Targeted Antigen Delivery to DEC-205+ Dendritic Cells for Tolerogenic Vaccination

Dendritic cells (DCs) and Foxp3-expressing CD4⁺ regulatory T (Treg) cells play non-redundant roles in the maintenance of peripheral tolerance to self-antigens, thereby preventing fatal autoimmunity. A common hallmark of intra- and extra-thymic Treg cell lineage commitment is the induction of Foxp3 expression as a consequence of appropriate T cell receptor engagement with MHC class II:agonist ligand. It has now become increasingly clear that agonist ligand presentation by immature DCs in the steady state induces T cell tolerance by both recessive and dominant mechanisms, rather than promoting productive T helper cell responses. In this context, the ability of steady-state DCs to promote the extrathymic conversion of initially naïve CD4⁺Foxp3^- T cells into Foxp3⁺ Treg cells is of particular interest as it provides novel perspectives to enhance antigen-specific Treg cell function in clinical settings of unwanted immunity, such as β-cell autoimmunity.     

Evaluation of the Effect of Inactivated Transmissible Gastroenteritis Virus Vaccine with Nano Silicon on the Phenotype and Function of Porcine Dendritic Cells

A transmissible gastroenteritis virus (TGEV) is a porcine enteropathogenic coronavirus, causing acute swine enteric disease especially in suckling piglets. Mesoporous silica nanoparticles (MSNs) are safe vaccine adjuvant, which could enhance immune responses. Our previous research confirmed that nano silicon had immune-enhancing effects with inactivated TGEV vaccine. In this study, we further clarified the immune-enhancing mechanism of the inactivated TGEV vaccine with MSNs on porcine dendritic cells (DCs). Our results indicated that the inactivated TGEV vaccine with MSNs strongly enhanced the activation of the DCs. Expressions of TLR3, TLR5, TLR7, TLR9, and TLR10, cytokines IFN-α, IL-1β, IL-6, IL-12, and TNF-α, cytokine receptor CCR-7 of immature DCs were characterized and showed themselves to be significantly higher in the inactivated TGEV vaccine with the MSN group. In summary, the inactivated TGEV vaccine with MSNs has effects on the phenotype and function of porcine DCs, which helps to better understand the immune-enhancing mechanism.     

Solar-Simulated Ultraviolet Radiation Induces Abnormal Maturation and Defective Chemotaxis of Dendritic Cells

Exposure to ultraviolet (UV) light induces immunosuppression. Different evidences indicate that this phenomenon is mainly a consequence of the effect of UV light on skin dendritic cells (DC). To investigate the cellular and molecular basis of this type of immunosuppression, we assessed in vitro the effect of solar-simulated UV radiation on the phenotypic and functional characteristics of human monocyte-derived DC and Langerhans-like DC. UV radiation induced a decreased expression of molecules involved in antigen capture as DC-SIGN and the mannose receptor. This effect was accompanied by a diminished endocytic capacity, an enhanced expression of molecules involved in antigen presentation such as major histocompatibility complex-II and CD86, and a significant increase in their capability to stimulate T cells. Furthermore, irradiated DC failed to acquire a full mature phenotype upon treatment with lipopolysaccharide. On the other hand, solar-simulated radiation induced the secretion of tumor necrosis factor-αand interleukin (IL)-10 by DC, but no IL-12. Interestingly, solar-simulated UV radiation also caused an altered migratory phenotype, with an increased expression of CXCR4, and a lack of induction of CCR7, thus correlating with a high chemotactic response to stromal cell-derived factor 1(SDF-1) (CXCL12), but not to secondary lymphoid tissue chemokine (SLC) (CCL21). These data indicate that solar-simulated UV radiation induces a defective maturation and an anomalous migratory phenotype of DC.     

