成人骨髓间充质干细胞抑制异体淋巴细胞增殖的实验研究

目的研究成人骨髓间充质干细胞（BMMSCs）的免疫调节作用。方法从成人骨髓中分离和培养间充质干细胞，并通过形态的均一性及流式细胞术检测其表面标志以鉴定纯度。将分离培养的间充质干细胞接种于96孔板中，经丝裂霉素处理后，与异体淋巴细胞共同培养3d；并以单独培养的异体淋巴细胞作为对照组。各组经培养后加入植物血凝素（PHA）刺激72h，用MTT还原法测定细胞增殖率，观察成人骨髓BMMSCs对异体淋巴细胞增殖转化的影响。结果成人BMMSCs与PHA诱导的异体淋巴细胞共同培养组抑制了细胞增殖，其增殖转化抑制率为对照组的60．68％，配对t检验结果显示，两组差异有统计学意义（P〈0．01）。结论成人BMMSCs可以抑制PHA诱导的异体淋巴细胞增殖转化，对同种异体免疫反应具有负调节作用。     

骨髓间充质干细胞体外对1型糖尿病大鼠淋巴细胞的免疫调节作用

目的 观察骨髓间充质干细胞(MSCs)体外对1型糖尿病(T1DM)大鼠淋巴细胞表型及增殖能力的影响,探讨其抑制淋巴细胞增殖的机制.方法 分离、培养和鉴定大鼠MSCs,噻唑蓝(MIT)比色法观察该细胞对淋巴细胞增殖能力的影响,应用流式细胞术分析MSCs对植物血凝素(PHA)作用下淋巴细胞凋亡,周期水平和CD4+CD25+调节性T细胞亚群(CD4+CD25+Tregs)比例的影响.结果 大鼠MSCs表型为CD29+、CD90+、CD106+、CD34-、CD45-,对PHA刺激的淋巴细胞增殖有抑制作用,以淋巴细胞:MSCs为1∶1时(C组)抑制作用最强;共培养体系中,大部分淋巴细胞处于G0/G1期;C组淋巴细胞凋亡水平(58.05±0.89)%显著高于对照组(43.35±0.86)%(P＜0.05);CD4+CD25+Tregs的比例C组(22.76±1.15)%显著高于对照组(5.80±0.68)%(P＜0.05).结论 MSCs体外可显著抑制PHA刺激的T1DM大鼠淋巴细胞的增殖,其机制与CD4+CD25+Tregs比例增高密切相关.

Abstract：

Objective To observe the effects of bone marrow mesenchymal stem cells (MSCs) on the lymphocytes of rats with type 1 diabetes mellitus (T1DM) in vitro, and investigate the inhibitory effect of MSCs on lymphocytes proliferation and the underlying mechanism. Methods MSCs were isolated from SD rats, cultured in vitro, purified and then identified by testing the phenotypes with flow cytometry (FCM). The third-generation MSCs were planted in 24-well plates. After treated with mitomycin C, MSCs were co-cultured for 72 h with the T1 DM rat's lymphocytes activated by phytohemagglutinin (PHA). The proliferation of lymphocyte was measured by methyl thiazol tetrazolium (MTT) method. FCM analysis was done to investigate the apoptosis, cell cycle and the proportion of CD4+ CD25+ regulatory T cells of the T1 DM rat's lymphocytes after co-cultivation. Results The phenotypes of MSCs from normal SD rats were CD29 + , CD90 +, CD106 + , CD34-, CD45 -. MSCs obviously inhibited the lymphocyte proliferation stimco-culture system, most of the lymphocytes were arrested at G0/G1 phase. The apoptosis rate of lymphocytes (58.05 ± 0. 89)% in group C was increased significantly as compared with the control group (43.35± 0.86 ) % ( P ＜ 0. 05 ) as well as the proportion of CD4 + CD25 + regulatory T cells (22.76 ± 1.15 ) % vs (5.80 ± 0. 68) %. Conclusion In vitro, MSCs can obviously inhibit the T1 DM rat' s lymphocytes proliferation stimulated with PHA via increasing the proportion of CD4 + CD25 + regulatory T cells.     

