Polymorphisms of CCL3L1/CCR5 genes and recurrence of hepatitis B in liver transplant recipients

BACKGROUND:The genetic diversity of chemokines and chemokine receptors has been associated with the outcome of hepatitis B virus infection.The aim of this study was to evaluate whether the copy number variation in the CCL3L1 gene and the polymorphisms of CCR5Δ32 and CCR5-2459A→G (rs1799987) are associated with recurrent hepatitis B in liver transplantation for hepatitis B virus infection-related end stage liver disease.METHODS:A total of 185 transplant recipients were enrolled in this study.The genomic DNA was extracted from whole blood,the copy number of the CCL3L1 gene was determined by a quantitative real-time PCR based assay,CCR5Δ32 was detected by a sizing PCR method,and a single-nucleotide polymorphism in CCR5-2459 was detected by restriction fragment length polymorphism PCR.RESULTS:No CCR5Δ32 mutation was detected in any of the individuals from China.Neither copy number variation nor polymorphism in CCR5-2459 was associated with post-transplant reinfection with hepatitis B virus.However,patients with fewer copies (<4) of the CCL3L1 gene compared with the population median in combination with the CCR5G allele had a significantly higher risk for recurrent hepatitis B (odds ratio=1.93,95% CI:1.00-3.69;P=0.047).CONCLUSION:Patients possessing the compound decreased functional genotype of both CCL3L1 and CCR5 genes might be more likely to have recurrence of hepatitis B after transplantation.

Constituents of the anti-asthma herbal formula ASHMI^(TM) synergistically inhibit IL-4 and IL-5 secretion by murine Th2 memory cells,and eotaxin by human lung fibroblasts in vitro

OBJECTIVE： Anti-asthma herbal medicine intervention （ASHMITM）, a combination of three tradi- tional Chinese medicinal herbs developed in our laboratory, has demonstrated efficacy in both mouse models of allergic asthma, and a double-blind placebo-controlled clinical trial in patients with asthma. This study was designed to determine if the anti-inflammatory effects of individual herbal constituents of ASHMITM exhibited synergy. METHODS： Effects of ASHMI and its components aqueous extracts of Lingzhi （Ganoderma lucidum）, Kushen （Sophora flavescens） and Gancao （Glycyrrhiza uralensis）, on Th2 cytokine secretion by murine memory Th2 cells （D10.G4.1） and eotaxin-1 secretion by human lung fibroblast （HLF-1） cells were determined by measuring levels in culture supernatants by enzyme- linked immunosorbent assay. Potential synergistic effects were determined by computing interaction indices from concentration-effect curve parameters. RESULTS： Individual Lingzhi, Kushen and Gancao extracts and ASHMI （the combination of individual extracts） inhibited production of interleukin （IL）-4 and IL-5 by murine memory Th2 cells and eotaxin-1 production by HLF-1 cells. The mean 25%-inhibitory-concentration （IC2s） values （mg/mL） forASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 30.9, 79.4, 123, and 64.6, respectively; for IL-5 production were 30.2, 263, 123.2 and 100, respectively; for eotaxin-1 were 13.2, 16.2, 30.2, and 25.1, respectively. The IC50values （mg/mL） for ASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 158.5, 239.9, 446.7, and 281.8, respectively; for eotaxin-1 were 38.1, 33.1, 100, and 158.5, respectively. The interaction indices of ASHMI constituents at IC25 were 0.35 for IL-4, 0.21 for IL-5 and 0.59 for eotaxin-l. The interaction indices at IC^0 values were 0.50 for IL-4 and 0.62 for eotaxin-1 inhibition. Inhibition of IL-5 did not reach IC^0 values. All interaction indices were below 1 which indicated synergy. CONCLUSION： By comparing the interaction index values, we find that constituents in ASHMITM synergistically inhibited eotaxin-1 production as well as Th2 cytokine production.

