Effects of low concentrations of cadmium on immunoglobulin E production by human B lymphocytes in vitro

Exposure to cadmium (Cd) can cause a variety of biological effects including alterations of immune responses in animals and humans. Both immunosuppression and immunoenhancement have been reported. The present study was aimed at investigating the consequences of exposure to Cd on the human immunoglobulin (Ig) E synthesis, using purified peripheral blood B lymphocytes and IL-4 and anti-human CD40 monoclonal antibody (a-CD40 mAb) as stimuli. Low concentrations of Cd (0.1-10 microM) markedly inhibited production of IgE in a concentration-dependent manner. IgG production, in contrast to IgE, showed a tendency towards being enhanced by Cd, although with a certain individual variability; IgM production was not affected. Cd failed to alter immediate surface expression of the activation markers CD69 and CD23 indicating that early activation events were not impaired. However, the portion of activated B cells was diminished by Cd after stimulation for more than 24 h, paralleled by a concomitant decrease in viability and a subsequent reduction in proliferation. These data suggest that the mechanism of Cd action on activated B cells involved pathways that interrupted an effectively initiated cell activation and induced a cytotoxic signal. Results from this study thus provide further evidence for and new information on the immunotoxic and immunomodulatory effects of Cd on human immune responses.     

Modulation of human alveolar macrophage properties by ozone exposure in vitro.

We have investigated changes in human alveolar macrophage (HAM) function after exposure in vitro to ozone (O3) (0.1-1.0 ppm for 2-4 hr). The functions studied reflect concern that O3 is detrimental to host defense mechanisms in the bronchoalveolar spaces. Exposure of HAM to O3 caused a concentration-dependent increase in release of prostaglandin E2 (PGE2), an important modulator of inflammation, phagocytosis, and oxidative burst. Although phagocytosis of particulate immune complexes was decreased by O3, we found no change in the quantity of Fc receptors and complement receptors on the HAM surface. Superoxide (O2-) production in response to phorbol ester was reduced after exposure of HAM to O3 while the basal O2- release in response to plastic adherence was not affected. Growth inhibition of the opportunistic yeast Cryptococcus neoformans by HAM was not affected by O3 exposure. The production of inflammatory mediators and immune modulators such as tumor necrosis factor-alpha, interleukin 1, and interleukin 6 were not induced by exposure to O3. However, compared to controls, O3- exposed HAM produced significantly lower levels of these cytokines when stimulated with bacterial lipopolysaccharide (LPS). Two-dimensional gel electrophoretic analysis of proteins made by HAM following in vitro exposure to O3 identified 11 proteins whose rate of synthesis was significantly altered. Thus, these studies show that exposure to O3 alters the functional competence of HAM. While there is a minimal effect on protein expression or synthesis, the responses of HAM to particulate immune complexes, to bacterial LPS, and to PMA are impaired. The release of arachidonic acid and PGE2 suggest that the effect of O3 is primarily targeted to the HAM cell membrane. These changes may ultimately result in increased susceptibility to inhaled infectious agents in the O3-exposed individual.     

Toxic effects of ozone on human cells in vitro,exposed by gas diffusion through teflon film

An exposure system has been developed in which direct interaction between ozone and cultured cells can be realized by gas diffusion through thin teflon films supporting the cells growing under normal tissue culture conditions (Alink et al., Chemosphere 2, 63–73, 1979). Confluent monolayers of human alveolar type II cells (A549) were exposed to 0.1 or 0.2 μg ozone/culture dish (growth area 12 cm²) in 2.5 h. Viability and plating efficiency were decreased at both concentrations, as compared to control cells. After exposure to 0.1 μg ozone the superoxide dismutase (SOD) activity was higher than in unexposed cells due to an increase of the mitochondrial component of SOD. Morphological studies of the cells after exposure to ozone, using light microscopy, scanning and transmission electron microscopy, revealed alterations like a rounded shape, disappearance of microvilli and disintegration of the cytoplasm. In spite of these effects, the plasma membrane showed no ultrastructural damage, suggesting that the primary reaction with ozone had taken place at the molecular level. These results further indicate that exposure of cultured cells to ozone by means of gas diffusion through teflon films may contribute to the evaluation of the toxic effects of this pollutant in vitro.     

The effect of methotrexate on in vitro immunoglobulin production

The effect of Methotrexate (MTX) on pokeweed mitogen (PWM) induced immunoglobulin (Ig) production in vitro was studied over a range of 10(-4) - 10(-12)M MTX, using an enzyme-linked-immunosorbent assay (ELISA). MTX, at a concentration of 5 X 10(-9)M profoundly decreased all classes of Ig production, IgM greater than IgG greater than IgA. Plasma cell numbers, identified using polyclonal antihuman immunoglobulin, demonstrated a similar sensitivity to MTX. There was a paradoxical increase in IgM and IgG production at 5 X 10(-11)M MTX. Clinical implications of these findings are discussed.     

