强直性脊柱炎患者外周血T细胞亚群的变化及意义

目的探讨强直性脊柱炎（AS）患者外周血中T细胞亚群的变化及其意义。方法采用流式细胞仪（FCM）检测外周血淋巴细胞表面CD3、CD4、CD8及其胞质内细胞因子IFN-γ和IL-4的表达。结果与正常对照组相比,As患者外周血Th细胞（CD;CD4＋）百分率无显著差异,Tc细胞（CD;CD;）明显升高（P〈0．05）;Th1细胞（CD3^＋CD4^＋IFN-γ^＋）百分率明显升高,Th2细胞（CD3^＋CD4^＋IL4^＋）无显著性差异;Te1细胞（CD3^＋CD8^＋IFN-γ^＋）百分率明显降低,T02细胞（CD3^＋CD8^＋IL4^＋）无显著性差异。结论AS患者外周血T细胞Th1／Th2、Tcl／Tc2亚群比例失衡,呈Th1、Tc2优势型。     

T细胞亚群Ⅰ和Ⅱ型细胞比例与急性移植物抗宿主病相关性研究

目的：研究T细胞亚群Ⅰ、Ⅱ型细胞比例在异基因造血于细胞移植（allo-HSCT）早期的变化，探讨其在急性移植物抗宿主病（aGVHD）发生中的作用。方法：移植后7、14、21、28d采取21例allo-HSCT受者外周血，加入佛波醇酯、伊能霉素、莫能霉素体外培养4h。3色荧光标记后用流式细胞仪检测T细胞亚群Ⅰ、Ⅱ型细胞比例。结果：发生aGVHD组Ⅰ型T细胞比例明显高于无aGVHD组。发生aGVHD时Th1、Tc1和CD4^＋IL-2^＋T细胞比例明显高于发生前和治疗后。与轻度相比，中、重度aGVHD发生时Tc1、Tc2和CD8^＋IL-2^＋T细胞比例更加升高，Th2细胞比例明显下降。移植早期CD3^＋干扰素-γ（INF-γ）^＋T细胞比例≥8％者aGVHD的发生率增加。移植后第7天Th1细胞比例〉5．5％者发生中、重度aGVHD的危险性显著增高（P〈0．05）。aGVHD腹泻者Th1细胞比例显著高于非aGVHD腹泻者（P〈0．05）。结论：Th1和Tc1细胞比例在aGVHD发生时明显升高，因此移植早期动态检测T细胞亚群的Ⅰ型细胞比例有助于aGVHD的诊治。     

慢性HBV感染患者外周血T淋巴细胞免疫应答及其临床意义

目的探讨Th1／Th2、Tc1／Tc2亚群比例在慢性HBV感染的临床分型中的变化,以及在慢性HBV感染免疫病理损伤中的作用。方法用PMA、Ionomycin作为刺激剂,采用流式细胞仪（FACS）胞内细胞因子法对慢性HBV感染者外周血CD3＋CD8＋T细胞和CD3＋CD8-T细胞内IFN～y和IL-4的表达进行分析,比较慢性肝炎、肝炎肝硬化（活动性）、慢性重症肝炎各组Th1／Th2、Tc1／Tc2亚群比例变化。结果慢性肝炎、肝炎肝硬化（活动性）、慢性重症肝炎患者的Th1、Tc1细胞均高于正常对照组。慢性重症肝炎组Th1、Tc1显著高于慢性肝炎、正常对照组（P〈0．01）,慢性重症肝炎组Tc1显著高于活动性肝炎后肝硬化组（P〈0．05）。肝炎肝硬化（活动性）组Tc1显著高于正常对照组（P〈0．05）。Th1、Tc1细胞随着慢性乙型肝炎肝脏炎症活动的加剧而增高。而Th2、Tc2细胞则在各组中均无显著性差异（P〉0．05）。慢性重症肝炎患者恢复期Th1和Tc1细胞显著低于治疗前水平（P〈0．01）。结论在慢性HBV感染中,机体免疫平衡偏向Th1类反应,Th1、Tc1细胞与肝脏炎症活动严重程度呈正相关。提示Th1和Tc1细胞在慢性乙型肝炎肝脏病理发生中可能起了重要作用。     

