对过继免疫治疗中外周血单个核细胞采集术的探讨

目的观察细胞表面分化抗原CD117在急性髓细胞白血病(AML)与急性淋巴细胞白血病(ALL)的表达差异及其意义,评价其作为髓系抗原的特异性。方法采用CD45/SSC双参数散点图设门法进行三色流式细胞术分析。直接免疫荧光标记法标记20种细胞表面分化抗原,经流式细胞仪测定,对286例白血病患者骨髓或外周血白血病细胞CD117及其他表面抗原的表达结果进行分析。结果CD117在ALL中表达率极低,仅占2%,在AML中表达率为56.9%,2者差异有统计学意义(P<0.05)。在AML各型中,CD117在M3亚型中的表达率最低,为23.1%。本组数据统计结果显示CD117的髓系特异度为0.98(SE=0.014),髓系敏感度为0.57(SE=0.04),统计结果表明CD117比CD13、CD33更具髓系特异性(P<0.05)。结论与CD13和CD33相比较,CD117在作为髓系标记的敏感度不如前两者高,但它更具有髓系特异度,因而可作为排除ALL及辅助诊断AML的标志。     

NK细胞过继免疫治疗HIV感染研究进展

NK细胞是天然免疫中的重要效应细胞,在机体早期抗病毒、抗肿瘤的免疫应答中起关键作用。NK细胞因其主要组织相容性复合体非限制性、泛特异性识别和杀伤靶细胞及快速应答等特点成为免疫治疗研究热点。随着对NK细胞表型、功能及作用机制研究的不断深入,NK细胞过继免疫治疗也被尝试应用于HIV感染治疗。本文从NK细胞生物学特性、NK细胞在HIV感染中的作用以及NK细胞过继免疫治疗等方面来阐述NK细胞过继治疗在HIV感染领域应用的机遇与挑战。     

末梢血单个核细胞被HCV感染

我们调查了丙型肝炎患者末稍血单个核细胞(PBMC)被HCV感染的情况。用逆转录——套式PCR法检测了HCV RNA正、负链。22例受试者的PBMC中检出HCV RNA正链12例(54.5％),其中5例(22.7％)负链也阳性。结果提示,在丙肝患者的PBMC中存在着HCV的活动性感染和复制,表明了HCV的肝细胞外感染。     

晚期非小细胞肺癌自体T细胞过继免疫治疗后T细胞受体变化

目的探讨肺癌患者经自体T细胞过继在治疗后T细胞受体(TCR)的变化特征及临床意义。方法用分子生物学方法对晚期非小细胞肺癌放化疗结合细胞治疗的患者,免疫细胞治疗前和治疗3个疗程结束后检测TCR表达的变化分析。结果 21例患者中的12例在CD4+TCR基因家族中产生克隆化改变,而这种改变发生在CD8+TCR基因家族有16例。结论外周血TCR克隆化分析可能成为自体T细胞过继免疫治疗的疗效评价指标。     

乙型肝炎病毒感染对外周血单个核细胞分泌功能的影响

目的:观察乙型肝炎病毒(HBV)感染后外周血单个核细胞(PBMC)分泌功能的动态变化,为病毒免疫实验提供参考。方法:分离健康人PBMC,分为空白对照组和病毒干预组。空白对照组PBMC正常培养,病毒干预组PBMC加入HepG 2.2.15培养上清液中浓缩的HBV共培养。分别在培养4 h、8 h、12 h、24 h、36 h、48 h后收集培养上清液进行细胞因子(CK)检测。观察细胞因子随时间变化曲线,分析病毒刺激对PBMC分泌功能的影响。结果:病毒干预后IL-2的含量随时间变化呈上升趋势,其余各因子含量随时间变化呈先上升后下降趋势,IL-4、IL-17A、IFN-γ、TNF-ɑ分泌高峰在8 h, IL-6分泌高峰在36 h, IL-10分泌高峰在24 h;空白对照组PBMC中IL-4、IFN-γ各时间点含量高于病毒干预组(P<0.05),IL-6、TNF-ɑ各时间点含量低于病毒干预组(P<0.05);IL-2、IL-10、IL-17A、含量除个别时间点均低于病毒干预组(P<0.05)。结论:病毒刺激后各细胞因子的分泌高峰不同,可根据各自分泌特点选择恰当的研究时间点;HB...     

