Human thioredoxin-1 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor production.

BACKGROUND & AIMS: Thioredoxin-1 (TRX) is a small multifunctional protein with antioxidative and redox-regulating functions. In this study, we investigated the significance of TRX in patients with inflammatory bowel disease (IBD) and the ability and mechanism to ameliorate experimental colitis. METHODS: Serum TRX and macrophage migration inhibitory factor (MIF) levels were measured in patients with IBD. The effects of TRX were evaluated in a dextran sulfate sodium (DSS)-induced colitis model by comparing TRX-overexpressing transgenic (TRX-TG) and control mice. We further evaluated the effect of recombinant human TRX (rhTRX) administration on DSS-induced colitis and colonic inflammation of interleukin (IL)-10 knockout (IL-10 KO) mice. Colonic inflammation was examined clinically and histologically. Proinflammatory cytokine levels were examined in colonic tissues, and MIF levels were measured in colonic tissues and sera in mice. The effect of TRX on MIF production was also analyzed in vitro. RESULTS: Serum TRX and MIF levels were significantly higher in patients with IBD than normal controls, and TRX levels correlated with disease activity. TRX significantly ameliorated DSS-induced colitis and colonic inflammation of IL-10 KO mice. Increase of tumor necrosis factor-alpha and interferon-gamma in colonic tissues was significantly suppressed in TRX-TG mice compared with wild-type mice. MIF levels in colonic tissues and sera were significantly lower in TRX-TG mice than in wild-type mice, irrespective of DSS administration. Anti-TRX treatment exacerbated DSS-induced colitis. In vitro studies demonstrated that rhTRX suppressed MIF production in human monocyte cells. CONCLUSIONS: TRX might have a potential as a novel therapeutic agent for the treatment of IBD.

Overexpression of redox-active protein thioredoxin-1 prevents development of chronic pancreatitis in mice.

Chronic pancreatitis (CP) is considered to result from repetitive pancreatic injury, and sustained production of various proinflammatory cytokines and chemokines are closely involved in its pathogenesis. Monocyte chemoattractant protein 1 (MCP-1), a member of the CC chemokine family, is believed to contribute to the progression of CP through monocyte/macrophage recruitment. This study aimed to clarify the protective role of thioredoxin-1 (TRX-1), a redox-regulating protein with antioxidative activity, in MCP-1 production and pancreatic fibrosis using a CP model in transgenic mice overexpressing TRX-1 (TRX-1-TG mice) and wildtype C57BL/6 mice. Experimental CP was induced by repeated administration of cerulein and lipopolysaccharide for 6 weeks. In TRX-1-TG mice, pancreatic atrophy was ameliorated, and histologically detectable inflammatory cell infiltration, glandular atrophy, and pseudotubular complex formation were suppressed. Overexpression of TRX-1 also attenuated pancreatic fibrosis and suppressed the activation of pancreatic stellate cells. Serum levels of MCP-1 and pancreatic expression of transforming growth factor-beta, platelet-derived growth factor, and MCP-1 were reduced in TRX-1-TG mice compared with levels in wild-type mice. Overexpression of TRX-1 also reduced H(2)O(2)-induced MCP-1 production in isolated pancreatic acinar cells. These results indicate that TRX-1 can potentially attenuate pancreatic fibrosis via the suppression of oxidative stress and MCP-1-mediated chronic inflammation.

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.

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.

Protective roles of redox-active protein thioredoxin-1 for severe acute pancreatitis.

Severe acute pancreatitis is a disease with high mortality, and infiltration of inflammatory cells and reactive oxygen species have a crucial role in the pathophysiology of this disease. Thioredoxin-1 (TRX-1) is an endogenous redox-active multifunctional protein with antioxidant and anti-inflammatory effects. TRX-1 is induced in various inflammatory conditions and shows cytoprotective effects. The aim of the present study was to clarify the protective roles of TRX-1 in the host defense mechanism against severe acute pancreatitis. Experimental acute pancreatitis was induced by intraperitoneal administration of cerulein, a CCK analog, and aggravated by lipopolysaccharide injection in transgenic mice overexpressing human TRX-1 (hTRX-1) and control C57BL/6 mice. Transgenic overexpression of hTRX-1 strikingly attenuated the severity of experimental acute pancreatitis. TRX-1 overexpression suppressed neutrophil infiltration as determined by myeloperoxidase activity, oxidative stress as determined by malondialdehyde concentration, and cytoplasmic degradation of inhibitor of kappaB-alpha, thereby suppressing proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6; a neutrophil chemoattractant, keratinocyte-derived chemokine; and inducible nitric oxide synthase in the pancreas. Administration of recombinant hTRX-1 also suppressed neutrophil infiltration, reduced the inflammation of the pancreas and the lung, and improved the mortality rate. The present study suggests that TRX-1 has potent antioxidant and anti-inflammatory actions in experimental acute pancreatitis and might be a new therapeutic strategy to improve the prognosis of severe acute pancreatitis.