Evaluation of the Immunomodulatory Effect of Curdlan on Maturation and Function of Mouse Spleen-Derived Dendritic Cells

Background: T helper 1 and T helper 17 cells play important roles in immunity against foreign invaders. Differentiation of these Th subsets is affected by state of maturation and cytokines that are produced by dendritic cells (DCs). Curdlan is a linear (1→3)-β- glucan and has shown activity against tumors and infectious agents. Objective: This study aims to investigate whether curdlan plays its role through affecting the maturation and cytokine production by DCs. Methods: DCs were isolated from the spleen of BALB/c mice by MACS method. After an overnight culture of DCs in the presence of curdlan, the expression levels of CD40, CD86, and MHC-II molecules were determined by flow cytometry. The production of cytokines involved in Th1 and Th17 cell differentiation (IL-12 and IL-6, respectively) was also evaluated by ELISA. Lipopolysaccharide (LPS) treated and untreated cells were considered as positive and negative controls, respectively. Results: The results of this study did not show a significant difference in the levels of surface expression of CD40 (p=0.82), CD86 (p=0.79), and MHC class II (p=0.84) molecules upon exposure to curdlan. However, LPS increased the intensity of CD40 expression on dendritic cells (p=0.04). In addition, it was revealed that curdlan-exposed DCs are not able to produce a significant amount of IL-6 and IL-12 cytokines. Conversely, LPS-treated DCs were able to make a significant amount of IL-12 (p=0.005). Conclusion: The results of the present study suggest that curdlan has no effect on Th1 or Th17 differentiation while LPS may induce Th1 deviation by induction of CD40 expression and IL-12 production.     

LPS诱导的DCs成熟在小鼠免疫耐受中的作用研究

目的探讨LPS诱导DCs成熟对于过敏原致耐受中的作用。方法分离、培养、纯化DCs,加入LPS100 ng/m l诱导DCs成熟,然后加入OVA共同孵育24 h后收集DCs,按每鼠106/m l气管内接种。以PBS作为阴性对照,以未加LPS刺激、与OVA共同培养的DCs气管内接种作为阳性对照。接种次日后将小鼠置于雾化箱内以1%OVA雾化,连续5 d。结果 LPS刺激后DCs对OVA的吞噬能力下降（其吞噬能力在30 m in时,分别为75.7%和34%;60 m in分别下降为71%和29.7%）,将这些DCs接种于气管内不能诱发典型的肺部过敏性炎症,与注射PBS相似;而未经LPS处理的DCs与OVA共培养后再接种于气管,经雾化激发后可引起典型的过敏性炎症。结论 LPS诱导的DCs成熟后对过敏原的递呈能力下降可能是机体免疫耐受中的主要原因。     

益生菌对炎症和免疫系统影响的研究进展

益生菌有利于人体健康,现在不但作为食品添加剂广泛地使用,还可应用于胃肠炎的治疗、胃肠管道癌症的防治以及术后恢复等.益生菌是如何调节免疫系统和影响炎症？益生菌可否作为治疗疾病的手段？本文将做进一步的讨论.     

钾离子通道开放剂停搏液对未成熟心肌电生理特性的影响

目的观察钾离子通道开放剂加入St.Thomas Ⅱ液内,能否改善缺血后未成熟心肌的保护作用.方法未成熟新西兰大耳白兔(2～3周)24只随机分为对照组、处理组和预处理组(各组n＝8),取离体心脏乳头肌标本.对照组充氧台氏液灌注平衡60分,St.Thomas Ⅱ号液灌注停跳30分,充氧台氏液复灌60分.处理组将钾离子通道开放剂Nicorandil加入St.Thomas Ⅱ号液灌注停跳30分.预处理组使用特异性钾离子通道阻滞剂Glibenclamide在平衡期后15分预处理乳头肌标本.利用传统玻璃微电极技术记录心肌细胞动作电位变化.结果①处理组乳头肌停跳后静息电位低于对照组和预处理组,有显著性差异(P＜0.05).②处理组停跳时间短于其他2组,有显著性差异(P＜0.05).③复灌早期处理组50%动作电位时程(APD50、)、90%动作电位时程(APD90)短于停跳前,有显著性差异(P＜0.01),而对照组和预处理组APD50、APD90都明显延长,有显著性差异(P＜0.05),但2组间无显著性差异.④再灌注后处理组动作电位振幅(APA)、动作电位超射值(OS)及动作电位最大复极速度(Vmax)的恢复优于对照组和预处理组,有显著性差异(P＜0.05),而对照组和预处理组间无显著性差异.结论钾离子通道开放剂Nicorandil停搏液可以引起心肌细胞停跳在超极化状态,减轻心肌缺血再灌注损伤,提高再灌注后心肌功能的恢复率.     

