膜截流分子量在新型生物人工肝免疫安全性中的作用研究

目的 探讨膜截流分子量对新型生物人工肝(bioartificial liver,BAL)支持系统免疫安全性的影响.方法 应用D氨基半乳糖静脉滴注比格犬建立肝功能衰竭模型.急性肝功能衰竭犬根据BAL隔离膜截流分子量的大小分为两组:A组:200 kD组;B组:1200 kD组.两组均接受新型2次BAL治疗,每次6h.观察各组体内IgG、IgM、50％补体溶血单位(CH50)变化及反应器内抗体漏过情况.结果 在第一次BAL治疗后两组的IgG和IgM水平均没有发生明显的变化.在第2次治疗后第7天,1200 kD组的IgG和IgM水平明显升高,200 kD组IgG和IgM水平仍未出现明显上升或下降.在第一次BAL治疗后,两组CH50都出现暂时性的下降,在治疗后1h达到最低值,而后缓步上升,在7d后恢复至治疗前水平.反应器内培养液中IgG、IgM及CH50检测结果显示,1200 kD组在治疗结束后IgG、IgM及CH50含量显著高于200 kD组.结论 膜截流分子量可能是影响BAL异种免疫排斥的重要因素之一.     

异氟醚对人舌癌细胞基质金属蛋白酶-2表达的影响

目的 评价异氟醚对人舌癌细胞(Tea8113细胞)基质金属蛋白酶-2表达的影响.方法 取对数生长期的Tea8113细胞,制成单细胞悬液,接种于96孔培养板(5×103/ml,200 μl/孔)、6孔培养板(1×105/ml,5 ml/孔)、培养皿(5×105/ml,5 ml/皿)和Transwell小室[上层小室加入细胞悬液200μl(细胞密度5×105/ml),下层小室加入10％血清培养基500 μl],常规培养待细胞完全贴壁后进行实验.采用随机数字表法,将细胞分为3组(n=30):对照组(C组)、2％异氟醚2h组(Ⅰ1组)、2％异氟醚4h组(Ⅰ2组).C组不给予异氟醚,Ⅰ1组和Ⅰ2组细胞培养液中加入2％异氟醚分别孵育细胞2或4h.采用MTT法检测细胞活力,流式细胞术检测细胞凋亡率;以细胞划痕实验及Transwell小室实验检测细胞水平迁移和侵袭能力;采用免疫细胞化学及RT-PCR检测细胞MMP-2及其mRNA表达水平.结果 与C组比较,Ⅰ1组、Ⅰ2组细胞活力和细胞水平迁移率升高,细胞凋亡率降低,MMP-2及其mRNA表达上调,Ⅰ2组透膜细胞数增多(P＜0.05).与Ⅰ1组比较,Ⅰ2组细胞活力和细胞水平迁移率升高,细胞凋亡率降低,透膜细胞数增多,MMP-2及其mRNA表达上调(P＜0.05).结论 异氟醚可能通过上调人舌癌Tca8113细胞的MMP-2表达来促进癌细胞的迁移侵袭能力.     

