Immunomodulation by ethanolic extract of Boerhaavia diffusa roots

We have earlier reported that ethanolic extract of Boerhaavia diffusa, a plant used in Indian traditional system of medicine, significantly inhibits the cell proliferation. This led us to evaluate the immunomodulatory properties of this plant extract on various in vitro tests such as human natural killer (NK) cell cytotoxicity, production of nitric oxide (NO) in mouse macrophage cells, RAW 264.7, interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), intracytoplasmic interferon-gamma (IFN-gamma) and expression of various cell surface markers on human peripheral blood mononuclear cells (PBMCs). Ethanolic extracts of B. diffusa roots inhibited human NK cell cytotoxicity in vitro, production of NO in mouse macrophage cells, IL-2 and TNF-alpha in human PBMCs. Intracytoplasmic IFN-gamma and cell surface markers such as CD16, CD25, and HLA-DR did not get affected on treatment with B. diffusa extract. Our study demonstrates immunosuppressive potential of ethanolic extract of B. diffusa.     

Effect of topical immunomodulators on acute allergic inflammation and bronchial hyperresponsiveness in sensitised rats

We examined the effects of different immunomodulators administered topically on asthmatic responses in a rat model of asthma. Sensitised Brown-Norway rats were administered rapamycin, SAR943 (32-deoxorapamycin), IMM125 (a hydroxyethyl derivative of D-serine(8)-cyclosporine), and budesonide by intratracheal instillation 1 h prior to allergen challenge. Allergen exposure induced bronchial hyperresponsiveness, accumulation of inflammatory cells in bronchoalveolar lavage fluid, and also an increase in eosinophils and CD2+, CD4+ and CD8+ T cells in the airways. Interleukin-2, interleukin-4, interleukin-5, interleukin-10, and interferon-gamma mRNA expression was upregulated by allergen exposure. Budesonide abolished airway inflammation, suppressed the mRNA expression for interleukin-2, interleukin-4, and interleukin-5 (P<0.03), and bronchial hyperresponsiveness (P<0.05). IMM125 suppressed airway infiltration of eosinophils, and CD8+ T cells (P<0.02), and prevented the upregulated mRNA expression for interleukin-4, interleukin-5, and interferon-gamma (P<0.02). Rapamycin suppressed CD8+ T cell infiltration in airway submucosa (P<0.03), and mRNA expression for interleukin-2 (p<0.002), while SAR943 suppressed interleukin-2, interleukin-4, and interferon-gamma mRNA (P<0.05). IMM125, rapamycin and SAR943 did not alter airway submucosal CD2+ and CD4+ T cell infiltration, and bronchial hyperresponsiveness. CD8+ T cells, in contrast to CD4+ T cells, are more susceptible to the inhibition by IMM125 and rapamycin, which also caused greater suppression of Th1 compared to Th2 cytokine mRNA expression. In this acute model of allergic inflammation, differential modulation of Th1 and Th2 cytokines may determine the effects of various immunomodulators on airway inflammation and bronchial hyperresponsiveness.     

FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells

To understand the mechanism of action of FK506 (Tacrolimus) on neutrophil chemotaxis, we examined its effect on human neutrophil chemotaxis and neutrophil chemoattractant production by peripheral blood mononuclear cells. FK506 and cyclosporin A had no direct suppressive effect on neutrophil chemotaxis induced by interleukin-8, leukotriene B(4), complement 5a (C5a), zymosan-activated serum and formyl-Met-Leu-Phe (fMLP). FK506 and cyclosporin A only slightly suppressed the chemotactic activity of platelet-activating factor (PAF). Dexamethasone did not inhibit the chemotactic activity of any chemoattractant. The supernatant of peripheral blood mononuclear cells stimulated with anti-CD3 and CD2 antibodies induced neutrophil chemotaxis. FK506 and cyclosporin A suppressed the chemotactic activity of the supernatant in parallel to the suppression of interleukin-8 production by peripheral blood mononuclear cells. Anti-interleukin-8 antibody completely suppressed the chemotactic activity of the supernatant without drugs. These studies indicate that FK506 may exert a beneficial effect on human inflammatory diseases by suppressing neutrophil chemotaxis secondary to inhibition of chemoattractant (for example, interleukin-8) production by leukocytes.     