Immunomodulatory Effects of Mice Mesenchymal Stem Cells on Maturation and Activation of Dendritic Cells

Background: Mesenchymal stem cells (MSCs) possess a wide range of immunomodulatory functions mostly in immune cells including dendritic cells (DCs). DCs are the key cells in the immune response and play an important role in initiating cell-mediated immunity. Objective: To evaluate the immunomodulatory effects of MSCs supernatant on maturation and function of DCs. Methods: Bone marrow derived mice MSCs were isolated and cultured. Twenty-four, forty-eight and seventy-two hours after passage 6, supernatants were collected and MSCs were assessed by cytometric analysis for the expression of CD34, CD44, CD45 and SCA-1. Splenic DCs were isolated using MACS and then co-cultured with MSCs supernatant. Expression of CD86, CD40 and MHC-II on DCs were also evaluated by cytometry. H 3-thymidine incorporation by proliferating T cells was determined in two separate MLR assay settings. In one setting, DCs were co-cultured with T cells in the presence of MSCs supernatant, and in the other setting DCs were treated with MSCs supernatant and then were co-cultured with T cells. Production of IL-12, IL-6 and IL-10 cytokines was measured in the supernatant of DCs treated with MSCs supernatant. We also measured IFN- γ and IL-4 levels in MLR supernatant. Results: The results showed that 72h MSCs supernatant could decrease the expression of MHC-II and CD86. The T cell proliferation was inhibited in the presence of MSCs supernatant and MSCs supernatant treated DCs as demonstrated by MLR assay. A significant increase in IL-4 level and a non significant decrease in IFN- γ level in MLR supernatant were observed. However, IL-6, IL-10 and IL-12 production did not change significantly. Conclusion: MSCs supernatant has a time dependent effect on the maturation of DCs. Also, it could alter cytokine production from responding T cells toward Th2. Generally, the findings of this study supported the immunomodulatory effect of MSCs supernatant on DCs maturation and function.     

The Immunostimulatory Effect of Lactic Acid Bacteria in a Rat Model

Background: Probiotics are “live”, beneficial microbes that provide important health benefits in their hosts. There is significant interest in the modulation and regulation of the immune function by probiotics. Objective: To investigate the immunomodulatory effects of a probiotic mixture, including Lactobacillus and Bifidobacterium species, by detecting serum cytokine and immunoglobulin levels. Methods: The rats were randomly divided into 4 groups. The first group was “Control group” and other 3 groups were probiotic application groups who received different doses of probiotics. The probiotic mixture included 12 probiotic bacteria, mostly Lactobacillus and Bifidobacterium strains. Probiotic mixture was administered to rats for 12 consecutive days. TNF-α, TGF-β, IL-1-β, IL-6, and IL-10 levels as well as serum IgG and IgA concentrations were detected in the sera after 12 days. Results: Probiotics led to a decrease in the levels of TNF-α, IL-6 and TGF-β; however, they led to increase in the serum levels of IL-10, IgG and IgA. There were significant differences between control group and probiotic application groups (p<0.05). Conclusion: These data suggest that the commensal microbiota are important for stimulating both proinflammatory and regulatory responses in order to rapidly clear infections and minimize inflammation-associated tissue damage.     

青春型双歧杆菌对哮喘儿童树突状细胞功能的影响

目的研究青春型双歧杆菌对过敏性哮喘儿童外周血单个核细胞来源的树突状细胞（dendritic calls,DC）表面共刺激分子表达及其细胞因子分泌的影响。方法从15例过敏性哮喘儿童和15例非哮喘儿童的外周血单个核细胞诱导生成未成熟DC．与青春型双歧杆菌共培养48小时后．用流式细胞仪检测DC表面CD86和HLA-DR分子的表达,用ELISA方法检测培养上清中自细胞介素（IL）-10、IL-12和IFN-γ的水平。结果经双歧杆菌刺激后,哮喘儿童DC表面CD86表达明显增高（P〈0．05）,DC分泌IL-12和IFN-γ水平明显增高;而双歧杆菌刺激对非哮喘儿童的CD86和HLA-DR表达无明显影响,但可使其DC分泌IL-12及IL-10水平明显增高。结论青春型双歧杆菌既可以通过上调CD86的表达,促进DC成熟;又可刺激DC分泌IL-12和IFN-γ,改变Th2优势分化,纠正Th1/Th2失衡,这可能是益生菌防治变态反应性疾病的机制之一。     

