Decreased somatostatin is related to the hypersensitivity of intestinal epithelia to LPS via upregulated TLR4-TBK1 pathway in rats chronically exposed to ethanol

Chronic alcoholics are predisposed to the development of a systemic inflammatory response syndrome, which is usually triggered in the gut. This study aimed to investigate in rats the role of intestinal epithelial inflammatory responsiveness in the susceptibility of alcoholics to excessive inflammation. Thirty Wistar rats were randomly divided into three groups: 10 rats killed immediately after acclimation (baseline control), 10 rats treated with 25% (vol/vol) ethanol for 6 months (ethanol group), and 10 rats given double-distilled water until killed simultaneously with the ethanol group (9-month control). The intestinal microflora, the epithelial histology and ultrastructure, the level of Toll-like receptor 4 (TLR4), TANK-binding kinase-1 (TBK1), and activated nuclear factor-κB (NF-κB) in the intestinal mucosa, and somatostatin (SST) levels in plasma and small intestine were evaluated in each group. Isolated intestinal epithelia from each rat were used to examine lipopolysaccharide (LPS) responsiveness with or without SST pretreatment by quantification of TLR4, TBK1, activated NF-κB, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Compared with both the control groups, the amount of mucosal Escherichia coli in the ethanol group was not changed, whereas the number of intestinal lactobacilli in the ethanol group was significantly reduced (P < .05). Mild inflammatory injury and upregulation of TLR4 and TBK1 were observed in the intestinal mucosa of the ethanol group. The LPS-enhanced in vitro expression of TLR4, TBK1, and production of IFN-γ and TNF-α in isolated intestinal epithelia of the ethanol group were significantly higher than those in either control group (P < .05) and were dramatically inhibited by SST (P < .05), whereas NF-κB was activated by LPS only in the control groups. The plasma and intestinal levels of SST in the ethanol group were significantly lower than those in either control group (P < .05). These findings suggest that impairment of intestinal SST production by chronic ethanol administration leading to upregulation of the TLR4-TBK1 pathway may be one of the mechanisms underlying the LPS hypersensitivity of intestinal epithelia.     

脂多糖经MyD88/NF-κB信号途径抑制Bewo细胞中乙型肝炎病毒复制

目的 探讨Toll样受体4(TLR4)配体脂多糖(LPS)抑制人滋养层细胞Bewo中乙型肝炎病毒( HBV)复制的作用机制,为防治HBV宫内感染提供依据.方法 首先将2μg 1.3倍HBV全基因重组质粒pcDNA3.1(+)-HBV1.3转染Bewo细胞12h后,以TLR4配体LPS处理3d.尔后用LPS处理Bewo细胞,观察IFN-β、TNF-α表达的动力学、NF-κB拮抗剂二硫代氨基甲酸吡咯烷( PDTC)对LPS诱导Bewo细胞产生细胞因子的作用.采用微粒子酶免疫分析法(MEIA)和荧光定量PCR法分别检测HBsAg、HBeAg和HBV DNA水平,并以ELISA和RT-PCR分别检测IFN-β、TNF-α水平及TIR结构域的转接蛋白(TRIF)、髓样分化蛋白(MyD88)表达.结果 与对照组比较,LPS可显著抑制Bewo细胞中HBV复制(P＜0.01),且LPS可显著诱导Bewo细胞产生TNF-α(P＜0.05),呈时间和剂量依赖性.PDTC可抑制LPS诱导细胞产生TNF-α,显著低于对照组(P＜0.01),但对IFN-β无显著作用(P＞0.05).与对照组比较,LPS可诱导HBV重组质粒转染的Bewo细胞表达MyD88(P＜0.01).结论 通过MyD88/NF-κB信号途径诱导Bewo细胞产生TNF-α,TLR4配体LPS可显著抑制HBV复制.     

