系统性红斑狼疮PBMCs c-myc mRNA的表达及其与疾病活动性相关性研究

目的:探讨系统性红斑狼疮(SLE)外周血单个核细胞(PBMCs)的c-myc mRNA表达在SLE发病中的作用.方法:从33例SLE患者及20例健康对照组外周静脉血中分离PBMCs,提取总RNA作模板,按文献设计合成c-myc引物,以β-actin作内参,应用半定量逆转录-聚合酶链反应(RT-PCR)检测SLE患者及健康对照的PBMCs c-myc mRNA表达水平并进行组间比较,用系统性红斑狼疮疾病活动性指数(SLEDAI)评定每例患者疾病活动性,并对SLE患者PBMCs的c-myc mRNA表达水平与SLEDAI进行相关分析.结果:c-myc及β-actin的RT-PCR扩增产物电泳显示分别为268和163 bp条带.SLE患者PBMCs的c-myc mRNA相对表达量为0.58±0.26,而正常对照的相对表达量为0.07 ±0.04,两组差别有显著性(t'=11.024,P=0.000).25例活动期SLE患者的c-myc mRNA相对表达量为0.62±0.25,而8例缓解稳定期SLE患者的c-myc mRNA相对表达量为0.25 ±0.01,组间差别也有显著性(t'=7.86,P=0.000).SLE患者PBMCs的c-myc mRNA相对表达量与SLEDAI呈正相关(r =0.865 1,P＜0.05).结论:SLE患者PBMCs的c-myc mRNA表达异常,c-myc mRNA表达水平与SLE疾病活动评分指数呈直线正相关关系.c-myc mRNA表达水平可作为判断SLE疾病活动性的一个指标.     

催乳素对小鼠脾脏CD11c+树突状细胞合成细胞因子的调控

本研究采用逆转录-多聚酶链反应(RT-PCR)方法,在mRNA水平上了解不同浓度的催乳素(PRL)对小鼠脾脏树突状细胞CD11c+(spleen CD11c-positive dendritic cells,SDC)合成细胞因子的影响。结果表明,低(0.01 nmol/L)、中浓度(0.1nmol/L)的PRL可以上调IL-6、IL-10、IL-12和TNF-α的水平而高浓度(1 nmol/L)则降低它们的表达(IL-12除外)。这提示PRL可能通过改变抗原提呈细胞SDC细胞因子的合成,进而参与调节机体的生理或病理性免疫反应。     

Cerebral capillary endothelial cells are covered by the VEGF-expressing foot processes of astrocytes

Molecules that have crucial functions in both nervous and vascular systems have attracted keen attention recently, and the name "angioneurins" has been proposed. The most striking example of angioneurins is vascular endothelial growth factor A (VEGF), which was originally identified as a key regulator of angiogenesis and has only recently been found to have important functions in the nervous system. In this study, we compared VEGF expression in the vasculature in the brain with that in the aorta and the vasculature in the kidney in mice. In larger vessels containing smooth muscle cells, VEGF was expressed by smooth muscle cells covering the lining of endothelial cells, both in and outside the brain. In cerebral capillaries lacking smooth muscle cells, endothelial cells were closely covered by VEGF-expressing foot processes of astrocytes, whereas capillaries were surrounded by VEGF-expressing processes of podocytes in the renal glomeruli. We also found that cultured cerebral microvessel endothelial cells do not express VEGF, whereas cultured cortical astrocytes do express VEGF.     

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques.     

