Kazunori Sugi

Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells

BACKGROUND & AIMS Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression. METHODS hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate-phalloidin analysis. RESULTS Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine. CONCLUSIONS hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.

Fecal eosinophil granule-derived proteins reflect disease activity in inflammatory bowel disease

OBJECTIVES:The aims of this study were: 1) to examine whether the fecal levels of eosinophil granule-derived proteins reflect disease activity in inflammatory bowel disease (IBD); and 2) to examine the extracellular release of these proteins from eosinophils and their stability in feces by an in vitro study.METHODS:We investigated 42 patients with ulcerative colitis (UC), 37 patients with Crohns disease (CD), and 29 control subjects. The stool samples were collected at 4°C over 48 h and were homogenized. The fecal levels of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured by radioimmunoassay. Fecal Hb (Hb), α1-antitrypsin (AT), and lactoferrin (Lf) were also measured by ELISA.RESULTS:Fecal ECP and EPX concentrations were significantly increased in both active UC and active CD compared to inactive UC and inactive CD, respectively. Fecal EPX concentration correlated with the fecal Hb, AT, and Lf concentrations more closely than fecal ECP concentration. Even in the inactive stage, CD patients who relapsed within the following 3 months showed higher fecal ECP and EPX concentrations compared to the patients who did not. EPX was released extracellularly more efficiently than ECP (18.6%vs 6.3%, after incubation for 15 min at 25°C). EPX was more stable in the feces than ECP.CONCLUSIONS:The measurement of eosinophil granule-derived proteins in feces is useful for evaluating disease activity and predicting relapse in patients with IBD. EPX may be more suitable than ECP as a fecal eosinophil marker.

Immunochemical detection of human lactoferrin in feces as a new marker for inflammatory gastrointestinal disorders and colon cancer

We have developed a new immunochemical test for fecal lactoferrin (LF) utilizing an enzyme-linked immunosorbent assay (ELISA). The ELISA had a sensitivity of about 10 micrograms/L of lactoferrin and the measurable range was 10.0-1000.0 micrograms/L (1.0-100.0 micrograms LF/g feces). The stability of lactoferrin in feces was greater than that of myeloperoxidase and leucocyte elastase. The fecal concentration of lactoferrin (mean +/- SD) in 35 normal subjects was 0.75 +/- 0.83 microgram/g feces, whereas that in 24 patients with colon cancer was 74.4 +/- 88.3 micrograms/g feces. The fecal lactoferrin concentration of 38 patient with active ulcerative colitis was 307.4 +/- 233.9 micrograms/g feces, and that in 36 patients with active Crohns disease was 191.7 +/- 231.1 micrograms/g feces. The ELISA for human fecal lactoferrin might be useful in the diagnosis of colon disease.

Intestinal Protein Loss and Bleeding Assessed by Fecal Hemoglobin, Transferrin, Albumin, and Alpha-1-Antitrypsin Levels in Patients with Colorectal Diseases

Four fecal proteins (hemoglobin, transferrin, albumin, and alpha 1-antitrypsin) were measured by enzyme-linked immunosorbent assay (ELISA) in patients with colorectal diseases. Levels of all 4 proteins were significantly increased in patients with colonic cancer and ulcerative colitis (UC) compared to levels in control subjects, while fecal alpha 1-antitrypsin was particularly elevated in colonic Crohns disease (CD). That is, the fecal protein pattern of CD was distinct from those of colonic polyps, colonic cancer, and UC. To investigate whether levels of these fecal proteins reflect disease activity in UC and CD, comparative evaluation of fecal proteins in the active and inactive phases was performed. In UC, differences in the fecal concentrations of all 4 proteins were significant between the active and inactive phases of the disease. In CD, however, the difference in alpha 1-antitrypsin concentration was significant. Our results suggest that measurements of these 4 fecal proteins would be useful in the screening of colorectal diseases. In addition, these markers can also be used as indicators of disease activity in inflammatory bowel diseases.

Antineutrophil cytoplasmic antibodies in Japanese patients with inflammatory bowel disease: prevalence and recognition of putative antigens

Objective:Our aim was to investigate the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in Japanese patients with ulcerative colitis (UC) and Crohns disease (CD), and the putative antigens recognized by perinuclear staining pattern ANCA (p-ANCA)–positive sera.Methods:Sera from UC n = 52 and CD n = 43 patients, and from healthy controls n = 74 were studied. The indirect immunofluorescence (IIF) method was used for the detection of ANCA and its binding pattern. p-ANCA–positive sera were studied further for putative antigens. ELISAs using lactoferrin (Lf), myeloperoxidase (MPO), and cathepsin G (Cat G) as antigens were performed.Results:ANCA was positive in 40 of the 52 (76.9%) UC (p-ANCA in 33) and in 32 of the 43 (74.4%) CD (p-ANCA in 31) patients. UC and CD patients showed significantly higher titers of p-ANCA than controls; however, no significant difference was observed between UC and CD. In UC, 23, 17, and nine of the 33 patients with p-ANCA–positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. In CD, 21, 20, and 11 of the 31 patients with p-ANCA–positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. Fourteen of the UC and six of the CD patients showed reactivity with two different antigens, and seven of the UC and 11 of the CD patients showed reactivity with all three antigens. The presence of anti-Lf and anti-MPO antibodies was further confirmed by Western blotting.Conclusions:ANCA is useful in distinguishing patients with IBD from normal subjects but is not sufficient for the differential diagnosis of CD and UC. p-ANCA reactivity might be derived from the recognition of heterogeneous neutrophil-associated antigens.

Medium-chain fatty acids stimulate interleukin-8 production in Caco-2 cells with different mechanisms from long-chain fatty acids

Background and Aim: It has been suggested that dietary fat exacerbates intestinal inflammation. We investigated the effect of fatty acids on interleukin (IL)‐8 production in a human intestinal epithelial cell line (Caco‐2).

