Masanori Koyabu

Analysis of regulatory T cells and IgG4-positive plasma cells among patients of IgG4-related sclerosing cholangitis and autoimmune liver diseases

ObjectivesPatients with autoimmune pancreatitis (AIP) characteristically show elevated serum levels of immunoglobulin G4 (IgG4) and abundant infiltration of IgG4-positive plasmacytes in the involved organs. The most common involved organ showing extrapancreatic lesions is the bile duct, which exhibits sclerosing cholangitis (SC). However, the role of IgG4 in the development of IgG4-related SC (IgG4-SC) remains unclear. To clarify the role of IgG4 in IgG4-SC, we have performed an immunohistochemical analysis of the bile duct.MethodsLaboratory and immunohistochemical findings of liver biopsy specimens obtained from patients with IgG4-SC, primary sclerosing cholangitis (PSC), autoimmune hepatitis (AIH), and primary biliary cirrhosis (PBC) were compared. The biopsy specimens were first stained with anti-IgG1, anti-IgG4, and anti-Foxp3 (forkhead box P3) antibodies, and the ratio of IgG4-, IgG1-, and Foxp3-positive cells, respectively, to infiltrated mononuclear cells (IgG4/Mono, IgG1/Mono, Foxp3/Mono) was assessed.ResultsThe ratio of IgG4/IgG1-positive plasma cells was significantly higher in specimens obtained from patients with IgG4-SC than in those from patients with PSC, AIH, and PBC. The Foxp3/Mono ratio in patients with PBC was significantly higher than that in patients with IgG4-SC and PSC. In patients with IgG4-SC, the number of Foxp3-positive cells was significantly correlated with the number of IgG4-positive cells; in the other patient groups, there was no correlation.ConclusionsThe IgG4/IgG1 ratio in the liver may be a useful marker for differential diagnosis of IgG4-SC and PSC. In IgG4-SC, abundant infiltration of regulatory T cells (Tregs) may affect the switching of B cells to IgG4-producing plasmacytes, and there is a possibility that the function of Tregs is different in IgG4-SC and other liver diseases (PSC, AIH, and PBC).

Involvement of inducible costimulator- and interleukin 10-positive regulatory T cells in the development of IgG4-related autoimmune pancreatitis.

Objectives: Immunoglobulin G4 (IgG4)-related autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are immunologic and histological abnormalities, including increased serum IgG4 levels and the infiltration of IgG4-positive plasmacytes. However, the role of IgG4 is unclear. Recently, regulatory T cells (Tregs) were reported to contribute to the development of various autoimmune diseases as well as in B-cell shifting to IgG4-producing plasmacytes. We studied Tregs in the pancreas and peripheral blood. Methods: We recruited 44 patients with IgG4-related AIP. For comparison, we recruited 37 patients with other pancreatic diseases and 27 healthy subjects as controls. We studied infiltrating cells in the pancreas by immunohistochemistry and analyzed inducible costimulator-positive Tregs and interleukin 10-positive Tregs in the peripheral blood by flow cytometry. Results: The ratio of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono) in AIP patients was significantly higher than in patients with alcoholic chronic pancreatitis. In AIP, Foxp3/Mono and IgG4/Mono were positively correlated. Inducible costimulator-positive Tregs were significantly higher in AIP patients than in the patients with other pancreatic diseases and the healthy control group. Interleukin 10-positive Tregs were significantly higher in AIP patients than in the healthy control group. Conclusions: Increased quantities of inducible costimulator-positive Tregs may influence IgG4 production in IgG4-related AIP.