流式细胞仪检测急性病毒性心肌炎T淋巴细胞亚群及NK细胞的意义

目的观察急性病毒性心肌炎T淋巴细胞亚群及自然杀伤（NK）细胞的意义，探讨其发病机制。方法采用流式细胞术对急性病毒性心肌炎病人（观察组）进行CD3、CD4、CD8和NK细胞淋巴细胞亚群检测，并设正常人群作对照组。结果观察组CD8和NK明显高于对照组（P〈0．01），CD4和Th／Tc比值明显低于对照组（P〈0．01），观察组CD3的变化不明显（P〉0．05）。结论急性病毒性心肌炎病人淋巴细胞亚群中CD4、CD8和NK细胞亚群异常增殖；而且Th／Tc比例失衡，揭示细胞免疫功能紊乱与急性病毒性心肌炎有关。     

IL-17对日本血吸虫感染抗体及Th应答的影响

目的观察IL-17在日本血吸虫感染小鼠中对抗体及Th细胞免疫应答的影响。方法在小鼠日本血吸虫感染前后,连续多次腹腔注射rm-IL-17细胞因子（实验组1）或PBS（对照组1）,连续多次腹腔注射anti-IL-17中和抗体（实验组2）或同型抗体（对照组2）。运用流式细胞术,采用表面分子、胞内因子同时染色的三色标记法,对日本血吸虫感染小鼠脾脏、淋巴结中的Th17、Th1和Th2细胞亚群占CD4＋T细胞的比例进行观察;同时采用ELISA法检测各组小鼠血清中血吸虫抗原特异性总IgG及亚类IgG1和IgG2a的水平。结果实验组1 CD4＋T细胞中的Th17、Th1和Th2细胞亚群的比例分别是对照组1的1.09倍（P（0.05）、1.17倍（P（0.05）和1.15倍（P（0.05）;实验组2 CD4＋T细胞中的Th17、Th1和Th2细胞亚群的比例分别是对照组2的1.01倍（P（0.05）、1.11倍（P（0.05）和0.98倍（P（0.05）。rm-IL-17注射后仅可溶性虫卵抗原（SEA）特异性IgG1水平显著下降（P（0.05）、可溶性成虫抗原（SWA）特异性IgG2a的水平升高（P（0.05）,其他抗体水平均无...     

百令胶囊对2型糖尿病肾病患者细胞免疫功能的影响

目的　探讨百令胶囊对2型糖尿病肾病患者外周血淋巴细胞亚群分布的影响。方法　108例患者随机分为治疗组和对照组,每组54例,另选择40例健康体检者作为健康组。对照组予常规治疗,治疗组加服百令胶囊,12周为一疗程。测定主要生物化学指标,流式细胞术检测患者T细胞（CD3＋、CD3＋CD4＋、CD3＋CD8＋）、B细胞（CD3－CD19＋）、NK细胞（CD3－CD16＋CD56＋）、NKT细胞（CD3＋CD16＋CD56＋）分布水平,并与健康组比较。结果　两组治疗后血清胱抑素C（CysC）、24　h尿蛋白定量显著降低（P<0.05）,且治疗组降低更为明显（P<0.05）。治疗前两组患者淋巴细胞亚群分布与健康组相比均有显著性差异（P<0.05）,即存在细胞免疫功能紊乱;治疗后,对照组淋巴细胞亚群分布无明显改变,治疗组CD3＋T细胞亚群、CD3＋CD4＋T细胞亚群和CD3＋CD8＋T细胞亚群、CD3－CD16＋CD56＋NK细胞亚群及CD3＋CD16＋CD56＋NKT细胞亚群比例上升,CD3－CD19＋B细胞亚群比例下降,CD4＋/CD8＋比值明显降低（P<0.05）,且与对照组相比均有显著性差异（P<0.05）。结论　百令胶囊可明显改善2型糖尿病肾病患者细胞免疫功能紊乱。     

