In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL‐2 production in human T‐cells

Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)‐2 production in the human Jurkat T‐cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD‐3/anti CD‐28, or anti CD‐3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml^?1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml^?1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD‐3 exhibited decreased IL‐2 production beginning at 50 µg PFOS ml^?1 and 5 µg PFOS ml^?1 respectively, but stimulation with anti‐CD3/anti‐CD28 resulted in no changes compared with the control. Addition of the PPAR‐alpha antagonist GW6471 to PFOS‐dosed cells stimulated with PHA/PMA resulted in decreases in IL‐2 production starting at 50 µg PFOS ml^?1, which suggests PFOS affected T‐cell IL‐2 production via PPAR‐alpha‐independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL‐2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL‐2 production, but PFOS suppressed IL‐2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL‐2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.     

Inhibitory effect of epirubicin-loaded lipid microbubbles with conjugated anti-ABCG2 antibody combined with therapeutic ultrasound on multiple myeloma cancer stem cells

Ultrasound-targeted microbubble destruction (UTMD) technique is thought to improve the chemotherapeutic agent delivery from microbubbles (MBs) in tumor tissues and reduce the side effects in non-tumor tissues. Multiple myeloma (MM) is a bone marrow cancer and remains to be an incurable disease. In this study, we used the UTMD technique to investigate the inhibitory effect of our developed novel reagent on MM cancer stem cells (CD138^?CD34^?MM CSCs) that are MM cells with CD138^?CD34^? phenotypes, responsible for MM-initiating potential, drug resistance and eventual relapse. The preparatory steps of novel reagent was first epirubicin (EPI)-loaded in the lipid MBs that was consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-biotin, dipalmitoyl-phosphatidylglycerol and 25-NBD-cholesterol, then anti-ABCG2 monoclonal antibody (mAb) was conjugated onto the MB surface to form EPI-MBs+mAb. CD138^?CD34^?MM CSCs were isolated from human MM RPMI 8226 cell line by the magnetic associated cell sorting method. The results showed that the attenuated proliferation, migration and invasion ability, and increased apoptosis were observed when MM CSCs were incubated with a various agents. EPI-MBs+mAb combined with therapeutic ultrasound significantly promoted the MM CSC apoptosis compared with EPI, EPI-MBs alone or EPI-MBs+mAb without ultrasound exposure. These results suggest that the developed EPI-MBs+mAb combined with therapeutic ultrasound remarkably induced MM CSC apoptosis in vitro.     

Growth factor receptors in hematopoietic stem cells: EPH family expression in CD34+ and CD133+ cell populations from mobilized peripheral blood

Cell-surface antigen expression of hematopoietic stem cells has a crucial role in characterizing cell subpopulation with distinct functional properties. The Eph receptors are the largest receptor tyrosine kinase family being involved in processes like vascular remodelling during development and physiological and pathological angiogenesis. Some Eph/Ephrin members are expressed in hematopoietic cells. The ability to isolate purified cell populations co-expressing CD34 and CD133 antigens as most commonly used markers for identification of hematopoietic progenitors has provided the opportunity to identify their surface-receptor profile. As positively expressed CD34 and CD133 cells take place not only in hematopoietic but also in endothelial differentiation, we aimed to define the Eph/Ephrin characteristic of these cells and relate these findings to new therapy strategies. Positive selections of CD34 and CD133 cells from PBPC in lymphoma patients were performed using magnetic beads and AutoMACS (Miltenyi Biotec) device. The purity of isolated cells was tested by flow cytometry. Immunocytochemistry was used to assess the Eph/Ephrin expression profile of positively selected samples. Our study revealed that all samples (10 from CD34+ and 8 from CD133+ cells) expressed one or more of Eph/Ephrin antigens in different proportions. All CD34+ cell samples, and 6 of 8 in the CD133+ cell fraction were strongly immunoreactive for EphA2. EphB2 was strongly expressed in all CD133+ cases, but 50% of the CD34 positive group lacked or weakly expressed this receptor. EphB4 was negative in 9 of 10 CD34+ cases and in all CD133+ cells. Thus, we have shown the surface marker profile of positively selected CD34 and CD133 cells in leukapheresis samples from lymphoma patients with regard to Eph/Ephrin receptors and discussed their biological clinical potential.     