Toll样受体7激动剂对荷瘤小鼠肾癌的抑制作用及免疫机制

目的　研究Toll样受体7激动剂Imiquimod对荷瘤小鼠肾癌的抑制作用及免疫机制。方法　用Renca肾癌细胞构建BABL/c小鼠肾癌模型,实验分为control组、imiquimod组和sorafenib组。观察3组小鼠肿瘤体积及重量、抑瘤率,qPCR法检测肿瘤组织中炎性相关因子肿瘤坏死因子α（Tumor necrosis factor-α,TNF-α）、白细胞介素1β（Interleukin-1β,IL-1β）、白细胞介素6（Interleukin-6,IL-6）、干扰素α（Interferon-α,IFN-α）、干扰素β（Interferon-β,IFN-β）、干扰素γ（Interferon-γ,IFN-γ）及Toll样受体通路相关因子Toll样受体7（Toll-like receptor 7,TLR7）、髓样分化因子88（Myeloid differentiation primary response gene 88,MyD88）、核因子κB （Nuclear factor κB,NF-κB）mRNA表达,Western blot法检测瘤组织中TLR7、MyD88、NF-κB蛋白表达。流式细胞术检测脾脏中CD4+T、CD8+T、CD4+IFN-γ和CD8+IFN-γ细胞比例。结果　与control组比较,imiquimod组和sorafenib组小鼠的肿瘤体积及瘤体重量降低（P＜0.05）;与control组相比,imiquimod组TNF-α、IL-6、IL-1β、IFN-α、IFN-β、IFN-γ及TLR7、MyD88、NF-κB mRNA水平升高（P＜0.05）,而sorafenib组各因子略有上升（P＞0.05）;与control组和sorafenib组比较,imiquimod组CD4+T、CD8+T细胞比例有不同程度的升高（P＞0.05）,CD4+IFN-γ和CD8+IFN-γ细胞比例增加（P＜0.05）。结论　Toll样受体7激动剂通过活化TLR7-MyD88-NF-κB信号刺激多种炎性细胞因子分泌,并上调CD4+T、CD8+T比例及功能,对荷瘤小鼠肾癌发挥抑制作用。     

芩百清肺浓缩丸治疗肺炎支原体感染大鼠模型细胞因子表达水平变化

目的分析芩百清肺浓缩丸治疗肺炎支原体(MP)感染大鼠细胞因子表达水平变化,进一步探究芩百清肺浓缩丸对其免疫机制及炎症反应的调控。方法 SPF级成年雄性Wistar大鼠(90~110 g)60只随机分为4组:正常对照组、肺炎支原体肺炎模型(MP组)、芩百清肺浓缩丸组及阿奇霉素组。通过经鼻反复感染MP构建感染模型,对照组鼻内滴入等体积支原体改良培养基。芩百清肺浓缩丸组及阿奇霉素组从MP感染日起连续4天分别给与芩百清肺浓缩丸3.2 g/kg及阿奇霉素25 mg/kg,每日1次,模型组给予等量生理盐水灌胃。给药后第5天抽取血样、分离血清检测各组大鼠血清中肿瘤坏死因子-α(TNF-α)及γ干扰素(IFN-γ)水平,留取大鼠肺组织检测Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子-κB(NF-κB)蛋白表达水平。结果与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组大鼠血清中TNF-α含量显著升高(均P<0.05),与MP组比较,芩百清肺浓缩丸组及阿奇霉素组TNF-α水平显著降低(均P<0.05),但芩百清肺浓缩丸组与阿奇霉素组TNF-α水平无显著性差异;与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组IFN-γ含量均呈不同程度的降低(均P<0.05),与MP组比较,芩百清肺浓缩丸组及阿奇霉素组的IFN-γ水平均升高(均P<0.05),但芩百清肺浓缩丸组与阿奇霉素组IFN-γ水平无显著性差异;肺组织实时荧光PCR(qPCR)结果显示,与对照组比较,MP组、芩百清肺浓缩丸组及阿奇霉素组的TLR4、MyD88、 NF-κB mRNA表达水平均呈不同程度的升高(均P<0.05),与MP组比较,芩百清肺浓缩丸组和阿奇霉素组上述指标水平显著降低(均P<0.05),但芩百清肺浓缩丸组和阿奇霉素组上述指标水平无显著性差异。免疫印迹(WB)结果示TLR4、MyD88、 NF-κB的蛋白表达变化趋势与mRNA一致。结论芩百清肺浓缩丸可降低MP感染大鼠TLR4、MyD88、NF-κB、TNF-α的表达水平,增加IFN-γ的水平,从而减轻MP肺炎的炎症反应。     

