Rational design of hypoallergens applied to the major cat allergen Fel d 1

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.     

The effect of 2,3-dimercaptopropane sodium sulfonate on mercury retention in rats in relation to age

The effectiveness of DMPS (sodium 2,3-dimercaptopropane-1-sulfonate) in reducing inorganic mercury retention was studied in 2-, 6-, and 28-week-old albino rats. ²⁰³Hg was administered IP. The chelating agent DMPS was administered by IP injection at a dose of 250 mol/kg body weight three times, 1 day after ²⁰³Hg administration and at 24 h intervals thereafter. The whole body retention determined 1, 2, 3, and 6 days after ²⁰³Hg administration showed that DMPS decreased the body retention of mercury in all age groups, being about twice as effective in adult compared to suckling rats. The reduced effectiveness was due to the reduced efficacy of DMPS in reducing kidney retention in young animals. In other organs the effectiveness of DMPS was not age dependent. These and previous results obtained with different chelating agents and other metals indicate that age might be an important factor in chelation therapy in general.     

Long non-coding RNA LINC01116 accelerates the progression of keloid formation by regulating miR-203/SMAD5 axis

BackgroundEmerging evidence reveals the importance of long non-coding RNAs (lncRNAs) in the development and progression of keloid formation. However, the roles and molecular mechanism of lncRNA LINC01116 in the progression of keloid formation remain largely unknown.MethodsThe expression levels of LINC01116, microRNA-203 (miR-203) and SMAD family member 5 (SMAD5) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation, migration and invasion were detected by Cell counting Kit-8 (CCK-8) assay and transwell assay. Flow cytometry and western blot assay were used to examine cell apoptosis and extracellular matrix (ECM) production. The interaction between miR-203 and LINC01116 or SMAD5 was predicted by bioinformatics analysis and verified by dual-luciferase reporter and RNA Immunoprecipitation (RIP) and RNA pull-down assays.ResultsLINC01116 and SMAD5 were upregulated while miR-203 was downregulated in keloid tissues and keloid fibroblasts. LINC01116 knockdown suppressed the proliferation, migration, invasion, and ECM production but induced apoptosis in keloid fibroblasts through enhancing miR-203 and inhibiting SMAD5. Moreover, SMAD5 was identified as a direct target of miR-203 and miR-203 could directly bind to LINC01116. Besides, LINC01116 regulated SMAD5 expression by targeting miR-203.ConclusionDownregulation of LINC01116 inhibited the progression of keloid formation by regulating miR-203/SMAD5 axis, which might provide a novel target for keloid therapy.     

黄芪-丹参药对通过下调miR-466b-5p改善高血压肾损害

目的：探讨黄芪-丹参药对通过调节微小核糖核酸-466b-5P（miR-466b-5p）改善高血压肾损害的机制。方法：基于微RNA（miRNA）测序寻找自发性高血压大鼠与Wistar-京都鼠（WKY）的差异基因,构建腺相关病毒转染小鼠,24只C57BL/6小鼠,随机选取12只尾静脉注射小鼠腺相关病毒miR-466b-5P（rAAV-miR-466b-5P）构建miR-466b-5p过表达组,余12只作为空白对照组注射腺相关病毒-9（AAV-9）空载体,转染6周后再各随机分为2组,每组6只,A组（黄芪-丹参+miR-466b-5P过表达组）和C组（黄芪-丹参+空白对照组）以黄芪配方颗粒：2.036 g/kg+丹参配方颗粒：0.255 g/kg灌胃;B组（miR-466b-5P过表达组）和D组（空白对照组）以相同体积生理盐水灌胃;灌胃28 d后观察各组小鼠尿β2-微球蛋白（β2-MG）、N-乙酰-β-D-葡萄糖苷酶（NAG）、微量白蛋白（mALB）,血清胱抑素C（Cys-C）、血管紧张素Ⅱ（AngⅡ）、C反应蛋白（CRP）水平和肾脏病理,进行实时荧光定量(RT-qPCR)与蛋白质印迹法(Western Blotting)寻找其靶基因。结果：1）基于前期miRNA测序选定了miR-466b-5p;2）与D组比较,B组小鼠尿β2-MG、NAG、mALB、血清Cys-C、AngⅡ、CRP均显著升高（P<0.01）;与B组比较,A组小鼠血清Cys-C、CRP明显升高（P<0.05）,尿β2-MG、NAG、mALB、血清AngⅡ无明显改变（P>0.05）;3）与D组比较,B组小鼠出现明显肾小球硬化、小管纤维化,A组较B组有明显改善;4）与D组比较,B组小鼠透明质酸酶2（HAS2）明显下调,而经黄芪-丹参干预后有所回调。结论：miR-466b-5p的过表达会导致高血压肾损害的发生,黄芪-丹参药对通过靶向HAS2下调miR-466b-5p改善高血压肾损害。     

