B7-1、B7-2在牙龈卟啉单胞菌脂多糖诱导的T淋巴细胞反应中的作用

目的：研究B7?1、B7?2分子对牙龈卟啉单胞菌（ Porphyromonas gingivalis,P． g）脂多糖诱导的T淋巴细胞反应的影响。方法：分别从基因敲除小鼠（ B7?1 KO组、B7?2 KO组、B7?1／2 KO组）和野生小鼠（ WT组）骨髓培养获得原代树突状细胞（ dendritic cell,DCs）并经P． g脂多糖刺激后,与T细胞混合培养。 CCK8试剂盒评价T细胞增殖,ELISA检测培养液中IFN?γ、IL?4及IL?17的水平,荧光定量PCR检测T细胞IFN?γ、IL?4、IL?17、GATA?3、T?bet及RORγt的转录水平。结果：CCK8检测显示各组T细胞增殖水平无差异。 ELISA及荧光定量PCR显示与WT组比较,B7?1 KO组IFN?γ降低,IL?4升高,IL?17无变化,T?bet降低,GATA?3升高,RORγt无变化;B7?2 KO组IFN?γ升高,IL?4降低,IL?17降低,T?bet升高,GATA?3降低,RORγt降低;B7?1／2 KO组各因子变化趋势介于B7?1 KO组和B7?2 KO组之间。结论：B7?1及B7?2分子对P． g脂多糖刺激的T细胞增殖无明显影响。但B7?1分子可促进T细胞向Th1型分化,抑制Th2型分化,与Th17型分化相关性不大;而B7?2分子抑制T细胞向Th1型分化,促进Th2型及Th17型分化。     

Biological and molecular characterization of PNH-like lymphocytes emerging after Campath-1H therapy

Campath-1H, an anti-CD52 monoclonal antibody, is therapeutically active in lymphoproliferative and autoimmune diseases. After Campath-1H therapy, lymphocytes with a paroxysmal nocturnal haemoglobinuria (PNH) phenotype have been reported to emerge. We characterized a PNH-like lymphocyte population emerging after Campath-1H therapy, in a patient with fludarabine refractory B-cell chronic lymphocytic leukaemia (B-CLL). We demonstrated a reduction in PIG-A mRNA levels compared with controls, and of all cytokines tested [interleukin (IL)-4, IL-13, IL-2, interferon(IFN)-gamma, IL-6, IL-10, and tumour necrosis factor (TNF)-alpha], except transforming growth factor (TGF)-beta. Given the inhibitory activity of TGF-beta, its elevated levels may contribute to the selective pressure of Campath-1H, leading to the emergence of PNH-like lymphocytes.     

Biochemical and immunologic abnormalities in peripheral blood T lymphocytes of patients with hemophilia A

Subset distribution, ecto-5'nucleotidase (5'NT) activity, and the generation of allospecific cytotoxic T lymphocytes (CTL) were investigated in peripheral blood T lymphocytes from 39 hemophilia A patients divided into three groups: group A and group B (HIV-patients with CD4:CD8 ratio less than 1 and greater than 1 respectively), and group C (HIV + patients). 5'NT activity was significantly decreased compared with healthy controls in groups B and C. In group B, this deficiency was attributable to expansion of CD8 + CD11 + suppressor cells. In group C, activated (HLA-DR+) CD8+ cells were also present and contributed to 5'NT deficiency. The suppressor cell expansion seemed to be mainly related to AHF infusions, whereas HLA-DR expression was related to HIV infection. CD11+ and HLA-DR+ cells were also expanded in the CD4+ subpopulation of all three groups, whereas CD4+ CD28+ lymphocytes were significantly decreased in group C only. Lastly, alloreactive cytotoxicity was decreased in group B and was normal in groups A and C.     