双歧杆菌黏附素对应激大鼠肠黏膜核因子-κB和细胞因子的影响

目的 研究双歧杆菌黏附素对大鼠肠黏膜核因子-κB （NF-κB）和细胞因子的影响.方法 将48只实验大鼠按随机数字表法分成应激组、黏附素组,每组24只,以束缚为应激条件建立应激模型,造模后持续实验8d.两组大鼠肠内营养供给量为热量125.4 kJ/（kg·d）,氮量0.2 g/（kg·d）.黏附素组大鼠喂养肠内营养加双歧杆菌黏附素5 mg/（kg·d）,应激组大鼠喂养肠内营养加等量的生理盐水5 mg/（kg·d）.测定造模前、造模后、实验第3天和第8天两组大鼠肠黏膜NF-κB定性表达结果以及肠黏膜和血浆NF-κB、白细胞介素-10 （IL-10）、肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）定量表达结果.透射电镜下观察小肠黏膜形态学变化.结果 （1） NF-κB的表达：造模前、造模后、实验第3天、实验第8天应激组肠黏膜NF-κB的阳性表达率分别为O、79.2％ （19/24）、63.5％（15/24）、66.7％ （16/24）,黏附素组分别为O、68.4％ （17/24）、55.7％ （14/24）、45.8％ （11/24）;与造模前比较,造模后两组肠黏膜NF-κB表达明显上调（均P=0.000）,实验第3天和实验第8天,两组肠黏膜细胞核NF-κB表达阳性率仍明显高于造模前（均P=0.000）,与造模后和应激组比较,实验第8天黏附素组肠黏膜NF-κB表达明显下调（P值分别为0.015、0.021）.（2） TNF-α、IFN-γ与IL-10的定量表达：与造模前比较,造模后应激组与黏附素组大鼠肠黏膜TNF-α[应激组：（154.63±17.52）、（198.72±26.59） pg/g,黏附素组：（154.63±17.52）、（201.45±28.16） pg/g]、IFN-γ[应激组：（39.47±5.76）、（55.32±5.93） pg/g,黏附素组：（39.47±5.76）、（60.75±7.68）pg/g]浓度量和血浆TNF-α[应激组：（83.31±9.78）、（117.64±15.37） ng/L,黏附素组：（83.31±9.78）、（114.82±13.78） ng/L]、IFN-γ[（应激组：（17.35±2.62）、（28.73±4.17） ng/L,黏附素组：（17.35±2.62）、（30.56±4.85） ng/L]浓度明显升高（均P＜0.05）;实验第3天和第8天,应激组大鼠肠黏膜IFN-γ[（58.16±7.38）、（56.37 ±7.29） pg/g]、TNF-α[（215.76±31.54）、（211.83±33.61） pg/g]浓度和血浆IFN-γ[（29.35±4.76）、（30.25±3.67） ng/L]、TNF-α浓度[（125.71±17.38）、（141.26±19.65） ng/L]明显高于造模前（P均＜0.05）;实验第3天和第8天,黏附素组大鼠肠黏膜TNF-α[（165.43±24.58）、（171.57±26.87） pg/g]、IFN-γ[（42.35±4.92）、（40.58±4.65） pg/g]和血浆TNF-α[（103.96±13.68）、（94.53±12.66） ng/L]、IFN-γ[（20.78±2.84）、（19.65±2.45） ng/L]水平明显低于造模后（P均＜0.05）,而IL-10[肠黏膜：（62.82±8.34）、（75.16±9.65） pg/g,血浆：（43.32±5.28）、（55.64±6.87） ng/L]水平明显高于造模后（P均＜0.05）;与应激组比较,实验第3天和第8天,黏附素组大鼠肠黏膜TNF-α、IFN-γ和血浆IFN-γ、TNF-tα水平明显降低（P均＜0.05）,而IL-10水平明显升高（P均＜0.05）.（3）组织形态学观察：实验第8天,在电镜下可见黏附素组较造模后和应激组回肠绒毛、隐窝结构显著恢复.应激组较造模后肠黏膜绒毛、隐窝结构有一定程度的损害,固有膜水肿,膜内有炎性细胞浸润.结论 双歧杆菌黏附素通过调节肠黏膜炎性递质和细胞因子释放,对应激后肠黏膜损伤修复有一定作用.     

