IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

苦参碱抑制游离脂肪酸诱导小鼠肝实质细胞脂肪变性

目的：探讨苦参碱对游离脂肪酸（free fatty acids,FFA）诱导的小鼠肝实质细胞脂肪变性的影响。方法Ⅳ型胶原酶灌注分离 BALB／c 小鼠原代肝实质细胞并进行体外培养。实验分对照组（CG）,高脂组（FG）及苦参碱组（MG）。通过体外测定细胞内三酰甘油的含量。用油红染色的方法观察肝实质细胞的脂肪样变性。Western 印迹检测细胞内MAPK 相关信号通路磷酸化的变化。实时定量 PCR 检测细胞中与脂肪化密切相关的 miR-122的表达和相关靶基因的表达改变。结果与高脂组比较,苦参碱组肝实质细胞内三酰甘油含量显著降低,脂肪颗粒明显减少,脂肪变性得到改善,表明苦参碱能够抑制 FFAs 诱导的 JNK、p38通路磷酸化。Q-PCR 结果表明苦参碱能促进肝实质细胞内 miR-122的表达,并降低脂肪化相关基因 Fas、Acc1的表达。结论苦参碱能显著改善高脂诱发的肝细胞脂肪样变性,并抑制 JNK、P38通路磷酸化,其机制可能同 miR-122通路相关。     

A new polymorphism for the RI22H mutation in hereditary pancreatitis

BACKGROUND AND AIMS: Hereditary pancreatitis (HP) is a rare form of recurrent acute and chronic pancreatitis. Mutations in the cationic trypsinogen (protease serine 1, PRSS1) gene have been identified as causing HP. The R122H (previously known as R117H) mutation is the commonest and can be detected by a single and rapid polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) based technique using the AflIII enzyme. This test however may give a false negative result in the presence of a neutral polymorphism within the enzyme recognition site. The frequency of this event was examined by sequencing studies in patients with HP and in healthy controls. METHODS: Of 60 families identified by the UK and Ireland consortium of EUROPAC (European Registry for Hereditary Pancreatitis and Familial Pancreatic Cancer), 51 were screened for R122H, N29I, and A16V mutations using standard techniques, and by sequencing of all five exons of cationic trypsinogen. RESULTS: Twelve families had the N29I mutation, one family had A16V and, on standard testing, 15 families had the R122H mutation. An additional family with the R122H mutation was found on direct sequencing. The false negative result was due to a neutral polymorphism C-->T at the third base of the codon, not affecting the amino acid coded for, destroying the AflIII restriction site. This polymorphism was not observed in 50 DNA specimens (100 chromosomes) from controls nor from 50 individuals from PRSS1 mutation negative HP families. A novel mutation specific PCR was developed to avoid this pitfall. CONCLUSIONS: One of the 16 families with HP and an R122H mutation contained a polymorphism affecting the AflIII restriction site. Adoption of an alternative R122H assay is important for genetic studies in individuals with apparent HP.     

The diagnostic utility of microRNA 222-3p,microRNA 21-5p,and microRNA 122-5p for HCV-related hepatocellular carcinoma and its relation to direct-acting antiviral therapy

Background and study aimsRecent reports have emphasized the increased risk of hepatocellular carcinoma (HCC) post direct-acting antiviral (DAAs) therapy in chronic hepatitis C virus (HCV) patients. Unfortunately, reliable diagnostic markers for HCC are still lacking. In this context, serum microRNAs have become promising diagnostic targets. Thus, the current study aims to elaborate the diagnostic utility of microRNA 122-5p, microRNA 21-5p, and microRNA 222-3p in the serum of Egyptian patients presenting with HCV infection and HCC post DAA therapy.Patients and methodsQiagen specific microRNA assays were utilized to assess the expression levels of the chosen microRNAs in the serum samples collected from 3 groups: (1) 50 patients with HCV-related HCC, (2) 50 patients with HCC post DAA therapy, and 20 healthy control.ResultsThe mean serum values of microRNA 21-5p and microRNA 122-5p were significantly lower in the HCC post DAA therapy group than in both the group with HCC without prior exposure to DAAs (P < 0.001) and control group (P 0.05 and 0.02, respectively). A significant upregulation was observed for both microRNA 21-5p and microRNA 122-5p in the HCV-related HCC group compared with the control group (P < 0.001 and = 0.011, respectively). On the other hand, the mean serum value of microRNA 222-3p was significantly raised in the HCC post DAA therapy group than in the control group (P = 0.007), whereas no statistically significant difference was observed between both groups with HCC and between the group with HCV-related HCC without prior exposure to DAAs and control group.ConclusionThis is the first study to introduce microRNA 21-5p, microRNA 122-5p and microRNA 222-3p as noninvasive biomarker candidates for HCC post DAA therapy. Their altered expression among treatment-naive HCC and HCC post DAA therapy might assume a different microRNA profiling in both HCC groups.     