肺癌患者外周血细胞亚群及其与临床关系分析

目的分析肺癌患者外周血T细胞亚群及NK细胞活性的表达,研究其与肺癌患者临床分期及化疗的关系。方法分析2009年6月至2010年7月苏州大学附属第一医院收治的89例肺癌患者及30例健康体检者外周血中流式细胞仪（flowcytometry,FCM）检测的T细胞亚群及NK细胞活性及血中CEA含量。结果Ⅰ~Ⅱ期、不同病理分化级别及CEA正常范围内的肺癌患者外周血各细胞亚群分布与健康对照组无统计学差异;Ⅲ~Ⅲ期及CEA升高肺癌患者CD4＋、CD4＋/CD8＋、CD2＋8细胞比值下降（P〈0.05）,CD8＋、CD4＋、CD2＋5细胞比值升高（P〈0.05）,NK细胞比例较健康对照组无统计学差异。化疗2周后患者外周血CD3＋、CD4＋、CD4＋/CD8＋、CD2＋8比值降低（P〈0.01）,CD8＋、CD4＋、CD2＋5细胞比值升高（P〈0.01）,NK细胞比例升高。结论肺癌患者免疫功能存在一定抑制,Ⅲ~Ⅳ期及CEA升高的患者免疫抑制更明显,化疗后患者各细胞亚群分布较前并未得到改善,甚至恶化。     

食管癌患者外周血淋巴细胞亚群分析

背景：食管癌患者外周血淋巴细胞亚群与临床病理参数的相关性尚存在争议.目的：分析食管癌患者外周血淋巴细胞亚群的变化情况.方法：以流式细胞术检测食管癌荷瘤组（n=207）、非荷瘤组（n=33）和正常对照组（n=96）的外周血淋巴细胞亚群,并与临床病理参数行相关性分析.结果：荷瘤组患者外周血CD3＋、CD4＋、CD8＋、CD19＋细胞比例较正常对照组明显下降（P＜0.01或P＜0.05）,CD4＋/CD8＋比值、CD25＋、CD44＋、自然杀伤细胞比例较正常对照组明显升高（P＜0.01）.Ⅰ/Ⅱ期与Ⅲ/Ⅳ期、低分化与中分化、淋巴结阴性与阳性亚组间外周血淋巴细胞亚群差异无统计学意义（P＞0.05）.非荷瘤组患者外周血CD3＋、CD4＋细胞比例与正常对照组相比差异无统计学意义（P＞0.05）,其余淋巴细胞亚群比例与正常对照组相比差异有统计学意义（P＜0.05）.结论：食管癌患者外周血淋巴细胞亚群比例明显异常,提示机体免疫系统紊乱,但与肿瘤临床分期和恶性程度并不相关：食管癌患者的免疫功能于术后第3-4周开始恢复,CD3＋、CD4＋细胞是早期监测食管癌患者机体免疫状态的敏感指标.     

