多发性骨髓瘤患者基质细胞衍生因子-1α、CD44v6表达的研究

目的 探讨基质细胞衍生因子-1α(SDF-1α)、CD44v6(一种变异的CD44受体)在多发性骨髓瘤(MM)中的表达水平及其与病情进展的关系.方法 用酶联免疫吸附试验(ELISA)检测24例MM患者[14例初发和复发MM患者(初发和复发MM组),10例病情稳定MM患者(病情稳定MM组)]和15位健康骨髓移植供者或非肿瘤良性贫血患者(对照组)的骨髓单个核细胞(MNC)和骨髓基质细胞(BMSC)培养上清的SDF-1 α、CD44v6水平.结果 初发和复发MM组MNC培养上清的SDF-1α、CD44v6表达水平[(7232.41±2644.97)pg/ml和(34.34±13.20)ng/ml]显著高于病情稳定MM组[(2315.49±748.29)pg/ml和(15.69±5.28)ng/m1](t=6.25、t=7.82;均P＜0.05)和对照组[(1149.52±636.06)pg/ml和(4.85±3.62)ng/ml](t=4.60、t=7.61;均P＜0.05).病情稳定MM组SDF-1α、CD44v6水平显著高于对照绀(t=2.99、t=4.87;均P＜0.05).9例初发和复发MM组的BMSC与人类骨髓瘤细胞系细胞U266加入rhIL-6进行混合培养后,SDF-1 α水平[(6180.25±5925.38)pg/ml]显著高于5例对照组BMSC[(1021.13±358.65)pg/ml]和9例初发和复发MM组[(1004.07±727.36)pg/ml](t=2.66、t=2.42;均P＜0.05).而其他BMSC各组问的SDF-1α水平差异无统计学意义(P＞0.05).SDF-1 α与CD44v6两者表达水平呈正相关(r=0.51,P=0.03).结论 SDF-1 α、CD44v6水平升高与MM的病情进展或发病有关,也可能与MM的肿瘤浸润过程有关;而这些体内过程可能需骨髓瘤细胞和BMSC与IL-6、SDF-1α和CD44v6等因素协同完成.     

rAAV/CEA转染树突状细胞诱导特异性CTL杀伤MCF-7细胞系CD44+ CD24-/low乳腺癌干细胞

目的:携带癌胚抗原(carcinocmbryonic antigen,CEA)基因的重组人腺相关病毒(reconstructive human adeno-association virus,rh-AAV)感染树突状细胞(dendritic cell,DC)诱导获得抗原特异性细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL),体外检测其对CD44+CD24-/low乳腺癌干细胞的杀伤效果.方法:分离供者外周血单个核细胞,以细胞因子白介素-4(interleukin-4,IL-4)、粒细胞-巨噬细胞克隆刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)和肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)诱导培养获得DC,细胞因子白介素-2(interleukin-2,IL-2),刺激培养获得T淋巴细胞.含CEA基因的rh-AAV感染培养DC,诱导成熟后将DC和T细胞混合培养获得CTL细胞.流式分选MCF-7和MA-MDB-231细胞系中CD44+CD24-/low乳腺癌干细胞,MTT法检测CTL细胞对CD44+CD24-/low乳腺癌干细胞的杀伤效果.结果:MCF-7和MDA-MB-231中CD44+/CD24-/low群比例分别为5.1%和76.3%.CEA基因转染DC诱导的CTL细胞对表达CEA抗原的MCF-7杀伤率为46.5%±15.0%,与未转染组相比,差异有统计学意义(P=0.009);对MCF-7细胞中分选的CD44+CD24-/low乳腺癌干细胞的杀伤率为44.7%±28.2%,明显高于未转染组.对非乳腺癌干细胞杀伤率为50.6%±22.2%,与未转染组相比,差异有统计学意义(P=0.05).在不表达CEA基因的MDA-MB-231乳腺癌细胞,CEA转染诱导的CTL细胞对未分选细胞、分选的CD44+/CD24-/low亚群和非干细胞亚群的杀伤率与未转染的对照组相比,差异均无统计学意义(P＞0.05).结论:携带CEA基因rh-AAV转染DC诱导产生的抗原特异性CTL细胞可杀伤表达CEA的乳腺癌细胞,对CD44+CD24-/low乳腺癌干细胞也具有一定的杀伤活性,提示免疫治疗可能是治疗乳腺癌干细胞潜在有效的手段.     