IgG4 cholangiopathy – Current concept, diagnosis, and pathogenesis

IgG4 related cholangiopathy, a distinctive type of cholangitis of unknown origin, is characterized by increased serum levels of IgG4, massive infiltration of IgG4-positive plasma cells with storiform fibrosis and/or obliterative phlebitis in the thickened bile duct wall, and good response to steroids. Patients with IgG4-cholangiopathy are frequently associated with autoimmune pancreatitis; IgG4-cholangiopathy is recognized as a biliary manifestation of IgG4-related disease. This condition can be diagnosed by a combination of imaging, serology, histopathology, and steroid responsiveness; however, cholangiographic features are often difficult to differentiate from primary sclerosing cholangitis, pancreatic cancer, or cholangiocarcinoma. The Japanese clinical diagnostic criteria for IgG4-related sclerosing cholangitis established in 2012 are useful in the diagnosis of IgG4-cholangiopathy. Although the precise pathogenic mechanism remains unclear, the development of IgG4-cholangiopathy may involve: susceptible genetic factors, abnormal innate and acquired immunity, decreased naïve regulatory T cells, and specific B cell responses. Further studies on genetic backgrounds, disease specific antigens, and the role of IgG4 are necessary to clarify the pathogenesis.

Regulatory T Cells in Type 1 Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) is a newly recognized pancreatic disorder. Recently, International Consensus Diagnostic Criteria for AIP (ICDC) was published. In this ICDC, AIP was classified into Type 1 and Type 2. Patients with Type 1 AIP have several immunologic and histologic abnormalities specific to the disease, including increased levels of serum IgG4 and storiform fibrosis with infiltration of lymphocytes and IgG4-positive plasmacytes in the involved organs. Among the involved organs showing extrapancreatic lesions, the bile duct is the most common, exhibiting sclerosing cholangitis (IgG4-SC). However, the role of IgG4 is unclear. Recently, it has been reported that regulatory T cells (Tregs) are involved in both the development of various autoimmune diseases and the shift of B cells toward IgG4, producing plasmacytes. Our study showed that Tregs were increased in the pancreas with Type 1 AIP and IgG4-SC compared with control. In the patients with Type 1 AIP and IgG4-SC, the numbers of infiltrated Tregs were significantly positively correlated with IgG4-positive plasma cells. In Type 1 AIP, inducible costimulatory molecule (ICOS)+ and IL-10+ Tregs significantly increased compared with control groups. Our data suggest that increased quantities of ICOS+ Tregs may influence IgG4 production via IL-10 in Type 1 AIP.

Primary sclerosing cholangitis with elevated serum IgG4 levels and/or infiltration of abundant IgG4-positive plasma cells

Immunoglobin G4-related sclerosing cholangitis (IgG4-SC) is recognized as one of the systemic sclerosing diseases characterized by abundant IgG4-positive plasma cells with effective steroid therapy. On the other hand, primary sclerosing cholangitis (PSC), recognized as a sclerosing cholangitis of unknown origin without steroid efficacy, has been often clinically confused with IgG4-SC. To date, the prognosis of IgG4-SC is unclear, while the prognosis of PSC is well known to be poor. Therefore, it is clinically very important to be able to distinguish IgG4-SC from PSC. However, at the present time it still remains unclear whether PSC may sometimes be misdiagnosed as IgG4-SC or not. Herein, we report three rare cases of PSC with elevated serum IgG4 levels and/or an infiltration of abundant IgG4-positive plasma cells in the liver: a young male with ulcerative colitis (UC), and elderly female and a young female, each with elevated serum IgG4 levels. The first two patients showed infiltration of abundant IgG4-positive plasma cells in the portal area of the liver without response to steroid therapy. From our experiences, we emphasize that some patients with PSC, who do not respond to steroid therapy, show elevated serum IgG4 levels and/or infiltration of abundant IgG4-positive plasma cells, although the mechanism still remains unclear.

The effective therapy of cyclosporine A with drug delivery system in experimental colitis

