P-selectin-dependent monocyte recruitment through platelet interaction in intestinal microvessels of LPS-treated mice.

BACKGROUND: Although platelets or monocytes are thought to be involved in intestinal inflammation, there has been no report on whether platelets can modulate monocyte recruitment in intestinal microvessels. The objective of this study was to determine whether blockade of platelet adhesion attenuates monocyte recruitment in inflamed murine intestinal microvessels. METHODS: Monocytes and platelet-rich plasma were obtained from C57B6/J mice. Interaction of monocytes and platelets with intestinal microvessels was observed under an intravital microscope. Lipopolysaccharide (LPS) was administered intraperitoneally. The effects of anti-P-selectin or anti-platelets antibody treatments or phosphodiesterase (PDE) inhibitors (PDE-3 and PDE-2/4 inhibitor) treatments were also studied. RESULTS: LPS-treatment increased the rolling and adhesion of both platelets and monocytes. Pretreatment with an anti-P-selectin antibody inhibited the increased platelet adhesion to venular walls and also attenuated the monocyte adhesion. A PDE-2/4 inhibitor (ibuzilast) also ameliorated both platelet and monocyte adhesion. A PDE-3 inhibitor (cilostazol) ameliorated only monocyte adhesion without directly affecting the adhesion of platelets to microvessels. CONCLUSIONS: We observed inhibition of platelets adhesion attenuated monocytes recruitment in intestinal microvessels. Attenuation of LPS induced monocyte adhesion by a specific PDE-3 inhibitor suggests that P-selectin on activated platelets may play an important role through monocyte and platelet interaction.

Omega-3 fatty acids exacerbate DSS-induced colitis through decreased adiponectin in colonic subepithelial myofibroblasts.

BACKGROUND: Although the immunoregulatory effects of omega-3 fatty acid and adiponectin have been postulated, their role in intestinal inflammation is controversial. The aim of this study was to determine whether dietary fat intake influences activity of colonic inflammation through modulating this system. METHODS: C57BL/6 mice received dextran sulfate sodium for induction of colitis. Mice were fed a control diet, omega-3 fat-rich diet, omega-6 fat-rich diet, or saturated fat-rich diet. Some mice were administered a peroxisome proliferator activated receptor-gamma; agonist, pioglitazone. Messenger RNA expression of adiponectin and its receptors were analyzed. Adiponectin expression in colonic mucosa of ulcerative colitis patients was also analyzed. RESULTS: The receptors for adiponectin were found to be ubiquitously expressed in epithelial cells, intraepithelial lymphocytes, lamina proprial mononuclear cells, and subepithelial myofibroblasts from colonic tissue, but adiponectin was only expressed in myofibroblasts. Induction of colitis significantly decreased the expression of adiponectin in colonic mucosa. The omega-3 fat diet group, but not the other fat diet groups, showed exacerbated colitis with a further decrease of adiponectin expression. Pioglitazone treatment ameliorated the level of decrease in adiponectin expression and improved colonic inflammation induced by the omega-3 fat-rich diet. In patients with ulcerative colitis, the expression level of adiponectin in colonic mucosa was also decreased compared with that in control mucosa. CONCLUSIONS: Adiponectin was found to be expressed in myofibroblasts. Adiponectin expression was significantly suppressed by induction of colitis, and aggravation of colitis after exposure to omega-3 fat may be due to a further decrease in the expression level of adiponectin.

Increased expression of long pentraxin PTX3 in inflammatory bowel diseases.

The aims of this study were to investigate the expression of pentraxin-3 in inflamed gastrointestinal tissue in patients with inflammatory bowel diseases and to elucidate the usefulness of plasma pentraxin-3 level as an inflammation marker in patients with inflammatory bowel diseases. Pentraxin-3 immunoreactivity was found in infiltrating neutrophils and vessels in the inflamed gut. Plasma pentraxin-3 concentration in patients with active inflammatory bowel diseases was significantly higher than that of normal subjects and patients with inactive inflammatory bowel diseases. Significant positive correlations of clinical disease activity with plasma pentraxin-3 concentration and serum CRP concentration were found in patients with inflammatory bowel diseases. Pentraxin-3 is directly produced from the inflamed gut in inflammatory bowel diseases. In conclusion, plasma pentraxin-3 concentration is a useful marker for understanding the disease activity in patients with inflammatory bowel diseases.

Pressure loading and ethanol exposure differentially modulate rat hepatic stellate cell activation.

