移植物化学修饰与阻断 OX40-OX40L途径预防异基因骨髓移植小鼠移植物抗宿主病的研究

目的 观察甲氧基聚乙二醇-琥珀酰亚胺丙酸酸酯(mPEG-SPA)与抗OX40L单抗预处理移植物对小鼠异基因骨髓移植后急性移植物抗宿主病(aGVHD)的影响及其机制.方法 供、受鼠的脾淋巴细胞体外混合培养,加入抗OX40L单抗孵育培养后,再用mPEG-SPA化学修饰,与C57BL/6供鼠骨髓细胞混合后移植给经致死量全身照射的BALB/c受鼠.同时设相应的对照组.观察移植后受鼠aGVHD的临床表现、病理学改变、存活时间及生存率等,检测移植鼠T淋巴细胞亚群,细胞因子的变化及异基因嵌合体.结果 ①单纯骨髓移植对照组小鼠均出现aGVHD临床表现,17 d内全部死于aGVHD,存活时间为(12.1±5.5)d;而mPEG-SPA和抗OX40L单抗单独或联合预处理组小鼠一般状态较好,部分出现aGVHD表现,且较单纯骨髓移植对照组症状轻,皮肤、肝脏、小肠病理表现均较单纯骨髓移植对照组明显减轻,mPEG-SPA和抗OX40L单抗单独或联合预处理组小鼠存活时间分别为(36.2±24.9)、(32.0±24.8)和(44.3±23.2)d,均较单纯骨髓移植对照组明显延长(P＜0.05),其中抗OX40L mPEG-SPA联合预处理组小鼠平均存活时间最长(P＜0.05),mPEG-SPA单独预处理组和抗OX40L单独预处理组牛存率分别为50.0%、41.7%,明显高于单纯骨髓移植组,OX40L mPEGSPA联合预处理组生存率最高,为66.7%.②移植后对照组小鼠血清IFN-γ浓度升高,在+10～+15d时达高峰;而mPEG-SPA和抗OX40L单抗单独或联合预处理组降低,+10 d时降至最低,OX40LmPEG-SPA联合预处理组降低最明显(P＜0.01).移植后对照组IL4、IL-10浓度轻度降低,而mPEG-SPA和抗OX40L单抗单独或联合预处理组均明显升高(P＜0.05),+10～+15 d达高峰,以联合组升高最明显(P＜0.01).③mPEG-SPA和抗OX40L单抗单独或联合预处理组+60 d时异基因嵌合率为95%～100%,证实为完全供者型植入.结论 mPEG-SPA与抗OX40L单抗联合应用能够阻断T细胞激活的抗原和共刺激双信号通路,协同抑制T细胞的增殖活性,促进Th0细胞向Th2细胞分化,从而明显减轻小鼠骨髓移植中aGVHD的发生.     

甲氧基聚乙二醇遮蔽猕猴红细胞表面类人B抗原的研究

目的　观察mPEG对猕猴类人B抗原的修饰效果及mPEG修饰后的红细胞给动物输血的安全性。方法　用 3mg/ml的mPEG修饰遮蔽猕猴红细胞表面的类人B血型抗原 ,吸收放散法检测猕猴血型及mPEG对猕猴红细胞血型的修饰结果 ;红细胞变形仪等检测mPEG修饰对红细胞的变形能力、结构与功能的影响 ;荧光素标记 ,自体回输实验检测红细胞存活率 ;异体回输实验观察修饰后红细胞对受血猴的血液、尿液的影响。结果　mPEG修饰可以遮蔽猕猴类人B抗原的抗原性 ,不影响所修饰红细胞结构、功能及体内存活 ;mPEG修饰的类人B型红细胞输给类人A型血的猕猴 ,未发生输血反应 ;受血猴的血细胞、血液生化及尿液各项指标与输血前相比 ,无明显变化。结论　mPEG修饰可遮蔽猕猴红细胞表面类人B抗原的抗原性 ,mPEG修饰红细胞在受血猴体内是安全的。     

D40—CD40L在移植免疫中的研究进展

CD40－CD40L交联在体液免疫和细胞免疫中是一条重要的细胞信号传导途径，参与了体液免疫和细胞免疫的调节。近年在骨髓移植和器官移植中应用anti-CD40/CD40L单克隆抗体或基因敲除等手段来干预CD40－CD40L的异常表达，发现阻断CD40－CD40L共刺激信号可诱导免疫耐受和抑制移植物抗宿主反应等。因此阻断CD40－CD40L共刺激途径将成为临床移植领域中防治排斥反应的新途径。本文就CD40／CD40L及其交联作用在移植免疫中的研究进展作一综述。     