Cyclosporine A (CyA) is a useful immunosuppressive agent for steroid-dependent or steroid-refractory ulcerative colitis. However, side effects have been reported in clinical trials of ulcerative colitis treated with CyA. Biodegradable microspheres (MS) have been investigated as drug delivery system. We evaluated the effect of a drug delivery system with poly(d,l-lactic acid)-MS containing CyA. Colitis was induced in C57BL/6 mice by 3% dextran sulfate sodium (DSS). Mice with DSS-induced colitis were treated with oral administration of CyA or CyA-MS: CyA (0.2 mg/kg/day)-MS; CyA (2 mg/kg/kg)-MS). Serum levels of CyA were significantly less elevated after oral administration of CyA (2 mg/kg/day)-MS compared with CyA (2 mg/kg/day) (CyA (2 mg/kg/day), 44.7 ± 0.8 ng/ml; CyA (2 mg/kg/day)-MS, 7.7 ± 1.3 ng/ml). The body weight at day 10 was significantly recovered in the mice treated with CyA (0.2 mg/kg/day)-MS and CyA (2 mg/kg/day)-MS compared with CyA (0). The histological score and myeloperoxidase activity in the mice treated with CyA-MS was significantly lower than CyA (0). Gene expressions of interleukin-1β (IL-1β), IL-6, and CXCL1 in the mice treated with CyA (0.2 mg/kg/day)-MS and CyA (2 mg/kg/day)-MS were downregulated compared with CyA (0)-MS. CyA-MS might be possible to treat ulcerative colitis effectively by decreasing the total dosage without the elevation of the serum level or the side effects of CyA.

Possible Involvement of Foxp3(+) Regulatory T Cells in the Development of Immune-Mediated Pancreatitis in MRL/Mp Mice Treated with Polyinosinic:Polycytidylic Acid.

Objectives. This study was conducted to clarify whether or not Tregs are involved in the development of immune-mediated pancreatitis in MRL/Mp mice as an AIP (autoimmune pancreatitis) model, in order to understand more clearly the pathogenic mechanism of AIP. Methods. We compared the immunohistochemical features of pancreatic forkhead box P3 (Foxp3) in the administration of poly I:C in MRL/Mp mice and two types of control mice (BALB/c and C57BL/6). As a contrast, we analyzed three mouse models of pancreatitis without autoimmune mechanism (Cerulein-, Ligation-, and Ligation + Cerulein-treated mice). After staining these specimens, we compared the ratios of Foxp3-positive cells to infiltrated mononuclear cells (Foxp3/Mono). Results. Our immunohistochemical study of Foxp3 revealed that the infiltration of Foxp3-positive cells increased in poly I:C-treated MRL/Mp mice. The histopathological score of pancreatitis showed no difference among poly I:C-treated MRL/Mp, Ligation-, and Ligation + Cerulein-treated mice; however, the Foxp3/Mono ratio in poly I:C-treated MRL/Mp mice was significantly increased compared with Ligation- and Ligation + Cerulein-treated mice. Conclusions. MRL/Mp mice treated with poly I:C showed early development of pancreatitis with abundant infiltration of Foxp3-positive cells. There may be a possibility that Tregs are involved in the development of pancreatitis in these mice.

Techniques of ERCP with a conventional endoscope in pancreatoduodenectomy anatomy

both reported success rates in excess of 80%. A third study assessed removable metal stenting in older patients with a median age of 81 years (range, 68-95 years) in whom medical comorbidities added to procedural difficulty. Finally, Everson et al used metal stents in 12 cases of irreversible coagulopathy, which limited the use of advanced extraction techniques. In that study, only 3 of the successful cases required mechanical lithotripsy for stone removal. Prospective studies would be valuable to further assess the role of removable metal stenting in the treatment of CBD stones. However, current data indicate satisfactory success rates and acceptable safety profiles in patients with complex stone disease.

M1398 Possible Role of Regulatory T Cells in Producing IgG4 in the Involved Organs With Autoimmune Pancreatitis

