Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC

Background Proteitis of hen egg white are common ingredients of food and difficult to eliminate. Allergens of egg while induce allergic symptoms among relatively high numbers of palients suffering from food allergy. B cell epitopes to hen egg white tnajor allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Objectives Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105–122 and 323–339 were investigated. Methods Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Results Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105–122 are reported for the first time. The cell lines were enriched CD4⁺/CD8⁺ T cells (CD2⁺ 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Conclusions Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105–122 and 323–339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.     

肝豆灵对Wilson病模型大鼠肝组织miRNA-122表达的影响

目的研究肝豆灵对Wilson病(Wilsons disease,WD)大鼠肝组织miRNA-122表达的影响,探究其保护肝脏的机制。方法将24只Wistar大鼠随机分为空白对照组、模型组、青霉胺组和肝豆灵组。铜负荷喂养8周后,分别用青霉胺、肝豆灵以成人等效剂量灌胃,空白对照组及模型组均以等量生理盐水灌胃,每组均灌胃4周。观察各组大鼠肝功能及肝组织miRNA-122的表达水平。结果肝豆灵和青霉胺均可显著降低WD大鼠异常升高的血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、总胆红素(total bilirubin,TBil)(P0.05,或P0.01),显著升高WD大鼠肝组织异常降低的miRNA-122表达水平(P0.01)。肝豆灵降低血清ALT、AST和TBil的作用及升高肝组织miRNA-122表达的作用显著优于青霉胺(P0.05,或P0.01)。结论肝豆灵保护WD大鼠肝功能的作用与其上调肝组织miRNA-122的表达有关。     

MicroRNA-122作用于HBx影响乙肝病毒复制

为了寻找与HBV感染相关的microRNA并初步探讨其作用机制,首先,我们前期研究发现在转染HBV表达质粒的HepG2细胞内,miR-122上调表达,推测miR-122与HBV的感染有关;在此基础上,本研究进一步将miR-122和表达HBV的质粒pCH9-HBV1.1共转染HepG2细胞,Southern blot检测发现miR-122可抑制HBV的复制。利用计算机软件miRanda预测表明,HBx可能是miR-122的作用靶序列;进一步利用荧光素酶报告基因系统和Western blot检测靶基因HBx蛋白表达改变,验证miR-122对HBx表达的调节作用。最终推测miR-122可能通过调节HBx的表达而影响HBV的复制。     

MicroRNA-122 sensitizes HCC cancer cells to adriamycin and vincristine through modulating expression of MDR and inducing cell cycle arrest

Hepatocellular carcinoma (HCC) is a hypervascular cancer characterized by rapid progression as well as resistance to conventional chemotherapy. It has been shown that microRNAs play critical roles in pathogenesis of HCC. MicroRNA-122 (miR-122) is a liver-specific microRNA and is frequently downregulated in HCC. In the present study, we investigated whether restoration of miR-122 in HCC cells could render cells sensitive to chemotherapeutic agents adriamycin (ADM) or vincristine (VCR). Our data showed that overexpression of miR-122 in HCC cells induced by adenovirus expressing miR-122 could render cell sensitive to ADM or VCR. Analysis of cell cycle distribution showed that the anti-proliferative effect of miR-122 is associated with increase of cell number in the G2/M phase. Moreover, treatment with Ad-miR122 and ADM or VCR resulted in high accumulation of HCC cells in G2/M phase. We further demonstrated that overexpression of miR-122 could modulate the sensitivity of the HCC cells to chemotherapeutic drugs through downregulating MDR related genes MDR-1, GST-π, and MRP, antiapoptotic gene Bcl-w and cell cycle related gene cyclin B1. Taken together, our findings demonstrated that combination of Ad-miR122 with chemotherapeutic agents inhibited HCC cell growth by inducing G2/M arrest and that this arrest is associated, at least in part, with reduced expression of MDR related genes and Cyclin B1.     