小儿急性淋巴细胞白血病血清CD3+ CD4- CD8- T细胞和T细胞亚群的变化及其临床意义

目的 探讨小儿急性淋巴细胞白血病(ALL)外周血CD3+ CD4- CD8- T细胞(DNT细胞)及T淋巴细胞亚群的变化及意义.方法 采用免疫荧光流式细胞术检测30例ALL患儿及24例健康小儿外周血DNT细胞及T淋巴细胞亚群CD3+ T细胞、CD3+ CD4+ T细胞及CD3+ CD8+ T细胞水平.结果 DNT细胞、CD3+ T细胞、CD3+ CD4+ T细胞百分比均明显低于对照组,分别为(4.93±3.75)%比(8.19±3.21)%(t=3.4,P<0.01):(49.99 ±11.70)%比(64.13 ±11.39)%(t=4.1,P<0.01);(28.30 ±7.56)%比(34.61 ±6.43)%(t=3.2,P<0.01);而CD3+ CD8+ T细胞百分比明显高于对照组,为(31.19±9.89)%比(24.33 ±4.24)%(t=3.1,P<0.01).结论 ALL患儿外周血DNT细胞及T亚群的变化提示患儿体内存在免疫功能紊乱,这可能与ALL的发病机制有关.     

淋巴细胞功能相关抗原-1/细胞间黏附分子-1介导的细胞因子诱导的杀伤细胞体外抑瘤机制

目的 探讨淋巴细胞功能相关抗原-1(LFA-1)/细胞间黏附分子-1(ICAM-1)介导的细胞因子诱导杀伤细胞(CIK)的体外抑瘤机制.方法 从白血病患儿外周血分离淋巴细胞,经过干扰素-γ(IFN-γ)、抗CD3单克隆抗体(CD3McAb)、白细胞介素-2(IL-2)诱导并与树突状细胞(DC)共培养,获得大量的DC-CIK.在经10、20μg/ml等不同质量浓度小鼠抗人LFA-1单克隆抗体处理后,采用MTT法研究DC-CIK细胞对多种白血病细胞株的杀伤活性,RT-PCR与Western blotting方法检测GATA-3和T-bet基因表达水平的变化.ELISA方法测定DC-CIK细胞释放细胞因子IL-12、IFN-γ、肿瘤坏死因子-α(TNF-α)的表达水平.结果 诱导后的DC-CIK细胞形态规则,经不同浓度的LFA-1单克隆抗体处理后,MTT结果:20μg/ml LFA-1单克隆抗体封闭组DC-CIK细胞对B95细胞杀伤作用下降最为明显(t=10.138,P＜0.05);RT-PCR与Western blotting结果:20μg/ml LFA-1单克隆抗体封闭的B95细胞组,GATA-3基因mRNA水平和蛋白水平表达增加最为明显(t=16.386,P＜0.05;t=22.652,P＜0.05);同时T-bet基因mRNA水平和蛋白水平表达降低最为明显(t=17.728,P＜0.05;t=17.452,P＜0.05);ELISA结果:20μg/ml LFA-1单克隆抗体封闭的B95细胞组中细胞因子IL-12、IFN-γ、TNF-α分泌水平下降最为明显(t=21.621,P＜0.05;t=13.739,P＜0.05;t=15.278,P＜0.05).结论 GATA-3和T-bet基因参与了LFA-1/ICAM-1介导的DC-CIK抑瘤途径,并且通过分泌Th1型细胞因子IL-12、IFN-γ、TNF-α等发挥抑瘤作用.     