大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

目的 观察胰腺癌微环境对树突状细胞(DC)成熟的影响及功能变化并探讨胰腺肿瘤细胞免疫逃逸的机制.方法 培养树突状细胞,加入粒细胞巨噬细胞集落刺激因子(rmGM-CSF)40μg/L、白细胞介素(IL)-4 40μg/L,培养到第6天时得到大量的未成熟树突状细胞(imDC),加入大鼠胰腺癌细胞(AR42J cell)培养上清液诱导,流式细胞术检测DC的表面分子CD86、CD80的表达(n=6),观察能否延缓或阻断imDC的成熟及其在脂多糖(LPS)刺激后这种作用能否被逆转.并观察AR42J细胞培养上清诱导的Dc对同种异体混合淋巴细胞增殖.结果 加入胰腺癌癌细胞上清液培养的DC,与正常成熟的DC比较,CD80+CD86+阳性率由(70.88±3.60)%降至(7.15±0.71)%,LPS刺激后DC细胞的表面分子CD80+CD86+表达仍然较低(7.43±1.05)%,表明胰腺癌细胞培养上清液对DC的成熟有阻断作用.AR42J细胞上清诱导培养的imDC组刺激同种异体混合淋巴细胞增殖的强度显著低于正常培养的imDC组刺激同种异体混合淋巴细胞增殖的强度.结论 体外大鼠胰腺癌细胞培养上清液可以诱导DC处于不成熟状态,且这种不成熟状态不容易被逆转.     

成骨诱导后的骨髓间充质干细胞对外周血单个核细胞增殖的免疫调节作用

目的:探索骨髓间充质干细胞(BMSCs)及成骨诱导后的骨髓间充质干细胞(OM-BMSCs)对同种异体的外周血单个核细胞(PBMCs)的免疫调节作用。方法:分离比格犬BMSCs,成骨诱导培养基(ost eogenic medium,OM)进行成骨诱导,建立BMSCs、OM-BMSCs与同种异体PBMCs的共培养体系,分三种实验条件,共9组。A组:5×104受体PBMCs加入5×104供体PBMCs共培养;B组:A组组分加入已铺被1×104/孔BMSCs的96孔板;C组:A组组分加入已铺被1×104/孔OM-BMSCs的96孔板;D组:5×104受体PBMCs加入植物血凝素A(PHA)刺激物;E组、F组:将D组组分按B、C组方式处理;G组:5×104受体PBMCs加入IL-2炎症因子;H组、I组:G组组分按B、C组方式处理。A、D、G组分别为三个实验条件的对照组,单独接种1×104/孔BMSCs、OM-BMSCs作为空白对照组Bl ank1组、Bl ank2组。以上各组分别培养120h后,应用Cell Titer 96 Aqueous检测各组PBMCs的增殖情况。结果:在各实验条件下与相应对照组相比较,BMSCs与OM-BMSCs均能抑制双向混合淋巴细胞反应(MLR)中PBMCs的增殖,均能抑制经PHA刺激后PBMCs的增殖,均能抑制经IL-2刺激后PBMCs的增殖(P<0.01)。结论:BMSCs及成骨分化的BMSCs对刺激细胞、PHA、IL-2刺激的同种异体PBMSs的增殖均表现为抑制效应,成骨分化后的骨髓间充质干细胞对同种异体免疫反应仍具有免疫调节作用。     

