不同来源人造血干/祖细胞在NOD/SCID小鼠体内归巢能力的差异

目的 比较不同来源的人造血干／祖细胞在NOD／SCID小鼠体内归巢能力的差异性，并探讨其体内归巢能力与膜表面归巢相关分子CXCR4表达水平的相关性。方法 采用流式细胞术（FACS）检测新鲜脐血、冻存脐血、动员后外周血（mPB）及骨髓来源的CD34^＋细胞表面CXCR4表达水平；将荧光染料CFSE标记的人CD34^＋细胞移植人接受照射的NOD／SCID小鼠，移植后20小时检测已归巢于NOD／SCID小鼠骨髓及脾脏中不同来源的人CD34^＋细胞，计算其相应的骨髓及脾脏归巢效率；并将小鼠股骨制成组织切片，荧光显微镜下观察人CD34^＋细胞在小鼠骨髓腔内的分布。结果 新鲜脐血、冻存脐血、mPB和骨髓CD34^＋细胞膜表面CXCR4表达阳性率分别为（49.52±1．12）％。（46．12±2．29）％，（48．50±2．48）％和（65．39±1．27）％，CD34^＋细胞在NOD／SCID小鼠骨髓的归巢效率分别为（3．00±0．44）％，（2．84±0．46）％，（4．06±0．70）％及（5．76±0．52）％；在脾脏的归巢率分别为（1．88±0．12）％，（1．80±0．15）％，（1．90±0．22）％，（2．16±0．34）％。归巢的CD34^＋细胞主要分布于小鼠股骨的骨内膜区域。结论 脐血CD34^＋细胞膜表面CXCR4水平低于mPB和骨髓。经冻存复苏后脐血CD34^＋细胞膜表面CXCR4水平略有下调。脐血CD34^＋细胞在照射NOD／SCID小鼠的骨髓归巢效率低于mPB和骨髓。     

骨髓增生异常综合征患者外周血树突细胞数量、亚群及其表面协同刺激分子表达的研究

目的 检测骨髓增生异常综合征（MDS）患者外周血树突细胞（DC）总量、亚群（pDC和mDC）比例及其表面协同刺激分子（CD80、CD86和CIMO）表达，探讨MDS患者细胞免疫异常的形成机制。方法 选取38例MDS患者及19名正常对照，采用荧光素单克隆抗体标记法和流式细胞术检测MDS患者外周血中Lin1^—HLA-DR^＋细胞（DC）、Lin1^-HLA—DR^＋CD123^＋细胞（pDC）、Lin1—HLA—DR^＋CD11c^＋细胞（mDC）的数量以及CD80、CD86和CIMO在DC膜上的表达。结果低危MDS组、高危MDS组和正常对照组外周血DC总量分别为（33．7±7．0）×10^6／L、（56．3±29．0）×10^6／L和（12．1±1．4）×10^6／L（P〈0．05），pDC数量分另U为（12．6±4．1）×10^6／L、（3．6±1．0）×10^6／L和（6．6±0．7）×10^6／L（P〉0．05），mDC数量分别为（16．7±6．3）×10^6／L、（28．7±17．6）×10^6／L和（5．5±0．9）×10^6／L（P〈0．05）。低危MDS组、高危MDS组和正常对照组DC占外周血单个核细胞（PBMNC）百分比分别为（2．37±0．53）％、（3．58±1．39）％和（0．68±0．08）％（P〈0．05），pDC占PBMNC百分比分别为（0．82±0．29）％、（0．31±0．06）％和（0．37±0．04）％（P〉0．05），mDC占PBMNC百分比分别为（0．96±0．35）％、（1．51±0．70）％和（0．32±0．05）％（P〈0．05）。低危MDS组、高危MDS组和正常对照组外周血中CD80^＋DC分别为（30．6±11．8）×10^6／L、（2．3±0．9）×10^6／L和（2．3±0．6）×10^6／L（P〈0．05），CD86^＋DC分别为（25．1±7．4）×10^6／L、（12．4±6．3）×10^6／L和（6．2±3．2）×10^6／L（P〈0．05），CD40^＋DC分别为（2．8±1．0）×10^6／L、（1．5±0．9）×10^6／L和（3．2±2．3）×10^6／L（P〉0．05）。结论 MDS患者外周血中DC数量增多、比例异常；以mDC增多为主，pDC无明显增多；MDS患者DC高表达CD80和CD86，CD40表达不增高。提示MDS患者诱导抗肿瘤细胞免疫的抗原呈递细胞（pDC）数量及功能不足；而与正常造血克隆炎性损伤有关的抗原呈递细胞（mDC）却增多。这可能是导致MDS患者细胞免疫异常的重要机制之一。     

