ground were awakened to the dynamic factors that needed to be considered in interpreting results; clinicians were resolved to dissect arthritic conditions further on clinical grounds so that both disciplines could work together in attempting a more careful correlation of clinical, laboratory, and radiological changes with precise pathological features of the diseases studied.The next conference in the series will be held on 4 May 1989 at Harrogate, and the subject will be announced shortly. In an appropriately selected phase system 8-33% of lymphocytes from normal individuals are found in a discrete peak, having a partition ratio that consists of cells with high affinity for the upper, positively charged phase. This subpopulation contains virtually all the LGLs of the unfractionated lymphocytes6 and is highly enriched with natural killer and antibody dependent cellular cytotoxic activity.7 Further confirming this enrichment is our finding that cells with the natural killer antigens Leu 7 and HNK-1 are found almost exclusively in the peak with a high partition ratio (including those HNK-1+ lymphocytes that coexpress the suppressor/ cytotoxic T cell antigen, CD8).8To examine the partitioning of lymphocytes from subjects with rheumatoid arthritis we studied 13 patients with this disease and compared them with 32 control subjects. Most of the patients were men from a Veterans Administration Hospital clinic who had relatively severe disease. Peripheral blood mononuclear cells were isolated on Ficoll-Hypaque cushions and depleted of monocytes by velocity sedimentation at unit gravity on a Bont apparatus.9 The lymphocytes were then subfractionated in a charge sensitive dextran-polyethylene glycol aqueous phase system by countercurrent distribution (a multistep extraction procedure) as previously described.' "I (The phase system used contained 5% (w/w) dextran, 4% (w/w) polyethylene glycol, 130 mM NaCl, 150 mM sodium phosphate buffer pH 7-4, and 5% (w/w) heat inactivated fetal bovine serum.) The distribution of lymphocytes was plotted as the number of cells in different cavities along the extraction train. Two peaks were obtained and the relative percentage of cells in the two peaks was determined by plotting curves on heavy bond paper, cutting out the two peaks, and weighing them.When countercurrent distribution curves of lymphocytes from patients with rheumatoid arthritis were compared with those from normal individuals we found that the patients had a modest but statistically significant increase in the peak with a high partition ratio. In the 13 patients with rheumatoid arthritis (RA) 18-6 (0-80)% (mean (SD)) of lymphocytes were found in this peak compared with only 15-2 (4 8)% in the 32 controls (p<0-05). Although the patients with RA and normal subjects had comparable percentages of cells with LGL morphology in this peak 53-4 (8-9)
Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems suitable for the separation by partitioning of cells based on subtle differences in their membrane surface properties. Human peripheral blood mononuclear cells were obtained by centrifuging blood over Hypaque-Ficoll and removing the cells from the cushion. Clean lymphocytes were then isolated by velocity sedimentation of the mononuclear cells. Lymphocytes were subjected to countercurrent distribution (i.e., a multiple extraction procedure) in a two-polymer aqueous phase system which separates cells primarily on the bases of surface charge-associated properties. It was found that a small subpopulation of cells with highest partition coefficient (presumably highest surface charge-associated properties) contain virtually all of the natural killer (NK) and K cell activities.
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