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Islet fibrosis, pancreatic β-cell dysfunction, and β-cell apoptosis are features of pancreatic diabetes and type 2 diabetes; however, the underlying mechanisms remain largely unknown. We hypothesized that pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, might affect the phenotype of pancreatic β-cells. α-Smooth muscle actin (a marker of activated PSC)-positive cells were found within and around the fibrotic islets. Indirect co-culture with PSCs reduced insulin expression and induced apoptosis in RIN-5F pancreatic β-cells. Induction of β-cell apoptosis was associated with activation of the caspase pathway and mitochondrial depolarization. Diphenylene iodonium, an inhibitor of PSC activation, inhibited islet fibrosis and protected islets in vivo. Our findings suggest a novel mechanism linking PSCs, islet fibrosis, and diabetes mellitus.

There is accumulating evidence that pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, promote the progression of pancreatic cancer. The interactions between PSCs and pancreatic cancer have attracted substantial attention as a novel therapeutic target for the treatment of pancreatic cancer. We examined here the effects of olmesartan, an angiotensin II type I receptor blocker, on pancreatic cancer-associated fibrosis using a subcutaneous tumor model developed by co-injection of pancreatic cancer cells with PSCs in nude mice. Co-injection of pancreatic cancer cells AsPC-1 with PSCs increased the size of tumors compared with AsPC-1 cells alone. Olmesartan administrated at 10 mg/kg in drinking water inhibited the growth of subcutaneous tumors derived from the co-injection, but not those derived from mono-injection. This effect was accompanied by decreased expression of α-smooth muscle actin (a marker of activated PSCs) and collagen deposition. The inhibitory effect of olmesartan was also observed even if it was administrated after significant development of subcutaneous tumors. In addition, olmesartan decreased cell growth and type I collagen production in PSCs in vitro. These results suggest that olmesartan inhibited the growth of tumors by targeting stellate cell activities, and that olmesartan might be useful as an anti-fibrosis therapy in pancreatic cancer.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis associated with chronic pancreatitis and pancreatic cancer. Connexins (Cxs) allow direct intercellular communications as components of gap junction but also play important roles in the regulation of cell proliferation, cell differentiation, and tissue development. We here examined the expression of Cxs and Cx-mediated regulation of cell functions in PSCs.Human PSCs were isolated from patients undergoing operation for chronic pancreatitis or pancreatic cancer. The expression of Cxs was examined by reverse transcription polymerase chain reaction, Western blotting, and immunofluorescent staining. The roles of Cxs in PSC functions were examined by using carbenoxolone, a broad-spectrum Cx inhibitor, and small interfering RNA for Cx43.Human activated PSCs expressed a variety of Cxs including Cx43 both in vitro and in vivo. Carbenoxolone inhibited platelet-derived growth factor-BB-induced proliferation and migration, and type I collagen expression in PSCs. In addition, carbenoxolone inhibited the activation of quiescent PSCs to a myofibroblastlike phenotype. Decreased Cx43 expression by small interfering RNA resulted in decreased proliferation and type I collagen expression.Pancreatic stellate cells expressed a variety of Cxs. Connexins, especially Cx43, might regulate the cell functions and activation of PSCs.

Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR-126 is down-regulated in IDA compared with IPMA or IPMC, and miR-126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA, and IPMC, we newly identified miR-197 as an up-regulated miRNA specifically in IDA. Expression of miR-197 in pancreatic cancer cells resulted in the induction of epithelial-mesenchymal transition (EMT) along with the down-regulation of p120 catenin which is a putative target of miR-197. Direct interaction between miR-197 and p120 catenin mRNA sequence was confirmed by 3'UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR-197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR-197 in the regulation of p120 catenin. This miR-197/p120 catenin axis could be a novel therapeutic target.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis associated with pancreatic ductal adenocarcinoma (PDAC). Kindlin-2 is a focal adhesion protein that regulates the activation of integrins. This study aimed to clarify the role of kindlin-2 in PSCs in pancreatic cancer. Kindlin-2 expression in 79 resected pancreatic cancer tissues was examined by immunohistochemical staining. Kindlin-2-knockdown immortalized human PSCs were established using small interfering RNA. Pancreatic cancer cells were treated with conditioned media of PSCs, and the cell proliferation and migration were examined. SUIT-2 pancreatic cancer cells were subcutaneously injected into nude mice alone or with PSCs and the size of the tumors was monitored. Kindlin-2 expression was observed in PDAC and the peritumoral stroma. Stromal kindlin-2 expression was associated with shorter recurrence-free survival time after R0 resection. Knockdown of kindlin-2 resulted in decreased proliferation, migration, and cytokine expression in PSCs. The PSC-induced proliferation and migration of pancreatic cancer cells were suppressed by kindlin-2 knockdown in PSCs. In vivo, co-injection of PSCs increased the size of the tumors, but this effect was abolished by kindlin-2 knockdown in PSCs. In conclusion, kindlin-2 in PSCs promoted the progression of pancreatic cancer.

