汉滩病毒感染HUVEC细胞系炎症介质表达的检测

目的 检测汉滩病毒陈株感染HUVEC细胞系ECV304中炎症介质的表达。方法 用汉滩病毒陈株感染HUVEC细胞系ECV304,取感染后不同时间的细胞提取细胞总RNA,用半定量RT－PCR检测感染细胞中ICAM－1、eNOS和IL－6基因的表达,并将扩增的ICAM－1和eNOS基因克隆,测定核苷酸序列。结果LACM－1的表达产物在病毒感染后较未感染细胞有明显升高,序列分析结果表明扩增的基因为特异性扩增产物;用扩增eNOS的引物从病毒感染细胞可扩增出表达产物,从未感染细胞不能扩增出任何产物,但序列分析结果表明扩增的基因与一新基因高度同源。结论 汉滩病毒陈株感染ECV304细胞可上调炎症某些介质的表达。     

Herpes simplex virus 1 induced LOX-1 expression in an endothelial cell line, ECV 304

Infections, such as by Chlamydophilia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori, have been shown to be involved in atherogenesis. Herpes simplex virus I (HSV-1) could infect vascular endothelial cells, and it has been shown that, when endothelial cells were activated with oxidized LDL (oxLDL), a number of cellular events are occurred, leading to endothelial cell dysfunction. Since LOX-1 is a major receptor for oxLDL on endothelial cells and its expression was increased in atherosclerosis, we investigated whether HSV1 infection can lead to the increase expression of LOX-1 in endothelial cells. LOX-1 mRNA expression determined by RT-PCR and LOX-1 promoter activity measured by luciferase assay were increased in endothelial cells following HSV-1 infection. This suggests that one of the mechanisms by which HSV-1 is involved in atherogenesis maybe the enhanced uptake of oxLDL via the increased expression of LOX-1 in endothelial cells.     

Orientia tsutsugamushi inhibits apoptosis of macrophages by retarding intracellular calcium release

Orientia tsutsugamushi shows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited by O. tsutsugamushi infection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected with O. tsutsugamushi. Antiapoptotic activities of O. tsutsugamushi in infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.     

Cellular invasion of Orientia tsutsugamushi requires initial interaction with cell surface heparan sulfate

Role of transmembrane heparan sulfate proteoglycans on invasion of Orientia tsutsugamushi into host cells was investigated. Pretreatment with heparan sulfate and heparin inhibited the infection of O. tsutsugamushi for L cell, mouse fibroblast, whereas other glycosaminoglycans had little effect. These same treatments were also shown to reduce the infection in a dose-dependent manner, and enzymatic treatment of cells with heparitinase, but not chondroitinase ABC, inhibited the infection. In addition, mutant cell lines of Chinese hamster ovarian cell defective in heparan sulfate synthesis but not chondrotin sulfate synthesis and defective in all glycosaminoglycan synthesis showed marked reduction in susceptibility to infection by O. tsutsugamushi. Also mutant cell lines, which express heparan sulfate proteoglycans at low level, showed intermediate level of infectivity. Finally O. tsutsugamushi bind to(35)S-labelled heparin. Collectively, these findings provide strong evidence that heparan sulfate proteoglycans contribute to the attachment of O. tsutsugamushi to the cells.     

A serosurvey of Orientia tsutsugamushi from patients with scrub typhus

Many countries where scrub typhus is endemic use their own cutoff values for antibody titres to differentiate between cured cases and current infections. To establish an antibody titre cutoff value, one needs to investigate the seroprevalence in endemic areas, and the duration of the increase in titre after complete cure. We conducted a follow-up study of anti-Orientia tsutsugamushi antibody titres using indirect immunofluorescence assays (IFA) and passive haemagglutination assays (PHA) in patients with scrub typhus. After the onset of symptoms, IgM antibody titres increased gradually over 2–3 weeks, peaked at about 4 weeks, and started to decrease rapidly between 4 and 5 weeks. At 1-year follow-up, the median IgM value was 1:10. Out of 77 patients who were tested at that time, 36 (47%) had IgM titres ≥1:20, and none had titres exceeding 1:80. Over the first 2 weeks, IgG antibody titres increased sharply, peaked at about 4 weeks and decreased rather gradually thereafter, with a median titre of 1:128 maintained up to the 18th month. At 1-year follow-up, five out of 77 patients (6.5%) had titres ≥1:1,024 and 57% had titres ≥1:128. Based on these results, a cutoff value of ≥1:160 for IgM antibody should differentiate between previous and current infections in endemic areas such as Korea and Japan, where scrub typhus occurs mainly in the autumn.     

Expression and regulation of endothelial nitric oxide synthase by vascular endothelial growth factor in ECV 304 cells

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with VEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to VEGF(165), eNOS activity and cell growth were increased by approximately two-fold in the VEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, VEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.     

Detection of new genotypes of Orientia tsutsugamushi infecting humans in Thailand.

