Tim-3及其配体Galectin-9与病毒感染的关系

T细胞免疫球蛋白黏蛋白3(Tim-3)基因是Tim家族成员,与其配体半乳凝素-9(Galectin-9)结合可显著抑制T细胞的活化和增殖,并调节细胞因子的表达和分泌.Tim-3/Galectin-9参与免疫反应的调节及抗病毒免疫反应,阻断此通路可促进CD8+T细胞应答和对病毒的控制.因此,Tim-3/Galectin-9通路可能与病毒感染慢性化有关.本文对Tim-3及其配体的生物学特性、功能及在病毒感染中的研究近况进行综述.     

Elevation of soluble VCAM-1 plasma levels in children with acute dengue virus infection of varying severity

Approximately 1,000 million infections with dengue viruses are estimated to occur annually. The majority of the cases develop mild disease, whereas only small proportion of the infected individuals develop severe hemorrhagic manifestations at the end of the acute phase of illness. In this study, the value of plasma levels of vascular cell adhesion molecule 1 (VCAM-1) in the pathogenesis and prognosis of dengue illness was investigated in children with dengue infections of varying severity. The plasma levels of soluble VCAM-1 (sVCAM-1) were measured in serial plasma samples obtained from 168 children aged between 7 months and 14 years with confirmed dengue infection. Of those children, 71 were suffering from dengue fever, 30 were suffering from dengue hemorrhagic fever, and 67 were suffering from dengue shock syndrome. Plasma samples obtained from 21 patients with febrile illness other than dengue served as controls. A commercially available kit (R&D Systems, Oxon, UK) was used to measure the levels of sVCAM-1 in plasma samples. sVCAM-1 was elevated during acute dengue infection, and significantly elevated among dengue shock syndrome patients as compared to dengue fever or dengue hemorrhagic fever patients (P < 0.05). Statistical analysis revealed that sVCAM-1 was associated with dengue disease severity and the time post infection (acute vs. convalescent phase) and not with age, sex, or previous exposure of the patients to dengue infection. A significant difference was found in the plasma levels of sVCAM-1 between dengue shock syndrome and dengue fever patients, however, the prognostic value of this marker in the acute stage of dengue illness proved to be limited. These data also favor to study the further elucidation of the role of sVCAM-1 in the pathogenesis of dengue infections.     

小鼠抗登革病毒2型单克隆抗体的制备及鉴定

目的 制备小鼠抗登革病毒2型(TR1751株)的单克隆抗体(Monoclonal antibody mAb)，并对其性质进行鉴定。方法免疫原为病毒感染的Blabfc乳鼠脑组织，匀浆后离心，去除细胞成分。免疫Blab／c小鼠，将小鼠脾细胞和SP2／0细胞融合后，通过ELISA、间接免疫荧光染色、噬斑减少中和实验(Plaque reduction neutralization test，PRNT)及Western blot进行筛选和鉴定。结果筛选到I1、I9和B152 3株杂交瘤细胞系，其分泌的mAb均为识别登革病毒E蛋白的中和抗体。结论获得了3株分泌小鼠抗登革病毒2型mAb的杂交瘤细胞系，利用所制备的抗体，为进一步研究登革病毒致病机理和开发疫苗提供了免疫学工具。     

Transient CD4/CD8 ratio inversion and aberrant immune activation during dengue virus infection

The immune status after dengue virus infection was studied in dengue patients from an outbreak of serotype 3 dengue virus infection in the southern part of Taiwan during November and December 1998. Consecutive blood samples from 29 dengue patients, of whom 21 had dengue fever and 8 had dengue hemorrhagic fever/dengue shock syndrome, were collected, and the immunophenotypes of the peripheral blood mononuclear cells were determined by flow cytometry. The early activation marker CD69 appeared on lymphocytes and monocytes at day 4 after the onset of fever, and declined afterward. However, a transient reverse in the CD4/CD8 ratio occurred at days 6-10 after the onset of fever. The CD4/CD8 ratio inversion was manifested in 10 of 29 dengue patients and was encountered more frequently in dengue hemorrhagic fever/dengue shock syndrome than in dengue fever patients. Analysis of the clinical blood cell count of these 10 cases showed that increase of immature neutrophils developed at fever days 5-6, CD4(dim) or CD8(dim) monocytosis at days 6-7, and atypical lymphocytosis at days 8-10 after the onset of fever. Serum IL-6 was found at either day 7 or day 9-11. The PHA-stimulated T-cell response was depressed as well. These changes in immune parameters indicate aberrant immune activation during dengue virus infection and might be involved in the pathogenesis of dengue virus infection.     