ObjectiveCorticosteroid has been established as the standard therapy for autoimmune pancreatitis (AIP), but the requirement for maintenance corticosteroid therapy is controversial. We conducted a randomised controlled trial to clarify the efficacy of maintenance corticosteroid therapy in patients with AIP.DesignWe conducted a multicentre, tertiary setting, randomised controlled trial. After the induction of remission with the initial oral prednisolone (PSL) treatment, maintenance therapy with PSL at 5–7.5 mg/day was continued for 3 years or withdrawn at 26 weeks. The primary endpoint was relapse-free survival over 3 years and the secondary endpoint was serious corticosteroid-related complications. All analyses were performed on an intention-to-treat basis.ResultsBetween April 2009 and March 2012, 49 patients with AIP were randomly assigned to the maintenance therapy group (n=30) or the cessation group (n=19). Baseline characteristics were not different between the two groups. Relapses occurred within 3 years in 11 out of 19 (57.9%) patients assigned to the cessation group, and in 7 of 30 (23.3%) patients in the maintenance therapy group. The relapse rate over 3 years was significantly lower in the maintenance therapy group than that in the cessation group (p=0.011). The relapse-free survival was significantly longer in the maintenance therapy group than that in the cessation group (p=0.007). No serious corticosteroid-related complications requiring discontinuation of PSL were observed.ConclusionsMaintenance corticosteroid therapy for 3 years may decrease relapses in patients with AIP compared with those who discontinued the therapy at 26 weeks.Trial registration numberUMIN000001818; Results.

Background and study aim: Self-expandable metallic stents (SEMSs) are used for palliation in patients with malignant perihilar biliary strictures. However, recurrent biliary obstruction occasionally causes cholangitis and jaundice. This study aimed to identify risk factors for recurrent biliary obstruction in such patients. Methods: Data from consecutive patients with malignant perihilar biliary strictures treated with endoscopic placement of SEMSs between 2007 and 2014 in Tohoku University Hospital were retrospectively reviewed. Risk factors for recurrent biliary obstruction were calculated using the Cox proportional hazards models (with hazard ratios [HRs] and 95 % confidence interval [95 %CIs]), and SEMS patency period was examined using the Kaplan – Meier method. SEMS patency was defined as the period between SEMS insertion and the development of recurrent biliary obstruction. Results: 104 patients were included. Median survival time was 281 days; and 85 patients died during a median follow-up period of 320 days. Recurrent biliary obstruction occurred in 35 patients. Median SEMS patency period was 549 days. Multivariable analyses showed that: compared with bile duct carcinoma, gallbladder carcinoma was associated with shorter SEMS patency (HR 8.18, 95 %CI 2.41 – 26.83); patency of left-sided SEMS was inferior to that of bilateral (HR 0.5, 95 %CI 0.32 – 0.93) and right-sided SEMS (HR 0.1, 95 %CI 0.02 – 0.65). Cholangitis before SEMS placement increased the risk of recurrent biliary obstruction (HR 11.44; 95 %CI 4.48 – 32.35) and reduced the SEMS patency period (746 vs. 210 days). Conclusion: Gallbladder carcinoma, left-sided stent placement, and cholangitis before SEMS placement are risk factors for recurrent biliary obstruction after SEMS placement.

International audience; A hybrid allele between the carboxyl ester lipase gene (CEL) and its pseudogene, CELP (called CEL–HYB), generated by non-allelic homologous recombination between CEL intron 10 and CELP intron 10′, was found to increase susceptibility to chronic pancreatitis in a case–control study of patients of European ancestry. We attempted to replicate this finding in 3 independent cohorts from China, Japan, and India, but failed to detect the CEL–HYB allele in any of these populations. The CEL–HYB allele might therefore be an ethnic-specific risk factor for chronic pancreatitis. An alternative hybrid allele (CEL–HYB2) was identified in all 3 Asian populations (1.7% combined carrier frequency), but was not associated with chronic pancreatitis.

