Characterization of a tumor necrosis factor-alpha inhibitor activity in cancer patients.

To evaluate the feasibility of tumor necrosis factor-alpha (TNF-alpha) treatment of lung cancer patients, we chose the malignant cells contained in their pleural effusions as a first convenient target. We found, however, that a TNF-alpha inhibitor (TNF-alpha I) activity was present in both patient sera and pleural fluids. We therefore compared the TNF-alpha I activity present in patients with benign or malignant pleural effusions using a bioassay of TNF-alpha inhibition and partially characterized it. A high TNF-alpha I activity characterizes cancer patients with sera levels twice as high as the control level measured for blood bank donors (2.54 +/- 1.28 versus 1.19 +/- 0.38) and with even higher levels in pleural fluids (3.75 +/- 1.83). In contrast, patients with benign pleural effusions present similar levels of TNF-alpha I activity, at about the control level, in both their sera and pleural fluids (1.37 +/- 0.98 versus 1.16 +/- 0.85). A high TNF-alpha I activity is consistently found in cancer patients but is only released in vitro by leukocytes. It is most likely related to recently purified TNF-alpha inhibitors that, as soluble shed fragments of TNF receptors, may function as traps for TNF molecules. This study suggests that tumors may evade TNF cytotoxic action by modulating systemic levels of TNF and implies a reassessment of TNF therapy in cancer patients.     

Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion

We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-kappaB activity in vitro and in vivo, which we used to drive expression of a NF-kappaB-dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-kappaB-dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-kappaB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-kappaB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-alpha levels. These studies indicate that tumor NF-kappaB activity regulates pleural tumor progression. This reproducible model of MPE can be used to further study the influence of specific host and tumor factors on the pathogenesis of MPE and evaluate new therapeutic strategies.     

Clinical evaluation and tissue distribution of CA125 in patients with pleural effusion

CA125 in serum and pleural effusion was measured in 51 patients with malignant effusion and 38 patients with benign effusion, and the tissue distribution of CA125 was investigated by immunohistochemical technique. The 51 malignant effusions were secondary to primary lung cancer. The 38 benign effusions were taken from 23 patients with tuberculous pleurisy, 9 patients with empyema, 5 patients with congestive heart failure and one patient with nephrosis. In the mean level and the positive rate of serum CA125, no significant difference was shown between primary lung cancer and tuberculosis or the other benign diseases. The mean level of CA125 in pleural effusion of primary lung cancer was significantly higher than that in pleural effusion of tuberculosis (p less than 0.01), and showed a tendency to increase compared to that in pleural effusion of the other benign diseases (p less than 0.1). The mean level of CA125 in pleural effusion of tuberculosis was significantly lower than that in the other benign diseases (p less than 0.02). The positive rate of CA125 in malignant effusion was 43.1% and the diagnostic specificity of it was 86.7%. CA125 was detected in carcinoma cells and activated mesothelial cells in pleural effusion and mesothelial cells of normal pleural tissue by immunohistochemical staining. These results suggest that the measurement of CA125 in pleural effusion is useful for differential diagnosis of the malignant effusion from the benign effusion and that CA125 in pleural effusion of pleuritis carcinomatosa is produced by not only carcinoma cells but also activated mesothelial cells.     

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.     

胸腹水细胞端粒酶活性定性和定量检测

目的探讨端粒酶活性定量检测在诊断良恶性胸腹水中的应用价值。方法采用TRAP-银染定性方法和rrRAP-PicoGreen定量方法,对102例已确诊患者的胸腹水细胞进行端粒酶活性分析。结果恶性胸腹水细胞端粒酶活性明显高于良性胸腹水细胞,其定性检测诊断率明显高于细胞病理学。乳腺癌患者胸腹水细胞的端粒酶活性明显高于卵巢癌、肝癌患者胸腹水细胞的端粒酶活性;肺癌患者胸腹水细胞端粒酶活性明显高于肝癌。在良性胸腹水中,感染性胸腹水细胞端粒酶活性高于非感染性胸腹水。结论恶性胸腹水细胞端粒酶活性明显升高。端粒酶活性定量检测较定性检测更敏感、简便,对良恶性胸腹水的诊断和鉴别诊断有一定应用价值。     

TNF revisited: TNF-independent antitumor activity in sera of mice sensitized with Propionibacterium acnes and challenged with lipopolysaccharide