蝙蝠葛碱在体外对轻度修饰的脂蛋白诱导人单核细胞血小板源生长因子B蛋白量的影响

从正常人血中分离单核细胞, 分别加入25 mg·L^-1脂蛋白(LDL, VLDL)及经Cu²⁺轻度氧化修饰脂蛋白(MM-LDL, MM-VLDL)进行培养. 应用免疫细胞化学技术观察蝙蝠葛碱对脂蛋白诱导单核细胞 产生血小板源生长因子B(PDGF-B)蛋白的影响. 结果表明, LDL和VLDL对单核细胞PDGF-B量无明显影响, MM-LDL, MM-VLDL明显增强单核细胞PDGF-B量, 而蝙蝠葛碱对此有显著抑制作用. 结果提示, 蝙蝠葛碱抗动脉粥样硬化作用可能与抑制PDGF的作用有关.     

人肝癌细胞系对葫芦素B的体外摄取经时过程

目的:考察人肝癌细胞系HepG-2细胞对葫芦素B摄取过程的经时变化规律。方法:采用MTT法确定药物摄取剂量与时间,细胞培养技术考察药物摄取过程,高效液相色谱-二极管阵列检测(HPLC-DAD)法检测药物摄取量,并对摄取的量-时过程进行分析。结果:确定葫芦素B摄取为10μg.mL-1,摄取时间4 h。在0.5 h前,细胞摄取速度较平缓,0.5～1 h摄取速度呈指数增加趋势,1 h后摄取速度放缓,2 h到达最大量,摄取率30.37%,2 h后细胞摄取葫芦素B的量逐渐减小。结论:摄取过程表现出一定的剂量与时间相关性;建立的方法能够用于HepG-2细胞对葫芦素B的摄取研究,亦可为其他药物的摄取研究提供借鉴。     

Dose-dependent modulation of the in vitro cytokine production of human immune competent cells by lead salts.

Lead pollution constitutes a major health problem that has been intensively debated. To reveal its effects on the immune response, the influence of lead on the in vitro cytokine production of human peripheral mononuclear blood cells was investigated. Isolated cells were exposed to lead acetate or lead chloride for 24 h in the presence of either heat-killed Salmonella enteritidis (hk-SE) or monoclonal antibodies (anti-CD3, anti-CD28, anti-CD40) as cell activators. Our results showed that while higher lead doses are toxic, lower ones evoke immunomodulatory effects. All tested lead doses significantly reduced cell vitality and/or proliferation and affected secretion of proinflammatory, T helper cell type (T(H))1 and T(H)2 cytokines. Expression of interferon (IFN)-gamma, interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha was reduced at lower lead doses in both models of cell stimulation. Although hk-SE failed to induce detectable IL-4 levels, monoclonal antibody-induced IL-4, IL-6, and IL-10 secretion increased in the presence of lower lead doses. Also, levels of hk-SE-induced IL-10 and IL-6 secretion were increased at lower lead doses. Thus, exposure to lower doses leads to suppression of the T(H)1 cytokine IFN-gamma and the proinflammatory cytokines TNF-alpha and IL-1beta. The elevated production of IL-4 and/or IL-10 can induce and maintain a T(H)2 immune response and might contribute to increased susceptibility to pathologic agents as well as the incidence of allergic hypersensitivity and/or T(H)2-dominated autoimmune diseases.     

磷酸化cAMP反应元件结合蛋白对发育期人牙乳头细胞增殖和分化的影响

目的 构建CBP抑制表达慢病毒载体,探讨CBP有效表达沉默后对体外培养人牙乳头细胞(human dental papilla cells,HDPC)的增殖及分化影响.方法 利用基因重组技术构建重组慢病毒建立牙乳头细胞沉默稳转细胞,采用CCK-8检测细胞增殖的变化,采用检测分化相关蛋白的表达评价CBP对HDPC分化能力的影响.结果 测序结果提示成功构建四组人CBP基因的重组慢病毒;转染HDPC后,通过RT-PCR和蛋白质印迹法筛选获得有效靶点的重组慢病毒;CCK-8检测结果表明HDPC在CBP表达下调后增殖活性受到抑制;慢病毒介导CBP沉默后影响了HDPCs相关分化蛋白ColI、OCN、OPN的表达,较对照组显著下调(P＜0.05);牙乳头细胞的ALP活性在转染重组慢病毒后5,7,14 d后显著降低(P＜0.05);结论 成功构建了高效CBP抑制表达载体,CBP沉默可抑制HDPCs的增殖和分化.     

