当归多糖对小鼠腹腔巨噬细胞释放细胞效应分子的诱导作用

目的 :观察当归多糖对小鼠腹腔巨噬细胞产生细胞毒效应分子的影响。方法 :从BALB/c小鼠的腹腔分离巨噬细胞 ,并进行原代细胞培养。采用MTT比色法及紫外分光光度法 ,检测当归多糖对腹腔巨噬细胞释放一氧化氮 (NO)、肿瘤坏死因子α(TNF α)、活性氧 (ROS)以及诱导型一氧化氮合酶 (iNOS)和溶菌酶 (LSZ)活性的影响。结果 :当归多糖可激活巨噬细胞释放NO、TNF α和ROS等效应分子 ,并显著提高LSZ的活性。当归多糖可能通过提高巨噬细胞iNOS的活性而增加NO的释放量 ,但其与脂多糖 (LPS)无协同促进NO释放的作用。当归多糖体外无直接杀伤肿瘤细胞的作用 ,但其与巨噬细胞共孵育的培养上清具有杀伤L92 9细胞的作用。结论 :当归多糖可促进巨噬细胞释放NO、TNF α及ROS等细胞效应分子 ,并可通过作用于巨噬细胞而促进TNF α的分泌 ,发挥间接的抗肿瘤免疫作用     

SNAP-23对NK细胞杀伤效应的调控

目的探讨SNAP-23对NK细胞杀伤效应的调控作用。方法通过核酸转染仪转染SNAP-23 shRNA（short hairpin RNA）质粒阻断NK92细胞中SNAP-23蛋白的表达，分别利用乳酸脱氢酶（LDH）释放实验和β-己糖氨酶释放实验检测SNAP-23表达下降对NK细胞杀伤效应和胞吐效应的影响。结果核转染仪可将shRNA质粒成功转入NK92细胞，所设计的3条SNAP-23的shRNA能抑制SNAP-23的蛋白表达，继而导致NK92细胞的杀伤活性和胞吐效应下降。结论SNAP-23通过调控NK细胞的胞吐效应而参与NK细胞杀伤效应的调控。     

细胞因子调节人肝癌细胞ICAM-1分子的表达及其对CD3AK杀伤功能的影响

应用基因重组人干扰素α（IFN-α）、干扰素γ（IFN-γ）和肿瘤坏死因子（TNF）体外处理人肝癌细胞系H-7402,ABC-CELISA法检测各处理组和对照组细胞表面细胞间粘附分子-1（ICAM-1）的表达水平,用 LDH释放法观察CD3单克隆抗体活化的杀伤细胞（CD3AK）对各处理条件下H-7402细胞的杀伤活性。结果表明:经TNF,IFN-α处理的肿瘤细胞ICAM-1表达水平较对照组显著增高,同时对CD3AK细胞的杀伤敏感性亦显著增强,IFN-γ虽能够提高靶细胞ICAM-1分子的表达,但CD3AK的杀伤活性并未因此增强。抗ICAM-1和抗白细胞功能相关抗原LFA-1单抗能够有效地降低CD3AK对靶细胞的杀伤作用,并且能够阻断IFN-α、TNF对CD3AK细胞杀伤效应的促进作用。提示ICAM-1/LFA-1参与了CD3AK粘附杀伤肿瘤细胞过程,TNF、IFN-α的促杀伤效应与肿瘤细胞表面ICAM-1分子的表达水平升高有关。     

H33342释放法检测NK细胞和单核细胞的杀伤活性

本文采用DNA特异性荧光染料Hoechst dye No 33342(H33342)标记肿瘤细胞技术,建立了自动微量荧光分析法,检测NK细胞和单核细胞介导的杀伤效应。结果表明,损伤的靶细胞其DNA断裂后释放到上清液中,采用微量荧光仪可定量测定上清液中的荧光强度。效应细胞的杀伤活性与靶细胞释放的荧光强度呈正相关性。此外,该法的检测敏感性与经典的~(51)Cr释放法基本相似,是检测细胞杀伤活性的一种新型方法。     

