Immunomodulatory effects of Ziziphora tenuior L. extract on the dendritic cells

Background

Ziziphora tenuior L. (Kakuti in Persian) is used in traditional medicine for treatment of gastrointestinal disorders as carminative and analgesic plant. The other usages of this plant are included treatment of diarrhea and nausea. Therefore in the present study we evaluated the immunomodulatory effects of the ethanolic extract of this plant on the dendritic cells (DCs).

Results

Ziziphora tenuior L. extract significantly (p = 0.002) increased the level of surface expression of CD40 as an important co-stimulatory marker on DCs compared to the control. However this extract did not change CD86 and MHC-II molecules, so it could promote DCs phenotypic maturation. Treatment of DCs with the extract resulted in slightly increased of the production of (IL-12); however, this change was not significant. In addition, the ability of treated DCs to stimulate allogenic T cells proliferation and cytokines secretion was examined in the co-cuture of these cells with T cells in mixed lymphocyte reaction (MLR). Z. tenuior L. at the 100 μg/ml concentration inhibited the proliferation of allogenic T cells and also significantly (P < 0.001) increased the level of IL-10. Moreover, the extract at 10–100 μg/ml concentration caused slightly increase in IFN-γ production and decreased IL-4 cytokines but these changes were not significant.

Conclusions

These findings indicated that Z. tenuior L. extract can modulate immune response by induction of CD40 expression on DCs and cytokine production; whereas it can inhibit T cell stimulating activity of DCs in high concentration. These findings possibly in part explain the traditional use of this plant in treatment of immune-mediated disorders. However future studies are needed.     

Galactomannan from Caesalpinia spinosa induces phenotypic and functional maturation of human dendritic cells

Plant polysaccharides present an interesting potential as immunomodulators, particularly in the induction of antitumoral responses, principally because of their molecular complexity and low in vivo toxicity. Activation of dendritic cells (DCs) could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. Herein, we investigated the immunomodulatory activity of a neutral plant polysaccharide Galactomannan on human monocyte-derived DCs (MDDC). MDDCs were stimulated with Galactomannan (GLM) from Caesalpinia spinosa and both phenotypic and functional activities were assessed by flow cytometry and real-time PCR. The phagocytic ability of MDDCs was determined by using E-coli pHrodo particles and induction of T-lymphocyte allostimulation was determined after T-cell staining with carboxyfluorescein succinimidyl ester (CFSE). In MDDCs, purified Galactomannan induced phenotypic maturation revealed by increased expression of CD83, CD86, CD206, and HLA-DR. Functional experiments showed the loss of particulate antigen uptake in Galactomannan-stimulated DCs and increased alloantigen presentation capacity. Finally, Galactomannan increased protein and mRNA levels of pro-inflammatory cytokines including IL-1β, IL-6, IL-8, IL-12p70, and TNF-α. These data reveal that Galactomannan obtained from Caesalpinia spinosa promotes effective activation of MDDCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting.     

异丙酚对树突细胞炎症因子分泌的影响

目的 观察异丙酚对树突细胞成熟和分泌细胞因子的影响.方法 取6～8周C57BL/6野生型老鼠(雌雄不限)的骨髓细胞,在FMS样酪氨酸激酶3(Fh3)配基的作用下诱导分化为树突细胞,利用脂多糖刺激树突细胞成熟并检测异丙酚对树突细胞表面成熟标记分子表达和分泌细胞因子的影响.结果 100μM的异丙酚能明显抑制脂多糖诱导的IL-6和IL-12的分泌,同时提高IL-4的分泌.脂多糖能使树突细胞表面表达的成熟分子CD40、CD80和CD86表达量明显上升,200μM异丙酚不影响脂多糖介导的树突细胞成熟分子的表达.结论 异丙酚不影响脂多糖介导的树突细胞的成熟但影响它的分泌从而抑制机体的免疫反应.     

Anti-hepatotoxic activity of cichotyboside,a sesquiterpene glycoside from the seeds of Cichorium intybus

The seeds of Cichorium intybus L. (Asteraceae) afforded a new guaianolide sesquiterpene glycoside, cichotyboside, which was characterized as 2α, 6β, 7β, 15-tetrahydroxy-1 (10), 4 (5)-diene-guaian-9α, 12-olide-7-O-β-caffoyl-15-O-β-D-glucoside (1) by means of spectral methods. Cichotyboside (1) exhibited a significant anti-hepatotoxic activity against CCl₄ induced toxicity in Wistar rats, wherein it reduced the elevated levels of liver enzymes such as serum glutamate oxaloacetate transaminase (SGOT) by 52 units/ml; SGPT 38 units/ml; ALKP 24.97 units/ml and 7.54 g/dl, 5.48 g/dl increase in total protein and albumin, respectively. It was observed that cichotyboside (1) decreased the level of ALKP comparable with that of standard drug silymarin, exhibiting an 88% decrease in comparison to silymarin (92%) and increased the level of total albumin 85% in comparison to silymarin (89%) against intoxicated control. Whereas, the levels of SGOT and SGPT were also decreased considerably in comparison to standard and intoxicated control.     