对用于包被冠状动脉支架的抗CD34单克隆抗体的生物学指标的质量控制研究

目的:对鼠源抗CD34单克隆抗体的结合活性以及包被在冠状动脉支架上的抗体量和牢固度等技术指标进行评价。方法:采用流式细胞术测定抗CD34单克隆抗体结合KG-1 a细胞的活性;酶联免疫显色方法测定支架上抗体包被量;体外模拟冲刷方法测定支架上抗体牢固度;采用显微镜计数方法测定包被抗CD34抗体支架体外捕获CD34(+)细胞的能力。结果:在抗CD34 M cAb浓度为1μg.mL-1时,细胞平均阳性率为96.34%;支架上抗体含量为50.97 ng.cm-2,而且体外模拟冲刷后支架上抗体含量无显著变化;包被抗CD34单抗的冠状动脉支架捕获细胞数为4万个.cm-2。结论:该方法可用于体外检测抗CD34单克隆抗体包被冠状动脉支架的生物学参数。     

体外阻断CD137-CD137L途径抑制移植物抗宿主病的免疫效应

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径抑制供鼠淋巴细胞的免疫反应。方法应用雄性供鼠BALB/c脾淋巴细胞为反应细胞,雌性受鼠C57BL/6脾淋巴细胞为刺激细胞,进行混合淋巴细胞培养(MLC)。设单抗实验组(加抗CD137L单抗)和对照组(不加抗CD137L单抗),初次和再次MLC第1、3、5、7天采用流式细胞术检测CD3+CD4+T细胞、CD3+CD8+T细胞;RT-PCR法检测培养的细胞IL-2、IFN-γ、IL-4、IL-10的水平。结果实验组CD3+CD8+T细胞明显低于对照组(P<0.01);实验组IL-2、IFN-γ水平明显低于对照组(P<0.01);实验组IL-10表达明显高于对照组(P<0.01)。结论体外MLC中,应用抗CD137L单抗孵育供鼠脾T淋巴细胞主要抑制供鼠CD3+CD8+T细胞的增殖,抑制Ⅰ类细胞因子IFN-γ及IL-2的表达,促进Ⅱ类细胞因子IL-10的表达。     

人脐血CD34^ 内皮祖细胞的体外分化

目的：研究人脐血CD34^ 细胞群体中内皮祖细胞在体外分化为内皮细胞的过程中,干细胞标志以及内皮细胞表型随时间的变化。方法：将免疫磁珠细胞分选法（MACS）得到的CD34^ 细胞体外培养于纤连蛋白和无纤连蛋白处理的培养皿中,以免疫细胞化学鉴定贴壁细胞的内皮标志Flk-1和vWF,并以流式细胞仪分析其干细胞标志AC133。结果：贴壁细胞的内皮标志Flk-1和vWF是逐步出现的,d3时有27.0%贴壁细胞表达Flk-1,vWF不表达,d7时已100%表达vWF和Flk-1,纤连蛋白促进贴壁细胞内皮标志Flk-1和vWF的表达,d3时的表达百分率分别为34.0%和47.0%,d7时Flk-1和vWF的表达均为100%,在培养过程中,AC133阳性细胞的比例迅速下降,但纤连蛋白对AC133的表达无显著影响。结论：在内皮祖细胞分化的过程中,干细胞标志迅速消失,向内皮细胞分化,内皮细胞的表型是逐步出现的,纤连蛋白促进内皮祖细胞的分化。     