CD28/CTLA-4共刺激分子在再生障碍性贫血免疫发病机制中的作用

目的 研究CD28、CTLA-4在再生障碍性贫血（AA）免疫发病机制中的可能作用。方法 以流式细胞术分别分析23例发病期AA、10例恢复期AA患者和15名正常人骨髓细胞中CD3^＋CD4^＋T细胞上CD28、CTLA-4表达率，Th1、Th2细胞比例，评价CD28、CTLA4表达与Th1／Th2、中性粒细胞绝对值（ANC）之间的关系。结果 ①正常对照组CD3^＋CD4^＋T细胞上CD28、cTLA4表达率及CD28^＋／CTLA-4^＋比值分别为（31．40±10．83）％、（2．45±1．30）％、17．02±13．44，AA患者发病期分别为（39．84±10．89）％、（1．43±0．67）％、43．04±37．61，AA患者恢复期分别为（22．00±9．08）％、（3．46±2．26）％、10．49±7．80；与正常对照组比较，AA患者发病期CD28显著升高（P〈0．05），CTLA-4显著下降（P=〈0．01），CD28^＋／CTLA4^＋比值相应地亦显著升高（P〈0．05），而恢复期上述数值与正常对照组比较，差异无统计学意义（P〉0．05）。②正常对照组Th1、Th2细胞、Th1／Th2比值分别为（4．21±2．11）％、（1．99±1．27）％、2．46±1．28，AA发病期为（11．13±4．96）％、（2．46±1．65）％、5．20±1．98，恢复期分别为（5．39±4．29）％、（2．53±2．41）％、2．87±1．43；与正常对照组比较，AA发病期Th1增高，Th1、Th2平衡向Thl偏移（P值均〈0．01），恢复期与正常对照组比较，差异无统计学意义。③AA患者CD28^＋／cTLA4^＋比值与Thl／Th2比值呈正相关（P〈0．05），ANC与CD3^＋CD4^＋CD28^＋T细胞数呈负相关（P〈0．01），与CD3^＋CD4^＋CTLA-4^＋T细胞数呈正相关（P〈0．01）。结论 ①AA发病期患者骨髓CD4^＋T细胞表面共刺激分子表达异常，CD28升高，而CTLA-4下降，AA患者骨髓T细胞处于“预激活”状态；②CD28／CTLA-4比值与Th1／T12．比值呈正相关提示：CD28、CTLA-4表达失衡可引起Th细胞格局偏移，Th1型细胞增多。③ANC和CD28、CTLA-4间相关分析进一步说明CD28、CTLA-4与AA的发生、发展有关。     

CD4+CD25+调节性T细胞在特发性血小板减少性紫癜患者中的变化

目的探讨CD4^＋CD25^＋T细胞在特发性血小板减少性紫癜（ITP）患者发病机制中的作用。方法应用流式细胞术检测ITP患者外周血CD4^＋CD25^＋T细胞、CD4^＋CD25^highT细胞、CD4^＋FOXP3^＋T细胞、CD4^＋CD25^＋FOXP3^＋T细胞的数量；实时荧光定量PCR检测外周血FOXP3 mRNA的表达水平。将ITP患者和正常人CD4^＋CD25^highT细胞与自身CD4^＋CD25^-T细胞混合培养，检测CD4^＋CD25^high T细胞免疫抑制功能。结果ITP患者外周血中CD4^＋CD25^＋T细胞约占CD4^＋T细胞的（15．64±5．82）％，明显高于正常对照组（9．30±3．95）％（P〈0．01），CD4^＋CD25^high T细胞比例为（1．53±0．66）％，与对照组[（1．36±0．55）％]比较差异无统计学意义（P〉0．05）；CD4^＋FOXP3^＋T细胞和CD4^＋CD25^＋FOXP3^＋T细胞分别为（1．82±1．42）％和（1．25±0．94）％，均明显低于对照组[（3．90±1．37）％和（2．65±0．92）％]（P值均〈0．01）。ITP患者外周血FOXP3 mRNA表达水平较正常人明显下调（P〈0．01），CD4^＋CD25^high T细胞的抑制活性较正常人减弱（P〈0．01）。结论ITP患者中CD4^＋CD25^＋T细胞FOXP3表达水平降低，抑制活性减弱。     

