Mitogenic Factor from Chronically Infected Guinea Pigs

A lymphokine produced by antigen stimulated lymphocytes, induces blastogenesis in cultures of lymphocytes which are not sensitive to the specific antigen. The in vitro production of this factor (MF) was accomplished utilizing Peritonea exudate (PE) cells from Coccidioides immitis infected guinea pigs. Production of MF by lymphoid cultures paralleled skin test reactivity of the donor animal. Removal of adherent cells from the PE population did not decrease the production of MF; conversely, a more significant production of MF was effected by the adherent cell depleted populations. Maximal production of MF was achieved at non-adherent cell concentrations from 4 × 10⁶ to 8 × 10⁶ cells/ml. Cell concentrations below 4 × 10⁶/ml produced material which inhibited DM synthesis in test cultures. MF was separated from the inhibitory substance(s) by column chromatography of the crude preparations on Sephadex G-75. Inhibitor(s) eluted in the void volume (Vo), and the MF eluted in an effluent volume (Ve) which was greater than the total bed volume (Vt) suggesting that MF is adsorbed by Sephadex beads.     

异种(猪)脱细胞真皮基质粉对表皮细胞和成纤维细胞的促进作用

目的观察冷冻研磨获得的脱细胞真皮基质粉(ADMP)生物学性状,探索高纯度且保留生物活性的异种(猪)ADMP对表皮细胞和成纤维细胞生长的支持及在皮肤创伤修复中的应用。 方法选取幼龄长白猪背部皮肤,经生物酶法脱细胞、冷冻干燥后经过不同冷冻研磨程序得到异种(猪)ADMP,再由⁶⁰钴辐照获得无菌异种(猪)ADMP;通过扫描电子显微镜和透射电子显微镜观察异种(猪)ADMP颗粒大小及胶原形态,确定适合应用的研磨程序;通过MTT法检测HaCaT细胞和BJ人皮肤成纤维细胞在不同浓度异种(猪)ADMP包被下的黏附作用,确定异种(猪)ADMP实现细胞最大黏附的浓度范围;DMEM培养液基添加异种(猪)ADMP为实验组,单纯DMEM培养液基为对照组,使用实时细胞分析技术分析BJ人皮肤成纤维细胞迁移指数,验证异种(猪)ADMP对成纤维细胞的促迁移作用;原代表皮细胞接种在异种(猪)ADMP培养液8 d,绘制增殖曲线,检测异种(猪)ADMP对原代表皮细胞的促增殖作用,同时免疫荧光检测原代表皮细胞表面成熟与分化蛋白的表达。对数据进行Student′s t－检验。 结果1、2、3、5、7、10次循环研磨获得异种(猪)ADMP,肉眼可见全部为白色、无块状,其中1、2、3次循环呈较粗颗粒;5、7、10次循环呈现集中均匀趋势,粒径分布在0～12 μm之间。其中5次循环粒径(13.00±2.10) μm与7次循环[(6.00±0.96) μm比较,差异有统计学意义(t＝6.093,P＝0.0002)。异种(猪)ADMP透射电子显微镜下发现,不同循环次数获得的异种(猪)ADMP均能保留大量胶原分子。BJ人成纤维细胞和HaCaT细胞随包被浓度的增加,细胞黏附作用不断增强,浓度高于750 μg/cm²,无明显变化。实时细胞分析技术检测BJ人皮肤成纤维细胞在DMEM培养液基对照组培养液72 h后的迁移指数为1.21±0.10,而异种(猪)ADMP培养液基试验组的迁移指数为3.66±0.11,两组比较差异有统计学意义(t＝22.24,P＝0.002)。在异种(猪)ADMP培养液的原代表皮细胞形态统一增殖迅速,对照组出现一定比例的分化细胞,试验组与对照组第5天细胞数分别为(4.11±0.28)×10⁵、(1.10±0.12)×10⁵个,两组比较差异有统计学意义(t＝13.51,P＝0.005);第8天细胞数分别为(5.00±0.48)×10⁵、(3.05±0.30)×10⁵个,两组比较差异有统计学意义(t＝4.87,P＝0.039),增殖曲线显示试验组在接种即日培养液至第5天,一直处于指数增长期。荧光染色中异种(猪)ADMP培养液的原代表皮细胞E-cad全部表达,少量表达角蛋白10,整合素α6几乎全部强阳性,角蛋白14全部表达并有部分强阳性,Ki-67抗原表达率高。 结论通过生物酶解和冷冻研磨获得的异种(猪)ADMP对参与皮肤创伤修复最重要的表皮细胞和成纤维细胞,具有促进其增殖和迁移的作用,未来可进一步应用于皮肤创伤修复。     