OBJECTIVES The usefulness of early severity assessment of acute pancreatitis (AP) by contrast-enhanced computed tomography (CECT) was investigated. METHODS Data were obtained from a 2007 nationwide survey in Japan. Clinical data of 983 patients with AP were analyzed. All were examined by CECT on the day of admission. RESULTS Early findings of CECT demonstrated that low enhanced pancreatic parenchyma (LEPP) was associated with the incidence of organ failure (OF), multiple OF, and infectious complications as well as mortality (P < 0.0001). Next, patients were further divided into 4 groups according to the CECT findings, which focused on the LEPP and peripancreatic collections (PPCs). The LEPP/PPC (+/+) group was characterized as high morbidity and high mortality. The incidence of OF (28.2%), multiple OF (15.5%), and mortality (11.4%) in patients assigned to the (+/+) group was significantly higher than in those assigned to the other groups. The incidence of infectious complications was significantly higher in patients assigned to the (+/+) group (16.7%), the (+/-) group (9.0%), and the (-/+) group (7.0%) than those assigned to the (-/-) group (1.8%). CONCLUSIONS The detection of LEPP and PPC was a useful CECT finding for the early assessment of the severity of AP.

The serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor (PSTI), is a peptide secreted by pancreatic acinar cells. Genetic studies have shown an association between SPINK1 gene variants and chronic pancreatitis or recurrent acute pancreatitis. The aim of this study was to clarify whether the SPINK1 variants affect the level of serum PSTI.One hundred sixty-three patients with chronic pancreatitis or recurrent acute pancreatitis and 73 healthy controls were recruited. Serum PSTI concentrations were determined with a commercial radioimmunoassay kit.Ten patients with the p.N34S variant, 7 with the IVS3+2TC variant, two with both the p.N34S and the IVS3+2TC variants, and one with the novel missense p.P45S variant in the SPINK1 gene were identified. The serum PSTI level in patients with no SPINK1 variants was 14.3 ± 9.6 ng/ml (mean ± SD), and that in healthy controls was 10.7 ± 2.2 ng/ml. The PSTI level in patients carrying the IVS3+2TC variant (5.1 ± 3.4 ng/ml), but not in those with the p.N34S variant (8.9 ± 3.5 ng/ml), was significantly lower than that in the patients without the SPINK1 variants and the healthy controls. The serum PSTI level in the patient with the p.P45S variant was 4.9 ng/ml. Low levels of serum PSTI (6.0 ng/ml) showed sensitivity of 80 %, specificity of 97 %, and accuracy of 96 % in the differentiation of IVS3+2TC and p.P45S carriers from non-carriers.Serum PSTI levels were decreased in patients with the IVS3+2TC and p.P45S variants of the SPINK1 gene.

A nationwide survey was conducted to clarify the epidemiological features of patients with chronic pancreatitis (CP) in Japan.Two sequential surveys were conducted. In the first survey, both the prevalence and incidence of CP in Japan in 2007 were estimated by a questionnaire, which was mailed to 3027 randomly chosen Japanese facilities. In the second survey, the second questionnaire was then mailed to 1110 facilities selected by the first survey to clarify the clinicoepidemiological features of the patients.The estimated annual prevalence of CP was 36.9 per 100,000; 53.2 in males and 21.2 in females. The estimated annual incidence was 11.9 per 100,000. The prevalence and the incidence of CP gradually increased in Japan as compared to former surveys. The sex ratio (male/female) of definitive and probable CP patients was 4.5, with a mean age of 59.4 years; 59.2 years in males and 60.2 years in females. Alcoholic (69.7%) was most the common and idiopathic (21.0%) was the second most common cause of CP. The proportion of alcoholic CP increased as compared to the 55.5% found in 1994. The clinical features of overall Japanese patients with CP were: abdominal pain (60.6%), malabsorbtion (12.2%), diabetes mellitus (39.7%) and pancreatolithiasis (75.7%). Alcoholic patients were characterized by high morbidity as compared to nonalcoholic patients: abdominal pain (alcoholic 65.0% vs nonalcoholic 53.0%, p0.0001), diabetes mellitus (44.8% vs 31.4%, p0.0001) and pancreatolithiasis (84.0% vs 60.8%, p0.0001).The prevalence and the incidence of CP, especially alcoholic CP, have been increasing in Japan.

The epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry of metastatic disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entire picture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targeting multiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductal adenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, and human pancreatic cancer cell line to elucidate essential miRNAs which regulate invasive growth of pancreatic cancer cells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reduced expression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to target ADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreatic cancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 3′ untranslated region assay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted in reduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first time that the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells. Mol Cancer Res; 10(1); 3–10. ©2011 AACR.

Objectives The aim of this study was to clarify the pancreatic blood perfusion in patients with autoimmune pancreatitis (AIP) and the changes after steroid treatment. Methods Perfusion computed tomography was performed in 11 patients with AIP and 12 control subjects. Pancreatic volumetric blood flow (F(V)), volume of distribution (V(D)), and blood transit time τ were determined from a single-compartment kinetic model. Nine patients with AIP were reexamined by perfusion computed tomography after corticosteroid administration. Results The pancreatic F(V) values of the 11 patients with AIP (82.7/min) were significantly lower than those of control subjects (163.5/min, P = 0.0006). On the other hand, the pancreatic V(D) and τ values were not significantly different between AIP and normal. After steroid treatment, the F(V) values of 9 reexamined patients with AIP (76.2/min) were significantly elevated (109.8/min, P = 0.0391). However, the changes of the values after the treatment differed in degree among individuals. The values of 4 patients were dramatically elevated to greater than 100/min, whereas those of 4 other patients did not improve well. The value of the remaining patient whose initial F(V) value was normal (168.09/min) did not change after the treatment. Conclusions Pancreatic volumetric perfusion was attenuated in AIP patients. The perfusion was improved after the steroid treatment.

A nationwide epidemiological survey was conducted to estimate the number of patients treated for acute pancreatitis (AP) in 2007 in Japan and to clarify the clinicoepidemiological features of AP.In the first survey, a simple questionnaire was used to inquire about the number of patients with AP who visited the hospital in the year 2007. This questionnaire was directly mailed to the heads of 3027 facilities. The second questionnaire was forwarded to those facilities from which patients with AP were reported on the first questionnaire.The estimated total number of patients treated for AP in 2007 was 57,560 (95% confidence interval, 48,571-66,549), with an overall prevalence rate of 45.1 per 100,000 population. The sex ratio (male-female) of the patients was 2.0, with a mean age of 56.6 years in men and 64.6 years in women. Alcoholic AP was most common in men and gallstone AP in women. The overall mortality rate of AP was 1.9% and, in severe cases, 8.0%.The number of patients with AP increased about 3-fold during this decade (19,500 in 1998 to 57,560 in 2007), and the mortality rate of AP was reduced from 7.4% in 1998 and 2.9% in 2003 to 1.9% in 2007.

Patients with branch duct type intraductal papillary mucinous neoplasm (BD-IPMN) without invasion usually show favorable prognosis. However, the prognosis becomes poor when the IPMN lesions give rise to invasive carcinoma cells. In addition, recent studies have revealed that BD-IPMN is frequently complicated by common type pancreatic ductal carcinoma. Thus, the prognosis of BD-IPMN depends on the occurrence of these two types of invasive carcinoma. However, little is known about the risk factors for the development of these invasive carcinomas in BD-IPMN. This study aims to identify the factors which predict the development of invasive carcinoma in BD-IPMN. Invasive pancreatic carcinoma associating with BD-IPMN was classified as invasive IPMN group (invasive carcinoma derived directly from IPMN lesions) and concomitant group (common type of invasive carcinoma concomitant with BD-IPMN). The relation between the incidence of each type of invasive carcinoma in BD-IPMN and the clinicopathological parameters was retrospectively analyzed. There were 12 patients with invasive IPMN and 7 patients with concomitant cancer in 159 patients with BD-IPMN. Diameter of dilated branch (P

Catalytic light-off of a stream of NO, H2, CO in an excess O2 has been studied over various metal oxides loading 1 wt% Pt. Because a low-surface area Y2O3 (

We synthesized nanoporous carbon containing noble metal particles via the co-intercalation of noble metal precursors and sucrose into montmorillonite (mont) and the subsequent sulfuric acid-catalyzed carbonization. A TEM image of the Ru-containing porous carbon material (Ru-C/mont) showed Ru particles of uniform particle sizes (1–2 nm). Residual sulfur species were also observed in Ru-C/mont even after heating in N 2 flow at 1073 K. The FT-IR spectra of Ru-C/mont showed peaks corresponding to the asymmetric S O stretching mode of SO 3 − as well as to the asymmetric and symmetric stretching modes of Si O Al. SO 3 − and Si O Al acted as acid sites. The catalytic activity of Ru-C/mont in cyclopentene hydration was investigated. Cyclopentene hydration proceeded using C/mont as catalyst even without Ru incorporation. However, the catalytic activity of the synthesized carbon material increased in the presence of Ru. The cyclopentanol yield was higher using Ru-C/mont than using H-ZSM-5 or Nb 2 O 5 · n H 2 O as a catalyst. Thus, the coexistence of acid sites and Ru plays a key role in cyclopentanol formation.

