OX40/OX40L与急性冠状动脉综合征

急性冠状动脉综合征为一类慢性炎症性疾病,T细胞的激活是急性冠状动脉综合征的形成和发展的重要因素.OX40/OX40L是机体炎症反应中一对重要的信号通路,它们相互作用能促进CD4 T细胞的活化、增殖、迁移,因此OX40/OX40L可能参与急性冠状动脉综合征的形成.     

OX40/OX40 ligand interactions in T-cell regulation and asthma

The OX40 receptor is preferentially expressed by T cells, and its cognate ligand OX40L is primarily expressed by antigen-presenting cells such as dendritic cells following activation by thymic stromal lymphopoietin (TSLP). TSLP is released by the bronchial epithelium, airway smooth muscle, and some inflammatory cells in response to numerous insults such as allergens, viruses, and physical damage. OX40L is a costimulatory molecule that plays a sentinel role in the adaptive immune response by promoting T helper (Th) 2 polarization of naive T cells within the lymph node. These polarized T cells produce Th2 cytokines such as IL-4, IL-5, and IL-13, which have been implicated particularly in allergic eosinophilic asthma. Animal models have positioned both TSLP and OX40/OX40L as critical in the development of airway inflammation and hyperreactivity. In human disease, there is good evidence that TSLP is upregulated in asthma, but there are limited data to demonstrate overexpression of OX40 or OX40L in disease. Targeting the OX40/OX40L axis or TSLP presents a novel therapeutic strategy that has the potential of modifying the disease process and, therefore, impacting on its natural history. Whether this approach can demonstrate efficacy in established disease rather than at disease onset is unknown. Biologic therapies directed toward OX40/OX40L are in early phases of development, and results from these studies are eagerly awaited.     

OX40 Signaling Renders Adult T-Cell Leukemia Cells Resistant to Fas-Induced Apoptosis

We reported previously that OX40, a member of the tumor necrosis factor receptor family, is expressed constitutively on fresh leukemia/lymphoma cells isolated from patients with adult T-cell leukemia (ATL). In this study, we tested whether OX40 signaling affects the Fas-mediated apoptosis of fresh ATL cells isolated from 7 patients (3 acute type, 3 chronic type, and 1 smoldering type). In all these patients, the coculture of ATL cells with MMCE/OX40 ligand gp34, a stable human gp34 transfectant of a mouse epithelial cell line, resulted in a decrease in the percentage of apoptotic cells after treatment with anti-Fas monoclonal antibody, compared to coculture with MMCE/mock controls. Similar findings were obtained in OX40(+)- human T-cell leukemia virus type I-transformed T-cell lines. To elucidate the molecular mechanism of this phenomenon, we used Kit225/OX40, a stable OX40 transfectant of an IL-2-dependent T-cell line, and its deletion mutant, Kit225/del-OX40, in which the intracytoplasmic domain of OX40 had been deleted. Coculture with MMCE/gp34 inhibited the apoptosis of Kit225/OX40, but Kit225/del-OX40 apoptosis was hardly affected. These results suggest that ATL cells may escape Fas-mediated destruction of the immune system through OX40 signaling.     

Mesenchymal Stem Cells Do Not Suppress Lymphoblastic Leukemic Cell Line Proliferation

Background: Several studies have demonstrated the immunosuppresive effects of mes-enchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this re-gard. Objective: To investigate if adipose derived MSCs could inhibit Jurkat lym-phoblastic leukemia T cell proliferation during coculture. Methods: Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial charac-terization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were la-beled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increas-ing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results: Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, ini-tial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Conclusion: Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of differ-ent sources are needed to fully characterize the immunological properties of MSCs be-fore planning clinical applications.     

OX40/OX40L分子在炎症性肠病免疫病理机制中的作用

OX40/OX40L是一对重要的协同刺激分子,它们能增强CD 4~ T细胞的扩增、成熟和产生效应功能,还可使T细胞免于凋亡,并促进记忆性T细胞产生。因此,OX40/OX40L在机体的免疫应答和多种疾病中起重要作用。目前的研究表明免疫异常是炎症性肠病发病的重要因素,此文就OX40/OX40L的生物学功能及其在IBD中的作用作一综述。     