Immunostimulation-Inhibition of Tumor Cell Growth in Vitro Utilizing Tumor Target Cells Labeled with 125I-Iodo-Deoxyuridine

Immune stimulation-inhibition of normal and sensitized syngeneic, allogeneic and xenogeneic lymphocytes with the B16 melanoma was tested by an in vitro assay. Various numbers of lymphocytes were mixed with non-labeled or with ¹²⁵IUDR-labeled B₁₆ cells incubated for 2 hours on a rotating platform and plated into culture dishes. One, 2 or 3 days later the dishes were fixed and viable tumor cells counted either by microscopy or by radioactive monitoring. Sensitized but not normal lymphocytes at ratios up to 1:1000 repeatedly and significantly enhanced the plating efficiency and growth of the target cells. At higher lymphocyte doses, colony inhibition was evident. Numbers of viable target cells calculated from radioactive monitoring agreed closely with visual counts of the cultures. It appears that the technique utilizing ¹²⁵IUDR-labeled cells affords a relatively easy, fast and accurate assay for in vitro studies of immune stimulation-inhibition of target growth.     

Lack of expression of the T-cell marker XTLA-1 in Xenopus tropicalis: exploitation in thymus restoration studies.

Flow cytometric studies employing the monoclonal antibody XT-1 reveal that, in contrast to other strains and species of Xenopus examined, thymocytes and splenocytes from X. tropicalis do not express the T lineage-specific antigen XTLA-1. Thymus dependence of XTLA-1 expression in splenocytes is confirmed in X. laevis by early thymectomy experiments. When X. tropicalis larval thymus is implanted to thymectomized X. borealis larvae, histological studies reveal extensive colonisation by host-derived (quinacrine-positive) cells following metamorphosis. Flow cytometry of propidium iodide-stained nuclei shows that within 3 months of xenothymus implantation, 90% of cells within the implant originate from the recipient; donor cells were undetectable in recipient spleens at this time. The emergence of near normal levels and distribution of XTLA-1 positive cells within xenogeneic thymus implants within 3 months of implantation is illustrated by flow cytometric observations and through immunofluorescence analysis of cryostat sections. These kinetic studies indicate that immigrant host cells require sojourn within the foreign thymus environment before they express the T-cell marker.     

The role of CD4 and CD8 cells in normal F1 hybrid host in the resistance to lethal graft-versus-host disease induction by transfer of parent spleen cells

Transfer of a certain number of C57BL/6 (B6) spleen cells into (BALB/cxB6)F₁ (CB6F₁) nu/nu mice, which are deficient in T cells, causes lethal graft-versus-host disease (GVHD) in the recipients. However, when normal CB6F₁ mice are used as recipients, lethal GVHD does not occur. Using this lethal GVHD system, we investigated which roles CD4⁺ and CD8⁺ T cells play in the resistance to lethal GVHD induction by parent cell transfer in the normal F₁ hybrid host. Lethal GVHD induction by B6 spleen cells in CB6F₁ nu/nu mice was blocked by prior reconstitution of the recipients with normal syngeneic spleen cells. In addition, all nu/nu mice reconstituted with syngeneic CD8⁺ spleen cells developed lethal GVHD, whereas none of the nu/nu mice reconstituted with CD4⁺ cells did. Both spleen weight and number of spleen cells in the former prominently decreased in contrast to the slight increase (peak at 15 weeks) seen in the latter after transfer of donor spleen cells. H-2D^d− Thy1.2⁺ cells, which are considered to derive from donor B6 T cells, existed in the spleen from the CD4⁺ spleen cell-reconstituted GVHD mice, peaking at 5 weeks then gradually decreasing after transfer of donor cells. However, they disappeared in the normal spleen cell-reconstituted GVHD mice 5 weeks later. These findings suggest that CD4⁺ cells in the normal F₁ hybrid host play a critical role in the resistance to lethal GVHD induction by parent spleen cell transfer, although CD8⁺ cells are required for the prompt elimination of donor cells.     

