Masanori Asada

Identification of a novel autoantibody against pancreatic secretory trypsin inhibitor in patients with autoimmune pancreatitis.

Objectives: Although autoimmune pancreatitis (AIP) has been recently recognized as a new disease entity of chronic pancreatitis, the clinical diagnosis of the disease remains disputed. Autoantibodies against carbonic anhydrase II and lactoferrin are detected in most patients with AIP, but not in about 10%. We undertook this study to determine whether additional autoantibodies are present in the serum level of AIP patients. Methods: We recruited 26 patients with AIP for the study. For comparison, we also recruited 53 patients with various pancreatic diseases and 12 healthy subjects. We immunoscreened human pancreatic cDNA library using patients sera. Positive clones were analyzed by DNA sequencing and were constructed into a pGEX-4T-1 expression vector. The recombinant proteins were used as antigens in enzyme-linked immunosorbent assay to screen the subjects sera for autoantibodies. Results: We cloned a cDNA encoding the pancreatic secretory trypsin inhibitor (PSTI). Among 26 patients with AIP, autoantibodies against PSTI were significantly positive in 11 (42.3%) by western blotting and in 8 (30.8%) by enzyme-linked immunosorbent assay, respectively. However, none of control subjects was positive for anti-PSTI antibodies. Conclusions: These findings suggest that PSTI may be related to the pathogenesis of AIP, and autoantibodies against PSTI can be a useful diagnostic marker for the disease.Abbreviations: AIP - autoimmune pancreatitis, PSTI - pancreatic secretory trypsin inhibitor, CA-II - carbonic anhydrase II, LF - lactoferrin, TBST - Tris-buffered saline containing 0.05% Tween 20, SDS-PAGE - sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ELISA-enzyme-linked immunosorbent assay, OD - optical density, ANA - antinuclear antibody, AMA - antimitochondrial antibody, RF - rheumatoid factor

Endoscopic management of biliary strictures after duct-to-duct biliary reconstruction in right-lobe living-donor liver transplantation

Background. The aims of this study were to characterize the features of the biliary strictures that occur after duct-to-duct biliary reconstruction during right-lobe living-donor liver transplantation (LDLT) and to evaluate the feasibility of correcting such stricture endoscopically by inserting an “inside stent,” that is, a short internal stent, above the sphincter of Oddi. Methods. Biliary stricture occurred in 26 (35.6%) of 73 consecutive patients who underwent right-lobe LDLT with duct-to-duct biliary reconstruction from July 1999 through October 2001 and survived for more than 3 months. Of the 26 patients who had biliary stricture, 22 were referred for endoscopic retrograde cholangiography (ERC) and 4 for percutaneous cholangiography. Results. ERC disclosed biliary stricture in 19 (86.4%) of the 22 patients who underwent the procedure. One patient had an unbranched stricture, 16 had a fork-shaped stricture, 1 had a trident-shaped stricture, and 1 had a stricture with more than three branches. Fourteen (73.7%) of the patients with strictures were treated endoscopically by inserting inside stents ranging from 7 F to 12 F in size, three underwent a Roux-en-Y hepaticojejunostomy to repair their stricture, and two were closely observed as outpatients. Of the 14 patients who were treated with the inside-stent, only 1 had acute cholangitis immediately after the procedure and underwent a Roux-en-Y hepaticojejunostomy. The other 13 patients who were treated with the inside stent have not required surgical repair for as long as an average of 586 days. Conclusion. Endoscopic placement of an inside stent is useful for treating biliary strictures in patients who have undergone right-lobe LDLT with duct-to-duct reconstruction.