Interleukin-7 activates intestinal lymphocytes

Human intestinal lymphocytes, particularly intraepithelial lymphocytes, proliferate minimally to some agents, like mitogens and stimuli of the CD3 pathway. Thisin vitro finding may be due, in part, to a loss of factors foundin vivo. Three T-cell growth factors, IL-7, IL-9, and IL-12, were tested for their ability to stimulate the proliferation of intestinal lymphocytes. Both intraepithelial lymphocytes and lamina propria lymphocytes proliferated more vigorously to IL-7 than to IL-9 or IL-12, and only IL-7 increased stimulation through the CD3 pathway. The IL-7-induced response was IL-2-dependent: IL-2 receptors appeared on both intestinal lymphocyte types, and antibody to the IL-2 receptor blocked IL-7-induced proliferation. Both CD4⁺ and CD8⁺ T-cell subsets responded to this cytokine as shown by phenotype-depletion experiments and constancy in the CD4/CD8 ratios after culture with IL-7. In addition, the T-cell receptor and subsets responded equally well to IL-7. This newly described selective proliferative response of intestinal lymphocytes to IL-7, but not to IL-9 or IL-12, requires no preactivation and may enhance, growthin vivo.This work was supported by grants from the Crohn's & Colitis Foundation of America and the National Institutes of Health (DK42166).     

High levels of antiphospholipid antibodies are associated with cytomegalovirus infection in unrelated bone marrow and cord blood allogeneic stem cell transplantation

Antiphospholipid antibodies (APA) are a family of autoimmune and alloimmune immunoglobulins recognizing protein-phospholipid complexes in in vitro laboratory test systems. These antibodies have been associated with several conditions (malignancies, autoimmune diseases, infections, use of drugs); moreover, a syndrome capable of inducing thromboembolic disease has recently been associated with the presence of these antibodies. The aim of this prospective study was to investigate the levels of APA in subjects affected by haematological malignancies undergoing allogeneic haematopoietic stem cell transplantation (ASCT). Between March 1996 and December 1997, 32 patients undergoing ASCT were studied prospectively until day +180 from transplant. The mean values of IgG and IgM anticardiolipin antibodies (ACA) increased in recipients of stem cells from anunrelated donor, and a statistically significant difference inACA IgG mean value between unrelated and related transplanted patients was demonstrated between days +95 and +180. All of the subjects who received stem cells from an unrelated donor had APA levels higher than the mean normal value +3 SD vs. 35% of those receiving stem cells from a related donor (P < 0.01). The reason for such a difference may be a result of the different incidence in documented cytomegalovirus (CMV) infection in the two groups (83% vs. 23%; P < 0.01), as indicated by the significant correlation between APA positivity and CMV infection (P < 0.05). No relationship was found between APA, conditioning regimen and acute or chronic graft vs. host disease (GVHD). Moreover, we did not observe any thromboembolic disorder or veno occlusive disease (VOD).     

砷与5-氮胞苷对人淋巴细胞DNA损伤的联合作用

为探讨砷和5－氮胞苷对人淋巴细胞DNA损伤及修复的联合作用，应用单细胞凝胶电泳（SCGE）技术比较研究了5－氮胞苷与砷同时和前后作用于人类淋巴细胞产生的联合毒性，结果显示10μmol/L5-氮胞苷和10μmol/L砷单独处理人淋巴细胞2h引起明显的DNA泳动（彗星尾），但两试剂引起的DNA泳动（彗星尾）间无显差异，5－氮胞苷前处理与砷后处理2h引起的彗星尾与其单独处理组比较非常显，砷前处理与5－氮胞苷后处理引起的彗星尾与其单独处理组比较无显性差异，但较对照组差异显，10μmol/L5-氮胞苷和10μmol/L砷分别单独处理2h引起了人淋巴细胞显的DNA损伤（链断裂），5－氮胞苷与砷在对淋巴细胞DNA的损伤上表现为单纯相加作用。5－氮胞苷前处理显增加了细胞对砷的基因毒性的敏感性，或砷后处理显增加了5－氮胞苷引起的DNA损伤，5－氮胞苷后处理2h显抑制了细胞对砷所致DNA损伤的修复。     

The endocytic pathway for H-ferritin established in live MOLT-4 cells by laser scanning confocal microscopy

Summary. We have studied the properties of eosinophils from 26 patients with malignant melanoma and 16 patients with renal cell carcinoma (RCC) who were entered into a phase II clinical trial using various schedules of low-dose rhIL-2 immunotherapy. Eosinophilia was observed in 65% of melanoma patients and 100% of renal patients when receiving rhIL-2 therapy.
The eosinophil count increased up to 20-fold approximately 5 d after the appearance of lymphocyte activation markers. This would be consistent with eosinophil kinetics and the release of soluble mediators, for example IL-5, from lymphocytes. Eosinophils from eosinophilic patients became hypodense compared to their pre-treatment levels as demonstrated by sedimentation through a discontinuous metrizamide density gradient; they also showed an increased expression of CD4, CD25 and CD11b cell surface activation markers. Eosinophil count could not be correlated to either patient survival or response to therapy in melanoma patients: however, patients with renal cell carcinoma demonstrated a significant correlation ( P 0·05) between eosinophil count and survival but not with clinical response. Therefore the maximum eosinophil count achieved during rhIL-2 therapy is of prognostic significance in patients with renal cell carcinoma.     