OX40-OX40L相互作用对小鼠平滑肌细胞亲环素A表达的影响

目的　观察OX40-OX40配体（OX40L）相互作用对小鼠主动脉血管平滑肌细胞（VSMC）亲环素A（CyPA）表达的影响。方法　小鼠VSMC采用组织块贴壁法原代培养；应用实时荧光定量PCR和流式细胞术检测OX40L　mRNA和蛋白表达水平；细胞内、外CyPA水平分别采用实时荧光定量PCR及Western　blot方法检测。结果肿瘤坏死因子α＋脂多糖联合处理小鼠VSMC，细胞OX40L　mRNA和蛋白表达呈时间依赖性，mRNA表达在24　h（5.967±0.252比1.000±0.000，P<0.05）达到高峰，蛋白表达在36　h（61.900±2.551比20.967±0.451，P<0.05）增加最显著；可溶性OX40处理VSMC后，CyPA　mRNA表达呈时间依赖性并在90　min达到高峰（1.799±0.098比1.000±0.000，P<0.05），2　h后细胞培养上清中可检测到高水平的CyPA（1.119±0.059比0.281±0.038，P<0.05），而细胞内CyPA表达较对照组无明显差异；抗OX40L预处理细胞可显著抑制可溶性OX40诱导的CyPA　mRNA（1.105±0.091比1.799±0.098，P<0.05）及蛋白（0.635±0.040比1.119±0.059，P<0.05）表达。结论　OX40-OX40L相互作用可影响VSMC　CyPA的分泌。     

Interacting Cell Populations Affecting Granulopoietic Colony Formation by Normal and Leukemic Human Marrow Cells

Marrow from 28 nonleukemic individualswas separated by adherence to glass orplastic into nonadherent (NA) and adherentpopulations. The NA populations werefound to be more dependent for colonyformation in culture on added colonystimulating activity (CSA) than unseparated marrow suspensions, and thereforeproved useful for CSA assays. Quantitativereconstitution procedures were used toassay CSA-producing cells. Either increasing numbers of irradiated unseparatedmarrow, or adherent cells derived fromvarying numbers of marrow cells, wereused to restore colony-forming efficiencyto NA populations. Assay procedures forCSA-producing cells were applied to fourpatients with acute leukemia prior totreatment. In all four instances, a defect inCSA-producing cells was demonstrated.

Submitted on January 22, 1973 Revised on March 21, 1973 Accepted on April 26, 1973     

人脐带血LAK细胞克隆化及其对白血病细胞的杀伤效应

重组人白细胞介素Ⅱ(rhlL-2)刺激下,脐带血单个核细胞在半固体培养中可增殖形成淋巴细胞克隆.大约20％的克隆细胞具有明显的LAK活性,对NK敏感的K562、NK抵抗的H7402及HL60、KG-1、Jurket(?)等多种白血病细胞均有强烈的杀伤作用,对HL60白血病祖细胞增殖(CFU-L)也有显著的抑制作用.     

Growth of Leukemic Cells in Culture

Peripheral blood cells from three patients with leukemia in relapse increased in numbers in short-term suspension cultures. This increase wasdependent on the presence of eitherfeeder populations containing a highpercentage of blast cells or conditioned medium derived from suchpopulations. Aneuploid cells present indirect marrow preparations were alsoprevalent in the cultures after 10 days.The apparent specificity of the increase for leukemic populations mayprovide a new approach to the studyof leukemic blast cells.

Submitted on April 7, 1972 Revised on July 13, 1972 Accepted on July 31, 1972     

OX40L/OX40基因组变异与早发冠心病风险之间的关系

目的:研究OX40L/OX40基因组变异与早发冠心病风险之间的关系。方法:用序列特异性引物—聚合酶链反应技术对243例年龄55岁早发急性冠状动脉(冠脉)综合征(PACS)的患者为PACS组、209例早发稳定性心绞痛(PSAP)为PSAP组和138例对照组检测OX40L基因rs3850641 A/G变异位点和OX40基因rs2298212 G/A位点变异的基因型、等位基因及其单倍型;用冠脉造影测量病变血管支数和狭窄程度积分;用酶链免疫吸附法(ELISA)测试血浆可溶性OX40L(sOX40L)、血管细胞黏附分子1(VCAM-1);用散射比浊法测试血浆C反应蛋白(CRP)水平;分析OX40L/OX40基因变异与冠心病风险、冠脉病变程度和血浆3个细胞因子之间的关系。结果:OX40L GG基因型和G等位基因发生PACS风险比AA基因型和A等位基因显著增高(OR=1.99,1.83;P=0.021,P=0.0001);而OX40 A等位基因比G等位基因发生PACS风险显著降低(OR=0.49,P=0.002);单倍型GGGG比AAGG(野生型)的PACS风险显著增加(OR=2.11,P=0.033);在4种高危因素(高血压、高脂血症、糖尿病和吸烟)被校正后OX40L G等位基因和GG基因型及单倍型GGGG与PACS风险仍具有独立关联性(OR=1.97、1.78、2.01,P=0.026、0.001、0.038),OX40 A等位基因和AA基因型对PACS风险仍有独立的关联性(OR=0.31、0.45;P=0.043、0.0001);OX40L基因型GG+AG比AA型血管病变支数≥3支的百分率和狭窄程度积分均显著增加;OX40基因型AA+GA比GG型血管病变支数≥3支的百分率和狭窄程度积分显著减低;差异均有统计学意义(P0.05)。基因型变异与炎性细胞因子表达:OX40L基因型GG+AG比AA型血浆可溶性OX40L、C反应蛋白、血管细胞黏附分子1水平均显著增高;OX40基因型AA+GA比GG型三指标显著降低。差异均有统计学意义(P0.05)。结论:OX40L/OX40基因组变异可能与PACS风险、冠脉病变程度及炎性因子的表达的正反调节有关。     

