Ken-ichi Katsu

Therapeutic effect of intracolonically administered nuclear factor kappa B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis.

Cytokines such as IL‐1, tumour necrosis factor‐alpha (TNF‐α), IL‐6 and IL‐8 are increased in inflamed colonic mucosa after administration of mouse DSS. Nuclear factor κB (NF‐κB) is a transcription factor which regulates the expression of these cytokine genes. The effect of intracolonically administered NF‐κB (p65) antisense phosphorothioate oligonucleotide was examined in mouse DSS‐induced colitis using drinking water containing 5% DSS. When antisense oligonucleotide was given on day 0, the disease activity index (DAI) representing clinical symptoms improved and the histological score decreased; furthermore, IL‐1, IL‐6, and TNF‐α concentrations in rectal mucosa were lower compared with the control group. Clinical and histological improvement was also observed when antisense oligonucleotide was begun on day 2 but not on day 7. In addition, the distribution of antisense oligonucleotides was investigated by confocal laser microscopy. In colonic mucosa, oligonucleotides were predominantly localized to cells in the lamina propria, but also in the epithelium. Western blot analysis using homogenized rectal mucosa showed the decreased expression of NF‐κB p65 in the antisense oligonucleotide‐treated group, although it was increased in the colitis group. These results suggest that intracolonic administration of NF‐κB antisense oligonucleotide may be effective in ulcerative colitis.

Inhibition of dextran sulphate sodium (DSS)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules (endothelial leucocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1)).

We examined the effect of intracolonic administration of anti‐adhesion molecule antibodies on DSS‐induced colitis in mice. Immunohistochemical staining in mice with colitis showed increased expression of ELAM‐1 and ICAM‐1 on endothelial cells of vessels in the lamina propria and submucosa at sites of inflamed lesions. Intracolonic administration of anti‐ELAM‐1 or anti‐ICAM‐1 antibody decreased bloody stools, anaemia, and histologically evident damage, as well as myeloperoxidase activity and IL‐1β content. We concluded that adhesion molecule expression is important in the development of DSS‐induced colitis in mice and that intracolonic administration of anti‐adhesion molecule antibodies, especially anti‐ELAM‐1 antibody, effectively inhibits the colonic inflammation. Intracolonic administration of anti‐adhesion molecule antibodies may show therapeutic promise in ulcerative colitis.

Balloon-occluded retrograde transvenous obliteration of high risk gastric fundal varices.

OBJECTIVE:Balloon-occluded retrograde transvenous obliteration is an effective new method for treating gastric fundal varices, but subsequent occurrence of esophageal varices creates a problem. The relationship between portal hemodynamics and the occurrence of esophageal varices after prophylactic balloon-occluded retrograde transvenous obliteration was investigated.METHODS:Ten cirrhotic patients considered to have high risk gastric fundal varices underwent angiography. Six patients showed a communication between blood flow in gastric wall vessels and that in the gastrorenal shunt (type I), whereas the others (type II) did not. Depending on the flow direction in the left gastric vein, the two groups were further divided into hepatopetal (a) and hepatofugal (b) subgroups. The therapeutic effect on portal hemodynamics and the relationship between pretreatment portal hemodynamics and posttreatment occurrence of esophageal varices were investigated.RESULTS:Fundal varices disappeared endoscopically in all 10 patients and the gastrorenal shunt was also occluded after the procedure. No patient showed worsening of liver function or systemic complications during follow-up. The increase in portal blood flow was more significant in type Ib patients than in the others. Esophageal varices occurred in all type I patients, and as to those in type Ib, high risk varices developed within 6 months after treatment. On the other hand, esophageal varices did not occur in type II patients.CONCLUSIONS:This procedure was effective for treating gastric fundal varices. However, type Ib patients are likely to develop high risk esophageal varices after occlusion of the gastrorenal shunt.

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.

Expression of the EP4 Prostaglandin E2 Receptor Subtype with Rat Dextran Sodium Sulphate Colitis: Colitis Suppression by a Selective Agonist, ONO-AE1-329

Expression of the EP4 receptor, a prostaglandin (PG)E2 receptor subtype, as well as disease suppression by the administration of a selective EP4 agonist (ONO‐AE1‐329) was investigated in the colorectal mucosa of rats with dextran sodium sulphate (DSS)‐induced colitis. Rats were given drinking water containing 3% DSS for 2 weeks. Expression of EP4 receptor mRNA was barely detectable under normal conditions according to reverse transcription‐polymerase chain reaction (RT‐PCR). By 1 week after the initial administration of DSS, the receptor mRNA was strongly expressed. After ONO‐AE1‐329 was administered intracolonically to rats with DSS colitis for 7 consecutive days, erosion and ulceration decreased. Peripheral white blood cell (WBC) counts became less elevated. Interleukin (IL)‐1β and growth‐regulated gene product/cytokine‐induced neutrophil chemoattractant (GRO/CINC‐1) concentrations in colorectal mucosa were lower than in colitis control group (IL‐1β: 12.8 ± 4.6 and 30.8 ± 6.2 µg/mg protein, P < 0.05; GRO/CINC‐1: 15.5 ± 3.0 and 39.2 ± 5.4 µg/mg protein, P < 0.05), and the expression of the corresponding cytokine mRNA was strongly suppressed. IL‐10 concentration was higher than in control group (14.5 ± 1.7 and 7.9 ± 1.2 µg/mg, P < 0.05), and the mRNA was more strongly expressed. These results suggest that the EP4 receptor is important in colonic inflammation, and that PGE2 suppresses DSS colitis at least partly via the EP4 receptor and the above cytokine changes. Intracolonic administration of selective EP4 agonist might have therapeutic applicability in inflammatory bowel disease such as ulcerative colitis.