IA- Macrophages and Cytokine Networks Contribute to Tumor-Induced Suppression of CD4+ Autoreactive T Cells

Tumor growth changes the functions and phenotypes of macrophages (Mφ) and T cells. Suppression of CD4⁺ T cell autoresponses during tumor growth was contributed primarily by Mφ. Tumor-induced alterations in the abilities of these cells to mediate autorecognition were assessed through syngeneic mixed lymphocyte reaction (SMLR) assays. Tumor-bearing host (TBH) Mφ were significantly more suppressive (60-90%) than normal host (NH) Mφ, and this suppression was caused partly by reduced la expression. TBH la^- Mφ were significantly more suppressive (50-80%) than their NH counterparts. The suppression mechanism was controlled partly by prostaglandin E₂ (PGE₂), because treating cultures with indomethacin and titrated NH and TBH la^- Mφ led to increased T-cell responsiveness, although responsiveness never reached levels of assays containing unseparated Mφ. Blocking studies using anti-interferon-gamma (anti-IFN-γ) monoclonal antibodies (mAb), anti-interleukin 4 (anti-IL-4) mAb, and indomethacin suggested that IFN-γ, IL-4, and PGE₂ contributed to tumor-induced Mφ-mediated suppression. Our results suggested that a quantitative shift in Mφ phenotype and a qualitative shift in Mφ function in addition to differences in cytokine-directed accessory activities are partly responsible for tumor-induced suppression CD4⁺ T cell autoresponses.     

自体骨髓间充质干细胞联合外周血干细胞移植治疗非霍奇金淋巴瘤2例

背景：为了解决造血微环境受损导致造血重建延迟或失败这一常见难题,国内外研究开始尝试外周血干细胞移植联合骨髓间充质干细胞治疗。 目的：探讨自体骨髓间充质干细胞联合外周血干细胞移植治疗非霍奇金淋巴瘤的安全性和疗效。 方法：对2例确诊为非霍奇金淋巴瘤的患者,采用R-CHOP方案(利妥昔单抗、环磷酰胺、长春新碱、泼尼松)化疗5或6个周期。在自体外周血干细胞动员前取自体骨髓培养骨髓间充质干细胞。动员方案为环磷酰胺、粒细胞集落刺激因子或利妥昔单抗、环磷酰胺、粒细胞集落刺激因子。预处理方案为利妥昔单抗、环磷酰胺、足叶乙甙或利妥昔单抗、环磷酰胺、脂质体阿霉素、地塞米松。回输单个核细胞2.98×10⁶/kg,3.84×10⁸/kg,骨髓间充质干细胞为3.8×10⁶/kg,3.96×10⁶/kg。 结果与结论：例1移植后10 d白细胞下降至最低值,为0.1×10⁹ L^-1,中性粒细胞为0×10⁹ L^-1,移植后12 d 血小板下降至最低值45×10⁹ L^-1,外周血象恢复正常时间为移植后15 d。例2移植后白细胞和血小板低谷时间为移植后5 d,外周血象恢复正常时间为移植后9 d。移植相关并发症为急性上呼吸道感染,外痔感染,经过相应处理感染控制。结果说明自体骨髓间充质干细胞联合外周血干细胞移植治疗后造血重建快,肿块或肿大淋巴结消失,近期疗效可,长期疗效有待进一步观察。     

Surface-aminated electrospun nanofibers enhance adhesion and expansion of human umbilical cord blood hematopoietic stem/progenitor cells

Interaction between hematopoietic stem/progenitor cells (HSPCs) and their extra cellular matrix components is an integral part of the signaling control for HSPC survival, proliferation and differentiation. We hypothesized that both substrate topographical cues and biochemical cues could act synergistically with cytokine supplementation to improve ex vivo expansion of HSPCs. In this study, we compared the ex vivo expansion of human umbilical cord blood CD34⁺ cells on unmodified, hydroxylated, carboxylated and aminated nanofibers and films. Results from 10-day expansion cultures showed that aminated nanofiber mesh and film were most efficient in supporting the expansion of the CD34⁺CD45⁺ cells (195-fold and 178-fold, respectively), as compared to tissue culture polystyrene (50-fold, p<0.05). In particular, aminated nanofiber meshes supported a higher degree of cell adhesion and percentage of HSPCs, as compared to aminated films. SEM imaging revealed the discrete colonies of cells proliferating and interacting with the aminated nanofibers. This study highlights the potential of a biomaterials approach to influence the proliferation and differentiation of HSPCs ex vivo.     

