Relation between adenosine deaminase activities and cytokine-producing T cells in women with preeclampsia

OBJECTIVES: To investigate the relation between plasma adenosine deaminase (ADA) activity and cytokine-producing T cells in preeclampsia. DESIGN AND METHODS: Plasma ADA activity and the proportions of cytokine-producing T cells were measured in peripheral blood of 28 women with normal pregnancies and preeclampsia. The proportion of CD4-positive T cells producing interferon-gamma and interleukin-4 were analyzed by flow cytometry. RESULTS: Plasma ADA activity in preeclampsia was significantly higher than that in normal pregnancy (p < 0.05). In preeclampsia, the proportion of interferon-gamma producing cells significantly increased, and the proportion of interleukin-4 producing cells was significantly decreased compared with normal pregnancy. A significant correlation was found between ADA activity and the proportion of interferon-gamma producing cells in preeclampsia (r = 0.58, p < 0.05). CONCLUSIONS: Increased plasma ADA activity in preeclampsia may be in part associated with changes in the proportion of interferon-gamma secreting cells.     

Induction of apoptosis of cytokine-producing bladder cancer cells by adenovirus-mediated IkappaBalpha overexpression

We investigated whether the cell growth and apoptosis of multiple cytokine-producing bladder cancer cells can be regulated by nuclear factor kappaB (NF-kappaB). The bladder cancer cell line KU-19-19, obtained from a 76-year-old man who demonstrated marked leukocytosis, produces multiple cytokines and demonstrates autocrine growth by granulocyte colony-stimulating factor (G-CSF). Electrophoretic mobility shift assay (EMSA) revealed that NF-kappaB was activated in KU-19-19 but not in other bladder cancer cell lines (KU-1, KU-7, or T-24, respectively). The inhibition of NF-kappaB DNA-binding activity with adenovirus vectors expressing the stable form of the NF-kappaB inhibitor IkappaBalpha (multiplicity of infection [MOI] of 10) inhibited growth and induced apoptosis of KU-19-19, but not KU-1, KU-7, or T-24. The production of several cytokines was suppressed significantly in KU-19-19 by this gene delivery. Although dexamethasone (10 microM) could also suppress cytokine production, it did not induce dramatic cell death in KU-19-19 because it could not inhibit NF-kappaB activation stably and strongly. These results suggest that NF-kappaB activation maintains the cell viability as well as regulates cytokine production in cytokine-producing cancer cells and therefore these in vitro experiments support a rationale for preclinical in vivo studies to demonstrate growth inhibition in established tumors.     

Expansion of cytokine-producing CD4-CD8- T cells associated with abnormal Fas expression and hypereosinophilia

The mechanisms of sustained overproduction of eosinophils in the idiopathic hypereosinophilic syndrome and in some human immunodeficiency virus (HIV)-1-infected individuals are largely unknown. We hypothesized that T cells may release soluble products that regulate eosinophilia in these patients, as has been previously shown in bronchial asthma. We identified one patient with idiopathic hypereosinophilic syndrome and one HIV-1-infected individual with associated hypereosinophilia who demonstrated high numbers of CD4-CD8- T cells in peripheral blood. CD4-CD8- T cells from both patients, although highly activated, did not express functional Fas receptors. In one case, the lack of functional Fas receptors was associated with failure of Fas mRNA and protein expression, and in another, expression of a soluble form of the Fas molecule that may have antagonized normal signaling of Fas ligand. In contrast to the recently described lymphoproliferative/autoimmune syndrome, which is characterized by accumulation of CD4-CD8- T cells and mutations within the Fas gene, this study suggests somatic variations in Fas expression and function quite late in life. Both genetic and somatic abnormalities in regulation of the Fas gene are therefore associated with failures to undergo T cell apoptosis. Furthermore, the expanded population of CD4- CD8- T cells from both patients elaborated cytokines with antiapoptotic properties for eosinophils, indicating a major role of these T cells in the development of eosinophilia. Thus, this study demonstrates a sequential dysregulation of apoptosis in different cell types.     