Serum cytokine/chemokine profiles in patients with dengue fever (DF) and dengue hemorrhagic fever (FHD) by using protein array

BackgroundDENV infection can induce different clinical manifestations varying from mild forms to dengue fever (DF) or the severe hemorrhagic fever (DHF). Several factors are involved in the progression from DF to DHF. No marker is available to predict this progression. Such biomarker could allow a suitable medical care at the beginning of the infection, improving patient prognosis.ObjectivesThe aim of this study was to compare the serum expression levels of acute phase proteins in a well-established cohort of dengue fever (DF) and dengue hemorrhagic fever (DHF) patients, in order to individuate a prognostic marker of diseases severity.Study designThe serum levels of 36 cytokines, chemokines and acute phase proteins were determined in DF and DHF patients and compared to healthy volunteers using a multiplex protein array and near-infrared (NIR) fluorescence detection. Serum levels of IL-1ra, IL-23, MIF, sCD40 ligand, IP-10 and GRO-α were also determined by ELISA.ResultsAt the early stages of infection, GRO-α and IP-10 expression levels were different in DF compared to DHF patients. Besides, GRO-α was positively correlated with platelet counts and IP-10 was negatively correlated with total protein levels.ConclusionsThese findings suggest that high levels of GRO-α during acute DENV infection may be associated with a good prognosis, while high levels of IP-10 may be a warning sign of infection severity.     

Association of FCGR2A p.R131H and CCL2 c.-2518 A > G gene variants with thrombocytopenia in patients with dengue virus infection

FCGR2A and CCL2 gene variants are important in dengue pathogenesis and were investigated in 122 dengue patients (DENs) [89 dengue fever (DF) and 33 dengue hemorrhagic fever (DHF)] and 107 healthy controls (HCs) to find out their association with severity of dengue. Genotype frequencies of FCGR2A p.R131H and CCL2 c.-2518 A > G polymorphisms were not different between DF, DHF and HC. Significantly higher frequency of R/R genotype of FCGR2A p.R131H was observed in DEN cases with thrombocytopenia (TP) while the G/G genotype of CCL2 c.-2518 A > G was observed only in DEN cases with TP (p < 0.005). These results suggest that FCGR2A and CCL2 gene variants were associated with the risk of TP in dengue infections.     

Maturation of dengue virus nonstructural protein 4B in monocytes enhances production of dengue hemorrhagic fever-associated chemokines and cytokines

High levels of viremia and chemokines and cytokines underlie the progression of severe dengue disease. Dengue virus (DENV) preferentially infects peripheral blood monocytes, which secrete elevated levels of immunomediators in patients with severe disease. Further, DENV nonstructural proteins (NS) are capable of modifying intracellular signaling, including interferon inhibition. We demonstrate that peak secretions of immunomediators such as IL-6, IL-8, IP-10, TNFα or IFNγ in DENV-infected monocytes correlate with maximum virus production and NS4B and NS5 are primarily responsible for the induction of immunomediators. Furthermore, we demonstrate that sequential NS4AB processing initiated by the viral protease NS2B3(pro) and via the intermediate 2KNS4B significantly enhances immunomediator induction. While the 2K-signal peptide is not essential for immunomediator induction, it plays a synergistic role with NS4B. These data suggest that NS4B maturation is important during innate immune signaling in DENV-infected monocytes. Given similar NS4B topologies and polyprotein processing across flaviviruses, NS4B may be an attractive target for developing Flavivirus-wide therapeutic interventions.     

