Kazue Tabata

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.

Lactoferrin in whole gut lavage fluid as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins

OBJECTIVES:We investigated which neutrophil-derived proteins in whole gut lavage fluid (WGLF) most accurately reflect disease activity in inflammatory bowel disease.METHODS:WGLF was obtained from patients undergoing whole gut lavage as a bowel preparation for colonoscopy. Twenty-seven patients with ulcerative colitis (UC), 23 patients with Crohns disease (CD), and 35 control subjects were examined. The concentrations of lactoferrin, polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase, and lysozyme in WGLF were measured by ELISA. For the assessment of stability, WGLF samples were stored at 37°C for various periods.RESULTS:In UC, the concentrations of lactoferrin, myeloperoxidase, and lysozyme in WGLF had good correlations with colonoscopic grading. Zero, 12, five, and 10 of 28 samples from active UC patients showed normal concentrations of lactoferrin, PMN-E, myeloperoxidase, and lysozyme, respectively. In CD, the concentrations of lactoferrin and myeloperoxidase had good correlations with the Crohns disease activity index. Thirteen and seven of 36 samples from inactive CD patients (Crohns disease activity index ≤ 150) showed high concentrations of lactoferrin and myeloperoxidase, respectively. Most of them (11/13, 6/7) were found to have ulceration by colonoscopy or small bowel x-ray. The ratio of the lactoferrin concentration in the WGLF supernatant to that in total WGLF was highest among these proteins in all disease groups and control subjects. Lactoferrin and myeloperoxidase showed good stability in WGLF, whereas PMN-E and lysozyme did not.CONCLUSION:Lactoferrin is the most suitable of these proteins for use as a neutrophil-derived WGLF marker of intestinal inflammation.

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).

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.

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.