益生菌治疗大鼠实验性结肠炎效果观察及对T细胞亚群的影响

目的评价益生菌对大鼠实验性结肠炎的疗效,以及研究治疗后体内T细胞亚群变化情况。方法选择北京大学人民医院消化内科与北京医院特需医疗科于2004年9月至2005年2月建立实验性结肠炎模型,设立不同剂量益生菌治疗组、阴性对照组和阳性对照治疗组(泼尼松治疗组及柳氮磺胺吡啶治疗组),组织学积分评定疗效;流式细胞仪检测外周血、脾脏和结肠CD4 、CD8 T细胞亚群变化情况。结果大剂量益生菌对治疗大鼠实验性结肠炎有效;结肠炎大鼠结肠内CD4 、CD8 T细胞均增加,益生菌治疗后回落,而CD4 /CD8 比值及外周循环T细胞亚群无变化。结论大剂量益生菌治疗大鼠实验性结肠炎有效,可能与免疫调节机制有关。     

ADMA对单核细胞来源的树突状细胞成熟和免疫的影响

目的:通过观察非对称性二甲基精氨酸(ADMA)对树突状细胞(dendritic cells,DCs)成熟及免疫的影响来探讨AS形成的可能机制。方法:贴壁法分离人外周血单核细胞,在含重组人粒-巨噬细胞集落刺激因子(rhGM-CSF 20ng/ml)和重组人白细胞介素-4(rhIL-4,10ng/ml)的完全培养基中培养,五天后收集imDC,用1、8、16umol/L的ADMA干预未成熟DC 24h。用流式细胞术检测DC细胞表面分子的表达、吞噬能力及DC的凋亡,用混合淋巴细胞反应检测成熟DC刺激T淋巴细胞增殖的能力,用ELISA检测DC细胞因子的分泌。结果:生理浓度ADMA并不刺激DC成熟及分化;但病理浓度ADMA抑制DC成熟;抑制DC诱导的T淋巴细胞增殖;诱导DC凋亡:抑制DC分泌IL-12细胞因子、TNF-α及IL-10细胞因子。结论:生理浓度ADMA并不刺激DC成熟及分化;但病理浓度AD-MA抑制DC成熟和免疫。     

益生菌治疗大鼠实验性结肠炎效果观察及对T细胞亚群的影响

目的评价益生菌对大鼠实验性结肠炎的疗效，以及研究治疗后体内T细胞亚群变化情况。方法选择北京大学人民医院消化内科与北京医院特需医疗科于2004年9月至2005年2月建立实验性结肠炎模型，设立不同剂量益生菌治疗组、阴性对照组和阳性对照治疗组（泼尼松治疗组及柳氮磺胺吡啶治疗组）。组织学积分评定疗效；流式细胞仪检测外周血、脾脏和结肠CD4^＋、CD8^＋T细胞亚群变化情况。结果大剂量益生菌对治疗大鼠实验性结肠炎有效；结肠炎大鼠结肠内CD4^＋、CD8^＋T细胞均增加，益生菌治疗后回落，而CD4^＋／CD8^＋比值及外周循环T细胞亚群无变化。结论大剂量益生菌治疗大鼠实验性结肠炎有效，可能与免疫调节机制有关。     