Ethanol may cause an increase in sinusoidal pressure accompanied by portal hypertension. Hepatic stellate cells (HSCs) located in hepatic sinusoids may therefore be frequently exposed to dual stimulations of mechanical pressure and ethanol exposure in alcoholic liver injury. In this study, the effects of pressure loading and ethanol exposure on activation of rat cultured HSCs were investigated using an in vitro pressure-inducing apparatus. HSCs were cultured in media containing ethanol (0-100 mM) under different pressures (1-40 mmHg). Morphological changes and migration index were determined. We also determined the expression levels of alpha-smooth muscle actin (alpha-SMA) and mitogen-activated protein kinases (MAPKs) by Western blot analysis and the level of collagen IV and transforming growth factor beta1 (TGF-beta1) by ELISA. Pressure loading alone induced up-regulation of alpha-SMA via the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-jun N-terminal kinase (JNK) signaling pathways, prolonged extension of marginal length, and increased production of collagen IV. In contrast, ethanol exposure alone increased only extension of marginal length and cell migration. Dual stimulations of pressure loading and ethanol exposure enhanced the production of TGF-beta1 and migration index. The TGF-beta1-dependent p38 MAPK pathway may operate for production of extracellular matrix (ECM) or enhanced migration in the case of dual stimulations. In conclusion, static pressure loading is an important factor directly accelerating the activation of HSCs. Although increased sinusoidal pressure and ethanol exposure might differentially modulate HSC activation, both stimuli are involved in an additive manner in some situations.

Increased pentosidine, an advanced glycation end-product, in urine and tissue reflects disease activity in inflammatory bowel diseases.

BACKGROUND: Under inflammatory conditions with strong oxidative stresses, advanced glycation end-products (AGE), carbonyl compounds, are produced. The concentration of pentosidine, an AGE, reportedly correlates with complications of diabetes mellitus and worsening of rheumatoid arthritis, but its role in the pathogenesis of inflammatory bowel diseases (IBD) is unclear. METHODS: Immunohistochemistry was performed with antibodies against pentosidine, and 8-OH-2-deoxyguanosine. The urinary concentration of pentosidine was also quantified by enzyme-linked immunosorbent assay method. RESULTS: Pentosidine expression was up-regulated in the inflamed tissue of IBD. The expression of both pentosidine and 8-OH-2-deoxyguanosine was similar and increased in the inflamed epithelium and infiltrating cells (neutrophils and lymphocytes). The urinary concentration of pentosidine in active ulcerative colitis was significantly greater than that in inactive ulcerative colitis (0.12+/-0.15 vs 0.021+/-0.011 microg/mg of Cr, P<0.05), and was greater in active Crohn's disease than in inactive Crohn's disease (0.071+/-0.086 vs 0.039+/-0.023 microg/mg of Cr). CONCLUSIONS: The urinary pentosidine level correlated with the activity of ulcerative colitis and may be a marker for disease activity in ulcerative colitis.

Effects of intratumoral injection of CCL2 on monocyte-endothelial cell interactions in mouse pancreatic cancer.

OBJECTIVE: Although monocyte infiltration is an important aspect of the host response to tumor growth, the mechanisms of recruitment and their impact on tumor growth are still unknown. The authors studied monocyte-endothelial interaction and the effect of chemokine CCL2 in orthotopic mouse pancreatic cancer. METHODS: The rolling and adhesion of labeled monocytes in peritumoral and intratumoral areas were assessed by using an intravital microscope. Further, the effects of intratumoral injection or superfusion of CCL2 on in situ recruitment of monocytes and other immune cells and adhesion molecules were investigated. RESULTS: More monocytes were recruited in the peritumoral area than in the intratumoral area with increased vascular interaction, and the effect was more apparent by intratumoral CCL2 injection than superfusion. In both CCL2-treated groups infiltration of CD11b(+), CD68(+), and CD4(+) cells were increased, but the magnitude of increase was larger in intratumoral injection. Quantitative RT-PCR for the tumor tissue revealed that ICAM-1 expression was increased by the injection of CCL2. CONCLUSION: These results show intratumoral injection of CCL2 induces effective interaction between monocytes and endothelial cells in the peritumoral area of pancreatic cancer accompanied by the upregulation of ICAM-1 and may possibly become a tool for immunotherapy by promoting the infiltration of immune cells in cancers.

Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases.

