Tanshinlactone A from Salvia miltiorrhiza modulates interleukin-2 and interferon-gamma gene expression

Salvia miltiorrhiza Bunge (Tanshen), a traditional Chinese herbal medicine, is popularly used to treat cardiovascular disorders. In the present study, effects of tanshinlactone A (C(16)H(12)O(4); M.W. 268), newly discovered from Salvia miltiorrhiza, on phytohemagglutinin (PHA)-stimulated cell proliferation were investigated in human peripheral blood mononuclear cells (PBMC). The results indicated that tanshinlactone A inhibited PBMC proliferation activated with PHA with an IC(50) of 15.6+/-1.9 microM. Cell viability test indicated that inhibitory effects of tanshinlactone A on PBMC proliferation were not through direct cytotoxicity. Furthermore, tanshinlactone A significantly decreased the interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) gene expression in PHA-activated PBMC. It reduced the phosphorylation of mitogen-activated protein kinases (MAPK) involving extracellular signal-regulated protein kinase (ERK), P38, and c-Jun NH(2)-terminal kinase (JNK) in PHA-treated PBMC. We suggested that the inhibitory effects of tanshinlactone A on PHA-induced PBMC proliferation, appeared to be mediated, at least in part, through reduction of MAPK activation and IL-2 and IFN-gamma production. Therefore, data demonstrate for the first time that tanshinlactone A is likely an immunomodulatory agent for PBMC.     

Vandellia cordifolia regulated cell proliferation and cytokines production in human mononuclear cells

Vandellia cordifolia (V. cordifolia) used for treatment inflammation in traditional Chinese medicine was selected for immunopharmacological activity test. The effects of V. cordifolia extracted fractions on human mononuclear cells (HMNC) proliferation were determined by tritiated thymidine uptake. The results indicated that VC-ME fraction suppressed HMNC proliferation activated with phytohemagglutinin (PHA) and stimulated cell cycle progression was arrested at the G0/G1 stage. The inhibitory mechanisms may involve the blocking of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production, since VC-ME suppressed IL-2 and IFN-gamma production of HMNC in a dose-dependent manner. Therefore, it is suggested that immunomodulatory agents are contained in V. cordifolia.     

Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordyceps cicadae

The effects of Cordyceps cicadae extracted fractions on human mononuclear cells (HMNC) proliferation were determined by tritiated thymidine uptake. The results indicated that aqueous methanol (50%) extracts of C. cicadae ascocarps portion (CC-1-2) enhanced HMNC proliferation activated with phytohemagglutinin (PHA) with an EC(50) of 13.8+/-4.6 micro g/ml. By contrast, the methanol (100%) extracts of C. cicadae insect-body portion (CC-2-1) suppressed HMNC proliferation stimulated by PHA with an IC(50) of 32.5+/-5.2 micro g/ml. Cell viability test indicated that inhibitory effects of CC-2-1 on HMNC proliferation were not through direct cytotoxicity. The action mechanisms of CC-1-2 and CC-2-1 may involve the regulation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in HMNC. Since CC-2-1 suppressed IL-2 and IFN-gamma production in HMNC induced with PHA. The CC-1-2 enhanced IL-2 and IFN-gamma production of HMNC stimulated with PHA in a concentration dependent manner. Therefore, the results demonstrated that C. cicadae contained growth modulators for HMNC.     

Immunomodulatory proanthocyanidins from Ecdysanthera utilis

Two new A-type proanthocyanidins have been isolated from Ecdysanthera utilis and identified as epicatechin-(4beta-->8,2beta-->O-->7)-epicatechin-(4beta-->8)-epicatechin (5) and epicatechin-(4beta-->8)-epicatechin-(4beta-->8,2beta-->O-->7)-epicatechin-(4beta-->8)-epicatechin (6), respectively. The structure-related components epicatechin (1), procyanidin B2 (2), proanthocyanidin A1 (3), proanthocyanidin A2 (4), and aesculitannin C (7) were also isolated. All of these compounds were identified and evaluated for immunopharmacological activity. Human peripheral blood mononuclear cells (PBMC) were used as target cells, and cell proliferation was determined by (3)H-thymidine uptake. The results indicated that compound 3 suppressed PBMC proliferation activated with phytohemagglutinin (PHA). The inhibitory mechanisms may involve the blocking of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production, since compound 3 attenuated IL-2 and IFN-gamma production of PBMC in a dose-dependent manner. Therefore, it is suggested that immunomodulatory agents are present in E. utilis.     

Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: a study on human leukocytes

AIM OF THE STUDY: Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of "Marcela". Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). MATERIALS AND METHODS: Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. RESULTS: In PBMCs, Achyrocline satureioides infusion in the 0.06-0.24microg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-gamma and interleukin (IL)-4. Lower concentrations of the infusion (0.006-0.03microg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-gamma and IL-4 and decreased the ratio IFN-gamma/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations > or =0.06microg/ml QE. On the contrary, in the 0.0012-0.03microg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. CONCLUSIONS: The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.     

当归多糖组分促进淋巴细胞增殖及对IL-2和IFN-γ的诱生作用

目的:观察当归多糖及其3个组分对淋巴细胞增殖和细胞因子IL-2、IFN-γ的影响.方法:机械分散法制备小鼠单个脾细胞悬液;MTT法检测细胞增殖;酶联免疫法测定培养上清液中IL-2和IFN-γ的浓度.结果:当归多糖及其3个组分在30～100 μg/ml剂量范围内,能显著促进脾细胞的增殖;其中组分AP-3能够显著促进巨噬细胞、混合淋巴细胞的增殖反应,剂量依赖性地增加细胞上清液中IL-2和IFN-γ的浓度.结论:当归多糖能够活化巨噬细胞和T细胞,还可以通过细胞因子IL-2、IFN￣γ发挥免疫调节作用.     

Cissampelos sympodialis Eichl. leaf extract increases the production of IL-10 by concanavalin-A-treated BALB/c spleen cells

The effect of the aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl. (Menispermaceae) leaves (AFL) on Concanavalin-A (Con-A)-activated spleen cell proliferation and cytokine (IL-2, IL-4, IL-10 and IFN-gamma) secretion were analyzed. BALB/c spleen cells were treated, in vitro, with different concentrations, ranging from 6.25 to 400 microg/ml of AFL either in the presence or the absence of 5 microg/ml of the mitogen Con-A. It was observed that concentrations of the AFL higher then 50 microg/ml were toxic to the cells and concentrations ranging from 6.25 to 50 microg/ml decreased the lymphocyte proliferative response in the presence of the mitogen. This inhibition of mitogen induced proliferation was not reverted by the addition of exogenous recombinant IL-2. The AFL did not significantly inhibit IL-2 secretion. In the presence of AFL there was a reduction in the levels of secreted IFN-gamma while the production of both IL-10 and IL-4 were increased. AFL did not induce the production of any of the cytokines analyzed, in the absence of Con-A. It is suggested that increased IL-10 production down regulates IFN-gamma secretion and T cell proliferative responses.     

The effect of liu-wei-di-huang wan on cytokine gene expression from human peripheral blood lymphocytes

Liu-Wei-Di-Huang Wan (LWDHW) has been used by traditional Chinese doctors to treat asthma patients. This study was to examine the potential effect of this decoction on the regulation of T helper (Th)1- and Th2-type cytokine gene expression in vitro. Peripheral blood mononuclear cells (PBMC) were activated with mitogen for 24 hours in the presence or absence of LWDHW extracts. Concentrations of different cytokines in the culture supernatants were determined with ELISA. RNA isolated from cultured cells was subjected to RT-PCR analysis. The results showed that the expression of all cytokines (Th2-type: IL-4, IL-5, IL-10, or IL-13 and Th1-type: IL-2 and IFN-gamma) examined was inhibited at both RNA and protein levels by LWDHW. Since the cell viability was similar in all cultures, the reduction of cytokine production was not due to the toxicity of LWDHW. Moreover, the cells either retained or increased their capacity to respond to mitogen stimulation after incubation with the LWDHW decoction. Therefore, the data suggest that LWDHW functioned directly on cytokine gene expression from activated PBMC.     