体外阻断CD137-CD137L途径控制小鼠移植物抗宿主病的研究

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径控制小鼠异基因骨髓移植（allo-BMT）后急性移植物抗宿主病（aGVHD）及其机制。方法供、受鼠的淋巴细胞体外混合培养，分别加入抗CD137L单抗或不加抗CD137L单抗培养后与供鼠骨髓细胞混合移植给受鼠。采用流式细胞术检测移植后受鼠T细胞亚群的变化，RT-PCR法检测细胞因子mRNA表达水平的变化，观察移植后受鼠GVHD的临床及病理改变。结果与未用抗CD137L单抗组相比，应用抗CD137L单抗组CD3^＋CD8^＋T细胞明显降低（P＜0．01）；IFN-γ表达水平明显减低（P＜0．01），IL-10的表达水平明显升高（P＜0．01）。移植后未预防GVHD组（A组）小鼠移植后15d内均死于aGVHD。采用甲氨蝶呤＋环孢素预防GVHD组（B组）小鼠100％发生aGVHD，但临床及病理改变程度较A组轻，平均存活时间[（9．5±2．5）d]较A组[（7．5±1．5）d]略有延长。采用抗CD137L单抗预防GVHD组（C组）受鼠aGVHD的发生率为70％，程度比A、B两组轻，与A、B两组相比生存率明显提高（P＜0．01），平均存活时间[（16．0±2．5）d]明显延长（P＜0．01），30％的小鼠生存时间大于30d。结论抗CD137L单抗体外阻断CD137-CD137L共刺激途径能有效控制小鼠GVHD，可能与影响Ⅰ类和Ⅱ类T细胞因子平衡有关。     

血清OX40L、Occludin与急性缺血性脑卒中患者病情程度及预后的相关性

目的 探讨急性缺血性脑卒中患者血清OX40配体(OX40L)、闭合蛋白(Occludin)的水平,分析其与患者病情严重程度及预后的关系。方法 选取2016年1月至2020年1月在该院接受治疗的135例急性缺血性脑卒中患者为观察组。另选取同期该院体检健康者100例为对照组。采用酶联免疫吸附试验检测血清OX40L、Occludin水平差异。患者出院后6个月采用改良Rankin量表(mRS)评估预后状况,并分为预后良好组93例(mRS评分≤2分)和预后不良组42例(mRS评分>2分)。采用多因素Logistic回归分析影响急性缺血性脑卒中患者预后不良的危险因素。结果 观察组血清OX40L、Occludin水平与对照组相比,差异有统计学意义(P<0.05)。急性缺血性脑卒中重度组血清OX40L、Occludin水平高于中度组、轻度组,中度组血清OX40L、Occludin水平高于轻度组,差异有统计学意义(P<0.05)。预后不良组血清OX40L、Occludin水平明显高于预后良好组,差异有统计学意义(P<0.05)。多因素Logistic回归分析结果显示,美国国立卫生...     

CD40-CD40L在移植免疫中的研究进展

CD40-CD40L交联在体液免疫和细胞免疫中是一条重要的细胞信号传导途径,参与了体液免疫和细胞免疫的调节。近年在骨髓移植和器官移植中应用anti-CD40/CD40L单克隆抗体或基因敲除等手段来干预CD40-CD40L的异常表达,发现阻断CD40-CD40L共刺激信号可诱导免疫耐受和抑制移植物抗宿主反应等,因此阻断CD40-CD40L共刺激途径将成为临床移植领域中防治排斥反应的新途径。本文就CD40/CD40L及其交联作用在移植免疫中的研究进展作一综述。     

CD40一CD40L在移植免疫中的研究进展

CD40-CD40L交联在体液免疫和细胞免疫中是一条重要的细胞信号传导途径,参与了体液免疫和细胞免疫的调节.近年在骨髓移植和器官移植中应用anti-CD40/CD40L单克隆抗体或基因敲除等手段来干预CD40-CD40L的异常表达,发现阻断CD40-CD40L共刺激信号可诱导免疫耐受和抑制移植物抗宿主反应等,因此阻断CD40-CD40L共刺激途径将成为临床移植领域中防治排斥反应的新途径.本文就CD40/CD40L及其交联作用在移植免疫中的研究进展作一综述.     

