Stimulation of megakaryocytopoiesis by acute thrombocytopenia in rats

Rats were made acutely thrombocytopenic by injection of antiplatelet serum. Marrow sections and squash preparations were made at intervals during 120 hr. Determinations were made of mitotic index, stage of maturation, ploidy level, and cell size of megakaryocytes; number and size of platelets were measured. Increased endomitosis among megakaryocytes was followed by an increase in the proportion of immature megakaryocytes, a greater average ploidy level of recognized megakaryocytes, and larger megakaryocytes. Maximum changes in these several parameters occurred between 32 and 72 hr after induction of thrombocytopenia. By 120 hr all megakarocyte parameters were near normal. For about 3 days, beginning at about 36 hr, platelet numbers increased rapidly. Average platelet size rose and returned to normal within about 60 hr. Changes in ploidy and size of megakaryocytes were measured in the immature and mature maturation stages. The results suggest that the initial stimulus in response to acute thrombocytopenia acts primarily on diploid precursors, programming them to mature into a population of megakaryocytes with an average ploidy approximately one level greater than in normal rats and a proportionate increase in cell size. The larger megakaryocytes presumably produce more platelets, accounting for a major part of the increased rate of platelet production. Since the changes in megakaryocytes begin to reverse before circulating platelet numbers have reached the normal level, reversal of the stimulus appears to be initiated by some change other than platelet mass.     

Bone matrix degradation by the plasminogen activation system. Possible mechanism of bone destruction in arthritis

The observed increase in urokinase-type plasminogen activator (u-PA) and its receptor (u-PAR) in synovial tissue of patients with rheumatoid arthritis (RA) suggests pathophysiological involvement of the plasminogen activation (PA) system in inflammatory joint disease. In the present study, we investigated the capacity of the PA system to degrade non-mineralized and mineralized bone-like matrix in vitro as a model for bone destruction. Transfected mouse LB6 cell lines, that expressed either human u-PA or u-PAR, were cultured separately and simultaneously on radiolabelled bone matrix in the presence of plasminogen. Osteoblast-like murine calvarial MC3T3-E1 cells were used to produce a well-characterized, highly organized bone-like matrix, that could be mineralized in the presence of beta-glycerol phosphate. Bone matrix degradation was followed by the release of radioactivity in the culture medium. u-PA-producing cells, in contrast to u-PAR- producing cells, degraded both non-mineralized and mineralized bone matrix. This effect could be inhibited by anti-u-PA antibodies, as well as by tranexamic acid and by aprotinin, indicating that the degrading activity is u-PA mediated and plasmin dependent. Co-cultivation of a small portion of u-PA-producing cells with u-PAR-expressing cells resulted in a marked increase in degradation activity. Reduction of this potentiating effect by suramin or the amino-terminal fragment of u- PA, both competitive inhibitors of u-PA receptor binding, shows that this synergistic effect is due to binding of u-PA to u-PAR. u-PAR must be cell associated, as binding of u-PA to a soluble u-PAR prevented this enhancement. The capability of the PA system to degrade bone matrix in vitro, and the previously demonstrated increased expression of u-PA and u-PAR in synovial tissue of patients with RA, further support a role for the PA system in the development of bone erosions.      

胰岛素在严重烧伤大鼠急性肺损伤中的保护作用

目的:观察胰岛素对严重烧伤所致的急性肺损伤的保护效应。方法:实验于2006-01/08在解放军第四军医大学西京医院全军烧伤中心实验室完成。取成年雄性SD大鼠36只,随机分成3组,每组12只:①烫伤 胰岛素组:水浴锅94℃,20s,制备30%总体表面积全层皮肤烫伤模型,伤后即刻腹腔注射生理盐水40mL/kg,并皮下注射胰岛素3U/kg。②烫伤组:造模和腹腔注射同烫伤 胰岛素组,皮下注射等体积生理盐水。③假烫组:用室温水浴模拟烫伤过程,伤后不给予补液。烫伤24h后,收集动脉血测定超氧化物歧化酶活性,收集支气管肺泡灌洗液测定蛋白含量,取肺组织进行苏木精-伊红染色观察病理变化,并测定髓过氧化物酶活性,同时电镜观察胸主动脉血管内皮细胞变化情况。结果:36只大鼠进入结果分析。①肺病理变化:烫伤 胰岛素组肺脏渗出和水肿较烫伤组明显减轻。②肺泡灌洗液中蛋白的质量浓度:烫伤组和烫伤 胰岛素组高于假烫组[(702.9±169.5),(486.5±149.2),(240.5±140.7)mg/L,P<0.05],烫伤 胰岛素组低于烫伤组(P<0.05)。③肺脏髓过氧化物酶活性:烫伤 胰岛素组低于烫伤组[(36.01±8.17),(59.51±12.50)nkat/g,P<0.05],与假烫组比较差异不显著。④血清超氧化物歧化酶活性:烫伤组和烫伤 胰岛素组低于假烫组[(2.27±0.18),(2.63±0.19),(2.81±0.21)mkat/L,P<0.01,0.05],烫伤 胰岛素组高于烫伤组(P<0.01)。⑤电镜下可见烫伤 胰岛素组内皮细胞损伤较烫伤组轻。结论:严重烧伤早期外源性胰岛素干预后可减轻急性肺损伤,具有明显的肺脏保护作用,这种作用可能与胰岛素的内皮细胞保护效应有关。     