人BMSCs分化过程中免疫能力的实验研究

目的研究人BMSCs在成骨、成软骨及成脂分化过程中的免疫原性及其对外周血单个核细胞(peripheral blood mononuclear cell,PBMC)增殖的抑制能力。方法取健康志愿者骨髓分离培养BMSCs,分别进行成骨、成软骨和成脂诱导分化7、14、21 d。通过流式细胞术检测未分化及分化过程中BMSCs的人类白细胞抗原(human leukocyte antigen,HLA)Ⅰ类及Ⅱ类分子表达水平的改变。分离培养健康志愿者PBMC,按10∶1比例将PBMC与BMSCs共培养5 d,通过流式细胞术检测未分化及分化过程中BMSCs对PBMC增殖的抑制能力变化。结果未分化BMSCs表达HLA-Ⅰ类分子,基本不表达HLA-Ⅱ类分子。成骨、成软骨分化过程中各时间点HLA-Ⅰ、Ⅱ类分子表达无明显改变(P0.05),且HLA-Ⅱ类分子表达水平均较低。成脂分化14、21 d HLA-Ⅰ类分子表达逐渐升高,与0、7 d比较差异有统计学意义(P0.05);成脂分化7、14、21 d HLA-Ⅱ类分子表达也逐渐出现并升高,均显著高于0 d(P0.05)。PBMC在无抗CD3、CD28微球刺激下无明显增殖,加入抗CD3、CD28微球后显著增殖,未分化BMSCs能显著抑制PBMC的增殖。成骨、成软骨分化过程中BMSCs抑制PBMC增殖能力无明显改变(P0.05)。成脂分化7、14、21 d,BMSCs抑制PBMC增殖能力逐渐减弱,各时间点间差异均有统计学意义(P0.05)。结论人BMSCs成骨、成软骨分化过程中仍具有低免疫原性和较强的免疫抑制能力,可作为同种异体组织工程修复和生物治疗的细胞;而成脂分化后免疫原性升高、免疫抑制能力降低,不适合作为同种异体组织工程修复和生物治疗的细胞。     

腺病毒载体转染人未成熟树突状细胞对其成熟特性的影响

目的观察重组腺病毒载体AdEASY-增强型绿色荧光蛋白(EGFP)转染人未成熟树突状细胞(imDC)后,其表型特征及免疫学功能的变化,并探讨白细胞介素(IL)10对腺病毒转染诱导imDC成熟的抑制作用。方法贴壁法分离人脐带血来源的单核细胞,利用重组粒细胞巨噬细胞集落刺激因子(GM-CSF)和IL-4诱导分化imDC。对照组为常规培养的imDC,转染组用AdEASY-EGFP转染imDC,IL-10组用IL-10处理转染后细胞。流式细胞仪检测细胞表面成熟标志[CD86、CD83和人类白细胞DR抗原(HLA-DR)],混合淋巴细胞反应(MLR)检测其刺激同种异体未致敏T淋巴细胞的增殖能力。结果腺病毒转染imDC后,转染组细胞成熟表型表达率分别为CD86:46±10、CD83: 38±7、HLA-DR:82±10,均较对照组(10±7、8±3、68±8)显著上调,且刺激T淋巴细胞增殖的能力显著加强(SI>2．0)。IL-10组处理的imDC上述表型表达率分别为CD86:8±5、CD83:9±3、HLA-DR: 63±12,与对照组比较差异无统计学意义(P>0．05),其刺激T淋巴细胞增殖的能力也明显下降。结论腺病毒能有效转染imDC,但在转染后有促进其成熟的趋势;用IL-10能有效抑制该成熟状态。     