Sera of mice sensitized with bacteria and subsequently challenged with lipopolysaccharide promote hemorrhagic necrosis of tumors in vivo and display cytotoxic activity against tumor cells in vitro, which has been attributed to the induction of tumor necrosis factor (TNF). Here, we describe the induction of a previously unrecognized antitumor activity in such sera, which is distinct from TNF but displays tumor-specific cytocidal activity in vitro as well as potent tumor-regressing activity in vivo. Biochemical analysis of this activity yielded a molecular mass of approximately 150 kDa, closely resembling a novel tumoricidal factor of murine macrophages (Mphi) termed MTC 170 (Mphi tumor cytotoxin, approximate molecular mass 170 kDa), which we have previously proposed to constitute a major effector pathway for the destruction of tumor cells by activated Mphi.     

Inhibition of tumor necrosis factor-mediated lysis by spleen cell-conditioned medium

A spleen cell-elaborated factor which inhibits both TNF and natural cytotoxic cell lytic activity has been described. Production of this factor is dependent on prostaglandins, and inhibitory activity is abrogated by protein synthesis inhibitors. Furthermore, it was demonstrated that the factor renders TNF sensitive cells refractory to TNF rather than acting on TNF directly. Here we show that the factor is produced by spleen cells from both BALB/c and C57bl/6 mice and inhibits TNF mediated lysis of various TNF sensitive cells. The inhibitory factor has a molecular weight greater than 30 kDal and is heat labile, suggesting it is a protein. Furthermore, the factor is produced by high density spleen cells (e.g. cells of the myeloid lineage). Although it is not clear which cell type(s) is responsible for the production of this factor, these findings suggest that high density spleen cells, which do not produce TNF, can modulate TNF lytic activity via this factor.     

Elevated IL-5 levels in pleural fluid of patients with paragonimiasis westermani

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) in pleural effusions. Samples were obtained from 11 patients with Paragonimus westermani infection. In addition, samples from 12 patients with pleural transudates, 16 with tuberculous pleurisy, seven with empyema and 20 with lung cancer were also examined. Eosinophilia was remarkable in peripheral blood (range 4-34%, median 23.4%) and pleural fluid (range 0-95%, median 71%) of paragonimiasis patients. IL-5 concentrations in pleural effusions of paragonimiasis were markedly higher than those in other groups. Although marked elevation of GM-CSF and IFN-gamma levels was observed in pleural effusion of empyema and tuberculosis patients, it was marginal in the pleural effusion of paragonimiasis patients. In paragonimiasis patients, IL-5 levels in the pleural effusion correlated well with the percentage of eosinophils in peripheral blood and pleural fluid. Such a correlation was not observed between GM-CSF levels in pleural effusion and percentages of eosinophils in pleural fluid or peripheral blood. Our findings suggest that in paragonimiasis IL-5 in the local inflammatory site is particularly important in mediating eosinophilia in peripheral blood and pleural effusion.     

p16基因启动子甲基化联合检测在肺癌早期诊断中的临床意义

目的： 检测可疑肺癌患者外周血血浆、痰液、支气管肺泡灌洗液(BALF)、胸水以及活检组织中p16基因启动子的甲基化状态, 探讨p16基因启动子的甲基化检测在肺癌早期诊断中的意义。方法: 利用甲基化特异性PCR（MSP）检测可疑肺癌患者外周血血浆、痰液、BALF、胸水以及活检组织中p16基因启动子的甲基化状态。结果: 经病理证实,67例可疑肺癌患者中42例确认为肺癌。p16基因启动子甲基化阳性率在肺癌患者的血浆、痰液、BALF、胸水和活检组织中依次为52.4%（22/42）、47.6%（20/42）、59.5%（25/42）、71.4%（10/14）和61.9%（26/42）;25例良性病变患者中除1例在血液、1例在胸水中检测到异常甲基化外,其余标本中均未检测到p16基因启动子的异常甲基化(P<0. 05)。p16基因甲基化阳性检出率与肺癌的组织类型、临床分期、病理分级以及有无淋巴结和远处转移无关(P>0.05)。结论: MSP检测肺癌患者外周血血浆、痰液、BALF、胸水以及活检组织中p16基因启动子的甲基化状态, 是一项很有潜力的肺癌早期诊断技术。     

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non‐small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine‐induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real‐time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL‐expressing cells was evaluated by flow cytometry, and caspase‐8 and caspase‐3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine‐activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti‐Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane‐bound FasL (mFasL) and of mFasL‐positive apoptotic H292 cells, as well as caspase‐8 and caspase‐3 cleavage. Moreover, gemcitabine increased CH11‐induced caspase‐8 and caspase‐3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine‐treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up‐regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas‐dependent apoptosis mediated by caspase‐8 and caspase‐3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine‐induced lung cancer cell killing.     