PCR screening of blood specimens taken from 195 patients with serologically confirmed scrub typhus in three Thai provinces detected the 56-kDa protein-encoding gene from Orientia tsutsugamushi in ten (5%) patients. Significant genetic diversity was found among the ten amplicons, with nine new genotypes identified that were different from those found previously in Thailand. Phylogenetically, the ten sequences obtained in the present study and sequences from 71 strains characterised previously were distributed into several clusters that included the Karp, Gilliam, Kuroki, Saitama, Kawasaki and Kato clusters. Two of the new genotypes found in the present study clearly belonged to the Karp cluster. However, the other new genotypes formed three different clusters, including one cluster that appeared to be distant from all previously known clusters, and which may therefore be representative of a previously undescribed serotype. Other genotypes formed two other clusters that may also be associated with undescribed serotypes.     

Risk-factors for human infection with Orientia tsutsugamushi: a case-control study in Korea.

A case-control study was conducted involving 156 patients with scrub typhus and 130 controls. Three factors were associated significantly with the risk of developing scrub typhus: engaging in fruit farming (OR 2.44; 95% CI 1.04-5.69), gathering chestnuts (OR 2.05; 95% CI 1.09-3.87) and taking breaks in areas adjacent to agricultural operations (OR 3.06; 95% CI 1.50-6.22). In contrast, receiving information or educational materials concerning the prevention of scrub typhus had a protective effect (OR 0.45; 95% CI 0.24-0.83). These results suggest that a health education programme will lower the risk of developing scrub typhus when applied to high-risk groups.     

Endothelial cell formation of focal adhesions on hydrophilic plasma polymers.

Endothelial cell (EC) formation and distribution of both actin stress fibers and focal contacts on hydrophilic plasma polymers derived from gamma-butyrolactone (GBL) and n-vinylpyrrolidone (NVP) were examined to determine their ability to support endothelial cell growth in comparison to fibronectin. One hour after seeding, cells adhered and spread moderately on fibronectin with the development of defined actin stress fibers and focal adhesions compared to NVP and GBL, on which the cells were spread with poorly developed stress fibers and a perinuclear localization of vinculin. At 3 h, cells continue to spread more on fibronectin and NVP than GBL, and the cells on fibronectin had well-defined stress fibers terminating with sharp spikes of vinculin, typical of focal adhesions. At this time point, paxillin, a signaling component of focal adhesion complex, was predominantly localized at the focal contacts for well-spread EC on fibronectin and NVP, whereas it was almost entirely concentrated in the perinuclear region of less-spread cells on GBL. However, by 24h, cells were much more spread on all three surfaces with defined stress fibers and focal contacts although EC expression of vinculin and paxillin was moderate on GBL compared to fibronectin and NVP. These results suggest that EC can form cytoskeletal structures necessary for cell survival on plasma polymers, especially on more hydrophilic NVP, which could be exploited as interface material for seeding endothelial cells.     

Vertical transmission of Orientia tsutsugamushi in two lines of naturally infected Leptotrombidium deliense (Acari: Trombiculidae)

Vertical transmission of Orientia tsutsugamushi (Hayashi), the etiologic agent for scrub typhus, was studied in two lines of naturally infected Leptotrombidium deliense Walch. In one line of mites originating from a single adult (V3M), the rate of filial transmission was 100% for the first two laboratory generations, but declined to 86.6% in the third laboratory generation. The vertical infection rate in this line of mites was 100% for the parental generation, but declined to 95.6% for the F1 generation and 88.6% for F2. The transmission of O. tsutsugamushi in another line of L. deliense (V3F) was less efficient than mites originating from V3M. In the initial laboratory generation of V3F a filial transmission rate of 100% was recorded. However, none of the F2 generation of this line transmitted rickettsiae to mice (Mus musculus L.), resulting in a filial transmission rate of 0%. Transmission of O. tsutsugamushi to mice by progeny from cohort larvae originally from the same adult (V3F) was also studied in the laboratory and these were found to be relatively poor transmitters of rickettsiae. The filial infection rate of F2 larvae was 60%, F3 was 88.8%, and F4 was 55.9%. The biology of infected L. deliense was studied and compared with uninfected mites reared under the same laboratory conditions. The results showed that infected female L. deliense laid approximately the same or more eggs as uninfected adults. The rate of development of the progeny of infected L. deliense was not significantly different from uninfected mites.     

Microarray analyses of differentially expressed human genes and biological processes in ECV304 cells infected with rubella virus

Changes in mRNA expression levels of ECV304 cells infected with the wild-type rubella strain were analyzed using a microarray system representing 18,716 human genes. Four hundred eighty-seven genes exhibited differential expression levels; 456 of these genes were up-regulated while 31 genes were down-regulated. We identified 53 biological processes that were significantly relevant to the RV-infection. Among these biological processes, 52 were one-gene processes and one was a process involving five genes: IFNA21 (interferon, alpha 21), interferon stimulated exonuclease gene 20 kDa (ISG20), zinc finger protein 175 (ZNF175), tripartite motif-containing 22 (TRIM22), and MX2 [myxovirus (influenza virus) resistance 2 (mouse)]. Except for ZNF175, gene annotation indicated four of these genes encoded interferon or interferon-induced genes. These results suggest that genes relevant to interferon-regulated pathways may be involved in the pathogenesis of rubella.     