"Tien-Hsien liquid" can modulate antigen-stimulated cytokine production by T-cells isolated from patients with recurrent aphthous ulcerations

Recurrent aphthous ulcerations (RAU) represent a common oral mucosal disease with altered humoral and cellular immunities. Tien-Hsien liquid (THL) is an extract of Chinese medicinal herbs with immunomodulating effects. Our previous study found that THL can modulate the antigen-stimulated proliferative response of peripheral blood mononuclear cells and T-cells isolated from RAU patients. In this study, we further tested whether THL can modulate the antigen-stimulated cytokine production by T-cells isolated from RAU patients. To achieve this goal, T-cells isolated from 19 RAU patients were incubated with phytohemagglutinin (PHA), glutaraldehyde-inactivated tetanus toxoid (TT), glucosyltransferase D (GtfD), or antigens of Streptococcus mutans in the presence or absence of THL. The levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), IL-6, or IL-10 in the supernatants of T-cell cultures were measured by cytokine enzyme-linked immunosorbent assay (ELISA) kits. We found that THL significantly increased the PHA- or TT-stimulated TNF-alpha, IL-6, and IL-10 production by T-cells isolated from RAU patients. However, THL could also significantly decrease the TT-stimulated IL-2 production, the GtfD-stimulated IL-2, TNF-alpha, IL-6 and IL-10 production, and the S. mutans-stimulated IFN-gamma, TNF-alpha, and IL-10 production by T-cells isolated from RAU patients. These results indicate that THL can modulate the antigen-stimulated cytokine production by T-cells isolated from RAU patients. Because RAU is probably a Thl-mediated disease with elevated levels of IL-2, IFN-gamma, TNF-alpha and IL-6 in either the patient's sera or oral lesions and these increased levels of cytokines can be reduced by THL, we suggest that THL may be a potential immunoceutical agent for treatment of RAU.     

雷公藤多甙对哮喘患者Th1、Th2细胞因子产生的影响

目的：了解雷公藤多甙对哮喘患者Th1,TH2细胞因子的影响，进一步探讨雷公藤多甙治疗哮喘的,机制。方法：选12例中，重度哮喘患者，口服雷公藤多甙每日40mg 或60mg治疗4周，治疗前后取外公藤甲素处理24h，收集培养上清，用酶联免疫吸附试验（ELISA）对患者血清及PBMC培养上清IL－2，IL－4，IL－5，γ干扰素（IFN－γ）水平进行检测。结果：（1）雷公藤多甙治疗后哮喘患者血清中IL－2，IL－4，IL－5水平较治疗前降低（P＜0．01），而IFN-γ治疗前后均在检测灵敏度（25pg/ml)以下。（2）雷公藤甲素处理后哮喘患者PBMC分泌IL－2，IL－4，IL－5减少（P＜0．01，P＜0．05），而IFN－γ也在检测灵敏度以下。结论：雷公藤多甙对哮喘患者Th2细胞因子的产生具有明显的抑制作用，是治疗哮喘的重要机制，但雷公藤多甙对Th1细胞因子的产生也有抑制作用，说明雷公藤抑制Th2,Th1 细胞因子产生的作用无特异性。     

补肾方对慢性乙型肝炎T细胞亚群及其功能的影响

目的观察补肾方对慢性乙型肝炎患者免疫功能状态的调节作用。方法收集HBeAg阳性ALT异常者30例,给予补肾方治疗,用药6个月,于用药前、用药3个月、6个月时分别检测下列指标。采用罗氏荧光定量PCR法检测HBVDNA,外周血单个核细胞(peripheral blood mononuclear cell,PBMC)加入HBeAg、HBcAg与植物血凝素(Phytohemagglutinin,PHA)培养48h,检测培养上清液中IL-10、IFN-γ,血液中CD4^+、CD8^+、CD8^+CD28^+、CD8^+CD28^-和CD28^+T细胞的比例及加入HBeAg、HBcAg与PHA培养48h后培养细胞中CD8^+CD28^+的比例。结果补肾方治疗有效组在治疗3个月后,PBMC培养中IFN-γ较治疗前明显上升,IL-10明显下降(P<0．05,P<0．01);CD8^+CD28^+T、CD28^+T、培养后CD8^+CD28^+T细胞较治疗前明显上升,CD8^+CD28^-T细胞明显下降(P<0．05或P<0．01)。结论补肾方能明显增强Th1型细胞因子的表达,降低Th2型细胞因子的表达,促进CD8^+(Cytotoxic T lymphocyte,CTL)的表达,这可能是使HBVDNA复制得到抑制的机制之一.     