感染期子宫颈癌U14细胞荷瘤小鼠巨噬细胞分泌CCL5受到抑制的机制

目的：探讨感染期子宫颈癌U14细胞荷瘤小鼠巨噬细胞分泌CC型趋化因子配体5（CCL5）受到抑制的机制。方法取6~8周龄的雌性C57BL/6小鼠随机（采用随机数字表法）分为4组，每组6只。无瘤组：皮下注射DMEM培养基；荷瘤组：皮下注射子宫颈癌细胞系U14细胞；无瘤＋脂多糖（LPS；用于诱导小鼠感染）组：皮下注射DMEM培养基，腹腔内注射LPS；荷瘤＋LPS组：皮下注射U14细胞，腹腔内注射LPS。（1）采用ELISA法及荧光定量PCR技术检测各组小鼠血清及巨噬细胞中CCL5和前列腺素E2（PGE2）的表达。（2）提取各组荷瘤小鼠血清制备肿瘤条件培养基（TCM），体外诱导巨噬细胞，检测诱导前、后各组上清液和巨噬细胞中CCL5、PGE2和环磷酸腺苷（cAMP）的表达。（3）荷瘤＋LPS组选用环氧合酶2（COX-2）抑制剂——NS398阻断PGE2的产生（即荷瘤＋LPS＋NS398组），蛋白激酶A（PKA）阻滞剂——H89阻断cAMP/PKA信号通路（即荷瘤＋LPS＋H89组），分别制备TCM并检测两组上清液和巨噬细胞中CCL5、PGE2和cAMP的表达。结果（1）荷瘤组小鼠血清中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（151±35）pg/ml、1.0]均明显低于无瘤组[分别为（691±85）pg/ml、4.5±0.8；P〈0.05]；其血清中PGE2蛋白含量及巨噬细胞中PGE2 mRNA的表达水平[分别为（1198±83）pg/ml、5.8±0.8]均明显高于无瘤组[分别为（187±25）pg/ml、1.0；P〈0.05]。无瘤＋LPS组小鼠血清中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（4049±141）pg/ml、31.5±2.0]均高于明显高于荷瘤＋LPS组[分别为（1951±71）pg/ml、12.1±2.8；P〈0.05]；其血清中PGE2蛋白含量及巨噬细胞中PGE2 mRNA的表达水平[分别为（676±70）pg/ml、3.4±0.4]均低于荷瘤＋LPS组[分别为（2550±382）pg/ml、11.6±0.9；P〈0.05]。（2）各组小鼠血清制备的TCM在体外培养巨噬细胞后，荷瘤组上清液中CCL5、PGE2、cAMP蛋白含量[分别为（1626±177）、（790±156）、（164±30）pg/ml]及巨噬细胞中CCL5、PGE2、cAMP mRNA的表达水平（分别为28.6±1.2、1.7±0.3、1.6±0.3）均明显高于无瘤组[其蛋白含量分别为（27±3）、（448±115）、（118±25）pg/ml，其mRNA的表达水平均为1.0；P〈0.05]。无瘤＋LPS组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（10475±742）pg/ml、212.0±5.7]均明显高于荷瘤＋LPS组[分别为（6375±530）pg/ml、142.3±2.5；P〈0.05]；无瘤＋LPS组上清液中PGE2、cAMP蛋白含量[分别为（2438±95）、（340±13）pg/ml]及巨噬细胞中PGE2、cAMP mRNA的表达水平（分别为4.3±0.7、4.1±0.4）均明显低于荷瘤＋LPS组[其蛋白含量分别为（3441±163）、（542±42）pg/ml，其mRNA的表达水分别为5.9±0.3、5.4±0.5；P〈0.05]。（3）与荷瘤＋LPS组比较，荷瘤＋LPS＋NS398组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（7691±269）pg/ml、159.0±8.9]明显升高（P〈0.05），而上清液中PGE2、cAMP蛋白含量及巨噬细胞中PGE2、cAMP mRNA的表达水平均明显降低（P〈0.05）；荷瘤＋LPS＋H89组上清液中CCL5蛋白含量及巨噬细胞中CCL5 mRNA的表达水平[分别为（8375±520）pg/ml、177.0±8.8]进一步升高（P〈0.05），而上清液中PGE2、cAMP蛋白含量及巨噬细胞中PGE2、cAMP mRNA的表达水平均进一步降低（P〈0.05）。结论感染期子宫颈癌U14细胞荷瘤小鼠血清可在体外抑制巨噬细胞分泌CCL5，这种抑制效应可能是通过cAMP/PKA通路而受PGE2的调控。