A Case of Autoimmune Pancreatitis Associated with Sclerosing Cholangitis, Retroperitoneal Fibrosis and Sjögren’s Syndrome

We report a very rare case of autoimmune pancreatitis (AIP) associated with sclerosing cholangitis, retroperitoneal fibrosis and Sjögren’s syndrome. The patient had an enlarged pancreas, and autoantibodies were detected in the serum. Serum IgG and IgG4 concentrations were also elevated. Endoscopic retrograde cholangiopancreatography revealed an irregular narrowing of the main pancreatic duct from the head to the body and sclerotic change in the intrapancreatic common bile duct, which later extended to the intrahepatic bile ducts. In addition, histological examination of the liver revealed lymphocytic sclerosis around the bile ducts, similar to the histology in the pancreas of AIP. Retroperitoneal tumors were diagnosed as retroperitoneal fibrosis by histological examination. Serological and functional abnormalities suggestive of Sjögren’s syndrome were detected, and histological findings of the lip were compatible with Sjögren’s syndrome. Immunohistochemistry of each lesion disclosed that most of the infiltrating lymphocytes were T cells with similar levels of both CD4+ and CD8+ cells. Moreover, some of the infiltrating plasma cells were positive for anti-IgG4 monoclonal antibody. These diseases were dramatically improved by steroid therapy. Although the pathophysiology of AIP is still unclear, the present case suggests a common pathophysiological mechanism for AIP, sclerosing cholangitis, retroperitoneal fibrosis and Sjögren’s syndrome.

Involvement of myeloid dendritic cells in the development of gastric secondary lymphoid follicles in Helicobacter pylori-infected neonatally thymectomized BALB/c mice

ABSTRACT We previously described an animal model of Helicobacter pylori-induced follicular gastritis in neonatally thymectomized (nTx) mice. However, it is still not clear whether antigen-presenting dendritic cells (DCs) in the stomach have a role in the development of secondary follicles in H. pylori-infected nTx mice. We investigated the distribution of DC subsets using this model and examined their roles. To identify lymphoid and myeloid DCs, sections were stained with anti-CD11c (pan-DC marker) in combination with anti-CD8α (lymphoid DC marker) or anti-CD11b (myeloid DC marker) and were examined with a confocal microscope. Expression of macrophage inflammatory protein 3α (MIP-3α), which chemoattracts immature DCs, was analyzed by real-time PCR and immunohistochemistry. Follicular dendritic cells (FDCs) were stained with anti-SKY28 antibodies. In noninfected nTx mice, a few myeloid and lymphoid DCs were observed in the bottom portion of the lamina propria, whereas in H. pylori-infected nTx mice, there was an increased influx of myeloid DCs throughout the lamina propria. FDC staining was also observed in the stomachs of members of the infected group. MIP-3α gene expression was upregulated in the infected nTx group, and the immunohistochemistry analysis revealed MIP-3α-positive epithelial cells. These data suggest that H. pylori infection upregulates MIP-3α gene expression in gastric epithelial cells and induces an influx of myeloid DCs in the lamina propria of the gastric mucosa in nTx mice. Myeloid DCs and FDCs might contribute to the development of gastric secondary lymphoid follicles in H. pylori-infected nTx mice.

Specific antibodies against recombinant protein of insertion element 900 of Mycobacterium avium subspecies paratuberculosis in Japanese patients with Crohn's disease

Background: Mycobacterial avium subspecies paratuberculosis (MAP) infection has been hypothesized as an etiological factor of Crohns disease (CD). However, the involvement of MAP in the pathophysiology of CD is controversial. The aim of this study is to investigate whether MAP is involved in the pathogenesis of CD with the glutathione S‐transferase fusion recombinant protein encoding a portion of insertion element (IS) 900 (IS900‐GST), which is specific for MAP. Methods: Serum samples from the patients with CD (n = 50), ulcerative colitis (n = 40), colonic tuberculosis (n = 20), and non‐IBD controls (n = 44), were applied for solid‐phase enzyme‐linked immunosorbent assay (ELISA) to detect antibodies against MAP and Saccharomyces cerevisiae. IS900‐GST, which was made by the pGST‐4T‐2 vector inserted with polymerase chain reaction‐amplified IS900DNA, was used as an antigen of MAP. Moreover, we studied the relationship between antibodies against IS900‐GST and clinical characteristics. Results: ELISA showed that the serum level of immunoglobulin G and immunoglobulin A antibodies against IS900‐GST (anti‐IS900) in patients with CD were significantly higher than those with ulcerative colitis, colonic tuberculosis, and control subjects. The levels of anti‐IS900 tended to be higher in CD patients with small intestinal involvement than with colonic involvement alone. Anti‐IS900 in patients with penetrating‐ and stricture‐type CD was significantly higher than with inflammatory‐type CD. Furthermore, a negative correlation was found between the titer of anti‐IS900 and disease duration. Anti‐IS900 was not associated with surgical treatment nor was it associated with the use of immunosuppressants. No significant correlation was observed between the serum levels of anti‐IS900 and anti‐S cerevisiae antibody. Conclusions: This is the first demonstration of the ELISA system of detecting antibodies against IS900 in IBD patients. MAP could be involved in the pathophysiology of Japanese patients with CD.

