Rapid method for isolation of normal human peripheral blood eosinophils on discontinuous Percoll gradients and comparison with neutrophils

Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin- activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu- [3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu- Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.     

Effects on the buoyant density of rabbit platelets of ADP and agents that increase the concentration of cyclic AMP

Rabbit platelets were aggregated by adenosine diphosphate (ADP), allowed to deaggregate and then separated into density subpopulations by centrifugation through discontinuous Stractan density gradients. Although ADP causes little or no release of the contents of the amine storage granules of rabbit platelets, ADP caused a decrease in platelet density as compared with control platelets subjected to the same procedures except for exposure to ADP. The density change persisted for at least four hours. The apparent size of platelets stimulated with ADP increased initially, but returned to control values during a one-hour period. A similar decrease in platelet density was observed with an albumin density gradient. Under conditions in which aggregation did not occur in response to ADP with ethylenediaminetetraacetic acid (EDTA) in the medium, little or no decrease in platelet density was observed. Agglutination with polylysine did not change platelet density. Thus, not only agents such as thrombin and plasmin that cause the release of the contents of the platelet granules decrease platelet density, but ADP also has this effect. Platelets would be exposed to all of these stimuli during thromboembolic processes, and their effect on platelets may account for the decrease in platelet density observed previously in experiments with rabbits with indwelling aortic catheters. Agents that increase the concentration of cyclic AMP (cAMP) in platelets (PGE1, adenosine, dibutyryl cAMP, forskolin, and papaverine) also decreased platelet density. This effect persisted when the platelets were washed and resuspended in fresh medium and was also demonstrable in plasma. Platelet size was gradually increased by prostaglandin E1 (PGE1) which maintains platelets in a disc shape and does not cause the release of granule contents, indicating that the decrease in platelet density caused by PGE1 may be attributable to platelet swelling.     

尖头梅花髓内针与带锁髓内针治疗胫腓骨骨折比较

目的　比较改良的尖头梅花针与带锁髓内针治疗胫腓骨干骨折的临床综合疗效 ,正确选取内固定物。方法　分别用改良的尖头梅花针与带锁髓内钉内固定治疗胫腓骨干骨折 74例 ,比较两组的综合疗效。结果　改良梅花针组 48例 ,轻度外旋畸形 2例 ,无骨不愈合 ,平均骨折愈合时间 3.5个月 ,平均手术时间 40 mim ,住院费用 30 0 0元 (人民币 ) ,优良率93.75 % ;带锁髓内钉组 2 1例 ,发生骨延期愈合 2例 ,平均骨折愈合时间 5个月 ,平均手术时间 6 0 mim ,住院费用 5 5 0 0元(人民币 ) ,优良率 95 .2 4%。除胫骨远端下 1/ 3或严重的粉碎性骨折外 ,两组疗效无明显差异 (P >0 .0 5 )。结论　改良尖梅花针内固定操作简单、价格低廉、疗效确切 ,仍有较高的临床使用价值 ;带锁髓内钉能满足胫骨各种类型的骨折 ,扩大了髓内固定的适应症 ,随着成本的降低 ,已逐渐成为治疗胫腓骨骨折髓内固定的方向。     

Interleukin-4 enhances the survival of severe combined immunodeficient mice engrafted with human B-cell precursor leukemia

Human interleukin-4 (huIL-4) has been shown to inhibit the growth in vitro of cells from patients with acute lymphoblastic leukemia (ALL). With the aim of determining whether this cytokine might be useful in the treatment of patients with ALL, the effects of huIL-4 on human B- cell precursor ALL engrafted in severe combined immunodeficient (SCID) mice were examined. The inhibition of [3H] thymidine uptake of primary ALL cells by huIL-4 was maintained following engraftment and passage of leukemia in SCID mice. Five of seven xenograft leukemias showed significant inhibition in vitro by huIL-4 at concentrations as low as 0.5 ng/mL; furthermore, huIL-4 counteracted the proliferative effects of IL-7. When used to treat two human leukemias engrafted in SCID mice, huIL-4 200 microgram/kg/d, as a continuous 14-day subcutaneous infusion, suppressed the appearance of circulating lymphoblasts and extended survival of mice by 39% and 108%, respectively, the first demonstration of IL-4 activity against human leukemia in vivo. The mean steady-state huIL-4 level in mouse plasma during the infusion was 1.46 ng/mL (SEM +/- 0.14 ng/mL), which was similar to concentrations found to be effective in vitro. ALL cells obtained from mice relapsing after huIL-4 treatment continued to show inhibition by the cytokine in vitro. These data suggest that IL-4 may be useful in the treatment of patients with ALL.