探讨从骨髓血中分离单个核细胞方法

自体骨髓间充质干细胞移植修复中枢神经及骨髓损伤在临床应用取得了新的进展,由于采集骨髓血中含有以血浆为主的多种成份,单纯采用离心法分离单个核细胞难以达到分离效果,笔者采用美国进口羟乙基淀粉做红细胞沉降剂,实验选择羟乙基淀粉与骨髓血之间合适体积比,从中探讨其对单个核细胞回收率的影响.     

催乳素对Graves 病甲状腺细胞与自体外周血单个核细胞体外相互作用的影响

目的　研究催乳素对Graves病 (GD)甲状腺细胞与自体外周血单个核细胞 (PBMC)在体外共同培养时相互作用的影响。方法　利用免疫荧光染色和流式细胞仪等 ,测定在不同羊催乳素(oPRL)水平进行共同培养时PBMC的活化、增殖反应和甲状腺细胞对人类白细胞抗原基因复合体(HLA) DR及CD4 0 的表达。结果　oPRL浓度为 2 0 0 μg/L时与GD甲状腺细胞共同培养的PBMC中CD4 CD2 5 细胞百分率 [(13 0 8± 2 5 4) % ,P <0 0 1]和增殖指数 [(17 82± 3 0 2 ) % ,P <0 0 1]及 10 0 0μg/L时的增殖指数 [(16 5 7± 2 5 6 ) % ,P <0 0 5 ]均较 0 [分别 (10 15± 2 6 0 ) %和 (14 38± 2 6 4) % ]、12 5及 5 0 μg/L时有显著增加。在 2 0 0和 10 0 0 μg/L时相应甲状腺细胞中CD4 0 细胞百分率 [(4 8 2 5± 6 6 3) % ,(5 2 2 8± 6 94) % ]和平均荧光强度 (dMF ;42 94± 10 2 4,49 5 1± 12 34)明显低于 0 [(5 8 38± 6 6 2 ) %和 6 7 30± 2 0 2 0 ]、12 5及 5 0 μg/L剂量组。而在 5 0 μg/L时其HLA DR 细胞百分率[(4 6 79± 7 5 1) % ,P <0 0 1]和dMF(2 1 0 2± 5 43 ,P <0 0 1)显著高于 0 [(33 5 1± 8 5 8) %和 13 91±3 88]、12 5、2 0 0及 10 0 0 μg/L剂量组。除此以外 ,oPRL各剂量组间差异均     

CD3 AK细胞过继免疫治疗晚期恶性肿瘤76例疗效观察

分离76例晚期恶性肿瘤患者外周血单个核细胞,采用抗CD3Ab、IL-2等诱导自体抗CD3单克隆抗体激活的杀伤细胞（CD3AK细胞）,将CD3AK细胞用生理盐水洗涤3次后混悬于含2%白蛋白的250ml生理盐水中回输给患者。观察疗效、患者一般情况、治疗前后T细胞亚群水平及毒副作用等。结果本组有效率（CR＋PR＋MR）为68.42%;患者食欲、睡眠、体质量改善,疼痛减轻;CD８^＋较治疗前下降,CD3^＋、CD4^＋和CD4^＋/CD8^＋均较治疗前显著升高（P均〈0.05）;治疗过程中4例（5.26%）出现畏寒、发热（体温37.8～38.6℃）,自行缓解,未出现其他不良反应。认为CD3AK细胞过继免疫治疗能有效控制肿瘤,提高患者细胞免疫水平,改善生活质量,且无明显副作用。     