Functional roles of CCL5/RANTES in liver disease

Inflammation,which is mediated by leukocyte trafficking and activation,plays a prominent role in the pathogenesis of acute and chronic liver injury.Chemokines are critical mediators involved in the migration of leukocytes into the diseased liver via binding to their G protein-coupled receptors.CeC motif ligand 5(CCL5)belongs to the CC-chemokine family and is secreted by several hepatic cell pop-ulations including hepatocytes,macrophages,hepatic stellate cells,and endothelial cells upon activation.CCL5 regulates the recruitment and migration of T cells(via CCR5)and NK cells(via CCR1).Moreover,CCL5 activates and stimulates T cell proliferation and cytokine production,sequentially regulating in-flammatory responses.Accumulating studies have identified crucial effects of CCL5 both in liver-disease patients and in experimental models,in which CCL5 is elevated and displays distinct effects according to pathological conditions.In this review,we discussed the crucial functions of CCL5 in liver diseases,including acute liver failure,hepatic ischemia-reperfusion injury,acute liver failure,acute and viral hepatitis,alcoholic liver disease,non-alcoholic fatty liver disease,fibrosis,and hepatocellular carcinoma.Continued understanding the roles of CCL5 in liver disease and their mechanisms of activation are indispensable for the development of effective clinical therapeutics.

应用血清RANTES诊断严重脓毒症及预后判断的价值评价

目的：检测严重脓毒症患者血清中受激活调节正常T细胞表达和分泌因子（RANTES）水平，并评价其对严重脓毒症的诊断和预后价值。方法选取严重脓毒症患者40例为严重脓毒症组（SS组），同期门诊体检者20例为对照组，收集临床及实验室参数，计算APACHEⅡ评分和 DIC 评分，采用酶联免疫吸附测定（ELISA）方法检测血清 RANTES 的水平。结果 SS组患者血清RANTES水平[（3175.91±1341.78）pg/ml]较对照组[（5374.27±927.87）pg/ml]下降（P＜0.05）。相关分析显示RANTES与WBC、PLT、AST、TBIL、Cr、PT、APTT、PCT、APACHEⅡ评分和DIC评分均呈显著负相关（P＜0.05），显示RANTES诊断严重脓毒症的AUCSS＝0.917，95%CI 0.817-0.993（P＜0.05），判断严重脓毒症死亡的AUCdeath＝0.786，95%CI 0.650-0.922（P＜0.05）。结论严重脓毒症患者的血清RANTES水平明显降低，并死亡率升高，对严重脓毒症的诊断和预后判断有较好的价值。

阿维A对寻常型银屑病患者趋化因子RANTES和MCP-1的影响

目的观察阿维A对寻常型银屑病患者血清中和外周血单一核细胞(PBMC)内RANTES和MCP-1表达水平的影响。方法分离并提取30例健康对照者及48例阿维A治疗前、后银屑病患者的血清及PBMC,血清采用ELISA检测其中RANTES和MCP-1的分泌水平,PBMC接受实时荧光定量PCR法检测细胞内RANTES和MCP-1的mRNA的水平,并进行临床PASI评分。结果经ELISA检测发现,寻常型银屑病患者血清中RANTES和MCP-1水平明显高于健康对照组(P<0.01),应用阿维A治疗后,两因子水平均显著降低(P<0.01);QRT-PCR检测发现,患者PBMC中RANTES和MCP-1的mRNA相对表达量均明显高于健康对照组(P<0.01);治疗后PBMC中RANTES和MCP-1均显著降低(P<0.01)。治疗后患者PASI评分明显下降,且治疗前后银屑病患者PASI评分差值与血清RANTES和MCP-1水平差值均呈正相关性(r1=0.469,P<0.01;r2=0.431,P<0.01),与PBMC中RANTES mRNA表达量差值呈正相关(r=0.484,P<0.01)。结论阿维A对寻常型银屑病的疗效显著,降低CC型趋化因子RANTES和MCP-1的水平可能是阿维A治疗银屑病的机制之一。