| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1998: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Background : In ulcerative colitis, the colonic mucosa exhibited the marked infiltration of activated eoslnophils with the secretoly form of eosinophil cationic protein(ECP), one of the eosinophll granule proteins. Although it Is strongly suggested that ECP is closely associated with the inflammatory process of ulcerative colitis, the direct effect of ECP to the intestine remains obscure.
To elucidate the mucosal damage caused by ECP, purification of ECP from normal human eosinophils was performed, and then, to examine the effect of ECP on the contraction of the smooth muscle of the glnea pig small intestine.
Methods : 1. Purification of ECP : ECF was purified from the granules of human buffy coat eosinophils obtained from healthy Individuals. Method of Peterson et al. (1988) was followed for purification The procedure included 1) pretreatment : (1) separation of leukocytes by Dextran treatment from approximately 2l buffy coats of normal subjects, (2) separation of granules by homogenizi
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ng of leukocytes, and (3) 20-fold concentration of the granules by ultra-filtration, and 2) cabin chromatography : (1) gel filtration on Superdex 75, (2) ion exchange chromatography on SOURCE.and (3) chelating chromatography on zinc-chelating Sepharose. ECP was measured by RIA (Pharmacla ECP RIA kit). 2. Examinatlon of the contraction : The strips of the guinea pig small intestine (2cm long were removed from a region 10cm proximal from the lleo-cecal Junction) were placed in a oragan bath In the presence of Krebs-Ringer solution. ApproxImately 600 mg of resting tension was applied and was kept constant. ECP1O mug/l was added to the oragan bath, and mechanical activity was recorded by means of an isometric transducer. In addition, examination whether atropine inhibits the contraction effect of ECP was performed.
Results : 1. Purification of ECP : The ECP activity at the final stage of separating process showed a definite I)COk at fractions No. 47(the concentration of ECP was 4292 mug/l) and No. 48 (that was 3805 mug/l) ECP was existent in the area of molecular weight l6kD to 21.4kD in electrophoresis, and it was considered that a high degree of ECP purification was available. 2. Examination of the contraction : After addition of 10 mug/l ECP, which was the cut off concentration of normal serum, to the organ bath, the transient contraction was observed. The width of the contraction Inducted by ECP was approximately 13% of that of inducted by 10^-^6 M acetylcholine. Furthermore, the ECP contraction effect to the smooth muscle of guinea-pig were inhibited partially by atropine at preliminary experiment.
Conclusion : This is the first demonstration of the effect of ECP to the intestine. ECP conrtacts the smooth of the guipan pig intestene and may contribute the pathopbysiology of ulcerative colitis. Less
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