新城鸡瘟病毒瘤苗对小鼠H22肝癌的免疫防治效应及其机理的研究

目的:研究新城鸡瘟病毒(NDV-L)修饰的肿瘤细胞对小鼠肝癌是否具有免疫防治作用。方法:主要采用透射电镜,免疫荧光和噻唑蓝比色法。结果:生理盐水及单纯活NDV-L对照组小鼠全部长瘤死亡,平均存活时间MST分别为16.8d和22.6d;经NDV-L修饰的瘤苗及未经NDV-L修饰的瘤苗免疫组小鼠MST分别为32.4d和19.6d。免疫治疗实验组小鼠NDV-L修饰的H22瘤苗能诱导出较强的抗肿瘤免疫效应,其抑瘤率(57％),生存率(90％),MST(44.0d)和肺转移率(20％)均比其它各组疗效明显。结论:NDV-L修饰的肿瘤细胞对小鼠肝癌具有免疫防治效应。     

抗体依赖性细胞介导的细胞毒作用杀伤机制的扫描电镜研究

作者采用CBA鼠的脾细胞为效应细胞、抗体致敏的鸡红细胞为靶细胞,结合~51Cr释放试验在扫描电镜下观察了抗体依赖性细胞介导的细胞毒作用(ADCC)的杀伤机制。发现在ADCC过程早期,效应细胞借其表面绒毛状突起与靶细胞膜紧密结合。随后,效应细胞的绒毛状突起插入靶细胞,靶细胞扭曲、变形、损伤。尤其注意到,在与效应细胞绒毛状突起结合的部位,靶细胞膜呈现“卷须样”变化,提示靶细胞的损伤有机械性因素参与。     

γ射线辐照外周血单个核细胞联合ApoG2体外杀伤PC-3细胞的效应

目的：观察低剂量辐射外周血单个核细胞联合棉酚衍生物ApoG2对体外培养人前列腺癌PC-3细胞的杀伤作用。方法：采用1Gyγ射线照射人外周血单个核细胞,辐射剂量率为17Gy／min,实验设对照组、照射及未照射的外周血单个核细胞与PC．3细胞共培养组、ApoG2处理组及照射的外周血单个核细胞与ApoG2联合作用组。利用吖啶橙／溴化乙啶（AO／EB）荧光双染色法及MTT法,观察外周血单个核细胞和／或ApoG2对肿瘤细胞的杀伤情况。结果：辐照的外周血单个核细胞及ApoG2处理组对PC-3的杀伤作用明显增强,杀伤活性明显高于未辐照组（P〈0．05）,而联合作用组的杀伤活性明显高于辐照组及Ap0G2处理组（（P〈O．01）。结论：低剂量辐射外周血单个核细胞联合ApoG2可以明显提高其体外的抗肿瘤效应。     

9.1C3分子抑制杀伤性细胞的细胞毒作用及其机制

目的 探讨９．１Ｃ３分子对杀伤细胞功能的影响。方法 以新鲜分离的ＰＢＭＣ或混合淋巴细胞培养活化的淋巴细胞为效应细胞,采用重导向杀伤实验（Ｒｅｄｉｒｅｃｔｅｄ ｋｉｌｌｉｎｇ ａｓｓａｙ,ＲＫＡ）观察９．１Ｃ３抗体效应细胞杀伤Ｐ８１５细胞作用及其杀伤过程中ＴＮＦ－α分泌的影响。结果 ９．１Ｃ抗体能显著抑制杀伤细胞对靶细胞的细胞毒作用,并使效应细胞分泌ＴＮＦ－α水平降低。结论：９．１Ｃ３分子是一种抑制     

免疫性肝损伤中诱导型一氧化氮合酶的细胞来源

采用胶原酶-链霉蛋白酶灌流法对免疫性肝损伤大鼠肝实质细胞与枯否细胞进行分离与原代培养,Griess反应法检测细胞培养上清中一氧化氮（NO）生成量的变化。结果显示在刺激条件及细胞数量同等情况下,肝实质细胞NO生成量显著高于枯否细胞;肿瘤坏死因子（TNFα）单克隆抗体可拮抗枯否细胞由细菌脂多糖（LPS）刺激所致NO生成的增加,而对卡介苗（BCG）所致NO生成无显著影响。提示免疫性肝损伤中NO生成主要源于肝实质细胞;LPS通过使枯否细胞释放TNFα对NO生成进行调节。     

