自体外周血CD34+细胞移植后巨细胞病毒感染的检测*

背景：巨细胞病毒活动性感染，尤其是巨细胞病毒肺炎死亡率极高，并易合并细菌、真菌、原虫等感染，直接影响患者的长期存活。因此，寻找一种早期、敏感的巨细胞病毒检测方法显得特别重要。 目的：探讨酶联免疫吸附法、免疫组织化学法及流式细胞仪检测在自体外周血造血干细胞移植后巨细胞病毒感染的早期诊断价值。 设计、时间及地点：对比观察，病例来自2002/2005南方医科大学南方医院。 对象：选择行自体外周血CD34＋细胞移植的系统性红斑狼疮(16例)和天疱疮(3例)患者19例，其中男5例，女14例，年龄11~38岁。所有受试者均无糖尿病、哮喘、荨麻疹、湿疹、炎症性肠病和其他风湿病。 方法：分别于移植前、移植后3，6，12，24个月进行外周血采集。 主要观察指标：应用酶联免疫吸附法、免疫组织化学法及流式细胞仪检测19例接受自体外周血造血干细胞移植患者移植前、移植后3，6，12，24个月时的抗巨细胞病毒抗体及巨细胞病毒抗原。 结果：19例患者均进入结果分析。血清学检测显示，全部标本抗巨细胞病毒IgG全部阳性，阳性率100%。抗巨细胞病毒IgM阳性3例，阳性率3.2%。免疫组织化学法检测巨细胞病毒抗原阳性14例，阳性率14.7%。流式细胞仪检测巨细胞病毒抗原阳性13例，阳性率13.7%。4种检测巨细胞病毒感染方法的阳性率存在显著差异(χ2=261.929，P < 0.01)。 结论：自体外周血造血干细胞移植后存在不同程度的巨细胞病毒感染，临床上开展流式细胞仪检测巨细胞病毒感染具有重要意义。     

Autologous peripheral blood stem cell transplantation for chronic acquired demyelinating neuropathy

Six patients with chronic acquired demyelinating neuropathy (CADP) were treated with autologous peripheral blood stem cell transplantation (PBSCT). Two with polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome improved–improvement was sustained in one but relapsed and required repeat transplant in the other. Two of the three with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and one with an IgM paraprotein and antibodies to nerve improved–of the responders, one relapsed after 18 months and the other was in remission after 6 months. Four developed neutropenic septicemia and pneumonia. The role of PBSCT in CADP refractory to other treatment deserves further investigation but the serious adverse events and lack of sustained response in some patients emphasize the need for caution.     

Neurological and functional recovery in human stroke are associated with peripheral blood CD34+ cell mobilization

BACKGROUND: A spontaneous mobilization of Peripheral Blood-Mononuclear CD34+ Cells (PB-MNC-CD34+) has recently been reported in human myocardial infarction and found to be related to improved heart function and survival. However, nothing is known regarding a possible relation between PB-MNC-CD34+ mobilization and neurological recovery in human acute cerebral ischemia. METHODS AND RESULTS: PB-MNC-CD34+ were determined daily after an acute cerebral ischemic attack for 14 days in 25 patients with acute ischemic stroke and compared with controls. Results indicated that stroke was followed by large and bursting mobilizations of PB-MNC-CD34+. The amplitude of the mobilizations was similar to those observed in Granulocyte Colony Stimulating Factor (G-CSF) conditioned aplastic patients following myeloablative therapy before leukapheresis and autologous bone graft. The extent of PB-MNC-CD34+ mobilization in each patient was directly related to neurological and functional recoveries as assessed by NIH Stroke Scale, and modified Rankin Scale respectively. CONCLUSIONS: The mobilization of PB-MNC-CD34+ cells might be predictive of neurological and functional recovery.     

Hyperbaric oxygen therapy combined with Schwann cell transplantation promotes spinal cord injury recovery

Schwann cell transplantation and hyperbaric oxygen therapy each promote recovery from spinal cord injury, but it remains unclear whether their combination improves therapeutic results more than monotherapy. To investigate this, we used Schwann cell transplantation via the tail vein, hyperbaric oxygen therapy, or their combination, in rat models of spinal cord contusion injury. The combined treatment was more effective in improving hindlimb motor function than either treatment alone; injured spinal tissue showed a greater number of neurite-like structures in the injured spinal tissue, somatosensory and motor evoked potential latencies were notably shorter, and their amplitudes greater, after combination therapy than after monotherapy. These findings indicate that Schwann cell transplantation combined with hyperbaric oxygen therapy is more effective than either treatment alone in promoting the recovery of spinal cord in rats after injury.     

