SI11Endolyn (CD164) Modulates the CXCL12-Mediated Migration of Umbilical Cord Blood CD133+ and Jurkat T Cells

Stem cells offer a promising approach to the treatment of myocardial infarction and prevention of heart failure. We have used iron-labelling of bone marrow stromal cells (BMSCs) to noninvasively track cell location in the infarcted rat heart over 16 weeks using cine-MRI and to isolate the BMSCs from the grafted hearts using the magnetic properties of the donor cells. BMSCs were isolated from rat bone marrow, characterised by flow cytometry, transduced with lentiviral vectors expressing GFP and labelled with iron particles. BMSCs were injected into the infarct periphery immediately following coronary artery ligation and rat hearts were imaged at 1, 4, 10 and 16 weeks post-infarction. Signal voids caused by the iron particles in the BMSCs were detected in all rats at all time points. In mildly infarcted hearts, the volume of the signal void decreased over the 16 weeks, whilst the signal void volume did not decrease significantly in severely infarcted hearts. High resolution 3D MR microscopy identified hypointense regions at the same position as in vivo . Donor cells containing iron particles and expressing GFP were identified in MR-targeted heart sections after magnetic cell separation from digested hearts. In conclusion, MRI can be used to track cells labelled with iron particles in damaged tissue for at least 16 weeks after injection and to guide tissue sectioning by accurately identifying regions of cell engraftment. The magnetic properties of the iron labelled donor cells can be used for their isolation from host tissue to enable further characterisation.     

SI32Management of Anaemia in MDS

The myelodysplastic syndromes (MDS) are clonal disorders of haemopoeisis. They share characteristic morphological abnormalities of the blood and bone marrow and a risk of evolution to acute leukaemia which varies according to the subtype of MDS and the prognostic score (IPSS). Overall annual incidence rates based on European studies are between 3 and 4 per 100 000 but the rate dramatically increases with age, exceeding 30 per 100 000 for patients over 80 years. It is therefore mainly a disease seen in older patients, where curative treatments are not an option, and supportive care is the reality. The majority of patients are anaemic at diagnosis and red cell transfusions are the mainstay of supportive care in those symptomatic. The aim is to achieve a haemoglobin level to maximise the patient's quality of life (QOL), which should be tailored to the individual. Red cell transfusions do however have risks associated, including the development of red cell antibodies and iron overload in multi‐transfused patients. Iron chelation therapy, due to its mode of delivery, further impacts on QOL. Alternatives to transfusions include growth factors and disease modifying agents. The aim of using Erythropoietin (EPO), is to improve the overall QOL by avoiding fluctuations in the haemoglobin levels. It also has the benefit therefore of avoiding the risks of transfusion. Unfortunately, it has been shown that those who would benefit most, are least likely to respond. Overall response rates (100% reduction in transfusion requirements) are in the order of 16–24%. Other options to avoid multiple transfusions are by modifying the disease. There are a number of approaches available including stem cell transplantation, immunosuppression, demethylating agents (azacitidine, decitabine) and thalidomide analogues (Lenalidomide). These should be tailored to the individual patient and where possible patients should be treated within clinical research protocols.     

LPS刺激来自人单核细胞的树突状细胞在调节自体CD4+CD25+T细胞活性作用研究

树突状细胞（DC）是现今被认为最具潜能的专职抗原呈递细胞.应用不同方法在体内诱导细胞毒T细胞（CTL）来识别肿瘤相关抗原的肿瘤免疫治疗研究已有报告.然而,在体内免疫治疗的有效性可能仅限制在局部或系统抑制CTL产生和功能.为了检测LPS刺激人单核细胞的DC来抑制自体CD4^＋CD25^＋T细胞能力,使用HLA-A2限制性p53264-272肽作为肿瘤抗原,用LPS（DC-LPS^＋）或不用LPS（DC-LPS^-）产生的DC分别与自体T细胞共同培养.结果显示：在DC-LPS^＋活化的T细胞的CD4^＋CD25^＋T细胞群比DC-LPS^-活化的T细胞要低.这个结果提示,DC-LPS^＋与CD4^＋CD25^＋T细胞群有关联,而且这种特性可能是由于T细胞对肿瘤相关抗原的调节作用.     

