CD163基因敲除iPAMs的构建及其感染PRRSV的特征分析

[目的]获得分化抗原163(cluster of differentiation 163,CD163)基因敲除的永生化猪肺泡巨噬细胞系(immortalized porcine alveolar macrophages,iPAMs),并从细胞水平研究CD163蛋白在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)入侵过程中的作用。[方法]将靶向CD 163基因第7外显子的sgRNA质粒(pX330-sgCD163)转染至iPAMs,转染48 h后通过有限稀释法筛选单克隆细胞并进行基因型鉴定,通过Western blotting和脱靶分析来筛选CD 163基因敲除的iPAMs;通过实时荧光定量PCR、Western blotting、间接免疫荧光试验(indirect immunofluorescence assay,IFA)和半数细胞培养物感染量(50%tissue culture infective dose,TCID 50)等试验分析CD 163基因敲除的iPAMs感染PRRSV的特征。[结果]测序结果表明,100株单克隆细胞中有1株CD 163双等位基因敲除的iPAMs,敲除效率为1%;Western blotting结果显示,CD 163基因敲除的iPAMs中检测不到CD163蛋白的表达,且在预测的脱靶位点未检测到脱靶效应。实时荧光定量PCR结果表明,与野生型iPAMs组相比,CD 163基因敲除的iPAMs组病毒拷贝数极显著降低(P<0.01);Western blotting和IFA结果表明,在接毒24 h后的CD 163基因敲除的iPAMs组中未检测到PRRSV-N蛋白的表达;TCID 50试验结果同样显示,在CD 163基因敲除的iPAMs组中未观察到细胞病变。[结论]本研究利用CRISPR/Cas9编辑系统成功构建了CD 163基因敲除的iPAMs,该细胞系可以完全抵抗PRRSV感染。该细胞系的建立为后续研究PRRSV与CD163蛋白的互作机制提供了新型试验材料。

CD8 T cell response in a phase I study of therapeutic vaccination of advanced NSCLC with allogeneic tumor cells secreting endoplasmic reticulum-chaperone gp96-Ig-peptide complexes

Antigen containing, allogeneic cells secreting the genetically modified protein and peptide-chaperone gp96-Ig cross, prime and expand antigen specific CD8 T cells with therapeutic antitumor activity in mice. In a first in man phase I study, we now report the results of therapeutic vaccination of non-small cell lung cancer (NSCLC) patients with an established, allogeneic non-small cell lung adenocarcinoma cell line secreting gp96-Ig. Advanced NSCLC-patients stage IIIB or IV of any histological subtype were enrolled and treated with up to 36 vaccinations over the course of 18 weeks. Primary endpoint was safety, secondary endpoints tumor response and overall survival. Measurement of tumor antigen specific CD8 CTL responses is precluded by the lack of known NSCLC associated antigens. Therefore, we measured patient CD8 T cell-IFN-γ responses to allo-antigens of the vaccine cells as surrogate for tumor antigen specific CD8 CTL. In 7 of 18 treated patients tumor growth was stabilized, however none of the 18 patients had an objective tumor response by RECIST criteria. Of 15 patients evaluable for immune response, 11 responded to vaccination with more than twofold increase in CD8-IFN-γ frequency above baseline. These patients had a median survival time of 16.5 months. Four patients who had no CD8 response above base line had survival times from 2.1 to 6.7 months. Our data are consistent with the concept that generation of CD8 CTL by therapeutic vaccination may delay tumor growth and progression and mediate prolonged survival even in the absence of objective tumor responses. Further studies will be required to test this concept and promising result.

IL-8基因沉默后猪血管内皮细胞相关细胞因子表达的变化

为了探究内皮源IL-8对猪血管内皮细胞其他相关细胞因子表达的影响,解析IL-8在内皮细胞中作用,利用siRNA干扰技术对猪血管内皮细胞(VEC)中IL-8基因进行沉默,于干扰后不同时间收集细胞及上清,应用荧光定量PCR技术和ELISA检测VEC中各细胞因子的变化。结果显示,在mRNA水平上,促炎因子IL-6和促内皮生长因子(VEGF)在12h上调显著,且在4hVEGF显著上调;ICAM-1和VCAM-1在4h显著上调,其中ICAM-1在48h极显著下调,72h又显著上调;巨噬细胞集落刺激因子(M-CSF)几个时间段均极显著高于对照组。蛋白水平上,VEGF除48h外均显著高于对照组,M-CSF在4h后均显著低于对照组。以上数据表明,内皮源IL-8变化可能影响VEC增殖、迁移以及对造血干细胞分化。

