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.

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.

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.

A liver tolerates a portal antigen by generating CD11c+ cells, which select Fas ligand+ Th2 cells via apoptosis.

Administration of an antigen (Ag) per oral route leads to apoptosis of Ag-specific CD4(+) T cells and to development of Th2 cells expressing Fas ligand (FasL) in the liver. We determined whether presentation of an ingested Ag in the liver alone was enough to select these FasL(+)Th2 cells and explored how this selection was achieved in the liver. Ovalbumin (OVA) administered orally was colocalized with class II(+) cells in the periportal and parenchymal area of the liver. On coculture with naive OVA-specific CD4(+) T cells, hepatic CD11c(+) cells from mice fed OVA generated Ag-specific Th2 cells. This was achieved by apoptosis of CD4(+) T cells, decrease of interleukin 12 (IL-12) secretion, and increase of IL-18 secretion by the CD11c(+) cells. Addition of IL-12 to this coculture prevented apoptosis of the CD4(+) T cells, which was associated with up-modulation of IL-2 receptor beta chain expression. Administration of IL-12 to mice fed OVA prevented apoptosis of OVA-specific CD4(+) T cells in the liver. Moreover, adoptive transfer of hepatic CD11c(+) cells from mice fed OVA together with OVA-specific CD4(+) T cells led to development of Th2 cells as well as apoptosis of the transferred CD4(+) T cells in the lymph nodes of the recipient mice on immunization with OVA. In conclusion, presentation of an ingested Ag by hepatic CD11c(+) cells selects Th2 cells resistant to apoptosis in the liver, which is mediated in part by down-regulation of IL-12 secretion by the former cells.

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.

Experimental immune-mediated pancreatitis in neonatally thymectomized mice immunized with carbonic anhydrase II and lactoferrin.

We previously reported that autoantibodies against carbonic anhydrase II and lactoferrin are frequently identified in patients with autoimmune-related pancreatitis. To clarify the role of carbonic anhydrase II and lactoferrin, we created animal models of autoimmune pancreatitis by immunizing neonatally thymectomized mice with carbonic anhydrase II and lactoferrin and also by transferring immunized spleen cells to nude mice. Neonatally thymectomized BALB/c mice were immunized with carbonic anhydrase II or lactoferrin followed by three booster injections (n = 10 in each group). We transferred whole, CD4+, or CD8+ spleen cells prepared from immunized neonatally thymectomized mice to nude mice (n = 5 in each group). Gene expression of IFN-gamma and IL-4 was investigated using semiquantitative reverse transcription-polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining was used to examine apoptosis. In immunized neonatally thymectomized mice, the prevalence of inflammation was significantly higher in the pancreas. Inflammation was present in all mice receiving whole or CD4+ cells. There was no change in any of the mice receiving CD8+ cells or nonimmunized spleen cells. Carbonic anhydrase II or lactoferrin-immunized mice had apoptotic duct cells or acinar cells, respectively. Expression of the IFN-gamma gene was up-regulated in each group. Similar findings were observed in the salivary glands and liver. An immunologic mechanism against carbonic anhydrase II or lactoferrin is involved in the pathogenesis of these pancreatitis models, in which the effector cells are Th1-type CD4+ T cells.

New cytokine delivery system using gelatin microspheres containing interleukin-10 for experimental inflammatory bowel disease.

Interleukin (IL)-10 is an anti-inflammatory cytokine that suppresses the T helper 1 immune response and down-regulates macrophages and monocytes. The therapeutic effect of systemic administration of IL-10 for patients with inflammatory bowel disease, however, has not been satisfactory. We examined whether rectal administration of gelatin microspheres (GM) containing IL-10 (GM-IL-10) prevents colitis in IL-10-deficient (IL-10(-/-)) mice. GM-IL-10 and IL-10 alone were administered rectally. The colon was examined macroscopically and microscopically. IL-12 mRNA expression and CD40 expression in Mac-1-positive cells were also examined. Macroscopic and microscopic examination revealed marked improvement of colitis in IL-10(-/-) mice treated with GM-IL-10. mRNA expression of IL-12 in Mac-1-positive cells in GM-IL-10-treated mice was significantly decreased compared with that in the mice treated with IL-10 alone. Additionally, CD40 expression in Mac-1-positive cells in GM-IL-10-treated mice was decreased more prominently than in mice treated with IL-10 alone. The therapeutic effects of GM-IL-10 were associated with decreased expression of IL-12 mRNA and down-regulation of CD40 expression in Mac-1-positive cells. GM-IL-10 might be useful for treatment of patients with inflammatory bowel disease.