Helicobacter felis-induced gastritis was suppressed in mice overexpressing thioredoxin-1

Thioredoxin-1 (TRX-1) is a redox-active protein involved in scavenging reactive oxygen species and regulating redox-sensitive transcription factors. TRX-1 is induced in various inflammatory conditions and shows cytoprotective action. We investigated the roles of TRX-1 in the host defense mechanism against Helicobacter felis (H. felis) infection. Transgenic (TG) mice overexpressing human TRX-1 and wild-type (WT) mice were orally inoculated with H. felis. After 2 months, histology, oxidative damage, and gene expression of several cytokines, including macrophage inflammatory protein-2 (MIP-2), a murine equivalent to interleukin (IL)-8, in the gastric mucosa were investigated. Furthermore, the effects of TRX-1 on oxidative stress and neutrophil migration were studied both in vivo and in vitro. The gastric mucosa was thickened in H. felis-infected WT mice, but not in infected TRX-1-TG mice. Histologically, all H. felis-infected WT mice developed moderate-to-severe gastritis, whereas the development of gastritis was significantly suppressed in infected TRX-1-TG mice. Oxidative damage markers, 8-hydroxy-2′-deoxyguanosine and malondialdehyde, increased in the stomach of infected WT mice, but not TRX-1-TG mice. Upregulation of IL-1β and tumor necrosis factor-α gene expression in H. felis-infected TRX-1-TG mice was significantly lower than in WT mice. However, upregulation of MIP-2 and IL-7 was not different between the two groups. TRX-1 suppressed oxidative cytotoxicity and DNA damage, and inhibited neutrophil migration both in vivo and in vitro. The present study suggests that overexpression of TRX-1 suppresses H. felis-induced gastritis by inhibiting chemotaxis of neutrophils and reducing oxidative stress.

Analysis of humoral immune response in experimental autoimmune pancreatitis in mice.

Objectives: To study the autoimmune response in MRL/Mp mice, which spontaneously develop pancreatitis in the exocrine pancreatic tissue. Methods: Six-week-old female mice were injected intraperitoneally with polyinosinic polycytidylic acid at a dose of 5 mg/kg of body weight twice a week for up to 12 weeks. The mice were serially killed, and the severity of their pancreatitis was graded with a histological scoring system. Immunohistological examinations were performed, and the serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Results: The administration of polyinosinic polycytidylic acid accelerated the development of pancreatitis, with abundant infiltration of B220+ B cells and CD138+ plasmacytes. Various autoantibodies directed against autoantigens, including carbonic anhydrase II and lactoferrin, were detected but none against glutamic acid decarboxylase. Of these, autoantibodies directed against the pancreatic secretory trypsin inhibitor (PSTI; 91.7%) were more prevalent than those against carbonic anhydrase II (33.3%) or lactoferrin (45.8%). Determination of the epitope of the anti-PSTI antibody showed that most immunoreactivity was directed at the site on PSTI that is active in the suppression of trypsin activity. Conclusions: The autoimmune response to PSTI protein may induce a failure of PSTI activity, resulting in the activation of trypsinogen and the subsequent disease progression.