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-1beta and tumor necrosis factor-alpha 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.

Therapeutic effects of rectal administration of basic fibroblast growth factor on experimental murine colitis.

BACKGROUND & AIMS: Basic fibroblast growth factor (bFGF) is a promising therapeutic agent for various diseases. It remains unclear, however, whether bFGF is effective for the treatment of inflammatory bowel disease. The aim of this study was to examine the efficacy of bFGF on 2 experimental murine colitis models and to investigate its molecular mechanisms. METHODS: We evaluated the effects of human recombinant bFGF (hrbFGF) on mice with dextran sulfate sodium (DSS)-induced colitis and mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis as well as normal mice. Body weight, survival rate, and histologic findings of the colonic tissues were examined. Gene expression of tumor necrosis factor (TNF)-alpha, cyclooxygenase (COX)-2, transforming growth factor (TGF)-beta, mucin 2 (MUC2), intestinal trefoil factor (ITF), and vascular endothelial growth factor (VEGF) in the colonic tissues was determined. The proliferation activity of hrbFGF on the colonic epithelium was evaluated by immunohistochemistry. RESULTS: Rectal administration of hrbFGF ameliorated DSS-induced colitis in a dose-dependent manner. Gene expression of TNF-alpha was significantly reduced in the colonic tissues of mice with DSS-induced colitis treated with hrbFGF, whereas MUC2 and ITF messenger RNA expression was up-regulated. Rectal administration of hrbFGF significantly improved the survival rate of mice with TNBS-induced colitis and partially ameliorated colitis. hrbFGF significantly increased the number of Ki-67-positive cells in the colonic epithelium of normal mice, and up-regulated the gene expression of COX-2, TGF-beta, MUC2, ITF, and VEGF in the colonic tissues. CONCLUSIONS: Rectal administration of bFGF might be a promising option for the treatment of inflammatory bowel disease.

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.

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 10(8) 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-X(L) 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.

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

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-CD8alpha (lymphoid DC marker) or anti-CD11b (myeloid DC marker) and were examined with a confocal microscope. Expression of macrophage inflammatory protein 3alpha (MIP-3alpha), 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-3alpha gene expression was upregulated in the infected nTx group, and the immunohistochemistry analysis revealed MIP-3alpha-positive epithelial cells. These data suggest that H. pylori infection upregulates MIP-3alpha 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.

Interaction of Helicobacter pylori-induced follicular gastritis and autoimmune gastritis in BALB/c mice with post-thymectomy autoimmune gastritis.

BACKGROUND: There is still controversy about the relationship between Helicobacter pylori infection and autoimmune gastritis. The aim of this study was to clarify whether or not H. pylori infection interacts with the development of autoimmune gastritis. METHODS: Neonatally thymectomized BALB/c mice with autoimmune gastritis received orally administered H. pylori and were examined histologically and serologically. The T-helper (Th)1/Th2 immune balance in the microenvironment of the stomach was evaluated by reverse-transcriptase-polymerase chain reaction (RT-PCR) for interferon (IFN)-gamma and interleukin (IL)-4. RESULTS: Uninfected mice showed disappearance of parietal cells, and upregulation of IFN-Gamma, but no germinal center formation. The infected neonatally thymectomized mice showed follicular gastritis, preserved parietal cells, decreased serum anti-parietal antibodies, and upregulation of IL-4 and IFN-gamma. CONCLUSIONS: H. pylori infection changes the microenvironment of the gastric mucosa by inducing a Th2 immune response in addition to a Th1 response, and regresses autoimmune gastritis in neonatally thymectomized BALB/c mice.