树突状细胞瘤苗诱导的抗神经胶质瘤作用体外实验研究

目的 :研究负载自体神经胶质瘤抗原的树突状细胞 (DCs)瘤苗在体外诱导的特异性细胞毒性淋巴细胞 (CTL)对神经胶质瘤细胞的杀伤效应。方法 :以组合酶消化法从新鲜神经胶质瘤手术标本中获取神经胶质瘤细胞 ,冻融制备神经胶质瘤抗原。GM -CSF、IL -4体外诱导外周血单个核细胞 (PBMC)获得DCs并负载神经胶质瘤抗原 ,继而以其刺激自体T淋巴细胞制备神经胶质瘤抗原特异性CTL ;用CytoTox96TM检测CTL对患自身神经胶质瘤细胞体外杀伤效应。结果 :负载神经胶质瘤抗原DCs诱导的特异性CTL对患者自身神经胶质瘤细胞的杀伤率达88 17 % ,显著高于LAK细胞的杀伤率 (P<0 05)。且其对同种不同分化类型的神经胶质瘤细胞株 (P<0 01)。结论 :负载神经胶质瘤抗原的DCs体外可诱导出高效而特异的抗神经胶质瘤效应 ,提示以DCs为中心的肿瘤生物治疗作用可望提高神经胶质瘤综合治疗水平     

脂质体-鱼精蛋白-DNA复合物的制备及对树突状细胞的成熟诱导

目的研究脂质体-鱼精蛋白-DNA复合物(LPD)的制备方法及对树突状细胞(DC2.4)的成熟诱导作用。方法薄膜-超声分散法制备空白的阳离子脂质体,再与鱼精蛋白-DNA复合物室温孵育形成LPD;测定其粒径和电位。流式细胞仪检测鼠源树突状细胞系DC2.4的甘露糖受体表达水平;用DC2.4表面标记分子CD80、CD40、CD86和MHC-2的表达水平,考查LPD对树突状细胞的成熟诱导作用。结果 LPD的最佳比例为DDAB-鱼精蛋白-DNA(2∶1.5∶1);LPD粒径为84.28±0.56 nm,Zeta电位为27.33±1.23 mV。通过FITC-ManBSA的结合作用检测到DC2.4表面有83%的甘露糖受体表达。LPD明显上调DC2.4表面标记分子的表达水平(P<0.05)。结论 LPD制备工艺简单,是一个良好的疫苗佐剂。     

Trichothecenes NIV and DON modulate the maturation of murine dendritic cells

Nivalenol (NIV) and Deoxynivalenol (DON), mycotoxins of the trichothecene family are considered very common food contaminants. In this work, we investigated whether the immunotoxic effects ascribed to these trichothecenes may be mediated by perturbations in the activity of dendritic cells (DCs). Murine bone marrow-derived DCs were used to evaluate the effects of NIV and DON on the LPS-induced maturation process. We found that the expression of the class II MHC and of the accessory CD11c molecules, but not of the costimulatory CD86 marker, was down-regulated by NIV and DON exposure in LPS-treated DCs, as well as nitric oxide (NO) production. Interestingly, NIV, but not DON, induced DC necrosis. Moreover, the analysis of the cytokine pattern showed that IL-12 and IL-10 expressions induced by LPS exposure were suppressed by both trichothecenes in a dose-dependent fashion. On the other hand, the secretion of the proinflammatory cytokine TNF-α was increased as a direct consequence of DON and NIV exposure. Taken together, our data indicated that the immunotoxicity of NIV and DON was related to the capacity of both trichothecenes to interfere with phenotypic and functional features of maturing DCs.     

Caffeic amide derivatives inhibit allergen-induced bone marrow-derived dendritic cell maturation

Background

Caffeic amides are derivatives of caffeic acid, which have antioxidant and anti-inflammatory properties, and high in vivo stability. The therapeutic effect of caffeic amides on allergic diseases, and especially on the maturation of bone marrow-derived dendritic cells (BM-DCs), remains unclear. In this study, we investigated the therapeutic potential of caffeic amides on allergic diseases by evaluating the maturation of DCs and evaluated their potential in inducing the differentiation of T_H2 cells.