自身免疫病患者自体造血干细胞移植后CD4+CD45RA+及CD4+CD45RO+T辅助淋巴细胞亚群的改变

目的检测严重自体免疫病(SA ID)患者自身造血干细胞移植(AHSCT)前后外周血中CD 4 CD 45RA (na ive T h细胞)及CD 4 CD 45RO (记忆T h细胞)T辅助淋巴细胞亚群的变化,探讨na ive T h细胞和记忆T h细胞在AHSCT诱导自身免疫病缓解中的作用。方法使用三色流式细胞术检测SA ID患者AHSCT前1 w及移植后1~3 m o外周血中CD 4 CD 45RA 及CD 4 CD 45RO T辅助淋巴细胞亚群的改变,na ive T h细胞表型为CD 4 CD 45RA ,记忆T h细胞表型为CD 4 CD 45RO 。分析各细胞亚群占CD 4 T辅助淋巴细胞的百分比,并比较移植前后na ive T h、记忆T h细胞的变化。结果AHSCT后,患者外周血中记忆T h细胞显著增高,P<0.05;na ive T h细胞和CD 45RO CD 45RA 双阳性细胞均显著下降,P<0.05。结论AHSCT治疗SA ID患者,患者体内na ive T h细胞和CD 45RO CD 45RA 双阳性T h细胞百分率显著减少,这是AHSCT诱导SA ID缓解的机制之一。     

川崎病外周血中CD4＋CD25＋调节性T细胞与其他免疫细胞的关系研究

目的 探讨CD4＋CD25＋调节性T细胞在川崎病发病机制中作用.方法 以2006年12月～2008年2月入院的32例KD患儿为研究对象,以年龄匹配的30例儿童作为对照组（其中15例急性呼吸道感染的发热患儿为非KD发热组,15例健康体检患儿为健康组）,KD患儿分别于静脉注射丙种球蛋白（IVIG）治疗前和IVIG治疗后热退2～3天取外周血,用流式细胞仪测定外周血单个核细胞CD4＋CD25＋调节性T细胞及其他免疫细胞比例,并分析其关系.结果 急性期KD患儿外周血CD4＋CD25＋调节性T细胞比例与非KD发热患儿和健康对照组相比均明显降低（P〈0.01）.而IVIG治疗后热退2～3天,CD4＋CD25＋调节性T细胞比例明显升高,接近于正常水平.同时,急性期KD患儿外周血中,CD3＋CD4＋、CD19＋CD23＋细胞业群比例明显增高,而CD3＋CD8＋、CD16＋CD56＋细胞亚群比例明显下降.在IVIG治疗后热退2～3天,CD3＋CD4＋,CD19＋CD23＋细胞亚群比例均有明显的下降,CD3＋CD8＋细胞比例升高,但未见CD16＋CD56＋细胞比例的升高.且在KD急性期,CD3＋CD4＋、CD19＋CD23＋细胞亚群比例与CD4＋CD25＋调节性T细胞比例成负相关性（P〈0.01）,而CD3＋CD8＋、CD16＋CD56＋细胞亚群比例则与其没有明显的相关性（P〉0.05）.在IVIG治疗后热退2～3天,CD3＋CD4＋、CD19＋CD23＋细胞亚群比例仍与CD4＋CD25＋凋节性T细胞比例呈负相关（P〈0.01）.结论 CD4＋CD25＋调节性T细胞可能通过对体内免疫细胞的调控参与了KD的发病机制.     

SI30The Naughties–Recruiting Donors in the Decade of Donor Deferrals

Donor deferral rates continue to rise inexorably. Currently in Scotland more than a third of new donors are deferred, and nearly 20% of all donors attending. This reflects not only some major precautionary measures, e.g. the exclusion of donors with a history of transfusion since 1980, but also the increasing stringency and effectiveness of our selection procedures. The CSD SAC has been working to make the donor selection guidelines more evidence based, but often there is little or no evidence to guide decision making. Donors are frequently upset and angry at what they see as arbitrary decisions. While they are not always right, and cannot be considered to have a right to donate, their reaction may be triggered by the paternalistic nature of our approach to this issue. While accepting that we must do nothing to jeopardise recipient safety, an argument can be constructed for putting our relationship with potential donors on a more ‘modern’ footing, perhaps by defining a ‘contract’ which allows for a definition of, and acceptance of, the risks of donation on a ‘partnership’ basis. It is also fair to say that improvements in recipient safety due to the extreme sensitivity of donation testing have as yet had no impact on donor selection. This is not unreasonable but as and when pathogen inactivation becomes a reality it will perhaps provide an opportunity to review donor selection criteria for which we should be prepared. The threat that testing for vCJD might drastically reduce the available donor base should provide the stimulus for us to begin to contemplate a ‘paradigm shift’ in donor selection.     

