严重烧伤患者外周血树突细胞的变化及临床意义

目的 探讨严重烧伤患者外周血树突细胞(DC)的变化及与烧伤严重程度和脓毒症发生的关系.方法 22例严重烧伤患者根据脓毒症的诊断分为烧伤组(10例)和烧伤脓毒症组(12例);按烧伤总体表面积(TBSA)分为TBSA Ⅰ组(TBSA 30％～50％,14例)和TBSA Ⅱ组(TBSA 51％～80％,8例).采集患者伤后1、3、7、14和20 d外周静脉血,用流式细胞仪检测外周血中髓样树突细胞(mDC,Lineage1- HLA-DR+CD11e+)和浆样树突细胞(pDC,Lineage1-HLA-DR +CD123+)两种DC亚型的数量.以同期10例健康体检者作为对照.结果健康对照组外周血mDC为(0.450±0.150)％,pDC为(0.241±0.084)％.与健康对照组比较,烧伤组患者伤后1、3、7d外周血mDC[(0.257±0.116)％、(0.274±0.086)％、(0.317±0.056)％]和pDC[(0.122±0.058)％、(0.165±0.051)％、(0.177±0.024)％]均显著减少(均P＜0.05),14 d、20 d时mDC、pDC数量恢复至正常水平;烧伤脓毒症组患者伤后1d外周血mDC[(0.230±0.090)％]和pDC[(0.114±0.071)％]即较烧伤组进一步减少(均P＜0.05),至20 d时外周血mDC和pDC仍显著低于烧伤组[mDC:(0.246±0.076)％比(0.412±0.097)％; pDC:(0.097±0.032)％比(0.203±0.039)％,均P＜0.05].与健康对照组比较,TBSA I组患者伤后1、3、7d外周血mDC[(0.266±0.062)％、(0.289±0.071)％、(0.351±0.054)％]和pDC[(0.131±0.025)％、(0.163±0.037)％、(0.178±0.038)％]均显著减少(均P＜0.05),14 d、20 d时mDC、pDC数量恢复至正常水平;TBSAⅡ组患者伤后1d外周血mDC[(0.227±0.070)％]和pDC[(o.112±0.047)％]即较TBSA Ⅰ组进一步减少(均P＜0.05),至20 d外周血mDC和pDC仍显著低于TBSA Ⅰ组[mDC:(0.297±0.072)％比(0.423±0.046)％;pDC:(0.107±0.061)％比(0.197±0.042)％,均P＜0.05].结论严重烧伤患者早期外周血mDC和pDC数量均减少,烧伤程度越严重DC数量减少越明显;外周血DC数量减少导致烧伤后机体免疫功能低下,数量减少明显者易并发脓毒症;烧伤后定期检测外周血DC数量可以作为脓毒症的早期预警指标.     

HLA单倍体相合造血干细胞移植后外周血树突细胞重建

目的探讨HLA单倍体相合造血干细胞移植（HSCT）后外周血树突细胞（DC）亚群重建的特点和临床意义。方法选择21例HLA单倍体相合的HSCT患者进行研究，12名健康供者作为对照。流式细胞术检测外周血DC亚群重建及其与急性移植物抗宿主病（aGVHD）的关系。结果白血病患者外周血DC、髓样树突细胞（MDC）和髓样树突细胞1型（MDC1）的百分比分别为0．13％、0．05％和0．03％，绝对值分别为7．89×10^6／L、1．52×10^6／L和1．33×10^6／L；健康对照百分比分别为0．76％、0．65％和0．44％，绝对值分别为42．66×10^6／L、35．56×10^6／L和26．70×10^6／L，白血病患者均明显低于健康对照（P＜0．05）。而白血病患者髓样树突细胞2型（MDC2）、浆样树突细胞（PDC）的百分比分别为0．01％和0．07％，绝对值分别为0．15×10^6／L和2．60×10^6／L；健康对照百分比分别为0．44％和0．14％，绝对值分别为2．40×10^6／L和8．50×10^6／L，两组比较，差异无统计学意义（P＞0．05）；MDC／PDC比值明显减低（P=0．001）。移植后14天（＋14天）MDC1、MDC2及PDC绝对值分别为1．45×10^6／L、0．32×10^6／L和1．60×10^6／L，达到患者移植前水平，但＋100天内均未能恢复正常水平；MDC／PDC比值＋14天（1．40）与移植前（0．50）相比，差异有统计学意义（P=0．024），至＋30天恢复正常（P=0．602）。＋14天高PDC、DC组和低MDC／PDC比值组Ⅱ～Ⅳ度aGVHD的累计发生率较低PDC、DC组和高MDC／PDC比值组明显增高（P＜0．05）。结论白血病患者移植前外周血DC百分比、绝对值及MDC／PDC比值均减少；移植后MDC／PDC比值恢复较快，＋30天恢复正常，其他指标恢复较慢，＋100天内均未恢复正常；且恢复情况与aGVHD相关。     