Trans-10, cis-12-conjugated linoleic acid modulates NF-κB activation and TNF-α production in porcine peripheral blood mononuclear cells via a PPARγ-dependent pathway

The activation of PPARγ by ligands, including conjugated linoleic acid (CLA) isomers, plays an important role in the immune response. Among CLA isomers, trans-10, cis-12 (t10c12)-CLA is known to participate in the modulation of pro-inflammatory cytokine secretion. The aim of the present study was to assess the effect of t10c12-CLA on PPARγ activation, NF-κB activation and TNF-α expression in lipopolysaccharide (LPS)-naive and LPS-stimulated porcine peripheral blood mononuclear cells (PBMC). In addition, the effect of PPARγ inhibition on NF-κB activation and TNF-α expression in porcine PBMC was examined. t10c12-CLA was found to increase TNF-α expression and NF-κB activity in LPS-naive porcine PBMC. In contrast, t10c12-CLA decreased TNF-α expression and NF-κB activity in LPS-stimulated porcine PBMC. t10c12-CLA up-regulated PPARγ activity and mRNA expression in both LPS-naive and LPS-stimulated porcine PBMC. GW9662, a PPARγ antagonist, completely negated the modulating effects of t10c12-CLA on TNF-α expression and NF-κB activity in both LPS-naive and LPS-stimulated porcine PBMC. These results suggest that t10c12-CLA can modulate TNF-α production and NF-κB activation by a PPARγ-dependent pathway in porcine PBMC.     

Evaluation of the immune responses against reduced doses of Brucella abortus S19 (calfhood) vaccine in water buffaloes (Bubalus bubalis), India

BackgroundBrucella abortus S19 is the most widely used vaccine for the prevention of bovine brucellosis which remains the reference vaccine to which many other vaccine/s are compared. Considering the larger vaccination coverage by reduced dose of vaccine, the study aimed to compare reduced graded doses (1/10th, 1/20th and 1/100th) with standard dose of S19 vaccine (40 × 10⁹CFU /dose) to determine the effective immunizing dose in water buffaloes.MethodsA total of 25 female buffalo calves (Bubalus bubalis) in the age group of 4–5 months were equally grouped into five animals each in four test and one control groups and given with specified vaccine dose. The blood samples were collected on post vaccination days 14, 28, 45, 60, 90 and 120 for assessing innate (TNF-α and IL-12), humoral (IgG antibodies against Brucella LPS) and cell mediated immune responses (IFN-γ, CD4 + and CD8 + counts).ResultsThe full dose, 1/10th and 1/20th reduced doses of S19 vaccine was capable of eliciting pathogen-specific antibody response, vaccine induced secretion of IL-12, TNF-α and IFN-γ with CD4 + and CD8 + effector T cell responses. Persistence of antibody and magnitude of immune responses were found dose dependent.ConclusionComparable immune responses were noticed with 1/10th reduced dose similar to standard dose. With this observation, decline of antibody titre will reduce the number of false positives and reduced dose of vaccine will facilitate larger vaccination coverage in the country.     