免疫功能测定及免疫干预在呼吸机相关性肺炎患者的临床意义

目的 探讨免疫功能及免疫调理对呼吸机相关性肺炎(VAP)患者治疗反应及预后的评价.方法 对2011年1月至2012年3月入住内科重症监护病房(MICU)且资料完整的40例VAP,按治疗28 d转归分为存活组(24例)和死亡组(16例)；并进行临床肺部感染评分(GPIS).于确诊后第1天清晨留取外周静脉血,分别送检T淋巴细胞计数及免疫球蛋白检测.T淋巴细胞(CD3+)、辅助性T淋巴细胞(CD4+)、抑制性T淋巴细胞(CD8+)计数采用流式细胞仪检测、免疫球蛋白IgA、IgM、IgG采用免疫散射比浊法检测.同期选取20名健康人作为健康对照组.对其中30例VAP患者随机分为乌司他丁治疗组15例,对照组15例,于治疗前和治疗第7天留取外周血,分别送检T淋巴细胞计数及免疫球蛋白检测.结果 (1)死亡组外周血CD3+、CD4+、CD8+细胞计数、CD4 +/CD8+比值[(280.32±169.58)×106/L、(212.56±122.99)×106/L、(132.73±56.74)×106/L、1.48 ±0.82]和存活组外周血CD3+、CD4+、CD8+细胞计数、CD4+/CD8+比值[(485.05±209.18)× 106/L、(352.05±116.41)×106/L、(245.68±68.69)×106/L、2.02±1.06]均低于健康对照组[(1183±639.18)×106/L、(631±321.64)×106/L、(525±221.67)×106/L、2.78±1.04],差异有统计学意义(P＜0.05)；死亡组以上指标也明显低于存活组,差异有统计学意义(P＜0.05)；(2)死亡组患者血清免疫球蛋白IgG[(10.76±4.52) g/L]明显低于存活组[(13.65±6.34) g/L]及健康对照组[(14.39 ±7.47) g/L],差异有统计学意义(P＜0.05)；(3)30例VAP患者给予免疫干预后,治疗组外周血CD3+、CD4+细胞计数[(676.26±220.78)×106/L、(358.87±133.53)×106/L]明显高于治疗前水平[(429.28±130.46)×106/L、(216.85±106.32)×106/L],也明显高于对照组[(451.32±150.78)×106/L、(278.74 ±75.57)×106/L,P＜0.05].(4) CPIS评分与外周血CD3+、CD4+细胞计数呈负相关(r=-0.689、-0.594,P＜0.01).结论 VAP患者存在细胞免疫功能及体液免疫功能低下,给予乌司他J免疫治疗后,细胞免疫功能增强；CPIS评分与细胞免疫呈负相关,免疫功能监测可预测VAP患者的病情程度、治疗效果及预后.     