影响外周血CD34+细胞纯化的相关因素

背景：造血干细胞具有良好的自我复制和更新的能力,CD34+细胞具备有造血干细胞的标志。 目的：分析影响外周血CD34+细胞纯化的因素。 方法：90例患者,经粒细胞集落刺激因子5 μg/(kg•d)动员1~3 d后,应用COBE血细胞分离机采集外周血单个核细胞液80~100 mL,经Clini MACS免疫磁珠分选技术纯化CD34+细胞。 结果与结论：90例CD34+细胞平均数为(1.73±1.15)×107,经流式细胞仪分析,CD34+细胞阳性率大于80%。COBE血细胞分离机单次收集的循环血量在980~1 100 mL时,利于CD34+细胞收集(P=0.005);动员后白细胞浓度在(16~21)×109 L-1时,利于CD34+细胞收集(P < 0.05);中间细胞和淋巴细胞总比例超11%时,利于CD34+细胞收集(P < 0.05);单个核细胞液血小板小于2 100×109 L-1时,利于CD34+细胞的收集(P < 0.05);年龄小于16岁,CD34+细胞数高(P=0.003);CD34+细胞抗体的温度、磁性标记及细胞处理时离心力的大小,均有影响。结果提示,经Clini MACS免疫磁珠细胞分选技术纯化的CD34+细胞能满足临床需要,实验稳定性好,重复性好;注重相关因素的影响,可提高纯化的CD34+细胞数量。     

肾移植患者外周血中的CD4+ CD25+ CD127low/-调节性T细胞

背景：越来越多的实验在分析免疫耐受标志,以期能够更好地辅助患者进行移植后免疫抑制治疗。 目的：分析肾移植后患者外周血中CD4+ CD25+ CD127low/-调节性T细胞在肾移植免疫耐受中的作用。 方法：采集62例肾移植后患者(急性排斥反应组22例,移植稳定组40例)及20例健康对照者的外周抗凝血,经免疫染色,应用流式细胞仪分析CD4+ CD25+ CD127low/-调节性T细胞所占CD4+ T细胞百分含量,同时采用ELISA方法检测患者血清中白细胞介素2和白细胞介素10的质量浓度。 结果与结论：移植稳定组中CD4+ CD25+ CD127low/-调节性T细胞所占CD4+ T细胞百分含量显著高于健康对照组和急性反应排斥组(P < 0.01);CD4+ CD25+ CD127low/-调节性T细胞百分含量与白细胞介素2呈显著负相关(P < 0.05),与白细胞介素10呈显著正相关(P < 0.01)。提示CD4+ CD25+ CD127low/-调节性T细胞在肾移植后免疫耐受的机制中发挥了一定作用。     

Edaravone promotes functional recovery after mechanical peripheral nerve injury

Edaravone has been shown to reduce ischemia/reperfusion-induced peripheral nerve injury. However, the therapeutic effect of edaravone on peripheral nerve injury caused by mechanical factors is unknown. In the present study, we established a peripheral nerve injury model by crushing the sciatic nerve using hemostatic forceps, and then administered edaravone 3 mg/kg intraperitoneally. The sciatic functional index and superoxide dismutase activity of the sciatic nerve were increased, and the malondialdehyde level was decreased in animals in the edaravone group compared with those in the model group. Bcl-2 expression was increased, but Bax expression was decreased in anterior horn cells of the L4–6 spinal cord segments. These results indicated that edaravone has a neuroprotective effect following peripheral nerve injury caused by mechanical factors through alleviating free radical damage to cells and inhibiting lipid peroxidation, as well as regulating apoptosis-related protein expression.     

Pleiotrophin promotes functional recovery after neural transplantation in rats

Pleiotrophin promotes survival of dopaminergic neurons in vitro. To investigate whether pleiotrophin promotes survival of grafted dopaminergic neurons in vivo, donor cells from ventral mesencephalon were treated with pleiotrophin (100 ng/ml) during cell preparation and grafted into striatum of hemi-Parkinson model rats. Functional recovery in methamphetamine-induced rotations was improved, and more tyrosine hydroxylase-positive cells survived in the striatum in the pleiotrophin-treated group. Pleiotrophin addition to cells just before transplantation also resulted in better functional recovery; however, no caspase-3 activation was seen during cell preparation. Interestingly, the effect of pleiotrophin on the survival was additive to that of glial-cell line-derived neutropic factor. These results revealed that pleiotrophin had effects on donor cells in neural transplantation in vivo.     