rhG-CSF动员的骨髓和外周血干细胞混合移植物首次采集时供者外周血单核细胞计数反映混合采集物中CD34^＋细胞含量

本研究探讨人重组粒细胞集落刺激因子（rhG-CSF）动员的骨髓和外周血干细胞混合移植健康供者首次干细胞采集时供者外周血单核细胞数量与骨影外周血混合采集物中CD34^＋细胞数量的关系。对70名亲缘健康供者均皮下注射rhG-CSF5μg／（kg·d）,连续5天。第4天和第5天分别采集骨髓和外周血干细胞。首次干细胞采集时用EX2100血细胞分析仪检测供者外周血细胞计数,同时应用流式细胞仪测定骨髓和外周血混合采集物中的CD34^＋细胞数量。结果表明：rhG—CSF动员的70例供者首次干细胞采集时外周血单核细胞的数量为（1．15±0．60）×10^9／L;骨髓和外周血采集物中的CD34^＋细胞总量分别是（5．854±2．93）×10^7和（1．33±0．77）×10^8;骨髓和外周血混合采集物的CD34^＋细胞总量是（1．92±0．86）×10^8。Pearson和Spearman分析显示首次干细胞采集时供者外周血单核细胞计数（×10^9／L）与骨髓采集物中CD34^＋细胞的总量（相关系数：r=0．265,P=0．027）、外周血采集物中CD34^＋细胞总量（r=0．340,P=0．004）以及混合移植物中CD34^＋细胞的总量（r=0．398,P：0．001）均存在显著的相关性;多因素分析表明,首次干细胞采集时供者外周血单核细胞计数与骨髓采集物、外周血采集物以及混合移植物中CD34^＋细胞的总量均呈正相关关系（P值分别为0．027、0．004和0．001）。首次干细胞采集时供者外周血单核细胞数量对混合移植物中CD34^＋细胞总量预测的敏感性是71％,特异性是70％（P=0．007）。结论：rhG-CSF动员的骨髓和外周血混合移植健康供者首次干细胞采集时外周血单核细胞计数可有效预测输注给受者的CD34^＋细胞总量即采集效果。     

动员后外周血中CD34+细胞去除前后对改善肢体缺血疗效的研究

目的对动员的外周血单个核细胞（PBMNC）和动员的去除CD34^＋细胞的PBMNC移植治疗裸鼠下肢缺血的疗效进行比较，以探讨PBMNC的治疗机制。方法经过单采获得G-CSF动员的PBMNC后，一部分通过CD34磁珠抗体分选得到去除CD34^＋细胞的PBMNC。动员的PBMNC和动员的去除CD34^＋细胞的PBMNC荧光标记后按1×10^6细胞或相应体积的PBS分别局部肌肉注射移植到裸鼠缺血下肢。观察下肢血流灌注以及毛细血管密度。用ELISA法检测下肢肌肉的血管内皮生长因子（VEGF）表达，并进一步观察表达的VEGF是否由移植细胞分泌。结果PBMNC移植后缺血下肢血流明显恢复，毛细血管密度明显增加，但去除CD34^＋细胞的PBMNC移植组疗效有所下降。细胞移植后4周，PBMNC组的血流灌注由（20．3±4．2）％恢复为（96．4±5．6）％，对照组仅恢复为（71．3±4．4）％（P〈0．01），去除CD34^＋细胞组恢复为（83．8±5．2）％（P〈0．05）。PBMNC组血管密度为（521±47）／mm^2，去除CD34^＋细胞组为（3964-21）／mm^2（P〈0．05），但仍高于对照组[（276±43）／mm^2]（P〈0．05）。在PBMNC组可以观察到移植的细胞整合到缺血的毛细血管壁。缺血肌肉VEGF的表达明显升高，其共表达VEGF和移植的单个核细胞。结论移植G-CSF动员的PBMNC不但可以通过干细胞整合到血管壁的机制促进血管生长，还可以通过提供细胞因子的机制促进血管生长。去除CD34^＋细胞削弱了动员的PBMNC移植治疗肢体缺血的血管新生效应。     

