Masashi Taguchi

Inhibition of transforming growth factor beta decreases pancreatic fibrosis and protects the pancreas against chronic injury in mice.

Transforming growth factor-β (TGF-β) is an important cytokine in the fibrogenesis in many organs, including the pancreas. Using an adenoviral vector expressing the entire extracellular domain of type II human TGF-β receptor (AdTβ-ExR), we investigated whether inhibition of TGF-β action is effective against persistent pancreatic fibrosis, and whether it exerts a beneficial effect on the pancreas in the process of chronic injury. To induce chronic pancreatic injury and pancreatic fibrosis, mice were subjected to three episodes of acute pancreatitis induced by six intraperitoneal injections of 50 μg/kg body weight cerulein at hourly intervals, per week for 3 consecutive weeks. Mice were infected once with AdTβ-ExR, or with a control adenoviral vector expressing bacterial β-galactosidase (AdLacZ). Pancreatic fibrosis was evaluated by histology and hydroxyproline content. Activation of pancreatic stellate cells (PSCs) was assessed by immunostaining for α-smooth muscle actin. Apoptosis and proliferation of acinar cells were assessed by immunostaining of ssDNA and Ki-67, respectively. Three-week cerulein injection induced pancreatic fibrosis and pancreatic atrophy with proliferation of activated PSCs. In AdTβ-ExR-injected mice, but not AdLacZ-injected mice, pancreatic fibrosis was significantly attenuated. This finding was accompanied by a reduction of activated PSCs. AdTβ-ExR, but not AdLacZ, significantly increased pancreas weight after chronic pancreatic injury. AdTβ-ExR did not change the proportion of proliferating acinar cells, whereas it reduced the number of apoptotic acinar cells. Our results demonstrate that inhibition of TGF-β action not only decreases pancreatic fibrosis but also protects the pancreas against chronic injury by preventing acinar cell apoptosis.

Induction of PDX‐1‐positive cells in the main duct during regeneration after acute necrotizing pancreatitis in rats

Pancreatic regeneration involves two pathways; proliferation and differentiation of pancreatic progenitor cells, which probably exist in pancreatic ductal epithelium, and replication of pre‐existing differentiated acinar, islet, and ductal epithelial cells. During pancreatic development, differentiated cells arise from the ductal progenitor cells expressing the pancreatic/duodenal homeobox‐1 (PDX‐1) homeodomain transcription factor. The aims of this study were to characterize cell proliferation and differentiation during regeneration after acute necrotizing pancreatitis and to evaluate the role of PDX‐1‐positive stem cells. Necrotizing pancreatitis was induced in rats by retrograde intraductal infusion of sodium taurocholate. Cell types were classified into five categories: main, large, and small ductal epithelial cells, tubular complexes and acinar cells. Each category was scored using a 5‐bromo‐2′‐deoxyuridine (BrdU) labelling index (LI) at various time points after induction of pancreatitis. Tissue sections were also immunostained for PDX‐1 to determine the source of pancreatic stem cells. Acinar necrosis was observed at 24 h after induction of pancreatitis and most lobules were filled with tubular complexes on day 5. Subsequently, newly formed acinar cells were observed on day 7, but the lobular architecture returned to normal appearance on day 28. Proliferation started in the main and large ducts at 24 h; marked mitotic activity was evident in small ductal epithelial cells and tubular complexes on day 3, and in acinar cells on day 7. At 24 h after induction of pancreatitis, epithelial cells of the main duct with PDX‐1‐positive nuclei were greatly increased, simultaneously with the peak LI of BrdU. These results suggest that regeneration after necrotizing pancreatitis involves proliferation and differentiation of pancreatic progenitor cells, and that ductal epithelial cells with PDX‐1‐positive nuclei may contribute to the differentiation of pancreatic stem cells in the main duct. Copyright

Randomised controlled trial of long-term maintenance corticosteroid therapy in patients with autoimmune pancreatitis

Objective Corticosteroid 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. Design We 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. Results Between 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. Conclusions Maintenance corticosteroid therapy for 3 years may decrease relapses in patients with AIP compared with those who discontinued the therapy at 26 weeks. Trial registration number UMIN000001818; Results.

Green tea polyphenol (–)-epigallocatechin-3-gallate inhibits ethanol-induced activation of pancreatic stellate cells

Background  Activated pancreatic stellate cells (PSCs) play a central role in the pathogenesis of pancreatic fibrogenesis and inflammation. Ethanol, a major cause of chronic pancreatitis, directly induces PSC activation and oxidative stress. Inhibition of PSC activation or stimulation to PSC might be an effective therapeutic strategy for the prevention of pancreatic fibrosis, and (–)‐epigallocatechin‐3‐gallate (EGCG), a major component of green tea extracts, is a potent antioxidant of polyphenols. Therefore, we examined the mechanisms through which ethanol induces oxidative stress on PSCs and evaluated the effect of EGCG on activation and cell functions of ethanol‐stimulated PSCs.