非透析终末期尿毒症患者血清对外周血单个核细胞TNF－α分泌的影响

目的探讨非透析终末期尿毒症（ESRD）患者混合血清及不同分子质量血清对外周血单个核细胞（PBMCs）肿瘤坏死因子α（TNF－α）分泌的影响。方法分离非透析ESRD患者及正常人混合血清，并采用超滤离心装置分离非透析ESRD患者不同分子质量血清，同时用Ficoll密度梯度离心分离非透析ESRD患者和正常人的PBMCs，将正常人或非透析ESRD患者混合及不同分子质量的血清与非透析ESRD患者、正常人的PBMCs共同培养，用ELISA法检测血清和培养上清液中TNF-α浓度。结果①非透析ESRD患者外周血TNF-α含量显著高于正常人（P〈0．01）；②与正常人混合血清刺激PBMCs相比，非透析ESRD患者混合血清刺激PBMCs分泌TNF-α的水平明显升高（P〈0．05）；③非透析ESRD患者大分子质量血清刺激同源PBMCs分泌TNF-α水平较中、小分子高（P〈0．05）。结论尿毒症毒素尤其是大分子尿毒症毒素可能是导致非透析ESRD患者微炎症状态的独立危险因素。     

Proliferation of human peripheral blood mononuclear cells during calcium channel blockade

To evaluate the role of intracellular calcium and particularly Ca²⁺ uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil—which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels—on the proliferation of human peripheral blood mononuclear cells (PBMC) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of ³H-thymidine, ³H-uridine, and ³H-leucine incorporation into control and concanavalin A-stimulated PBMC in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 μmol/L), and of the intracellular calcium antagonist TMB-8 or the calmodulin antagonist W-7 (1, 10, 25, or 50 μmol/L) was assayed in cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures was also determined in mibefradil- or nifedipine-treated control or stimulated cells. Mibefradil and nifedipine reduced the cell number and the ³H-thymidine, ³H-uridine, or ³H-leucine incorporation or the de novo DNA, RNA, or protein synthesis in control and concanavalin A-stimulated human PBMC in a concentration-dependent manner. Mibefradil exhibited a more pronounced inhibition than nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent upon the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine were added 1 to 7 h after cell stimulation. However, regardless of time of addition, TMB-8 and W-7 caused a persistent inhibition of the proliferation of human PBMC. Our data show that mibefradil had a more pronounced inhibitory effect on the proliferation of human PBMC than nifedipine and that this inhibitory effect on de novo DNA synthesis was dependent upon the timing of treatment with both drugs. Mibefradil and nifedipine also reduce RNA and protein synthesis in human PBMC.

Therefore, administration of these calcium channel blockers to inhibit cellular proliferation might be most beneficial at anatomic sites where cellular proliferation is not already an active process, while being ineffective in the presence of ongoing active proliferation, as suggested by some prospective studies.     

Distribution of integrins on human peripheral blood mononuclear cells

The receptors for fibronectin (FN-R) and vitronectin (VN-R) belong to a family of integral membrane glycoproteins known to be involved in cell- extracellular matrix and cell-cell interactions named integrins (FN-R = beta 1 integrin and VN-R = beta 3 integrin). Adhesion studies using FN- coated plastic dishes and highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) showed a strong binding of monocytes and T lymphocytes to FN but virtually no binding of B cells to FN. Binding of monocytes and T cells to FN could be partially inhibited by a hexapeptide (GRGDSP) containing the adhesive peptide sequence Arg-Gly- Asp (RGD) as well as by an anti-FN-R antibody. The distribution of beta 1 and beta 3 integrin complexes on PBMCs was characterized by immunoprecipitation of detergent extracts of 125I-labeled cells using polyclonal antibodies against these two receptors. Two surface polypeptides corresponding to the alpha and beta chains of FN-R and VN- R were found on all three cell types. To characterize these receptors further, monoclonal antibodies (MoAbs) against the very late antigens (VLAs) 1, 3, and 5 were used for immunoprecipitation studies. Monocytes and T cells reacted with VLA 5 that was previously identified as the human FN receptor, whereas no labeling with anti-VLA 5 could be shown for B cells. When cell populations were cultured in 10% human serum for 24 hours, an increase in beta 1-integrin+ monocytes and T cells was observed. The number of beta 3-integrin+ cells remained essentially unchanged. The presence of beta 1 and beta 3 integrins on monocytes as well as on T and B lymphocytes may be of significance in the ability of these cells to interact with each other and participate in hematopoiesis and certain immune reactions.     

Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells

OBJECTIVES: To test whether the active metabolite of leflunomide (LEF-M), in addition to blocking the proliferation of activated lymphocytes by inhibiting dihydro-orotate dehydrogenase (DHODH), influences the transendothelial migration (TEM) of peripheral blood mononuclear cells (PBMC). METHODS: In an in vitro model of PBMC transmigration through an endothelial cell (EC) barrier, PBMC were re-collected in three groups: cells not adherent to the EC, cells bound to, and cells which had migrated through, the EC layer. Experiments in which cells were pretreated with LEF-M (in the absence or in the presence of uridine) were compared with parallel experiments in the presence of medium alone. RESULTS: Preincubation of EC with LEF-M led to a 36 (SEM 16)% reduction in PBMC TEM (p<0.05). Likewise, preincubation of PBMC induced a reduction in their TEM of 39 (9)% (p<0.005). Incubation of both PBMC and EC with LEF-M had an additive effect (mean reduction of 48 (6)%, p<0.005). Incubation of PBMC with LEF-M also decreased monocytic CD44 expression (p<0.005) and PBMC-hyaluronan binding (p<0.05). Incubation of cells with LEF-M and uridine in addition to LEF-M reversed the inhibition of migration, suggesting that the observed effects were due to DHODH inhibition. Fluorocytometric analysis of PBMC subsets within the migrated population showed a decrease of monocytes, but not of B or T cells, after LEF-M treatment. CONCLUSIONS: LEF-M reduces monocytic adhesion molecule expression and TEM and may thus interfere with monocyte and EC activities in RA. Thus, the clinical effects of leflunomide may, at least in part, be due to blocking cell traffic into the inflamed synovia.     

Antiproliferative effects of methotrexate on peripheral blood mononuclear cells

Methotrexate was added to cultured mononuclear cells from the peripheral blood of normal individuals and patients with rheumatoid arthritis (RA) to study the drug's effects on mononuclear cell proliferation and antibody synthesis. In the presence of methotrexate, marked antiproliferative effects (to levels less than 15% of baseline) were seen with 3H-deoxyuridine, but not with 3H-thymidine, as the marker of cell division. This difference was not due to altered kinetics of proliferation or the presence of salvage nucleotides in the culture medium. The absence of suppression of antibody production preactivated by pokeweed mitogen in vitro and the low levels of suppression of spontaneous IgM rheumatoid factor production by blood mononuclear cells from RA patients suggested a relative resistance of activated cells to the effects of methotrexate. The effects of methotrexate on both cell proliferation and antibody synthesis were completely reversed by the addition of high concentrations of exogenous folinic acid. The results suggest that methotrexate has effects on immunocompetent cells that may contribute to the efficacy of this drug in the treatment of RA and other autoimmune diseases.     