Human Cripto-1 (CR-1) is a cell membrane protein that is overexpressed in several different types of human carcinomas. In the present study we investigated the mechanisms that regulate the expression of CR-1 gene in cancer cells. We cloned a 2,481 bp 5'-flanking region of the human CR-1 gene into a luciferase reporter vector and transfected NTERA-2 human embryonal carcinoma cells and LS174-T colon cancer cells to test for promoter activity. Activity of CR-1 promoter in both cell lines was modulated by two TGF-beta family members, TGF-beta1 and BMP-4. In particular, TGF-beta1 significantly up-regulated CR-1 promoter activity, whereas a dramatic reduction in CR-1 promoter activity was observed with BMP-4 in NTERA-2 and LS174-T cells. Changes in the CR-1 promoter activity following TGF-beta1 and BMP-4 treatments correlated with changes in CR-1 mRNA and protein expression in NTERA-2 and LS174-T cells. We also identified three Smad binding elements (SBEs) within the CR-1 promoter and point mutation of SBE1 (-2,197/-2,189) significantly reduced response of the CR-1 promoter to both TGF-beta1 and BMP-4 in NTERA-2 and LS174-T cells. Chromatin immunoprecipitation assay also demonstrated binding of Smad-4 to a CR-1 promoter DNA sequence containing SBE1 in LS174-T cells. Finally, BMP-4 inhibited migration of LS174-T cells and F9 mouse embryonal carcinoma cells by downregulation of CR-1 protein. In conclusion, these results suggest a differential modulation of CR-1 gene expression in embryonal and colon cancer cells by two different members of the TGF-beta family.

Periostin is a secretory protein that has been suggested to function as a cell adhesion molecule and promote the invasiveness or growth rate of tumors. However, little is known about the association of its expression and epithelial to mesenchymal transition (EMT), which is considered to play a crucial role in cancer cell metastasis. Thus, the authors investigated whether periostin could be involved in the process of EMT and the role of this gene in pancreatic cancer development. The expression of periostin was observed mainly in stromal cells but very little in cancer cells by immunohistochemistry and real-time RT-PCR. In vitro, pancreatic stellate cells (PSCs) exhibited a much higher basal expression of periostin compared with cancer cells. Periostin secreted in the supernatant from 293T cells that expressed periostin (approximately 150 ng/ml) inhibited the migration of pancreatic cancer cells. Coculture assay revealed that periostin expression in PSC was induced by pancreatic cancer cells. To assess the direct role of periostin in pancreatic cancer cells, the authors generated pancreatic cancer cell lines that stably express periostin. The induced expression of periostin (to 150 ng/ml) altered the morphology of cancer cells, changing them from mesenchymal to epithelial phenotypes with the induction of epithelial markers and a reduction of mesenchymal markers, and showed reduced cell migration in vitro and formed smaller tumors as well as suppressed metastasis in vivo. On the other hand, high concentration of recombinant periostin (1 microg/ml) promoted cell migration with AKT activation. The findings suggest that periostin has biphasic effect on the development of pancreatic cancer.

膵癌早期発見のためには膵発癌機構を解明することが不可欠である．そのためには，遺伝子解析も作成法も容易である動物モデルの開発が必要と考えられる．我々は化学発癌物質DMBA投与によってマウス膵発癌モデルを作成し，それがヒト膵癌研究に応用できるか否かを検討した．DMBA処理2週後からマウス膵にtubular complexが観察され，1ヶ月でPanIN類似病変，2ヶ月後3ヶ月後にかけて癌病変の形成が認められた．癌は肉腫様形態を呈したがcytokeratin陽性，vimentin, chymotrypsin陰性で膵管由来の癌と考えられた．過形成病変および癌病変において，ヒト膵癌同様，悪性度の進行に伴って，smad4発現の消失，cyclin D1, p53発現の増強，Notchシグナルの活性化が認められた．しかし，ヒトで最も初期にまた最も高頻度に異常の認められるK-ras遺伝子の変異は確認されなかった．これらの事から，本マウスモデルは，多くのヒト膵癌とは初期の発癌過程は異なるが進展過程に関与する遺伝子異常には類似性がみられ，ヒトにおける発癌過程の後期から癌進展過程の研究に適用できる可能性が示唆された．