Tissue specific susceptibility of alpha-adrenoceptor mediated vasoconstriction to nifedipine

Summary The influence of the calcium antagonist nifedipine on ₁- and ₁-adrenoceptor vasoconstrictor effects was investigated in vitro. Changes in tension were monitored isometrically on helical strips of canine circumflex coronary and saphenous arteries suspended in 10 ml organ baths and of saphenous veins superfused with Krebs-Henseleit solution. Distinction between ₁- and ₂-adrenoceptor was made by using selective -adrenoceptor blocking drugs such as rauwolscine, yohimbine, corynanthine and prazosin, and the agonists noradrenaline, phenylephrine and guanfacine. In venous and both arterial vascular smooth muscles, the contractile process could be triggered by stimulation of both ₁- and ₂-like adrenoceptors. Nifedipine inhibited the venoconstrictor response to the ₂-agonist guanfacine, leaving that to the ₁-agonist phenylephrine unchanged. In saphenous arteries, nifedipine in addition to guanfacine also antagonized constrictor responses to phenylephrine, though to a significantly weaker extent. In circumflex coronary arteries, nifedipine was equally potent in antagonizing responses to both ₁- and ₂-adrenoceptor stimulation.It is suggested that the susceptibility of -adrenoceptormediated vasoconstrictor effects to blockade by calcium antagonists depends not only on the subtype of -adrenoceptor but, in addition, on the type and origin of vascular smooth muscle and may be a reflection of tissue variations in intracellular calcium stores.     

MiR-206 inhibits Head and neck squamous cell carcinoma cell progression by targeting HDAC6 via PTEN/AKT/mTOR pathway

As a kind of endogenous noncoding small RNA, microRNA (miRNA) plays important roles of regulation to various physiological functions, while its affections on senescence of human Head and neck squamous cell carcinoma (HNSCC) are still unknown. The objective of this study was to investigate the effect of miR-206 in HNSCC tissues, adjacent normal tissues and cell lines, and explore its biological functions in HNSCC.In our study, the level of miR-206 in HNSCC tissues and adjacent normal tissues was detected via real-time qPCR. The effect of miR-206 on cell proliferation was tested by MTT assay, colony formation and cell cycle assays. In order to explore the effect of miR-206 on HNSCC cell migration and invasion, we performed wound healing assays and transwell assays. Luciferase reporter assays were designed to identify the interaction between 3′UTR of HDAC6 and miR-206. The level of signaling pathway-related proteins was determined by western blot. The expression of miR-206 was found to be observably decreased in HNSCC tissues and cell lines through real time-PCR. Restoration of miR-206 weaked cell proliferation, invasion and migration in HNSCC cells and the cell cycle was arrest in S phase. Further explores have shown that miR-206 could inhibit HNSCC cells proliferation by targeting the HDAC6 via PTEN/AKT/mTOR pathway. Taken together, our results demonstrated that miR-206 plays a critical role in HNSCC progression by targeting HDAC6 via PTEN/AKT/mTOR pathway, which might be a potential target for diagnostic and therapeutic in HNSCC.     

Silencing of MicroRNA-21 confers the sensitivity to tamoxifen and fulvestrant by enhancing autophagic cell death through inhibition of the PI3K-AKT-mTOR pathway in breast cancer cells

Tamoxifen (TAM) and fulvestrant (FUL) represent the major adjuvant therapy to estrogen receptor-alpha positive (ER⁺) breast cancer patients. However, endocrine resistance to TAM and FUL is a great impediment for successful treatment. We hypothesized that miR-21 might alter the sensitivity of breast cancer cells to TAM or FUL by regulating cell autophagy. Using the ER⁺ breast cancer cells, we knockdown miR-21.by transfection with miR-21 inhibitor, then the cells were exposed to TAM or FUL and the percentages of apoptosis and autophagy were determined. Knockdown of miR-21 significantly increased the TAM or FUL-induced apoptosis in ER⁺ breast cancer cells. Further, silencing of miR-21 in MCF-7 cells enhanced cell autophagy at both basal and TAM or FUL-induced level. The increase of autophagy in miR-21-knockdown MCF-7 cells was also indicated by increase of beclin-1, LC3-II and increased GFP-LC3 dots. Importantly, knockdown of miR-21 contributed to autophagic cell death, which is responsible for part of TAM induced cell death in miR-21 inhibitor-transfected cells. Further analysis suggested that miR-21 inhibitor enhance autophagic cell death through inhibition of PI3K-AKT-mTOR pathway. MiR-21 coordinated the function of autophagy and apoptosis by targeting Phosphatase and tensin homolog (PTEN) through inhibition of PI3K-AKT-mTOR pathway. In conclusion, silencing of miR-21 increased the sensitivity of ER⁺ breast cancer cells to TAM or FUL by increasing autophagic cell death. Targeting autophagy-related miRNAs is a potential strategy for overcoming endocrine resistance to TAM and FUL.     