Mutagenic DNA Polymerase in Human Leukemic Cells

Evidence is presented that DNA polymerases from human leukemic cells are mutagenic. Nucleic acid-free extracts of acute lymphoblastic leukemic cells polymerized about 10-times more dCTP using poly(dA-dT).poly(dA-dT) as a template than did extracts from normal lymphocytes. Mutagenic DNA polymerases could perhaps play an important role in tumor progression.     

Studies of Leukemic Cell Populations in Culture

A suspension culture technique for thestudy of peripheral blood cells from patients with leukemia is described. Cellsfrom patients in relapse were preservedby freezing with dimethylsulfoxide at-70°C to ensure the availability of thesame population of cells for repeated experiments of varying designs. Cells fromone AML patient proliferated only in thepresence of phytohemagglutinin (PHA) orleukocyte-conditioned medium (LCM).Cells taken at a later date, after commencement of chemotherapy, failed toproliferate in response to PHA, althoughthe responsiveness to LCM was retained.Proliferation of the cells obtained afterchemotherapy could also be stimulated byculture supernatants from the originalPHA-responsive cells. Eight other patientsstudied showed varying responses to LCMand PHA. It is proposed that cells fromleukemic patients are made up of two interacting populations, one which producesstimulatory factors and another which responds to these factors by proliferation.Delineation of these two populations andthe factors produced may provide a betterunderstanding of leukemic cell proliferation.

Submitted on October 1, 1973 Revised on January 28, 1974 Accepted on February 22, 1974     

Concurrent Pancreatic and Renal Leukemic Cell Infiltration

Pancreatic involvement in acute lymphoblastic leukemia (ALL) is uncommon more so in adults. It can present as obstructive jaundice, pancreatitis or can be asymptomatic. We report here the clinical and imaging features in a 28 years old man with B cell ALL with simultaneous involvement of pancreas and bilateral kidneys. Computed tomography of abdomen showed diffuse infiltration of pancreas by multiple tiny hypodense lesions and multiple hypodense lesions in both kidneys. Although leukemic involvement of pancreas is unusual and our patient was asymptomatic, one should consider the possibility of pancreatic infiltration in a leukemic patient presenting with pancreatic enlargement, cholestatic jaundice or pancreatitis.     

OX40 costimulation turns off Foxp3+ Tregs

OX40 is a recently identified T-cell costimulatory molecule that belongs to the TNF/TNFR superfamily. OX40 can be expressed by both activated T effector cells and Foxp3(+) Tregs. It is well known that OX40 delivers a potent costimulatory signal to T effector cells, but very little is known about the role of OX40 in regulating the suppressor properties of Foxp3(+) Tregs and the de novo generation of new inducible Foxp3(+) Tregs from T effector cells. In the present study, we found, by using a newly created foxp3gfp knockin model, that OX40 was dispensable for the genesis and suppressor functions of naturally arising CD4(+)Foxp3(+) Tregs, but stimulating OX40 on the Foxp3(+) Tregs abrogated their ability to suppress T effector cell proliferation, IFN-gamma production, and T effector cell-mediated allograft rejection. OX40 costimulation did not significantly affect proliferation and survival of the naturally arising Foxp3(+) Tregs, but profoundly inhibited Foxp3 gene expression. Importantly, OX40 costimulation to T effector cells prevented the induction of new inducible Foxp3(+) Tregs from T effector cells. Our study identified OX40 as a key negative regulator of Foxp3(+) Tregs and may have important clinical implications in models of transplantation and autoimmunity.     