Insulin binding to target cells

Summary Following Yalow and Berson's basic research on the binding of polvpeptide hormones to plasma proteins, an integrated picture of hormone-receptor interaction and biological activity has been proposed for insulin in experimental models and in man. The extracellular interaction of the insulin molecule with the cell membrane structure modifies the intracellular metabolism, and it has been suggested that this occurs through the activation of a second messenger or the trans. duction of an insulin fragment into the cell. The use of monoiodoinsulin has made it possible to perform a series of experiments on cells isolated from the blood (monocytes) or from the tissues (adipocytes) and on plasma membranes prepared by ultracentrifugation. The existence of specific receptor sites for insulin in all cases, both in animals and in man, has been confirmed by mathe matical analysis of the binding curves; their non-linear course as plotted by Scatchard's method, may depend on negative cooperation or on different classes of receptor. From an evaluation of recent studies on human obesity, particularly on adipocytes and circulating monocytes, a new approach to the problem of ‘insulin resistance’ in obesity has been proposed, and this has shown that a reduction in the number of receptors on the target cells may contribute to peripheral insulin insensitivity. This phenomenon, which seems to be characteristic of the static phase of obesity, is reversible during fasting or weight reduction and may be a compensatory mechanism for hyper-insulinaemia. These results are an example of the significance and applicability of the experimental method of detecting insulin binding to target cells, and suggest a wide application in different endocrinological fields.     

Oxidant-induced DNA damage of target cells.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.     

The effect of pulse treatment of target cells with guinea pig lymphotoxin and the nature of its binding to target cells.

In order to investigate the binding of guinea pig lymphotoxin (GLT) to target cells, the reduction of survival ratio (S) after the pulsed exposure of target cells to GLT at 4 degrees C and subsequent incubation at 37 degrees C for 24 hr was examined in detail. The dose response of GLT, and the relation between the S value and the length of pulse treatment demonstrated the saturation and equilibrium of the GLT-binding. It was also shown that the GLT once bound to the target cells dissociated very slowly. Several sugars possessing a terminal beta-galactosyl residue had an inhibitory activity against the effect of pulse treatment. This suggests that GLT binds to receptors on the target cell surface, probably glycoproteins having beta-galactosyl residues as terminal sugar residues. The trypsin treatment of GLT-pulsed target cells restored survival ratios and this restoration was dependent upon the time of trypsin treatment. This suggests that the stage of the association of GLT to target cells changes from trypsin-sensitive into trypsin-insensitive.     

Recycling and target binding capacity of human natural killer cells

By combining a newly established single-cell cytotoxicity assay in agarose (16) with estimations of the maximum natural killer (NK) potential (Vmax) by 51Cr release that percentage of target-binding cells (TBC), the fraction of active killers among TBC, the kinetics of single-cell cytotoxicity, and the recycling of effector cells was studied. Using nylon wool-passed peripheral lymphocytes, approximately 10% of the cells will bind to NK- susceptible target cell lines. Most of these have receptors for IgG. Some 50% will go on to kill T cell targets and some 20% to kill the standard target cell K-562. As the individual NK cell is shown to have the capacity to recycle, i.e., to kill more than one target cell in the 3-h test period, and as recycling seems to vary between individuals, there is no consistent correlation between the number of TBC and 51Cr-release values. It seems as if the single-cell cytotoxicity assay, as presently performed in agarose, is a valuable complement to Vmax determinations by 51Cr-release to study the different steps involved in the cytolytic process: recognition, enzyme activation, and effector cell recycling. The discrimination between these steps will probably be necessary to define mechanisms influencing NK cells in different disease states as well as in learning more about the normal function and regulation of the human NK system.     