Up-regulated microRNA-155 expression is associated with poor prognosis in cervical cancer patients

BackgroundMicroRNAs (miRNAs) play important roles in tumor development and progression. The purposes of the study was to investigate the role of miR-155 in cervical cancer.MethodsQuantitative real-time RT-PCR (qRT-PCR) was performed to examine miR-155 expression in cervical cancer tissues and adjacent non-cancerous tissues. The association with overall survival of patients was analyzed by Kaplan-Meier survival analysis. Small interfering RNA (siRNA) was used to suppress miR-155 expression in cervical cancer cells. In vitro assays were performed to further explore the biological functions of miR-155 in cervical cancer.ResultsWe found that miR-155 expression was markedly up-regulated in cervical cancer tissues and correlated with FIGO stage, lymph nodes metastasis, vascular invasion and HPV. Patients with high miR-155 expression level had poorer overall survival than those with low miR-155 expression. Furthermore, multivariate Cox regression analysis suggested that increased miR-155 was an independent prognostic indicator for cervical cancer (P = 0.007; HR = 2.320; 95%CI: 1.259–4.276). Moreover, knockdown of miR-155 was demonstrated to inhibit cell proliferation, migration, and invasion in vitro.ConclusionOur study presents that miR-155 is a novel molecule involved in cervical cancer progression, which provide a potential prognostic biomarker and therapeutic target.     

慢性淋巴细胞性白血病患者miR-9-3甲基化异常及其意义

目的本研究旨在探讨microRNA-9-3(miR-9-3)在慢性淋巴细胞性白血病骨髓细胞中的甲基化异常及其意义。方法采用甲基化特异性聚合酶链反应(MSP)技术检测8例正常骨髓组织、78例新确诊的慢性淋巴细胞性白血病患者和7种白血病细胞株甲基化水平。结果正常对照组miR-9-3呈未甲基化状态;7种白血病细胞株中有5种呈未甲基化状态(71.4%);78例慢性淋巴细胞性白血病患者中65例呈miR-9-3甲基化,MSP阳性率为83%。5-氮杂-2'-脱氧胞苷(5-Aza Dc)处理白血病细胞株后I83-E95和WAC3CD5+细胞株miR-9-3呈未甲基化状态。结论慢性淋巴细胞性白血病患者存在miR-9-3表达受抑,可能与其基因甲基化异常有关。而miR-9-3甲基化在激活慢性淋巴细胞性白血病患者NF-κB1信号通路的作用值得进一步研究。     

脑膜瘤中miR-200b和ZEB1的表达水平变化及其与预后的关系

目的 探讨脑膜瘤组织中miR-200b、ZEB1表达水平变化与脑膜瘤患者预后的关系。方法 选择2013年5月-2014年12月在本院行神经外科手术治疗的脑膜瘤患者42例,采用qRT-PCR法检测各研究对象脑膜瘤组织与瘤旁组织中miR-200b表达水平,采用免疫组织化学染色法检测ZEB1水平,分析miR-200b、ZEB1表达水平与脑膜瘤临床病理特征的相关性,并对出院后的脑膜瘤患者进行随访,分析miR-200b、ZEB1表达水平变化与脑膜瘤患者预后的关系。结果 脑膜瘤组织miR-200b表达水平显著低于瘤旁组织(P<0.05); 脑膜瘤组织ZEB1阳性表达率高于瘤旁组织(P<0.05); miR-200b、ZEB1表达水平与肿瘤分期有关(P<0.05); miR-200b高表达组的5年无瘤生存率显著高于miR-200b低表达组,ZEB1阳性表达组的5年无瘤生存率显著低于ZEB1阴性表达组(P<0.05); miR-200b低表达、ZEB1阳性表达可能是脑膜瘤患者不良预后的独立危险因素。结论 在脑膜瘤组织中miR-200b低表达、ZEB1阳性表达,且与患者无瘤生存期有关,可作为判断脑膜瘤患者预后的潜在标志物。