婴幼儿急性上呼吸道感染淋巴细胞亚群的改变

目的研究婴幼儿上呼吸道感染时细胞免疫状况的变化情况。方法采用流式细胞仪检测急性上呼吸道感染患儿及正常对照组外周血Th1和Th2的百分率，CD4^＋与CD8^＋细胞百分率，CD4^＋／CD8^＋比值及CD19^＋和CD19^＋CD23^＋的表达水平，以探讨婴幼儿急性上呼吸道感染T淋巴细胞亚群的变化及B淋巴细胞的活化状况。结果上呼吸道感染的患儿的外周血Th1，Th2的细胞百分率（13．15%±6．23%及4．57％±3．10％）均显著高于对照组（7．14％±5．09％及2．03％±0．95％），患儿组CD4^＋细胞百分率亦较对照组为高（P〈0．05）；CD8^＋，CD4^＋／CD8^＋比值均与正常对照组之间无统计学差异（P〉0．05），CD19^＋与CD19^＋CD23^＋的表达阳性率无明显升高。结论婴幼儿上呼吸道感染时可活化T辅助淋巴细胞亚群，表现为Th1扫Th2细胞百分率均增加，Th1和Th2细胞免疫反应增强，而B淋巴细胞状况无明显改变。     

粒细胞集落刺激因子在自体外周血干细胞移植治疗缺血性下肢血管病中的作用

目的：观察缺血性下肢血管病患者进行自体外周血干细胞移植时。应用粒细胞集落刺激因子后细胞成分的变化以及对自身身体状况的近期影响。 方法：选取2004-11／2005—04解放军第四六三医院内分泌科收治的126例接受粒细胞集落刺激因子动员的缺血性下肢血管病患者，全部接受皮下注射粒细胞集落刺激因子5-12μg（kg&;#183;d），连续4-5d。为防止血黏度增加引起心脑血管意外，在干细胞动员的同时应用低分子肝素钙5000u，皮下注射1次／d。连续4～5d。每天监测血细胞计数和凝血3项。同时采用流式细胞仪监测外周血中CD34^＋细胞数。观察并记录动员后及采集过程中、后出现的毒副反应。 结果：按意向处理分析。实验纳入126例缺血性下肢血管病患者。全部进入结果分析。①全部患者动员过程中外周血象的变化：粒细胞集落刺激因子动员前白细胞数量为（5．35&;#177;1．64）&;#215;10^9L^-1，动员第5天为（42．17&;#177;18．56）&;#215;10^9L^-1．第6天为（44．23&;#177;17．47）&;#215;10^9L^-1。动员后比动员前提高5～13倍（P〈0．01）；血红蛋白和血小板动员前后无明显变化。（爹全部患者动员后采集细胞悬液的情况：37例患者于动员第5天进行采集，单个核细胞数值和CD34^＋百分数分别为（432．68&;#177;89．36）&;#215;10^9L^-1和（0．87&;#177;0．38）％；其余89例均于第6天进行采集，单个核细胞数值和CD34^＋百分数分别为（463．71&;#177;58．33）&;#215;10^9L^-1和（0．90&;#177;0.35）％，两者基本相近（P〉0．05）。③性别、年龄和体质量对单个核细胞数值和CD34^＋细胞百分数的影响：男性患者采集的单个核细胞数值高于女性患者（P〈0．05）．而单位体质量的CD34^＋细胞数值男女基本相近；以年龄55岁为界，大于55岁和小于55岁的患者差异显著（P〈0．05）；高体质量患者采集的单个核细胞数值高于低体质量患者（P〈0．05），而单位体质量的CD34^＋细胞数值基本相似。④不良事件和副反应：主要的不良反应有骨痛、周身肌肉酸痛、乏力、头痛、失眠、食欲下降、恶心呕吐、低热。采集过程中可能出现口周、面部或四肢麻木，一般停药2～4d症状即可消失。 结论：缺血性下肢血管病患者进行自体外周血干细胞移植时。粒细胞集落刺激因子作为有效动员剂，可有效动员单个核细胞和CD34^＋细胞。绝大部分患者能够耐受．但廊任用一定剂量的抗凝剂预防不良反席的发毕．     