粒细胞集落刺激因子对供体造血干细胞移植物中树突状细胞的影响

目的 探讨粒细胞集落刺激因子(G-CSF)对正常异基因造血干细胞移植供者外周血与骨髓移植物中Ⅰ型树突状细胞(DC1)、Ⅱ型树突状细胞(DC2)的数量及DC2/DC1比例的影响.方法 以G-CSF每天10μg/kg动员5 d后,以流式细胞术(FCM)检测11例G-CSF动员的异基因外周血造血干细胞移植物及20例G-CSF动员的异基因骨髓移植物中的DC1、DC2数量及DC2/DC1比例,并与8例正常供者动员前外周血及10例健康者动员前骨髓进行比较.结果 动员前后骨髓DC2由14.37×106/L增至29.68×106/L(t=2.433,P=0.022),而骨髓DC1分别为13.77×10a/L和18.88×106/L(t=0.625,P=0.541);DC2/DC1比例在动员后为1.83±0.81,较动员前的1.12±0.32明显升高(t=2.685,P=0.013).正常供者以G-CSF动员前、后移植物中外周血DC2数量分别为14.92×106/L和26.76×106/L(t=2.390,P=0.029),DC2/DC1比例分别为1.00±0.37和2.02±1.43(t=2.158,P=0.044),但外周血DC1分别为14.21×106/L和18.02×106/L(t=0.625,P=0.541).结论 移植前以G-CSF动员正常异基因干细胞移植供者,可选择性提高外周血及骨髓移植物中DC2的数量,而DC1数量无明显增加.     

供者外周血干细胞动员采集198例临床分析

目的 研究外周血造血干细胞移植( PBSCT)供者外周血干细胞动员采集的方法及其效 果。方法 198名健康供者每天皮下注射重组人粒细胞集落刺激因子(rhG-CSF)(5～10) μg/kg进行外周血干细胞动员,第5天开始采集。采用血细胞分析仪行单个核细胞( MNC)计数,流式细胞术(FCM)行CD34+细胞计数。分析供者性别、身高、年龄、采集当天外周血白细胞(WBC)计数对动员采集效果的影响。结果所有供者均成功动员采集,采集当天的MNC计数平均为(4.19±1.96)×108/kg,CD34+细胞计数平均为(2.98±1.40) ×106/kg; MNC和CD34+细胞计数与供者性别、身高、年龄无关;采集当天外周血WBC计数与MNC、CD34+细胞计数呈正相关(r= 0.9201,P=0.0035;r=0.8420,P= 0.0149);采集当天外周血WBC计数≥20.0×109/L的供者比＜20.0×109/L的供者采集效果更显著(F=4.688,P= 0.0013;F= 4.622,P=0.0006)。结论rhG-CSF动员的健康供者采集当天外周血WBC计数是一项预测CD34+细胞采集数量的简单、可行的指标。     

难治性淋巴瘤患者外周血T淋巴细胞亚群与自然杀伤细胞活性的检测及其临床意义

目的 探讨外周血T淋巴细胞亚群及自然杀伤(NK)细胞活性水平与难治性淋巴瘤的相关性.方法 采用流式细胞术(FCM)检测60例初治淋巴瘤患者化疗前外周血T淋巴细胞亚群水平与NK细胞的活性,化疗后随访分为难治组30例、有效组30例,以20名健康者为健康对照组.结果 淋巴瘤患者组化疗前外周血CD+4、CD+4/CD+8、NK细胞数比健康对照组低(30.17±8.63与46.52±1.39,t=12.218,P＜0.05;0.86±0.45与1.64±0.05,t=11.225,P＜0.05;12.39±7.08与19.29±0.84,t=6.365,P＜0.05),CD+3、CD+8细胞数比健康对照组高(76.14±10.71与70.48±1.44,t=-3.439,P＜0.05;40.28±14.03与28.35±0.73,t=-5.625,P＜0.05).难治组化疗前外周血CD+4、CD+4/CD+8、NK细胞数比有效组低(27.70±7.81与33.13±8.82,t=2.163,P=0.036;0.67±0.27与1.10±0.52,t=3.272,P=0.003;9.87±6.60与15.40±6.58,t=2.771,P=0.008),而CD+3、CD+8细胞数比有效组高(79.67±8.18与71.91±12.00,t=-2.540,P=0.015;44.70±13.99与34.98±12.41,t=-2.416,P=0.020).结论 淋巴瘤初治患者化疗前外周血T淋巴细胞亚群水平及NK细胞活性的检测,对判断、预测容易转归为难治的患者可能有一定的参考价值.     