氮网球花定碱盐酸盐对活化T细胞增殖的抑制作用

目的 探究氮网球花定碱盐酸盐(NMHC)抑制活化T细胞增殖的作用机制.方法 Ficoll法提取分离健康人外周血单个核细胞(PBMC),免疫磁珠法纯化出PBMC中的T细胞,抗人CD3/CD28抗体活化纯化后的静息T细胞.NMHC 0.125,0.5,2和8μmol·L-1与静息T细胞作用,用流式细胞术检测细胞毒性(96 ...     

青藤碱对类风湿关节炎树突状细胞CD80mRNA、CD86mRNA表达的影响

目的观察中药单体青藤碱对体外培养类风湿关节炎(RA)树突状细胞CD80mRNA、CD86mRNA表达的影响。方法分离10例RA患者外周血单核细胞,分为青藤碱高、中、低剂量组和空白对照组,采用重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素-4联合刺激,半定量逆转录聚合酶链反应检测CD80mRNA、CD86mRNA表达的改变。结果与空白对照组比较,经青藤碱高、中、低剂量干预的树突状细胞CD80mRNA、CD86mRNA表达减少(P<0.01或P<0.05),青藤碱3个剂量组之间比较表达存在差异(P<0.05)。结论青藤碱可能通过抑制树突状细胞CD80mRNA、CD86mRNA的表达,阻断对其T细胞的协同刺激而达到治疗RA的作用。     

Cytokine modulation and suppression of liver injury by a novel analogue of thalidomide

Thalidomide has been shown to reduce the production of tumor necrosis factor-alpha (TNF-alpha), a cytokine with deleterious pathophysiologic effects in various diseases. In search of thalidomide analogues with improved TNF-alpha inhibiting properties, 5-ethyl-1-phenyl-5-(3,4,5,6-tetrafluorophthalimido)barbituric acid (TFBA) was found to be superior to thalidomide. Besides TNF-alpha, TFBA also suppressed interleukin-6 and interleukin-10 production of isolated monocytes. The possibility that TFBA exerts its action by increasing levels of cAMP via inhibition of phosphodiesterase-4 activity was excluded. TFBA had no influence on T cell proliferation; neither did it inhibit TNF-alpha production in peripheral blood mononuclear cells stimulated by anti-CD3 monoclonal antibody. When applied to mice treated with D-galactosamine and lipopolysaccharide, TFBA prevented a rise in serum TNF-alpha, had no effect on interleukin-6 levels and led to an increase in interleukin-10 production. The changes in cytokine production observed in vitro and in vivo were reflected by similar changes in the mRNA expression. Moreover, TFBA significantly reduced liver transaminase levels in D-galactosamine/lipopolysaccharide-treated mice and thus efficiently protected the animals from liver injury. Thus, according to its properties, TFBA has the potential of modulating an immune response by acting as an anti-inflammatory agent.     

肝细胞癌患者外周血CD95和OX40L的表达

目的:研究肝细胞癌(HCC)患者外周血CD95和OX40L mRNA的表达情况,并探讨其临床意义.方法:将研究对象的淋巴细胞与相应荧光标记的单克隆抗体反应,经洗涤、固定后,应用双色流式细胞术分析T淋巴细胞表面CD95~+CD3~+的表达量.采用荧光定量逆转录聚合酶链反应(FQ-RT-PCR)方法检测外周血单个核细胞共刺激分子OX40L mRNA的表达水平.结果:HCC患者外周血CD95~+CD3~+的表达水平明显高于健康对照组,差异有统计学意义[(34±20)%vs(20±7)%,t=2.960,P＜0.01];HCC患者外周血单个核细胞OX40L mRNA表达水平明显低于健康对照组,差异有统计学意义(0.46±0.36vs 0.86±0.70,t=2.302,P＜0.05).结论:HCC患者外周血CD95及OX40L分子的异常表达在HCC的发生中起重要作用.     