Effect of neutralizing transforming growth factor beta1 on the immune response against Mycobacterium tuberculosis in guinea pigs

Transforming growth factor beta (TGF-beta) is a cytokine which has been shown to suppress the antimycobacterial immune responses of humans and experimental animals. In this study, the contributions of TGF-beta to cytokine production in vivo were investigated by using the established guinea pig model of tuberculous pleurisy. Mycobacterium bovis BCG-vaccinated guinea pigs were injected intrapleurally with heat-killed virulent Mycobacterium tuberculosis. Eight days following induction of an antigen-specific pleural effusion, guinea pigs were injected intrapleurally with anti-TGF-beta1 or isotype control antibody. The following day, pleural exudates were removed, and the fluid volume and characteristics of the infiltrating cells were determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis factor (TNF) protein levels by using appropriate bioassays. RNA from pleural effusion cells was examined to determine TGF-beta1, TNF-alpha, IFN-gamma, and interleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleural effusion lymphocytes were examined in response to concanavalin A and purified protein derivative (PPD) in vitro. Treatment with anti-TGF-beta1 resulted in decreased pleural fluid volume and decreased cell numbers in the pleural space along with an increased percentage of lymphocytes and a decreased percentage of neutrophils. The bioactive TNF protein levels in pleural fluid were increased in guinea pigs treated with anti-TGF-beta1, while the bioactive IFN protein concentrations were not altered. Expression of TGF-beta1 and TNF-alpha mRNA was significantly increased following TGF-beta1 neutralization. Finally, PPD-induced proliferative responses of pleural cells from anti-TGF-beta1-treated animals were significantly enhanced. Thus, TGF-beta1 may be involved in the resolution of this local, mycobacterial antigen-specific inflammatory response.     

胸水和血清中CEA、CYFRA21-1、TPS联合检测对良恶性胸水及肺癌的诊断价值

目的:探讨胸水和血清肿瘤标志物癌胚抗原(CEA),细胞角蛋白片段21-1(CYFRA21-1) ,组织多肽特异性抗原(TPS)在肺癌诊断中的临床意义.方法:应用电化学发光法和ELISA分别测定78例肺癌患者,45例肺部良性疾病胸水和血清CEA、CYFRA21-1、TPS水平,结果:肺癌组胸水CEA、CYFRA21-1、TPS水平明显高于良性疾病胸水组(P<0.01);肺癌组血清CEA、TPS水平明显高于良性疾病血清组(P<0.05,P<0.01);在不同病理类型肺癌中3种肺癌肿瘤标志物升高的程度均有所不同;恶性胸水组中肿瘤标志物的含量与同期血清中的含量相比,出现更早且浓度更高,尤以TPS升高最为明显.单项检测中,胸水TPS的敏感性最高,联合检测中,胸水TPS CYFRA21-1 CEA的敏感性和准确性最高.结论:CEA、CYFRA21-1、TPS三项联合检测对良恶性胸水的鉴别有较好的诊断价值. 胸水中3种肺癌标志物的联合检测较血清有更高的敏感性,准确性.其临床价值优于血清.     

Amebic lung abscess with coexisting lung adenocarcinoma: a unusual case of amebiasis

Amebic lung abscess with concurrent lung cancer, but without either a liver abscess or amebic colitis, is extremely uncommon. Here, we report a 70-year-old man presenting with pulmonary amebiasis and coexisting lung adenocarcinoma. During his first-time hospitalization, the diagnosis of lung amebiasis was confirmed by morphological observation and PCR in formalin-fixed and paraffin-embedded sediments of pleural effusion. Almost four months later, the patient was readmitted to hospital for similar complaints. On readmission, lung adenocarcinoma was diagnosed by liquid-based sputum cytology and thought to be delayed because coexisting amebic lung abscess. This case demonstrated that sediments of pleural effusion may be used for further pathological examination after routine cytology has shown negative results. At the same time, we concluded that lung cancer may easily go undetected in the patients with pulmonary amebiasis and repetitive evaluation by cytology and imaging follow-up are useful to find potential cancer.     