GM—CSF在登革病毒感染人单核细胞中的作用

以正常人外周血和脐带血单核细胞作为登革Ⅱ型病毒（Ｄ２Ｖ）感染的靶细胞，研究了粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）对Ｄ２Ｖ复制的影响以及单核细胞感染Ｄ２Ｖ后ＧＭ－ＣＳＦ产生能力的变化。结果表明，ＧＭ－ＣＳＦ能增加Ｄ２Ｖ的复制，Ｄ２Ｖ感染能引起人单核细胞产生ＧＭ－ＣＳＦ，它们的量与Ｄ２Ｖ感染量呈正相关。结果提示，ＧＭ－ＣＳＦ在病毒感染过程中起重要作用。     

重组登革病毒E蛋白结构域Ⅲ抑制登革病毒感染的初步研究

目的 了解原核表达的登革病毒(dengue virus, DV)的E蛋白结构域Ⅲ直接抑制登革病毒感染及其抗体的中和作用.方法 在大肠埃希菌中表达1~4型登革病毒E蛋白结构域Ⅲ(EⅢ).重组蛋白纯化后,进行阻断DV-2感染BHK~21细胞试验.用重组蛋白制备免疫血清,检测抗体中和作用.结果 在大肠埃希菌中成功表达了1-4型登革病毒E蛋白结构域埃希菌,4型重组E蛋白结构域Ⅲ均能够阻断2型DV感染,4型重组蛋白的免疫血清均能中和2型DV,但中和抗体效价不同.结论 原核表达的登革病毒结构域Ⅲ可以直接抑制病毒感染,所产生的抗体具有中和作用.直接抑制和中和抗体均对同型病毒作用较强.     

The pathology of dengue hemorrhagic fever

An estimated 2.5 billion people are at risk of dengue infection, and of the 100 million cases of dengue fever per year, up to 500,000 develop dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the life-threatening forms of the infection. The large majority of DHF/DSS occurs as the result of a secondary infection with a different serotype of the virus. While not completely understood, there is evidence that the target cells include dendritic reticulum cells, monocytes, lymphocytes, hepatocytes, and vascular endothelial cells. Viral replication appears to occur in dendritic cells, monocytes, and possibly circulating lymphoid cells, and damage to these and other target cells occurs through immune-mediated mechanisms related to cross-reacting antibodies and cytokines released by dendritic cells, monocytes, and vascular endothelium. There is evidence of a concomitant cellular activation as well as immune suppression during the infection. The activation of memory T cells results in cascades of inflammatory cytokines, including tumor necrosis factor-alpha, interleukins (IL-2, IL-6, and IL-8), and other chemical mediators that increase vascular endothelial permeability or trigger death of target cells through apoptosis. Pathological studies in humans are uncommon, and a suitable animal model of DHF/DSS does not exist. The current treatment of DHF/DSS is symptomatic, and prevention is through vector control. As such, there is a great impetus for the development of vaccines and novel therapeutic molecules to impede viral replication in infected cells or counteract the effects of specific inflammatory mediators on target cells. The role of genetics in relation to resistance to DHF/DSS also requires clarification.     