抗CD40L单抗4F1体外诱导再生障碍性贫血患者T细胞免疫沉默和促进造血恢复的研究

目前认为，原发性再生障碍性贫血（再障）的主要发病机制为T细胞介导的骨髓特异性自身免疫反应。根据免疫学基本原理，共刺激信号是抗原特异性T细胞活化过程中的必要条件。虽然对再障共刺激信号系统的改变认识仍非常有限，但已有证据表明，患者的共刺激信号系统存在异常。CD40／CD40L是共刺激信号系统中一个重要的调节分子对。我们通过观察抗CD40L单抗4FI对再障患者骨髓T淋巴细胞表型及集落形成的影响，研究其对再障T淋巴细胞异常免疫反应的抑制和造血恢复的促进作用。     

体外阻断CD40-CD40L共刺激途径减轻小鼠异基因骨髓移植中移植物抗宿主病的研究

目的在异基因骨髓移植移植物抗宿主病(GVHD)小鼠模型中,观察抗CD40L单克隆抗体(单抗)体外预处理供鼠T细胞输注,观察其减轻GVHD的作用并探讨其作用机制.方法供鼠(C57BL/6H-2b)脾T细胞作为反应细胞,受鼠(BALB/cH-2d)脾细胞作为刺激细胞,分别加抗CD40L单抗或不加单抗进行混合培养,培养第5天的细胞作为经体外诱导后的脾T淋巴细胞,分别混合供鼠骨髓细胞后移植给接受8.0 Gy全身照射预处理的受鼠.比较受鼠GVHD发生和造血重建.在移植后不同时间点采用流式细胞仪检测受鼠脾细胞中T细胞表型的改变,用ELISA法检测外周血血清中Th1和Th2类细胞因子的水平.结果移植后对照组受鼠均于25 d内死于GVHD,实验组GVHD的发生率为20%,与对照组小鼠相比,存活率和存活时间明显增高和延长(P<0.01);存活的实验组小鼠(8只)第40天时骨髓细胞中H-2Db阳性细胞为(93.54±2.32)%.实验组小鼠CD3+CD4+、CD3+CD8+、CD4+CD25+、CD4+CD69+、CD8+CD25+、CD4+CD40L+和CD8+CD69+T细胞比例明显低于对照组(P<0.05),CD8+CD40L+和CD4+CD45RA+T细胞比例在两组中变化差异无显著性(P>0.05).实验组受鼠血清中细胞因子水平明显低于对照组(P<0.05).结论应用抗CD40L单抗体外孵育的脾T细胞与骨髓细胞混合移植,可明显减少GVHD的发生率,且不影响供鼠造血干细胞的植入,CD4+和CD8+T细胞对异体抗原发生耐受,耐受化T 细胞的活化障碍发生于活化的早期和成熟阶段,同时抑制了Th1和Th2类细胞因子分泌,为抗CD40L单抗应用于临床移植预防GVHD提供了实验依据.     

阻断CD40／CD40L通道诱导同种胰岛移植免疫耐受的研究

目的：观察阻断CD40／CD40L共刺激通路对同种胰岛移植免疫反应的影响,探讨CD40L单克隆抗体对诱导同种移植免疫耐受的作用。方法：清洁级Wistar大鼠,STZ 60mg／kg诱导1型糖尿病模型,随机分为三组,A组：糖尿病大鼠,B组：糖尿病大鼠＋胰岛细胞＋抗CD40L抗体,C组：糖尿病大鼠＋胰岛细胞。B组在移植前24h注入抗CD40L抗体每只20μg。同种胰岛细胞来自SD大鼠,胶原酶V消化法和Ficoll400密度梯度离心法分离纯化胰岛细胞,收获量为（3．49±0．23）×10^5 IEQ／kg,将同种胰岛细胞按12000IEQ／kg导入受体,移植后每天和每周相同时间点检测血糖和体重变化,3d、7d、15d、22d后检测白介素-2的表达。结果：同种异体胰岛移植可使1型糖尿病大鼠的血糖降至正常并维持一段时间,A组为（23．84±2．74）mmol／L,B组（8．15±1．40）mmol／L,维持（18．12±4．12）d,C组（7．42±2．55）mmol／L,维持（8．00±1．30）d,后两组正常血糖维持天数比较,P〈0．001,有统计学意义。体重变化比较：非移植的A组随着时间的延长,体重不断下降,B、C组至移植后体重有所回升,但移植4周后C组的体重再次下降。移植7d、15d后,IL-2的表达B组和C组比较有显著性差异,B组为（0．1280±0．0104）和（0．1235±0．0073）,C组为（0．1744±0．0096）和（0．1667±0．0098）（P〈0．001）,移植22d后两组比较差异无显著性。结论：CD40L单克隆抗体阻断CD40／CD40L共刺激通路显著延长移植的同种胰岛细胞的存活时间,诱导机体产生针对胰岛移植物的免疫耐受状态。     

