Tsutomu Teranishi

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).

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.

Original ContributionsAntineutrophil 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 Crohn’s 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 by 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). METHODS The cells were cultured as monolayers on microporous membranes in culture inserts. Oleic acid (OA), capric acid (CA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were applied to the apical compartment of Caco-2 cell monolayers. The concentration of IL-8 in the basolateral medium was measured by using enzyme-linked immunosorbent assay, and the expression of IL-8 mRNA was measured by using competitive reverse transcription--polymerase chain reaction. Protein kinase C inhibitors (GF109203X and calphostin C) and H-7 (a protein kinase inhibitor) were used to study the mechanisms by which IL-8 production is stimulated. RESULTS Both OA and CA enhanced IL-8 production (approximately fivefold), whereas DHA and EPA did not. Both OA and CA also enhanced IL-1-induced IL-8 production. The onset of OA-induced IL-8 production was delayed compared with that of CA-induced IL-8 production. Both OA and CA enhanced IL-8 mRNA expression (approximately fivefold) after 6 and 3 h, respectively. The protein kinase inhibitor (H-7) reduced both OA- and CA-induced IL-8 production by 88.0 and 85.9%, respectively. The protein kinase C inhibitors (GF109203X and calphostin C) reduced OA-induced IL-8 production by 29.3 and 54.5%, respectively, but showed no effect on CA-induced IL-8 production. CONCLUSIONS These findings suggest that not only OA but also CA stimulates IL-8 production in intestinal epithelial cells, and the mechanisms of action differ between OA and CA.

Systemic endotoxemia in patients with inflammatory bowel disease: Its relation to metronidazole administration and liver injury

Systemic endotoxemia has been reported in patients with inflammatory bowel disease (IBD). However, it is still controversial whether systemic endotoxemia occurs frequently in IBD. It has been reported that metronidazole-treated mice had increased rates of translocation of intestinal bacteria, however, it is unknown whether this is true in humans. Liver dysfunction is relatively frequent in patients with IBD and the etiology is occasionally unknown. Aims: 1) To evaluate plasma endotoxin levels in patients with IBD. 2) To investigate the effect of metronidazole administration on plasma endotoxin levels in IBD. 3) To investigate the relationship between liver injury and plasma endotoxin levels in IBD. Methods: We studied 52 patients with ulcerative colitis (UC), 34 with Crohns disease (CD) and 31 control subjects. No subjects had evidence of liver injury. Plasma endotoxin levels were measured by a conventional method, Toxicolor (TOX) and an endotoxin-specific method, Endospecy (ES). In 10 of the patients, the endotoxin levels were determined again after oral metronidazole treatment (750mg/day) for two weeks. When the patient showed liver injury of unknown etiology during the clinical course, the end0toxin levels were examined again. Results: The plasma endotoxin levels measured by the ES method were 9.5 -+ 7.5 pg/ml (mean SD), 6.2 4.1, 6.9-+ 4.5, 6.8-,-4.1, and 3.6-+ 1.6 in patients with active UC, inactive UC, active CD, inactive CD, and in control subjects, respectively. By the TOX method, the respective plasma endotoxin levels were 23.0 ± 29.5, 9.9 ± 6.3, 17.4 ± 21.8, 12.2 ± 6.6, and 7.7 ± 5.3. With both methods, plasma endotoxin levels were significantly higher in the patients with active UC or CD than in the control subjects. There was a significant difference in endotoxin levels between the active and inactive phases in UC but not in CD. The plasma endotoxin levels were not significantly influenced by oral metronidazole treatment (7.5 ± 5.2 vs. 7.7 ± 3.2 pg/ml by the ES method, n=10). The plasma endotoxin levels did not increase after the development of liver injury (8.5 ± 9.4 vs. 8.9 ± 9.0 pg/ml by the ES method, n=5). Conclusions: These results suggest that systemic endotoxemia frequently occurs in patients with IBD. There was no relation of plasma endotoxin level to metronidazole administration or liver injury in patients with IBD.

The levels and the structure of fecal α 1-antitrypsin in patients with inflammatory bowel disease

Fecal ct 1-antitrypsin (ct 1-AT) clearance is now a standard test for the assessment of intestinal protein loss. In patients with inflammatory bowel disease(IBD), the sources of fecal a 1-AT remains uncleared and molecular heterogeneity of fecal a 1-AT has been reported. AIMS: 1) To examine the levels and the structure of a 1-AT in feces and sera of patients with IBD. 2) To examine the synthesis and secretion of a 1-AT in intestinal epithelial cells and hepatocytes. METHODS: We studied 36 patients with ulcerative colitis(UC), 26 patients with Crohns disease(CD), and 20 control subjects. The stool samples were collected at 4 °C over a period of 48 hours and were homogenized. We also used human intestinal epithelial cell lines, Caco-2 and HT-29, and a hepatocellular cell line, HepG2. These cells were stimulated with interleukin-6 (IL-6), interleukin-1 13 and tumor necrosis factor a. The level of <x 1-AT protein was measured by ELISA, and the expression of <x 1-AT mRNA by RT-PCR. The a 1-AT molecules were examined by Western blotting and the reactivity with Concanavalin A (Con A) was studied by Con Aa 1-AT Ab sandwich EIA. RESULTS: 1) Fecal ct 1-AT concentrations were significantly higher in active UC and CD (medians 1465, and 3482 lag/g respectively) than in inactive UC, CD and control subjects (medians 411,651, and 327 laglg respectively). In all disease groups and control subjects, serum a 1-AT showed an apparent molecular weight (MW) of-52 kDa and fecal a 1-AT showed fragments of various sizes. In active UC and CD, the apparent MW of the main band of fecal ct I-AT was higher than in inactive patients and control subjects. In active UC and CD, ConA-bound ct 1-AT was rich in the feces. 2) The synthesis and secretion of a 1-AT were increased after stimulation with IL-6 (10, 100 ng/ml) in Caco-2 cells, and HT-29 cells as well as HepG2. The percentage of ct I-AT released extracellularly was also increased. The level of ¢t 1-AT mRNA expression reached a peak within 120 mins of stimulation of IL-6. Neither IL-1 13 nor TNF a was a potent stimulant. CONCLUSIONS: An increase of fecal a 1-AT level is at least partly due to an increase of its secretion from intestinal epithelial cells in patients with intestinal inflammation. The structural analysis of fecal a I-AT is useful for assessment of disease activity in IBD.