance and factors related to mature DCs have not been addressed in metastatic liver tumors. In surgically resected, paraffin-embedded tissue sections from 70 patients with colorectal liver metastasis, CD83 (a marker of mature DCs) positive cells and cancer cells positive for the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were counted. Expression of gp96, which is considered to participate in the maturation of DCs, was also evaluated. CD83-positive cells were observed predominantly in the cancer invasive margin. Patients with CD83-positive cell counts of <2 per field had a significantly poorer prognosis (5-year survival rate 47.5% vs 23.1%; P=0.0184). Patients with >0.83% apoptotic cancer cells had significantly higher numbers of CD83-positive cells (7.3 +/- 7.3 vs 4.0 +/- 5.1; P=0.039). Patients with immunohistochemically positive gp96 expression in tumors had significantly higher numbers of CD83-positive cells than those with negative gp96 expression (6.0 +/- 6.5 vs 1.4 +/- 2.3; P=0.0108). Patients with metachronous occurrence of liver metastasis had significantly higher numbers of CD83 positive cells than those with synchronous detection (6.3 +/- 6.5 vs 3.9 +/- 5.9; P=0.0313). Although the number of apoptotic cancer cells, degree of tumor gp96 expression, and synchronous or metachronous occurrence of liver metastasis did not directly influence patient outcome, they did influence the number of CD83-positive cells in the cancer invasive margin, which was a significant prognostic factor in patients with colorectal liver metastasis.

Adenocarcinoma↗