甲氧基聚乙二醇修饰红细胞解决自身免疫性疾病所致配血困难研究

目的观察红细胞(RBC)经甲氧基聚乙二醇-琥珀酰亚胺丙酸酯(mPEG-SPA)修饰后对血型抗原的遮蔽作用,评估其解决自身免疫性疾病等所致配血困难的效果。方法以mPEG-SPA体外修饰正常人RBC,用微柱凝胶血型卡检测对A、B、RhD、RhC、RhE血型抗原的免疫遮蔽作用,并比较不同分子量不同浓度mPEG-SPA的遮蔽效果;观察修饰前后RBC的变形性、渗透脆性、自身溶血率,判断修饰对RBC功能的影响;比较修饰前后自身抗体阳性患者血清与相同ABO血型标本交叉配血时的凝集情况。结果mPEG-SPA修饰不影响RBC的变形性、渗透脆性、自身溶血率等特性,对A和B血型抗原基本没有遮蔽作用,而分子量20000的mPEG-SPA浓度达2mmol/L或分子量10000的mPEG-SPA为6mmol/L时,能有效地遮蔽RhD、RhC和RhE抗原,可有效消除受血者自身抗体和其他相应抗体所致的凝集现象。结论mPEG-SPA修饰可有效遮蔽Rh血型抗原并解决自身抗体所致的配血困难。     

Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol

Lysostaphin is a 27-kDa endopeptidase that enzymatically disrupts the cell wall of Staphylococcus aureus and is a promising candidate for treating S. aureus blood-borne infections. It would be extremely useful to define conditions that would both increase lysostaphin's in vivo half-life to allow for more effective tissue distribution and reduce its immunogenicity. Conjugation of polyethylene glycol (PEG) to lysostaphin (PEGylation) was investigated as a means to accomplish these goals. Rather than using linear forms of PEG, branched PEGs were chosen as the initial candidates because their large spatial volumes prevent entry of the polymer into the enzyme's active sites, which could potentially reduce enzymatic function. Enzymatic activity for most PEGylated lysostaphins was reduced, but these compounds were still considerably active compared to unconjugated lysostaphin, with conjugates that had lower degrees of PEG modification having greater activity than those with higher degrees. PEGylated lysostaphin injected intravenously had a serum drug half-life of up to 24 h and resulted in much higher plasma drug concentrations than an equal dose of unconjugated lysostaphin, which had a half-life of less than 1 h. Finally, reduced binding affinity was shown for PEGylated lysostaphin in an antilysostaphin capture enzyme-linked immunosorbent assay, with some PEG-lysostaphin conjugates having binding affinities that were reduced more than 10-fold compared to unconjugated lysostaphin. These findings demonstrate that PEGylation of lysostaphin, while diminishing its S. aureus killing activity, results in prolonged serum drug persistence and reduced antibody binding. These features should significantly enhance lysostaphin's therapeutic value as an intravenous "antibiotic" against S. aureus.     

Long-term humoral unresponsiveness in vivo, induced by treatment with monoclonal antibody against L3T4

mAbs directed against the L3T4 molecule administered in vivo caused a severe and long lasting helper cell depletion in mice. Regeneration of the L3T4+ subpopulation occurred gradually (2-3 mo) after a single antibody treatment. Experiments were designed to examine the humoral immunocompetence of such anti-L3T4-treated animals during and after regeneration of the L3T4+ T cell subset. The animals were injected with anti-L3T4, immunized with soluble antigen, and challenged with antigen every 2 wk. Antibody responses to two antigens, sperm whale myoglobin (SpWMb) and KLH, which differ with regard to their immunogenicity, were compared. The lack of humoral immune responsiveness to either of these two antigens shorty after anti-L3T4 treatment responsiveness to either of these two antigens shortly after anti-L3T4 treatment was probably due to clonal depletion. The anti-L3T4-induced immunosuppressive effect on antibody production seemed to be determined in part by the preexisting T cell repertoire, as was suggested by the recovery of responsiveness to the highly immunogenic antigen KLH and the transient inhibitory effect of anti-L3T4 treatment in primed animals. The regenerating L3T4+ T cell subpopulation was relatively incompetent in initiating B cell responses. More than 40% of the L3T4+ T cell compartment had to recover to provide help for the production of anti-KLH antibodies, whereas elimination of 90% of the L3T4+ helper cells did not inhibit a primary anti-KLH response. Evidence for a heterogeneous composition of the L3T4+ subset came from experiments using rIL-2 in vivo. The addition of rIL-2 during early helper cell depletion improved the recovery of the humoral responsiveness without apparently affecting the kinetics of the regeneration of L3T4+ T cells. Interestingly, humoral unresponsiveness to the weakly immunogenic antigen SpWMb persisted for at least 120 d. This long lasting unresponsiveness could not be explained by clonal depletion, and suggested as one possibility that the presence of antigen during regeneration of the L3T4+ helper cell population may have influenced the ultimate T cell repertoire.     