复合益生菌制剂对急性坏死性胰腺炎大鼠的保护作用

背景:肠道细菌易位是重症急性胰腺炎(SAP)时胰腺坏死感染的主要来源,因此保护肠黏膜屏障对SAP的治疗具有重要意义。目的:探讨复合益生菌制剂对急性坏死性胰腺炎(ANP)大鼠肠黏膜屏障和胰腺损伤的保护作用。方法:50只SPF级大鼠随机分为假手术组(n=10)、ANP模型组(n=20)和益生菌干预组(n=20)。采用胰腺被膜下均匀注射牛磺胆酸钠制备ANP模型,干预组术前30 min以双歧杆菌四联活菌片溶液灌胃。术后6 h采集标本,检测血淀粉酶、二胺氧化酶(DAO)、TNF-α水平,观察胰腺组织病理学表现和末端回肠组织超微结构。结果:ANP模型组血淀粉酶、DAO、TNF-α水平和胰腺组织学评分均显著高于假手术组(P<0.05),益生菌干预组各项指标均较ANP模型组有所改善(P<0.05)。假手术组末端回肠黏膜结构完整;ANP模型组回肠黏膜上皮细胞微绒毛萎缩、排列稀疏,细胞间连接松弛;益生菌干预组微绒毛稍稀疏,细胞间连接紧密度较ANP模型组增高。结论:复合益生菌制剂对ANP大鼠具有保护作用,不仅能减轻肠黏膜损伤,保护肠黏膜屏障功能,还能减轻胰腺局部损伤和全身性炎症反应。     

Effect of Lactobacillus fermentum on serum lipids in subjects with elevated serum cholesterol

Background and AimsThere is increasing interest in the use of natural therapies to reduce elevated low density lipoprotein (LDL) cholesterol. This study assessed the effects of PCC^® Lactobacillus fermentum on LDL cholesterol and other lipid fractions.Methods and resultsThis was a single centre, double blind, placebo-controlled, parallel design trial in volunteers having total cholesterol ≥4 mmol/L. Subjects (n = 46) were randomised to receive either Lactobacillus fermentum 2 capsules twice daily (each capsule containing 2 × 10⁹ colony forming units) or matching placebo for a period of 10 weeks. Main outcome measures were percentage changes in LDL cholesterol and other lipids, changes in liver enzymes and other safety tests. Two subjects withdrew early in the study, 1 for personal reasons and 1 because of bowel discomfort. Three other subjects experienced some bowel discomfort but still completed the study. LDL cholesterol showed a modest downward trend on both Lactobacillus fermentum and placebo of 7.0% and 5.2% respectively. This trend did not reach statistical significance over time, nor was there a significant difference between the treatment arms. There were no significant changes over time or between treatments noted in total cholesterol, high density lipoprotein cholesterol or triglycerides. There were no significant changes in liver enzymes or other safety parameters with time or between treatments.ConclusionLactobacillus fermentum did not appear to produce a major change in serum lipid fractions, but a small effect cannot be excluded.     

益生菌对重症患者呼吸机相关性肺炎的预防作用及肠道功能的影响

目的:观察益生菌制剂对重症监护病房(ICU)危重症患者呼吸机相关性肺炎(VAP)的预防效果及肠道功能的影响。方法:本试验为前瞻、随机、对照研究,将符合入组条件的155例患者随机分为对照组和试验组。2组患者给予相同的处理措施,在此基础上,试验组给予连续3周的益生菌(双歧杆菌、乳酸杆菌混合物)补充。比较2组患者VAP的发生率、肠道菌群变化及消化道症状发生率等。结果:共120例患者完成随访,其中对照组58例,试验组62例。试验组VAP的发生率低于对照组(38.7%比62.1%, χ2=6.541, P<0.05)。试验组口咽部及胃部微生物定植的总检出率低于对照组(51.6%比89.7%, χ2=20.052, P<0.05)。腹内压升高、腹泻、肠鸣音减弱或消失的发生率在试验组明显降低( χ2值分别为8.439、10.849、13.327, P值均<0.05)。试验组患者肠道中乳酸杆菌比例升高。试验组机械通气时间和ICU住院时间较对照组明显缩短( t值分别为8.051、6.538, P值均<0.05)。 结论:益生菌制剂能够明显提高患者肠道中乳酸杆菌比例,显著降低ICU危重症患者VAP发生率,并降低胃肠道症状的发生,减少患者机械通气时间和ICU住院时间,具有临床推广使用的价值。     

