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3H thymidine (1 μCi per well) during the last 15 hours before being harvested onto filters.
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Total cellular RNA was isolated from gut tissue and cells with guanidine isothiocyanate and phenol. Constructs encoding standard cytokine mRNAs [L. Eckmann, J. Fierer, M. F. Kagnoff. J. Immunol. 156, 2894 (1996)] and a KGF RNA construct [M. Bajaj-Elliott, R. Poulsom, S. L. F. Pender, N. C. Wathen, T. T. MacDonald, J. Clin. Invent. 102, 1473 (1998)] were used for competitive quantitative reverse transcription polymerase chain reaction (RT-PCR). The KGF construct and primers were designed to allow us to detect both mouse and human KGF.
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0344364331
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note
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Using the same culture conditions as detailed in (9), spleen cells gave a response to CD3 of 6939 ± 915 cpm (background, 1507 ± 808). CD3 plus int280 (5 μg/ml) boosted responses to 16,162 ± 2108, whereas the response to CD3 plus int280CS was only 9853 ± 650.
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18
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0344364329
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note
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2. The density of positive cells in the lamina propria was determined by image analysis.
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0344364328
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note
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Resident colonic lamina propria T cells did not respond to CD3 stimulation in vitro nor did they respond to int280 alone over a dose range of 1 to 25 μg/ml, but they did give a proliferative response to Con A (5 μg/ml). This response was not enhanced by int280. However, int280 strongly costimulates T cells from the colonic lymphoid follicles. In response to CD3 activation [details as in (9)], colonic lymphoid follicle T cells gave 9097 ± 1327 cpm, but in the presence of int280 the CD3 response increased to 30,252 ± 4678 cpm.
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note
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G. F. and G. D. are supported by the Wellcome Trust. L.M.H and N.S.G. are supported by the Crohn's in Childhood Research Association. We thank S. Daniell for the purification of int280 and M.F. Kagnoff for the gift of the plasmids for cytokine RT-PCR.
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