胃癌根治术病人围术期异体输血后外周血单核细胞CD40L表达的变化

目的 研究胃癌根治术病人围手术期异体输血外周血单核细胞(PBMCs)白细胞分化抗原40配体(CD40L)表达的变化。方法 胃癌根治术病人30例,随机分为3组,每组10例。A组围术期不输血,B组围术期输入去白细胞的全血,C组围术期输入异体全血。另选10例健康人作为对照。分别在手术前、术后2、5、10 d采外周静脉血5 ml,用Ficoll分离液梯度离心法分离出PBMCs和血浆,将PBMCs置于自身血浆环境中,并在植物血凝素(PHA,20 mg/L)的刺激下进行培养,48 h后收获细胞,用流式细胞术检测CD40L表达。结果 健康人外周血未受PHA刺激时检测不到CD40L的表达,经PHA刺激后CD40L 细胞占CD4 T细胞的百分数为1.7％±0.4％,与三组胃癌病人术前比较差异无显著性(P>0.05)。与术前比较,B组术后2 d PBMCs CD40L表达升高(P<0.05),C组术后各时点升高(P<0.05);与A组比较,B组术后2 d升高(P<0.05),C组术后各时点升高(P<0.05);与B组比较,C组术后各时点升高(P<0.05)。结论 围手术期异体输血可造成免疫抑制,输异体血后CD40L表达增加,且输全血比输去白细胞的全血更明显。围手术期成分输血优于输注全血。     

小鼠不成熟CD8α+树突状细胞的免疫抑制功能的体外实验

目的 观察不成熟CD8α+树突状细胞(DC)的体外免疫抑制功能.方法 取C57BL/6(H-2b)小鼠和Balb/c(H-2d)小鼠的骨髓和脾脏,制备不成熟CD8α+DC和脾淋巴细胞,并经丝裂霉素处理.采用甲基噻唑基四唑(MTT)法,实验设混合淋巴细胞反应(MLR)组(阳性对照组)、MLR加不同密度的同系CD8α+DC组、MLR加不同密度同种异型CD8α+DC组、MLR加不同密度CD8α+DC的培养上清组、CD8α+DC加同系T淋巴细胞组及阴性对照组.MLR组按刺激细胞/反应细胞10∶1建立.CD8α+DC按与反应细胞比例0.2∶1、0.5∶1、0.8∶1和1∶1的梯度加入MLR中分别建立同系CD8α+D组和同种异型CD8α+D组;MLR中加入不同密度(1×105/ml～5×106/ml)CD8α+DC的培养上清液建立CD8α+DC上清组;CD8α+DC与同系淋巴细胞共培养建立CD8α+DC加同系T淋巴细胞组;以反应细胞2×105/孔作为阴性对照.采用酶联免疫吸附试验法检测MLR加同系CD8α+DC组(1∶1)上清液中γ干扰素(IF-γ)和白细胞介素10(IL-10)的浓度.结果 同系和同种异型不成熟CD8α+DC对MLR均有抑制作用(P＜0.05),二者间差异无统计学意义(P＞0.05).抑制作用随DC比例的增加而增强,当CD8α+DC/反应细胞比例大于0.2时显示抑制作用(P＜0.05),比例为1时抑制作用最强.CD8α+DC体外刺激同系淋巴细胞增殖的能力较弱,当DC与T淋巴细胞的比值大于2时,显示出一定的刺激作用(P＜0.05),其培养上清液对MLR也有抑制作用(P＜0.05),其中密度5×105/ml的细胞培养上清液抑制作用最强.MLR加同系CD8α+DC组(1∶1)上清液中IL-10的含量为(451.9±12.2)pg/ml,IFN-γ的含量为(1.0±1.2)pg/ml.结论 不成熟CD8α+DC体外具有免疫抑制或诱导免疫耐受的功能,可产生较高水平的IL-10,CD8α+DC及其培养上清液均可抑制MLR.