日本血吸虫感染ICR小鼠后Tc1和Tc2细胞的动态观察及其功能分析

目的 目的 观察日本血吸虫感染ICR小鼠后不同时期T淋巴细胞中杀伤性T细胞 （Tc） 亚型Tc1和Tc2比例的动态变化, 并分析两者分别与Th1和Th2细胞比例变化的相关性。 方法 方法 分别取日本血吸虫感染0、 3、 5、 8、 13周的小鼠脾脏淋巴细胞, 采用流式细胞术检测各个时期Tc1、 Tc2和Th1、 Th2细胞亚群分别占T淋巴细胞的比例。 结果 结果 与0周对照比较, 感染3、 5、 8、 13周小鼠T淋巴细胞中Tc1细胞比例显著增高 （P均< 0.01）, 感染5、 8、 13周小鼠T淋巴细胞中Tc2细胞比例显著增高 （P均< 0.01）。比较Tc1和Tc2细胞比例在相同时间点的增速发现, 感染3周时Tc1细胞比例增高最显著, 而感染5周时Tc2细胞比例的增高最显著。感染各个时间点CD3+ T细胞中的Th1细胞比例与Tc1细胞比例平行增长, 且两者呈正相关（r = 0.978, P = 0.004）; CD3+ T细胞中的Th2细胞比例与Tc2细胞比例平行增长, 且两者呈正相关 （r = 0.974, P = 0.005）。体外可溶性成虫抗原 （SWA） 刺激脾细胞可优势增加T淋巴细胞中的Tc1细胞比例 （P < 0.01）, 而可溶性虫卵抗原 （SEA） 刺激脾细胞可优势增加T淋巴细胞中的Tc2细胞比例 （P < 0.01）。 结论 结论 日本血吸虫感染不同阶段, 小鼠T淋巴细胞中的Tc1 和Tc2细胞比例均显著升高, 并分别与Th1和Th2细胞比例呈正相关。在感染3周时主要以Tc1细胞比例升高为主, 而感染 5周时主要以Tc2细胞比例升高为主。SWA可优势诱导Tc1细胞, 而SEA可优势诱导Tc2细胞。     

骨髓间充质干细胞对外周血T淋巴细胞亚群影响的体外实验研究

目的：探讨骨髓间充质干细胞（MSC）在体外对T淋巴细胞亚群及其分泌的细胞内因子的影响。方法：通过Ficoll—Hypaque梯度密度离心法分离出正常人骨髓单个核细胞，体外培养扩增MSC，获取第3代细胞。将其按照不同的比例加入双向混合淋巴细胞培养（MLC）体系中，在第1，3，5天，采用MTT比色法检测各组MLC中的T淋巴细胞的增殖情况，再用流式细胞术分析各组T细胞亚群及其内因子白介素（IL）-4和干扰素（IFN）-γ的分泌变化情况。结果：加入了MSC的MLC体系中，MSC对T淋巴细胞的增殖抑制具有剂量依赖性，且随着时间延长，抑制程度增强；其中，CD4^＋T细胞亚群受抑不如CD8^＋T细胞亚群显著；而根据T细胞内因子的变化，Th1和Tc1的比率较对照组有显著降低，而Th2和Tc2的比率却有轻度上升。结论：MSC在体外能够明显抑制T淋巴细胞的增殖，尤其是CD8^＋T细胞（CTL）。除此之外，它还能降低MLC体系中的Th1Tc1，升高Th2和Te2。所以在临床上可能有减轻移植后急性移植物抗宿主病（aGVHD）的发生，而保留移植物抗白血病（GVL）的潜力。     

Effect of interleukin (IL)-12 and IL-15 on apoptosis and proliferation of umbilical cord blood mononuclear cells

Decreased graft-versus-host disease (GVHD) in cord blood (CB) transplantation may be attributed to the immunological immaturity and susceptibility to apoptosis of CB mononuclear cells (MNCs). Cytokines like interleukin (IL)-12 and IL-15 may be used for in vivoadministration or ex vivo expansion of lymphoid cells for more rapid recovery following stem cell transplant, and for providing a graft-versus-leukemia (GVL) effect. We investigated the effects of IL-12 and IL-15, alone or in combination on apoptosis and proliferation of both CB and adult peripheral blood (APB) MNCs, with particular emphasis on CB CD4+, CD8+ and CD56+ lymphocyte subpopulations. The results of our study indicated that: (1) the combination of IL-12+IL-15 resulted in a greater degree of CB and APB apoptosis than either cytokine alone; (2) the level of both spontaneous and cytokine-induced apoptosis by IL-12 and/or IL-15 are greater in CB MNCs than in APB MNCs using TUNEL assays; (3) IL-15 is superior to IL-12 in enhancing the proliferative response in CB and APB MNCs; (4) the combination of IL-12+IL-15, but not either cytokine alone, significantly enhanced apoptosis in CD8+ and CD56+ CB subsets, but not in CD4+ CB subsets; (5) IL-12 or IL-15 alone resulted in increased proliferation in CD4+, CD8+ and CD56+ CB subsets, with IL-12+IL-15 producing the greatest increase of proliferation in all three CB subsets; and (6) IL-15 and/or IL-12 significantly upregulate Fas (CD95) expression on CB T and NK cells. These findings may have therapeutic implications when designing cytokine therapy in patients receiving CB transplant.     