A case of metastatic breast cancer with outgrowth of HER2-negative cells after eradication of HER2-positive cells by humanized anti-HER2 monoclonal antibody (trastuzumab) combined with docetaxel

A 38-year-old woman with cancer of the left breast underwent a modified radical mastectomy with lymph node dissection. Twenty-one months later, massive liver metastases and pleural carcinomatosis occurred. The liver metastases responded completely to chemotherapy with trastuzumab combined with docetaxel, but the pleural carcinomatosis was refractory to the therapy. Fluorescence in situ hybridization showed that both the primary tumor and the metastatic tumors of the lymph nodes were composed of HER2 amplification-positive and HER2 amplification-negative cancer cells. This analysis also detected a single cell with HER2 amplification in the pleural effusion that was taken at the completion of the chemotherapy, but four follow-up tests showed no amplified cells. It is speculated that in the liver metastases, the trastuzumab was cytotoxic to both HER2-amplified and nonamplified cancer cells and may have acted through its antiangiogenic effect. However, in the pleural effusion, the effect of trastuzumab was more specific to HER2-amplified cells and caused outgrowth of cancer cells lacking expression of HER2 receptors.     

肿瘤坏死因子(TNF)的小鼠体内外抗Ehrlich腹水瘤作用及其机理的初探

家兔经BCG和LPS联合刺激后,其血清中产生一种细胞毒因子,称肿瘤坏死因子(Tumor necrosis factor,TNF),该因子对L_(?)细胞及其它种属的肿瘤细胞,如Ehrlich腹水瘤细胞有较强的细胞毒效应。对人胚肺二倍体细胞则不表现细胞毒作用。单独使用BCG或LPS后,家兔血清均有一定的对L_(?)细胞的细胞毒性;联合使用,则家兔血清中细胞毒性显著提高。体外TNF与Ehrlich腹水瘤细胞共同孵育2、6h后,台酚兰染色表明细胞基本存活,但其肿瘤移植能力明显降低。TNF对Ehrlich腹水瘤的体内抑瘤作用,以攻击肿瘤前两天用药效果为佳。TNF注入小鼠腹腔后,能明显提高小鼠腹腔巨噬细胞的吞噬功能。     

A comparison of eosinophil-activating factor (EAF) with other monokines and lymphokines

Monocytes from moderately eosinophilic individuals secrete material that enhances the cytotoxic activity of eosinophils against antibody-coated schistosomula of Schistosoma mansoni. This material is not a single substance, but can be fractionated into several active components of different size and different charge. Gel filtration of mononuclear cell supernatants separated the eosinophil-activating activity into a major component of molecular mass of 40 kDa and a minor component of molecular mass of less than 10 kDa. The major component exhibited further heterogeneity on fractionation by high performance liquid chromatography. The bulk of the eosinophil-activating activity could be separated from both colony-stimulating factor (CSF) alpha activity and from tumor necrosis factor (TNF) activity. However, human recombinant CSF alpha (GM-CSF), human recombinant TNF and rabbit tumor necrosis serum all had eosinophil-activating activity when tested against schistosomula. Eosinophils were not activated by interleukin 1, interleukin 2, interferon-alpha, lipopolysaccharide or phorbol myristate acetate.     

Tumour necrosis factor-like activity on paraformaldehyde-fixed monocyte monolayers.

A tumour necrosis factor (TNF)-related protein appears to be associated in part with the monocyte surface membrane, since paraformaldehyde-fixed monocyte monolayers induced cytolysis of WEHI 164 clone 13 cells. Cytolysis was inhibited by neutralizing antisera raised against recombinant TNF (rTNF) and against the monocyte-derived cytotoxic factor, CF, which presumably is identical to natural TNF. Moreover, crude monocyte membrane preparations also contained cytotoxic activity, which was inhibited by antisera raised against rTNF and CF, and this activity was associated with particulate structures as it did not pass through filters with a pore size of 0.2 micron. Gamma-interferon (gamma-IFN)-activated monocytes fixed with paraformaldehyde had more TNF-like activity than fixed unactivated monocytes, and the crude membrane preparations from gamma-IFN-activated monocytes contained about five times more TNF-like activity than preparations from unactivated monocytes.