Down-Regulation of CD25 Expression on the Surface of Activated Tumor-Infiltrating Lymphocytes in Human Cervical Carcinoma

ABSTRACT: To investigate the activation status of tumor-infiltrating lymphocytes (TILs) within the tumor milieu of human cervical carcinoma, we quantitatively measured and compared the activation markers on lymphocyte subpopulations which infiltrating normal and neoplastic cervix. A total of 20 patients with stage IA to IIA cervical cancer (cancer group) and 10 women with normal cervix (control group) were enrolled in this study. Mononuclear cells were isolated from tissue specimens by mechanical dispersal technique and three-color flow cytometry was utilized for the quantification of activation markers on lymphocyte subsets. Compared with control group, lymphocytes isolated from cancer tissue consisted of higher proportions of B cells (7.23% ± 4.49% vs. 3.67% ± 3.19%, P = 0.016) and T cells (72.33% ± 8.70% vs. 53.15% ± 17.36%, P = 0.004), but an inverted CD4:CD8 ratio (0.74 ± 0.27 vs. 1.14 ± 0.28, P = 0.002) and decreased NK cells (7.53% ± 4.33% vs. 16.00% ± 11.82%, P = 0.035). Low expression of CD25, but not CD69 and HLA-DR was observed on both CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells derived from cervical cancer (P < 0.0001). Further dual activation marker analysis demonstrated that the expression of CD25 was dissociated from CD69 and HLA-DR on the same TILs in cancer tissue (P < 0.001). TILs in the tumor microenvironment can be functionally inhibited and lose the ability of clonal proliferation due to depressed expression of CD25.     

自体外周血干细胞动员和采集并联合化疗方案治疗儿童神经母细胞瘤及原始神经外胚层肿瘤35例

背景：自体外周血干细胞移植是目前治疗恶性实体瘤的重要方法之一,干细胞动员与采集是决定造血重建的重要因素。 目的：主要评价环磷酰胺,吡柔比星,长春新碱动员方案对儿童神经母细胞瘤及原始神经外胚层肿瘤自体外周血造血干细胞移植动员采集的临床效果。 方法：对35例患儿,确诊神经母细胞瘤 30例,原始神经外胚层肿瘤 5例,采用CDV化疗方案动员,观察采集干细胞效果。 结果与结论：所有病例化疗后第4~9天(平均6.5 d)白细胞< 2×10⁹ L^-1,给予粒细胞刺激因子5~10 mg/kg刺激造血,化疗后13~19 d(平均15.5 d)至白细胞> 5×10⁹L^-1后开始采集。所有病例均采集到足够的单个核细胞数和CD34⁺细胞,总采集次数1~4次,平均2.1次,单个核细胞：(6.1±1.2)×10⁸/kg,CD34⁺细胞为(5.3±0.8)×10⁶,锥虫蓝拒染率：99.5%(99%~100%),动员并发症少,患儿均能耐受。其中25例进行自体外周血干细胞移植后均获快速造血功能重建,白细胞开始回升(中性粒细胞绝对值>0.5×10⁹ L^-1)时间为移植后10~20 d(平均14d)血红蛋白恢复(> 80 g/L)的时间为移植后10~30 d(平均18 d),血小板恢复(> 20×10⁹ L^-1)时间为移植后12~35 d(平均20 d)。结果提示CDV 方案可以安全有效地完成神经母细胞瘤及原始神经外胚层肿瘤患儿自体外周血干细胞动员和采集。     

Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

血红素加氧酶1在间充质干细胞上调哮喘患者外周血调节性T细胞中的作用

背景：上调CD4⁺CD25⁺CD127^low/-调节性T细胞是目前治疗哮喘的新靶点。骨髓间充质干细胞在体外可上调正常人外周血的调节性T细胞,但机制尚未明确。 目的：观察血红素加氧酶1对间充质干细胞上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞的影响。 方法：分别加入0,15,30,45,60 μmol/L氯化高铁血红素和0,5,10,15,20 μmol/L锌-原卟啉对骨髓间充质干细胞进行预处理,荧光定量PCR检测各组间充质干细胞中血红素加氧酶1 mRNA的表达量。密度梯度离心法分离哮喘急性发作期患者和健康对照者的外周血单个核细胞,分别与经氯化高铁血红素预处理、经锌-原卟啉预处理、不经预处理的间充质干细胞共孵育,加入植物血凝素反应72 h,流式细胞仪检测各组CD4⁺CD25⁺CD127^low/-调节性T细胞占CD4⁺T细胞的比例。 结果与结论：人骨髓间充质干细胞中有血红素加氧酶1表达,其表达在体外可被诱导和抑制。间充质干细胞中血红素加氧酶1 mRNA的表达量随氯化高铁血红素浓度增加而增加(P < 0.05),随锌-原卟啉浓度增加而减少(P < 0.05),具有剂量依赖性。间充质干细胞在体外可提高哮喘患者和健康对照者CD4⁺CD25⁺CD127^low/-调节性T细胞的水平(P < 0.01),诱导间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞水平提高(P < 0.01),抑制间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞的水平降低(P < 0.01),但仍高于对照组(P < 0.01)。提示骨髓间充质干细胞部分通过血红素加氧酶1上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞。     

Human purified CD8+ T cells: Ex vivo expansion model to generate a maximum yield of functional cytotoxic cells

CD8⁺ T cells are a critical component of the cellular immune response. They play an important role in the control of viral infection and eliminating cells with malignant potential. However, attempts to generate and expand human CD8⁺ T cells in vitro for an adoptive immunotherapy have been conducted with limitation of the very low frequency of CD8⁺ T cells in blood. Therefore, several expansion protocols have been developed to obtain large and efficient numbers of human CD8⁺ T cells for use in adoptive immunotherapies. In this study various common culture conditions using different cytokines IL-2, IL-4, IL-7, IL-10, IL-12 and IL-15 and autologous feeders and sera were investigated to expand human purified CD8⁺ T cells. The importance and the influence of these factors on the growth and phenotype of CD8⁺ T cell were assessed by serially sampling cultures using flow cytometry. We demonstrated that combination of IL-2 (50U/ml) and autologous feeders induced maximal CD8⁺ T cell proliferation (40-50 folds) compared to other cytokines. Immunophenotypic analysis of cultured cells showed that expanded CD8⁺ T cells were activated and differentiated. Furthermore our expansion model also demonstrated that expanded CD8⁺ T cells are functionally cytotoxic active by killing Allogeneic LCLs cells. In conclusion, we have developed a reliable, simple method that uses minimal cell numbers to generate a high yield of functional cytotoxic CD8⁺ T cells, which can be used for the development of cellular immunotherapies.     

供体凋亡淋巴细胞预输注对猪肝移植受体CD4+T、CD8+T、CD4+CD25+调节性T细胞的影响

背景：凋亡细胞能够主动调节机体的免疫功能,并能通过调节机体细胞免疫和体液免疫的途径诱导免疫耐受,但这些结果只在大鼠肝脏移植模型中证实。 目的：探讨通过⁶⁰Co γ射线体外处理后的供体淋巴细胞预输注诱导猪肝移植特异性免疫耐受的作用中,对淋巴细胞亚群的影响。 方法：建立非转流小型猪原位肝移植模型。将受体猪随机摸球法均分为2组：空白对照组,受体猪无特殊处理,行肝移植;淋巴细胞组：受体猪在肝移植前7 d经耳静脉注射⁶⁰Co γ射线处理过的5×10⁸个供体淋巴细胞。观察两组受体猪移植后的存活时间,移植后T淋巴细胞亚型CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr变化及病理。 结果与结论：移植后3 d,两组病理活检均呈急性中、重度排斥反应;移植后6 d,两组均呈急性重度排斥反应。移植后1,3,6 d CD4⁺T、CD8⁺T、CD4⁺CD25⁺Tr升降趋势,两组间差异无显著意义(P > 0.05)。提示,⁶⁰Co γ射线体外处理过的淋巴细胞预输注未能够诱导猪同种异体肝移植特异性免疫耐受,未能引起T淋巴细胞亚群变化有关。     