Musca domestica pupae lectin improves the immunomodulatory activity of macrophages by activating nuclear factor-κB

In this study, Musca domestica pupae lectin (MPL) was screened for its immunomodulatory effect on macrophages. The phagocytosis of macrophages was improved significantly when they were treated with MPL: remarkable changes were observed in the morphology of the cells, the metabolic abilities of DNA and RNA were enhanced, and the production of hepatin was increased. Meanwhile, compared with the control group, not only the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ) in macrophages, but also the productions of proteins, were strongly induced by MPL; these effects were inhibited by pyrrolidine dithiocarbamate. Further study suggested that MPL could increase the nuclear factor-κB (NF-κB) p65 level in the nucleus. Overall, these results indicate that the improving immunomodulatory activity induced by MPL is mainly due to the increasing productions of TNF-α, IL-6, and IFN-γ and that the activation of macrophage by MPL is partly mediated via the NF-κB pathway.     

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin

Background

Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1^-/- and PPAR-α^-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.

Results

Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.

Conclusions

The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.     

Changes in cytokine levels during admission and mortality in acute alcoholic hepatitis

Cytokine levels are raised in acute alcoholic hepatitis. However, there are disparate results regarding the duration of altered plasma levels, and there are also discrepancies about the relation of changes during the first 15 days after admission with short-term (in-hospital) or long-term mortality. In 56 patients with acute alcoholic hepatitis we found that IL-8, IL-4, Interferon-γ (IFN-γ), malondialdehyde and C-reactive protein remained higher in patients than in 18 age- and sex-matched controls at admission, at the 7th day and at the 15th day after admission. Moreover, IL-4 levels (and to a lesser extent, IL-10 and IFN-γ ones) increased along the three determinations. However, comparing patients who died during the admission with those who did not, there were no statistically significant differences, but there was a nearly significant trend for MDA (Z = 1.89; p = 0.059), with higher levels among those who died. When changes between the first and the second determinations were compared with long-term survival, only IL-8 and IFN-γ showed a relation with mortality. IFN-γ values increased among those who survived and decreased among those who died (p = 0.048). IFN-γ values at the first determination also showed a relation with long-term mortality, especially when patients with IFN-γ values in the first quartile were compared with those of the 4th one (log rank = 5.64; p = 0.018; Breslow = 4.64; p = 0.031). Besides Interferon-γ, only C-reactive protein showed differences between the first and the 4th quartile regarding mortality (Log rank = 4.50; p = 0.034; Breslow 4.33; p = 0.038). In contrast with other studies, no relation was found between TNF-α or IL-6 and mortality.     

The immunological effect of revaccination with Bacille Calmette-Guérin vaccine at 19 months of age

Background

Bacille Calmette-Guérin (BCG) vaccination has important non-specific immune effects. In a randomized trial in Guinea-Bissau, BCG revaccination was associated with significantly increased survival in children who received diphtheria-tetanus-pertussis (DTP)-booster vaccine before enrolment and in children who did not receive micronutrient supplementation (MN). Within the trial we assessed the immunological effects of BCG revaccination.

Methods

Children were randomized to BCG or nothing. Blood was sampled 6–11 weeks after randomization (early sample group) or 5–9 months later (late sample group). In vitro cytokine responses (interferon (IFN)-γ, interleukin (IL)-13, tumor-necrosis-factor (TNF)-α, and IL-10) were assessed in whole blood cultures stimulated with lipopolysaccharide (LPS), purified protein derivative (PPD) or phytohaemagglutinin (PHA). Effect-modification by sex, DTP-booster vaccination and MN was studied.

Results

Cytokines were measured in 345 infants. BCG was associated with significantly increased IFN-γ (geometric mean ratio (GMR) = 4.54 (95% confidence interval: 3.13–6.58)) and IL-13 (GMR = 1.43 (1.00–2.05)) PPD responses, the effect being strongest in the early sample group. Across all three conditions BCG tended to increase IL-10 (LPS, PHA, PPD: GMR = 1.20, 1.12, 1.20), most pronounced in the late sample group. BCG reduced the TNF-α/IL-10 ratio in boys with DTP-booster at bleeding and increased it in those without (interaction test: p = 0.03). In children without MN, BCG was associated with reduced TNF-α response in the early sample group (p = 0.006), and increased IL-10 in the late sample group (p = 0.03).