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.

Estimation of Mucosal Inflammatory Mediators in Rat DSS-Induced Colitis

In order to investigate the mucosal injury mechanism in UC, we made dextran sulfate sodium (DSS)-induced colitis in rat and examined pathological findings, MPO activity, PGE2 level, and local mRNA expression and secretion of IL-1β, TNF-α, GRO/CINC-1 and IL-10 in DSS colitis mucosa. Moreover, we estimated the correlation between the severity of mucosal damage and changes of these local inflammatory mediators’ values. Neutrophil infiltration was marked and MPO activity was locally increased in proportion to the severity of mucosal damage. The mRNA expression and secretion of IL-1β, GRO/CINC-1 and IL-10 were increased. Especially, the secretions of IL-1β and GRO/CINC-1 were increased in proportion to the severity of mucosal damage. However, those of TNF-α were not increased in the colitis mucosa. An abnormal macrophage function and the presence of macrophage subtypes producing different cytokines would be predicted from our TNF-α data. The lesion was less severe in the colonic mucosa with higher levels of endogenous PGE2, while it was more severe in the colonic mucosa with lower levels of endogenous PGE2, implicating this compound as an inhibitory factor against the development of inflammation in the affected mucosa. Our results suggest that PGE2 might have therapeutic applicability to UC.

A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis

BACKGROUND We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.

Evaluation of Gastric Motor Activity in the Elderly by Electrogastrography and the 13C-Acetate Breath Test

Background: Elderly people frequently have symptoms of fullness and appetite loss due to impaired gastric motor activity. These symptoms may cause malnutrition, immunosuppression and other complications. Objective: The effects of aging and daily activity on gastric motility in the elderly were investigated by electrogastrography and the 13C-acetate breath test. Methods: We enrolled seven active elderly subjects (active elderly group), seven elderly subjects staying at a geriatric facility who had reduced mental and physical capacities (inactive elderly group) and seven healthy young volunteers (young group). Electrogastrography was recorded before and after ingestion of a 13C-acetate-mixed liquid meal. Expired air was sampled every 10 min after the meal to measure the 13CO2 concentration. Results: The ratio of the incidence of the 3-cpm wave (gastric intrinsic frequency) during the postprandial period compared to the fasting state was reduced in both elderly groups compared to young subjects, and the reduction was greater in the inactive elderly than in the active elderly group. The ratio of the amplitude of the peak frequency during the postprandial period to that in the fasting state (power ratio) was also lower in the elderly groups. The time of peak 13CO2 expiration was delayed in the active elderly and more so in the inactive elderly group. Conclusion: Postprandial peristalsis and gastric contractile force are reduced in the elderly, and gastric emptying is delayed indicating a reduction in gastric motor activity.

EP1 and EP4 Receptors Mediate Exocytosis Evoked by Prostaglandin E2 in Guinea‐Pig Antral Mucous Cells

Effects of prostaglandin E2 (PGE2) on exocytosis of mucin were studied in mucous cells isolated from guinea‐pig antrum using video‐microscopy. Stimulation with PGE2 elicited a sustained increase in the frequency of exocytotic events in a dose‐dependent manner, which was under regulation by both Ca2+ and cAMP. Stimulation with a selective prostanoid EP4 receptor agonist (ONO‐AEI‐329, 10 μM), which activates cAMP signals, elicited a sustained increase in the frequency of exocytotic events (30% of that evoked by 1 μM PGE2). Stimulation with an EP1 agonist (17‐P‐T‐PGE2, 1 μM), which activates Ca2+ signals, increased the frequency of exocytotic events to a lesser extent (5% of that evoked by 1 μM PGE2), while addition of an EP1 antagonist (ONO‐8713, 10 μM) decreased the frequency of exocytotic events (approximately 40% of that evoked by 1 μM PGE2). However, addition of the EP1 agonist potentiated the frequency of exocytotic events evoked by the EP4 agonist or forskolin (which elevates cAMP levels) and increased the sensitivity of the exocytotic events to forskolin. These results suggest that the Ca2+ signal activated via the EP1 receptor potentiates the cAMP‐regulated exocytotic events activated via the EP4 receptor during PGE2 stimulation, by increasing the sensitivity of the exocytotic response to cAMP. In conclusion, exocytotic events in PGE2‐stimulated antral mucous cells were regulated by interactions between EP1 and EP4 receptors.