Elevated levels of IL-8 in dengue hemorrhagic fever

Dengue virus causes dengue fever, a mild febrile illness, and at times dengue hemorrhagic fever (DHF), a severe illness the pathogenesis of which is not fully understood. Given the crucial roles played by interleukin-8 (IL-8) as a chemoattractant cytokine and in inflammatory processes, levels of circulating IL-8 in the sera and IL-8 mRNA in the peripheral blood mononuclear cells (PBMC) were measured in 99 patients of a recent dengue epidemic that occurred in India in 1996 and in 21 normal healthy controls. Twenty-six of the patients had dengue fever (DF) and the remaining 73 were diagnosed as having different grades of DHF. All the control normal sera were negative for IL-8, so were their PBMC for IL-8 mRNA. Increased levels of IL-8 in the sera and IL-8 mRNA in their PBMC were observed in patients with severe illness of DHF grades III and IV. Only two out of 26 patients of DF and one out of 10 DHF grade I patient were positive for IL-8 and all three deteriorated to DHF grade IV within 24 hr. All six patients of DHF grade IV who died had higher serum level of IL-8 above 200 pg/ml, the highest being 5,568 pg/ml in one patient; the presence of mRNA for IL-8 was very high in all patients. A striking correlation was observed between increased levels of IL-8 and severe DHF, with greater levels in patients with increased grade of the disease and death. These results suggest that IL-8 may have an important role and may be an indicator of increasing severity of the disease and death. J. Med. Virol. 56:280–285, 1998. © 1998 Wiley-Liss, Inc.     

IL-6与登革病毒感染相互关系的实验研究

目的：研究ＩＬ－６对登革Ⅱ型病毒（Ｄ２Ｖ）复制的影响以及细胞感染Ｄ２Ｖ后ＩＬ－６产生能力的变化。方法：以人单核细胞为感染模型，用微量蚀斑法测定病毒滴度，用ＥＬＩＳＡ法测定ＩＬ－６含量。结果：重组ＩＬ－６能增强Ｄ２Ｖ的复制，Ｄ２Ｖ感染能引起人单核细胞产生ＩＬ－６，它们的水平与Ｄ２Ｖ感染量正相关。结论：ＩＬ－２在登革病毒感染中起重要的作用。     

2018年杭州市登革热流行病学及病原特征研究

目的:了解2018年杭州市登革热疫情的流行病学及病原特征。方法:应用RT-PCR方法检测血清标本中的登革病毒核酸及其型别,并对阳性标本进行病毒分离、 E基因扩增、序列测定及进化分析;描述病例的时间、人群和地区分布特征。 结果:2018年杭州市共检测登革热病例80例,其中输入性病例55例,本地病例2...     

广州市2002-2006年登革热病例特征

目的分析2002-2006年广州市1342例登革热（dengue fever，DF）患者病例特征，以期更好地做好DF的防治工作。方法对我院2002-2006年收治的1342例DF患者的资料进行回顾性分析，用C6／36细胞分离血液登革病毒（dengue virus，DV），逆转录聚合酶链反应与基因测序法鉴定病毒。结果1342例患者平均年龄为34．4岁，分布无明显性别差异。大多数患者有明显的病毒血症，主要临床表现为发热（100％）、头痛（85．9％）、肌肉酸痛（64．5％）、骨痛（46．6％）和皮疹（65．9％）等。实验室检查外周血白细胞及血小板减少者分别占66．0％和61．3％，丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）升高分别占69．0％和85．7％，部分患者（28．4％）出现血钾降低，除2例诊断为登革出血热外，均诊断为典型登革热。病原学检查血清登革病毒抗体IgM（DF-IgM）阳性率为90％，其中病程5天内累积阳性率为31．3％，8天内为86．4％，经病毒分离与鉴定证实均为DV—Ⅰ型感染。结论广州市2002-2006年流行的DF均为DV—Ⅰ型所致，绝大多数病例符合DF的典型临床表现，肝损害为常见的并发症，登革出血热极少见。     

Galectin-9(Tim-3L)显著减弱小鼠移植排斥

目的 探究Galectin-9(即T cell immunoglobulin domain and mucin domain protein-3 ligand,Tim-3L)的真核定位与功能,及其对同种异基因排斥反应的影响.方法 PCR扩增Galectin-9片段,插入到pEGFP-N1质粒,转染中国仓鼠卵巢癌细胞系(CHO)行G418筛选,分选增强型绿色荧光蛋白(EGFP)阳性细胞.免疫组化检测Galectin-9的表达,Alamar Blue法检测稳定转染Galectin-9的CHO细胞及其新鲜培养上清对脾细胞增殖的影响,ELISA检测IL-2水平.给予BALB/c→C57BL/6心脏移植受鼠Galectin-9蛋白100μg×7 d.结果 Galectin-9表达于细胞浆,稳定转染Galectin-9-EGFP的CHO细胞上清抑制淋巴细胞增殖和IL-2的产生.上述效应可被Tim-3-Fc融合蛋白逆转.Galectin-9干预的心脏移植物中位生存时间明显延长[(22.7±1.2)d,(7.2±0.4)d)].结论 真核细胞中Galectin-9可通过分泌形式负调节同种异基因免疫;Galectin-9有望成为新型的抗排斥药物.     