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (omega-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

The NO-H 2 -O 2 reaction was studied over supported bimetallic catalysts, Pt-Mo and Pt-W, which were prepared by coexchange of hydrotalcite-like Mg-Al double layered hydroxides by Pt(NO 2 ) 4 2− , MoO 4 2− , and/or WO 4 2− and subsequent heating at 600 °C in H 2 . The Pt–Mo interaction could obviously be seen when the catalyst after reduction treatment was exposed to a mixture of NO and H 2 in the absence of O 2 . The Pt-HT catalyst showed the almost complete NO conversion at 70 °C, whereas the Pt-Mo-HT showed a negligible conversion. Upon exposure to O 2 , however, Pt-Mo-HT exhibited the NO conversion at the lowest temperature of ≥30 °C, compared to ≥60 °C required for Pt-HT. EXAFS/XANES, XPS and IR results suggested that the role of Mo is very sensitive to the oxidation state, i.e., oxidized Mo species residing in Pt particles are postulated to retard the oxidative adsorption of NO as NO 3 and promote the catalytic conversion of NO to N 2 O at low temperatures.

The catalytic NO–H 2 –O 2 reaction at low temperatures (50–130 °C) has been investigated over Pt-supported Mg–Al binary oxides, which were prepared from PtCl 6 2− and Pt(NO 2 ) 4 2− –exchanged hydrotalcite(HT)-like compounds by heating at 600 °C in H 2 . Although the NO x conversion of both catalysts was similarly high (>80%) at around 70 °C, the Pt(NO 2 ) 4 2− –HT catalyst exhibited the higher selectivity to N 2 (53% N 2 and 47% N 2 O), compared to 12% N 2 and 88% N 2 O for the PtCl 6 2− –HT catalyst. The XANES/EXAFS spectroscopy showed that Pt prepared from PtCl 6 2− –HT is partly oxidized due to the coordination of residual chlorides, whereas Pt(NO 2 ) 4 2− –HT yielded highly dispersed metallic Pt. Because of the presence of chloride strongly bound to Pt, the oxidative NO adsorption as NO 2 /NO 3 was inhibited for PtCl 6 2− –HT. The improved N 2 selectivity with an increase of oxidative NO adsorption is in accord with the catalytic property of Na–Pt/ZSM-5 in our previous work [M. Machida, T. Watanabe, Appl. Catal. B: Environ. 52 (2004) 281.], suggesting that the N 2 would be formed via NO 2 /NO 3 intermediates.

To establish pancreatic cancer in mice, dimethylbenzanthracene (DMBA) was administered into mice pancreata. The formation of tubular complex lesions was found in the pancreatic sections from 2 weeks after DMBA treatment. Abnormal tubular complex formations with ductal metaplasia were found from 1 month after the administration. By 3 months after DMBA injection into the pancreas, 6 of 10 mice showed visually recognizable tumors with precursor lesions of various types of cell atypia. In contrast, there were no visually or histologically detectable tumors in the placebo-treated animals. The expression profiles of smad 4, cyclin D1 and p53 in the DMBA-induced tumors were similar to those of human pancreatic cancer, suggesting that this would be a useful mouse model for studying the morphological and molecular mechanisms involved in pancreatic carcinogenesis. Immunohistochemical study using specific antibodies revealed that Notch-1 and Hes-1 were expressed in lesions ranging from tubular complexes to carcinoma in these chemically induced pancreatic tumors. Semiquantitative reverse transcription-polymerase chain reaction with microdissection demonstrated that Notch-1 expression was continuous from precursor lesions to carcinoma cells, whereas Pdx-1 expression was attenuated in carcinoma cells compared to precursor lesions. In addition, inhibition of the Notch signaling pathway by the gamma-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester reduced pancreatic cancer cell growth. Therefore, Notch signaling is required to form the tubular complexes and its continuous activation might lead to the transition from tubular complexes to premalignant or malignant lesions and carcinoma cell development in the pancreas.