Interferon gamma and tumour necrosis factor alpha are overexpressed in bone marrow T lymphocytes from paediatric patients with aplastic anaemia

Twelve paediatric patients with aplastic anaemia and two groups of normal control subjects underwent flow cytometric analysis for intracytoplasmic expression of gamma interferon (gamma-IFN) and tumour necrosis factor alpha (TNF-alpha) in bone marrow and peripheral blood CD4+ and CD8+ cells. The same cytokines were tested, by immunoassay, in culture supernatants from unstimulated bone marrow mononuclear cells (MNCs). Marrow CD4+ and CD8+ cells expressing gamma-IFN and TNF-alpha were significantly increased in the patients in comparison with normal control subjects (P from < 0.05 to < 0.0001 in the different cellular subsets). Patients' marrow CD4+ and CD8+ cells containing gamma-IFN and TNF-alpha were significantly increased when compared with the same cell fractions from paired peripheral blood samples (P from < 0.05 to < 0.0001 in the various cellular subsets). In the supernatant of marrow MNCs, gamma-IFN and TNF-alpha were detected in four out of eight and five out of eight cases, respectively, whereas neither cytokine was traceable in the control subjects. Patients' peripheral blood CD4+ and CD8+ cells containing gamma-IFN and TNF-alpha were not significantly increased in comparison with those from normal control subjects. Whereas patients with favourable and unfavourable outcomes had no significantly different proportions of marrow gamma-IFN+/CD4+ and gamma-IFN+/CD8+ cells, the percentages of marrow CD4+ and CD8+ cells containing TNF-alpha were significantly lower in subjects with favourable than in those with unfavourable outcome. Overall, these findings show that, in aplastic patients, T cells overexpressing gamma-IFN and TNF-alpha concentrate in the bone marrow and that intracytoplasmic expression of TNF-alpha in marrow CD4+ and CD8+ cells is associated with an unfavourable clinical course.     

Imbalance between expression of endothelin receptors A and B in terminal liver cirrhosis due to hepatitis C viral infection: Immunohistochemical study of autopsy cases

Background and Aim:  Expression of endothelin receptors in terminal liver cirrhosis is not well investigated. The aim of this study was to investigate the expression of the endothelin type A receptor (ETAR) and endothelin type B receptor (ETBR) immunohistochemically using paraffin-embedded tissue sections from patents with terminal liver cirrhosis (TLC), non-terminal liver cirrhosis (NTLC) and non-cirrhotic liver fibrosis (NCLF) caused by hepatitis C viral infection.
Methods:  Liver tissue sections from 38 autopsy cases, including 12 cases of NCLF (mild, moderate or severe liver fibrosis), 11 cases of NTLC and 15 cases of TLC, were stained using anti-ETAR and anti-ETBR antibodies after antigen retrieval. Double staining using antibodies to alpha-smooth muscle actin (ASMA) was also performed.
Results:  There were significantly fewer ETBR-positive cells in TLC compared with NTLC and NCLF. Numbers of ASMA-positive stellate cells expressing ETBR were also significantly lower in TLC. Therefore, the ETAR/ETBR ratio of sinusoidal cells is significantly higher in TLC than in NTLC and NCLF. ASMA-positive stellate cells showed similar evidence of ETAR and ETBR expression.
Conclusions:  There is a difference in ETAR and ETBR expression among TLC, NTLC and NCLF: the ETAR/ETBR ratio is increased in TLC due to a relative decrease in ETBR expression. This finding may be useful for the diagnosis of TLC with regard to circulatory disturbances in the liver.     