In vitro immunomodulatory activities of a newly concocted traditional Chinese medicine formula: VI-28

Previous studies have suggested that Vigconic VI-28, an anti-aging traditional Chinese medicine (TCM) formula containing Radix Ginseng and Cornu Cervi Pantotrichum, possesses immunological efficacy. This in vitro study further investigated the immunomodulatory effects of the hot water extracts of VI-28. The study included (1) colorimetric 5-bromo-2'-deoxy-uridine proliferation ELISA for estimating mitogenicity in human peripheral blood mononuclear cells (PBMC), (2) immunofluorescence staining for measuring the expression of IL-2 receptor alpha (CD25) on lymphocytes, (3) cytometric bead array (CBA) for quantifying cytokine liberation from PBMC, and (4) intracellular immunophenotyping for macrophage phagocytosis and hydrogen peroxide (H(2)O(2)) production from monocytes. The results demonstrated that VI-28 (1) could dose-dependently inhibited the proliferation of unstimulated and lipopolysaccharide-activated PBMC but enhanced the proliferation of phytohemagglutinin-activated PBMC at concentrations of <1 mg/mL, (2) significantly augmented the expression of CD25 on lymphocytes at concentrations of 0.4 mg/mL or above (p < 0.05), (3) dose dependently (0.1-1.0 mg/mL) activated macrophage phagocytosis and monocyte synthesis of H(2)O(2) and (4) significantly increased the production of cytokines IL-8, IL-10, IL-12 and IL-1beta at various concentrations of VI-28 (p < 0.05). The results suggest that VI-28 is a potential immunomodulator which probably acts through the activation of lymphocytes and monocytes.     

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??OBJECTIVE To determine the immunosuppressive activity of a novel benzothiazole derivative BD759 on T cell proliferation and its potential mode of action. METHODS T cell proliferation, CD25 expression and cell cycle distribution were measured by flow cytometer. Cytokine levels, including IL-2, IL-4, IL-6, IL-10, IL-17A and IFN-??, were determined by ELISA. RESULTS BD759 significantly inhibited human T cell proliferation, stimulated either by anti-CD3/anti-CD28 monoclonal antibodies or by an alloantigen, in a dose-dependent manner with IC₅₀ values of (3.5??0.7) and (3.3??0.9) ??mol??L^-1, respectively. No obvious cytotoxic effects of BD759 were observed on human resting na??ve T cells and peripheral blood mononuclear cells in our experimental conditions. Furthermore, BD759 did not inhibit CD25 expression or IL-2, IL-4 and IL-10 secretion, but inhibited IL-6, IL-17A and IFN-?? production and induced cell cycle arrest at the G₀/G₁ phase in activated T cells. CONCLUSION These data indicate that BD759 has no effect on T cell activation, but induces T cell cycling arrest at G₀/G₁ phase. BD759 also inhibits the secretion of inflammatory cytokines, such as IL-6, IL-17A and IFN-gamma. Thus, BD759 has the potential to be used as a lead compound for the design and development of new immunosuppressants for treating autoimmune diseases and preventing graft rejection.     