Background & Aim: Autoimmune pancreatitis (AIP) is a newly recognized pancreatic disorder. Patients with AIP have several immunologic and histologic abnormalities specific to the disease, including increased levels of serum IgG4 and storiform fibrosis with infiltration of lymphocytes and IgG4-positive plasmacytes in the involved organs. Among the involved organs showing extrapancreatic lesions, bile duct is the most common, exhibiting sclerosing cholangitis (IgG4-SC). However, the role of IgG4 is unclear in the pancreas and involved organs. Recently, it has been reported that regulatory T cells (Tregs) are involved in both the development of various autoimmune diseases and the shift of B-cell toward IgG4 producing plasmacytes. For this report, we analyzed the infiltrated Tregs and IgG4-positive plasma cells in the pancreas and liver by immunohistochemistory and IL-10 producing Tregs by flowcytometry to clarify the role of IgG4 and Tregs in AIP. Subjects and Methods: The 6 AIP patients were obtained from Kansai Medical University. 6 cases of chronic pancreatitis were served as disease control. Hepatic tissue was sectioned from 16 IgG4-SC patients and 26 patients with PSC. All AIP and 14 IgG4-SC patients were diagnosed by Asian criteria. One IgG4-SC patient was diagnosed by Mayo (HISORt) criteria and other was diagnosed by biopsy. We studied infiltrating IgG4-positive cells and Tregs in the pancreas and liver by immunohistochemistry. IL-10 producing Tregs were analyzed from peripheral blood by flow cytometry. Results: In the pancreas, Tregs were increased in the pancreas with AIP (24.6±18.0 cells/HPF) compared with controls (5.1±4.3 cells/HPF). The numbers of IgG4positive plasma cells were significantly higher in AIP (18.6±10.3 cells/HPF) than in controls (1.1±0.7 cells/HPF). However, there were no significant differences in CD3+, CD4+, or CD79+ cells between the AIP (CD3: 63.1±27.1, CD4: 48.2±32.9, CD79: 24.1±9.2 cells/ HPF) and control sections (CD3: 73.6±37.1, CD4: 34.0±23.8, CD79: 14.5±14.6 cells/HPF). In the liver, the numbers of IgG4-positive plasma cells were significantly higher in IgG4SC (16.6±7.7 cells/HPF) than in PSC (4.0±0.7 cells/HPF). Tregs in the patients with IgG4SC (5.4±1.5 cells/HPF) were also significantly increased compared with PSC (2.0±0.3 cells/ HPF). In the patients with IgG4-SC, the numbers of infiltrated Tregs were significantly positively correlated with IgG4-positive plasma cells (R=0.75). In patients with the untreated AIP, IL-10 producing Tregs and IgG4 also positively correlated (R=0.53). Conclusion: In AIP, abundant infiltration of Tregs may affect the switching of B cells to IgG4-producing plasmacytes.

S1279 Analysis of Pancreatic and Peripheral Regulatory T Cells in Patients with Autoimmune Pancreatitis

Background : Auto-immune pancreatitis (AIP) should be suspected in patients with unexplained pancreatitis. AIP is recognized according to HISORt criteria (including histology, pancreatic imaging, elevated serum IgG4 level and response to steroid therapy) as recently proposed by the Mayo Clinic. These criteria are highly selective and often difficult to fulfill. Previous studies suggested that carbonic anhydrase (CA) II is a target antigen in AIP. The aim of the present study was to evaluate the prevalence of serum antibodies to CA II in patients with AIP. Patients and Methods : 21 patients with AIP fulfilling HISORt criteria were retrospectively studied. An enzyme-linked immunosorbent assay was used to quantify serum antibodies to CA II. The concentrations of serum IgG4were measured by nephelemetry with a cut-off of 2 g/L. Thirty two patients with pancreatic adenocarcinoma or non-auto immune pancreatitis, and 96 healthy subjects served as controls. Results : 21 patients (13 males), median age 37 years [21-70] had AIP, histologically defined (57%) or with typical pancreatic imaging and response to steroids (43%). Pancreatic imaging revealed mass or irregular ducts in 71% and 48% of the cases, respectively. Four patients had ulcerative colitis. Serum IgG4 were elevated in 1 patient (5%). Serum antibodies to CA II were present in 2 patients (10%). Both had a pancreatic mass, and underwent pancreaticoduodenectomy. They developed post-operative, steroid-sensitive cholangitis. Serum antibodies to CA II were present in 3 healthy controls (3%) and in 1 subject with alcoholic chronic pancreatitis (3%). No patient with pancreatic cancer had serum antibodies to CA II. The sensitivity, specificity, positive and negative predictive values of antibodies to CA II for the diagnosis of AIP were 10%, 97%, 33%, et 87%, respectively. Conclusion : 10% of patients with well-defined AIP have serum antibodies to CA II in an European center. This prevalence is in accordance with previously reported studies, but lower than in Japanese series. The high specificity of CA II antibodies might be helpful for AIP diagnosis, particularly when serum IgG4 is normal.