Anti-CD40 Treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 mice induces activation of antigen presenting cells yet fails to overcome TCDD-induced suppression of allograft immunity

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.     

FK506 inhibits prostaglandin E2 production from synovial cells by suppressing peripheral blood mononuclear cells

FK506, an immunosuppressive drug for T cells, reduces pain in patients with rheumatoid arthritis. However, the mechanism for pain reduction remains uncharacterized. In this study, we investigated the effect of FK506 on prostaglandin E(2) (PGE(2)) production from synovial cells in vitro. Human synovial cells were cultured with supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD28 antibodies. Cultured synovial cells with PBMC supernatant produced a high amount of PGE(2) and FK506 inhibited PGE(2) induction from synovial cells. Culture supernatant contained interleukin-1beta (IL-1beta) and TNFalpha, and FK506 suppressed both in PBMC supernatant. Anti-IL-1beta neutralizing antibody, but not anti-TNFalpha neutralizing antibody, completely inhibited PGE(2) induction by PBMC supernatant. These results suggest that FK506 suppresses inflammation by inhibiting PGE(2) production from synovial cells through suppression of IL-1beta production from leukocytes.     

N-acetyl-D-glucosamine-coated polyamidoamine dendrimer promotes tumor-specific B cell responses via natural killer cell activation

N-acetyl-D-glucosamine-coated polyamidoamine dendrimer (GN8P), exerting high binding affinity to rodent recombinant NKR-P1A and NKR-P1C activating proteins, was shown previously to delay the development of rat colorectal carcinoma as well as mouse B16F10 melanoma, and to potentiate antigen-specific antibody formation in healthy C57BL/6 mice via NK cell stimulation. In this study, we investigated whether GN8P also modulates tumor-specific B cell responses. Serum anti-B16F10 melanoma IgG levels, IgG2a mRNA expression, antibody dependent cell-mediated cytotoxicity (ADCC), and counts of plasma as well as antigen presenting B cells were evaluated in tumor-bearing C57BL/6 mice treated with GN8P and in respective controls. To reveal the mechanism of GN8P effects, the synthesis of interferon-gamma (IFN-γ) and interleukin-4 (IL-4), cytokines involved in regulation of immunoglobulin class switch, was determined. The GN8P treatment significantly elevated IgG, and particularly IgG2a, response against B16F10 melanoma, which led to augmented ADCC reaction. The significant increase in production of IFN-γ, which is known to support IgG2a secretion, was observed solely in NK1.1 expressing cell populations, predominantly in NK cells. Moreover, GN8P raised the number of plasma cells, and promoted antigen presenting capacity of I-A/I-E-positive B lymphocytes by up-regulation of their CD80 and CD86 co-stimulatory molecule expression. These results indicate that GN8P-induced enhancement of tumor-specific antibody formation is triggered by NK cell activation, and contributes to complexity of anticancer immune response involving lectin-saccharide interaction.     

Increased circulating pro-inflammatory cytokines and imbalanced regulatory T-cell cytokines production in chronic idiopathic urticaria

The immunologic characterization of chronic idiopathic urticaria (CIU), mainly regarding cytokine profile needs more investigation. We examined circulating inflammatory cytokine levels, T-cell induced secretion, and cytokine mRNA expression in patients with CIU subjected to the intradermal autologous serum skin test (ASST). Increased levels of circulating pro-inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-12p70, and IL-6 have been observed in most of patients with CIU, together with an enhancement of IL-2 secretion following T-cell stimulation. Highlighting the inflammatory profile in CIU found in ASST positive, is the enhanced B-cell proliferative responsiveness and increased IL-17 secretion levels. ASST-positive patients also exhibited impaired IL-4 secretion associated with increased IL-10 production. Altered cytokine expression in patients with ASST-negative, was the down-modulation of spontaneous IL-10 mRNA expression levels in peripheral blood mononuclear cells. Our findings support the concept of immunologic dysregulation in CIU, revealing a systemic inflammatory profile associated with disturbed cytokine production by T cells, mainly related to IL-17 and IL-10 production.     