miR-122表达下调与脂肪性肝病的关系

目的探讨肝脂肪变时肝细胞株L-02 miR-122表达水平的变化。方法用油酸诱导法建立L-02脂肪变肝细胞株模型,于24、48、72 h用激光共聚焦显微镜检测细胞三酰甘油含量,提取细胞总RNA,逆转录为cDNA,用荧光定量PCR检测miR-122的表达,并与正常肝细胞株作对照。结果成功建立脂肪肝细胞株模型,随着油酸诱导时间延长,细胞内三酰甘油含量增加,miR-122在脂肪肝细胞表达下调,其拷贝数仅为正常肝细胞的1/16.68(P<0.05)。结论肝细胞株脂肪变时miR-122表达显著下降。     

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model

Objective： Intratumoral administration of adenoviral vector encoding herpes simplex virus （HSV） thymidine kinase （TK） gene （Ad-TK） followed by systemic ganciclovir （GCV） is an effective approach in treating experimental hepatocellular carcinoma （HCC）. However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy, miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. Methods： By inserting miR-122 target sequences （miR-122T） in the 3＇ untranslated region （UTR） ofTK gene, we constructed adenovirus （Ad） vectors expressing miR-122-regulated TK （Ad-TK-122T） and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Results： Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. Conclusions： miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.     

Serum transthyretin levels in senile systemic amyloidosis: effects of age,gender and ethnicity

Serum transthyretin (TTR) levels are reduced in familial amyloidotic polyneuropathy (FAP). A single study of patients with senile systemic amyloidosis (SSA) in Sweden found that those individuals also had a significantly lower mean serum TTR concentration than age- and gender-matched controls. To determine if the same phenomenon prevailed in an ethnically more heterogeneous population, we compared the serum TTR levels, as determined by ELISA, in 45 documented SSA patients with congestive heart failure, 20 AL patients with congestive heart failure and population controls. Serum TTR concentrations in the controls were influenced in a statistically significant manner by age, gender and ethnicity. Although it is unlikely that such differences are clinically relevant, they must be considered when assessing the meaning of serum TTR concentrations in any clinically defined population. The serum concentrations in patients with SSA did not differ from age, gender and ethnically matched controls or from a group of AL patients with significant clinical cardiac involvement. We also compared TTR concentrations in 12 African-Americans carrying the TTR V122I allele with those in 826 African-Americans who were homozygous wild type at the TTR locus. The TTR V122I carriers had significantly lower serum TTR concentrations than appropriate controls even though the majority of such individuals had not reached the age of clinical or anatomic risk, i.e. over 60. Thus, as in carriers of other TTR mutations the serum TTR level is lower than normal, despite having a much later appearance of clinical disease.     

Sensitive detection of hepatocellular injury in chronic hepatitis C patients with circulating hepatocyte‐derived microRNA‐122

As chronic hepatitis C patients with progressive disease can present themselves with normal ALT levels, more sensitive biomarkers are needed. MicroRNAs are newly discovered small noncoding RNAs that are stable and detectable in the circulation. We aimed to investigate the association between hepatocyte‐derived microRNAs in serum and liver injury in patients with chronic hepatitis C. The hepatocyte‐derived miR‐122 and miR‐192 were analysed in sera of 102 chronic HCV‐infected patients and 24 healthy controls. Serum levels of miR‐122 and miR‐192 correlated strongly with ALT (R = 0.67 and R = 0.65, respectively, P < 0.001 for both). Median levels of miR‐122 and miR‐192 in HCV‐infected patients were 23 times and 8 times higher as in healthy controls (P < 0.001 for both). Even within the HCV‐infected patients with a normal ALT (n = 38), the levels of miR‐122 and miR‐192 were 12 times and 4 times higher compared with healthy controls (P < 0.001 for both). Multivariate logistic regression analyses showed that only miR‐122 was a significant predictor of the presence of chronic HCV infection (P = 0.026). Importantly, miR‐122 was also superior in discriminating chronic HCV‐infected patients with a normal ALT from healthy controls compared with the ALT level (AUC = 0.97 vs AUC = 0.78, P = 0.007). In conclusion, our study confirmed that liver injury is associated with high levels of hepatocyte‐derived microRNAs in circulation and demonstrated that in particular miR‐122 is a sensitive marker to distinguish chronic hepatitis C patients from healthy controls. More sensitive blood markers would benefit especially those patients with minor levels of hepatocellular injury, who are not identified by current screening with ALT testing.