小鼠骨髓来源树突状细胞对T细胞的活化作用

目的 从小鼠骨髓中体外诱导、扩增树突状细胞(DCs),检测DCs对T细胞的活化作用.方法 从小鼠骨髓中分离单个核细胞(MNCs),在重组鼠粒细胞-巨噬细胞集落因子(rmGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)的诱导下培养扩增DCs.光学显微镜观察其形态特征,流式细胞仪分析其表型特征,混合淋巴细胞反应(MLR)检测DCs刺激同种异体T细胞增殖的能力.结果 MNCs经过rmGM-CSF、IL-4和TNF-α诱导培养12 d后,具有典型的DCs形态,高表达MHCⅡ类分子、CD11c、CD80、和CD86抗原.具有极强的激发刺激同种异体T细胞增殖的能力.结论 体外诱导、扩增DCs具有极强的激发刺激同种异体T细胞增殖的能力.     

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.     

黄芪多糖促进人树突状细胞体外分化成熟的实验研究

目的：研究黄芪多糖（APS）对人树突状细胞（DCs）体外分化成熟及存活时间的影响。方法：健康志愿者外周血单个核细胞培养2 h获得贴壁的单核细胞,加入含rhGM-CSF（1000 U/mL）r、hIL-4（500 U/mL）的无血清培养基,实验组再加入3种浓度的APS（1μg/mL,10μg/mL,100μg/mL）,对照组加入等体积生理盐水,一组细胞体外培养7 d,收集悬浮细胞获得普通DCs（Mo-DCs）及APS诱导的DCs（APS-DCs）,流式细胞仪测定细胞免疫表型,同种异体混和淋巴细胞反应检测DCs的免疫激活功能,另外一组细胞体外连续培养14 d,光镜下观察细胞形态及存活时间。结果：APS能够显著促进DCs表面免疫分子HLA-DR、CD83、CD80、CD86、CD40、CD54的表达,APS-DCs较Mo-DCs具有更强的T细胞体外激活能力,并显著延长DCs的体外存活时间达2～3 d。结论：APS可以促进DCs的成熟和延长DCs的存活时间,从而发挥增强机体免疫力的功能。     

树突状细胞联合细胞因子诱导的杀伤细胞体外抗肺癌的实验研究

目的探讨树突状细胞（DC）联合细胞因子诱导的杀伤细胞（CIK）在体内外能否有效的抗肺癌。方法正常人外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞,单个核细胞经贴附塑料平皿获得单核细胞,加入GM—CSF,IL-4及TNF—a诱导获得DC,悬浮细胞加入IFN—g,24h后IL-1a,IL-2及CD3McAb诱导获得CIK,将A549制备成肿瘤细胞冻融物．作为肿瘤抗原刺激DC,并将DC与CIK细胞联合培养（DC＋CIK,负载抗原的DC＋CIK）,以CIK细胞单独培养作为对照。CytotoxicityTOX96体外杀伤实验测定体外细胞毒活性。结果在培养的第15d．与负载抗原的DC共同培养的CIK与CIK细胞单独培养细胞相比,增殖速率明显提高[（23．4±2．3）倍vs（16．7±2．7）倍,P〈0．05],CD3＋CD56＋细胞表达水平也明显提高,[（64．3±3．6）％vs（43．9±2．1）％,P〈0．05],同时负载抗原的DC的CIK对A549细胞的体外下细胞毒活性增强。结论DC与CIK细胞共培养后可提高CIK细胞的增殖速率,提高CIK细胞表型的表达水平,增强CIK抗肺癌的活性．将来可作为一种临床有效的过继免疫治疗策略。     