Left gastric vein hemodynamics and variceal recurrence in patients undergoing prophylactic endoscopic ligation of high-risk esophageal varices.

BACKGROUND Early recurrence of esophageal varices remains problematic after endoscopic variceal ligation. To evaluate the efficacy of prophylactic endoscopic ligation for esophageal varices at high risk for bleeding, the relationship between left gastric vein hemodynamics and variceal recurrence was investigated. METHODS Thirty-five patients with cirrhosis underwent endoscopic variceal ligation. Angiography was performed in all patients before treatment and after eradication of varices to study left gastric vein hemodynamics. RESULTS Before treatment, 12 patients had hepatopetal flow in the left gastric vein (type I), 17 had hepatofugal flow (type II), and 6 had hepatofugal flow with an extra-esophageal shunt (type III). In type I and III patients, the direction of blood flow in the left gastric vein did not change after eradication of varices. Type II patients showed bi-directional flow in the left gastric vein after treatment. Varices recurred in all but one type II patient and in one type I patient during follow-up (mean 36.7 months). The 2-year recurrence-free rate was higher in type I patients (p = 0.0001) and type III patients (p = 0.0002) than in type II patients. CONCLUSIONS Prophylactic ligation seems to be a safe and useful procedure, especially in patients with type I or III hemodynamics in the left gastric vein before treatment.

Cyclosporine A inhibits interleukin-8 production in a human colon epithelial cell line (HT-29)

Intestinal epithelial cells produce various inflammatory mediators. However, the way in which immunosuppressive agents influence the production of these mediators by intestinal epithelial cells is not understood. The effects of cyclosporine A (CsA), tacrolimus (FK506), and dexamethasone (DEX) on cytokin-induced production of interleukin (IL)-8 in a human colonic cancer cell line (HT-29) were examined. HT-29 cells were stimulated with either IL-1β or tumor necrosis factor α (TNFα) together with CsA, FK506, or DEX. The presence of IL-8 protein was detected by enzyme-linked immunosorbent assay, and the expression of IL-8 messenger RNA (mRNA) by reversetranscription polymerase chain reaction. CsA (1, 5, and 10ng/ml) significantly reduced IL-1β-induced IL-8 production (by 32%, 41%, and 48%, respectively), and reduced TNFα-induced IL-8 production (by 21%, 42%, and 50%, respectively). FK506 or DEX had no effect on IL-1β- or TNFα-induced IL-8 production. The expression of IL-8 mRNA was also inhibited by CsA. These findings suggest that CsA may influence the production of inflammatory mediators in colonic cells in a different manner from FK506 and DEX.

Bile acids inhibit tumour necrosis factor α-induced interleukin-8 production in human colon epithelial cells

To clarify the regulatory mechanism of the production of various inflammatory mediators by intestinal epithelial cells, the effect of bile acids (tauroursodeoxycholate, TUDC; taurochenodeoxycholate, TCDC; and taurocholate, TC) on the cytokine‐induced production of interleukin (IL)‐8 in a human colon epithelial cell line (HT‐29) was examined. HT‐29 cells were incubated for 24 h in a culture medium containing tumour necrosis factor α (TNFα; 1 ng/mL) and/or interleukin (IL)‐1 β (1 ng/mL) in the presence or absence of bile acids. The IL‐8 concentration in the medium was measured by an enzyme‐linked immunosorbent assay. The binding assay of TNFα was performed using [125I]‐TNFα (100 pmol/L). Interleukin‐8 production during incubation with TNFα was markedly reduced in the presence of 0.5 and 1 mmol/L TUDC, 0.5 and 1 mmol/L TCDC and 0.5 and 1 mmol/L TC, by 56, 85, 86, 91, 37 and 70%, respectively. The IL‐8 production during incubation with IL‐1ß was not significantly reduced in the presence of these bile acids. The specific binding of TNFα to cells was inhibited 33, 47, and 14% by 1 mmol/L TUDC, TCDC and TC, respectively. These findings suggest that bile acids inhibit TNFα‐induced IL‐8 production by the colonic cells. The suppression may be partly due to inhibition of TNFα binding to the cells by bile acids.

Efficacy of prophylactic sclerotherapy in patients with hepatocellular carcinoma and varices negative for the red color sign

BACKGROUND A prospective randomized controlled study was performed to evaluate the usefulness of prophylactic endoscopic sclerotherapy in patients with hepatocellular carcinoma complicated by esophageal varices. METHODS The subjects included 58 patients with esophageal varices negative for the red color sign and hepatocellular carcinoma without tumor emboli in the portal trunk or primary portal branches. Patients were randomly assigned to prophylactic sclerotherapy (n = 29) or control (n = 29) groups, and their bleeding and survival rates were compared. RESULTS A mean of 3.0 sclerotherapy sessions was required for complete disappearance of varices in patients receiving prophylactic sclerotherapy. During the observation period, transcatheter arterial embolization for hepatocellular carcinoma was performed more often in patients with prophylactic sclerotherapy (mean 3.8 times) than in control patients (mean 2.0 times) (p < .05). Percutaneous ethanol injection therapy was performed more often in patients with prophylactic sclerotherapy than in controls (mean 8.1 times vs 5.0 times, respectively) (p < .05). The 3-year bleeding rates were 50% for the control group and 18% for the prophylactic sclerotherapy group (p < 0.05), and the 3-year survival rates were 16% for the control group and 37% for the therapy group (p < 0.05). CONCLUSIONS Prophylactic sclerotherapy improves survival in patients with hepatocellular carcinoma complicated by red color sign-negative esophageal varices without tumor emboli in the portal trunk or primary portal branches.