HCV 5′ NCR和结构蛋白编码序列的拼接及在pLNSX中的克隆

目的:构建丙型肝炎病毒(HCV)5′末端非编码区(5′NCR)和结构蛋白编码基因序列逆转录病毒重组体,用于探索控制HCV感染的新途径和基因治疗。方法:对多株HCV核酸序列进行同源性比较设计引物,逆转录聚合酶链式反应(RT-PCR)扩增5′NCR、C、E1和E2/NS14个编码区共5个片段,分别克隆。以连接聚合酶链反应(PCR)将这5个片段拼接为一连续的长2547nt的片段,含HCV完整的5′NCR和全部的结构蛋白编码序列。将此序列插入pGEM-Zf(+)载体,与逆转病毒pLNSX载体中,转化大肠杆菌DH5a、转化菌落经酶切、PCR和Southern杂交鉴定。结果:通过RT-PCR和连接聚合酶链式反应(PCR)扩增出2547nt含HCV完整的5′NCR和全部的结构蛋白编码序列,将此序列与pGEM-Zf(+)重组得重组体pHC2547,与逆转录病毒载体pLNSX重组得pL-HC。结论:成功构建了HCV逆转病毒重组体,以利于HCV的胞内基因表达调控研究,更为探索HCV分子致病机制和转基因动物及基因治疗提供可靠的物质基础。     

Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity

Three classic IBDV strains were previously isolated from commercial layer chicken flocks and shown to be phylogenetically related to vaccine strains but pathogenic in susceptible chickens. In this study, their viral genomes were sequenced and compared to sequences of vaccines being used in those flocks. The vaccine strains examined were sequenced directly from the manufacturer and had identical genome segment B sequences. Compared to these vaccines, the GA-1, H-30 and CS-2-35 isolates each had one silent mutation in the gene that encodes VP1. Compared to the two vaccines used at the time CS-2-35 was isolated, the segment A sequence of CS-2-35 contained numerous nucleotide and amino acid mutations suggesting the CS-2-35 virus was not closely related to these vaccines. This virus however did have amino acid mutations in VP2 that are reported to be necessary for replication in cell culture and lacked two of the three amino acid mutations previously shown to be necessary for virulence. These data suggest that CS-2-35 was a descendant from an attenuated strain of IBDV. When the segment A genomic sequences of the GA-1 and H-30 viruses were compared to the vaccines being used in those flocks they were most closely related to the attenuated D78 vaccine strain. In genome segment A, three nucleotide mutations in GA-1 and four in H-30 were observed compared to the D78 classic vaccine. These nucleotide mutations caused one amino acid (H253N) change in the GA-1 virus and two amino acids (H253Q and G259D) were different in the H-30 virus. In addition, both the GA-1 and H-30 viruses had the amino acid G76 in VP2 that appears to be unique to the vaccine D78. The data suggest that GA-1 and H-30 are genetically related and have a common ancestor even though they were isolated from geographically distant flocks. The evidence also suggests that GA-1, H-30 and CS-2-35 could be reversions from attenuated vaccine viruses or by coincidence genetically resemble classic IBDV vaccines. It should be noted that some of the classic virus vaccines were not being used according to the manufacturer's recommendations at the time the GA-1, H-30 and CS-2-35 strains were isolated. Together, the molecular and pathogenicity data indicate that a single amino acid mutation from Histidine (H) to Glutamine (Q) or Asparagine (N) at position 253 in VP2 will markedly increase the virulence of an attenuated IBDV.     

Rapid typing of the human neutrophil antigen 1a by the particle gel agglutination assay

The human neutrophil antigen 1a (HNA-1a) plays a major role in immune neutropenias and transfusion-associated lung injury. In this study, we describe a simple and rapid particle gel agglutination assay (PaGIA) for HNA-1a phenotyping. A commercially available monoclonal antibody (MoAb) to HNA-1a was biotinylated and coupled onto superparamagnetic streptavidin particles. Diluted anticoagulated whole blood samples from healthy blood donors ( n  = 147) were incubated with MoAb-coated particles, washed, transferred into an ID-gel card, and, subsequently, centrifuged. HNA-1a-positive samples resulted in a visible agglutination of the particles on top of the gel column and could be evaluated macroscopically. The results obtained by the new test were identical with those obtained by the polymerase chain reaction–sequence-specific priming technique that was performed in parallel. Seventy-four (50.3%) of the 147 samples were found to be HNA-1a positive. The HNA-1a PaGIA is both simple and safe and can be implemented in various laboratory settings.     