牙龈卟啉单胞菌脂多糖对树突状细胞成熟及功能影响的体外研究

目的 研究牙龈卟啉单胞菌脂多糖(P.gingivalis-LPS)的刺激对大鼠树突状细胞(DCs)成熟及功能的影响,为探索DCs在牙周炎的发生发展中的作用机制提供实验依据.方法 采用流式细胞学方法检测P.gingivalis-LPS和大肠杆菌脂多糖(E.coli-LPS)刺激下,CD11c+MHCⅡ+、CD11c+CD80+、CD11c+CD86+和CD11c+CD40+DCs的比率;采用ELISA法检测DCs分泌白介素-12(IL-12)、干扰素-γ(IFN-γ)、白介素-10(IL-10)和白介素-13(IL-13)的量.采用CCK8法检测与上述DCs共培养的CD4+T细胞的增殖;采用ELISA法检测T细胞分泌IL-2、IFN-γ、IL-10和IL-13的量.在上述的培养系统中加入Toll样受体4(TLR4)抑制剂(polymyxin B,PmB)或TLR2/TLR4抑制剂(oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl-choline,OxPAPC),观察TLR抑制剂对上述DCs成熟及功能的影响.结果 P.gingivalis-LPS与E.coli-LPS均能刺激DCs成熟.TLR4抑制剂明显抑制E.coli-LPS组DCs成熟和抗原提呈功能,对P.gingivalis-LPS组DCs成熟和抗原提呈功能没有显著抑制.TLR2/TLR4抑制剂对P.gingivalis-LPS组DCs成熟和抗原提呈功能显著抑制.P.gingivalis-LPS组DCs分泌IL-12和IFN-γ 的量低于E.coli-LPS组(P<0.05);P.gingivalis-LPS组DCs分泌IL-10和IL-13的量高于E.coli-LPS组(P<0.05).与P.gingivalis-LPS和E.coli-LPS共培养的DCs均能促进CD4+T细胞增殖.与P.gingivalis-LPS组DCs共培养的T细胞分泌IL-2和IFN-γ 的量低于E.coli-LPS组(P<0.05);其分泌IL-10的量高于E.coli-LPS组(P<0.05).结论 P.gingivalis-LPS能促进DCs的成熟和抗原提呈功能.P.gingivalis-LPS刺激下的DCs促进Th2型免疫应答;E.coli-LPS刺激下的DCs促进Th1型免疫应答.P.gingivalis-LPS通过TLR2通路刺激DCs成熟;E.coli-LPS通过TLR4通路刺激DCs成熟.     

In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells

Recent data suggest that some of the immunotoxic effects of the herbicide atrazine, a very widely used pesticide, may be due to perturbations in dendritic cell (DC) function. As consequences of atrazine exposure on the phenotypic and functional maturation of DC have not been studied, our objective was, using the murine DC line, JAWSII, to determine whether atrazine will interfere with DC maturation. First, we characterized the maturation of JAWSII cells in vitro by inducing them to mature in the presence of growth factors and selected maturational stimuli in vitro. Next, we exposed the DC cell line to a concentration range of atrazine and examined its effects on phenotypic and functional maturation of DC. Atrazine exposure interfered with the phenotypic and functional maturation of DC at non-cytotoxic concentrations. Among the phenotypic changes caused by atrazine exposure was a dose-dependent removal of surface MHC-I with a significant decrease being observed at 1 microM concentration. In addition, atrazine exposure decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. When, for comparative purposes, we exposed primary thymic DC to atrazine, MHC-I and CD11c expression was also decreased. Phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation as, albeit at higher concentrations, receptor-mediated antigen uptake was increased by atrazine. Thus, our data suggest that atrazine directly targets DC maturation and that toxicants such as atrazine that efficiently remove MHC-I molecules from the DC surface are likely to contribute to immune evasion.     

Inhibitory effect of cepharanthine on dendritic cell activation and function

Dendritic cells (DCs) are specialized antigen presenting cells that connect innate and adaptive immunity. DCs are considered as a major target for controlling excessive immune responses. In this study, the effect of cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, on murine DCs was examined in vitro. CEP inhibited antigen uptake by DCs at a concentration between 1 and 5 μg/ml. Although CEP did not inhibit the expression of costimulatory molecules and major histocompatibility complex (MHC) class I in DCs, the compound inhibited lipopolysaccharide (LPS)-induced DC maturation determined by the expression of costimulatory molecules and MHC class I. In addition, CEP could reduce the production of interleukin-6 and tumor necrosis factor-α in LPS-stimulated DCs. DCs treated with CEP were found to be a poor stimulator of allogeneic T cell proliferation and interferon-γ production from the cells. These results suggest that CEP may have great potential as an immunoregulatory agent against various autoimmune diseases and allergy.     

