Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

上海市徐汇区公共卫生从业人员弓形虫感染的血清学调查分析

随机抽取2010年12月~2011年3月上海市徐汇区公共卫生从业人员448名,采集静脉血,ELISA法检测血清弓形虫特异性IgG抗体,抽检的448名公共卫生从业人员中,弓形虫IgG阳性率为10.3%(46/448)。不同来源地(上海、安徽、江苏、河南和四川等)和性别人员之间感染率差异均无统计学意义(P>0.05)。≥30年龄组阳性率(14.9%,29/195)明显高于<30年龄组阳性率(6.7%,17/253)(P<0.05),30~39岁年龄组最高,为15.8%(16/101)。食品生产加工行业从业人员阳性率(12.6%,36/286)显著高于非食品生产加工行业从业人员阳性率(6.2%,10/162)(P<0.05)。     

浙江地区散发新型布尼亚病毒感染患者的临床特点及其基因序列

目的 分析发热伴血小板减少综合征布尼亚病毒(SFTSV)感染患者的临床特征、流行病学以及SFTSV基因序列.方法 收集2011年5月至7月在温州医学院附属舟山医院感染性疾病科收治的5例重症发热伴血小板减少综合征(SFTS)患者,均经PCR检测SFTSV核酸确诊.采用流式细胞仪(FCM)检测CD3+ CD4+、CD3+ CD8+T淋巴细胞,分离的病毒株测序并与GenBank比对.结果 SFTS患者典型临床表现为持续高热、全身肌肉酸痛、浅表淋巴结肿大、腹痛、腹泻,可伴消化道出血,在急性期WBC、PLT、CD3+CD4+T淋巴细胞呈进行性下降,最低分别为(0.97～2.00)×109/L,(12～42)×109/L和7.52％～20.39％.2例患者血清中分离出病毒,其基因序列与GenBank中的SFTSV进行比对,依赖RNA的RNA聚合酶基因与BX-2010、L-WWG、LN3、JS4、SD4、HN6和AH12的同源性为96％,糖蛋白基因的同源性为94％,而N蛋白基因与JS4、SD4和LN4的同源性为95％;分离的2株病毒的上述三种基因序列的同源性为99％.结论 SFTSV在浙江省有散发流行,以本土疫源性可能大,起病急,病情重,伴多脏器功能损害.     

酶靶试验:用溶组织素的免疫酶显色检测肠道的溶组织内阿米巴感染

探索一种高度敏感和特异的检测肠道溶组织内阿米巴感染的方法——酶靶(ENZYMEBA)试验。该法是以溶组织素的免疫酶显色为基础的。首先制备抗溶组织素抗体,即以纯化的溶组织素(主要是半胱氨酸蛋白酶)对新西兰白兔进行免疫,50μg酶加入0.5ml PBS,加福氏完全佐剂使其乳化,作     

Seroprevalence of Entamoeba histolytica infection in HIV-infected patients in China

Seroprevalence of Entamoeba histolytica infection in HIV-infected individuals from Shanghai city, Anhui province, and Henan province, China, was examined by enzyme-linked immunosorbent assay using crude antigen and a recombinant surface antigen, C-Igl, of the parasite. In 215 HIV-infected individuals, the positive rates for these antigens were 12.1% and 7.9%, respectively; these rates were significantly higher than the rates of 3.1% and 0.5%, respectively, in 191 patients with gastrointestinal symptoms who were not infected with HIV. There was no significant difference in seropositivity to E. histolytica between men and women. Seropositivity in HIV-infected individuals was higher in patients with a CD4(+) T cell count of < 200/microL. This is the first report showing a higher seroprevalence of E. histolytica infection in HIV-infected patients in China. Our results also suggest that HIV infection is a risk factor for infection with E. histolytica.     

间歇和连续静脉输注法莫替丁对消化性溃疡患者胃酸的影响

胃十二指肠出血通常由慢性消化性溃疡、应激性溃疡或局部胃黏膜因素导致的急性胃黏膜损伤 (ＡＧＭＬ)引起。对良性疾病导致的胃十二指肠出血的治疗分为保守疗法和手术疗法。但是 ,内镜下止血的进展以及有效的Ｈ2 受体拮抗剂 (Ｈ2 ＲＡ)和某些胃肠激素的应用使手术疗法在治疗胃十二指肠出血中的地位明显下降。Ｈ2 ＲＡ对上消化道出血的止血作用主要基于其抑酸作用 ,但此类药物控制应激性溃疡或ＡＧＭＬ时大出血的价值有限。因此将某些胃肠激素如生长抑素和促胰液素用于治疗这种大出血受到关注 ,且文献报道疗效很好。我们推测疗效的差异可能是…     

Lansoprazole ameliorates intestinal mucosal damage induced by ischemia-reperfusion in rats

AIM:To investigate the protective effect of lansoprazoleon ischemia and reperfusion(I/R)-induced rat intestinalmucosal injury in vivo.METHODS:Intestinal damage was induced by clampingboth the superior mesenteric artery and the celiac trunkfor 30 rain followed by reperfusion in male Sprague-Dawleyrats.Lansoprazole was given to rats intraperitoneally 1 hbefore vascular clamping.RESULTS:Both the intraluminal hemoglobin and proteinlevels,as indices of mucosal damage,significantlyincreased in I/R-groups comparion with those of sham-operation groups.These increases in intraluminal hemoglobinand protein levels were significantly inhibited by the treatmentwith lansoprazole at a dose of 1 mg/kg.Small intestineexposed to I/R resulted in mucosal inflammation that wascharacterized by significant increases in thiobarbituric acid-reactive substances(TBARS),tissue-associatedmyeloperoxidase activity(MPO),and mucosal content of ratcytokine-induced neutrophil chemoattractant-1(CINC-1).These increases in TBARS,MPO activities and CINC-1 contentin the intestinal mucosa after I/R were all inhibited bypretreatment with lansoprazole at a dose of 1 mg/kg.Furthermore,the CINC-1 mRNA expression was increasedduring intestinal I/R,and this increase in mRNA expressionwas inhibited by treatment with lansoprazole.CONCLUSION:Lansoprazole inhibits lipid peroxidation andreduces development of intestinal mucosal inflammationinduced by I/R in rats,suggesting that lansoprazole mayhave a therapeutic potential for I/R injury.     

A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection

The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resulted in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.     

Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages

Loss-of-function mutations in the murine dominant white spotting/c-kit locus affect a diverse array of biological processes and cell lineages and cause a range of phenotypes, including severe anemia, defective pigmentation, sterility, mast cell deficits, a lack of interstitial cells of Cajal, spatial learning memory deficits, and defects in peripheral nerve regeneration. Here we show that tyrosine residues 567 and 569 in the juxtamembrane (Jx) domain of the murine Kit receptor tyrosine kinase are crucial for the function of Kit in melanogenesis and mast cell development, but are dispensable for the normal development of erythroid, interstitial cells of Cajal and germ cells. Furthermore, adult mice lacking both tyrosines exhibit splenomegaly, dysregulation of B-cell and megakaryocyte development, and enlarged stomachs. Analysis of signal transduction events induced by the mutant receptors after ligand stimulation indicates that Jx tyrosine mutations diminish receptor autophosphorylation and selectively attenuate activation of extracellular signal-regulated kinase/mitogen-activated protein kinases. Together, these observations demonstrate that the Jx domain of Kit plays a cell-type specific regulatory role in vivo and illustrate how engineered mutations in Kit can be used to understand the complex biological and molecular events that result from activating a receptor tyrosine kinase.