富组蛋白1对人表皮细胞株HaCaT增殖和迁移功能的影响

目的 了解富组蛋白1( Hst1)对人表皮细胞株HaCaT增殖、迁移功能的影响. 方法 (1)常规培养HaCaT细胞,按照随机数字表法(分组方法下同)分为对照组与100、30、3μg/mL Hst1组以及10 ng/mL重组人表皮生长因子(rhEGF)组、30 μg/mL Hst1+ 10 ng/mL rhEGF组,每组样本数为27.对照组不添加刺激因素,后5组分别加入相应浓度Hst1和(或)rhEGF,培养24、48、72 h采用细胞计数法检测各组细胞增殖水平.(2)将HaCaT细胞分为对照组与100、30、3μg/mL Hst1组,每组样本数为27.对照组不添加刺激因素,后3组分别加入相应浓度Hst1,培养24、48、72 h采用流式细胞术检测各组细胞周期,计算增殖指数(PI).(3)将HaCaT细胞分为对照组、30 μg/mL Hst1组、10 ng/mL rhEGF组、30 μg/mL Hst1+10 ng/mL rhEGF组、15 μg/mL Hst1+5 ng/mL rhEGF组、15 iμg/mL Hst1+ 10 ng/mL rhEGF组,每组样本数为10.对照组不添加刺激因素,后5组分别加入相应浓度的Hst1和(或)rhEGF培养.将各组细胞分为2份,一份用丝裂霉素C处理2h,另一份不作处理,均行划痕实验,划痕后0(即刻)、16、24 h观测细胞迁移情况并计算划痕愈合面积百分比.对数据行方差分析、LSD-t检验或Dunnettt检验. 结果 (1)培养24 h,10 ng/mL rhEGF组、30 μg/mLHst1+ 10 ng/mL rhEGF组细胞数显著高于对照组(t值分别为3.813、5.410,P＜0.05或P＜0.01).与培养48 h时30、3 μg/mL Hst1组比较除外,其余各Hst1和(或)rhEGF处理组培养48、72 h细胞数均显著高于对照组(t值为7.754～24.979,P值均小于0.01).培养72 h,100 μg/mL Hst1组细胞数为(19.21±0.59)×104个,明显高于30 μg/mL Hst1组的(16.19±0.53) ×104个及3 μg/mL Hst1组的(15.38±0.13)×104个(t值分别为11.391、19.017,P值均小于0.01);30.μg/mLHst1+ 10 ng/mLrhEGF组细胞数高于30、3μg/mL Hst1组及10 ng/mL rhEGF组(t值为4.579 ～34.884,P＜0.05或P ＜0.01).各组细胞数均随培养时间的延长而增加.(2)与对照组培养24、48 h比较,100、30 μg/mL Hst1组G0/G1期细胞百分比降低,S期细胞百分比提高(30 μg/mL Hst1组与对照组培养24 h比较除外),PI值显著提高(t值为4.752 ～16.104,P值均小于0.01).3μg/mL Hst1组仅在培养48 h时PI值较对照组显著增加(t=4.609,P＜0.01).培养72 h,仅100 μg/mL Hst1组PI值显著高于对照组(t =8.005,P＜0.01).各Hst1处理组组间比较,各时相点随Hst1浓度降低,G0/G1期细胞百分比呈升高趋势,S期细胞百分比及PI值均呈下降趋势.各Hst1处理组组内比较,随着培养时间的延长,G0/G1期细胞百分比先降低后增加,S期细胞百分比、PI值均先增加后降低.(3)未用丝裂霉素C处理:划痕后16h,30 μg/mL Hst1组划痕愈合面积百分比为(75.9±3.9)％,显著高于对照组、10 ng/mL rhEGF组[(53.0±3.5)％、(61.7±2.5)％,t值分别为12.241、7.598,P值均小于0.01],低于30 μg/mL Hst1+10 ng/mL rhEGF组、15 μg/mL Hst1+5 ng/mL rhEGF组、15 μg/mLHst1+ 10 ng/mL rhEGF组[(95.0±4.1)％、(97.0±3.7)％、(80.5±5.9)％,t值为-11.324～-2.502,P＜0.05或P＜0.01].丝裂霉素C处理:划痕后16h,30 μg/mL Hst1组划痕愈合面积百分比为(54.1±4.5)％,高于对照组[(35.8±5.7)％,t =7.790,P＜0 01],但较未用丝裂霉素C处理时明显下降(t=- 10.863,P＜0.01);与其他Hst1和rhEGF联合处理组相近(t值为0.061 ～2.030,P值均大于0.05).未用丝裂霉素C处理时及丝裂霉素C处理后各组内划痕后24 h与16 h划痕愈合面积百分比相近(F值为0.856～3.062,P值均大于0.05). 结论 Hst1能促进HaCaT细胞增殖和迁移,与rhEGF联合应用时对细胞增殖有协同促进作用,但对细胞迁移无明显协同作用.     

超顺磁性纳米铁颗粒标记许旺细胞及体外磁共振示踪的实验研究

目的 研究超顺磁性纳米铁颗粒(SPIO)体外标记小鼠许旺细胞(SCs)及MRI成像示踪的可行性.方法 分离、纯化新生C57BL/6小鼠(5～7 d)的许旺细胞,取已分别用25.0 μg/ml、50.0 μg/ml浓度SPIO标记的0.5×106、1.0×106、5.0×106个许旺细胞,普鲁士蓝染色和透射电镜观察标记细胞内铁颗粒的分布情况,并用不同MRI扫描序列,测定标记的细胞群信号.结果 0.5×106、1.0×106、5.0×106小鼠许旺细胞与不同浓度的SPIO共同培养24 h后,普鲁士蓝染色见细胞内有许多蓝染的铁颗粒;透射电镜检查显示致密的铁颗粒位于细胞的吞噬泡或溶酶体中.体外MRI呈明显的低信号改变,以T2WI和GRE/30°序列改变最为明显.结论 SPIO可以标记小鼠许旺细胞,应用MRI可以对其进行体外示踪和监测.     