宫颈癌单克隆抗体AU14-1介导效应细胞对靶细胞的细胞毒

目的：研究单克隆抗体AU14-1介导的细胞毒作用对小鼠宫颈癌（U14）细胞的杀伤效果。 方法： 以小鼠淋巴因子激活的杀伤细胞（LAK）、脾淋巴细胞及巨噬细胞为效应细胞，应用MTT比色法测试在单克隆抗体AU14-1介导下对小鼠宫颈癌（U14）细胞的体外细胞毒作用。 结果： 3种效应细胞对于经单克隆抗体AU14-1处理的肿瘤细胞的杀伤率显著高于未经AU14-1处理的肿瘤细胞（P＜0.01）；以LAK的ADCC作用最强，巨噬细胞次之，脾淋巴细胞最弱（P＜0.01）。 结论： 单克隆抗体AU14-1能通过抗体依赖细胞介导细胞毒（ADCC）发挥淋巴样细胞、巨噬细胞及LAK细胞对靶肿瘤细胞的杀伤作用；提示特异性宫颈癌单克隆抗体AU14-1介导的ADCC在治疗宫颈癌中可能是一种有潜在价值的方法。     

Impairment of natural killer (NK) cytotoxic activity in hepatitis C virus (HCV) infection

In the present study, we evaluated the NK cell cytotoxic activity in a group of HCV-infected individuals. Although the number of NK cells present in the peripheral blood of the HCV-infected patients was comparable to non-infected individuals, spontaneous NK cytotoxicity was four-fold lower (P< 0.001) than in normal donors. This functional impairment was not overcome by depletion of adherent or B cells, and it was partially restored by short-term (18 h) stimulation with IL-2. However, long-term stimulation (72 h) with this lymphokine induced activated killer cell (LAK) activity comparable to normal controls. The reduction in NK cytotoxic response does not seem to be due to soluble suppressive factors, since incubation of normal peripheral blood mononuclear cells (PBMC) with infectious HCV serums for a 4-h period does not affect NK spontaneous cytotoxic activity. Successful in vitro infection of PBMC with HCV infectious serum also resulted in an impairment of NK cytotoxicity, suggesting that altered NK function is associated with HCV infection and may be responsible, at least in part, for the chronicity of the infection.     

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO.

Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO). We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO. Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production. Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules. Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release. Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells. These results indicate that M. arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.     

Cell-Mediated Immunity to Plasmodium falciparum Infection: Evidence against the Involvement of Cytotoxic Lymphocytes

Blood mononuclear cells (PBMC) recognizing soluble malaria antigens (SPag) are present in the peripheral blood of individuals clinically immune to malaria, and they proliferate after exposure to such antigens. To test whether these cells have effector activity against Plasmodium falciparum, we stimulated PBMC from malaria-immune donors by SPag and purified protein derivative (PPD) in culture for 7 days. The PBMC were then co-incubated with P. falciparum for 48 h, and parasitaemia was determined by microscopy. Parasite growth was only significantly impaired after incubation with PBMC stimulated by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized erythrocytes. These data support the hypothesis that activated monocytes are the most important effector cells in the peripheral blood of malaria immune individuals.     

Reversal of antibody-dependent cellular cytotoxicity suppression by cycloheximide.

Antibody-dependent cell-mediated cytotoxicity (ADCC) carried out by fresh and precultured peripheral blood mononuclear cells (PBMC) was compared. The results indicate that the cytotoxic capability was decreased in precultured PBMC, probably due to the effect of suppressor cells. The depletion of adherent cells before preincubation eliminated inhibitory effects, suggesting that the suppressor activity resided in this cell fraction. In addition, supernatants from adherent cell cultures were also able to suppress ADCC of non-adherent PBMC. Normal ADCC values were restored by supplementing the reaction medium with cycloheximide (Cy) 1 X 10(-3) M. The enhancing effect of Cy was exerted upon whole and adherent PBMC, while the opposite effect was observed when ADCC was carried out by non-adherent cells. The fact that Cy increased ADCC activity by precultured PBMC might rule out the possibility that suppression could be ascribed to trivial factors such as cell death, overcrowding or steric hindrance. The results presented in this report support the premise that ADCC is under active immunoregulation.     