Amino acids contributing to antigenic drift in the infectious bursal disease Birnavirus (IBDV)

We examined the effect of amino acids 222 and 254 on antigenicity of the variant Del-E strain of infectious bursal disease virus (IBDV). Using molecular epidemiology, we identified a virus designated as Del-E-222 that was identical to Del-E except for alanine at position 222. A second virus was generated using reverse genetics of the Del-E backbone to create Del-E-254 that contained an asparagine at amino acid 254. The Del-E-222 and Del-E-254 viruses were tested for their ability to escape neutralizing immunity provided by parenteral vaccination. The bursas from birds vaccinated with parental Del-E and challenged with Del-E-222 or Del-E-254 had macroscopic lesions typical of an IBDV infection, and their B-BW ratios were significantly smaller than the controls. Microscopic lesions included lymphocyte depletion and confirmed the ability of Del-E-222 and Del-E-254 to break through the immunity induced by the parental Del-E virus vaccination. Both mutations appear to be contributing to antigenic drift.     

Changes in beta-2 adrenergic receptor and AMP-activated protein kinase alpha-2 subunit in the rat vestibular nerve after labyrinthectomy

In the present study, to elucidate the role of vestibular ganglion (VG) after the unilateral labyrinthine damage, we examined quantitative changes in mRNA expression of beta-adrenergic receptors (bARs) and AMP-activated protein kinase alpha catalytic subunits (aAMPKs) in VG after unilateral labyrinthectomy (UL) in rats. Using the real-time PCR method, beta2 AR mRNA expression in bilateral VG and AMPK alpha2 mRNA expression in the ipsilateral VG were significantly up-regulated with the maximum increase at the postoperative 7 day and 1 day, respectively. The up-regulation of beta2 AR in bilateral VG was long-lasting until 28 days after UL and that of AMPK alpha2 in the ipsilateral VG was just transient within 7 days after UL. These mRNA changes were supported by immunohistochemical data. According to previous reports, both of bARs and aAMPKs could regulate mitochondrial uncoupling protein (UCP) mRNA expression in several kinds of tissues and therefore might have thermogenic neurotransmission and antioxidant neuroprotective roles in neuronal tissues. UL requires not only long-lasting response of VG for central vestibular neuro-plasticity around 2-4 weeks but rapid response of VG against apoptosis of peripheral vestibular epithelia-neuronal synapses. The present findings suggest that beta2 AR in bilateral VG and AMPK alpha2 in the ipsilateral VG might play important signaling roles after the unilateral labyrinthine damage.     

Toll-like receptor 2-mediated sequential activation of MyD88 and MAPKs contributes to lipopolysaccharide-induced sp-a gene expression in human alveolar epithelial cells

Surfactant proteins (SPs) produced by pulmonary epithelial cells participate in the regulation of sepsis-induced acute lung injury. Our previous study has shown that lipopolysaccharide (LPS), a Gram-negative bacterial outer membrane component, can regulate sp-a gene expression in human lung carcinoma type II epithelial A549 cells. This study was further designed to evaluate the signal-transducing mechanisms of LPS-induced sp-a gene expression. Exposure of A549 cells to LPS induced SP-A mRNA and protein production in time-dependent manners. Application of toll-like receptor 2 (TLR2) siRNA into A549 cells decreased the levels of this receptor and simultaneously inhibited LPS-induced SP-A mRNA expression. Sequentially, LPS enhanced phosphorylation of mitogen-activated protein kinase (MEK) 4 and c-Jun NH₂ terminal kinase 1 (JNK1) in time-dependent manners. Application of TLR2 siRNA decreased LPS-enhanced phosphorylation of MEK4 and JNK1. After knocking-down the translation of MyD88 by RNA interference, the LPS-triggered MEK4 phosphorylation was attenuated. Consequently, LPS augmented the translocation of c-Jun from the cytoplasm to nuclei without affecting c-Fos. Pretreatment of A549 cells with SP600125, an inhibitor of JNK1, significantly lowered LPS-induced SP-A mRNA production. Analyses of an electrophoretic mobility shift assay and a reporter gene further showed that LPS increased the transactivation activity of AP-1 in A549 cells. Therefore, the present study demonstrates that LPS can induce sp-a gene expression in human type II epithelial A549 cells through TLR2-mediated sequential activation of MyD88-MEK4-JNK1-AP-1.