原发性胆汁性肝硬化动物模型的建立及外周血T淋巴细胞表面CD40配体分析

目的用聚肌苷酸胞苷酸（polyinosinic polycytidylic acid，PolyI：C）注射C57BIL/6小鼠以研究建立原发性胆汁性肝硬化（PBC）动物模型，探讨CD40配体（CD40L）在外周血T淋巴细胞表面的表达，和在PBC中T淋巴细胞的活化情况。方法20只C57BIM6小鼠随机分对照组和模型组。将polyI：C以5ms／kg的剂量腹腔注射模型组小鼠，对照组注射PBS，每周2次，120d后处死，留取外周血和肝组织，间接免疫荧光法检测血清抗线粒体抗体（AMA），HE染色观察胆管病理变化，生化分析仪测定血清碱性磷酸酶（ALP）含量，流式细胞术分析外周血CD4^＋、CD8^＋T淋巴细胞所占比例及CD40L在T淋巴细胞表面的表达情况。结果PolyI：C注射120d后模型组小鼠均出现AMA阳性，肝组织小胆管周围有不同程度的炎性细胞浸润，血清ALP明显高于对照组（P〈0．001）；对照组胆管及血清ALP均未发生明显变化；模型组小鼠CD40L^＋／CD4^＋、CD40L^＋／CD8^＋T淋巴细胞[（4．35±0．34）％，（1．42±0．10）％）显著高于对照组[（0．78±0．10）％，（0．43±0．04）％，P〈0．01]；而模型组小鼠CD4^＋、CD8^＋T淋巴细胞所占比例[（25．83±1．80）％，（24．84±2．70）％]与对照组[（20．51±3．46）％，（17．84±1．53）％]比较，差异无统计学意义（P〉0．05）。结论PolyI：C腹腔注射C57BIM6小鼠120d可引起PBC病变，活化的CD4^＋、CD8^＋T淋巴细胞在PBC发病中起重要作用。     

G-CSF对慢性粒细胞白血病患者bcr/abl+-CD34+细胞增殖、分化的影响

目的了解G-CSF对bcr／abl^＋-CD34^＋细胞增殖、分化的影响。方法采集慢性粒细胞白血病（CML）患者的bcr／abl^＋-CD34^＋细胞，分别以0、10、100、1000ng／ml的G-CSF与之共培养，并以正常骨髓CD34’细胞为对照，通过锥虫蓝拒染法、流式细胞术和光学显微镜观察研究其细胞增殖、周期分布、抗原分化和形态变化特点。结果所有实验组bcr／abl^＋-CD34^＋细胞均明显增长，其中G-CSF10ng／ml组培养48、96h，其细胞数显著高于同期无G-CSF组（P〈0．05）；而正常CD34^＋细胞数只在G-CSF存在的情况下增长明显，其中G-CSF100ng／ml组培养48、96、144h细胞数均显著高于同期无G-CSF组（P值分别为〈0．05，0．01，0．01）；G-CSF10、100、1000ng／ml组的bcr／abl^＋-CD34^＋细胞培养144h其Go／G1期比例显著低于G-CSF空白组（P〈0．05）；而正常CD34^＋细胞G-CSF10、100、1000ng／ml组培养48和96h其Go／G1期比例均明显低于无G-CSF组（P〈0．01）；bcr／abl^＋-CD34^＋细胞及正常CD34^＋细胞CD34抗原表达均随培养时间延长而下降，伴随CD33和CDl3抗原先升后降，其变化与G-CSF浓度无关。但bcr／abl^＋-CD34^＋细胞的各抗原分化显著快于正常CD34^＋细胞。bcr／abl^＋-CD34^＋细胞和正常CD34^＋细胞均随着增殖分化表现出终末细胞的形态特征。结论G-CSF能促进bcr／abl^＋-CD34^＋细胞及正常CD34^＋细胞的增殖，但并非前者增殖的必要条件。bcr／abl^＋-CD34^＋细胞比正常CD34^＋细胞分化更快，但两类细胞的分化速度均与G-CSF浓度无关。     