In our study, we found that bone morphogenetic protein 4 (BMP4) has a novel effect as an inducer of epithelial-mesenchymal transition (EMT) on Panc-1 cells, a human pancreatic carcinoma cell line. BMP4-treated Panc-1 cells showed loose cell contacts and a scattered, fibroblast-like appearance along with E-cadherin downregulation, Vimentin upregulation and enhanced cell migration, which are characteristic of EMT. BMP4 treatment also induced homeobox gene MSX2 expression, which we previously showed to be associated with EMT in pancreatic carcinoma cells. BMP4 treatment activated the Smad signaling pathway, and extracellular signal-related kinase (ERK) and p38 mitogen-activated kinase (MAPK) pathways in these cells. MSX2 was markedly induced by BMP4 through the ERK and p38 MAPK pathways in collaboration with the Smad signaling pathway. The repression of E-cadherin, induction of Vimentin and enhanced cell migration disappeared when siRNA-based MSX2 downregulated pancreatic cancer cells were treated with BMP4. These findings indicate that BMP4 may be involved in pancreatic carcinoma development through the promotion of EMT and that MSX2 is indispensable to this process.

Genetic alterations in the carboxypeptidase A1 gene ( CPA1) are associated with early onset chronic pancreatitis (CP). Besides CPA1, there are two other human pancreatic carboxypeptidases (CPA2 and CPB1). Here we examined whether CPA2 and CPB1 alterations are associated with CP in Japan and Germany. All exons and flanking introns of CPA2 and CPB1 were sequenced in 477 Japanese patients with CP (234 alcoholic, 243 nonalcoholic) and in 497 German patients with nonalcoholic CP by targeted next-generation sequencing and/or Sanger sequencing. Secretion and enzymatic activity of CPA2 and CPB1 variants were determined after transfection into HEK 293T cells. We identified six nonsynonymous CPA2 variants (p.V67I, p.G166R, p.D168E, p.D173H, p.R237W, and p.G388S), eight nonsynonymous CPB1 alterations (p.S65G, p.N120S, p.D172E, p.R195H, p.D208N, p.F232L, p.A317V, and p.D364Y), and one splice-site variant (c.687+1G>T) in CPB1. Functional analysis revealed essentially complete loss of function in CPA2 variants p.R237W and p.G388S and CPB1 variants p.R110H and p.D364Y. None of the CPA2 or CPB1 variants, including those resulting in a marked loss of function, were overrepresented in patients with CP. In conclusion, CPA2 and CPB1 variants are not associated with CP.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis, a characteristic feature of pancreatic cancer. Although it is still controversial, previous studies have suggested that PSCs promote the progression of pancreatic cancer by regulating the cell functions of cancer cells. PSCs produce large amounts of IL-6, which promotes the accumulation of myeloid-derived suppressor cells via a signal transducers and activator of transcription 3 (STAT3)-dependent mechanism. But the role of IL-6/STAT3 pathway in the interaction between PSCs and pancreatic cancer cells remains largely unknown. To clarify the role of IL-6/STAT3 in the interaction between PSCs and cancer cells. Human pancreatic cancer cells (Panc-1 and SUIT-2 cells) were treated with conditioned medium of immortalized human PSCs (PSC-CM). The effects of PSC-CM and IL-6 neutralization on the mRNA expression profiles were examined using Agilent’s microarray. Activation of STAT3 was assessed by Western blotting using an anti-phospho-specific antibody. Cellular migration was examined by a two-chamber assay. The expression of markers related to epithelial–mesenchymal transition (EMT) was assessed by real-time reverse transcription PCR. PSC-CM induced the activation of STAT3 in pancreatic cancer cells. Neutralization of IL-6 suppressed the PSC-CM-induced upregulation of genes including complement factor B, lipocalin, and chemokine (C–C motif) ligand 20. Inhibition of IL-6/STAT3 pathway by anti-IL-6 antibody or a STAT3 inhibitor (NSC74859) inhibited the PSC-CM-induced migration and the expression of EMT-related markers (Snail and cadherin-2) in pancreatic cancer cells. IL-6/STAT3 pathway regulates the PSC-induced EMT and alterations in gene expression in pancreatic cancer cells.

Prognosis of pancreatic cancer remains dismal due to the resistance against conventional therapies. Metastasis and massive invasion toward surrounding organs hamper radical resection. Small part of entire cancer cells reveal resistance against chemotherapy or radiotherapy, increased tumorigenicity and migratory phenotype. These cells are called as cancer stem cells, as a counter part of normal stem cells. In pancreatic cancer, several cancer stem cell markers have been identified, which enabled detailed characterization of pancreatic cancer stem cells. Recent researches clarified that conventional chemotherapy itself could increase cancer cells with stem cell-phenotype, suggesting the necessity of cancer stem cell-targeting therapy. Based on these observations, pancreatic cancer stem cell-targeting therapies have been tested, which effectively eliminated cancer stem cell fraction and attenuated cancer progression in experimental models. Clinical efficacy of these therapies need to be evaluated, and cancer stem cell-targeting therapy will contribute to improve the prognosis of pancreatic cancer.