CD+4CD+25调节性T细胞在小儿急性白血病发病中的作用

目的 观察CD+4 CD+25调节性T(Treg)细胞在急性白血病患儿及非白血病患儿外周血中的变化,研究CD+4 CD+25 Treg细胞在小儿急性白血病发病中的作用.方法 采用流式细胞术检测急性白血病初诊患儿组20例及非白血病患儿对照组20例外周血CD+4 CD+25 Treg细胞的数量及比例.结果 初诊患儿组及对照组外周血CD+4 CD+25 CD-127 Treg细胞占CD+4 T细胞的比例分别为(11.57±1.04)%和(6.75±0.75)%,在初诊患儿组高于对照组(t=16.808,P<0.001).结论 急性白血病患儿外周血中CD+4 CD+25 CD-127 Treg细胞数量升高,提示CD+4 CD+25 Treg细胞可能在白血病的发生、发展中起一定作用.     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

目的 研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子(VEGF)及其受体( VEGFR)的影响。方法酶联免疫吸附法检测非细胞毒性浓度(5、10、20 ng/ml)青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果培养24、48 h后,无青蒿琥酯作用的SHI-1细胞培养上清液VEGF质量浓度分别为( 980.3±2.2)、(982.4±2.3) pg/ml,VEGFR-1表达率分别为(5.40±3.11)％和(4.45±2.85)％,VEGFR-2表达率分别为(13.90.± 2.26)％和（13.95±1.96）％。5、10、20 ng/ml青蒿琥酯作用24h后,SHI-1细胞培养上清液VEGF质量浓度分别为(234.6±1.8)、(114.9±1.6)、(108.8±1.5) pg/ml,作用48 h后分别为(62.3±1.7)、(60.9±1.6)、(32.7±1.7) pg/ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜ 0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义(均P＜ 0.05)。5、10、20 ng/ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为(4.30±2.21)％、(4.20±1.37)％和(3.90±1.86)％,作用48 h后分别为(3.80±2.87)％、(3.60±1.73)％和(3.00±1.82)％,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义(均P＞ 0.05);作用24h后,SHI-1细胞VEGFR-2阳性率分别为(4.40±1.15)％、(3.10±0.68)％和(1.10±0.72)％,作用48 h后分别为(3.00±1.68)％、(2.20±0.93)％和(0.60±0.92)％,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜ 0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞ 0.05）。结论SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

非清髓性异基因造血干细胞移植后早期自然杀伤细胞的重建

目的 研究非清髓性异基因造血干细胞移植(NAST)后早期自然杀伤(NK)细胞的重建情况.方法 分别用流式细胞术直接免疫荧光法、T细胞活化实验、四甲基偶氮唑蓝比色(MTT)法检测10例NAST后白血病患者及10位健康对照者细胞表面标志及体外免疫功能.结果 移植后28～35 d,患者外周血CD+3、CD+4细胞比例降低,分别为(2.03±15.60)%和(22.69±12.29)%,CD+8细胞比例正常或增高,平均为(29.26±8.99)%;T细胞对有丝分裂原反应减低,其增殖反应刺激指数为94.60±44.87;NK细胞比例在正常范围或增高,为(18.77±9.11)%;NK细胞表面IL-2R表达阳性率增高,平均为(3.71±2.23)%,和正常对照组的(2.05±0.94)%相比差异有统计学意义(t=2.116,P=0.044);部分患者NK细胞活性明显增强,移植组与健康对照组分别为(25.30±12.39)%和(16.60±3.53)%(t=2.135,P=0.047).结论 NK细胞在NAST后早期即恢复,当特异性免疫功能仍低下时,它可能在机体抗感染和抗肿瘤中发挥着重要作用.     