Active glycolytic metabolism in CD133(+) hepatocellular cancer stem cells: regulation by MIR-122

Although altered metabolic pathway is an important diagnostic maker and therapeutic target in cancer, it is poorly understood in cancer stem cells (CSCs). Here we show that the CD133 (+) hepatocellular CSCs have distinct metabolic properties, characterized by more active glycolysis over oxidative phosphorylation, compared to the CD133 (−) cells. Inhibition of PDK4 and LDHA markedly suppresses CD133 (+) stemness characteristics and overcome resistance to sorafenib (current chemotherapeutic agent for hepatocellular cancer). Addition of glucose or lactate to CD133 (−) cells promotes CSC phenotypes, as evidenced by increased CD133 (+) cell population, elevated stemness gene expression and enhanced spheroid formation. Furthermore, the liver-specific miRNA, miR-122, inhibits CSC phenotypes by regulating glycolysis through targeting PDK4. Our findings suggest that enhanced glycolysis is associated with CD133 (+) stem-like characteristics and that metabolic reprogramming through miR-122 or PDK4 may represent a novel therapeutic approach for the treatment of hepatocellular cancer.     

GPs抑制miRNA122表达及调节脂代谢酶活性降血脂作用研究

目的：探讨绞股蓝总苷（Gypenosides,GPs）能否抑制miRNA122表达及调节脂代谢酶活性发挥降血脂作用。方法：将48只健康雄性SD大鼠随机分为正常对照（Ｃ）组、高脂模型（M）组、辛伐他汀（S）组、GPs（G）组,除C组喂食普通饲料外,其余3组大鼠均喂食高脂饲料。将GPs溶于0.3%羧甲基纤维素钠（CMC-Na）溶液中,灌胃给药。Ｃ组和Ｍ组每天灌0.3% CMC-Na（1 mL/100 g）,G组每天灌GPs 160 mg·kg^-1,Ｓ组每天灌辛伐他汀5 mg·kg^-1,连续进行8周。最后一次给药后禁食12 h,用7%水合氯醛腹腔麻醉,取腹腔动脉血,测定血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）;称肝脏组织湿重,测定肝指数;提取肝脏总RNA,Real time-PCR测定肝脏miRNA-122的表达;另制备肝脏组织匀浆,测定肝酯酶（HL）、脂蛋白酯酶（LPL）、HMG-CoA还原酶活性;做离体胆固醇微胶粒形成实验。结果：与C组比较,M组大鼠血清TC、TG、LDL-C水平均显著升高（P<0.01）,HDL-C水平则明显下降（P<0.05）;与M组比较,S组、G组TC、TG、LDL-C水平均明显下降（P<0.05）,HDL-C水平则明显升高（P<0.05）;与M组大鼠比较,S组、G组肝指数均明显下降（P<0.05）;与M组比较,S组、G组大鼠肝脏miRNA-122的表达均明显下降（P<0.05）;与M组比较,S组、G组HMG-CoA还原酶活性明显降低（P<0.05）,HL、LPL活性明显升高（P<0.05）;GPs在一定程度上抑制肠道中胆固醇微胶粒的形成。结论：GPs可有效降低高脂血症大鼠血脂水平,减轻肝脏脂肪病变,其降脂机理与其抑制miRNA-122表达及调节脂代谢酶活性,抑制体内胆固醇微胶粒的形成有关。     

Circulating microRNA‐122 levels are important predictor of hepatitis B virus surface antigen seroclearance

It is currently unclear what impact serum microRNA‐122 (miR‐122) levels have on clearance of hepatitis B virus (HBV) surface antigen (HBsAg) in HBV‐infected patients who had not received antiviral therapy. The current study evaluated the impact of serum miR‐122 levels on HBsAg seroclearance in 367 consecutive HBV‐infected patients who had not received antiviral therapy between their initial and last visit, and investigated the predictive factors of HBsAg seroclearance. Cumulative HBsAg seroclearance rates were 13.5%, 32.0%, and 37.4% after 10, 20, and 30 years, respectively. The yearly incidence of HBsAg seroclearance over the investigated 30‐year period was 1.25%. A significant and strong correlation was observed between serum miR‐122 and HBsAg levels. Moreover, there was a significant correlation between serum miR‐122 levels and the levels of HBV DNA, hepatitis B e‐antigen, and HBV core‐related antigen. The HBsAg seroclearance rate in patients with a <1.0‐fold change of serum miR‐122 levels was significantly higher than in those with a ≥1.0‐fold change. Multivariate analysis identified age (≥30 years), HBV DNA levels (<2.2 log U/mL), HBV genotype (non‐C), and serum miR‐122 levels (<1.0‐fold change) as significant predictors of HBsAg seroclearance. Our results indicated that serum miR‐122 level is an important predictor of HBsAg seroclearance in Japanese patients who do not receive antiviral therapy. Understanding the complexity of the interactions among various virus‐related and host‐related factors could potentially help in the design of new therapies that enhance HBsAg seroclearance.     

Decreased level of intracellular cholesterol in peripheral blood mononuclear cells is associated with chronic hepatitis C virus infection

Peripheral blood mononuclear cells (PBMCs) constitute the main extrahepatic place of, hepatitis C virus (HCV) replication. We aimed to determine the impact of CHC infection and microRNA-, 122 expression on cholesterol expression in PBMCs. HCV RNA strand, intracellular cholesterol, HMGCoA, reductase and miR-122 expression in PBMC were determined in 54 CHC patients. The study shows that significant decrease of intracellular cholesterol level in PBMC (p = 0.000000), accompanied by serum hypocholesterolemia is the characteristic feature of chronic hepatitis C infection. Although, microRNA-122 expression was detectable in PBMCs of CHC patients (52.5%), the alteration of intracellular cholesterol level was independent of miR-122 expression.