Immunotherapy and enhanced antibody-dependent cell-mediated cytotoxicity using virally-infected target cells

We examined the ability of in vitro addition of Interleukin-2 (IL-2) to differentially enhance antibody-dependent cell mediated cytotoxicity (ADCC) utilizing cultured Epstein-Barr virus infected cells and gammaglobulin (Sandoglobulin). We found significant enhancement of ADCC when IL-2 was added. Chronic Epstein-Barr virus or Chronic Fatigue Syndrome patients in a therapeutic gammaglobulin program may benefit from IL-2 given in vivo.     

从单个细胞扩增靶基因片段的技术

目的 建立从人的单个细胞扩增特定靶基因片段进行基因诊断的技术。方法 利用显微操作技术挑取单个纤维母细胞，各置于0.2ml簿壁反应管的裂解液中，合成针对抑癌基因P53第5－9外显子（e）的引物，运用15mer高度随机引物或一个特异引物进行单个细胞的整修基因组DNA或特异靶片段的预扩增，再以其为模板进行巢式聚合酶链反应（PCR）扩增P53基因第e5-9的1900bp片段及含第5，6外显子的580bp片段，并用ABI－377测序仪测定其序列。结果 运用稀释至0.01ng的基因组DNA扩增1900bp片段，优化PCR的条件，分别从300个单个纤维母细胞扩增上述1900bp片段，10个获得1900bp片段，成功率为33％。拉坟580bp片段，则阳性率可达605。序列分析证明确为P53基因第5，6外显子序列。结论 运用本实验室建立的技术可从人的单个细胞扩增特定靶基因片段，并测定其序列，作出基因诊断。     

Mechanisms of hypochlorite injury of target cells.

HOCl, which is produced by the action of myeloperoxidase during the respiratory burst of stimulated neutrophils, was used as a cytotoxic reagent in P388D1 cells. Low concentrations of HOCl (10-20 microM) caused oxidation of plasma membrane sulfhydryls determined as decreased binding of iodoacetylated phycoerythrin. These same low concentrations of HOCl caused disturbance of various plasma membrane functions: they inactivated glucose and aminoisobutyric acid uptake, caused loss of cellular K+, and an increase in cell volume. It is likely that these changes were the consequence of plasma membrane SH-oxidation, since similar effects were observed with para-chloromercuriphenylsulfonate (pCMBS), a sulfhydryl reagent acting at the cell surface. Given in combination pCMBS and HOCl showed an additive effect. Higher doses of HOCl (greater than 50 microM) led to general oxidation of -SH, methionine and tryptophan residues, and formation of protein carbonyls. HOCl-induced loss of ATP and undegraded NAD was closely followed by cell lysis. In contrast, NAD degradation and ATP depletion caused by H2O2 preceded cell death by several hours. Formation of DNA strand breaks, a major factor of H2O2-induced injury, was not observed with HOCl. Thus targets of HOCl were distinct from those of H2O2 with the exception of glyceraldehyde-3-phosphate dehydrogenase, which was inactivated by both oxidants.     

LAK细胞对不同靶细胞的杀伤作用及其意义

我们研究了重组白细胞介素2(rIL2)活化的杀伤细胞(LAK)对K562细胞、Raji细胞、IIepG2细胞和2.2.15细胞的杀伤活性.结果 表明:LAK细胞对不同靶细胞,其杀伤活性不同.在诱导早期(3小时),对K562细胞有明显的杀伤活性;在诱导晚期(3天和7天),对Raji、HepG2和2.2.15细胞则有杀伤活性.揭示rIL2诱生的LAK细胞可能由不同的免疫杀伤细胞组成,在不同的诱导时期,活化不同的杀伤细胞.我们还建立HepG2 2.2.15细胞的靶细胞系统,可以在体外测定LAK细胞对HBV感染细胞的杀伤活性,亦可在体外筛选增强免疫杀伤细胞杀伤HBV感染细胞活性的药物.     