Disruption of the CXCR4/CXCL12 chemotactic interaction during hematopoietic stem cell mobilization induced by GCSF or cyclophosphamide

Hematopoietic progenitor cells (HPCs) normally reside in the bone marrow (BM) but can be mobilized into the peripheral blood (PB) after treatment with GCSF or chemotherapy. In previous studies, we showed that granulocyte precursors accumulate in the BM during mobilization induced by either GCSF or cyclophosphamide (CY), leading to the accumulation of active neutrophil proteases in this tissue. We now report that mobilization of HPCs by GCSF coincides in vivo with the cleavage of the N-terminus of the chemokine receptor CXCR4 on HPCs resident in the BM and mobilized into the PB. This cleavage of CXCR4 on mobilized HPCs results in the loss of chemotaxis in response to the CXCR4 ligand, the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). Furthermore, the concentration of SDF-1 decreased in vivo in the BM of mobilized mice, and this decrease coincided with the accumulation of serine proteases able to directly cleave and inactivate SDF-1. Since both SDF-1 and its receptor, CXCR4, are essential for the homing and retention of HPCs in the BM, the proteolytic degradation of SDF-1, together with that of CXCR4, could represent a critical step leading to the mobilization of HPCs into the PB in response to GCSF or CY.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function.

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

CD34+ cells and CD34+CD38- subset from mobilized blood show different patterns of adhesion molecules compared to those from steady-state blood, bone marrow, and cord blood

As suggested previously, a down-regulation of some cellular adhesion molecules (CAMs) on CD34(+) hematopoietic progenitor cells (HPC) may contribute to their egress from bone marrow (BM) to peripheral blood (PB) by decreasing their adhesion to BM stromal cells. Besides counting the percentage of CAM-positive cells, we decided to define clearly the antigen density (AgD) of the CAM on mobilized- and steady-state CD34(+) HPC using QIFIKIT calibration beads. Five sources of cells were compared: PB and BM from normal donors (nPB, nBM) cord blood (CB), mobilized PB obtained from leukapheresis products (LKP), and mobilized BM (mBM) samples. In our study the CAM-AgD was the lowest on CD34(+) cells in LKP which, on the contrary, contained the highest percentage of CD117(+), CD54(+), CD58(+) cell subsets. As for CB, a greater proportion of CD44(+) and CD62L(+) cells was observed in LKP than in other products. The LKP-CD34(+) cell population contained a greater percentage of CD11a(+) cells when compared to mBM, but the lowest percentage of CD49d(+) and CD49e(+) cells when compared to all products. The proportion of the CD34(+)CD38(-) immature subset expressing CD11a, CD44, CD54, or CD62L was greater in LKP than in mBM; the CD62L-AgD was higher in LKP than in mBM. This quantitative analysis clearly showed a downregulation of all CAM on LKP-CD34(+). The CD44, CD62L, CD11a, and CD54 AgD decrease appears to be specifically involved in the egress of the CD34(+) subsets into PB. The control of antigen density of these adhesion molecules is likely to be clinically important for effective mobilization of HPC as well as for rapid engraftment following HPC transplant.     

Decreased stroma adhesion capacity of CD34+ progenitor cells from mobilized peripheral blood is not lineage- or stage-specific and is associated with low beta 1 and beta 2 integrin expression

Molecular mechanisms leading to mobilization of hematopoietic cells from bone marrow (BM) to peripheral blood (PB) involve modulation of adhesion molecule expression on these cells that probably result in changes in adhesion capacity to the microenvironment. However, it is not clear whether these changes involve different stages or lineages of progenitor cells. In this study, we compared the capacity of mature and immature clonogenic progenitor cells from granulocyte colony-stimulating factor (G-CSF)-mobilized PB and normal BM CD34+ cells to adhere to complete marrow stroma. This functional capacity was assessed concurrently with molecular expression on CD34+ cells of integrins VLA-4 (alpha 4/beta 1), VLA-5 (alpha 5/beta 1), and LFA-1 (alpha L/beta 2) by interindividual (between mobilized PB and normal BM) and intraindividual (between mobilized PB and steady-state BM and PB in the same patient) analysis. The proportion of adherent clonogenic progenitor cells was significantly lower in PB than in BM, not only for total progenitor cells but also for mature and immature progenitor cells, and the difference was found for granulocytic and particularly for erythroid lineages. The lower adhesion capacity of PB CD34+ cells to stroma was associated with decreased expression (signal/noise MFI ratio) of integrin alpha 4, beta 1, alpha L, and beta 2 chains whereas that of alpha 5 chain did not differ from BM cells with the lowest expression level. Similar differences in integrin expression levels were also found between mobilized PB and steady-state BM CD34+ cells in the same patient except for the alpha L chain. Moreover, we demonstrated for the first time a strong positive correlation between mobilizing capacity and expression levels on mobilized CD34+ cells for the LFA-1 alpha L chain but not for VLA-4 or VLA-5. In conclusion, the decreased adhesion capacity of mobilized PB progenitor cells to stroma involves different maturation stages and different lineages. This is associated with down-regulation of integrins VLA-4 and LFA-1, but mobilizing capacity appears positively correlated with LFA-1 levels.     

