Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA‐E/human CD46 double‐transgenic pig limbs with human blood

Background

In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood.

Methods

Six pig forelimbs per group, respectively, stemming from either wild‐type (wt) or HLA‐E/hCD46 double‐transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells.

Results

Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA‐E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells.

Conclusions

Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell‐mediated rejection mechanisms of vascularized pig‐to‐human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA‐E/hCD46 in pig limbs provides partial protection from human NK cell‐mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.     

Food deprivation increases post-heparin lipoprotein lipase activity in humans

OBJECTIVE: To study the effect of fasting on lipoprotein lipase (LPL) activity in human post-heparin plasma, representing the functional pool of LPL. DESIGN: Fourteen healthy volunteers were recruited for the study. The subjects were fasted for 30 h. Activities of LPL and hepatic lipase (HL), and LPL mass, were measured in pre- and post-heparin plasma in the fed and in the fasted states, respectively. For comparison, LPL and HL activities were measured in pre- and post-heparin plasma from fed and 24-h-fasted guinea pigs. RESULTS: Fasting caused a significant drop in the levels of serum insulin, triglycerides and glucose in the human subjects. Post-heparin LPL activity increased from 79 +/- 6.4 mU mL-1 in the fed state to 112 +/- 10 mU mL-1 in the fasted state (P < 0.01), while LPL mass was 361 +/- 29 in the fed state and 383 +/- 28 in the fasted state, respectively (P = 0.6). In contrast, fasting of guinea pigs caused an 80% drop in post-heparin LPL activity. The effect of fasting on human and guinea pig post-heparin HL activity were moderate and statistically not significant. CONCLUSIONS: In animal models such as rats and guinea pigs, post-heparin LPL activity decreases on fasting, presumably due to down-regulation of adipose tissue LPL. In humans, fasting caused increased post-heparin LPL activity.     

骨髓间充质干细胞移植对豚鼠皮肤深Ⅱ度烧伤的治疗作用

目的探讨骨髓间充质干细胞 (MSCs)治疗皮肤深Ⅱ度烧伤的机制。方法体外培养扩增的MSCs悬液 ,以 2× 10 6/ml(A组 )和 2× 10 7/ml(B组 )两种细胞密度移植给皮肤深Ⅱ度烧的受体鼠创面 ,观察创面愈合速度 ,用免疫组织化学检测因子Ⅷ的表达。细胞移植后的第 15、3 0、5 0天 ,通过PCR方法检测创面皮肤组织中移植供体鼠的Y染色体基因表达。结果A、B组治疗侧的创面愈合速度均快于对照侧 ,A、B组治疗侧间无显著性差异。因子Ⅷ检测示第 15、3 0天时 ,A、B两组的微血管密度均多于对照侧 ,A、B组治疗侧间无显著性差异。PCR检测结果表明 ,第 15、3 0、5 0天时在部分受体鼠恢复的创面上有供体鼠Y染色体基因表达。结论骨髓MSCs对皮肤深Ⅱ度烧伤具有明显的疗效。     

微超声探头诊断食道隆起性病变的价值

目的 探讨经内镜微超声探头(TEMP)对食道隆起性病变的诊断价值。方法 对67例电子胃镜疑有食道隆起性病变者行经内镜微超声探头检查。结果 67例中平滑肌瘤8例，平滑肌肉瘤1例，早期和进展期癌各5例，囊肿24例，壁外压迫9例，食道静脉瘤6例，静脉曲张3例，炎症2例，异位胰腺1例，息肉2例。结论 经内镜微超声探头对食道隆起性病变有较大的诊断价值。     

Site potential for challenge attrition in mice, rats and guinea pigs vaccinated with irradiated cercariae of Schistosoma mansoni

The potential sites of attrition of a challenge population of schistosomes have been investigated in mice, rats and guinea pigs vaccinated with irradiated cercariae of Schistosoma mansoni, by the use of challenge regimens that permit sequential site elimination. Vaccinated mice showed significant immunity to a percutaneous cercarial challenge, but were only marginally resistant to an i.v. challenge with healthy lung stage worms. Vaccinated rats and guinea pigs differed from mice, in that they were able to mediate significant challenge attrition at post-skin sites. Healthy lung worms were subject to immune elimination by rats in the lungs, or perhaps en route to the liver, but not in the liver itself. In contrast, guinea pigs had the capacity to kill challenge lung worms injected into either the lungs or the liver. Interestingly, lung worms harvested by extended incubation were shown to be sub-optimal in terms of viability, since they were eliminated in significant numbers when injected i.v. into vaccinated mice. These data show that different hosts vaccinated in essentially the same manner differ in terms of their site potential for challenge attrition. It is emphasised however, that sites implicated by these experiments as having the capacity to mediate immune elimination are not necessarily the sites at which challenge attrition occurs under normal circumstances.     