阻断CD40/CD40L相互作用延长移植物抗宿主病小鼠存活时间

目的：克隆表达人CD40-Ig融合蛋白，并在小鼠移植物抗宿主病（GVHD）模型中研究利用其阻断CD40/CD40L相互作用的保护性效果。方法：利用RT-PCR技术自人Daudi细胞系中克隆CD40基因胞外区，并插入含有人IgG1Fc段基因的pIG1载体中，构建携带CD40-Fc融合基因的瞬时表达载体转染COS-7细胞，间接夹心ELISA法检测CD40-Ig融合蛋白的表达，再将CD4-Fc融合基因连接入pcDNA3.1的相应位点构建稳定表达载体转染CHO细胞，筛选分泌CD40-Ig融合蛋白的阳性重组CHO细胞并进行无血清大规模培养，收获培养上清利用ProteinA亲和层析法纯化融合蛋白。SDS-PAGE，Western blot和配基结合实验鉴定CD40-Ig的性质，将C57BL6/J（H-2^b)小鼠的脾细胞经尾静脉注射入亚致死剂量照射的BALB/c(H-2^d)小鼠体内建立急性GVHD模型。通过体内注射CD40-Ig融合蛋白评价其对急性GVHD小鼠的保护效果。结果：按上述方法分别构建了哺乳动物表达载体pIG/40Ig和p3.1/40Ig。ELISA和Western blot确定在COS-7和CHO细胞中表达了CO40-Ig融合蛋白，SDS-PAGE结果显示该蛋白具有通过二硫键结合的二聚体结构并以同源二聚体的形式存在，纯化的CD40-Ig可与CD40L结合，体内应用CD40-Ig融合蛋白治疗可延缓小鼠GVHD病情发展并显著延长小鼠的平均存活时间。结论：CD40/CD40L相互作用在GVHD的病理过程中可能扮演了十分重要的角色，提示人CD40-Ig融合蛋白在临床预防和治疗GVHD方面的巨大应用潜力。     

The Different Clinical Effects of Anti-BLyS, Anti-APRIL and Anti-CD20 Antibodies Point at a Critical Pathogenic Role of γ-Herpesvirus Infected B Cells in the Marmoset EAE Model

The robust and rapid clinical effect of depleting anti-CD20 monoclonal antibodies (mAb) in multiple sclerosis (MS) demonstrates a critical pathogenic contribution of B cells. The clinical effect of anti-CD20 mAb has been replicated in a relevant preclinical MS model, experimental autoimmune encephalomyelitis (EAE) in marmoset monkeys (Callithrix jacchus). By contrast, treatment with mAbs against two essential cytokines in B cell activation growth and survival, i.e. BlyS/BAFF and APRIL, was only partially effective. All three mAbs induced depletion of CD20+ B cells from the circulation, albeit with different kinetics and based on distinct mechanisms of action. In the current study we analyzed whether the different clinical effect of anti-CD20 mAb or the anti-BLyS and anti-APRIL mAbs is due to different depletion of B cells infected with the EBV of marmosets, CalHV3. Employing a novel PCR-based assay, half of the colony of group-housed marmosets was tested positive for CalHV3 DNA in secondary lymphoid organs. The same prevalence was observed in placebo-treated monkeys. In marmosets treated with anti-CD20 mAb the load of CalHV3 DNA in lymphoid organs was substantially reduced, while this was not observed in the monkeys treated with anti-BLyS or anti-APRIL mAbs. To examine the pathogenic role of virus-transformed B cells, we infused EBV-transformed B lymphoblastic cell (BLC) lines presenting the immunodominant MOG34-56 peptide. We observed in the recipients of MOG34-56 pulsed BLC, but not in their fraternal siblings infused with non-pulsed BLC, activation of anti-MOG34-56 T cells and meningeal inflammation. Collectively, the data show that among CD20+ B cells, the herpesvirus-transformed subset has a particularly important pathogenic role in the marmoset EAE model.     