组织蛋白酶B及其抑制剂Cystatin C在人腹主动脉瘤平滑肌细胞中的表达

目的 观察组织蛋白酶B及其抑制剂Cystatin C在人腹主动脉瘤平滑肌细胞中的表达情况,以探讨其在腹主动脉瘤发生发展中的作用.方法 对21例腹主动脉瘤患者和8例正常腹主动脉尸检者的腹主动脉血管标本行苏木精-伊红染色和免疫组织化学染色,观察组织蛋白酶B及其抑制剂Cystatin C对血管中膜的影响.结果 免疫组织化学染色可见组织蛋白酶B和Cystatin C分别在腹主动脉瘤、正常腹主动脉中免疫反应阳性,阳性定位于平滑肌细胞质;腹主动脉瘤平滑肌细胞中组织蛋白酶B阳性细胞平均光密度值明显增高,Cystatin C明显降低,与正常腹主动脉之间有显著差异(P<0.01).结论 腹主动脉瘤平滑肌细胞中组织蛋白酶B表达增强,Cystatin C表达下降,组织蛋白酶及其抑制剂Cystatin C间的不平衡可能引起动脉瘤壁细胞外基质广泛降解,从而导致腹主动脉瘤的发生和破裂.     

Cell-mediated suppression of megakaryocytopoiesis in acquired amegakaryocytic thrombocytopenic purpura

Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a disorder of hematopoiesis characterized by severe thrombocytopenia due to a selective reduction or total absence of megakaryocytes in an otherwise normal-appearing bone marrow. Although the development of autoantibodies directed against cells in the megakaryocyte progenitor cell pool has been implicated in the pathogenesis of this disorder, cell-mediated suppression of megakaryocytopoiesis has not been described. Accordingly, we report two cases of AATP in which in vitro suppression of megakaryocyte colony formation by autologous ancillary marrow cells was demonstrable. Light-density bone marrow mononuclear cells (MNCs) obtained from both patients were either plated directly into plasma clot cultures, or after first being depleted by adherent monocytes (M phi) or T lymphocytes using standard methodologies. In some experiments, the depleted ancillary marrow cells were recovered for autologous co-culture studies with the MNCs from which they had been depleted. Megakaryocyte colony formation was detected in the cultures using an indirect immunofluorescence assay with a rabbit anti- human platelet glycoprotein antiserum. Removal of M phi (n = 6), or T lymphocytes (n = 4) from normal marrow MNCs had no apparent effect on colony formation. In contrast, depleting T lymphocytes from the MNCs of patient 1 significantly augmented megakaryocyte colony formation; a similar effect was observed after depleting M phi from the MNCs of patient 2. This observed augmentation in colony formation could be abrogated by autologous co-culture with the putative suppressor cell at effector cell/target cell ratios of 1:10 in the case of T lymphocytes or 1:5 in the case of M phi. Neither suppression nor stimulation of megakaryocyte colony formation was observed after culturing normal MNCs with autologous T cells (n = 4) or M phi (n = 3) at similar or greater ratios. We also observed inhibition of megakaryocyte colony formation after culturing normal MNCs in the presence of tissue culture medium conditioned by the M phi of patient 2. This effect was shown to be specific for megakaryocytes since this same conditioned medium had no significant effect on BFU-E and CFU-E-derived colony formation by autologous marrow mononuclear cells. These results suggest that: both T cells and M phi are capable of exerting a regulatory effect on the proliferation of human megakaryocyte progenitor cells (CFU-Meg); in the case of M phi, a soluble factor elaborated by these cells may be responsible for suppressing CFU-Meg growth; and aberrant ancillary cell- megakaryocyte progenitor cell interactions may lead to clinically significant disease.     