Effect of cyclosporin A on the production of interferon by human peripheral blood leukocytes in vitro

Cyclosporin A (CsA) was assessed for its effect on the production of antiviral activity by human peripheral blood leukocytes (PBL). CsA markedly reduced the production of interferon-gamma (IFN-gamma) in response to stimulation with lectin mitogens, bacterial products, alloantigens, or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). CsA-mediated suppression of IFN-gamma secretion was dose-dependent and did not result from a shift of kinetics of the production of antiviral activity. The production of IFN-alpha in response to stimulation with Corynebacterium parvum (CP), viruses, and synthetic polynucleotides was not affected by the addition of CsA. These findings confirm earlier observations that CsA predominantly acts on T lymphocyte function. CsA may prove a valuable agent to study the role of IFN-gamma in the pathogenesis of virus-associated malignant lymphoproliferative disease.     

Generation of oxidative stress in human cutaneous models following in vitro ozone exposure.

Ozone, one of the main components of photochemical smog, represents an important source of environmental oxidative stress. The skin, being the outermost barrier of the body, is directly exposed to environmental oxidant toxicants. Skin sebum and cellular plasma membrane lipids contain polyunsaturated fatty acids which are primary targets for ozone and free radical attack induced lipid peroxides. These ozonation processes in skin can also generate aldehydes, hydroxyhydroperoxides and specific Criegee's ozonides. In order to evaluate in vitro human skin susceptibility to ozone, we have exposed cultured immortalized human keratinocytes (DK7-NR) and the reconstructed human epidermis Episkin to 10 ppm of ozone in a specific incubator. We measured the formation of protein carbonyls by an ELISA method and monitored the oxidative stress using the fluorogenic probe 2',7'-dichlorofluorescin-diacetate (DCFH-DA). Results showed a time-dependent increase of fluorescence levels (linked to oxidative stress) in both models exposed to ozone. Using this protocol, we investigated the protective potential of different products including vitamin C, a thiol derivative and a plant extract. All products dramatically reduced oxidative responses during ozone exposure. Decreases observed in fluorescence levels were between 60 and 90% as compared to non-protected controls. These results demonstrate: (a) cutaneous in vitro models are remarkably susceptible to oxidative stress generated by an environmental air pollutant as ozone, and (b) raw antioxidants, thiols and vitamin C were efficient products to prevent ozone induced cellular oxidative damage.     

吗啡对雌激素诱导的人正常乳腺细胞促增殖作用的影响

目的研究吗啡对雌激素诱导的体外培养的人正常乳腺细胞的促增殖作用的影响。方法培养人正常乳腺纤维细胞株,将培养好的细胞给予17-β雌二醇(E2)、吗啡及纳洛酮(特异性阿片受体拮抗剂)干预,分为4组:对照组(A组),E2组(B组),E2+吗啡组(C组),17-β雌二醇+吗啡+纳洛酮组(D组)。观察细胞的生长增殖情况并绘制生长曲线。结果第一代传代的乳腺细胞各组接种密度比较,差异无统计学意义(P>0.05)。接种用药第2、4、6、8天进行细胞计数,细胞数较对照组增加,C组细胞数较B组降低,差异有统计学意义(P<0.01)。D组与B组比较,差异无统计学意义(P>0.05)。结论吗啡可直接影响雌激素对乳腺的促增殖作用。     

Inhibition by DL-alpha-difluoromethylornithine of the pokeweed mitogen-induced immunoglobulin production in cultured human lymphocytes

The effects of DL-alpha-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, on immunoglobulin production were studied in vitro using human peripheral blood lymphocytes stimulated with pokeweed mitogen. DFMeOrn inhibits in a concentration-dependent manner the usual pokeweed mitogen-induced increases of polyamine contents (putrescine, spermidine, spermine) and of [3H]thymidine incorporation. In parallel with the reduction of polyamine content and of thymidine incorporation, IgG and IgM productions are diminished, a 70% decrease being observed at 5 mM DFMeOrn concentration. Therefore, it appears that inhibition of polyamine biosynthesis may ultimately interfere with the cellular immunologic response by blocking cell proliferation. These findings certainly deserve further consideration both under in vitro and in vivo conditions.     