人外周血树突状细胞的分离与鉴定

目的:体外分离培养并鉴定人外周血树突状细胞,并观察其抗原呈递功能。方法:实验于2005-05/2006-11在南方医科大学南方医院肿瘤中心生物治疗实验室完成。从人类白细胞抗原A2表达阳性的健康人外周血中分离获得单个核细胞。培养5h后洗涤贴壁细胞,加入含有10%人AB血清的RPMI1640培养基,及重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素4,于培养的第1,3,6天对树突状细胞的形态、表型进行分析,并定期检测树突状细胞的纯度与得率。抽取与以上树突状细胞不同来源的其他健康人外周血。经淋巴细胞分离液分离后,获取非贴壁细胞,用含10%人AB血清的1640培养基重悬,加入白细胞介素2继续孵育6d,作为同种异体T淋巴细胞。将树突状细胞分为两组,一组按常规方法培养6d,另一组在培养至第5天时加入黑色素瘤抗原基因A3编码的多肽继续培养24h。在经紫外线处理后的96孔板中,分别加入树突状细胞悬液1×104,5×103,2×103,1×103细胞/每孔,以自身T淋巴细胞作为对照,每孔设3个复孔,分别加入1×105淋巴细胞/每孔。评价树突状细胞刺激T淋巴细胞增殖的能力。结果:①单个核细胞体外培养至第6天,可获得大量、90.81%高纯度的树突状细胞,能够较高地表达21.8?1a、99.0%HLA-DR、63.4?80、18.9?83和80.6?86。②将诱导培养6d获得的两组树突状细胞作为刺激细胞,以不同的浓度与同种异体淋巴细胞混合,均可产生增殖反应;经过黑色素瘤抗原基因A3编码的多肽处理的各种比例的树突状细胞,较相应未经黑色素瘤抗原基因A3编码的多肽处理的树突状细胞激发淋巴细胞增殖的能力明显增强,浓度相对较高的树突状细胞刺激效果最明显,能够强烈地激发同种混合淋巴细胞增殖。结论:得到了一群较高程度表达CD83、CD86和HLA-DR分子、体外可强烈激发同种异体T淋巴细胞增殖的树突状细胞群。     

Low risk of viral infection after administration of vapor-heated factor VIII concentrate. International Investigator Group

A multicenter prospective study was carried out to evaluate whether a vapor-heated factor VIII concentrate transmitted blood-borne viral infections over a surveillance period of 15 months. Thirty-five patients with hemophilia and von Willebrand disease who had never received any blood components were treated. Twenty-eight were analyzed and found not to have non-A, non-B hepatitis. Sera from 20 of these 28 patients were also tested for the antibody to the hepatitis C virus. None had sero-converted during the follow-up period. None of the patients analyzed developed markers of the hepatitis B virus (n = 17) or the human immunodeficiency virus (n = 31). This vapor-heated factor VIII concentrate carries a low risk of transmitting hepatitis and human immunodeficiency virus infection.     

Allogeneic marrow grafting with partially mismatched, unrelated marrow donors

Forty patients with advanced hematologic malignancies or severe aplastic anemia received marrow grafts from partially mismatched, unrelated marrow donors. All patients were administered conventional prophylaxis for acute graft-v-host disease (GVHD) consisting of methotrexate and low-dose glucocorticoids. All but two patients who survived at least 30 days showed durable engraftment. Six patients survive 17+ to 36+ months following transplantation. Severe acute GVHD was seen in 47% of the patients; however, no direct correlation between GVHD and the degree of mismatching could be determined. Fatal infections were seen in 29 patients, and in the majority the infection occurred after the granulocyte count had risen to greater than 500 cells/microL. We conclude that the problems encountered in this pilot study can potentially be solved, and that further studies with this type of marrow grafting are warranted.