Induction of apoptosis in a T lymphoblastoid cell line infected with feline immunodeficiency virus

Summary The mechanism of cell death induced by feline immunodeficiency virus (FIV) infection was investigated in an interleukin 2(IL-2)-dependent T-lymphoblastoid cell line (MYA-1). DNA extracted from FIV-infected MYA-1 cells showed a ladder of nucleosomal DNA, indicating that the cytopathic effect (CPE) observed in these cells was due to apoptosis. Infection of MYA-1 cells with FIV was associated with suppression of the proliferative response of the cells to exogenous IL-2 prior to DNA fragmentation. These findings suggest that FIV-induced CPE in these T-lymhoblastoid cells is associated with apoptosis possibly due to a defect in the IL-2 signal transduction pathway.     

Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell line T24/83

Early reports indicated that ECV304 was a spontaneously-transformed line derived from a Japanese human umbilical vein endothelial cells (HUVEC) culture. Many morphological, immunochemical, and genetic studies provided further evidence that ECV304 was a valuable biomedical research tool and could be used to study processes that include angiogenesis in vitro and signal transduction by a variety of G protein-coupled receptors. However, several distinct differences between ECV304 and HUVEC are now apparent and recent reports have indicated genetic similarity between ECV304 and T24/83, a human bladder cancer cell line. To further assess the utility of ECV304 as a human endothelial cell model, we compared the functional responses of ECV304 and T24/83 to a range of G protein-coupled receptor agonists. We also used DNA fingerprinting to karyotype both ECV304 and T24/83. Both ATP and uridine triphosphate (UTP) stimulated inositol phosphate metabolism in ECV304 without alteration of cAMP levels. Comparative data using selective P2Y receptor agonists indicated that this response, leading to calcium mobilization from intracellular stores, was predominantly mediated by the activation of P2Y2 receptors. Similar responses were recorded from both ECV304 and T24/83 cells. ECV304 expressed a relatively high basal activity of NOS that was reduced by L-NAME and stimulated by P2Y2 receptor agonists. In contrast, P2Y2 receptor activation did not induce prostaglandin synthesis in ECV304. Both ECV304 and T24/83 express receptors for adenosine, adrenaline, and calcitonin, which stimulate adenylate cyclase. Proliferation of ECV304 and T24/83 cells, measured by the incorporation of [3H]thymidine into DNA, was largely serum-independent. This was in contrast to parallel experiments with porcine and bovine aortic endothelial cells that indicated a marked serum-dependent increase in DNA synthesis. Genetic analysis confirmed that ECV304 and T24/83 are identical. ECV304 displays some endothelial characteristics and is useful for the study of receptor pharmacology. However, ECV304 is not of HUVEC origin and is therefore an inappropriate cell line to study endothelial cell biology.     

Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.     

刺激ECV304细胞增殖的新基因EOLA1的克隆和功能研究

目的 扩增内皮细胞受内毒素刺激后上调表达的新序列标签ST55（GenBank No．BMl21646）全长eDNA序列并研究其结构和生物学功能。方法 应用快速扩增cDNA末端技术延伸ST55获得其全长cDNA序列，以Noithem杂交检测其组织分布，通过酵母双杂交筛选其胞内相互作用蛋白，在ECV304细胞中稳定转染目的基因并强制表达后观察细胞生长变化。结果 获得1个全长为1404碱基的cDNA序列，作为人类新mRNA被GenBank接受（AY074889），命名为内皮高表达脂多糖相关因子1（endothelial-overexpressed lipopolysaccharide-associated factor 1，EOLA1）。生物信息学分析显示EOLA1基因含5个外显子，定位于染色体Xq27．4，编码蛋白质由158个氨基酸组成，分子量为1789。Northern印迹显示EOLA1在人各组织和癌细胞系有不同的表达；以EOLA1 cDNA作为诱铒，进行酵母双杂交筛选人肝cDNA文库，鉴定了金属硫蛋白2A（metallothionein 2A，MT2A）为其相互作用蛋白并采用免疫共沉淀验证了这一结果。高表达EOLA1显著促进了ECV304细胞增殖。结论 EOLA1是人内皮活化相关新基因，EOLA1与MT2A的相互作用可能在炎症反应中细胞内保护方面发挥作用。     

Identification of the target cells of Orientia tsutsugamushi in human cases of scrub typhus.

Orientia tsutsugamushi is the etiologic agent of scrub typhus, a chigger-borne zoonosis that is a highly prevalent, life-threatening illness of greatest public health importance in tropical Asia and the islands of the western Pacific Ocean. The target cell of this bacterium is poorly defined in humans. In this study, O. tsutsugamushi were identified by immunohistochemistry using a rabbit polyclonal antibody raised against O. tsutsugamushi Karp strain in paraffin-embedded archived autopsy tissues of three patients with clinical suspicion of scrub typhus who died during World War II and the Vietnam War. Rickettsiae were located in endothelial cells in all of the organs evaluated, namely heart, lung, brain, kidney, pancreas, and skin, and within cardiac muscle cells and in macrophages located in liver and spleen. Electron microscopy confirmed the location of rickettsiae in endothelium and cardiac myocytes.