High glucose activates rat pancreatic stellate cells through protein kinase C and p38 mitogen-activated protein kinase pathway.

Objective: Hyperglycemia is implicated in fibrosis in many organs. Exocrine and endocrine pancreas are closely linked both anatomically and physiologically, and pathological conditions in the exocrine gland can cause impairment of endocrine function and vice versa. Chronic pancreatitis causes pancreatic fibrosis and sometimes results in diabetes mellitus. Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrogenesis. However, the effects of high glucose concentrations on PSC activation have not been fully elucidated. Methods: Cultured PSCs were incubated in the presence of various concentrations of glucose. Pancreatic stellate cell proliferation, &agr;-smooth muscle actin (&agr;-SMA) expression, and collagen production were determined by colorimetric conversion assay, Western blot analysis, and Sirius red dye binding assay, respectively. Results: High glucose concentrations significantly increased PSC proliferation, &agr;-SMA expression, and collagen type I production in PSCs. High glucose concentrations activated protein kinase C (PKC) in PSCs, and PKC inhibitor GF109203X inhibited glucose-stimulated PSC proliferation, &agr;-SMA expression, and collagen secretion. High glucose also activated p38 mitogen-activated protein kinase (MAPK) in PSCs, and p38 MAPK inhibitor SB203580 inhibited glucose-stimulated collagen secretion. Conclusions: Our results indicate that high glucose concentrations stimulate PSC activation via PKC-p38 MAP kinase pathway and suggest that high glucose may aggravate pancreatic fibrosis.

Body mass index influences the outcome of acute pancreatitis: an analysis based on the Japanese administrative database.

Objective This study aimed to investigate the relationship between body mass index (BMI) and risk of death in patients with acute pancreatitis (AP) using a Japanese national administrative database. Methods We analyzed a total of 6002 patients with AP. We collected patient information, including sex, age, BMI, severity of AP based on the Japan Pancreas Society scoring system, and prognosis. We classified BMI into 5 categories (underweight [BMI, <18.5], normal range [18.5–24.9], preobese [25–29.9], obese class I [30–34.9], and obese class II/III [>35]) and investigated the relationship between each category and risk of death in AP. Results There was a good correlation between the Japanese AP severity score and in-hospital mortality. Overall mortality of severe pancreatitis was 7.0% (n = 2245). Mortality in each BMI category was as follows: underweight, 6.4%; normal range, 3.6%; preobese, 2.4%; obese class I, 3.2%; and obese class II/III, 5.7%. Underweight and obese class II/III patients had significantly higher relative risk (RR) of death in AP compared with preobese patients after adjusting for sex, age, and severity of AP (RR, 2.7; 95% confidence interval, 1.6–4.5; and RR, 6.4; 95% confidence interval, 1.9–20.9, respectively). Conclusions Underweight or overweight was the independent risk factor for mortality in AP.

Persistent destruction of the basement membrane of the pancreatic duct contributes to progressive acinar atrophy in rats with experimentally-induced pancreatitis

Background and Aims: The imbalance between synthesis and degradation of extracellular matrix (ECM) proteins is a characteristic feature in chronic pancreatitis. We evaluated the relationship between type IV collagen structure in the basement membrane (BM) and the development of acinar atrophy or the regeneration from acinar injury. Methods: Three different models of pancreatitis were induced in rats by repetitive intraperitoneal injections of 500 mg/100 g body weight of arginine (Arg) or 20 μg/kg body weight of caerulein (Cn) or a single retrograde intraductal infusion of 40 μL/100 g body weight of 3% sodium taurocholate (NaTc). We examined the changes in type IV collagen structure by immunostaining, and the serial changes in the gelatinolytic activity of pro- and active matrix metalloproteinase-2 by zymography in these models of pancreatitis. Results: The pancreas appeared to be histologically normal on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc, whereas 85% to 90% of acinar tissue was replaced by fatty tissue and dilated pancreatic ducts on day 54 after the first intraperitoneal Arg injection. Immunoreactivity for type IV collagen appeared as a discontinuous line along the BM of ducts, vessels, tubular complexes, and acinar cells on day 40 in Arg-induced pancreatitis, whereas it was detected as a continuous line along the BM on day 35 in Cn-induced pancreatitis and on day 42 in NaTc-induced pancreatitits. Gelatinolytic activity of active MMP-2 increased significantly from day 13 to day 40 after the first intraperitoneal Arg injection, whereas it decreased to the baseline level on day 35 after the first intraperitoneal Cn injection and on day 42 after intraductal infusion of NaTc. Conclusions: Our findings indicate that a long-term increase in gelatinolytic activity of active MMP-2 in Arg-induced pancreatitis causes continuous disorganization of type IV collagen in the BM and progressive acinar atrophy, whereas a transient increase in gelatinolytic activity of active MMP-2 is involved in the regeneration of type IV collagen structure in the BM and recovery from acinar injury.