自身免疫病患者外周血CD34+细胞动员、采集和纯化研究

目的：探讨自身免疫病患外周血造血干细胞动员、采集和纯化的效果及安全性。方法：对17例确诊自身免疫病患予环磷酰胺＋粒细胞集落刺激因子（G－CSF）动员外周血干细胞，CliniMACS系统对其中11例患外周血单个核细胞进行CD34^ 细胞阳性分选，流式细胞仪检测分选前后CD34^ 细胞纯度及淋巴细胞各亚群。结果：1例动员中并发尿崩症，2例动员失败。11例分选后CD34^ 细胞纯度平均96．1％，回收率70．14％；CD3^ 、CD19^ 、CD14^ 细胞分别去除10^3/kg,10^2/kg,10^3/kg。结论：对自身免疫病患采用环磷酰胺＋G－CSF方案动员外周血干细胞方法可靠，安全性好；外周血干细胞的纯化方法可获得高纯度和高回收率的CD34^ 细胞，并极大限度的去除T、B等细胞。     

CD_(137)对宫颈癌患者CIK细胞功能调节的实验研究

目的探讨CD137mAb对宫颈癌患者CIK细胞的功能调节。方法分离宫颈癌患者外周血单个核细胞(PBMCs),常规CIK培养体系中加入CD137mAb为实验组(CD137-CIK组),同时对照组加入鼠IgG1同型对照(IgG1-CIK组)。动态观察CIK细胞体外增殖;流式检测CIK细胞凋亡率、细胞表型及CD3+CD56+细胞内因子的表达;LDH酶释放法检测CIK细胞杀伤活性。结果两组CIK细胞均有很强的增殖活性,实验组细胞浓度最高达(20.92±3.57)×106/mL,对照组为(14.02±2.68)×106/mL(P<0.05);第21天实验组和对照组CD3+CD56+细胞比例分别达到(35.48±5.46)%和(25.12±4.18)%(P<0.05);CD137-CIK组细胞凋亡坏死率明显低于IgG1-CIK组;实验组CIK细胞体外杀伤宫颈癌HeLa细胞株活性明显高于对照组(P<0.05);流式检测CD137-CIK组中CD3+CD56+细胞内IL-2,IFN-γ表达上调,IL-4,IL-10表达下调。结论 CD137mAb介导的共刺激信号可以显著提高宫颈癌患者CIK细胞的体外增殖活性和抗瘤作用。     

Formation and uptake of arylhydroxylamine-haptenated proteins in human dendritic cells.

Bioactivation of sulfonamides and the subsequent formation of haptenated proteins is believed to be a critical step in the development of hypersensitivity reactions to these drugs. Numerous lines of evidence suggest that the presence of such adducts in dendritic cells (DCs) migrating to draining lymph nodes is essential for the development of cutaneous reactions to xenobiotics. Our objective was to determine the ability of human DCs to form drug-protein covalent adducts when exposed to sulfamethoxazole (SMX), dapsone (DDS), or their arylhydroxylamine metabolites [sulfamethoxazole hydroxylamine (S-NOH) and dapsone hydroxylamine (D-NOH)] and to take up preformed adduct. Naive and immature CD34+ KG-1 cells were incubated with SMX, DDS, or metabolites. Formation of haptenated proteins was probed using confocal microscopy and ELISA. Cells were also incubated with preformed adduct (drug-bovine serum albumin conjugate), and uptake was determined using confocal microscopy. Both naive and immature KG-1 cells were able to bioactivate DDS, forming drug-protein adducts, whereas cells showed very little protein haptenation when exposed to SMX. Exposure to S-NOH or D-NOH resulted in protein haptenation in both cell types. Both immature and naive KG-1 cells were able to take up preformed haptenated proteins. Thus, DCs may acquire haptenated proteins associated with drugs via intracellular bioactivation, uptake of reactive metabolites, or uptake of adduct formed and released by adjacent cells (e.g., keratinocytes).     

Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line.