Augmented mobilization and collection of CD34+ hematopoietic cells from normal human volunteers stimulated with granulocyte-colony-stimulating factor by single-dose administration of AMD3100, a CXCR4 antagonist

BACKGROUND: AMD3100, a selective antagonist of CXCR4, rapidly mobilizes CD34+ hematopoietic progenitor cells (HPCs) from marrow to peripheral blood with minimal side effects. STUDY DESIGN AND METHODS: To further investigate potential clinical utility of AMD3100 for CD34+ cell mobilization and collection, a Phase I study in normal volunteers was performed examining single-dose administration of AMD3100 alone and in combination with a standard 5-day granulocyte-colony-stimulating factor (G-CSF) regimen. RESULTS: AMD3100 (160 microg/kg x 1 on Day 5) significantly increased both G-CSF-stimulated (10 microg/kg/day) mobilization of CD34+ cells (3.8-fold) and leukapheresis yield of CD34+ cells. Moreover, collection of CD34+ cells was comparable between individuals mobilized by a single-dose regimen of AMD3100 (240 microg/kg) and individuals mobilized with a 5-day regimen of G-CSF. AMD3100-mobilized leukapheresis products contained significantly greater numbers of T and B cells compared to G-CSF-stimulated leukapheresis products. CONCLUSION: These findings indicate that AMD3100 can be used alone or as an adjunct to G-CSF to mobilize cells for HPC transplantation.     

The use of chemokine receptor agonists in stem cell mobilization

INTRODUCTION: Pharmacological mobilization has been exploited as a means to obtain hematopoietic stem progenitor cells (HSPCs) for hematopoietic reconstitution. HSPCs mobilized from bone marrow into peripheral blood (PB) are a preferred source of stem cells for transplantation, because they are easily accessible and evidence indicates that they engraft faster after transplantation than HSPCs directly harvested from bone marrow (BM) or umbilical cord blood (UCB). AREAS COVERED: Since chemokine-chemokine receptor axes are involved in retention of HSPCs in the BM microenvironment, chemokine receptor agonists have been proposed as therapeutics to facilitate the mobilization process. These compounds include agonists of the CXCR4 receptor expressed on HSPCs (CTCE-0021 and ATI-2341) or chemokines binding to chemokine receptors expressed on granuclocytes and monocytes (e.g., CXCL2, also known as the growth-related oncogene protein-beta (Gro-β); CCL3, also known as macrophage inflammatory protein-1α (MIP-1α); or CXCL8, also known as IL-8) could be employed alone or in combination with other mobilizing agents (e.g., G-CSF or Plerixafor (AMD3100)). We discuss the current state of knowledge about chemokine receptor agonists and the rationale for their application in mobilization protocols. EXPERT OPINION: Evidence is accumulating that CXCR4 receptor agonists could be employed alone or with other agents as mobilizing drugs. In particular they may provide an alternative for patients that are poor mobilizers.     

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF-induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34(+) cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34(+) cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.     