Selective monitoring of vitamin D2 and D3 supplementation with a highly specific 25-hydroxyvitamin D3 immunoassay with negligible cross-reactivity to 25-hydroxyvitamin D2

Background

The effects of vitamin D₂ and D₃ supplementation on circulating concentrations of 25(OH)D₃ require reliable analytical tools for specific determination of 25(OH)D₃ and 25(OH)D₂. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D₂.

Methods

25(OH)D₃ concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D₃ daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D₂ daily for 3 months.

Results

This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D₂, (0.7%), and demonstrated a high correlation (r² = 0.93) with 25(OH)D₃ determined by HPLC. 25(OH)D₃ increased by 14% in subjects receiving vitamin D₃ for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D₃ levels in those receiving vitamin D₂.

Conclusions

We report that 25(OH)D₃ ELISA was used for evaluation of 25(OH)D₃ concentrations in subjects receiving vitamin D₂ and D₃ supplementation. The increase of 25(OH)D₃ in circulation with vitamin D3 supplementation and lack of increase with vitamin D₂ supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D₃ status in humans.     

SLE患者外周血Tc1/Tc2与CD4/CD25双阳性细胞关联性分析

目的分析SLE患者外周血CD8淋巴细胞分泌的细胞因子与CD4/CD25双阳性细胞反应格局与疾病的关联性.方法用三色免疫荧光单细胞染色法检测了11例SLE患者外周血CD8T细胞分泌的细胞因子与CD4/CD5双阳性细胞的表达.结果所检测11例SLE患者中有3例表现为Tc0,即同时分泌IFN-γ和IL-4的Tc细胞.4例以分泌IFN-γ为主,为Tc1,4例以分泌IL-4为主,为Tc2.同时测定了SLE病人与正常对照组CD4/CD25双阳性细胞群发现CD4/CD25双阳性细胞群在活动期SLE中显著低下[(17.54±4.11%)%,而对照组为(35.42±3.30)%].结论SLE患者外周血中CD8亚群表现为异质性,且与CD4/CD25双阳性细胞有关.至于其在SLE发病中的意义正在进一步研究中.     

CD4+CD25+CD127low/-调节性T细胞异常表达与多发性骨髓瘤的关联性分析

目的 检测多发性骨髓瘤(MM)患者和良性单克隆丙种球蛋白血症(MGUS)患者外周血及骨髓组织中CD4+CD25+CD127low/-调节性T(Treg)细胞表达水平,初探MM中调节性T细胞免疫功能状态以及与疾病发生、发展的关联性.方法 采用流式细胞术分别检测33例MM患者、44例MGUS患者和30例健康者外周血以及6例MM患者骨髓CD4+CD25+CD127low/-调节性T细胞水平.分别采用溴甲酚绿法、免疫散射比浊法检测MM患者血清清蛋白(Alb)、β2-微球蛋白(β2-MG)水平.结果 MM组外周血中CD4+CD25+CD127low/-细胞表达高于MGUS组及健康对照组,差异有统计学意义(P<0.05);MM中稳定期患者组外周血细胞水平较初诊患者组和复发、难治患者组升高,差异有统计学意义(P<0.05);但不同ISS分期的MM患者外周血细胞表达差异无统计学意义(P>0.05);而进展期MM患者中,CD4+CD25+CD127low/-T细胞表达在Ⅲ期高于Ⅰ期+Ⅱ期,差异有统计学意义(P<0.05);进一步研究发现,同一初诊MM患者骨髓CD4+CD25+CD127low/-T细胞水平低于其外周血T细胞水平,差异有统计学意义(P<0.05).结论 外周血CD4+CD25+CD127low/-T细胞表达异常可辅助鉴别良、恶性单克隆丙种球蛋白血症;监测MM的进展以及疾病的活跃程度,并为进展期MM患者及时进行临床干预提供实验依据.     

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。     

Induction of Immune Tolerance to FIX Following Muscular AAV Gene Transfer Is AAV-dose/FIX-level Dependent

Direct intramuscular injection (IM) of adeno-associated virus (AAV) has been proven a safe and potentially efficient procedure for gene therapy of many genetic diseases including hemophilia B. It is, however, contentious whether high antigen level induces tolerance or immunity to coagulation factor IX (FIX) following IM of AAV. We recently reported induction of FIX-specific immune tolerance by IM of AAV serotype one (AAV1) vector in mice. We hypothesize that the expression of high levels of FIX is critical to induction of FIX tolerance. In this study, we investigated the correlation among AAV dose, FIX expression, and tolerance induction. We observed that induction of immune tolerance or immunity to FIX was dependent on the dose of AAV1–human FIX (hFIX) given and the level of FIX antigen expressed in both normal and hemophilia mice. We then defined the minimum AAV1–hFIX dose and the lowest level of FIX needed for FIX tolerance. Different from hepatic AAV–hFIX gene transfer, we found that FIX tolerance induced by IM of AAV1 was not driven by regulatory T cells. These results provided further insight into the mechanism(s) of FIX tolerance, contributing to development of hemophilia gene therapy, and optimization of FIX tolerance induction protocols.     