PDE5 inhibitors promote recovery of peripheral neuropathy in diabetic mice

<正>Diabetes mellitus affects an estimated 422 million people worldwide.Peripheral neuropathy is one of the most common and disabling complications of diabetes.There is currently no effective treatment for diabetic neuropathy,even with diligent blood glucose     

同种异基因大鼠间充质干细胞移植对体内CD4+CD25+T细胞的影响**☆

背景：间充质干细胞的免疫抑制作用近年来逐渐得到证实并开始应用于临床,但相关研究成果主要来源于体外细胞实验。 目的：观察同种异基因大鼠骨髓间充质干细胞从静脉输入后对体内CD4+CD25+调节性T细胞的影响。 设计、时间及地点：以动物为对象的对照观察实验,于2008-03/09在南方医科大学珠江医院移植免疫研究所完成。 材料：Wistar大鼠6只用于制备骨髓间充质干细胞,SD大鼠20只作为输注骨髓间充质干细胞的受体鼠。 方法：从Wistar大鼠骨髓分离培养间充质干细胞,取第3~5代细胞进行实验。①体外淋巴细胞增殖实验：首先将间充质干细胞悬液从尾静脉注入SD大鼠体内10 d后脱臼处死,取大鼠脾脏制备脾淋巴细胞。实验分5组：分别为脾淋巴细胞/骨髓间充质干细胞为1∶10,1∶50,1∶100+ConA 5 μg组,脾淋巴细胞+ConA 5μg组（增殖组）,单纯淋巴细胞组(空白组),检测共培养体系中CD4+CD25+ / CD4+ T淋巴细胞比率。②体内注射骨髓间充质干细胞实验：将5×109 L-1,5×108 L-1,5×107 L-1间充质干细胞及PBS静脉输入SD大鼠体内,10 d后取受体鼠胸腺、脾脏、外周血检测CD4+CD25+ / CD4+ T淋巴细胞比率。 主要观察指标：①淋巴细胞增殖体系中CD4+CD25+/CD4+比率的变化。②SD大鼠胸腺、脾脏、外周血CD4+CD25+/CD4+比率的变化。 结果: ①体外淋巴细胞共培养体系中,1∶10组T细胞亚群CD4+ CD25+ / CD4+细胞百分率较增殖组显著升高（P < 0.01）。②体内输注间充质干细胞达5×109 L-1的受体鼠外周血和脾脏CD4+CD25+ / CD4+ T细胞比率上升,与输注PBS组相比有显著差异（P < 0.05）,而在胸腺中各组比率无明显差异。 结论：高浓度同种异基因大鼠间充质干细胞不仅可以在体外实验中增加CD4+CD25+ / CD4+ T细胞比率,静脉输入后仍具有相同作用。     

调节性T细胞在血管性痴呆患者外周血中表达的初步研究

目的探讨CD4+CD25+Foxp3+调节性T细胞(Treg)在血管性痴呆患者外周血中的表达情况。方法选择血管性痴呆患者48例,健康对照组40例,采取外周血标本,流式细胞学检测Treg占CD4+T细胞的比例,实时定量PCR检测外周血单个核细胞Foxp3 mRNA的水平,体外增殖抑制试验检测Treg的抑制功能,ELISA检测Treg抑制炎症因子的产生。结果与健康对照组相比,血管性痴呆患者CD4+CD25+Foxp3+Treg比例下降,Foxp3表达降低,并且Treg的抑制功能减低。结论 Treg在血管性痴呆患者外周血中表达降低,且功能减弱,提示Treg可能参与了血管性痴呆的发生发展。     

CD16+CD57- natural killer cells in multifocal motor neuropathy

We analyzed the CD16+CD57- lymphocyte subset, which is considered to have strong natural killer (NK) cell activity, in peripheral blood from patients with chronic immune-mediated neuropathies and patients with other neurological diseases. We found that the ratio of CD16+CD57- NK cells to total lymphocytes was increased in 4 of 6 patients with multifocal motor neuropathy (MMN) with persistent conduction block. Since the CD16 molecule is an Fc receptor for immunoglobulin G (IgG), high-dose intravenous immunoglobulin (IVIg) may interfere with CD16+CD57- NK cells via Fc receptor blockade. In addition, cyclophosphamide (Cy) is often used to suppress NK cells. Therefore, our findings may partly account for the effectiveness of IVIg or Cy, which is the current treatment of choice for MMN.     

Evidence that nerve growth factor promotes the recovery of peripheral neuropathy induced in mice by cisplatin: behavioral, structural and biochemical analysis

In this study we investigate the neurotoxic action of Cisplatin (6 micrograms/g body weight for 5 treatment cycles during 15 weeks with a total dose of 30 micrograms/g), an antitumor drug, and its effect on the level of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in peripheral tissues. We found that Cisplatin in adult rodents impairs peripheral sensory function and both sympathetic and sensory peripheral innervation as shown by the hot-plate response, catecholamine distribution and substance P immunoreactivity respectively. These changes are associated with decreased NGF in intestine, paws, and bladder while NGF increased in the spinal cord. Also BDNF decreased in bladder and paws and increased in spinal cord and intestine. To further investigate the role of NGF in the pathogenesis of Cisplatin-induced peripheral neuropathies a group of animals was injected with NGF (1 microgram/g every 4 days for 4 times) following Cisplatin treatment and evaluated for sensory function, sympathetic and sensory innervation and BDNF levels. Data demonstrated that exogenous NGF administration is able to restore biochemical, structural and functional changes induced by Cisplatin. These findings suggest that the reduction of NGF availability could be a cause of Cisplatin-induced peripheral neuropathies and that NGF exogenous administration could prevent or reduce Cisplatin neurotoxicity also in cancer patients, reducing the side effects of chemotherapy.     

Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation

The endothelial progenitor cells (EPCs) are responsible for postnatal vasculogenesis in physiological and pathological neovascularization and have been used for attenuating ischemic diseases. However, EPCs from umbilical cord blood (CB) were not well understood and the homing mechanisms of EPCs remain unclear. To determine the potential application of CB-derived EPCs, we established a culture system to induce the differentiation of CB cells into EPCs. Purified CB CD133(+) cells proliferated and, after further vascular endothelial growth factor receptor 2 (VEGFR-2) antibody purification, differentiated into EPCs expressing endothelial markers, such as VE-cadherin, VEGFR-2, CD31, von Willebrand factor (vWF) and Weibel-Palade bodies. These cells could also take up acetylated lower density lipoprotein (Ac-LDL) and bind Ulex europaeus agglutinin-1 (UEA-1).When expanded EPCs were transplanted via tail vein into nude mice, they incorporated into capillary networks in ischemic hindlimb, augmented neovascularization, and improved ischemic limb salvage. In addition, in ischemic tissue, there were elevated expressions of VEGF and stromal derived factor 1 alpha (SDF-1 alpha), both of which had chemotactic effect on EPCs. Moreover, P-/E-selectins was found on mouse ischemic endothelium and P-selectin glycoprotein ligand-1 (PSGL-1) on CB-derived EPCs. Neutralizing antibody against PSGL-1 blocked the homing of EPCs to ischemic area by 61%. These results demonstrate that CB CD133(+) cell-derived EPCs can be applied for therapeutic neovascularization in ischemic diseases, and reveal important roles of chemoattractants and adhesive molecules in the homing of EPCs.     

Autonomic and peripheral neuropathy in endstage liver disease and following liver transplantation

Severe chronic liver disease may be associated with a peripheral somatic and an autonomic neuropathy. There are only a limited number of reports on the incidence and features of these neuropathies. In addition the effects of liver transplantation on these neuropathies have not been well studied. We examined peripheral somatic and autonomic nerve function in 42 patients with endstage liver disease prior to transplantation and also examined the effect of liver transplantation on these neuropathies in 14 patients. Peripheral somatic neuropathy (93%) and autonomic neuropathy (50%) were common in patients with endstage liver disease and were more frequent than previously reported. Abnormalities improved in some patients after liver transplantation, particularly if there was return of normal hepatic function.     

CD4+T-bet+, CD4+pSTAT3+ and CD8+T-bet+ T cells accumulate in peripheral blood during NZB treatment

Circulating T cells and monocytes expressing T-bet, pSTAT1 and pSTAT3 increase in relapsing-remitting multiple sclerosis (RRMS) during relapse. Natalizumab (NZB) is an effective drug in RRMS, but exacerbation of the disease after its discontinuation has been described in some patients. The aim of this research was to study the effect of NZB treatment on circulating lymphomonocyte subpopulations expressing T-bet, pSTAT1, pSTAT3 and CD4+CD25+Foxp3+ regulatory T cells. Flow cytometry was used to evaluate the percentages of circulating CD4+ and CD8+ T cells, CD14+ monocytes and B cells expressing T-bet, pSTAT1, and pSTAT3, and CD4+CD25+Foxp3+ regulatory T cells from RRMS patients before and after 6-12 NZB infusions. In NZB-treated RRMS patients, the percentages of CD4+pSTAT1+ and CD8+pSTAT1+ T cells, CD14+pSTAT1+ monocytes, CD4+T-bet+, CD8+T-bet+ and CD4+pSTAT3+ T cells and CD14+pSTAT3+ monocytes increased after 12 drug infusions and were similar to those observed in untreated relapsing RRMS patients. Otherwise in vitro NZB exposure of peripheral blood mononuclear cells from untreated RRMS patients and controls had no effect. It was concluded that NZB treatment determines an accumulation of CD4+pSTAT1+, CD8+pSTAT1+, CD4+T-bet+, CD8+T-bet+ and CD4+STAT3+ T cells in peripheral blood that may account for the exacerbation of the disease observed in some patients after the discontinuation of the drug.