人脐带间充质干细胞的作用：细胞移植、免疫调节及充当基因治疗靶细胞

背景：脐带作为分娩废弃物,来源广泛,取材方便,不受任何伦理及法理的限制,因此人脐带来源间充质干细胞的发现突破了其他间充质干细胞来源的所有限制。
　　目的：综述人脐带来源间充质干细胞在软骨疾病、神经胶质瘤、缺血脑损伤、肺疾病、肝疾病和心肌梗死疾病模型中的应用情况。
　　方法：由第一作者在2014年3月检索了PUBMED数据库和万方数据库,检索词为Human umbilical cord mesenchymal stem cel s, disease models, cel therapy,以及人脐带间充质干细胞,动物模型,细胞治疗。共查到相关文献73篇,根据纳入标准选择35篇文献纳入研究。
　　结果与结论：人脐带来源间充质干细胞有相似于骨髓来源间充质干细胞的多系分化能力。对比骨髓间充质干细胞,人脐带来源间充质干细胞显示出更低的免疫原性。人脐带来源间充质干细胞对软骨疾病、神经胶质瘤、缺血脑损伤、肺疾病、肝疾病和心肌梗死动物模型都有一定的治疗效果,证明人脐带来源间充质干细胞可进行细胞移植治疗多种疾病。     

Virus and trinitrophenol hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells

Immune spleen cells from LCM virus-infected (CBA X C57BL/6)F1 radiation chimeras entirely repopulated with CBA-T6 lymphocytes were cytotoxic for allogeneic, LCM virus infected C57BL/6 mouse-derived target cells. Normal C57BL/6 targets were not lysed. CBA-T6 lymphocytes derived from (CBA X C57BL/6) radiation chimeras sensitized in vitro against TNP- conjugated C57BL/6 spleen cells lysed TNP-conjugated C57BL/6 targets. However normal C57BL/6 mouse-derived targets were not destroyed. The magnitude of virus-specific (or TNP-specific) cytotoxic responses against H-2 incompatible targets was lower compared to that against H-2 compatible targets. These data are considered to support and to extend the altered self concept, but are not consistent with the dual recognition concept.     

Interferon-induced resistance of tumor target cells against lysis by interleukin-2-activated killer cells

IFN-gamma has been shown to decrease the susceptibility of target cells to NK cell-mediated cytotoxicity. In this report, the effect of IFN-gamma on the sensitivity of target cells to killing by various human lymphocyte cytotoxic activities such as NK/K, IL-2-augmented NK/K cell activity, and IL-2-activated killer activity were studied. Although NK-sensitive K562 cells showed marked resistance to NK cell activity as previously reported, the resistance was overwhelmed by augmentation of NK activity with IL-2. IL-2-activated killer cell activity, which can lyse NK-resistant tumor cell lines upon culture in IL-2, showed decreased cytotoxicity against most of the IFN-gamma-treated target cells tested. By contrast, no decrease of target cell sensitivity to K cells was observed, even though K cells were treated with IL-2. These findings suggest that as far as NK-resistant tumor cells are concerned, an IFN-dependent mechanism inhibits the Fc receptor-independent mediators of tumor surveillance, but not Fc receptor-dependent ones. This should be considered when planning adoptive immunotherapy of IL-2-activated killer cells for human malignancy.     

Quantitation of influenza virus antigens on infected target cells and their recognition by cross-reactive cytotoxic T cells

Monoclonal antibody to type-A influenza virus matrix (M)-protein was used to quantitate the appearance of M-protein on abortively infected P815 cells. After 16 h of infection with different type-A viruses, only a low amount of M-protein appears on the surface of infected cells (approximately 10(3) site/cell) in contrast to approximately 10(5) hemagglutinin molecules on each cell surface. However, virus replication is required for M-protein appearance. Analysis of solubilized membranes purified from 16-h-infected cells shows approximately 10(4) M-protein molecule/cell in the plasma membrane, a content that is consistent with the observed low surface expression, and that indicates that most of the M-protein is localized internally. We found no evidence that cross-reactive cytotoxic T cells could recognize M-protein; neither monoclonal antibody or hyperimmune anti-M- protein antiserum could inhibit T cell killing, either alone or in combination with monoclonal anti-H-2 antibody. Taken together, the low level of M-protein appearance and lack of T cell blocking by anti-M- protein antibody leaves doubt that M-protein is the antigen recognized by cross-reactive cytotoxic T cells.     