Immunogenetic analysis of gastric MALT lymphoma-like lesions induced by Helicobacter pylori infection in neonatally thymectomized mice

Most gastric mucosa-associated lymphoid tissue (MALT) lymphomas are caused by Helicobacter pylori (H. pylori) infection. We previously reported that acquired lymphoid follicles with germinal centers were induced by H. pylori infection in neonatally thymectomized (nTx) mice. In the present study, we developed gastric MALT lymphoma-like lesions in nTx mice by long-term H. pylori infection, and performed immunogenetic analyses. BALB/c mice were thymectomized on the 3rd day after birth. At 6 weeks of age, mice were orally infected with 108 H. pylori and serially killed 2, 4, 6, and 12 months later. Normal BALB/c and noninfected nTx mice served as controls. Follicle formation occurred after 2 months of H. pylori infection in the nTx mice. Follicle formation and infiltration of intraepithelial lymphocytes progressed in a time-dependent manner. Lymphoepithelial lesions, a characteristic feature of MALT lymphoma, also occurred in a time-dependent manner (100% at 12 months). Serum immunoelectrophoresis revealed a monoclonal band (M-protein) in 30% (3/10) of mice 6 months after infection. M-protein-positive mice had amplification of one or two IgM and/or IgG heavy-chain genes in the gastric B lymphocytes, as determined with polymerase chain reaction, suggesting mono- or oligoclonality. Overexpression of Bcl-XL protein was immunohistologically observed in the infiltrating B lymphocytes and in some follicular B lymphocytes in 80% (8/10) of the cases at 12 months. Thus, H. pylori infection is involved in the development of gastric MALT lymphoma-like lesions in nTx mice. Our mouse model is useful for clarifying the pathogenetic mechanism of gastric MALT lymphoma by H. pylori infection.

Clinical significance of serum thioredoxin 1 levels in patients with acute pancreatitis

Objective: Thioredoxin 1 (TRX-1), a redox-regulating protein with antioxidant activity, is induced by oxidative stress, and serum TRX-1 levels are recognized as an oxidative-stress marker. The aim of this study was to clarify the clinical significance of serum TRX-1 levels in patients with acute pancreatitis (AP) and evaluate the usefulness of this measurement in assessing disease severity. Methods: Serum TRX-1 levels were determined on admission in 18 patients with severe AP and 36 patients with mild AP. We also investigated the relationship between serum TRX-1 levels and clinical and laboratory data. Results: The median serum TRX-1 levels on admission were 54.9 ng/mL in mild AP and 118.8 ng/mL in severe AP. When the cutoff value for TRX-1 in predicting severe AP was determined to be 100 ng/mL, its sensitivity, specificity, and accuracy were 83.3%, 94.4%, and 90.7%, respectively. A significant correlation was observed between serum TRX-1 levels and Ranson score (r = 0.674), C-reactive protein (r = 0.718), interleukin 6 (r = 0.712), leukocyte count (r = 0.642), and serum amylase (r = 0.436). Conclusions: Serum TRX-1 levels significantly correlate with AP severity. TRX-1 should constitute a reliable oxidative-stress marker for the evaluation of AP severity in relation to oxidative stress.

Inhibitory effects of Helicobacter pylori infection on murine autoimmune gastritis

Background and aim: Long term Helicobacter pylori infection leads to atrophic gastritis but the relation between H pylori infection and autoimmune related atrophic gastritis (AIG) remains unclear. We studied the effects of H pylori infection on the pathophysiology of AIG in mice. Materials and methods: BALB/c nu/nu mice (n=40) with or without H pylori infection received splenocytes from neonatally thymectomised mice to induce AIG. Half of the mice were orally infected with H pylori prior to AIG induction. Histological findings, and local and systemic immune responses were serially evaluated. Results: Two and six months after transfer, parietal cells in uninfected mice were depleted while those in infected mice were well preserved. The degree of gland atrophy (p<0.01), hyperplasia (p<0.01), gastric pH (p<0.05), and serum gastrin levels of infected mice were significantly lower than those of uninfected mice. Serum antiparietal cell antibody levels gradually decreased in infected mice, and were significantly lower than those of uninfected mice at six months (p<0.05). Real time polymerase chain reaction studies revealed significantly higher interleukin 4 (p<0.05) and transforming growth factor β (p<0.05) gene expression in the gastric mucosa in infected mice than in uninfected mice at both two and six months after AIG induction. Conclusions:H pylori infection inhibited the development of AIG in mice. Th2-type immune responses and transforming growth factor β in the gastric microenvironment might be involved in the inhibitory effects of H pylori infection on the development of AIG, in which Th1-type responses have an important role.