iNOS-mediated generation of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils

Infection due to implanted cardiovascular biomaterials is a serious complication initiated by bacterial adhesion to the surface of the implant. The release of reactive oxygen species by neutrophils, particularly superoxide anion, is a well-known bactericidal mechanism. Additionally, nitric oxide (NO) has also been identified as an important cytotoxic mediator in acute and chronic inflammatory responses with enhanced NO production by upregulation of inducible nitric oxide synthase (iNOS). The interaction of NO and superoxide anion will result in the formation of peroxynitrite (OONO-), a potent cytotoxic oxidant. In this study, we have shown that biomaterial-induced neutrophil activation does not cause upregulation of iNOS and activation of iNOS-mediated pathways. However, NO and O2- production does occur over time upon adhesion to a biomaterial and is modulated by biomaterial surface chemistry. With no stimulus, the polyethylene oxide-modified polyurethane induced greater neutrophil activation than did the control as indicated by the increased production of NO and O2- over time. Adherent-stimulated neutrophils generally produced lower amounts of NO over time in comparison with unstimulated cells. Furthermore, there is no evidence of peroxynitrite activity in unstimulated neutrophils adherent to the Elasthane 80A. However, upon stimulation with adherent Staphylococcus epidermidis, peroxynitrite formation did occur. Our results suggest that bactericidal mechanisms in neutrophils involving NO generation (NOS pathway) are further compromised than O2- producing pathways (NADPH oxidase) upon exposure to biomaterials, resulting in a diminished microbial killing capacity, which can increase the probability of device-centered infections.     

In vitro antihydatic action of IFN-gamma is dependent on the nitric oxide pathway.

Hydatidosis is a widely endemic helminthic disease vectored in human by the larval stage of the metacestode Echinococcus granulosus. It is characterized by the long-term coexistence of chronic infection with detectable humoral and cellular responses against the macroparasite. Previous studies demonstrated interferon-gamma (IFN-gamma) and nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we tested the hypothesis that NO production after IFN-gamma induction may constitute a host defense against E. granulosus. We also investigated the IFN-gamma effect on protoscolices (larval form of the parasite) viability in coculture with hydatid patients' peripheral blood mononuclear cells (PBMC). PBMCs from hydatic patients incubated with IFN-gamma (100 U/mL) alone are effective in the killing of protoscolices. This scolicidal activity is concomitant with elevation of nitrite levels. NO release and cytotoxic activity are inhibited by N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of the NO pathway and increased by L-arginine, an NO precursor, and tetrahydrobiopterin (BH4), a nitric oxide synthase (NOS) cofactor. Our results indicate that IFN-gamma mediated iNOS induction as one of host defense mechanism against human E. granulosus infection.     

Circulating soluble factor-inhibiting natural killer (NK) activity of fresh peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.     

Independence of the pattern of early cytokine release from autoregulation by nitric oxide.

The L-arginine-dependent tumor cell cytotoxicity produced by activated macrophages (M phi) may be mediated either directly by production of nitric oxide (NO), or by induction of NO synthesis in the tumor cell. The influence of M phi NO synthesis on the release of soluble cytotoxic mediators was investigated in this study. The synthesis of M phi NO, measured as nitrite, was detected 6 h after lipopolysaccharide (LPS)-triggering and reached a peak level by 44 h. A concurrent decrease in M phi viability beginning at 18 h after triggering was detected during a period of 72 h in culture. Both the production of NO and loss of viability correlated with the presence of L-arginine in the incubation media and was inhibited by NG-monomethyl-L-arginine (NMA). The medium in which LPS-triggered adherent peritoneal exudate cells were incubated was examined for the presence of tumor necrosis factor (TNF), gamma interferon (IFN-gamma), and the soluble mediators that induce mitochondrial respiratory inhibition in tumor cells. All of these effector molecules were released at peak levels into the conditioned supernatants within 12 h after LPS-triggering. The peak level obtained for each effector molecule was influenced by the media in which the M phi was incubated; however, no correlation was detected between the level of cytokines produced and the synthesis of nitrite by the M phi indicating that NO synthesis has no inhibiting effect on the initial burst of cytotoxic factors released.     