Elevated plasma levels of the long pentraxin, pentraxin 3, in severe dengue virus infections

C-reactive protein is one of the most widely used indicators of the response of acute-phase proteins. The measurement of C-reactive protein in dengue, however, is clinically not useful, because of marginally elevated levels and absent association with disease severity. The prototypic long pentraxin, pentraxin 3, is an acute phase protein that is structurally related but distinct from C-reactive protein which has proven to correlate with the severity of bacterial infection in critically ill patients. The potential involvement of pentraxin 3 in dengue and its aptitude to predict more severe disease or poor clinical outcome has not been studied previously. We therefore measured pentraxin 3 plasma levels in 44 dengue virus infected patients. Pentraxin 3 levels were strikingly higher when compared to C-reactive protein levels, with highest pentraxin 3 values observed in the first 7 days after the onset of symptoms. Median pentraxin 3 levels at admission and peak levels during follow up were higher in patients suffering from dengue shock syndrome (at admission: 119.3 ng/ml [interquartile range 61.8--188.7], peak values during follow up: 147.9 ng/ml [interquartile range 85.7--204.3]) compared to levels found in patients with dengue fever and dengue hemorrhagic fever (at admission: 59.0 ng/ml [interquartile range 28.6--100.3], P=0.040; peak values during follow up: 80.8 ng/ml [interquartile range 36.1--168.1], P=0.020). Our results indicate that pentraxin 3 seems to be a marker of infection better than C-reactive protein in dengue. The role of pentraxin 3 in the pathogenesis of dengue and its potential as an early prognostic indicator of disease severity needs further assessment.     

Steroidal saponins from the roots of Solanum sisymbriifolium Lam. (Solanaceae) have inhibitory activity against dengue virus and yellow fever virus

Dengue is the most important arthropod-borne viral disease worldwide. Infection with any of the four dengue virus (DENV) serotypes can be asymptomatic or lead to disease with clinical symptoms ranging from undifferentiated and self-limiting fever to severe dengue disease, which can be fatal in some cases. Currently, no specific antiviral compound is available for treating DENV. The aim of this study was to identify compounds in plants from Paraguayan folk medicine with inhibitory effects against DENV. We found high virucidal activity (50% maximal effective concentration (EC₅₀) value of 24.97 µg/mL) against DENV-2 in the ethanolic extract of the roots of Solanum sisymbriifolium Lam. (Solanaceae) without an evident cytotoxic effect on Vero E6 cells. Three saponins isolated from the root extract showed virucidal effects (EC₅₀ values ranging from 24.9 to 35.1 µg/mL) against DENV-2. Additionally, the saponins showed inhibitory activity against yellow fever virus (EC₅₀ values ranging from 126 to 302.6 µg/mL), the prototype virus of the Flavivirus genus, suggesting that they may also be effective against other members of this genus. Consequently, these saponins may be lead compounds for the development of antiviral agents.     

Rhabdomyolysis and severe muscular weakness in a traveler diagnosed with Alkhurma hemorrhagic fever virus infection

Alkhurma hemorrhagic fever virus (AHFV) is a tick-borne flavivirus with high case fatality rates, endemic in the Arabian Peninsula. Recently AHFV was detected in travelers returning from Egypt suggesting geographical spreading. We also report AHFV infection in a traveler ex Egypt, representing atypical symptoms of rhabdomyolysis and severe muscular weakness.