Cytotoxic activity against varicella-zoster virus-infected target cells after marrow transplantation

Cytotoxic activity by peripheral blood lymphocytes against varicella-zoster virus (VZV) infected matched and mis-matched fibroblasts and K562 targets was studied in 17 allogeneic marrow transplant recipients during the first 100 days after transplantation. Lysis of HLA mis-matched VZV infected target cells by patient's lymphocytes was significantly reduced after transplant compared to healthy normals (p less than 0.05). In contrast, lysis of K562 targets by peripheral blood lymphocytes from patients was similar or increased compared to controls. Three patients developed herpes zoster during the study. No significant difference was found in the lysis of VZV infected mis-matched target cells between patients who developed herpes zoster compared to those patients who did not. With the exception of in one patient shortly after the occurrence of herpes zoster, no HLA-restricted lysis could be detected. Depressed natural killer cell activity against VZV infected targets might be important for the development and outcome of VZV infection but studies in more patients and with longer follow-up time are needed.     

Unlicensed NK cells target neuroblastoma following anti-GD2 antibody treatment

Survival outcomes for patients with high-risk neuroblastoma (NB) have significantly improved with anti-disialoganglioside GD2 mAb therapy, which promotes NK cell activation through antibody-dependent cell-mediated cytotoxicity. NK cell activation requires an interaction between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands. NK cells lacking KIRs that are specific for self HLA are therefore "unlicensed" and hyporesponsive. mAb-treated NB patients lacking HLA class I ligands for their inhibitory KIRs have significantly higher survival rates, suggesting that NK cells expressing KIRs for non-self HLA are mediating tumor control in these individuals. We found that, in the presence of mAb, both licensed and unlicensed NK cells are highly activated in vitro. However, HLA class I expression on NB cell lines selectively inhibited licensed NK cell activity, permitting primarily unlicensed NK cells to mediate antibody-dependent cell-mediated cytotoxicity. These results indicate that unlicensed NK cells play a key antitumor role in patients undergoing mAb therapy via antibody-dependent cell-mediated cytotoxicity, thus explaining the potent "missing KIR ligand" benefit in patients with NB.     

Activation of secretion and surface alteration of cytolytic T-lymphocytes interacting with target cells

Cells obtained in mixed lymphocyte culture (MLC) and memory cells adsorbed on the surface of target cells (TC) were examined using scanning and transmission electron microscopy depending on the time of interaction with TC. Three types of lymphocytes were revealed: type I - cells of spherical shape with a smooth surface or an insignificant amount of microvilli; predominantly small and medium-sized lymphocytes contacting TC with non significant involvement of their surface or by several microvilli; type II - oval or round-shaped lymphocytes evenly covered with microvilli with considerably enlarged region of contact; type III cells - predominantly large lymphocytes and lymphoblasts flattened (spread) on TC, with multiple microvilli, ridge-like projections, and ruffles on their surface. TEM revealed activation of the secretory apparatus in the cytoplasm of such lymphocytes. With increased time of interaction, type III cells increase in number (from 8.6% after 10 min to 90.2% after 60 min of incubation). Memory cells show no morphologic signs of secretion in correlation with the absence of lysis of TC on which they are adsorbed. The surface of the lymphocytes adsorbed on the substrate with poly-L-lysin is not noticeably altered. It is suggested that 3 morphological types of lymphocytes correspond to 3 stages of secretion activation. Lymphocyte contact with TC surface is evidently a specific stimulus for activating secretory apparatus of CTL. SEM can be used for quantitation of activated lymphocytes.