粒细胞-巨噬细胞集落刺激因子基因修饰的树突状细胞疫苗增强体外抗肿瘤免疫效应

Objective:The aim of the study was to investigate whether dendritic cell (DC) precursors,recruited by injection of chemokine ligand 3 (CCL3),induce enhanced anti-tumor immunity after granulocyte-macrophage colony stimulating factor (GM-CSF) transfection in mice ex vivo.Methods:The 615 mice were injected with CCL3 via the tail vein.Freshly isolated B220–CD11c+ cells were cultured with cytokines.For adenoviral (Ad)-mediated gene transduction,DCs were transferred AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions,and detecting the expression of GM-CSF after transfection.The variation of GM-CSF gene-modified DCs were analyzed by morphological observation,phenotype analysis,and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by frozen and thawed method.The stimulated DCs vaccination induced T lymphocytes,and the killing effect of T cells to gastric cancer cells was assayed by MTT.INF-γ production was determined with the INF-γ ELISA kit.Results:B220–CD11c+ cells numbers increased after CCL3 injection.ELISA results showed that after GM-CSF gene modification,DC could produce high level of GM-CSF.When DCs were transferred AdGM-CSF gene at MOI equal to 1:100,GM-CSF level in culture supernatants reached saturation [(130.00 ± 12.61) pg/mL].After GM-CSF gene-modification,DCs tended to more maturated through morphological observation and were phenotypically identical to typical DC and gained the capacity to stimulate allogeneic T cells.T lymphocytes stimulated with DC transduced with GM-CSF gene showed the specific killing effect on gastric carcinoma cells and produced high level of INF-γ [(1245.00 ± 13.75) pg/mL].Conclusion:CCL3-recruited DCs modified by adenovirus-transducted GM-CSF could produce high level of GM-CSF,which tended to more maturated,and the capacity of activating allogeneic T lymphocytes proliferation was enhanced greatly.Moreover,they could stimulate specific cytotoxic T lymphocyte (CTL) to gastric cancer ex vivo.     

p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor

p40/LAIR-1, a member of the immunoglobulin superfamily, is a surface molecule broadly distributed among leukocytes which has been shown to down-regulate T and NK cell activation. In this study, we show that p40/LAIR-1 is highly expressed in CD14+ peripheral blood mononuclear cells (PBMC). When cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10-14 days, CD14+ cells acquired morphologic and phenotypic features (i.e. loss of CD14 and expression of CD80bright and CD86bright) typical of dendritic cells (DC) and lost the expression of p40/LAIR-1. Engagement of p40/LAIR-1 (but not of CD58) by specific monoclonal antibodies prevented CD14+ PBMC differentiation into DC; when cultured in the presence of GM- CSF upon p40/LAIR-1 cross-linking, the resulting cells were CD14+CD80(dull)CD86(dull) and displayed a macrophage-like morphology. We have recently demonstrated that peripheral blood CD14+ cells co-expressing the CD34 progenitor marker represent the circulating precursors of CD83+ DC. Herein we show that cross-linking of p40/LAIR-1 prevented the maturation of CD14+CD34+ cells into CD83+ DC. This effect appears to be consequent to the impairment of GM-CSF receptor-mediated activation signaling. Indeed, triggering of GM-CSF receptors in both CD14+ and CD14+CD34+ cells led to increases in the intracellular free calcium concentrations which were inhibited by p40/LAIR-1 engagement. Taken together, these data suggest a possible regulating role played by p40/LAIR-1 in the process of differentiation from peripheral blood precursors into DC induced by GM-CSF.     

Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation

Selective accumulation of eosinophils and activated CD4+ cells is now considered a central event in the pathogenesis of asthma, and this process is thought to be mediated by a number of cytokines including tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the Type 2 cytokines interleukin-4 (IL-4) and IL-5. To carry out a detailed time-course analysis of cellular changes in the bronchoalveolar lavage fluid (BAL), peripheral blood (PB), and bone marrow (BM), and of changes in the aforementioned cytokines in BAL and serum, Balb/c mice were sensitized by intraperitoneal injection with ovalbumin (OVA) adsorbed to aluminum hydroxide on two occasions 5 days apart, and were subjected to an OVA aerosol challenge 12 days after the second sensitization. This resulted in an airways inflammatory response characterized by early transient neutrophilia, marked eosinophilia, and, to a lesser extent, lymphocytosis in the BAL. Inflammatory events were first observed 3 h and 24 h after antigen challenge in the lung tissue and BAL, respectively, and lasted for 21 days. In the BM, we detected a 1.5- and 5-fold increase in the total number of cells and eosinophils, respectively, 4 days after the second sensitization. This was followed by a decrease, although BM eosinophilia remained clearly present at the time of antigen challenge. A second eosinopoietic event was observed in the BM shortly after challenge and reached a peak at day 3. BM cellularity returned to normal at day 21 after challenge. Serum OVA-specific IgE was first detected 3 days following the second sensitization (150 ng/ml). IgE levels then decreased but remained at the 75 ng/ml range at the time of the aerosol challenge. During the sensitization period, TNF-alpha (approximately 25 pg/ml), IL-4 (approximately 40 pg/ml), and IL-5 (approximately 250 pg/ml) were detected in serum, but not in the BAL fluid (BALF) and returned to background levels at the time of the antigen challenge. After antigen challenge, TNF-alpha, IL-4, IL-5, and GM-CSF were detected in serum. Peak levels were observed at 3 h (approximately 40 pg/ml), 3 h (approximately 120 pg/ml), 12 h (approximately 350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, and returned to background levels 24 h after challenge. In the BALF, we detected peak levels of TNF-alpha, IL-4, IL-5, and GM-CSF at 6 h (approximately 250 pg/ml), 24 h (approximately 140 pg/ml), 24 h (350 pg/ml), and 3 h (approximately 10 pg/ml), respectively, with a return to background levels 5 days after challenge. No IL-10 could be detected at any time point during sensitization or after challenge in either serum or BAL. We also detected approximately 40 pg/ml of interferon-gamma (IFN-gamma) in the serum of normal untreated mice. Serum IFN-gamma levels fluctuated during sensitization and after challenge, but never exceeded those observed in untreated mice. Thus, the cytokine profile observed in this experimental model of allergic inflammation is characterized by IL-4 and IL-5 dominance, with an apparently minor TNF-alpha and GM-CSF contribution and relatively low or undetectable levels of IFN-gamma and IL-10.     

CD19+ pro-B cells can give rise to dendritic cells in vitro

Dendritic cells (DC) have the specific capacity of initiating primary T cell responses and ultimately derive from precursors in bone marrow. DC were originally thought to be only of myeloid origin, and myeloid precursor cells could be induced to differentiate into functional DC in response to granulocyte-macrophage (GM)-CSF. However, early CD4low precursor cells from the thymus can also develop into DC when cultured in IL-1beta, IL-3, IL-7, TNF-alpha, stem cell factor, and Flt-3L. In that case, GM-CSF was not required. We now show that CD19+ pro-B cells develop into DC with T cell stimulatory properties when cultured under similar conditions. These pro-B cells acquired the DC-related markers CD11c and NLDC145/DEC205, along with CD80/B7-1, CD86/B7-2, and a high density of MHC class II Ags. The marrow-derived DC did not express CD4 or CD8alpha, which are markers related to thymic DC. These findings are consistent with a new pathway through which DC are generated from B lymphoid precursors.     

Mechanism of Immune Hyporesponsiveness Induced by Recipient-derived Immature Dendritic Cells in Liver Transplantation Rat