The upregulated expression of OX40/OX40L and their promotion of T cells proliferation in the murine model of asthma

Objective

To investigate whether the expression of OX40/OX40 ligand (OX40L) was upregulated in a murine model of asthma and their significance in the pathogenesis of asthma.

Methods

After an ovalbumin-sensitized/challenged murine model of asthma was established, the expressions of OX40, OX40L in peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage fluid (BALF) cell pellets were measured. Then T cell proliferation was analyzed by cell counting kit-8 (CCK8), and the protein levels of OX40 and OX40L in the lungs were determined by immunohistochemistry. The concentrations of IL-4 and IFN-γ in BALF and T cell culture supernatant were evaluated by ELISA.

Results

The percentages of CD4⁺OX40⁺, CD19⁺OX40L⁺, F4/80⁺OX40L⁺ in PBMCs and BALF cell pellets were higher in asthma group than in control group (all P<0.01). The proliferation capacity of T cells in asthma group was higher than that in control group (P<0.05). In asthma group, stimulation of OX40 by anti-OX40 mAb obviously promoted T cell proliferation and secretion of IL-4 and IFN-γ. Immunohistochemistry assay showed that OX40 and OX40L protein levels were higher in asthma group than those in control group (all P<0.05).

Conclusions

The expressions of OX40 and OX40L were upregulated in the murine asthmatic model. The upregulation of OX40/OX40L signals could induce the proliferation and cytokines secretion of T cells in asthmatic mice, indicating that OX40/OX40L signal was involved in the pathogenesis of asthma.     

Tissue Factor Activity of Normal and Leukemic Cells

Generation of procoagulant activity wasstudied in leukocytes and lymphocytesfrom normal human peripheral blood, leukocytes from patients with chronic myeloidleukemia (CML), chronic lymphatic leukemia (CLL), rabbit peripheral leukocytes,and macrophages harvested from the peritoneal cavity of rabbits. Marked procoagulant activity developed in peritonealmacrophages and rabbit peripheral leukocytes when incubated with endotoxin.Human peripheral blood leukocytes andlymphocytes also generated procoagulantactivity. When endotoxin was omitted theprocoagulant activity generated was considerably (3-12 times) lower. Leukocytesfrom patients with CML were less activethan normal leukocytes. Leukocytes frompatients with CLL generated very littleprocoagulant activity even after prolonged incubation. An antibody to rabbittissue factor neutralized the tissue factoractivity generated by rabbit leukocytes.

Submitted on January 12, 1973 Revised on March 30, 1973 Accepted on May 4, 1973     

N-RASMutations in Radiation-Induced Murine Leukemic Cells

ABSTRACT:N-rasmutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon-(3 γ-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of theN-rasgene: (i) a 128 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of theN-rasgene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GA→GA), 62 (CA→CA), or 70 (CA→CA) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CA→CA) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency ofN-rasmutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at theN-rasregion of the genome. More importantly,N-rasmutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding thatN-rasmutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence ofN-rasmutations. Alternatively,N-rasmay be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.     

Different Mechanisms of Resistance to Lymphokine-activated Killer (LAK) Cells in Leukemic Cell Sublines Selected by Etoposide or by LAK Cells

Two models of in vitro induction of human IM-9 lymphoblastoid cell line resistance to LAK cell-mediated killing were compared. IM-9 cell line variants were selected in vitro by prolonged exposure to LAK cells or to etoposide. Sensitivity to killing by LAK cells or by etoposide, conjugation with LAK cells and surface marker patterns were compared. Both LAK-cell-selected cell subline (IM-9/SL-15) and etoposide-selected cell subline (IM-9/ER) have acquired resistance to LAK cell-mediated death. IM-9/ER cells, but not IM-9/SL-15 cells, were 3.2-fold less sensitive to etoposide as compared to parental IM-9 cells. IM-9/SL-15 cells revealed decreased conjugation with LAK cells and augmented CDlla/CD18 surface molecule expression as compared to IM-9 line. In contrast, IM-9/ER cells displayed a level of conjugation with LAK cells equal to IM-9 cells, but loss of CD11a/C18 expression. Our results suggest different mechanisms of acquired resistance to LAK cells are operative in etoposide- or LAK-selected sublines, involving deterioration of LFA-1 molecule expression and altered conjugation properties, respectively.