Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"- terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.     

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3- dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL- 3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM- CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.     

Delineation of an immunodominant and human T-cell lymphotropic virus (HTLV)-specific epitope within the HTLV-I transmembrane glycoprotein

Antibody reactivity to the transmembrane region of human T-cell lymphotropic virus type I (HTLV-I) envelope, gp21, is observed in virtually all individuals infected with HTLV-I or HTLV-II. Recombinant proteins encoding selected portions of gp21 are described and used to define two immunogenic regions. The first epitope (designated GD21-I) contains amino acids 361 to 404 of the HTLV-I envelope and reacted with all of 54 sera from HTLV-I- and HTLV-II-infected individuals. The second epitope (designated BA21) expresses amino acids 397 to 430 of the HTLV-I envelope and was recognized by 33 of 54 HTLV antisera. To determine the specificity of GD21-I and BA21, sera from 17 HTLV- negative individuals with nonspecific reactivity to p21E were tested. None of these sera reacted with GD21-I, but 16 of 17 sera reacted with BA21. With virtually complete reactivity to sera from HTLV-infected individuals and no reactivity to sera from p21E-reactive uninfected individuals, GD21-I will be useful in immunoassays for the detection of HTLV infection.     

Use of recombinant human erythropoietin in allogeneic bone marrow transplant donor/recipient pairs

In an attempt to reduce or eliminate homologous red blood cell transfusion requirements during allogeneic bone marrow transplantation (BMT), we instituted a novel program whereby recombinant human erythropoietin was administered to pairs of BMT donors and recipients. Eleven recipients and their HLA-matched donors were enrolled. Donors treated with recombinant human erythropoietin (rHuEPO) were phlebotomized a median of 6 U (range, 4 to 11 U) of blood over a 5-week period. This donor-derived blood was available to the BMT donor or recipient as needed. Transplant recipients were also treated with rHuEPO post-BMT to hasten erythropoiesis. Five of 11 BMT recipients underwent transplant receiving only donor-derived red blood cell transfusion, compared with 0 of 11 concomitant control recipients (P = .04). In addition, the time to absolute reticulocyte count > or = 10(4)/microL was statistically shorter in the rHuEPO-treated recipient group. This study serves as a paradigm for hematopoietic growth factor use in allogeneic BMT to decrease or eliminate homologous transfusion exposures and to possibly hasten hematopoietic engraftment.     

CD8+ T Cell Recognition of Epitopes Within the Capsid of Adeno-associated Virus 8–based Gene Transfer Vectors Depends on Vectors' Genome

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8⁺ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8⁺ T cells. Our results clearly show that depending on the vectors'' genome, CD8⁺ T cells can detect such epitopes on AAV8''s capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.     

精神分裂症患者的社交技能训练小组活动方案

目的:通过社交技能训练让精神分裂症患者明确正确的沟通方式,锻炼自己的交谈能力,学会社会交往技巧,体会人与人之间的关系,学会分析解决社交过程中出现的问题,从而掌握社会交往的技能,防止或延缓发生严重的社会功能衰退。方法:选择早期精神分裂症患者(5年内),在住院治疗达到临床治愈出院后立即开始训练。活动以小组为单位(10人1组)进行,小组成员相对固定。前3个月每个月1次,以后每3个月1次,持续1年。活动过程中以游戏为主导,让组员在游戏中领会人际交往过程中的要领。具体训练方案包括6个方面:①训练一:语言表达能力,正确的沟通方式。②训练二:如何寻找帮助。③训练三:指导患者学习人际交往的基本技巧。④训练四:如何与人打交道。⑤训练五:合作。⑥训练六:社交问题的解决。结果:通过增加对社交时恐惧的暴露及社交技巧训练,对精神分裂症伴发社交恐惧症有效;但对精神分裂症意志活动减退所致社交时主动性不足效果较差。结论:对精神分裂症患者的社交训练应尽早进行,同时在设计精神分裂症社交训练时应加强对患者主动性不足的针对性。     

肺纤维化鼠Ⅰ型前胶原和Ⅲ型前胶原mRNA表达及芪丹颗粒剂的干预效应

目的:观察芪丹颗粒对博莱霉素致肺纤维化鼠的Ⅰ型前胶原和Ⅲ型前胶原mRNA表达的影响,探讨其作用机制。方法:实验于2004-10/2006-10在山东省医学科学院基础所病理实验室完成。实验材料:清洁级雄性SD大鼠160只,体质量180～210g。氢化可的松琥珀酸钠50mg/支;芪丹(颗粒剂)是由黄芪、丹参、川芎等中草药加工提纯后的粗提取物制成的颗粒剂,10g/包(1g干粉相当于原生药8g)。实验分组:160只SD大鼠按随机区组设计分为正常组20只、模型组40只、芪丹颗粒剂50只、氢化可的松50只。实验干预:正常组20只气管内灌注和灌胃均用生理盐水。其余大鼠经气管内一次性灌注博莱霉素A5按0.25mL左右(5mg/kg体质量)诱导大鼠肺间质纤维化。随机取40只为模型组。取芪丹颗粒剂组和氢化可地松组大鼠各30只,分别从造模后第2天灌注芪丹颗粒剂(3125mg/kg)和腹腔注射氢化可的松(25mg/kg),药物干预后第7,14,28天麻醉下处死动物。两组各余20只分别从造模14d后灌注芪丹颗粒剂和腹腔注射氢化可的松(用量同前),于第28、42天分别处死动物。实验评估:用苏木精-伊红评价肺组织病理学变化和原位杂交方法检测各组大鼠肺Ⅰ型和Ⅲ型前胶原mRNA表达。结果:160只大鼠全部进入结果分析。①大鼠肺脏大体标本观察:对照组肺组织各观察时间点无明显改变,模型组肺组织表面凸凹不平,部分肺叶体积缩小,表面见灰白色结节。氢化可的松组与模型组相似。芪丹颗粒剂组见部分肺叶表面不光滑及大小不等结节。②肺组织病理学观察:模型组7d肺泡腔内大量巨噬细胞淋巴细胞中性粒细胞浸润,肺间质成纤维细胞增殖,28d肺泡结构破坏肺泡内见大量胶原纤维和成纤维细胞。芪丹颗粒剂组肺泡炎及肺纤维化程度均明显轻于模型组和氢化可的松组(P<0.05)。③肺间质纤维化形成中Ⅰ型、Ⅲ型前胶原mRNA表达:原位杂交显示两种前胶原mRNA表达呈动态变化,早期肺泡炎以Ⅲ型前胶原mRNA大量增生为主,晚期纤维化期以Ⅰ型前胶原mRNA增生为主。芪丹颗粒剂组Ⅲ型前胶原mRNA的表达在第14天处于最高,至28d仍维持较高的水平,芪丹颗粒剂组Ⅲ型前胶原mRNA的表达高于模型组和氢化可的松组(P<0.05)。28d,Ⅰ型前胶原mRNA的表达在第二天给药芪丹颗粒剂组和氢化可地松组及第14天给药芪丹颗粒剂组和氢化可的松组组间差异均有显著性(P<0.05)。结论:大鼠肺纤维化的早期以Ⅲ型前胶原mRNA的表达为主,晚期纤维化期以Ⅰ型前胶原mRNA的大量表达为主。芪丹颗粒可减轻博莱霉素诱导的大鼠肺泡炎及肺纤维化的程度,其机制可能通过影响了Ⅰ型和Ⅲ型前胶原mRNA的代谢和表达,从而减慢肺间质纤维化的进程。芪丹颗粒对肺间质纤维化有治疗作用,且优于氢化可的松。