含白花蛇舌草血清对大鼠淋巴细胞和混合培养淋巴细胞增殖影响的实验研究

目的:研究含白花蛇舌草(Spreading Hedvotis Herb,简称SH)血清对植物血凝素(PHA)诱导大鼠脾淋巴细胞增殖、混合淋巴细胞培养(简称MLR)淋巴细胞增殖及对淋巴细胞分泌白细胞介素-2(IL-2)和γ-干扰素(IFN-γ)的影响。方法:大鼠20只,随机分为5组,每组4只,分为SH低剂量(4 g/kg)、中剂量(16 g/kg)、高剂量(24 g/kg)组,含环孢素A(CsA)血清组,按相应药物灌胃,空白血清组用等量生理盐水灌胃。MTT法测定PHA诱导的大鼠脾淋巴细胞和MLR淋巴细胞增殖,ELISA法测定PHA诱导淋巴细胞分泌IL-2、IFN-γ含量。结果:SH高剂量组可抑制PHA诱导大鼠脾淋巴细胞增殖,SH高剂量组、含CsA血清组与空白血清组比较,差异均有显著性意义(P0.05)。双向MLR体系中,各剂量组含SH血清均可抑制淋巴细胞增殖,其中SH高剂量组与空白血清组比较,差异有显著性意义(P0.05);在单向MLR体系中,各剂量组含SH血清可调节淋巴细胞增殖。SH中剂量组、SH高剂量组血清抑制脾淋巴细胞增殖,与空白血清组比较,差异均有显著性意义(P0.05)。含CsA血清组血清可抑制单向、双向MLR体系中淋巴细胞增殖,与空白血清组比较,差异均有显著性意义(P0.05)。SH中剂量组、SH高剂量组、含CsA血清组血清均可显著抑制大鼠脾淋巴细胞分泌IL-2、IFN-γ含量,与空白血清组比较,差异均有显著性意义(P0.05)。结论:SH能抑制淋巴细胞和混合培养淋巴细胞增殖,其作用机制可能与抑制淋巴细胞分泌IL-2、IFN-γ有关。     

Anti-inflammatory effects of an ethanolic extract from Clematis mandshurica Rupr

Clematis mandshurica Rupr (Ranunculaceae) roots are used in traditional Korean medicine to treat inflammation-related diseases. Therefore, we undertook to investigate their inhibitory effect on inflammation under non-cytotoxic conditions. The ethanolic extract of Clematis mandshurica at 100 microg/ml was found to significantly block the production of the pro-inflammatory mediators, nitric oxide (NO) and prostaglandin E(2) (PGE(2)), in lipopolysaccharide (LPS)/interferon(IFN)-gamma-stimulated mouse peritoneal macrophages, by up to 77% and 59%, respectively. In addition, it significantly inhibited cell proliferation and cytokine production (interleukin (IL)-2 and IFN-gamma) in splenocytes stimulated with Con A (concanavalin A; 5 microg/ml). Furthermore, when splenocytes from extract fed mice (200 mg/kg for 2 weeks) were activated with Con A, cell proliferation and the production of IL-2 and IFN-gamma were significantly inhibited. In addition, the extract reduced in vivo inflammation in oxazolone-induced delayed type hypersensitivity (DTH) model mice. Taken together, these data suggest that Clematis mandshurica is able to ameliorate inflammatory disease by exerting an anti-inflammatory effect in cases of proinflammatory and cell-mediated inflammation.     

灵芝水提取物对人免疫细胞及IL-6的调节作用

目的：观察灵芝水提取物对人免疫细胞及细胞因子的调节作用。方法：灵芝水提取物（ＧＬ－Ｗ）作用于人外周血淋巴细胞（ＰＢＬ）,用ＭＴＴ法及流式细胞仪技术检测。结果：１．ＧＬ－Ｗ以经植物凝集素（ＰＨＡ）活化的和未经ＰＨＡ活化的人ＰＢＬ均有促增殖效应,其中经ＰＨＡ活化的增殖反应显著（Ｐ〈０．０５）,细胞周期检测发现其主要促进细胞进入Ｓ期,表明ＧＬ－Ｗ可促进人ＰＢＬ的ＤＮＡ合成。２．选择最大促进增殖效诮浓度的     

Effects and mechanisms of total glucosides of paeony on synoviocytes activities in rat collagen-induced arthritis