Studies of osmotic diarrhea induced in normal subjects by ingestion of polyethylene glycol and lactulose.

The purpose of these studies was to gain insight into the pathophysiology of pure osmotic diarrhea and the osmotic diarrhea caused by carbohydrate malabsorption. Diarrhea was induced in normal volunteers by ingestion of polyethylene glycol (PEG), which is nonabsorbable, not metabolized by colonic bacteria, and carries no electrical charge. In PEG-induced diarrhea, (a) stool weight was directly correlated with the total mass of PEG ingested; (b) PEG contributed 40-60% of the osmolality of the fecal fluid, the remainder being contributed by other solutes either of dietary, endogenous, or bacterial origin; and (c) fecal sodium, potassium, and chloride were avidly conserved by the intestine, in spite of stool water losses exceeding 1,200 g/d. Diarrhea was also induced in normal subjects by ingestion of lactulose, a disaccharide that is not absorbed by the small intestine but is metabolized by colonic bacteria. In lactulose-induced diarrhea, (a) a maximum of approximate 80 g/d of lactulose was metabolized by colonic bacteria to noncarbohydrate moieties such as organic acids; (b) the organic acids were partially absorbed in the colon; (c) unabsorbed organic acids obligated the accumulation of inorganic cations (Na greater than Ca greater than K greater than Mg) in the diarrheal fluid; (d) diarrhea associated with low doses of lactulose was mainly due to unabsorbed organic acids and associated cations, whereas with larger doses of lactulose unmetabolized carbohydrates also played a major role; and (e) the net effect of bacterial metabolism of lactulose and partial absorption of organic acids on stool water output was done dependent. With low or moderate doses of lactulose, stool water losses were reduced by as much as 600 g/d (compared with equimolar osmotic loads of PEG); with large dose, the increment in osmotically active solutes within the lumen exceeded the increment of the ingested osmotic load, and the severity of diarrhea was augmented.     

Comparison of the polyethylene glycol antiglobulin test and the use of enzymes in antibody detection and identification

BACKGROUND: The polyethylene glycol indirect antiglobulin test for detection of red cell antibodies was compared with a proven, highly sensitive test system using papain. STUDY DESIGN AND METHODS: Parallel, prospective testing of 1508 samples with polyethylene glycol and with albumin and papain evaluated the sensitivity and specificity of polyethylene glycol. Retrospective analysis of antibody specificities was performed for the 2 years before and the 2 years after the institution of polyethylene glycol testing. RESULTS: Of 1508 prospective screens, 53 (3.5%) had discordant results: 5 were positive only in polyethylene glycol and 48 were positive only in albumin and papain. Upon antibody identification, the 5 samples that were positive only in polyethylene glycol showed 1 anti-D, 2 warm autoantibodies, and 2 false-positive results. The 48 samples that were positive only in albumin and papain showed 1 each of the following: anti-Le(b); anti-P1; anti-S; high-titer, low-avidity antibody; and cold autoantibody; there were 43 false-positive results. False-positive results totaled 12 (0.8%) with polyethylene glycol and 53 (3.5%) with albumin and papain. The retrospective analysis of antibody specificity with polyethylene glycol showed a significant increase in the detection of Fy(a) and/or Fy(b) (p < 0.0002) and Jk(b) (p < 0.0002) antibodies and a decrease in the detection of Le(a) and/or Le(b) antibodies (p < 0.0002). CONCLUSION: Polyethylene glycol retained the high sensitivity of the albumin and papain, while significantly lowering the number of false- positive results and decreasing the detection of antibodies of doubtful clinical significance.     

聚乙二醇衍生物修饰红细胞的保存性能研究

目的 评估经甲氧基聚乙二醇-琥珀酰亚胺丙酸酯(mPEG-SPA)修饰后红细胞(RBC)的保存性能.方法 以mPEG-SPA体外修饰健康人RBC;观察修饰前后RBC的变形性、渗透脆性、自身溶血率、磷脂酰丝氨酸和CD59,评估修饰对RBC保存性能的影响.结果 mPEG-SPA修饰后RBC的变形性略有下降、渗透脆性和自身溶血率有所增加;修饰后红细胞磷脂酰丝氨酸表达明显增加;红细胞CD59的表达严重下降,其表达率几近于零.结论 mPEG-SPA修饰后红细胞的保存性能下降,可能影响修饰后红细胞的寿命和输入体内的存活率.