A 50-year-old male was referred to our hospital for the evaluation of hyperproteinemia. Fluorodeoxyglucose positron emission tomography revealed high fluorodeoxyglucose uptake in the pancreas, bilateral lacrimal glands, submandibular glands, parotid glands, bilateral pulmonary hilar lymph nodes, and kidneys. Laboratory data showed an elevation of hepatobiliary enzymes, renal dysfunction, and remarkably high immunoglobulin (Ig) G levels, without elevated serum IgG4. Abdominal computed tomography revealed swelling of the pancreatic head and bilateral kidneys. Endoscopic retrograde cholangiopancreatography showed an irregular narrowing of the main pancreatic duct in the pancreatic head and stricture of the lower common bile duct. Histological examination by endoscopic ultrasonography-guided fine-needle aspiration revealed findings of lymphoplasmacytic sclerosing pancreatitis without IgG4-positive plasma cells. Abnormal laboratory values and the swelling of several organs were improved by the treatment with steroids. The patient was diagnosed as having type 1 autoimmune pancreatitis (AIP) based on the International Consensus Diagnostic Criteria. Therefore, we encountered a case of compatible type 1 AIP without elevated levels of serum IgG4 or IgG4-positive plasma cells. This case suggests that AIP phenotypes are not always associated with IgG4.

Purpose: Human Cripto-1 (CR-1), a cell membrane glycosylphosphatidylinositol-anchored glycoprotein that can also be cleaved from the membrane, is expressed at high levels in several different types of human tumors. We evaluated whether CR-1 is present in the plasma of patients with breast and colon cancer, and if it can represent a new biomarker for these malignancies. Experimental Design: We determined CR-1 plasma levels using a sandwich-type ELISA in 21 healthy volunteers, 54 patients with breast cancer, 33 patients with colon carcinoma, and 21 patients with benign breast lesions. Immunohistochemical analysis was also used to assess CR-1 expression in cancerous tissues. Results: Very low levels of CR-1 (mean ± SD) were detected in the plasma of healthy volunteers (0.32 ± 0.19 ng/mL). A statistically significant increase in the levels of plasma CR-1 was found in patients with colon carcinoma (4.68 ± 3.5 ng/mL) and in patients with breast carcinoma (2.97 ± 1.48 ng/mL; P < 0.001). Although moderate levels of plasma CR-1 were found in women with benign lesions of the breast (1.7 ± 0.99 ng/mL), these levels were significantly lower than in patients with breast cancer (P < 0.001). Finally, immunohistochemical analysis and real-time reverse transcription-PCR confirmed strong positivity for CR-1 in colon and/or breast tumor tissues. Conclusion: This study suggests that plasma CR-1 might represent a novel biomarker for the detection of breast and colon carcinomas.

Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3β, and dephosphorylated (DP)-β-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of β-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3β and increased nuclear localization of β-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3β and dephosphorylated (DP)-β-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or β-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3β, DP-β-catenin, and increased nuclear localization of β-catenin in human and MMTV-CR-1 mice leiomyosarcomas. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Thermal decomposition of PtCl6-exchanged hydrotalcite-like Mg0.74Al0.26(OH)2(NO3)0.26·0.36H2O (Pt−HT) in a stream of H2 has been investigated to demonstrate the catalytic role of intercalated Pt on the transformation to porous structures. Platinum species in the interlayer promoted the reaction between H2 and interlayer nitrate (NO3-) to yield N2 so as to give rise to a large surface area (≥200 m2 g-1) solid at as low as ca. 200 °C, compared to 400 °C required for the pristine HT. The XAFS analysis of the PtCl6-exchanged HT gave evidence that part of chloride (Cl-) ligands bound to Pt was replaced by NO3- during an exchange process. Since molecular hydrogen is activated by Pt, the reaction of nitrate ligand with hydrogen to form N2/H2O should generate a vacant site, which is successively occupied by NO3- ions lying in the interlayer. Such coordination/catalytic reduction cycles would accelerate the removal of NO3- surrounding Pt in the interlayer before dehydroxylation of brucite layers. This is in contra...

Clinical Impact of Elevated Serum Triglycerides in Acute Pancreatitis: Validation from the Nationwide Epidemiological Survey in Japan