Conclusion

BCG revaccination resulted in a strong IFN-γ response to PPD, which waned slightly over time. BCG also affected the pro-/anti-inflammatory balance, with reduced TNF-α and increased IL-10 responses to LPS, PHA and PPD. This effect depended on sex, DTP-booster vaccination and micronutrient supplementation, being most pronounced in children who had received DTP-booster before enrolment and children who had not received MN, i.e. the group of children which also had lower mortality after BCG revaccination.     

脂多糖经MyD88／NF-KB信号途径抑制Bewo细胞中乙型肝炎病毒复制

目的探讨Toll样受体4（TLR4）配体脂多糖（LPS）抑制人滋养层细胞Bewo中乙型肝炎病毒（rtBv）复制的作用机制,为防治HBV宫内感染提供依据。方法首先将2txg1．3倍HBV全基因重组质粒pcDNA3．1（＋）-HBV1．3转染Bewo细胞12h后,以TLR4配体LPS处理3d。尔后用LPS处理Bewo细胞,观察IFN—β、TNF-a表达的动力学、NF—KB拮抗剂二硫代氨基甲酸吡咯烷（PDTC）对LPS诱导Bewo细胞产生细胞因子的作用。采用微粒子酶免疫分析法（MEIA）和荧光定量PCR法分别检测HBsAg、HBeAg和HBVDNA水平,并以ELISA和RT—PCR分别检测IFN-β、TNF—a水平及TIR结构域的转接蛋白（TRIF）、髓样分化蛋白（MyD88）表达。结果与对照组比较,LPS可显著抑制Bewo细胞中HBV复制（P〈0．01）,且LPS可显著诱导Bewo细胞产生TNF-a（P〈0．05）,呈时间和剂量依赖性。PDTC可抑制LPS诱导细胞产生TNF-a,显著低于对照组（P〈0．01）,但对IFN—β无显著作用（P〉0．05）。与对照组比较,LPS可诱导HBV重组质粒转染的Bewo细胞表达MyD88（P〈0．01）。结论通过MyD88／NF—KB信号途径诱导Bewo细胞产生TNF—a,TLR4配体LPS可显著抑制HBV复制。     

Adjuvant-active aqueous extracts from Artemisia rupestris L. improve immune responses through TLR4 signaling pathway

Activating innate immunity by an adjuvant is required in vaccine development. The study aims to investigate adjuvant effects of aqueous extracts of Artemisia rupestris L. (AEAR) in vivo and in vitro. ICR mice were subcutaneously administered with antigen and AEAR at various doses to evaluate their immune responses of antibodies, dendritic cells (DCs), regulatory T cells (Treg), splenic lymphocyte, and cytokine. The evaluation results showed that AEAR could largely increase titers of antigen-specific antibodies (IgG, IgG₁, and IgG_2a) and T cell proliferation. AEAR also increased expression of IFN-γ in CD8⁺T cells as well as IL-4 and INF-γ expression in CD4⁺T cells. Expression levels of MHC-II, CD40, CD80, and CD86 on DCs were significantly elevated, whereas the Treg frequency was significantly decreased. AEAR (200 μg) showed remarkable adjuvant activity. Furthermore, AEAR enhanced MHC-II, CD40, CD80, and CD86 expression as well as the yields of TNF-α and IL-12 on DCs through toll-like receptor4 (TLR4) in vitro. Those results indicated that AEAR could serve as an efficacious immune stimulator for vaccines because it significantly enhanced specific immune responses by promoting DCs maturation and reduced Treg through TLR4 signaling pathway.     

Modulation of the nuclear factor-kappa B (NF-κB) signalling pathway by glutamine in peritoneal macrophages of a murine model of protein malnutrition

Background and aims

Protein malnutrition affects resistance to infection by impairing the inflammatory response, modifying the function of effector cells, such as macrophages. Recent studies have revealed that glutamine—a non-essential amino acid, which could become conditionally essential in some situations like trauma, infection, post-surgery and sepsis—is able to modulate the synthesis of cytokines. The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappa B (NF-κB) signalling pathway of peritoneal macrophages from malnourished mice.