Abstract：

Objective To observe the function of immature CD8α+ dentritic cells (DCs) in vitro. Methods The bone marrow and spleen of C57BL/6(H-2b) and Balb/c (H-2d) mice were got to prepare immature CD8α+ DCs and spleen lymphocytes,and treated by mytomycin. MTT test was used.MLR group, MLR plus variable density syngeneic CD8α+ DC group, MLR plus variable density allogeneic CD8α+ DC group,MLR plus variable density CD8α+ DC supernatant group,CD8α+ DC plus syngeneic T cell group and negative control group were established. MLR group was set up by responder cell ratio of 0.2,0.5,0.8,1.0,to build the MLR plus syngeneic and allogeneic CD8α+ DC experimental groups. Culture supernatant from different density (1 × 105/ml - 5 × 106/ml) of CD8α+DCs was added into MLR to build CD8α+ DC supernatant group. CD8α+ DCs were co-cultured with syngeneic T cells to build CD8α+ DCs plus syngeneic T cells group. 2 × 105/well responder cells served as the negative control group. ELISA was used to detect the concentrations of IFN-γ and IL-10 in the DCs could both suppress MLR (P＜0. 05), and the difference was not statistically significant (P＞0. 05). When CD8α+ DCs were increased, the suppressive effect was enhanced. When CD8α+ DC/responder cell ratio ＞0. 2, the inhibitory effect could be observed, and this effect reached the peak when the ratio was 1.0. The CD8α+ DCs had weak ability to stimulate syngeneic lymphocyte proliferation in vitro, and certain stimulating effect could be seen only when CD8α+ DC/responder cell ratio ＞2 (P＜0. 05). Its culture supernatant also showed suppressive effect (P＜0. 05), and the supernatant with a cell density of 5 × 105/ml showed the maximum effect. IL-10 concentration in the concentration was 1.0 ± 1.2 pg/ml. Conclusion The in vitro function of immature CD8α+ DCs was immunosuppression/tolerance,and they could secret high level of IL-10. The CD8α+ DCs and their culture supernatant could suppress MLR in vitro.     

大鼠树突状细胞CD86分子小干扰RNA慢病毒载体构建及其功能

目的 观察针对大鼠树突状细胞(DCs)表面共刺激分子CD86的小干扰RNA(siRNA)慢病毒载体转染骨髓来源大鼠DCs后,CD86分子mRNA水平变化及DC刺激T淋巴细胞增殖能力的变化.方法 将针对大鼠DCs表面共刺激分子CD86的siRNA慢病毒载体转染骨髓来源大鼠DCs,逆转录.聚合酶链反应(RT-PCR)方法 检测CD86分子mRNA水平,MLR检测转染后的树突状细胞刺激T淋巴细胞增殖能力的变化.结果 转染组CD86分子mRNA水平较未转染组及阴性对照组有明显下降.MLR中各实验组DC与细胞1:1、1:10、1:50混合72 h后492 nm吸光度值分别为,未转染组:0.876±0.117、0.582±0.085、0.472±0.034;阴性对照组:0.870±0.140、0.541±0.066、0.438±0.067;转染组:0.394±0.143、0.294±0.032、0.218±0.015.表明转染后的DCs刺激T淋巴细胞增殖的能力较未转染组及阴性对照组显著降低.结论 实验所用针对CD86分子的siRNA慢病毒载体,可以特异性的抑制大鼠树突状细胞CD86基因的表达,降低树突状细胞对T淋巴细胞的刺激增殖能力.     