The aim of the study was to investigate the effects of TGP, an active compound extracted from the roots of Paeonia lactiflora Pall, on the activities of synoviocytes in rats with collagen-induced arthritis (CIA) and its possible mechanisms. CIA was induced in male Sprague-Dawley (SD) rats immunized with chicken type II collagen (CII) in Freund's complete adjuvant (FCA). Synoviocytes proliferation was determined by 3-(4, 5-2dimethylthiazal-2yl) 2, 5-diphenyltetrazoliumbromide (MTT) assay. Tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), prostaglandin E(2) (PGE(2)) and cyclic adenosine monophosphate (cAMP) levels in synoviocytes were measured by radioimmunoassay (RIA). E-prostanoid (EP)(2) and EP(4) receptors were analyzed by Western blot analysis. The results showed that TGP significantly inhibited the proliferation of synoviocytes, decreased the production of IL-1, TNF-alpha and PGE(2) and elevated the levels of cAMP. Further study showed that TGP could up-regulate the expression of EP(2) and EP(4). These results indicated that TGP might exert its anti-inflammatory effects through inhibiting the production of pro-inflammatory mediators in synoviocytes of CIA rats, which might be associated with its ability to regulate cAMP-dependent EP(2)/EP(4)-mediated pathway.     

人参多糖对肾病患者外周血单核细胞诱生白细胞介素－2及其受体活性影响

通过体外研究证实，人参多糖对健康人和乙肝相关肾炎、肾病综合征和慢性肾功能哀竭患者外周血单核细胞诱生白细胞介素－２及其受体活性具有显著的促进作用，且在所试验的剂量范围内对健康人和三种肾病患者的作用均呈量效依赖性。毒性试验表明人参多糖对血细胞无毒性作用。认为人参作为一种有效生物反应调节剂，应用于临床具有重要的实用和开发价值。     

Immunomodulatory activity of biopolymeric fraction BOS 2000 from Boswellia serrata

Oral administration of BOS 2000 (1-10 mg/kg) elicited a dose related increase in the delayed hypersensitivity reaction (early 24 h and delayed 48 h) in mice. It also stimulated the IgM and IgG titre expressed in the form of plaques (PFC) and complement fixing antibody titre. The concentration of cytokines (IL-4, IFN-gamma and TNF-alpha) in serum with respect to T cell interactions, i.e. (CD4/CD8) and the proliferation of lymphocytes were significantly increased at 10 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of BOS 2000 in a dose-dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF-alpha and IFN-gamma production as a mode of action.     

Brazilian green propolis and its constituent, Artepillin C inhibits allogeneic activated human CD4 T cells expansion and activation

Ethnopharmacological relevance

Propolis has long been used as a popular folk medicine by various ethnic groups due to its wide spectrum of alleged biological and pharmaceutical properties including anti-microbial, anti-cancer and anti-inflammatory functions. All these can be linked to the modulation of immune function. Therefore, it will be relevant for us to find out whether there is any novel compound that can account for such action and the mechanism involved.

Aim of the study

We investigated the immune modulating effect of Brazilian green propolis (PBrazil) and its constituent Artepillin C (Art-C) by using mixed leukocytes reaction.

Materials and methods

The cytotoxic effect of Art-C on non-tumorigenic human liver cell line miHA and non-tumorigenic human kidney cell line HK-2 as well as human peripheral blood mononuclear cells (PBMCs) were measured by XTT cell proliferation assay. The effect of PBrazil and Art-C on T cell proliferation and activation were determined by using carboxyfluorescein succinimidyl ester (CFSE) and by CD25 expression, respectively. Cytokines including tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), interleukins such as IL-2, IL-17 were measured by intracellular cytokine staining and IL-10 was measured by ELISA. The effect of PBrazil and Art-C on regulatory T cells (Treg) induction was determined by the Foxp3 expression. The apoptotic effect of these compounds on CFSE labeled alloreactive T cells was measured by using Annexin V.

Results

Using mixed leukocytes reaction we demonstrated for the first time that both Art-C and PBrazil significantly inhibited the alloreactive CD4 T cell proliferation, activation, and suppressed the expressions of IL-2, IFN-γ and IL-17 in these alloreactive CD4 T cells. The inhibitions of Art-C and PBrazil on CD4 T cells were not due to direct cytotoxic effect on PBMC or inducing regulatory T cells differentiation. Both Art-C and PBrazil were found to selectively induce apoptosis in proliferating T cells. The anti-proliferative effect of Art-C and PBrazil were reversible and were also applied to the activated T cells.

Conclusions

In conclusion, our results indicated that Art-C and PBrazil can suppress alloreactive CD4 T cell responses in vitro, suggesting that Art-C could be used as a potential immunosuppressant, either solely or as adjunct agent in treating graft versus host disease.