鞘内注射曲马多对大鼠细胞免疫功能的影响

目的 评价鞘内注射曲马多对大鼠细胞免疫功能的影响.方法 32只成年雄性SD大鼠,体重250～300 g,采用改良Yaksh法进行鞘内置管成功后,随机分为4组(n=8):生理盐水组(NS组);不同剂量曲马多组(T1～3组)分别鞘内输注曲马多50、25、12.5μg/h.置管后5 d鞘内输注生理盐水或不同剂量曲马多,泵容量200 μl,泵速1μl/h,鞘内输注7 d后行福尔马林实验,采用痛级评分评价镇痛效果后处死大鼠,称取体重及脾脏重量,计算脾脏指数,并分离、培养脾脏淋巴细胞,甲基-3H胸腺嘧啶核苷掺入法检测脾脏T淋巴细胞增殖转化水平,乳酸脱氢酶释放法检测脾脏自然杀伤(NK)细胞活力.结果 与NS组比较,不同剂量曲马多组在福尔马林给药后5 min和20～60 min时痛级评分降低,T1组脾脏指数、T淋巴细胞增殖转化水平降低(P<0.05),T2组和T3组脾脏指数和T淋巴细胞增殖转化水平差异无统计学意义(P>0.05).不同剂量曲马多组NK细胞活力与NS组比较差异无统计学意义(P>0.05).结论 大鼠鞘内注射曲马多在产生良好的抗伤害作用时,曲马多12.5、25μg/h不抑制细胞免疫功能,较大剂量(50 μg/h)抑制细胞免疫功能.     

小鼠肝脏部分缺血再灌注损伤模型的建立

目的 建立小鼠肝脏部分缺血再灌注损伤模型并分析损伤评估指标的变化趋势.方法 采用96只7～8周龄的纯系C57BL/6雄性小鼠作为研究对象,建立70％肝脏缺血再灌注损伤模型.按照缺血时间将小鼠分为假手术组和缺血30、60、90 min组,每组24只.各组小鼠分别于再灌注后6、12、24和48 h处死.通过检测血清ALT、AST、TNF-α、IL-6和巨噬细胞炎性蛋白-2(MIP-2)水平以及病理组织学评分、细胞凋亡指数等方法评估各组小鼠肝组织的损伤情况.两独立样本比较采用t检验.结果 术后88只小鼠存活,8只死亡,造模成功率为91.7％ (88/96).假手术组、缺血30、60、90 min组ALT水平分别为(35±24) U/L、(1703±442) U/L、(5133±681) U/L和(8233±808) U/L,缺血30、60、90 min组ALT水平显著高于假手术组(t=6.54,12.97,17.56,P＜0.05);AST水平分别为(87±28) U/L、(2667 ±451) U/L、(6333±778)U/L和(9967±1168) U/L,缺血30、60、90 min组AST水平显著高于假手术组(t=9.89,13.89,14.65,P＜0.05);TNF-α水平分别为(14 ±5) μg/L、(83±14) μg/L、(133±17) μg/L和(202±21) μg/L,缺血30、60、90 min组TNF-α水平显著高于假手术组(t=7.78,11.82,15.34,P＜0.05);IL-6水平分别为(32 ±9) μg/L、(493±168) μg/L、(844±166) μg/L和(1345±198) μg/L,缺血30、60、90min组IL-6水平显著高于假手术组(=4.74,8.46,11.48,P＜0.05);MIP-2水平分别为(37±11) μg/L、(102±35) μg/L、(177±32)μg/L和(279±50) μg/L,缺血30、60、90 min组MIP-2水平显著高于假手术组(t=3.05,7.28,8.19,P＜0.05);细胞凋亡指数分别为1.7％±2.1％、22.7％±8.6％、54.3％±11.2％和76.3％±14.8％,缺血30、60、90 min组细胞凋亡指数显著高于假手术组(t=4.10,8.04,8.63,P＜0.05).在缺血时间相同的情况下,随着再灌注时间的延长,各监测指标呈“抛物线”样变化趋势.结论 小鼠肝脏部分缺血再灌注损伤模型能较好地反映小鼠肝组织的损伤情况.随着缺血时间的延长,小鼠肝脏的缺血再灌注损伤程度逐渐加重;随着再灌注时间的延长,ALT、AST、TNF-α、IL-6、MIP-2以及病理组织学评分和细胞凋亡指数均呈现“抛物线”样变化趋势.     