OBJECTIVES: PD-L1 (also B7-H1) and PD-L2 (also B7-DC) are ligands for programmed death-1 (PD-1), which is a member of the CD28/B7 superfamily of costimulatory molecules and plays an inhibitory role on the periphery. Impaired regulation of this system may cause disruption to self-tolerance leading to autoimmunity; however, the role of these molecules in the liver is unknown. Therefore, we examined the expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. METHODS: We examined the liver expression of these molecules in autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) with no previous medical treatment using immunohistochemical staining and real-time PCR, and compared with chronic hepatitis type C (CHC) as a control. RESULTS: Although PD-1, PD-L1, and PD-L2 were expressed in the liver in AIH, PBC, as well as CHC, the expressions were relatively lower in PBC. In AIH, despite more severe inflammation than in CHC, the expression of these molecules was not greater than in CHC, and when compared with the relative expression of PD-L1, PD-L2 was lower in AIH. PD-L1 and PD-L2 expressions were well correlated with the level of IFN-gamma; however, relatively decreased induction for PD-L1 and PD-L2 by IFN-gamma was observed in AIH or PBC than in CHC. CONCLUSION: Modulation of PD-1/PD-L1 and PD-L2 systems may play a role in the development of autoimmune liver diseases.

[A case of Churg-Strauss syndrome discovered with hematemesis].

A 68-years-old Japanese woman was hospitalized emergently because of hemorrhagic gastric ulcer. For the hospitalization period, elevated levels of white blood cell count, eosinophilic leucocyte count, serum IgE and positive MPO-ANCA were recognized. With considering clinical course and these laboratory findings, we diagnosed Churg-Strauss syndrome (CSS). Steroid therapy in combination with cyclophosphamide was effective. CSS is a rare disease, but we should discriminate this disease when we encounter gastrointestinal bleeding of unknown etiology, especially PPI-resistant gastric ulcer.

Differential modulation in the functions of intestinal dendritic cells by long- and medium-chain fatty acids.

BACKGROUND: Although dendritic cells (DCs) play significant roles in intestinal immune responses, little is known regarding the direct effects of luminal foods on DC functions in the intestinal mucosa. In this study, we examined the effects of fatty acids (FAs) with various chain length on the phagocytic function, antigen presentation, and chemotaxis of intestinal DCs. METHODS: DCs obtained from the thoracic duct lymph of mesenteric lymphadenectomized rats were cultured with long [arachidonic acid (AA) or oleic acid] or medium (octanoic acid) chain FAs with interleukin-4 and granulocyte macrophage-colony stimulating factor. Tumor necrosis factor-alpha was added in the maturation group. Phagocytic function was examined by the intake of fluorescent microbeads. The expression of cell surface molecules was determined by immunocytochemistry or fluorescence-activated cell sorting (FACS). Antigen presentation ability was evaluated by coincubating keyhole limpet hemocyanin (KLH)-sensitized spleen lymphocytes and KLH-pulsed DCs. Migratory ability of DCs toward the chemokines CC chemokine ligand (CCL) 20 and CCL21 was also assessed. RESULTS: There was a maturation-induced decrease in phagocytic function, and an increased expression of major histocompatibility complex (MHC) class II molecules. Exposure of DCs to both long- and medium-chain FAs maintained phagocytic ability. The expression of MHC class II molecules was significantly suppressed only by long-chain FAs. The expression of costimulatory factors was suppressed only by AA. Long- but not medium-chain FAs suppressed the antigen presentation ability of DCs induced by maturation. Chemotactic ability of mature DCs toward CCL21 was abrogated only by long-chain FAs. CONCLUSIONS: The data suggest that intraluminal exposure to long- and medium-chain FAs may differentially modulate the immune functions of intestinal DCs.

Jumbo biopsy is useful for the diagnosis of colonic prolapsing mucosal polyps with diverticulosis.

We report here a case of multiple prolapsing mucosal polyps with diverticulosis in the sigmoid colon. A 52-year-old man was admitted to our hospital because of bloody diarrhea. Colonoscopy and barium enema showed multiple diverticula, markedly thickened mucosal folds and polypoid lesions with mucus on the top of them in the sigmoid colon. Endoscopic ultrasonography showed thickening of the mucosal and submucosal layers. Several endoscopic biopsy specimens were taken from the polypoid lesions. Histological examination revealed only chronic inflammatory cell infiltration. In order to obtain a definite diagnosis, we performed endoscopic jumbo biopsy for the polypoid lesions after obtaining informed consent. Histological examination revealed marked lymphocyte infiltration, hemosiderin deposits and fibromuscular obliteration in the lamina propria, features similar to those of mucosal prolapsing syndrome. After anti-diarrhetic treatment, clinical findings were improved. Thus, jumbo biopsy is useful for diagnosis and treatment of prolapsing mucosal polyps.

Erythropoietin-induced proliferation of gastric mucosal cells.

AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of anti- erythropoietin antibody (P<0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Combination therapy with tumor-lysate pulsed dendritic cells and antiangiogenic drug TNP-470 for mouse pancreatic cancer.