急性心肌梗塞病人肝细胞生长因子的产生

目的     

Circulating Stem Cell Collection in Lymphoma and Myeloma after Mobilization with Cyclophosphamide and Granulocyte Colony-Stimulating Factor for Autologous Transplantation

We report the results of 72 leukapheresis procedures performed for autologous peripheral blood stem cell collection in 18 patients with lymphoma and myeloma, after combined mobilization with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF). The numbers of mononuclear cells (MNCs), CD34+ cells and granulocyte-macrophage colony-forming units (CFU-GM) either in the peripheral circulation (preleukapheresis sample) or in the product obtained from leukapheresis (leukapheresis sample) were evaluated. A highly superior proportion of CD34+ cells (14-fold) and CFU-GM (5-fold) resulted from the mobilization therapy. CFU-GM and CD34+ cells were highly enriched with respect to all MNCs (relative recoveries: 2.13, range 0.3–41, and 1.08, range 0.2–8.5, respectively) due to an additional mobilization effect by the leukapheresis procedure. Also, a relatively strong linear correlation between the three different parameters was found in the leukapheresis product (CD34+:CFU-GM, r = 0.81; MNCs:CD34, r = 0.69; MNCs:CFU-GM, r = 0.75; CFU-GM:CD34+, and MNCs, r = 0.85). Our data suggest that the number of MNCs and CD34+ cells obtained after combined mobilization with cyclophosphamide and G-CSF can be used as predictor of the number of granulomonocytic progenitors.     

Development of angiogenic cell and gene therapy by transplantation of umbilical cord blood with vascular endothelial growth factor gene.

Endothelial progenitor cells (EPCs) are present in the mononuclear cells (MNCs) of umbilical cord blood and peripheral blood. To establish the efficiency of angiogenic cell and gene therapies, we transfected the human vascular endothelial growth factor (hVEGF) gene into cord blood MNCs to enhance endothelialization. MNCs from cord blood and peripheral blood were isolated and transfected with pCR3 expressing hVEGF165 or GFP by the Hemagglutinating Virus of Japan (HVJ)-envelope and the cells were cultured in endothelium basal medium-2. The number of attached cells from cord blood was higher than that from peripheral blood. Attached cells expressed Flk-1, VE-cadherin, PECAM-1, CD34, and Tie-2. The increase in the number of attached cells was transient with the transfection of vascular endothelial growth factor (VEGF) gene early in the experimental period. Flt-1 mRNA was not expressed early in the culture period, but was expressed at 2 weeks after separation. VEGF gene transfer into MNCs at 12 days after separation, i.e., when Flt-1 mRNA was expressed continuously, increased the number of attached cells. We evaluated the effects of the transplantation of cord blood MNCs expressing the hVEGF gene on regional blood flow in an ischemic area in a rat model of chronic hindlimb ischemia. Blood flow was significantly improved in nude rats that received transplanted control MNCs. Transplantation of cord blood MNCs transfected with the hVEGF gene yielded greater improvements in blood flow. These results indicate that the hVEGF gene enhances endothelialization of EPCs, and that the transplantation of cord blood MNCs transfected with the VEGF gene may be feasible for the treatment of ischemic diseases as a type of angiogenic cell and gene therapy.     

Induction of calcitonin receptors by 1 alpha, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells

We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.     

Reconstruction of the immune system after unrelated or partially matched T-cell-depleted bone marrow transplantation in children: immunophenotypic analysis and factors affecting the speed of recovery

We prospectively studied immune reconstitution in 102 children who underwent T-lymphocyte depleted bone marrow transplants using either closely matched unrelated donors or partially matched familial donors by assaying total lymphocyte counts (TLC), T-cell subsets, B cells, and natural killer cells. TLC, CD3+, and CD4+ T-cell counts remained depressed until 2 to 3 years posttransplant, whereas CD8+ T-cell counts normalized by 18 months, resulting in an inverted CD4:CD8 ratio until 12 months posttransplant. Although the percentage of NK cells was elevated early posttransplant, their absolute numbers remained normal. CD20+ B cells were depressed until 12 to 18 months posttransplant. Factors affecting immunophenotypic recovery were analyzed by nonparametric statistics. Younger patients tended to have higher TLC posttransplant. Higher marrow cell doses were not associated with hastened immunophenotypic recovery. Graft-versus-host disease (GVHD) and/or its treatment significantly delayed the immune reconstitution of CD3+, CD4+, and CD20+ cells. The presence of cytomegalovirus was associated with increased CD8+ counts and a decrease in the percentages of CD4+ and CD20+ cells.