树突细胞联合CIK杀伤肺癌细胞的实验研究

目的 研究细胞因子诱导的杀伤细胞(CIK)与同源性树突细胞(DC)共同培养后增殖活性和细胞表型的变化,以及对SPC-A-1人肺癌细胞的杀伤作用.方法 通过采集健康志愿者外周血分离单个核细胞(PMBC),根据黏附特性的不同将贴壁细胞诱导为成熟的DC,同时将非黏附细胞诱导分化为CIK,在培养第9 d时将DC和CIK细胞按1∶20混合共培养3 d,同时以单纯CIK细胞为对照,运用LDH释放法检测对SPC-A-1的杀伤活性.结果 DC-CIK共培养后的增殖速度快于单纯的CIK细胞,培养12 d时CIK和DC-CIK的CD3+CD8+,CD3+CD56+双阳性率分别为51.2%±6.6%,21.4%±4.2%及70.7%±5.9%,39.6%±4.7%,表达差异有统计学显著性意义(P<0.001).在5∶1～20∶1效靶比范围内,DC-CIK对肺癌细胞的杀伤活性高于CIK(P<0.05),且随效靶比增加,杀伤活性增强.结论 DC-CIK的增殖活性和杀伤活性均高于单纯的CIK细胞.     

特发性血小板减少性紫癜患者外周血来源树突细胞的功能研究

特发性血小板减少性紫癜(ITP)是一种由于循环血中抗血小板抗体增多，导致血小板破坏增加而引起的自身免疫性疾病，其具体的发病机制尚不完全清楚。近年来大量研究显示，树突细胞(DC)功能的异常与自身免疫性疾病的发病关系密切。我们研究了ITP患者外周血来源DC的功能状态，以阐明DC在ITP发病机制中的作用。     

胚胎干细胞来源的树突细胞制备骨髓瘤疫苗的研究

树突细胞（DC）是体内功能最强大的专职抗原呈递细胞，由于其具有激活初始T淋巴细胞的能力，因而被认为是机体免疫的始动者，在抗肿瘤免疫方面发挥着重要作用。DC的免疫治疗亦被认为是最具前景的抗肿瘤治疗方法之一。我们利用胚胎干细胞（ESC）的全能性在体外诱导出具有正常表型及功能的DC用以制备骨髓瘤疫苗，并检测其抗肿瘤活性。     

乳腺癌患者大剂量环磷酰胺化疗方案联合G—CSF动员外周血造血干细胞

评价大剂量环磷酰胺化疗方案联合G-CSF对乳腺癌患者外周血造血干细胞的动员效果.动员方案为在常规联合化疗方案的基础上,提高环磷酰胺剂量2-4倍,化疗后当外周血WBC降至1.0×10 9/L以下时给予G-CSF,150微克/次,2次/日.在WBC恢复升高至5.0×10 9/L以上时,进行外周血造血干细胞采集.结果共有10例乳癌患者完成了上述方案的外周血造血干细胞动员,WBC降低的中位最低值为0.8(0.4-1.0)×10 9/L,造血干细胞的中位采集次数为2(2-3)次;采集的CD34+细胞中位数为6.43(1.99-18.75)×10 6/kg.结论提示,大剂量环磷酰胺化疗方案联合G-CSF是乳癌患者理想的外周血造血干细胞动员方案.     