Most cases of pancreatic cancer are inoperable when diagnosed. Since immunotherapy and antiangiogenic therapy have been reported to be promising for pancreatic cancer, we examined whether the combination of immunotherapy with dendritic cells (DCs) and the antiangiogenic drug TNP-470 induces tumor regression. Syngeneic mouse pancreatic adenocarcinoma cells were orthotopically inoculated into C57/BL6 mice. DCs with or without tumor lysate (TL) were administered i.p. at 4 and 5 weeks. TNP-470 was injected s.c. into tumor-bearing mice every other day from 4 weeks to 6 weeks. We compared anticancer effects in 6 groups: NT (no treatment), DC/TL- (DCs without TL), DC/TL+ (DCs pulsed with TL), TNP (TNP-470 alone), DC/TL-TNP (DC/TL- plus TNP-470) and DC/TL+TNP (DC/TL+ plus TNP-470). We measured tumor volume, mean vascular density (MVD) and vessel diameter by FITC-dextran using an intravital microscope; degrees of proliferation and apoptosis of cancer cells by PCNA and TUNEL; infiltrating lymphocytes and expression levels of VEGF and MMP-9 by immunohistochemistry and immunoblotting. Tumor volume and MVD were significantly suppressed in the treatment groups with prolonged survival rate, especially in the DC/TL+TNP group. There were no significant differences in apoptosis among the 6 groups except DC/TL+. The number of infiltrating CD4+ cells in the DC/TL+ group was higher than that in the NT group. VEGF expression was significantly suppressed in the treatment groups containing TNP-470, and MMP-9 was also suppressed in the groups containing DC/TL+. Our data suggested that TL-pulsed DCs combined with TNP-470 induced regression of mouse pancreatic cancer, possibly through induction of immune responses and suppression of angiogenesis.

Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis.

Nociceptin/orphanin (Noc/oFQ), endogenous agonist for nociceptin receptor (NOR), is thought to be a stimulator of neurogenic inflammation. We investigated the possible role of Noc/oFQ in the development of colitis using NOR-deficient mice treated with dextran sulfate sodium (DSS). Colitis was significantly improved in NOR-deficient mice against wild-type mice. Expression level of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and infiltrating cells also significantly decreased in NOR-deficient mice against wild-type mice. Nociceptin expression increased in wild-type mice after DSS treatment. These results suggest stimulation by Noc/oFQ deteriorates colonic inflammation via up-regulation of adhesion molecule.

Blockade of PSGL-1 attenuates CD14+ monocytic cell recruitment in intestinal mucosa and ameliorates ileitis in SAMP1/Yit mice.

The pathogenesis of Crohn's disease (CD) is not known. However, monocytes and macrophages are thought to play important roles in the development of mucosal inflammation. Therefore, in this study, we examined the role of monocyte-endothelial cell interactions in senescence-accelerated mouse P1 (SAMP1)/Yit mice, a murine model of spontaneous ileitis. Fluorescence-labeled CD14+ monocytic cells isolated from the spleen and mesenteric lymph nodes of AKR/J (control) mice were injected into the tail veins of recipient (AKR/J and SAMP1/Yit) mice, and migration in the postcapillary venules (PCV) of Peyer's patches, submucosal venules, and villus microvessels of the terminal ileum was monitored by using an intravital microscope. Rolling and adhesion of CD14+ monocytic cells in the PCV of Peyer's patches and microvessels of the terminal ileum were increased in SAMP1/Yit mice. An immunohistochemical study showed increased expression of P-selectin glycoprotein-1 (PSGL-1), P-selectin, and vascular cell adhesion molecule-1 in the terminal ileum of SAMP1/Yit mice. Antibodies against these three adhesion molecules significantly inhibited adhesion of CD14+ monocytic cells to the PCV of Peyer's patches and microvessels of the terminal ileum, treatment with an anti-PSGL-1 monoclonal antibody (mAb) showing the strongest suppressive effect. Anti-PSGL-1 mAb also attenuated T cell adhesion in microvessels of intestinal mucosa. In addition, periodical administration of an anti-PSGL-1 mAb for 7 weeks significantly ameliorated ileitis of SAMP1/Yit mice. The results suggest that PSGL-1-P-selectin interaction plays an important role in monocyte-endothelial cell interactions and the development of ileitis in a murine model of CD and that the blockade of this adhesion molecule may be a novel strategy for treating CD.

Melanoidin, a food protein-derived advanced maillard reaction product, suppresses Helicobacter pylori in vitro and in vivo.