五种益生菌菌株对脐血单核细胞表达CD4CD25的影响

目的:研究不同剂量的5种国内益生菌菌株(长双歧杆菌6-1株、婴儿双歧杆菌CGMCC313-1株、嗜酸乳杆菌YIT2004株、粪链球菌YIT0072株和酪酸梭状芽胞杆菌CGMCC313-2株)对脐血单核细胞(CBMC)的表达CD4、CD25的影响.方法:取7例健康孕妇的脐血分离出CBMC,分别与上述5种益生菌菌株,以菌和CBMC比例2:1(低)、20:1(中)和200:1(高)共培养24-36小时,同时设阴性对照(PBS)和阳性对照(脂多糖,LPS),采用流式细胞仪检测各组CBMC表面CD4CD25分子表达情况.结果:5种益生菌菌株中,除了高剂量的酪酸梭状芽胞杆菌株CGMCC313-2株组CBMC表达CD4CD25与阴性对照组表达有显著差异(P＜0.01)外,其余益生菌组表达CD4CD25与阴性对照组无明显差别(P＞0.05).结论:酪酸梭状芽胞杆菌CGMCC313-2株可影响Tn细胞分化为CD4+CD25+CBMC.     

慢性乙型肝炎外周血T淋巴细胞亚群CD4+/CD25+和CD8+/CD28-的研究

目的 通过对慢性乙型肝炎患者外周血中CD4+/CD25+和CD8+/CD28-调节性淋巴细胞的分析,探讨其与慢性乙型肝炎患者临床状态的关系.方法 采用流式细胞技术多色荧光分析法,对280例慢性乙型肝炎患者、28例慢性病毒携带者以及22例健康体检者外周血中CD4+/CD25+和CD8+/CD28-淋巴细胞测定.结果 慢性病毒携带者外周血中CD4+/CD25+调节性淋巴细胞为(3.23±1.37)%,明显高于慢性乙型肝炎患者(1.85±1.38)%和正常健康对照(1.59±0.54)%(P＜0.05),但慢性乙型肝炎患者与正常健康对照差别无统计学意义(P＞0.05);而且HBeAg阴性慢性乙型肝炎(1.66±1.09)%与HBeAg阳性慢性乙型肝炎(1.96±1.43)%比较,差别无统计学意义(P＞0.05).慢性乙型肝炎患者和慢性病毒携带者外周血中CD8+CD28-调节性淋巴细胞分别为(79.53±16.07)和89.30±5.12)%,均明显高于正常健康对照(63.93±7.85)%(P＜0.05);另外,HBeAg阳性慢性乙型肝炎组(87.47±17.82)%,也明显高于HBeAg阴性慢性乙型肝炎组(64.90±15.26)%(P＜0.05).结论 慢性病毒携带者处于病毒携带状态可能与体内调节性淋巴胞CD4+/CD25+和CD8+/CD28-的升高有关.     

SI08Protein Misfolding Cyclic Amplification of Disease‐Associated Prion Protein Using Human Autopsy Tissues

The protein misfolding cyclic amplification (PMCA) technique allows for the in vitro production of protease‐resistant prion protein (PrP^res) using a seed of disease‐associated prion protein (PrP^sc) to convert normal cellular prion protein (PrP^c). This system has been developed using brain tissue from scrapie infected hamsters to provide the seed and normal uninfected hamster brain to provide PrP^c substrate and the required conversion factors. These are mixed and subjected to repeated rounds of sonication and incubation. Extremely low concentrations of PrP^sc, such as those present in blood, have been detectable. In order to adapt this method for use in CJD research and in the development of diagnostic tests for human prion diseases it will be necessary to identify a source of human PrP^c and co‐factors that support amplification of human PrP^sc. We have tested whether human autopsy tissues held at the National CJD Surveillance Unit support PMCA. Using a seed extract from variant CJD brain mixed with homogenates of nonCJD neurological control brain we have been able to effect a >600‐fold increase in the detection limit of PrP^res, as determined by Western Blotting. Further development of this technique in concert with high sensitivity PrP^sc detection methods, such as conformation dependent immunoassay, may result in a method capable of acting as a confirmatory assay for disease‐associated prion protein in human tissues and biological fluids such as blood.     