Effect of N-nitrosodimethylamine on inducible nitric oxide synthase expression and production of nitric oxide by neutrophils and mononuclear cells: the role of JNK signalling pathway

In neutrophils (PMN) and mononuclear cells (PBMC), one of the enzymes responsible for nitric oxide (NO) synthesis is inducible nitric oxide synthase (iNOS). Changes in iNOS expression result from various signalling pathways including the mitogen-activated protein kinase (MAPK) pathway activated by endogenic and exogenic factors. N-nitrosodimethylamine (NDMA) is a xenobiotic with widespread occurrence in human environment and has an effect on cells of the immune system. The aim of this study was to determine the effect of NDMA on iNOS expression and NO production by human PMN and PBMC cells in the light of superoxide anion production by PMN cells. Moreover, the role of JNK and p38 pathways in NO production with involvement of iNOS was studied. Additionally, the function of JNK pathway in generation of superoxide anion was determined. Moreover, nitrotyrosine expression was studied in PMN and PBMC cells in the presence of NDMA. This work shows that NDMA increases iNOS expression and NO production by PMN and PBMC cells. In addition, elevated expression of phospho-JNK and phospho-p38, which are markers of JNK and p38 MAPK pathways activation, were observed. Lower iNOS expression and NO production by neutrophils exposed to extended action of NDMA were observed after application of inhibitor of JNK and p38 pathways. Lower phospho-p38 expression in PMN stimulated by NDMA was found as a result of arresting JNK pathway, whereas, application of inhibitor of p38 pathway resulted in enhanced phospho-JNK expression in PMN and PBMC cells. Increased ability to release superoxide anion by NDMA-stimulated PMN cells was observed. This ability was reduced after the application of inhibitor of JNK pathway. In PMN and PBMC cells exposed to NDMA, an increased expression of nitrotyrosine, which is dependant on JNK and p38 pathways that are activated by this particular xenobiotic, was observed. Generally, increased induction of iNOS related to elevated production of NO by PMN and PBMC cells exposed to NDMA may result in dysfunction of regulation of immunity responses controlled by this molecule in various conditions. Increased expression of nitrotyrosine in PMN and PBMC cells exposed to NDMA may affect their functions in an auto- and/or a paracrine way. Mutual interactions of JNK and p38 MAPK during the induction of iNOS expression in cells exposed to NDMA indicate complex mechanism of induction of iNOS synthase.     

Gene expression and production of tumour necrosis factor by a rat basophilic leukaemia cell line (RBL-2H3) with IgE receptor triggering

This study evaluated the gene expression of tumour necrosis factor (TNF) and the molecular weight of the cytotoxic factor in a subline of a rat basophilic leukaemia cell line, RBL-2H3. After IgE receptor triggering with a specific antigen that was associated with histamine release, cytotoxic activity in the cell lysates and supernatants increased for 2 hr during the culture of RBL-2H3 cells. Furthermore, calcium ionophore A23187 could induce release of histamine and cytotoxic activity from RBL-2H3 cells. However, compound 48/80, lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were unable to induce the release of either histamine or cytotoxic activity from the cells. These data suggested that, at least in part, there was a common pathway in histamine release and production of cytotoxic activity. A protein synthesis inhibitor, cycloheximide, did not affect histamine release, but inhibited the induction of cytotoxic activity. This cytotoxic activity from RBL-2H3 cells was completely neutralized by anti-mouse TNF rabbit serum. With Northern blot analysis, mouse TNF cDNA probe could hybridize with RNA isolated from RBL-2H3 cells. TNF mRNA was induced as early as 1 hr after stimulation with specific antigen and decreased by 4 hr. Moreover, the molecular weight (MW) of the released cytotoxic factor from RBL-2H3 cells triggered with IgE receptors was approximately 17,000 by SDS-PAGE, which was compatible to that of TNF. Thus, it is concluded that the gene expression and production of TNF occurred in RBL-2H3 cells after IgE receptor triggering in association with histamine release, suggesting that TNF produced by basophils and mast cells may play an important role in allergic reaction through its wide range of biological activity.