Co-localization of nestin and PDX-1 in small evaginations of the main pancreatic duct in adult rats.

Background and Aims: Pancreatic stem cells are powerful tools for future gene/cell therapy for diabetes, pancreatic carcinoma and chronic pancreatitis. However, it is unclear where the pancreatic stem cells exist in adult pancreas. To identify the pancreatic stem cells, we have focused on a homeodomain-containing transcription factor, pancreatic/duodenal homeobox-1 (PDX-1), and an intermediate filament protein, nestin, which are important candidates of pancreatic stem cell markers. Methods and results: We investigated the immunohistological localization for PDX-1 and nestin in adult rat pancreas. PDX-1 staining was detected in small evaginations of the main pancreatic duct and in nuclei of islet cells. Nestin staining was also detected in small evaginations of the main duct, islets and spindle-shaped cells in the connective tissue around the main duct. These spindle-shaped cells were also positive for α-smooth muscle actin, the marker of myofibroblasts. Confocal laser microscopy study showed that nestin and PDX-1 are co-localized not only in a subset of cells in small evaginations of the main duct but also in a few cells in the islets. These cells of the main duct were also positive for the ductal cell marker, cytokeratin 19. Conclusion: Our results suggest that the double positive cells for PDX-1 and nestin might be important candidates for pancreatic stem cells in adult rats.

Decreased production of immunoglobulin M and A in autoimmune pancreatitis

BackgroundAutoimmune pancreatitis (AIP) is a rare type of chronic pancreatitis caused by an autoimmune abnormality. It is well known that high serum concentrations of IgG4 are helpful for making a diagnosis of AIP; however, it is unclear whether there are abnormalities in the production of other immunoglobulins in AIP.MethodsWe examined the immune condition of AIP patients before and after glucocorticoid treatment, focusing on serum levels of IgG, IgG4, IgM and IgA, and compared the results with those in other hepato-pancreatic diseases, such as autoimmune hepatitis, primary biliary cirrhosis, chronic pancreatitis and pancreatic carcinoma.ResultsIgM and IgA were decreased in patients with untreated AIP. IgM and IgG or IgG4 were negatively correlated in patients with AIP. The ratios of IgG to IgM and IgG to IgA in patients with AIP were significantly increased compared with the other diseases. The diagnostic sensitivity of IgG to IgM and IgG to IgA was 0.800 and 0.950, and the specificity of each ratio was 0.703 and 0.728, respectively, in the differentiation of AIP from the other diseases. IgM was not significantly changed after glucocorticoid treatment in the patients with AIP, while IgG, IgG4 and IgA decreased.ConclusionsThe ratios of IgG to IgM and IgG to IgA may serve as novel diagnostic markers to differentiate AIP from other hepato-pancreatic diseases. Furthermore, low concentrations of IgM and IgA may be involved in the pathogenesis of AIP.

Long-term Overexpression of Membrane Type-1 Matrix Metalloproteinase and Matrix Metalloproteinase-2 in Oleic Acid-induced Pancreatitis in Rats

Introduction The matrix metalloproteinase (MMP) plays important roles in extracellular matrix turnover. However, little is known about the roles of MMP-2 and type IV collagen, and the relationship between MMP-2 and membrane type-1 matrix metalloproteinase (MT1-MMP) during progressive destruction of acinar cells in pancreatitis. Aims and Methodology To examine the serial changes in the expression and activity of MMP-2 and expression of MT1-MMP and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in rats after induction of pancreatitis by intraductal infusion of oleic acid, and to determine protein concentrations by Western blot analysis and localization of type IV collagen by immunostaining. Results Gelatinolytic activity of pro-and active MMP-2 and concentrations of MT1-MMP protein, as determined by zymography and Western blot analysis, respectively, increased significantly from 6 hours to day 42 after intraductal infusion of oleic acid. TIMP-2 mRNA expression was significantly higher than that at time 0 throughout the study period, and gelatinolytic activity of active MMP-2 increased from day 3 to day 42. In addition, immunoreactivity for type IV collagen was detected as a discontinuous line along the basement membranes of ducts, vessels, tubular complexes, and acinar cells. Conclusion Our findings indicate that long-term increases of gelatinolytic activity of active MMP-2 cause continuous disorganization of type IV collagen in basement membranes during progressive atrophy of pancreatic gland in oleic acid–induced pancreatitis, and that MT1-MMP and TIMP-2 work as activating factors during proMMP-2 activation. Moreover, basement membranes disorganization in the sustentacula of acinar cells and duct epithelia seems to participate in continuous derangement of acinar cells and duct epithelium.