It has been known that dendritic cells (DCs) including Langerhans cells (LCs) play a critical role in the skin sensitization process. Many attempts have been made to develop in vitro sensitization tests that employ DCs derived from peripheral blood mononuclear cells (PBMC-DC) or CD34+ hematopoietic progenitor cells (CD34+ HPC) purified from cord blood or bone marrow. However, the use of the DCs in in vitro methods has been difficult due to the nature of these cells such as low levels in the source and/or donor-to-donor variability. In our studies, we employed the human monocytic leukemia cell line, THP-1, in order to avoid some of these difficulties. At the start, we examined whether treatment of the cells with various cytokines could produce DCs from THP-1. Treatment of THP-1 cells with cytokines such as GM-CSF, IL-4, TNF-alpha, and/or PMA did induce some phenotypic changes in THP-1 cells that were characteristic of DCs. Subsequently, responses to a known sensitizer, dinitrochlorobenzene (DNCB), and a non-sensitizer, dimethyl sulfoxide (DMSO) or sodium lauryl sulfate (SLS), on the expression of co-stimulatory molecules, CD54 and CD86, were examined between the naive cells and the cytokine-treated cells. Interestingly, the naive THP-1 cells responded only to DNCB and the response to the sensitizer was more distinct than cytokine-treated THP-1 cells. Similar phenomena were also observed in the human myeloid leukemia cell line, KG-1. Furthermore, with treatment of DNCB, naive THP-1 cells showed augmented expression of HLA, CD80 and secretion of IL-1 beta. The response of THP-1 cells to a sensitizer was similar to that of LCs/DCs. Upon demonstrating the differentiation of monocyte cells in our system, we then evaluated a series of chemicals, including known sensitizers and non-sensitizers, for their potential to augment CD54 and CD86 expression on naive THP-1 cells. Indeed, known sensitizers such as PPD and 2-MBT significantly augmented CD54 and CD86 expression in a dose-dependent manner while non-sensitizers, such as SLS and methyl salicylate (MS), did not. To note, the metal allergens such as (NH(4))(2)[PtCl(4)], NiSO(4) and CoSO(4) augmented significantly only CD54 expression. Taking advantage of a cultured cell line, measurement of the co-stimulatory molecules, CD54 and CD86, on naive THP-1 cells following chemical exposure shows promise for the development of a simple, short-term in vitro sensitization test.     

17-beta-estradiol alters Jurkat lymphocyte cell cycling and induces apoptosis through suppression of Bcl-2 and cyclin A

In this investigation, the effects and potential mechanisms of female sex steroid action on proliferation, cell cycling, and apoptosis in Jurkat CD4 + T lymphocytes were examined. 17-beta-Estradiol (estrogen) inhibited Jurkat T cell proliferation, stimulated accumulation of cells in S and G2/M phases of the cell cycle, and induced apoptosis over 72 h in a dose-dependent manner. 4-Pregnene-3,20-dione (progesterone) did not induce redistribution of the cells in the cell cycle but did induce cytostasis and slightly increased apoptosis. Simultaneous staining with anti-BrDU and propidium iodide indicated that estrogen-treated Jurkat T cells proceeded through S phase prior to apoptosis. Progesterone halted cell cycle progression; cells did not progress through S phase or incorporate BrDU. Both hormones decreased the percentage of cells in S or G2/M expressing cyclin A protein, but did not affect cyclin D protein expression. Cyclin A mRNA was markedly decreased by estrogen. Bcl-2 protein and mRNA were also reduced in estrogen but not progesterone-treated Jurkat T lymphocytes. This data shows that high concentrations of estrogen or progesterone significantly suppress lymphoproliferation in association with suppression of cyclin A. Additionally, bcl-2 protein levels were suppressed in association with estrogen-induced apoptosis. These findings demonstrate direct, hormone-specific effects on lymphocytes that may provide insight into their role in immunomodulation or the development of autoimmunity.     

Experimental attempt to produce mRNA transfected dendritic cells derived from enriched CD34+ blood progenitor cells

It Peripheral blood progenitor enriched CD34+ cells (PBPC) are rather often used as stem cell background in cancer patients following high dose therapy. Keeping in mind that precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. The aim of the present study is to develop a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Various concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-a, SCF, Flt-3L and INF-a. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave equal results as serum-containing medium. Following incubation, the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELISPOT assay using mock-transfected DCs as control. The results of our study show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.     