Reduction of B cell turnover in chronic lymphocytic leukaemia

Whether chronic lymphocytic leukaemia (CLL) is a latent or a proliferating disease has been intensively debated. Whilst the dogma that CLL results from accumulation of dormant lymphocytes is supported by the unresponsiveness of leukaemic cells to antigens and polyclonal activators, recent in vivo kinetic measurements indicate that B lymphocytes do divide at significant rates in CLL. However, an important and still unanswered question is whether CLL cells proliferate faster or slower compared with their normal counterparts. This report addressed directly this point and compared B-cell kinetics in CLL subjects and healthy controls, using a pulse-chase approach based on incorporation of deuterium from 6,6-(2)H(2)-glucose into DNA. We confirmed that B cells proliferated at significant levels in CLL but found that the proliferation rates were reduced compared with healthy subjects (mean 0.47 vs. 1.31%/d respectively, P = 0.007), equivalent to an extended doubling time of circulating B cells (147 d vs. 53 d). In conclusion, CLL B cells proliferate at reduced levels compared with healthy controls. CLL is thus characterized by an aberrant B-cell kinetics with a decrease in cell turnover, an observation that may impact on elaboration of efficient therapeutic strategies.     

某省肿瘤医院不同年龄、性别的健康人外周血各类T细胞亚群和NK细胞的分布研究

目的调查不同年龄、性别的正常人外周血中各类T细胞亚群和NK细胞的分布。方法流式细胞仪检测193例健康者外周血的各类T细胞亚群和NK细胞在淋巴细胞、T细胞、或T细胞亚群中的百分比含量。结果 193例健康人按年龄分为青年（≤39岁）、中年（40-59岁）、老年（≥60岁）三个组。获得在不同年龄、性别组中T、T4、T8和NK细胞占外周血淋巴细胞中的平均值和标准差;Th、Tc、Ts、Treg、NKT、活化T细胞等T细胞亚群占外周血T细胞中的平均值和标准差;Treg细胞占T4细胞亚群中,Tc、Ts占T8细胞亚群中的平均值和标准差。结论本结果可作为健康人外周血细胞流式细胞术参数的正常参考值,供基础和临床研究参考。     

Silencing the expression of Cbl-b enhances the immune activation of T lymphocytes against RM-1 prostate cancer cells in vitro

BackgroundThe ubiquitin ligase Cbl-b potently modulates T lymphocyte immune responses and is critical in modulating tumor-induced immunosuppression. The influence of Cbl-b in modulating T lymphocyte activity against prostate cancer remains ill defined. We have determined the effects of silencing Cbl-b expression in T lymphocytes and their subsequent cytotoxic activity against prostate cancer cells.MethodsT lymphocytes were isolated from the spleens of C57BL/6 mice. Lipofectamine-directed transfection of T lymphocytes with specific small interfering RNA (siRNA) silenced Cbl-b expression, which was confirmed by Western immunoblotting. The siRNA species were chosen that promoted the greatest transfection efficiency and dampened Cbl-b expression in T lymphocytes. The expression of CD69, CD25, and CD71 by the transfected T lymphocytes was determined by flow cytometry. T lymphocyte proliferation was assessed by CCK-8 assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-β. The objective was to compare the cytotoxic activity of transfected T lymphocytes and nontransfected (i.e., negative control) T lymphocytes against the murine prostate cancer cell line target RM-1 in vitro.ResultsWe selected a specific siRNA that decreased T lymphocyte Cbl-b expression to 15%. The siRNA-transfected T lymphocytes showed higher proliferation; higher CD69, CD25, and CD71 expression (p < 0.001); and higher IL-2, IFN-γ, and TNF-β secretion (p < 0.05), compared to the nontransfected cells. Transfected T lymphocytes were also more potent at killing RM-1 prostate cancer cells, compared to the negative control in vitro.ConclusionSilencing Cbl-b significantly enhanced T lymphocyte function and T lymphocyte cytotoxicity activity against a model prostate cancer cell line in vitro. This study suggests a potentially novel immunotherapeutic strategy against prostate cancer.