高迁移率族蛋白B1对体外人肺动脉血管平滑肌细胞增殖、迁移和凋亡的影响

目的 评价高迁移率族蛋白B1(HMGB1)对体外培养人肺动脉血管平滑肌细胞(hPASMC)增殖、迁移和凋亡的影响.方法 体外培养hPASMC,调整细胞密度(2× 105个/ml)后接种到96孔板(100 μl/孔,2× 105个/ml)、6孔板(1ml/孔,2× 106个/ml)和改良24孔Boyden趋化小室(100μg/孔,5×103个/ml),采用随机数字表法,将其分为5组:对照组(C组)和不同浓度HMGB1组(H1组～H4组),分别在DMEM和含HMGB1 1、10、100、1000 ng/ml的DMEM培养液孵育.孵育24和48 h时,采用MTT法检测细胞增殖率,Boyden小室法检测透膜细胞数,TUNEL法检测hPASMC凋亡情况.结果 与C组比较,H1组～H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);与H1组比较,H2组～H4组细胞增殖率升高,H3组和H4组透膜细胞数增多(P＜0.05);与H2组比较,H3组和H4组细胞增殖率升高,透膜细胞数增多(P＜ 0.05);H3组和H4组间各指标比较差异无统计学意义(P＞0.05);与孵育24h时比较,各组孵育48 h时细胞增殖率升高(P＜0.05).各组细胞凋亡率比较差异无统计学意义(P＞ 0.05).结论 HMGB1可促进hPASMC的增殖和透膜迁移,可能参与肺损伤肺血管重构的发生.     

转化生长因子－β1基因修饰的不成熟树突状细胞的免疫生物学功能

目的 探讨转化生长因子(TGF) -β1基因转染后树突状细胞(DC)细胞表型和免疫生物学功能的变化.方法 利用大鼠骨髓细胞诱导、培养不成熟树突状细胞(imDC),以脂质体介导的pIRES2-EGFP-hTGF-β1转染imDC细胞,通过酶联免疫吸附试验(ELISA)和Western blot法检测转染后各组imDC培养上清TGF-β1蛋白表达;通过流式细胞法、免疫细胞化学法及混合淋巴细胞反应检测各组imDC表面分子的分子表型和免疫功能变化.结果 TGF-β1基因转染的imDC上清中均检测相应蛋白的高表达,TGF-β1基因转染组TGF-β1蛋白表达(252.75±11.31) μg/L均高于mDC组(11.19±2.29) μg/L、imDC组(35.18±6.17) μg/L、空载体组(33.67±4.61)μg／L;基因转染组的表面分子CD86、CD80及MHCⅡ表达明显低于空载体组和无任何转染的imDC组;基因转染组的imDC对T的增殖研究结果显示有显著抑制作用,低于空载体和无任何转染的imDC组.结论 TGF-β1基因成功导人imDC,基因改造的imDC不但能维持不成熟状态同时分泌免疫耐受抑制蛋白,致免疫耐受作用显著增强.     