Optimizing donor selection in order to establish a cord blood banking facility: maternal and obstetric factors impact

In this study, we analyzed the obstetric factors affecting total nucleated cells (TNC) content of cord blood units to establish the criteria for umbilical cord blood (UCB) donor selection in our geographic area. UCB was collected from normal uncomplicated pregnancies. In every case, following data were recorded: (1) gestation length; (2) type of delivery (cesarean or vaginal); and (3) newborn characteristics: weight and sex. For each sample, TNC content, percentage and number of CD34+ cells, and viability were analyzed. The results showed that TNC content increases with cord blood volume, gestational length and newborn weight. The mean blood volume and the mean TNC per unit were 42.37 ± 13.5 ml and 55.49 ± 19.4 × 10⁷, respectively. Stepwise regression analysis revealed a positive and significant correlation (r= 0.89) between these two variables. Meanwhile the CD34+ cell content remains unchanged in deliveries at 32–40 weeks of gestation. The mean CD34+ percentage obtained was 0.37 ± 0.06, and the total number of CD34+ cells was 4.827 ± 0.8204 × 10⁴ / mL UCB. Concluding, the maternal and obstetric factors have a significant impact on UCB cell quantity and quality. The main criteria for UCB collection and storage resulted to be: a gestational age higher than 36–40 weeks and newborn weight > 3200g; gestation number ≤ 2 and placental weight > 700g can be added to the standard criteria to improve the bank efficiency. Our results have also become helpful in evaluating stored UCB units to establish the adequacy for clinical transplant utilization.     

Autografting with peripheral blood CD34-positive cells following high-dose chemotherapy against breast cancer

We report autologous CD34+ cell transplantation performed in 3 cases of recurrent breast cancer. The hematological recovery in these cases was assessed by comparing with that in the previous cases of autologous hematopoietic stem cell transplantation performed with the same high-dose chemotherapy regimen. Patient 1 was a 32-year-old woman with pulmonary and skeletal metastases; patient 2, a 55-year-old woman with pulmonary metastases; and patient 3, a 48-year-old woman with hepatic metastases. On day 1, cyclophosphamide 1000 mg/m2 and epirubicin 130 mg/m2 were administered concurrently with granulocyte colony-stimulating factor, and peripheral blood stem cells were harvested on days 14-16. These stem cells were processed using anti-CD34 monoclonal antibody and an immunomagnetic bead device, Isolex 300i. The high-dose chemotherapy regimen consisted of cyclophosphamide 2000 mg/m2/day, div, and thiotepa 200 mg/m2/day, div on day -5, -4, and -3. The harvested CD34+ cells numbered 3.9 +/- 2.8 x 10(6)/kg (range: 0.73-7.8/10(6)/kg), and the CFU-GM, 8.3 +/- 5.6 x 10(5)/kg (range: 1.2-15.1/10(5)/kg). After the separation, the percent of CD34+ cells was 81.9 +/- 11.6% (range: 65.8-96.4%), the CD34+ cell yield, 71.8 +/- 30.2% (range: 46.0-129.6%), and the CFU-GM yield, 48.9 +/- 9.1% (range: 35.3-62.0%). At the time of transplantation, the number of nucleated cells was 0.55 +/- 0.31 x 10(5)/kg, and that of CFU-GM, 31.2 +/- 17.8 x 10(5)/kg. Comparison of the hematological recovery in these three cases with that in patients receiving an identical high-dose chemotherapy regimen revealed recovery rates significantly faster than in patients having bone marrow transplants, and approximately identical with that in peripheral blood stem cell transplantation cases.     