Methods

Two-month-old male Balb/c mice were submitted to protein-energy malnutrition (n = 10) with a low-protein diet containing 2 % protein, whereas control mice (n = 10) were fed a 12 % protein-containing diet. The haemogram and analysis of plasma glutamine and corticosterone were evaluated. Peritoneal macrophages were pre-treated in vitro with glutamine (0, 0.6, 2 and 10 mmol/L) for 24 h and then stimulated with 1.25 μg LPS for 30 min, and the synthesis of TNF-α and IL-1α and the expression of proteins related to the NF-κB pathway were evaluated.

Results

Malnourished animals had anaemia, leucopoenia, lower plasma glutamine and increased corticosterone levels. TNF-α production of macrophages stimulated with LPS was significantly lower in cells from malnourished animals when cultivated in supraphysiological (2 and 10 mmol/L) concentrations of glutamine. Further, glutamine has a dose-dependent effect on the activation of macrophages, in both groups, when stimulated with LPS, inducing a decrease in TNF-α and IL-1α production and negatively modulating the NF-κB signalling pathway.

Conclusions

These data lead us to infer that the protein malnutrition state interferes with the activation of macrophages and that higher glutamine concentrations, in vitro, have the capacity to act negatively in the NF-κB signalling pathway.     

Hepatitis B vaccine co-administration influences the heterologous effects of neonatal BCG vaccination in a sex-differential manner

IntroductionBacille Calmette-Guérin (BCG) and hepatitis B (HBV) vaccines are frequently given concomitantly at birth. Neonatal BCG vaccination induces off-target immunological effects. Whether HBV vaccine has immunomodulatory effects is unknown. As off-target effects might vary when vaccines are given simultaneously, this randomised controlled trial aimed to evaluate the influence of neonatal vaccination with BCG and/or HBV on heterologous immune responses.MethodsA total of 185 neonates in Australia were randomised to receive either neonatal BCG-Denmark vaccine, HBV vaccine, both (BCG + HBV group), or none (No vaccine group). In-vitro responses to heterologous stimulants were assessed 7 days after vaccination. The influence of (i) randomisation group and (ii) sex on interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), and tumour necrosis factor-alpha (TNF-α) responses was analysed using linear regression.ResultsOverall, BCG vaccination alone or with HBV co-administration reduced IFN-γ and MCP-1 responses to heterologous stimulants. HBV vaccination alone did not alter heterologous cytokine responses. In general, males produced more IFN-γ and TNF-α than females. We observed a sex-differential effect in relation to the influence of HBV co-administration on the effect of BCG on heterologous responses. Compared with males in the No vaccine group, males in the BCG + HBV group had lower IFN-γ and MCP-1 responses. In contrast, compared with females in the No vaccine group, females in the BCG group had higher IFN-γ response and lower MCP-1 responses.ConclusionNeonatal BCG vaccination resulted in lower cytokine responses to unrelated pathogens. HBV co-administration did not have a significant impact on responses overall but influenced the heterologous effects of neonatal BCG vaccination in a sex-differential manner.     

Modified apple polysaccharides suppress the migration and invasion of colorectal cancer cells induced by lipopolysaccharide

Metastasis is the major cause of death in colorectal cancer (CRC). In colitis-associated carcinogenesis, the activation of nuclear factor-κB (NF-κB) occurs via lipopolysaccharide (LPS) binding to the toll-like receptor 4 (TLR4). The LPS/TLR4/NF-κB pathway contributes to the development and metastasis of colitis-associated colon cancer. In the present study, we hypothesized that an extracted modified Fuji apple polysaccharide (MAP) would alter the LPS/TLR4/NF-κB pathway. Thus, we evaluated the effect of MAP in vitro on the LPS/TLR4/NF-κB pathway in CRC cells (HT-29 and SW620 cells). The results suggest that (i) MAP competed with LPS for binding to TLR4 to reduce LPS-induced NF-κB expression and (ii) MAP suppressed the nuclear translocation of NF-κB p65. MAP significantly decreased LPS-induced expression of TLR4, cyclooxygenase-2, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2, inducible nitric oxide synthase, and prostaglandin E2, and it increased the protein expression of the inhibitor of κBα and NF-κB p65 in cytoplasm when it was given in combination with LPS. These results indicate that MAP suppressed LPS-induced migration and invasiveness of CRC cells by targeting the LPS/TLR4/NF-κB pathway. Therefore, we propose that MAP has potential for the clinical prevention of CRC cell metastasis.     