树突细胞在多发性骨髓瘤免疫治疗中的作用

树突细胞(DC)是目前认为最有效的抗原提呈细胞,它是最早且直接调节免疫反应的细胞。多发性骨髓瘤(MM)分泌的单克隆免疫球蛋白(M-蛋白)具有独特型抗原决定簇,可作为肿瘤相关抗原(TAA),在DC的介导下,可激活体内独特型特异的MHC-Ⅰ类限制性Ⅰ型T细胞反应。以DC为基础的免疫治疗策略应用与MM,特别是干细胞移植后微小残瘤病变(MRD)的治疗,取得了较好结果。     

不同方式冻存的外周血干细胞复苏培养CIK细胞的实验研究

采集的PBSC悬液分外周血干细胞悬液冻存组和PBMNCs冻存组两组冷冻于-80℃保存。1个月后42℃复苏后,加入各种细胞因子(IFN-γ、IL-2、抗CD3单抗)诱导培养成CIK细胞,观察培养的细胞总数、培养总时间及细胞表型鉴定。经14~21d培养后,两组诱导扩增的CIK细胞总数无显著差异(P0.05),但在培养总时间上,PBMNCs冻存组为15.2±0.92d,干细胞悬液组为18.2±2.35d,差异有统计学意义(P0.05)。流式鉴定两组CIK细胞均可见总T细胞、CD8+T细胞、CD3+、CD56+细胞均较诱导前显著升高。通过在血细胞分离机分离采集单个核细胞时留取部分悬液冻存后复苏进行CIK细胞培养,可以获得有效的细胞输注数量,同时可作为一种补充手段,减少患者细胞采集时的痛苦和大量血液成分的丢失。     

树突细胞联合细胞因子诱导的杀伤细胞对耐药K562细胞的体外杀伤作用

目的研究细胞因子诱导的杀伤(CIK)细胞与同源树突细胞(DC)共培养后CIK细胞的增殖活性及表型的变化,并观察其对K562、K562/ADM细胞毒作用的影响。方法采集健康供者外周血单个核细胞(MNC)用于诱导培养CIK细胞及成熟DC,将成熟DC和CIK细胞混合培养,用MTT法检测DCCIK共培养细胞杀伤K562细胞及其耐药株的活性。结果在2.5～20.0效靶比范围内,CIK细胞对K562和K562/ADM细胞的杀伤率分别为(20.0±1.2)%～(61.1±2.2)%和(17.5±2.1)%～(45.2±3.3)%;DCCIK共培养细胞对K562和K562/ADM细胞的杀伤率分别为(25.2±2.3)%～(70.9±4.1)%和(22.4±2.7)%～(62.3±5.0)%。CIK细胞对K562敏感株和耐药株杀伤率的差异无统计学意义,DCCIK细胞对敏感株和耐药株的杀伤作用差异亦无统计学意义;DCCIK细胞对K562和K562/ADM细胞的杀伤活性均高于单纯CIK细胞组,差异有统计学意义(P<0.05)。结论DC与CIK共培养细胞的增殖活性和细胞毒活性高于CIK细胞。     

肿瘤细胞裂解物致敏树突状细胞对小鼠乳腺癌作用的研究

目的观察肿瘤细胞裂解物致敏树突状细胞 (DC)对小鼠乳腺癌的治疗作用。方法无菌取小鼠骨髓细胞 ,在体外培养条件下经细胞因子作用诱导为树突状细胞 (DC) ,用EMT6肿瘤抗原裂解物冲击致敏DC细胞 ,检测DC体外刺激活化淋巴细胞作用 ,以及经DC免疫产生的细胞毒T淋巴细胞 (CTL)体外杀伤肿瘤细胞的活性 ,观察致敏DC免疫对小鼠乳腺癌模型的治疗作用。结果镜下可见抗原致敏后的DC可吸引淋巴细胞聚集成团 ;致敏DC诱导生成的特异性CTL在体外对肿瘤细胞可产生杀伤作用 (与PBS对照组比较 ,P =0 .0 2 7) ,而未成熟DC细胞组和肿瘤抗原组与PBS对照组间无显著性差异 (P =0 .17,P =0 .0 72 ) ;经致敏DC注射免疫后 ,小鼠负荷肿瘤得到抑制 (与PBS对照组比较 ,P =0 .0 3 5 ) ,而单纯肿瘤抗原及未致敏DC免疫组与PBS对照组间无显著性差异 (P =0 .2 6,P =0 .11)。结论肿瘤抗原裂解物致敏的DC可有效递呈抗原并诱导淋巴细胞杀伤肿瘤细胞。     