冠状动脉内多普勒超声评价猪急性冠状动脉闭塞侧支循环状态及影响因素

目的评价猪急性闭塞冠状动脉侧支循环状态及硝酸甘油和酚妥拉明对其的影响。方法使用冠状动脉内多普勒超声测定14头小型家猪正常状态及急性闭塞后左回旋支的血流频谱,并观察冠状动脉内应用硝酸甘油和酚妥拉明对其的影响。结果急性闭塞10min时回旋支远端的平均峰值流速(APV)明显低于正常(0.97±1.05cm/s比25.78±4.36cm/s,P<0.01),应用硝酸甘油和酚妥拉明后梗死相关血管变为反向或双向血流,APV绝对值(8.32±1.53cm/s和8.54±2.15cm/s)较用药前明显增加(P<0.01);闭塞后90min时及重复应用上述两药后APV分别为0.99±1.14cm/s、9.02±1.47cm/s和8.95±2.36cm/s,闭塞90min较闭塞即刻差异无统计学意义,用两药后APV值差异无统计学意义(P均>0.05)。结论猪急性冠状动脉闭塞时侧支循环自身建立不足,硝酸甘油和酚妥拉明均可明显增加冠状动脉侧支循环血流。     

祖师麻止痛喷雾剂对豚鼠的长期毒性研究

目的:观察祖师麻止痛喷雾剂对豚鼠的长期毒性,为临床安全用药提供依据。方法:将50只豚鼠随机分为空白对照组、高剂量完整皮肤组、高剂量破损皮肤组、低剂量完整皮肤组、低剂量破损皮肤组,连续给药4周。随时观察记录其一般情况,用药后做血液学及血液生化指标检查,计算脏器系数并进行组织病理学检查。结果:祖师麻止痛喷雾剂各剂量组未见毒性反应,与空白对照组比较差异均无统计学意义(P>0.05),停药后也未见药物延迟性毒性反应。结论:长期给予豚鼠祖师麻止痛喷雾剂无明显毒性,推论临床拟用剂量安全。     

可植入微型轴流血泵溶血实验研究

目的通过自制轴流血泵进行体外、体内溶血实验,探讨血泵的溶血特性。方法轴流血泵置于充满新鲜羊血的模拟循环通路中,在输出流量为5L／min,后负荷压力为100mmHg的条件下,检测血泵体外溶血水平。在3只健康绵羊体内植入血泵进行在体辅助实验,并稳定运转24h,测定其在体溶血水平。结果可植入微型轴流血泵的体外溶血指数（normal index of haematolysis,NIH）为（0．01644&#177;0．00018）g／100L。在体实验中,血泵运行无机械故障,循环辅助后血浆游离血红蛋白（plasma free hemoglobin,FHb）及乳酸脱氢酶（lactate dehydrogenase,LDH）呈现上升趋势,平均最大值分别达（7．93&#177;0．71）g／L、（622．3&#177;22．5）U／L。结论血泵溶血性能在可接受范围内,接近于部分国外三代血泵溶血水平,初步具备进入动物辅助实验研究水平。     

延迟性和痕迹性条件反射建立过程中豚鼠眨眼反应特征参数的变化

目的研究延迟性和痕迹性条件反射建立过程中豚鼠眨眼反应特征参数的变化。方法采用500 ms正弦波纯音和100 ms束状氧气流的配对刺激,训练豚鼠建立延迟性和痕迹性眨眼条件反射。在此过程中,使用张力换能器记录豚鼠左侧眼轮匝肌活动,并使用我室编制的Matlab软件分析眨眼行为数据。结果2组豚鼠均分别能建立延迟性和痕迹性眨眼条件反射。但是,相对于痕迹性条件反射训练组,延迟性条件反射训练组豚鼠表现出了更快的条件眨眼反应习得率和更高的条件眨眼反应峰幅度(P<0.01)。2组豚鼠条件眨眼反应峰潜伏期以及非条件眨眼反应的峰幅度和峰潜伏期在10 d条件反射训练过程中变化均不显著(P>0.05)。结论在2类条件反射训练过程中,豚鼠眨眼反应特征参数的变化规律与其他常用啮齿类动物模型相类似。     

盐酸布替萘芬乳膏的离体透皮吸收研究

目的:研究盐酸布替萘芬乳膏对离体豚鼠皮的透皮吸收情况,并对自制乳膏与国内外已上市产品的离体透皮渗透量进行比较。方法:采用透皮吸收仪,皮片涂药后不同时间采样,用高效液相色谱法测定样品中盐酸布替萘芬的浓度。结果:在50%乙醇生理盐水释放液中,国外产品、国内产品和自制产品36 h透皮渗透量分别只有0.061 89,4.661,和0.289 7μg.cm-2,盐酸布替萘芬乳膏透皮渗透量很低,三者无显著性差异。结论:自制盐酸布替萘芬乳膏的豚鼠离体透皮吸收程度与国外同类品种相当。盐酸布替萘芬乳膏主成分大部分滞留在皮肤,只有极少数透过皮肤屏障,有利于其发挥局部治疗作用。