强直性脊柱炎患者骨髓间充质干细胞的生物学及免疫学特性

目的:探讨强直性脊柱炎(AS)患者骨髓间充质干细胞(BMSCs)的生物学及免疫学特性,为进一步阐明AS的发病机制和寻找新的治疗靶点提供理论依据。方法 :选取37例活动期AS患者(AS组),男34例,女3例,平均年龄(24.3±5.4)岁,HLA-B27均为阳性;49例健康志愿者作为对照组(HD组),男43例,女6例,平均年龄(25.7±4.9)岁;其中HLA-B27阴性44例(HD1组),HLA-B27阳性5例(HD2组)。从每例受检者髂后上棘穿刺采集骨髓组织,分离BMSCs,培养扩增至第3代,以1×104/ml的浓度接种于96孔板中,100μl/孔,从第1天开始每日取3孔进行细胞计数,共计12d,绘制生长曲线;每日取3孔经MTT处理后测定吸光度值,绘制细胞活力曲线,观察BMSCs的生物学特性。使用流式细胞仪检测各受检者BMSCs的细胞表面表型。将第3代BMSCs细胞接种于U形底96孔板,培养4h后使用60Co照射30Gy;取健康志愿者外周血采用密度梯度法分离单个核细胞(PBMCs),加入细胞培养液,计数后按BMSCs∶PBMCs 1∶20、1∶10、1∶5、1∶2、1∶1的比例接种于已接种BMSCs的96孔板,共培养5d,观察双向混合淋巴细胞反应情况;同样获取PBMCs,计数后同样以5个比例接种于96孔板,加入植物血凝素(PHA)4μg/ml,充分接触后共培养5d,观察淋巴细胞增殖反应情况。对组间进行统计学比较。结果:AS组、HD1和HD2组第3代BMSCs体外培养1~12d时的增殖能力、细胞活力无显著性差异(P>0.05);三组细胞表面表型均为高水平表达CD105、CD73和CD90,不表达CD45、CD34、CD14和HLA-DR。HD1组和HD2组BMSCs与不同比例PBMCs共培养的双向混合淋巴细胞反应和PHA刺激的淋巴细胞增殖反应均无显著性差异(P>0.05);AS组与HD组比较有显著性差异(P<0.05)。结论:AS患者BMSCs的生物学特性无明显改变,但其免疫调节功能明显下降,其可能在AS的发病机制中扮演重要角色。     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Dilemma of Membrane Biocompatibility and Reuse

Abstract: Numerous articles have been published on the multiple use of dialyzers and on the effect of different reprocessing chemicals and techniques on the dialyzer biocompatibility and performance. The results often appear contradictory, especially those comparing standard biocompatibility parameters. Despite this confusion, a discerning review of the published works allows certain limited conclusions to be drawn. Reprocessing of used hemodialyzers changes the biocompatibility profile of a dialyzer as defined by the parameters complement activation. leukopenia, and cytokine release. The effect of reprocessing depends on the chemicals and reprocessing technique applied and also on the type of membrane polymer being subjected to the reprocessing procedure. Reports of pyrogenic reactions indicate that the flux of the membrane also influences how suitable it is for safe reuse. An increased risk of allergic and pyrogenic reactions appears to be associated with dialyzer reuse. Furthermore, there has been a lack of investigations into the immunologic effect of the layer of adsorbed and chemically altered proteins that remains on the inner surface of reprocessed dialyzers. We conclude that the clinical benefit of dialyzer reuse cannot be generally accepted from a biocompatibility point of view.     

The analgesic effect of racemic ketamine in patients with chronic ischemic pain due to lower extremity arteriosclerosis obliterans

Background : Ketamine in sub-dissociative doses has been shown to have analgesic and phantom-Limb pain, where conventional treatment has often failed. Chronic ischemic pain due to lower extremity arteriosclerosis obliterans often responds poorly to analgesics, and the pain-generating mechanisms are not well understood.
Methods : Eight patients with rest pain in the lower extremity due to arteriosclerosis obliterans were given sub-dissociative doses of 0.15, 0.30, or 0.45 mg/kg racemic ketamine and morphine 10 mg as a 5-min infusion on four separate days in a cross-over, double-blind, randomised protocol. Plasma levels of (S)- and (R)-ketamine and their nor-metabolites were analysed with an enantioselective high-performance liquid chromatography (HPLC) method. Pain levels were evaluated with a visual analogue scale (VAS).
Results : Individual pain levels were highly variable during and after all the infusions but the pooled pain levels showed a dose-dependent analgesic effect of ketamine with a transient but complete pain relief in all patients at the highest dose (0.45 mg/ kg). Side-effects, mainly disturbed cognition and perception, were pronounced and dose-dependent. Morphine 10 mg had an analgesic peak at 20 min and 5/8 patients had complete pain relief. The remaining 3 patients also had high baseline pain scores, indicating a higher analgesic potency for the 0.30 and 0.45 mg/ kg ketamine doses than for morphine 10 mg.
Conclusion : We have demonstrated a potent dose-dependent analgesic effect of racemic ketamine in clinical ischemic pain. Due to a narrow therapeutic window, this analgesic effect is probably best utilised in combination with other analgesics.