Recent clinical development of dendritic cell-based immunotherapy for prostate cancer

Dendritic cells (DCs) are the most powerful immune cells that present antigens to T cells, resulting in a T cell response. Therapeutic strategies involving DCs have been explored in many malignancies, including prostate cancer. These strategies are designed to stimulate DC proliferation, promote antigen uptake and processing by DCs, or to directly provide antigen-loaded DCs. All approaches are designed to stimulate an antitumour T cell response leading to clinical benefit. Many approaches in prostate cancer have demonstrated successful induction of the desired immune response. Clinical effect has also been observed, prompting larger definitive trials.     

银屑病患者外周血树突状细胞功能的体外研究

目的研究寻常型银屑病患者外周血树突状细胞(DC)激活及促进T淋巴细胞分化的功能。方法用体外培养细胞法培养DC,流式细胞术检测DC细胞表面标志,ELISA法检测培养细胞上清中IL-12、IL-4、IFN-γ和IL-1β的含量。结果银屑病患者DC与正常人相比,其细胞表面标志CD83和CD86表达率明显升高(P<0.001),分泌IL-12明显增多(P<0.05),IL-4和IL-1β分泌明显减少(P<0.001),IFN-γ分泌无差异。结论寻常型银屑病患者外周血DC的抗原提呈能力和激活T淋巴细胞的功能增强,而且促进TH0向TH1分化的能力增强。     

冠心病患者外周血单个核细胞YKT6基因表达及其临床意义

目的：研究YKT6在冠心病（coronary heart disease,CHD）患者外周血单个核细胞中的表达及其与疾病发生、发展的关系.方法：分别采用半定量逆转录-聚合酶链反应（RT-PCR）和Westen blot检测40例CHD患者和37例健康者YKT6 mRNA和蛋白表达.结果：CHD患者外周血单个核细胞中YKT6 mRNA和蛋白表达均明显高于健康者（P＜0.05）.结论：YKT6表达与CHD发生存在一定相关性,在CHD发病过程中可能起重要作用,为CHD的诊断和预防提供了新线索.     

Natural killer activity of peripheral-blood mononuclear cells in breast cancer patients

Natural killer (NK) activity of immune cells plays a central role in host defense against cancer and virus-infected cells. Natural cytotoxic activity of peripheral-blood mononuclear cells was assessed by a Calcein-AM release assay in 89 subjects. In the present study, we here demonstrated that NK activities of peripheral-blood mononuclear cells (PBMCs) from breast cancer patients were significantly lower as compared with that of healthy individuals. There were significant differences in the NK activities of PBMCs from HER2-negative breast cancer patients as compared with HER2-positive patients. Our results suggest that NK activity of PBMCs is lower in breast cancer indicating a role for immunological natural host defense mechanisms against cancer.     

Maximizing dendritic cell migration in cancer immunotherapy

Background: The success of dendritic cell (DC)-based immunotherapy in inducing cellular immunity against tumors is highly dependent on accurate delivery and trafficking of the DC to T-cell-rich areas of secondary lymphoid tissues. Objective: To provide an overview of DC migration in vivo and how migration to peripheral lymph nodes might be improved to optimize DC therapy. Methods: We focused on DC migration in preclinical models and human skin explants and on clinical vaccination trials studying migration of in vitro-generated DC. Results/conclusions: DC migration requires an intricate interplay between the cell and its environment. To maximize migration for cellular therapy, it is important to optimize the generation of migratory DC as well as treatment strategies.     