Toll-like receptor expression in lupus peripheral blood mononuclear cells

OBJECTIVE: To investigate expression of members of the Toll-like receptor (TLR) family in peripheral blood mononuclear cells (PBMC) in patients with systemic lupus erythematosus (SLE). METHODS: We analyzed PBMC from 14 patients with SLE and 15 healthy subjects. The surface expressions of TLR2 and TLR4 and intracellular expression of TLR9 on PBMC were analyzed by flow cytometry. RESULTS: Although TLR4 expressions on CD14+ monocytes were not significantly different between healthy subjects and patients with SLE, TLR2 expressions on monocytes were reduced in patients with SLE compared to healthy subjects. Intracellular TLR9 expression levels of CD19+ B lymphocytes were significantly elevated in patients with SLE. However, the TLR9 expression levels of plasmacytoid dendritic cells were not significantly different between these patients and healthy subjects. CONCLUSION: Our results show that human peripheral blood B cells express TLR9 and that its expression is increased in patients with SLE. This upregulated expression of TLR9 in B cells may be related to the abnormal B cell hyperactivity in patients with SLE.     

Accumulation and release of isoferritins during incubation in vitro of human peripheral blood mononuclear cells

Ferritin concentration has been measured in peripheral blood mononuclear cells and in the incubation medium following in vitro culture. Antibodies to both heart and spleen ferritin were used. Mononuclear cells cultured in medium containing about 12 mumol Fe/l accumulate ferritin rapidly with an increase in the heart:spleen ferritin ratio from 3:1 to about 10:1. Higher concentrations of iron (100 mumol/l) produce an even greater effect. The accumulation of ferritin is prevented by the addition of desferrioxamine (2 mmol/l) to the incubation medium. Accumulation of ferritin appears to take place largely in monocytes. Phagocytosis of red blood cells also causes rapid accumulation of ferritin but without any change in the heart:spleen ratio. Small amounts of both spleen and heart type ferritin are released during incubation in an iron containing medium and following phagocytosis of red blood cells. Some concanavalin A binding ferritin is also released suggesting that phagocytic cells may be a source of the concanavalin A binding ferritin found in normal plasma.     

HLA-DP-unrestricted TNF-alpha release in beryllium-stimulated peripheral blood mononuclear cells.

Berylliosis is a granulomatous disorder of the lung caused by inhalation of beryllium (Be) and dominated by the accumulation of CD4+ T-helper (Th)1 memory T-cells proliferating in response to Be in the lower respiratory tract. Two gene markers have been associated with susceptibility to berylliosis: 1) the human leucocyte antigen (HLA)-DP gene whose allelic variants, carrying glutamate in position 69 of the beta-chain (HLA-DPGlu69), can bind Be directly and present it to interferon (IFN)-gamma releasing Th1 T-cell clones from patients with berylliosis; and 2) the cytokine gene tumour necrosis factor (TNF)-alpha which has been shown to increase berylliosis risk independent of HLA-DPGlu69. In order to determine whether TNF-alpha release was triggered by Th1 T-cell activation by Be stimulation in the context of HLA-DPGlu69 molecules, the proliferation of BeSO4-stimulated blood mononuclear cells and the release of IFN-gamma, TNF-alpha, RANTES (regulated on activation normal T-cell expressed and secreted), granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, IL-6, IL-8, IL-10 and IL-12 by BeSO4-stimulated blood mononuclear cells was quantified in 11 individuals with berylliosis using an anti-HLA-DP antibody as a probe for HLA-DP restricted T-cell activation. While proliferation and IFN-gamma release were completely abrogated by HLA-DP inhibition (inhibition with anti-HLA-DP monoclonal antibody (mAb): 88+/-16 and 77+/-16%, respectively; anti-HLA-DR: 29+/-38 and 14+/-10%, respectively), the release of TNF-alpha was not (inhibition with anti-HLA-DP mAb: 8.9+/-7.8%). No other cytokine was detected at significant levels. Moreover, Be was able to induce TNF-alpha production in healthy control subjects not exposed to Be in the absence of T-cell proliferation and IFN-gamma production. In conclusion, these data suggest that the tumour necrosis factor-alpha response of mononuclear cells is independent of the activation of beryllium-specific human leucocyte anitgen-DP restricted T-cells, which is consistent with the finding that the tumour necrosis factorA2 and the human leucocyte anitgen-DPGlu69 genetic markers are independently interacting in increasing berylliosis risk.