重组人粒细胞集落刺激因子动员对CD4+T淋巴细胞迁移和黏附功能的影响

目的探讨重组人粒细胞集落刺激因子(rhG-CSF)动员对CD4+T淋巴细胞表面分子CXCR4和淋巴细胞功能相关抗原1(LFA-1)所介导功能和相关信号机制的影响.方法在rhG-CSF动员前和动员后第5天抽取供者外周血,用三色荧光标记检测动员前后CD4+T淋巴细胞LFA-1和CXCR4的表达率,并应用免疫磁性分选法分离纯化CD4+T淋巴细胞,检测动员前后CD4+T淋巴细胞对基质细胞衍生因子1α(SDF-1α)的迁移能力和对细胞间黏附分子1(ICAM-1)的黏附能力.结果rhG-CSF动员前后CD4+T淋巴细胞的LFA-1(CD11a)和CXCR4表达率差异无统计学意义(P＞0.05),动员前后CD4+T细胞LFA-1表达率均为100%;动员前CD4+T淋巴细胞CXCR4表达率为(84.58±20.31)%,动员后为(81.23±22.46)%.动员前后CD4+T淋巴细胞向SDF-1α的4 h迁移率分别为(28.5±10.3)%和(31.2±8.9)%,差异无统计学意义(P＞0.05);动员前后CD4+T淋巴细胞在CD3单抗作用下对ICAM-1的黏附率分别为(85.59±14.21)%和(61.45±15.07)%,动员前显著高于动员后(P＜0.05).结论rhG-CSF动员不影响CD4+T淋巴细胞LFA-1和CXCR4的表达,但影响CD4+T淋巴细胞通过LFA-1对ICAM-1的黏附能力.     

CXCR-4 expression on bone marrow CD34+ cells prior to mobilization can predict mobilization adequacy in patients with hematologic malignancies

To investigate the mechanisms of mobilization and of the factors implicated in the homing of progenitors and possibly understand the reasons for unpredicted mobilization failure, we analyzed CXCR-4 (CD184) expression on bone marrow (BM) CD34+ cells prior to peripheral blood stem cell (PBSC) mobilization in 24 patients affected by hematologic malignancies (non-Hodgkin lymphoma, multiple myeloma, and acute myeloid leukemia). We wanted to determine whether the level of CXCR-4 expressed by hematopoietic stem cells could influence mobilization process and therefore could be considered a predictive factor for mobilization adequacy. These data were also compared with stromal cell function as assessed by colony forming unit-fibroblast (CFU-F) and CFU endothelial cells (CFU-En) assays and stromal layer confluence capacity exhibited by patients' BM cells. In this study, we also compared CXCR-4 expression on CD34+ cells from different sources and at different migration stages specifically bone marrow (BM), steady state peripheral blood (SSPB), fetal cord blood (FCB), cord blood (CB), and mobilized PBSC. Seven (29%) of the 24 patients undergoing mobilization failed to achieve an adequate number of CD34+ stem cells (5 x 10(6)/kg CD34+ cells) and showed a very high expression frequency of CXCR-4 on BM CD34(+) stem cells (mean number of positive cells, 97%) investigated before the mobilization regimen. We also found that high expression intensity per cell for CXCR-4 was associated with lower amounts of mobilized CD34+ cells whereas those patients (17 out of 24 patients, 71%) with lower expression intensity per cell of CD184 on BM CD34+ cells prior to mobilization harvested at least 5 x 10(6)/kg CD34+ cells. Setting a cut off of 5 x 10(6)/kg CD34+ cells harvested, patients mobilizing less had a mean value of 97% CD34+ cells expressing CXCR-4 with a relative mean channel fluorescence of 458 whereas patients mobilizing more than 5 x 10(6)/kg CD34+ progenitors showed a mean value of 59.8% CD34+/CXCR4+ cells with a relative mean channel fluorescence value of 305. Interestingly, in the poor mobilizers group, the marrow stromal microenvironment was found to be more severely damaged in comparison with that of good mobilizers. The comparative analysis of CXCR-4 expression showed no difference in percentage values between steady-state PB (87.4%) and BM (85.1%) stem cells whereas mobilized CD34+ stem cells have a lower expression frequency of CXCR-4 (71.6%) compared to that of progenitors from other sources. Fetal blood CD34+ stem cells had the lowest mean expression frequency of CD184 antigen (36.3%), while CB cells had the highest (94.8%). In conclusion, this study provides evidence that monitoring CXCR-4 CD34 double positive cells before mobilization can be regarded as a predictive factor for mobilization outcome, giving us directional cues for the choice of the best stem cell mobilization regimens.