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⁺ CD127^low/-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⁺ CD127^low/-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⁺ CD127^low/-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⁺ CD127^low/-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⁺ CD127^low/-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。     

Increased (E)-4-hydroxy-2-nonenal and asymmetric dimethylarginine concentrations and decreased nitric oxide concentrations in the plasma of patients with major depression

Background: (E)-4-Hydroxy-2-nonenal (HNE) is a highly electrophilic end-product of lipid peroxidation. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase (NOS). ADMA is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). DDAH contains a nucleophilic cysteine residue in its active site. There is an increase in lipid peroxidation in major depression. Major depression is associated with the development of coronary heart disease (CHD) and greatly increases morbidity and mortality. There is an increase in circulating ADMA in CHD and vascular risk factors. Objectives: To determine plasma HNE, ADME and nitric oxide (NO) concentrations in patients with major depression compared to normal volunteers and to examine the effect of HNE on ADMA formation and DDAH activity in cultured endothelial cells. Methods: The study was conducted in 25 patients with major depression (DSM-IV criteria) and 25 healthy control subjects. Plasma concentrations of HNE were determined as the O-pentafluorylbenzyl oxime using capillary column GC–MS and deuterated HNE as the internal standard; ADME by LC–MS–MS using ¹³C⁶- -arginine as the internal standard; and NO by GC–MS following reduction to nitrate and nitrite and derivatisation to the pentafluorobenzyl derivative using [¹⁵N]nitrate and [¹⁵N]nitrite as the internal standards. Human umbilical vein endothelial cells were incubated in serum-free medium in the presence of HNE. The concentration of ADMA in the medium was determined by LC–MS–MS. DDAH activity was determined by measuring -citrulline in endothelial cell lysates using LC–MS. Results: There was a significant increase in the plasma concentration of HNE (P<0.0001) and ADMA (P<0.0002) in patients with major depression. There was a significant decrease in the plasma concentration of NO (P<0.0001). A significant positive correlation was found between the plasma concentrations of HNE and ADMA (r=0.63, P<0.0001). A significant negative correlation was detected between the plasma concentrations of ADMA and NO (r=−0.595, P<0.0001). HNE significantly increased ADMA formation (P<0.0001) and significantly decreased DDAH activity (P<0.0001) in cultured endothelial cells. The effects of HNE on DDAH activity were significantly attenuated by the addition of glutathione (P<0.0001). Limitations: No allowance was made for the phase of the menstrual cycle which could influence plasma nitric oxide concentrations. Conclusions: There is an increase in circulating HNE in major depression. HNE inactivates the cysteine residue in the active site of endothelial DDAH leading to the accumulation of ADMA in the circulation. The ADMA then decreases the production of eNOS. This could reduce the amount of NO diffusing from cerebral blood vessels to nearby neurons and influence the release of neurotransmitters. ADMA also constricts cerebral blood vessels and may contribute to the decreased regional perfusion in major depression. The accumulation of ADMA could explain the increased risk of CHD in major depression. The preservation of DDAH activity and the reduction of ADMA accumulation may represent a novel therapeutic approach to the treatment of major depression.     

急性淋巴细胞白血病的免疫分型及CD4+CD25+T调节细胞检测

背景：在急性淋巴细胞白血病发病过程中,CD4⁺CD25⁺T调节细胞对机体免疫反应可能起着一定的调节作用。 目的：观察急性淋巴细胞白血病患者的免疫分型及外周血CD4+CD25+T调节细胞的变化情况。 方法：采用流式细胞仪对35例急性淋巴细胞白血病患者进行免疫分型,并检测外周血CD4⁺CD25⁺T调节细胞的数目,与18名健康对照作比较。 结果与结论：急性B细胞淋巴细胞白血病22例,急性T细胞淋巴细胞白血病13例;22例急性B细胞淋巴细胞白血病中CD19的阳性表达率最高(100%),而13例急性T细胞淋巴细胞白血病中CD7阳性表达率最高(100%)。急性B细胞淋巴细胞白血病患者外周血CD4⁺CD25⁺T调节细胞和急性T细胞淋巴细胞白血病患者差异无显著性意义(P > 0.05),但均高于健康对照(P < 0.05)。表明急性B细胞淋巴细胞白血病中CD19阳性表达率最高,急性T细胞淋巴细胞白血病中CD7阳性表达率最高,同时急性淋巴细胞白血病患者外周血CD4⁺CD25⁺T调节细胞水平显著增高。     