Objective To investigate the mechanism of immune hyporesponsiveness induced by donor-antigen-unloaded recipient-derived immature dendritic cell (imDC) of liver grafts in rats.Methods Forty Sprague-Dawley rats (donor) and forty male Wistar rats (recipient) were randomly divided into 4 groups:control,cyclosporine A (CsA),mature DC (mDC),and imDC groups respectively,with 10 donor rats and 10 recipient rats in each group.Recipient rats in CsA group were treated with 10 mg·kg-1.d-1 CsA starting day 2 after the transplantation.Recipients in the mDC or imDC groups were given Wistar rat derived mDCs (1×106/rat) or imDCs (1×106/rat) via dorsal vein of the penis respectively 1 day before the transplantation.In each group,5 recipients were kept for determination of survival time and the other 5 rats were executed at day 10 after transplantation.Blood samples were collected for the measurement of serum alanine aminotransferase (ALT),total bilirubin (TBIL),interleulkin 2 (IL-2),interferon gamma (IFN-γ),IL-4,and IL-10 levels,liver tissue was harvested for HE staining and acute rejection evaluation.Expression levels of Fas-L/Fas in the grafts were detected by immunohistochemical staining; and Western blot was used to detect the expression level of Scurfin.Results The survival time of CsA and imDC groups was significantly longer than that of control and mDC groups (all P<0.05).The levels of serum ALT and TBIL in the control group (2072.20±217.93 IU/L and 147.42±22.02 umol/L) and mDC group (2117.00±285.13 IU/L and 141.58+20.82 μmol/L) were significantly higher than those in the CsA group (59.68±13.48 IU/L and 15.40±2.13 umol/L) or imDC group (50.80±19.63 IU/L and 14.44±3.49 umol/L) (all P<0.05).In the CsA and imDC groups,the levels of IL-2 (22.52±3.75 pg/mL and 22.12±3.90 pg/mL) and IFN-γ (309.20125.19 pg/mL and 321.00±21.64 pg/mL) were significantly lower,but the levels of IL-4 (297.60±25.07 pg/mL and 277.00±22.47 pg/mL) and IL-10 (1226.00+140.49 pg/mL and 1423.00±106.39 pg/mL) were higher than those of the control (IL-2:147.78±12.80 pg/mL,IFN-γ:1758.60±106.22 pg/mL,IL-4:17.40+4.77 pg/mL,IL-10:81.00± 9.47 pg/mL) and mDC groups (IL-2:142.34±9.29 pg/mL,IFN-γ:1835.OOi82.63 pg/mL,IL-4:15.60+ 3.96 pg/mL,IL-10:68.80±11.23 pg/mL) (all P<0.01).The expression level of Scurfin protein on CD4+CD25+ T cells of the imDC group (1.34±0.29) was significantly higher than that in the control (0.72±0.13),CsA (0.37±0.11),and mDC groups (0.78±0.17) (all P<0.05).Conclusion Donor-antigen-unloaded recipient-derived imDC is an effective treatment in inducing immune hyporesponsiveness through induction of T cell apoptosis,shift in Thl/Th2 balance,and proliferation of regulatory T cell.     

Pharmacokinetic and pharmacodynamic studies of subcutaneously administered recombinant human interleukin-3 following chemotherapy for non-Hodgkin's lymphoma

The pharmacokinetics and the pharmacodynamic profile of subcutaneously administered recombinant human non-glycosylated interleukin-3 (rhIL-3) was studied in lymphoma patients after standard CHOP chemotherapy. 30 patients received 0.5, 1.0, 5.0, 7.5 and 10 micrograms/kg (six patients at each dose level) of rhIL-3 for 14 d. Serum rhIL-3 samples were obtained regularly, during the treatment and serially over a 24 h period on the first (cycle day 2) and the last (cycle day 15) day of rhIL-3 treatment for pharmacokinetic evaluation. Following s.c. injection on cycle day 2. the maximum rhIL-3 serum concentration ranged from 289 pg/ml (0.5 micrograms/kg) to 4690 pg/ml (10 micrograms/kg). Both the maximum serum concentration (R = 0.90. P < 0.0001) and the area under the serum concentration-time curve (R = 0.95, P < 0.0001) were related to dose. The elimination half-life T1/2 beta was 160 min for 0.5 micrograms/kg and 134 min for 10 micrograms/kg, with no apparent dose relationship. The systemic clearance of 3.0-6.0 ml/min/kg was comparable at all dose levels. No significant difference was noted between pharmacokinetic parameters on the first day of rhIL-3 and the last day of treatment, and no accumulation of the drug was noted throughout the study. The pharmacokinetic parameters correlated poorly to the clinical response of the growth factor. where dose in micrograms/kg seemed to be the most important single factor.     

Regulation of IFN-gamma production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF-/-) mice produce far lower serum levels of IFN-gamma in response to LPS than GM-CSF+/+ mice. CD4+ and CD8+ T cells from LPS-injected GM-CSF-/- mice showed a deficiency in IFN-gamma production and proliferative activity in response to IL-2 and IL-12, whereas IFN-gamma production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF-/- mice do not have an intrinsic defect in IFN-gamma production, because IL-12 injection induces the same high levels of IFN-gamma in GM-CSF-/- and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF-/- T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-gamma levels and T cell responses in LPS-injected GM-CSF-/- mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.     

In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.