Saponin-adjuvanted recombinant vaccines containing rCP00660, rCP09720 or rCP01850 proteins against Corynebacterium pseudotuberculosis infection in mice

PurposerCP01850, rCP09729 and rCP00660 proteins from Corynebacterium pseudotuberculosis, predicted as the three best targets to be used in vaccines against Caseous Lymphadenitis in mature epitope density (MED) analysis were tested as vaccinal targets in association to saponin as adjuvant.MethodologyrCP00660, rCP09720 and rCP01850 were expressed in E. coli and purified for immunization assay. Balb/c mice were divided into five groups of sixteen animals each. G1 was injected with saline solution (0.9% NaCl), G2 with saponin, G3, G4 and G5 with, respectively, rCP00660, rCP09720 and rCP01850 added by saponin. Two doses were administered within a 21-days interval, and blood samples were collected for IgG quantification. Twenty-one days after the last immunization, ten mice in each group were challenged with virulent C. pseudotuberculosis MIC-6 strain, and mortality was recorded for 40 days. Meanwhile six mice in each group were used for cytokine quantification by qPCR.ResultsG2, G3, G4 and G5 presented protection rates of 10, 30, 40 and 60%, respectively. In spite of levels of total IgG were higher in G4 and G5, production of IgG2a was higher than IgG1 for G5. G3, G4 and G5 presented significant high IFN-γ levels, however, only G5 showed high TNF-α while G3 and G4 showed high IL-17.ConclusionrCP01850 added by saponin was able to protect efficiently mice against C. pseudotuberculosis challenge, and to induce high IgG, IFN-γ and TNF-α levels. In spite of rCP00660 and rCP09720 had not same adequate protection levels, significant IgG, IFN-γ, and IL-17 levels and further studies aiming to improve protection rates should be conducted.     

基于TLR4信号通路探讨熊果酸改善大鼠酒精性肝损伤的作用机制

目的 探讨熊果酸对酒精性肝损伤大鼠TLR4信号通路的影响及其作用机制。方法 雄性SPF级SD大鼠40只,随机分为4组,每组10只,包括正常对照组、酒精模型组、熊果酸干预组和抑制剂干预组。干预7周,末次灌胃后,禁食不禁水12 h,处死大鼠,采用酶学实验检测肝脏生物标志物水平,HE染色进行肝组织病理学观察,ELISA法检测TNF - a、IL - 6、IL - Iβ水平,western blot法测定肝脏组织中TLR4、NF - κB p65和pNF - κB p65蛋白的表达水平。结果 酒精模型组大鼠血清内ALT、AST、CHE、TNF - α、IL - 6（34.95±2.03,128.76±10.91,137.85±17.31,70.37±20.30,31.52±3.95）水平均明显高于正常对照组（28.65±0.76,56.47±1.46,117.45±8.04,16.80±9.19,22.65±2.17）,熊果酸干预组与酒精模型组相比数值均有不同程度的下降(P＜0.05)。肝脏病理学观察结果显示酒精模型组肝索排列紊乱且肝细胞出现大量炎性因子浸润和脂肪空泡,熊果酸干预组和抑制剂干预组脂肪变性和炎性因子浸润状况均有减轻。与正常组相比酒精模型组大鼠肝脏组织TLR4、NF - κB p65、pNF - κB p65蛋白的表达水平有不同程度的升高(P＜0.05),熊果酸和抑制剂干预后都有不同程度的降低(P＜0.05)。结论 熊果酸对大鼠酒精性肝损伤具有保护作用,其机制可能是通过调控TLR4信号通路来实现的。