慢性乙型肝炎病毒感染对外周血单个核细胞中凋亡分子表达的影响及其与免疫功能的相关性

目的 研究慢性乙型肝炎病毒(HBV)感染对外周血单个核细胞中凋亡分子表达的影响及其与免疫功能的相关性.方法 选择76例慢性乙型肝炎(CHB)患者和体检的54例健康志愿者,分别作为CHB组和对照组,采集血清并测定凋亡分子含量、采集外周血单个核细胞并测定凋亡分子表达量以及免疫细胞含量.结果 CHB组患者血清中凋亡分子Fas、FasL、Caspase-3、Caspase-6、Caspase-8的含量以及外周血单个核细胞中Fas、FasL、Caspase-3、Caspase-6、Caspase-8的mRNA含量均显著高于对照组;CHB组患者外周血中CD3+ CD4+T细胞、CD3+ CD8+T细胞、CD16+ CD56+ NK细胞的百分比及CD4+/CD8+的比例均显著低于对照组;外周血单个核细胞中Fas、FasL、Caspase-3、Caspase-6、Caspase-8的mRNA含量与CD3+ CD4+T细胞、CD3 +CD8+T细胞、CD16+ CD56+自然杀伤(NK)细胞的百分比呈负相关.结论 慢性HBV感染能够增加外周血单个核细胞中凋亡分子的表达,进而造成T淋巴细胞和NK细胞凋亡、抑制细胞免疫应答和非特异性免疫应.     

Effects of glucocorticoids and cyclosporine on IL-2 and I kappa B alpha mRNA expression in human peripheral blood mononuclear cells

To evaluate molecular mechanisms that might account for the heterogeneity in the in vitro responsiveness of individual subjects' peripheral blood mononuclear cells (PBMC) to immunosuppressive drugs, the authors quantitated in normal human cells the suppressive effects of the glucocorticoids prednisolone and methylprednisolone and of cyclosporine on interleukin-2 (IL-2) mRNA expression and IL-2 production, as well as the stimulatory effect of these drugs on IkappaBalpha mRNA expression. As expected, cyclosporine was significantly more suppressive than either glucocorticoid of IL-2 mRNA expression and IL-2 production by mitogen-stimulated PBMC, with variable degrees of inhibition in cells from individual subjects. The authors confirmed in human PBMC the stimulation of IkappaBalphamRNA expression by the glucocorticoid reported by others in HeLa and transfected Jurkat cell lines. In addition, the authors observed a stimulatory effect on IkappaBalpha mRNA expression by cyclosporine as well in 8 of 10 PBMC preparations studied, suggesting a possible role of calcineurin in the regulation of IkappaBalpha production. Interindividual variability in the intracellular mechanisms of action, possibly based on molecular polymorphisms, might be one factor contributing to differences among patients in their clinical responses to treatment with such drugs.     

Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains.

The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.     

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.     

林可霉素对人外周血源性单核巨噬细胞免疫功能影响的体外研究

目的：研究林可霉素对于健康人外周血单核巨噬细胞表面分子表达的影响,探讨林可霉素对单核巨噬细胞免疫学功能的影响。方法：从健康人外周血中提取单核巨噬细胞体外培养,培养7d后将其分为霉素处理组和对照组,林可霉素处理组加入林可霉素,对照组加入0.9%氯化钠溶液,刺激48h后,流式细胞仪检测细胞表面CD80、MHCⅡ及Fas表达,ELISA法检测细胞培养上清液中细胞因子人干扰素-γ（IFN-γ）、肿瘤坏死因子α的浓度。结果：在林可霉素处理后,单核巨噬细胞抗原呈递相关分子CD80、MHCⅡ表达降低,细胞因子IFN-γ、TNF-α表达明显减少,其表面死亡受体Fas表达升高。结论：林可霉素可明显抑制单核巨噬细胞抗原呈递及细胞因子分泌等免疫功能,并诱导单核巨噬细胞死亡。