酸性复灌液对未成熟心肌内皮素和羟脯氨酸的影响

目的　观察不同pH值HEPES -KH复灌液对未成熟心肌内皮素和羟脯氨酸的影响。方法　采用Langen dorff离体灌注模型 ,分为 3组 ,每组 8只 ,正常对照组 (C) ,仅灌注pH 7 4HEPES -KH液 90min ;缺血 /再灌组 (I/R) ,灌流pH 7 4HEPES -KH液 2 0min后缺血 6 0min ,用pH 7 4HEPES-KH液恢复灌注 30min ;酸性灌注组 (E) ,缺血 6 0min后 ,先用pH 6 8HEPES -KH液灌注 5min ,然后换成pH7 1灌注 5min ,最后恢复pH 7 4灌注 2 0min。以心肌羟脯氨酸 (HP)和血清内皮素 (ET)含量作为观察指标。结果　E组HP含量高于I/R组 (P <0 0 5 ) ,ET含量低于I/R组 (P <0 0 5 )。结论　复灌初期应用梯度酸性复灌液有助于未成熟心肌间质的保护。     

Incomplete Killing And Enhanced Activation of Islet-Reactive CD8+ T Cells by FasL-Expressing Dendritic Cells Limits Protection from Diabetes

AIMS: Autologous dendritic cells (DC) are a promising tool for induction of cytotoxic CD8+ T cell immunity against tumors and chronic viral infections. When armed with the death-inducing Fas-ligand (FasL, CD195), DC attenuate delayed-type hypersensitivity reactions and allotransplant rejection by promoting activation-induced cell death in T cells. We investigated the possibility of using FasL-expressing DC to induce deletion of islet-reactive CD8+ T cells in vivo, and to prevent destruction of pancreatic islets in a model of autoimmune diabetes. METHODS: DC, propagated from mouse bone marrow cells, were purified and made to express FasL and islet-antigen via plasmid transfection. CD8+ T cells (OT-I cells) recognizing the antigen, ovalbumin, were adoptively transferred to transgenic mice expressing ovalbumin in islets (RIP-OVA^lo mice), and these mice were primed with ovalbumin. To test the potential of DC to prevent diabetes in this model, the mice were later intravenously vaccinated with the transfected DC. RESULTS: Transfected DC induced partial deletion of antigen-reactive CD8+ T cells in vivo and reduced the level of lymphocyte infiltration into pancreatic islets. Diabetes developed less frequently in vaccinated mice, but this effect was limited. Further in vitro analysis showed that FasL-expressing DC not only deleted many of the responding CD8+ T cells but also promoted the expansion of surviving cells and their IFN-γ production. CONCLUSIONS: FasL-expressing DC can also have stimulatory effects on CD8+ T cells warranting further investigation into the optimal design of tolerance-promoting DC-vaccination to prevent autoimmune diabetes.     

Effect of probiotic Lactobacillus rhamnosus GG intervention on global serum lipidomic profiles in healthy adults

AIM： To investigate the effect of three weeks＇ intervention with a probiotic Lactobacillus rhamnosus GG （LGG） bacteria on global serum lipidomic profiles and evaluate whether the changes in inflammatory variables （CRP, TNF-α and IL-6） are reflected in the global lipidomic profiles of healthy adults. METHODS： We performed UPLC/MS-based global lipidomic platform analysis of serum samples （n = 26） in a substudy of a randomised, double-blind, placebo- controlled 3-wk clinical intervention trial investigating the immunomodulatory effects of probiotics in healthy adults. RESULTS： A total of 407 lipids were identified, corresponding to 13 different lipid classes. Serum samples showed decreases in the levels of lysophosphatidylcholines （LysoGPCho）, sphingomyelins （SM） and several glycerophosphatidylcholines （GPCho）, while triacylglycerols （TAG） were mainly increased in the probiotic LGG group during the intervention. Among the inflammatory variables, IL-6 was moderately associated by changes in global lipidomic profiles, with the top-ranked lipid associated with IL-6 being the proinflammatory LysoGPCho （20：4）. There was a weak association between the lipidomic profiles and the two other inflammatory markers, TNF-~ and CRP. CONCLUSION： This was the first study to investigate the effects of probiotic intervention on global lipidomic profiles in humans. There are indications that probiotic LGG intervention may lead to changes in serum global lipid profiles, as reflected in decreased GPCho, LysoGPCho and SM as well as mainly increased TAG.