胃癌患者组织中树突状细胞亚型的表达及临床意义

目的探讨树突状细胞(DC)亚型在胃癌患者组织中的表达水平及其与临床病理特征及预后的关系。方法 202例随访胃癌患者中有140例符合纳入标准,用流式细胞术检测胃癌患者组织中DC亚型;免疫组织化学染色法检测140例胃癌患者癌组织和10例慢性胃炎组织中CD11c+DC的表达水平,并结合临床病理特征进行统计学分析。结果在胃癌组织中单核细胞来源DC(Mo DC)的表达水平为(6.3±6.3)%,而髓系来源的DC(m DC)及浆细胞来源DC(p DC)均不表达;胃癌组织中CD11c+DC表达水平与肿瘤大小、淋巴结转移、病理分级、TNM分期比较差异均有统计学意义(P均0.05);且其表达水平和TNM分期是胃癌患者预后的重要影响因素(P均0.05)。结论胃癌组织中浸润的DC细胞仅为Mo DC;CD11c+DC与胃癌发生和转移有关,可能参与调控肿瘤免疫应答。     

Potential approaches for more successful dendritic cell-based immunotherapy

Introduction: Dendritic cells (DCs) are the most important antigen-presenting cell population for activating antitumor T-cell responses; therefore, they offer a unique opportunity for specific targeting of tumors.

Areas covered: We will discuss the critical factors for the enhancement of DC vaccine efficacy: different DC subsets, types of in vitro DC manufacturing protocol, types of tumor antigen to be loaded and finally different adjuvants for activating them. We will cover potential combinatorial strategies with immunomodulatory therapies: depleting T-regulatory (Treg) cells, blocking VEGF and blocking inhibitory signals. Furthermore, recommendations to incorporate these criteria into DC-based tumor immunotherapy will be suggested.

Expert opinion: Monocyte-derived DCs are the most widely used DC subset in the clinic, whereas Langerhans cells and plasmacytoid DCs are two emerging DC subsets that are highly effective in eliciting cytotoxic T lymphocyte responses. Depending on the type of tumor antigens selected for loading DCs, it is important to optimize a protocol that will generate highly potent DCs. The future aim of DC-based immunotherapy is to combine it with one or more immunomodulatory therapies, for example, Treg cell depletion, VEGF blockage and T-cell checkpoint blockage, to elicit the most optimal antitumor immunity to induce long-term remission or even cure cancer patients.     

A changing world for DCvax: a PSMA loaded autologous dendritic cell vaccine for prostate cancer

Background: Northwest Therapeutics’ DCvax-prostate consists of autologous dendritic cells (DCs) loaded with prostate-specific membrane antigen (PSMA) peptides, administered intravenously. Phase I–II testing, a decade ago, showed clinical benefit and immunological response in some patients. More recently DCvax brain, a product using a similar DC platform showed encouraging Phase I–II results and sipleucel-T, a prostatic acid phosphatase (PAP)-directed DC immunotherapy had positive Phase III results. Objective: Features of the clinical setting into which a new immunotherapy could be introduced are discussed, to refine a perspective on DCvax-prostate in the context of evolving prostate cancer therapeutics. PSMA-directed therapeutics and immune anticancer technologies are reviewed, and the clinical and immunological correlative testing of DCvax-prostate is discussed. Methods: Clinical and preclinical data from peer-reviewed literature, meetings proceedings and manufacturer-provided information are considered. Conclusion: DCvax-prostate had encouraging early-phase trial results, but development and testing had stalled. As a more detailed understanding of patient-selection for capacity for anticancer immune response, the quantitation of immunological correlates, and the changing marketplace develop, it is appealing to consider a well tolerated, PSMA-directed autologous dendritic cell therapeutic product. Further clinical trial development of DCvax-prostate is warranted, and required if it is to find a relevant clinical application.     

Dendritic cell immunotherapy for breast cancer

Novel adjuvant therapies are urgently needed to complement the existing treatment options for breast cancer. The advent of the use of dendritic cells (DCs) for cancer immunotherapy provides a unique opportunity to overcome the relative non-immunogenic property of breast tumours and address the underlying immunodeficiency. To date, the success of this approach has been limited, possibly due to the targeting of specific tumour antigens that rapidly mutate and, thus, become undetectable to the immune system. A more efficient approach would include preparations encompassing multiple antigens, such as those provided by loading of whole tumour cells or tumour RNA. It is proposed that targeting mammary stem cells responsible for resistance to chemo/immunotherapy, through the expression of a broad array of wild-type and mutated tumour antigens in the context of DCs, will become a mainstay for immunotherapy of breast cancer.