Cultures derived from peripheral blood CD34+ progenitor cells of head and neck cancer patients and from cord blood are functionally different

Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects mediated, in part, by an increased number of immune suppressive CD34⁺ progenitor cells in their peripheral blood and tumor. One means of overcoming this immune suppression is to stimulate the CD34⁺ cells to differentiate into more mature, nonsuppressive progeny such as dendritic cells or monocytes. This study determined that CD34⁺ cells from the peripheral blood of HNSCC patients have the same potential to differentiate into dendritic cells as do human umbilical cord blood CD34⁺ cells following 12–16 days of culture with a cytokine cocktail. When compared functionally, the cultures that developed from CD34⁺ cells of cord blood were able to induce an allostimulatory response in naive T-cells, while the cultures that developed from patient CD34⁺ cells lacked allostimulatory ability. Both cultures expressed class II MHC (HLA-DR), but the proportion of cells expressing the costimulatory molecules CD80 and CD86 was significantly less in cultures that developed from HNSCC-patient CD34⁺ cells. Therefore, although the CD34⁺ cells from the peripheral blood of HNSCC patients can differentiate into dendritic cells, their allostimulatory capabilities are impaired, raising the question of their potential effectiveness in stimulating antitumor immune responses.     

间充质干细胞诱导的CD8α~+ Jagged2~(high) CD11b~(high)调节性树突状细胞防治小鼠aGVHD

目的:观察间充质干细胞诱导的CD8α+Jagged2highCD11bhigh调节性树突状细胞(MSC-DCregs),对小鼠急性移植物抗宿主病(a GVHD)的影响。方法:体外诱导MSC-DCregs。以C57BL/6(H-2b)小鼠为供鼠、接受清髓性全身照射(TBI)预处理的BALB/c(H-2d)小鼠为受鼠进行移植。分为以下5组:为正常对照组;TBI组;移植组:移植物为1×107骨髓细胞(BMCs);a GVHD组:移植物为1×107BMCs+1×107脾细胞(Sp Cs);MSC-DCregs组:移植物为1×107BMCs+1×107Sp Cs+1×106MSC-DCregs。监测植入情况、H2-Kb嵌合率、a GVHD评分、生存和病理学改变。结果:处理1 0 d后所有小鼠造血恢复,30 d后嵌合率检测转为供鼠型。a GVHD组和MSC-DCregs组中位生存期分别为27 d和33 d,MSC-DCregs组生存期较a GVHD组延长(P0.01),临床评分比a GVHD组低(P0.01),33 d的皮肤、肝脏病理学改变均优于a GVHD组。结论:MSC-DCregs具有防治a GVHD的作用,其相关作用机理待进一步研究。     

“Nk-Like” T Cytotoxicity Against B Lymphocytes in a Hypogammaglobulinemic Patient

Physiologically, cells with NK activity appear to exert a negative control on immunoglobulin production. The clinical association of large granular lymphocyte (LGL) proliferation with hypogammaglobulinemia suggests that these functional NK cells could also be involved in pathological situations.

We studied in vitro lymphocyte functions in a patient presenting LGL proliferation associated with hypogammaglobulinemia. The CD3⁺ CD8⁺ CD57⁺ CD16^- phenotype lymphocytes expressed a high NK type cytotoxicity towards K562 targets, suggesting that they may be considered as “NK-like” T cells. We cultured the patient peripheral blood mononuclear cells (PBMC) with control subject PBMC and with PBMC from two other subjects with B chronic lymphocytic leukemia (B- CLL) of the CD20⁺ CD21^- CD10^- phenotype. Patient PBMC exhibited a lytic activity on control PBMC and on the B lymphocytes of one of the two B- CLL but only in the presence of PWM. This activity was not exerted by the culture supernatant and required a cell - to - cell contact. We suggest that the hypogammaglobulinemia observed in this patient may be related to a cytotoxic effect exerted on B lymphocytes by a CD3⁺ CD8⁺ CD57⁺ CD16^- LGL proliferation.