BACKGROUND: Extracellular urease proteins located on the surface of Helicobacter pylori are gastric mucin-targeted adhesins, which play an important role in infection and colonization to the host. In this study we have determined the inhibitory activity of a variety of melanoidins, protein-derived advanced Maillard reaction products, ubiquitously found in heat-treated foods, on urease-gastric mucin adhesion. In addition, we have determined the anticolonization effect of melanoidin I, prepared by the Maillard reaction between casein and lactose, in an animal model and in human subjects infected with this bacterium. METHODS: The inhibitory activity of each compound was determined by a competitive binding assay of labeled gastric mucin to plate-immobilized urease. Melanoidin I was used in an in vivo trial using euthymic hairless mice as an infection model. Melanoidin I was consumed for 8 weeks by subjects infected with H. pylori. The [(13)C] urease breath test and H. pylori-specific antigen in the stool (HpSA) test were performed on subjects at week 0 and week 8. RESULTS: A variety of food protein-derived melanoidins strongly inhibited urease-gastric mucin adhesion in the concentration range of 10 micro g/ml to 100 micro g/ml. In particular, melanoidin I significantly (p <.05) suppressed colonization of H. pylori in mice when given for 10 weeks via the diets. Eight weeks daily intake of 3 g melanoidin I significantly (p <.05) decreased the optical density of HpSA in subjects. CONCLUSION: Foods containing protein-derived melanoidins may be an alternative to antibiotic-based therapy to prevent H. pylori that combines safety, ease of administration and efficacy.

Regulation of T-lymphocyte trafficking by ICAM-1, MAdCAM-1, and CCR7 in microcirculation of appendicular and intestinal lymphoid tissues.

OBJECTIVE: Although the appendix is recognized as an inductive site of intestinal inflammation, lymphocyte migration to lymphoid tissues of the appendix has not been characterized. The authors investigated if there are specific features in T-lymphocyte adhesion to microvessels of the appendix compared to mouse Peyer's patches (PPs). METHODS: T-lymphocyte interaction with postcapillary venules (PCVs) of lymph follicles of the appendix and PPs was observed using an intravital microscope. Antibodies against ICAM-1, MAdCAM-1, or anti-L-selectin were administered prior to lymphocyte administration, and in some experiments CCR7 on T-lymphocytes was desensitized by excess CCL21. RESULTS: The number of adhered T-lymphocytes reached the maximum value earlier in PCVs of PPs than in those of the appendix. T-lymphocyte adherence was significantly inhibited by anti-MAdCAM-1 at either the appendix or PPs, but adherence in the appendix was also significantly inhibited by anti-ICAM-1, suggesting a dependency on ICAM-1 in the appendix. Histologically, there was a significant ICAM-1 expression in the appendix. Desensitization of CCR7 suppressed T-cell adhesion in PCVs of the appendix and PPs to the same extent. CONCLUSION: ICAM-1 appeared to be more important in T-lymphocyte sticking in PCVs of the appendix compared with intestinal PPs, while MAdCAM-1 and CCR7 were shown to play important roles in T-lymphocyte adherence in all sites.

Adenosquamous pancreatic cancer producing parathyroid hormone-related protein.

An autopsy case of adenosquamous pancreatic cancer in a 61-year-old male patient with an elevated serum level of parathyroid hormone-related protein (PTH-rP) is reported. He was admitted to our hospital with a 1-month-long history of abdominal discomfort and progressive abdominal fullness. A computed tomography (CT) scan of the abdomen showed a retroperitoneal mass, approximately 10 cm in diameter, involving the pancreas, with round enhancement on contrast examination. Histological examination of a specimen taken by CT-guided needle biopsy suggested squamous cell carcinoma or transitional cell carcinoma. Laboratory data on admission revealed a high serum calcium level and high PTH-rP level. The calcium level initially responded to intravenous hydration, furosemide, calcitonin, and bisphosphonates, decreasing from 15.0 to 9.0 mg/dl. However, the hypercalcemia recurred after 10 days. The patient developed carcinomatous peritonitis and acute renal failure, and died on the 25th hospital day. Autopsy revealed a mass in the pancreatic body to tail, invading the retroperitoneum, with progressive carcinomatous peritonitis. Histological examination of the mass revealed infiltrating carcinoma, showing squamous differentiation with focal intracytoplasmic lumina formation, consistent with pancreatic adenosquamous carcinoma. Immunohistological examination showed positive staining for PTH-rP. Adenosquamous carcinoma of the pancreas is relatively rare; only a few cases associated with hypercalcemia and for which PTH-rP has been identified as a causative factor have been reported. This is the first case in which immunohistochemistry proved localized PTH-rP in adenosquamous pancreatic cancer cells, associated with persistent hypercalcemia.