IL-10在树突状细胞中的表达及效应研究

采用脂质体转染方法将 IL-10表达载体转入 DC,采用 ELISA和 SDS-PAGE鉴定转染质粒的 DC培养上清 ,用 MTT比色法检测 IL -10对同种细胞混合培养反应的影响。结果显示 ,EL ISA法检测证明将人 IL -10表达载体成功转入 DC,获得稳定表达 IL-10的 DC细胞株 ;IL-10可抑制单向混合淋巴细胞反应 ,且效应亦呈剂量依赖性。     

Cytoprotective effects of fucoidan,an algae-derived polysaccharide on 5-fluorouracil-treated dendritic cells

Although chemotherapeutic anticancer agents are effective, they also attack normal immune cells due to a lack of selectivity. 5-Fluorouracil (5-FU) is a representative anticancer agent that induces immunosuppression in cancer patients as a side effect. Fucoidan is an algae-derived sulfated polysaccharide that has recently been recognized as a hematopoietic mobilizer and immunomodulator. In this study, we investigated the cytoprotective effect of fucoidan on dendritic cells (DCs) against 5-FU-induced cellular damage. Several kinds of assays including flow cytometric analysis demonstrated the cytoprotective efficacy of fucoidan. In addition, fucoidan increased the expression of immune-related surface markers on and the alloproliferative capacity of DCs exposed to 5-FU. For investigating action mechanism, the expression levels of apoptosis-related molecules were measured. Taken together, the results of this study suggest that fucoidan, a marine-derived polysaccharide, has cytoprotective effects on DCs, the most potent antigen-presenting cell type, against 5-FU-induced cellular damage. These results provide valuable information to use fucoidan as an immunostimulatory agent for the chemotherapy of cancer patients.     

Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells

Aflatoxins (AFs) are harmful to animal and human health upon consumption of AF-contaminated feed or food. Among many forms of AFs, aflatoxin B₁ (AFB₁) is the most toxic and carcinogenic. In addition, AFB₁ impairs cell-mediated immunity, although the exact mechanism of this immunotoxicity is currently unknown. By far the most pivotal cells in the induction of immune responses are dendritic cells (DCs). These highly specialised cells dictate T-cell polarisation depending on the nature of the encountered antigens and environmental cues. To elucidate the effect of AFB₁ on the function of DCs, we used porcine monocyte-derived DCs (MoDCs) as a model system. A low dose of AFB₁ transiently reduced the phagocytic capacity of MoDCs. Furthermore, as compared to untreated MoDCs, AFB₁ significantly downregulated the cell surface expression of the co-stimulatory molecule CD40 at 12 h post treatment, while at 24 h the membrane expression levels of CD40 and the activation marker CD25 were significantly upregulated. Interestingly, the T-cell proliferation-inducing capacity of DCs was diminished upon AFB₁ treatment. In contrast, the cytokine secretion pattern of AFB₁-treated MoDCs was similar to mock-treated MoDCs. The results in this study indicate that a low level of AFB₁ dysregulates the antigen-presenting capacity of DCs, which could explain the observed immunotoxicity of this mycotoxin, and further stress the need to reduce AFB₁ levels in agricultural commodities.     

Antioxidative effects ofCichorium intybus root extract on LDL (Low Density Lipoprotein) oxidation

The water extract of Cichorium intybus (WECI) showed a remarkable antioxidative effect on LDL, and inhibitory effects on the production of thiobarbituric acid reactive substance and the Degradation of fatty acids in LDL. Vitamin E and unsaturated fatty acids in LDL were protected by adding WECI from the effects of metal catalyzed LDL oxidation. From the results obtained, we conclude that LDL oxidation is inhibited in vitro by the addition of WECI, and that LDL is protected by WECI from oxidative attack, as shown by agarose gel electrophoresis.     

Pidotimod promotes functional maturation of dendritic cells and displays adjuvant properties at the nasal mucosa level

Mucosal dendritic cells (DCs) are very important in the process of antigen presentation to T cells, playing a key role in the induction of primary and secondary immune responses. Pidotimod is a synthetic substance capable of modulating immune cell functions, but the effect of pidotimod on human DCs has not been investigated yet. Here we demonstrate the ability of pidotimod to induce DC maturation and up-regulate the expression of HLA-DR and co-stimulatory molecules CD83 and CD86, which are fundamental for communication with adaptative immunity cells. Pidotimod also stimulated DCs to release high amounts of pro-inflammatory molecules such as MCP-1 and TNF-α cytokines and to drive T cell proliferation and differentiation towards a Th1 phenotype. Moreover, we demonstrate that pidotimod in vivo promotes strong and specific humoral and cellular immune response when co-administered intranasally with a model antigen.Taken together our data suggest the possibility to use pidotimod as adjuvant molecule to facilitate the activation of the innate immune system as well as to promote an effective mucosal and systemic immune response.