The effects of Foxp3-expressing regulatory T cells expanded with CD28 superagonist antibody in DSS-induced mice colitis

Regulatory T (Treg) cells play an important role in the pathogenesis of inflammatory bowel disease (IBD). In the present study, we found that a superagonistic CD28-specific monoclonal antibody (supCD28mAb, D665) could preferentially stimulate expansion of CD4+Foxp3+ Treg cells. Foxp3(EGFP) mice were orally administrated with 3.5% DSS for 5days, and intraperitoneally injected supCD28mAb 1mg/mice in treated group. All of the mice were sacrificed on day 8, and both clinical and histological parameters showed that the severity of colitis was significantly reduced in treated group compared to controls. In treated group, the proportion of CD103, CD152 and CD62L expression on Foxp3+Treg cells in the spleen and mesenteric lymph node were higher than controls. Furthermore, qRT-PCR analysis showed that expression of anti-inflammatory cytokines such as IL-10, TGF-β was significantly increased in treated group. Taken together, our data demonstrated that supCD28mAb targets CD4+Foxp3+Treg cells expansion in vivo, maintains and enhances their regulatory functions, to reduce the damage of colon in dextran sulfate sodium (DSS)-induced mouse colitis by secreting a large amount of IL-10. It represents a major advance towards the therapeutic use of polyclonally activated Treg cells as cellular therapy for treatment of IBD.     

rhBMP-2m对辐射小鼠骨髓CD34~+细胞变化的影响

目的　探讨小鼠受到辐射后 ,重组人骨形成蛋白成熟肽 2 (rhBMP 2m)对骨髓CD34+ 细胞变化的影响。方法　通过60 Coγ射线照射 ,建立小鼠骨髓造血损伤模型 ,经rhBMP 2m连续治疗后 ,骨髓单个核细胞计数 ,通过流式细胞仪检测骨髓单个核细胞中CD34+ 细胞比例 ,比较对照组和治疗组之间的差别。结果　经rhBMP 2m治疗的动物 ,单个核细胞数和CD34+ 细胞比例均明显高于照射对照组 ,P <0 0 5 ,n=6。结论　对于辐射引起的小鼠骨髓造血损伤 ,rhBMP 2m能够增加造血细胞数量 ,提高骨髓单个核细胞中CD34+ 细胞的比例 ,加快骨髓造血系统的重建。     

Tezacitabine blocks tumor cells in G1 and S phases of the cell cycle and induces apoptotic cell death

Tezacitabine (FMdC) is a new cytostatic/cytotoxic agent widely investigated in clinical trials and on the cellular level. In a previous paper (3) we worked on human and murine leukemia (L-1210, HL-60, and MOLT-4) cells, and in this paper we investigated the influence of FMdC on the cell cycle and apoptosis in vitro of three other leukemias (CCRF-SB, KG-1, and Jurkat), and human solid tumor (carcinoma) cell lines (COLO-205, MCF-7, and PC-3). We found that FMdC induces the G1 (at concentrations higher than 10 nM). and S-phase (at low concentration) leaky block of the cell cycle. FMdC also effectively induces apoptotic death of cells by the caspase 3/7 pathway. We found also that FMdC induces intensive changes in the protein metabolism. These changes are correlated with the cell death.     

Complete dependence on CD4+ cells in late asthmatic response, but limited contribution of the cells to airway remodeling in sensitized mice

It is known that the late asthmatic response (LAR), a characteristic feature of asthma, is closely associated with CD4+ Th2 cell-mediated allergic inflammation. Airway remodeling is also a pathogenesis of asthma, but literature reporting roles of CD4+ cells in the remodeling is controversial. There has been no study that simultaneously assessed the roles of CD4+ cells in both LAR and airway remodeling. Sensitized mice were intratracheally challenged with ovalbumin 4 times. Treatment with an anti-CD4 monoclonal antibody (mAb) before the 1st challenge almost completely abolished increase in CD4+ cells in the tissues after the 4th challenge. The late phase increase in airway resistance after the 4th challenge was also completely inhibited by anti-CD4 mAb. Parameters of airway remodeling, subepithelial fibrosis and epithelial thickening were attenuated by treatment, whereas the inhibition was only 30% - 40%. Bronchial smooth muscle thickening was not affected. Because interleukin (IL)-5 production as well as eosinophilia was effectively suppressed by anti-CD4 mAb, the effect of anti-IL-5 mAb was also examined, resulting in no inhibition of airway remodeling. Collectively, although the LAR was completely dependent on CD4+ cell activation, airway remodeling was only partially dependent on the cell.