调节性T细胞的新标记CD4+CD25+CD127low/-在类风湿性关节炎中的应用

目的 选择用膜表面标志CD4+CD25+CD127low/-作为检测调节性T(Treg)细胞的指标,探讨其在类风湿性关节炎(RA)中的可能临床应用价值.方法 用流式细胞术检测正常人及RA患者外周血CD4+CD25high、CD4+ CD25+ FoxP3+ 和CD4+CD25+CD127low/- T细胞占CD4+T细胞的比例,分析CD4+CD25+CD127low/-与CD4+ CD25+ FoxP3+ 2群细胞比例之间的相关性.结果 正常人及RA患者外周血CD4+CD25+CD127low/-T细胞比例与CD4+ CD25+ FoxP3+T细胞比例之间呈显著正相关(r=0.694、0.768,P均＜0.01).RA患者外周血CD4+CD25high、CD4+ CD25+ FoxP3+及CD4+CD25+CD127low/-T细胞比例均显著低于正常人(P均＜0.01).结论 膜表面标志CD4+CD25+CD127low/-可以用来鉴定Treg细胞,RA患者外周血CD4+CD25+CD127low/-T细胞的明显减少可能是RA的发病机制之一.     

Change to AXB Phenotype in Genetic A2B Following Carcinoma of the Cervix

In a woman of A₂B genotype, phenotypic change from A₂B to A_xB is documented five yean after a successful operation for carcinoma of the cervix. Serum anti-A activity developed in addition to previously noted anti-A₁. There appears to be an absolute decrease in conversion of H to A substance, occurring independently of hematologic or other malignant disease.     

A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid Chromatographie step

A simple one step high-performance liquid Chromatographie (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D₃ (25-OHD₃) and 25-hydroxyvitamin D₂ (25-OHD₂) is described. Plasma (2–4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C₁₈ cartridge, washed with methanol: water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD₃, added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD₃ was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD₃) and between- (8.0% for 25-OHD₃) batch reproducibilities were satisfactory.     

P07Reduction of Allogeneic Red Cell Transfusion using Autologous Blood Cell Salvage in Knee Arthroplasty

Recent Hospital Transfusion Committee (HTC) audit at the Royal Bournemouth Hospital (RBH) confirmed an allogeneic red cell transfusion rate of 20% for primary Total Knee Replacement (TKR). Current policy at RBH states that when blood stocks reach 67% of normal (amber alert) then surgery with a >20% likelihood of blood transfusion will be cancelled. At current transfusion rates this would include primary TKR. Recent studies have shown a reduction in allogeneic transfusion rates when autologous transfusion drains are utilized. The purpose of this study was to see whether the current rate of allogeneic transfusion could be reduced with the introduction of the CellTransTM Autologous Knee Drainage Blood Transfusion System (ABT) in TKR at RBH. Over a 3 month period all patients undergoing primary, bilateral or revision knee arthroplasty received an ABT. Demographic data was collected from the orthopaedic pre‐assessment clinic. Following surgery further data was collected relating to volume of blood loss into the drain, volume of autologous blood re‐transfused, units of allogeneic blood required and the transfusion trigger, postoperative haemoglobin levels, infection rates and length of stay in hospital. We then compared this data set with retrospective data. Of 170 patients undergoing knee arthroplasty 141 received the ABT. The data collected was compared retrospectively with 169 patients from the previous 3 month period. We demonstrated a reduction in transfusion rates of 13% for primary TKR, 42% for bilateral TKR and 57% for revision TKR with the use of the ABT. In addition we demonstrated a reduction in total allogeneic blood use (99 units to 26 units) and a reduction in mean length of stay in hospital (8.6 days to 7.5 days) with the ABT. Further analysis of the data collected showed a 46% reduction in the allogeneic transfusion rate and a reduction in total allogeneic blood usage (99 units to 9 units) of anaemic patients presenting for surgery. This study has demonstrated a dramatic reduction in allogeneic blood transfusion rates with the use of the CellTransTM Autologous Blood Transfusion System. We have also shown a reduction in length of stay in hospital. Prior to the study primary total knee replacement would have been cancelled during times of limited blood availability (amber alert). The use of the ABT is good for the patient in reducing the need for allogeneic blood, and in addition has demonstrated a significant cost saving due to the reduced blood usage and potential prevention of cancelled operation lists.