关节腔内注射CoCrMo纳米微粒对雄性大鼠生殖功能的影响

目的 探讨关节腔内注射CoCrMo纳米微粒对雄性大鼠生殖功能的影响. 方法 利用直流电弧等离子体法制备CoCrMo纳米微粒.将75只8周龄雄性SD大鼠随机分为5组(每组15只):对照组膝关节腔内注射生理盐水,基础量组、低剂量组、中剂量组及高剂量组分别注射剂量为50、250、500、1000 μg/kg的CoCrMo纳米微粒生理盐水悬液,1次/周,连续10周.第11周处死动物,测定血清中Co、Cr离子含量和睾酮水平,检测附睾精子的活力、精子计数、精子畸形率及精子凋亡率,观察睾丸组织的病理变化. 结果 基础量组、低剂量组、中剂量组及高剂量组血清Co、Cr离子含量呈上升趋势,均明显高于对照组；高剂量组血清睾酮水平较对照组明显下降；中剂量组精子平均路径速度、鞭打频率,以及高剂量组精子平均路径速度、直线运动速度、曲线运动速度、鞭打频率明显低于对照组,以上各项目比较差异均有统计学意义(P＜0.05).中剂量组精子计数、精子畸形率、凋亡率平均分别为(62.24 ±21.33)×106/mL、12.1％±2.7％、12.4％ ±4.0％,高剂量组分别为(59.67±18.86)×106/mL、16.0％±3.8％、l2.9％±4.4％,与对照组[(83.11±23.58)×106/mL、6.2％±2.0％、6.1％±2.5％]比较差异均有统计学意义(P＜0.05).基础量组、低剂量组睾丸组织无明显病理改变,中、高剂量组睾丸组织发生病理改变. 结论 关节腔内注射中、高剂量的CoCrMo纳米微粒可能对雄性大鼠生殖功能造成不良影响.     

Effects of membrane molecular weight cutoff on performance of a novel bioartificial liver

Immunoisolation using semipermeable membranes has been incorporated into bioartificial liver (BAL) devices to separate cellular components of the recipient's immune system from the cells within the BAL device. This study was designed to explore the influence of membrane molecular weight cutoff on performance of the multilayer radial‐flow BAL using porcine hepatocytes cocultured with mesenchymal stem cells. In this study, healthy beagles underwent 6‐h treatment with a BAL containing membrane with 200 kDa retention rating or 1200 kDa retention rating. Functional markers of BAL performance were monitored before and after treatment, as well as cytotoxic immune response to BAL therapy. The results showed that hepatocyte performance levels such as albumin secretion, urea synthesis, and viability were all significantly higher in 200 kDa retention rating group compared with the 1200 kDa retention rating group after treatment (P < 0.05). Significant levels of canine proteins were detected in BAL medium from the 1200 kDa retention rating group. Fluorescence microscopy further verified that heavy deposition of canine IgG, IgM, and complement (C3) on coculture cells was obtained after BAL treatment in the 1200 kDa retention rating group. However, only trace deposits of canine immunoproteins were observed on coculture cells obtained from BAL in the 200 kDa retention rating group. Small membrane molecular weight cutoff of the BAL could reduce the transfer of xenoreactive antibodies into the BAL medium and improve the performance of the BAL.     

The influence of membrane molecular weight cutoff on a novel bioartificial liver

Given the xenogeneic immune reaction relevant to the molecular weight cutoff of the membrane of a bioartificial liver (BAL) system, we investigated the influence of membrane molecular weight cutoff in our BAL system in this study. Acute liver failure in beagles was induced by d-galactosamine administration. Eight beagles were divided into two groups by the membrane molecular weight cutoff of the plasma component separator. Group 1 beagles were treated with BAL containing 200 kDa retention rating membrane. Group 2 beagles were treated with BAL containing 1200 kDa retention rating membrane. Each group underwent two 6-h BAL treatments that were performed on day 1 and day 21. The hemodynamic and hematologic response, humoral immune responses, and cytotoxic immune response to BAL therapy were studied before and after treatments. All beagles remained hemodynamically and hematologically stable during BAL treatments. BAL treatment was associated with a significant decline in levels of complement; however, a longer time of level maintenance was observed in Group 2. Group 2 beagles experienced a significant increase in levels of IgG and IgM after two BAL treatments. Significant levels of canine proteins were detected in BAL medium from Group 2; only trace levels of canine proteins were detected in BAL medium from Group 1. The posttreatment viability of co-culture cells in Group 2 was lower compared with Group 1, and the viability of co-culture cells after treatments was associated with deposition of canine proteins on the cells. Xenogeneic immune response was influenced by membrane molecular weight cutoff in the BAL.     

Cytotoxic immune response to a xenogeneic bioartificial liver

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.     