Immune Responses to Trichloroethylene and Skin Gene Expression Profiles in Sprague Dawley Rats

Objective To characterize the immune reaction in SD rats exposed to trichloroethylene （TCE） and to identify the gene expression profiles involved in skin after TCE exposure. Methods Fifteen percent of TCE was injected intradermally into the rat back （100 μL/120 g） at intervals of 7 days. Whole blood was collected 24 h after the fifth or seventh intradermic administration of TCE. The percentages of CD4＋ and CD8＋ of T lymphocytes were measured by a flow cytometer. The concentrations of IFN-gamma and 1L-4 in the serum were semi-quantified by ELISA. Total RNAs of skin samples at 3 h or 24 h after the seventh dose of TCE in SD rats were extracted, and gene expression proftles of these tissues were analyszed by rat toxicology U34 array of Affymetrix. Results Obvious decline of CD4＋ in T lymphocytes was observed in the TCE-administer group. No significant concentration differences in IFN-gamma and IL-4 were found between TCE-treated and control rats. Gadd45a and Mel were significantly up regulated in skin tissue 24 h after TCE exposure. The expression regulation of immune response factors was as active as proteins associated with lipid metabolism and synthesis process in these skin samples of SD rats exposed to TCE. Conclusion T-helper type 1 cells mediate immune response can not be elicited in TCE-treated SD rats, but certain immune disorder can be induced.

Clinical significance of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression in t(8;21)acute myeloid leukemia

OriginalTranslation t(8;21)(q22;q22)acute myeloid leukemia(AML)is a highly heterogeneous hematological malignancy with a high relapse rate in China.Two leukemic myeloblast populations(CD34^(+)CD117^(dim) and CD34^(+)CD117^(bri))were previously identified in t(8;21)AML,and CD34^(+)CD117^(dim) cell proportion was determined as an independent factor for this disease outcome.Here,we examined the impact of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression on t(8;21)AML clinical prognosis.In this study,85 patients with t(8;21)AML were enrolled.The mRNA expression levels of CD34^(+)CD117^(dim)-associated genes(LGALS1,EMP3,and CRIP1)and CD34^(+)CD117^(bri)-associated genes(TRH,PLAC8,and IGLL1)were measured using quantitative reverse transcription PCR.Associations between gene expression and clinical outcomes were determined using Cox regression models.Results showed that patients with high LGALS1,EMP3,or CRIP1 expression had significantly inferior overall survival(OS),whereas those with high TRH or PLAC8 expression showed relatively favorable prognosis.Univariate analysis revealed that CD19,CD34^(+)CD117^(dim) proportion,KIT mutation,minimal residual disease(MRD),and expression levels of LGALS1,EMP3,CRIP1,TRH and PLAC8 were associated with OS.Multivariate analysis indicated that KIT mutation,MRD and CRIP1 and TRH expression levels were independent prognostic variables for OS.Identifying the clinical relevance of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression may provide new clinically prognostic markers for t(8;21)AML.

Disease Progression Patterns of SHIV-KB9 in Rhesus Macaques of Chinese Origin in Comparison with Indian Macaques

Objective To develop a model of SHIV-KB9/Chinese origin rhesus （Ch Rh） macaques for vaccine research and to compare the pathogenesis of SHIV-KB9 in Ch Rh macaques with that reported in Indian rhesus （Ind Rh） macaques. Methods Seven mamu-A＊01 negative Ch Rh macaques were inoculated intravenously with 1-10000 MID50 of SHIV-KB9. The monkeys were monitored for viral load, CD4, CDS, SHIV-specific antibody and virus genetic variation. The results were compared with those previously observed in Ind Rh macaques. Results As compared to that observed in Ind Rh macaques, SHIV-KB9 in Ch Rh macaques displayed three identical disease progression patterns. However, the primary pattern was not identical between the two subspecies. The level of plasma viremia differed in SHIV-KB9-infected Ch Rh macaques which exhibited different outcomes from those in Ind Rh macaques. Generally, the values of viral load and the maintenance of CD4^＋ T cells were associated with humoral responses. Otherwise, the viral genetic distances （divergence, diversity） were larger in animals （M419, M425） with their CD4^＋ T cells profoundly depleted. Conclusion The model of SHIV-KB9/Ch Rh macaques displays a relatively slow progression to AIDS compared with Ind Rh macaques, which may more accurately reflect the potential of candidate vaccines in humans.

Adenovirus induced acute hepatitis in non-human primates after liver-directed gene therapy

OBJECTIVE: To define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy. METHODS: Differences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining. RESULTS: The monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct. CONCLUSION: Immune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.