Effects of quinolones on interleukin 1 production in vitro by human monocytes

The new quinoline derivative antibiotics (quinolones), pefloxacin and ciprofloxacin at concentrations higher than 50 micrograms/ml inhibit the PHA response of the human mononuclear leukocytes in vitro. Since monocytes have been shown to be accessory cells for the activation of lymphocytes by mitogens, we investigated the effects of pefloxacin and ciprofloxacin on extracellular interleukin 1 (IL-1) and cell-associated IL-1 from lipopolysaccharide-stimulated human monocytes. Pefloxacin and ciprofloxacin decreased the extracellular IL-1 in a dose-dependent manner, while cell-associated IL-1 was not altered. These effects were observed even after a short period of incubation (1 or 2 h). No inhibitory activity against purified IL-1 or IL-2 could be demonstrated in the dialyzed supernatants from pefloxacin- or ciprofloxacin-treated monocytes. Neither pefloxacin nor ciprofloxacin modified the biological activity of preformed IL-1. The decrease of extracellular IL-1 induced by pefloxacin and ciprofloxacin could, in part, account for the observed decrease in the proliferative response of human mononuclear leukocytes to phytohemagglutinin, as extracellular IL-1 and proliferative response were positively correlated (at various concentrations of pefloxacin and ciprofloxacin). The decrease in extracellular IL-1 was not associated with any alteration in the expression of the HLA-DR antigen on the monocytes membrane. These data suggested that pefloxacin and ciprofloxacin could antagonize IL-1 production and release by lipopolysaccharide-stimulated monocytes. These quinolones could be interesting tools to study the production, processing, transport and release from the monocytes of IL-1.     

In vitro metabolism of citalopram by monoamine oxidase B in human blood.

The metabolism of the antidepressant citalopram (CIT) by monoamine oxidase B (MAO-B) was studied in vitro. In incubations with blood of nine healthy volunteers R-(P=0.015) and S-(P=0.0034) CIT propionic acid (CITPROP) production was correlated with the number of platelets. S-CITPROP production was 5.6 times higher than R-CITPROP production and in incubations containing the MAO-B inhibitor deprenyl, racemic CITPROP production was diminished to 9.1%. To our knowledge, this is the first time that MAO-B activity in blood is shown with an antidepressant as substrate. As MAO is strongly expressed in human brain, this observation suggests that this enzymatic system may be implicated in drug metabolism in the CNS.     

Effect of piceatannol in human monocyte-derived dendritic cells in vitro

Piceatannol is an anti-inflammatory, immunomodulatory, and antiproliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Dendritic cells (DCs) play a pivotal in the initiation of T-cell-mediated immune responses, making them an attractive cellular adjuvant for use in cancer vaccines. This study investigated the effect of piceatannol on the phenotypic and functional maturation of human monocyte-derived DCs in vitro. Human monocytes were cultured with GM-CSF and IL-4 for 6 days, followed by another 2 days in the presence of piceatannol or LPS. DCs harvested on day 8 were examined using functional assays. The expression levels of CD1a, CD80, CD83, and CD86 as expressed by mean fluorescence intensity (MFI) on DCs differentiated from immature DCs after culture with 1 muM of piceatannol for 2 days were enhanced and decreased endocytic activity. Piceatannol-treated DCs also displayed enhanced T-cell stimulatory capacity in a MLR, as measured by T-cell proliferation. Similar results were obtained with DCs differentiated with LPS from immature DCs. However, piceatannol did not inhibit phenotypic and functional maturation induced by LPS from immature DCs. Piceatannol-treated DCs induced the differentiation of naive T cells towards a helper T-cell type 1 (Th1) response at DCs/T (1:5) cells ratio depending on IL-12 secretion. These results demonstrate that piceatannol may be used on DC-based vaccine for cancer immunotherapy.     

The in vitro effects of Pb acetate on NO production by C6 glial cells.

The CNS neurotoxic effects of lead (Pb) are well documented; however, the molecular toxicity targets have not been clearly delineated. Astroglial cells, which are the most abundant cells in the brain and provide critical support to the neurons, are known to accumulate Pb. Although NO generated by inducible NO synthase (iNOS) in glial cells has been associated with many neurotoxic events, it can also serve to protect by modulating blood flow, increase antimicrobial and tumoricidal activities, and promote immune responses following injury or insult. The present investigations were designed to test the hypothesis that Pb exposure may perturb cytokine signal transduction pathways leading to NO production by astroglial cells. Pretreatment with Pb acetate (500 nM-10 microM) attenuated the generation of NO in a concentration-dependent manner up to 90%, and suppressed iNOS protein expression, as well as interfered with the homeostatic functions of calcium in the cytokine-induced NO signal transduction pathway. In addition, pretreatment with staurosporine, a serine-threonine kinase inhibitor, or KT5720, a specific protein kinase A inhibitor (PKA), inhibited cytokine-induced NO production in a concentration-dependent manner with IC(50) values of 26.3 and 346.7 nM, respectively. Therefore, Pb may impede events within the PKA signal transduction pathway; although, based on results from a gel shift assay, Pb does not directly affect PKA enzyme activity. Taken together, these results suggest the possibility that the suppressive effect of Pb acetate on cytokine-induced NO production in glial cells may be implicated in the neurophysiologic changes noted following occupational or environmental exposure to Pb.