Membrane pore size impacts performance of a xenogeneic bioartificial liver

BACKGROUND: We have developed a novel bioartificial liver (BAL) composed of porcine hepatocyte spheroids in a reservoir design. A semipermeable membrane is used to protect the spheroids from immune-mediated damage. This study was designed to assess the influence of membrane pore size on performance of the spheroid reservoir BAL. METHODS: Eight healthy dogs were studied during primary and secondary exposures to the spheroid reservoir BAL using membranes with small (10 nm) or large (200 nm) pores. BAL performance was assessed by multiple functional assays. Spheroids were examined microscopically before and after all BAL treatments. Titers of xenoreactive antibody were monitored until elective death of animals on day 42. RESULTS: Viability and functional performance of spheroids were significantly greater after all BAL treatments that used membranes with 10-nm versus 200-nm pores. Reduced performance in the 200 nm group was associated with 7.7-fold and 78.0-fold rise in xenoreactive antibody titers after first and second treatments, respectively. Dogs in the 10 nm group remained hemodynamically stable during all BAL treatments, whereas those in the 200 nm group experienced acute hypotension (P<0.001) during second BAL exposures. Microscopic examination of spheroids after BAL treatments indicated that deposition of canine proteins, including complement, was associated with reductions in both viability and functional performance of the BAL. CONCLUSIONS: The elicited immune response of healthy dogs to a xenogeneic BAL was blocked and BAL performance significantly improved by reducing the permeability of the BAL membrane.     

Membrane barrier of a porcine hepatocyte bioartificial liver

Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH₅₀ levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures. (Liver Transpl 2003;9:298-305.)     

Two dimensional protein patterns of bronchoalveolar lavage fluid from non-smokers, smokers, and subjects exposed to asbestos.

BACKGROUND: Bronchoalveolar lavage (BAL) fluid contains a large number of proteins which comprise a potential resource for studying respiratory effects due to occupational and environmental exposures. A study was undertaken to compare protein patterns of BAL fluid from non-smokers, smokers, and subjects exposed to asbestos. METHODS: BAL fluid samples were analysed with two dimensional gel electrophoresis (2-DE). The separated proteins were detected, quantified, and pattern-matched between different individuals with a computerised imaging system designed for evaluations of 2-DE patterns. RESULTS: About 200 different protein spots were detected in each sample of BAL fluid. As is the case with blood plasma, the BAL fluid samples contained large amounts of albumin, transferrin, and immunoglobulins. Higher levels of basic proteins were found in smokers than in non-smokers, while subjects exposed to asbestos had increased amounts of several high molecular weight proteins as well as basic proteins. Lower levels of albumin and higher levels of immunoglobulins were found in smokers than in non-smokers, while higher levels of transferrin were found in asbestos exposed subjects than in unexposed subjects. Moreover, in the group exposed to asbestos differences were found between patients with pleuritis and patients with pleural plaque, and one protein spot was found only in two patients with progressive pleural disease. CONCLUSION: These results suggest that both smokers and asbestos exposed subjects have significant changes in their airway protein expression compared with non-smokers and unexposed subjects. It is inferred that analysis of protein patterns in the BAL fluid with 2-DE may be used to detect and characterise, at a molecular level, respiratory effects due to occupational and environmental exposures.     

An Effective LDL Removal Filter for the Treatment of Hyperlipidemia

Abstract: On-line membrane plasma fractionation techniques have made semiselective removal of pathological macromolecules practical. However, several problems such as cryogel formation exist when the procedure is performed at ambient temperature. Cryogel formation takes place when heparinized plasma is cooled below 35°C and when it tends to occlude the pore structure of the secondary filter membrane resulting in a poor molecular cut off of the macromolecular filter. Thermofiltration is one of the on-line plasma fractionation techniques used when warming plasma from 37 to 42°C to prevent cryogel formation. Thermofiltration enhanced the performance of the lipofilter (Kuraray 4A) and demonstrated better molecular cut off between low density lipoprotein (LDL) cholesterol and high density lipoprotein (HDL) cholesterol than double filtration plasmapheresis (DFPP). An improved lipofilter (Kuraray 5A) has been developed and has shown better molecular cut off between LDL cholesterol and HDL cholesterol than the 4A filter. However, cryogel formation still occurred even using the 5A filter during the DFPP procedure. Thermofiltration maintains the performance of the secondary filter by preventing cryogel formation. Further studies are required to evaluate the enhanced performance of the 5 A filter by thermofiltration.     

Efficacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure

BACKGROUND: Fulminant hepatic failure is associated with a high mortality rate. Orthotopic liver transplantation is the only established treatment for patients who do not respond to medical management. A major limitation of this treatment is a shortage of donor organs, resulting in many patients dying while waiting for a transplant. An extracorporeal bioartificial liver (BAL) has the potential to provide temporary support for patients with fulminant hepatic failure (FHF) and for patients awaiting orthotopic liver transplantation. We developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer. MATERIALS AND METHODS: Twenty-four hours after cannulation of the left carotid artery and right jugular vein, FHF was induced in rats by administering 2 intraperitoneal injections of D-galactosamine (GalN) (1.2 g/kg) at a 12-h interval. The rats were connected to a BAL device 24 h after the first GalN injection and underwent extracorporeal perfusion for a duration of 10 h. Liver histology, liver-specific markers, and animal survival up to 168 h (7 days) were examined. RESULTS: Histologically, liver damage was reduced in the animal group treated with the hepatocyte-based BAL device. Significant reductions occurred in the plasma ammonia levels and prothrombin times in the group treated with the seeded BAL device. Animal survival in the group treated with the seeded BAL device was significantly higher (50.0%) than in the control animal group treated with an unseeded BAL device (11.1%). CONCLUSIONS: This flat-plate BAL with an internal membrane oxygenator and cultured porcine hepatocytes has yielded encouraging results in the treatment of rats with GalN-induced FHF.     

Mass Transfer Characterization of a New Polysulfone Membrane

A new polysulfone capillary membrane has been tested ex vivo for its clearance performance under diffusive, combined diffusive and convective, and pure convective solute mass transfer. As was expected, increasing convective mass transfer produced augmentation only of clearances of high molecular weight solutes, and to a much lesser extent than reported for other membranes. This behavior is explainable by the enormous diffusive clearance capacity of the new membrane, which nearly reaches its theoretical maximal value, as derived from a computer model. Based on the very high hydraulic permeability, which makes this membrane equally suitable for filtrative and diffusive procedures, no membrane solute retention was found up to a molecular weight of 5,000 daltons. Stable hemocompatibility parameters (leukocytes, thrombocyte, and complement measurements) demonstrated absence of activation by this membrane during treatment. It is concluded that since its clearance performance is near the theoretical maximal and it shows superior biocompatibility, the membrane exhibits negligible interaction with the transported solutes independent of the transport mode.     

Equine Culicoides Hypersensitivity: Evaluation of a Skin Test and of Humoral Response

Intradermal tests were carried out on 18 horses with clinical signs of Culicoides hypersensitivity (CHS) and 23 horses without clinical signs of CHS, and sera from these horses were analysed by SDS‐PAGE and Western blotting (W‐B). Intradermal injections of 0.1 ml of 25 μg/μl sterile Culicoides extract, 0.1 ml of 1 : 10 000 histamine (positive control) and 0.1 ml of physiological saline (negative control) were made in the dermis of the middle region of the neck. Analysis of reactions indicated that a 1 cm wheal and a skinfold thickness >10% at 24 h represented a valid cut‐off between horses with and without CHS. In these conditions the test, even in winter when clinical signs were absent, had 100% sensitivity and specificity. The W‐B was performed after running Culicoides extract on a 12% polyacrylamide gel. The test revealed the presence of several bands with molecular weight ranging from 6 to 200 kDa. In particular, a band of 65 kDa was predominantly found in hypersensitive horses by using an anti‐IgE antibody while in normal horses the same band was mainly detected by using an anti‐IgG antibody. Our results demonstrated that the skin test is a valid diagnostic test, with high sensitivity and specificity and that the band of about 65 kDa probably corresponds to the allergen involved in the pathogenesis of CHS.