Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.     

Cysteine Protease Cathepsins in Atherosclerosis and Abdominal Aortic Aneurysm

Extracellular matrix remodeling is an important mechanism in the initiation and progression of cardiovascular diseases. Cysteine protease cathepsins are among the important proteases that affect major events in the pathogenesis of atherosclerosis and abdominal aortic aneurysm, including smooth muscle cell transmigration through elastic lamina, macrophage foam cell formation, vascular cell and macrophage apoptosis, and plaque rupture. These events have been studied in cathepsin deficiencies and cathepsin inhibitor deficiencies in mice and have provided invaluable insights regarding the roles of cathepsins in cardiovascular diseases. Pharmacological inhibitions for cathepsins are under evaluation for other human diseases and may be used as clinical treatments for cardiovascular diseases in the near future. This article reviews different mechanisms for cathepsins in atherosclerosis and abdominal aortic aneurysm that could be targeted by selective cathepsin inhibitors.     

斯氏狸殖吸虫半胱氨酸蛋白酶基因克隆及由体定位

目的 克隆斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,并研究该酶在斯氏狸殖吸虫的表达部位。方法 通过RT-PCR方法扩增斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,TA克隆入pUCm-T载体,鉴定、测序;利用DNASIS程序推导其编码氨基酸序列,并比较分析与其相关虫种半胱氨酸蛋白酶氨基酸序列同源性。采用地高辛标记原位杂交技术检测该酶基因在斯氏狸殖吸虫成虫的表达及组织定位。结果 经RT-PCR和对阳性克隆鉴定、测序后获得一cDNA序列,长495bp;同源性分析结果显示,该序列与相关虫种的半胱氨酸蛋白酶存在较高同源性,组成半胱氨酸催化三联体的半胱氨酸、组胺酸和天冬酰胺残基高度保守。原位杂交结果显示斯氏狸殖吸虫成虫的肠管上皮呈阳性着色。结论 克隆获得了斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,该片段包含了与半胱氨酸蛋白酶活性和空间结构相关的重要基因位点。斯氏狸殖吸虫成虫半胱氨酸蛋白酶的表达部位主要为肠管上皮。     

斯氏狸殖吸虫半胱氨酸蛋白酶基因克隆及由体定位

目的克隆斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,并研究该酶在斯氏狸殖吸虫的表达部位。方法通过RT-PCR方法扩增斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,TA克隆入pUCm-T载体,鉴定、测序;利用DNASIS程序推导其编码氨基酸序列,并比较分析与其相关虫种半胱氨酸蛋白酶氨基酸序列同源性。采用地高辛标记原位杂交技术检测该酶基因在斯氏狸殖吸虫成虫的表达及组织定位。结果经RT-PCR和对阳性克隆鉴定、测序后获得一cDNA序列,长495bp;同源性分析结果显示,该序列与相关虫种的半胱氨酸蛋白酶存在较高同源性,组成半胱氨酸催化三联体的半胱氨酸、组胺酸和天冬酰胺残基高度保守。原位杂交结果显示斯氏狸殖吸虫成虫的肠管上皮呈阳性着色。结论克隆获得了斯氏狸殖吸虫成虫半胱氨酸蛋白酶cDNA片段,该片段包含了与半胱氨酸蛋白酶活性和空间结构相关的重要基因位点。斯氏狸殖吸虫成虫半胱氨酸蛋白酶的表达部位主要为肠管上皮。     

简单异尖线虫L-样半胱氨酸蛋白酶基因的克隆与表达

目的克隆简单异尖线虫L-样半胱氨酸蛋白酶基因(AsCP)全长,研究其表达特性。方法根据GenBank中简单异尖线虫表达序列标签L-样半胱氨酸蛋白酶基因的部分信息,设计特异引物,用cDNA末端快速扩增技术扩增3′端部分,获得基因全长序列。根据基因全长序列设计引物,以简单异尖线虫总RNA为模板,RT-PCR扩增AsCP基因编码序列,产物经EcoRⅠ和SalⅠ双酶切,克隆至表达载体pET32а(+),转化大肠埃希菌BL21(DE3)株,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达效果经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果3′端扩增片段大小为1211bp,拼接完整后基因全长1462bp,编码411个氨基酸,与秀丽隐杆线虫的L-半胱氨酸蛋白酶相似性达36.4%;重组载体pET32a(+)-AsCP经EcoRⅠ和SalⅠ双酶切后有一条约1150bp的条带,测序结果显示重组载体构建成功。SDS-PAGE结果表明,重组蛋白相对分子质量约为Mr60000(含6个组氨酸的标签),与目的蛋白相符。用不同浓度的IPTG诱导对表达量的影响很小,1mmol/LIPTG诱导2h后表达量达到最高水平。结论成功克隆并表达了L-样半胱氨酸蛋白酶。     

The Emerging Relevance of the Cysteine Protease Cathepsin S in Disease

Cathepsin S is a lysosomal cysteine protease that has been shown to play a key role in MHC class II antigen presentation. Consequently, it has been extensively evaluated as a therapeutic target in autoimmune diseases, such as rheumatoid arthritis and psoriasis. Additionally, clinical and mechanistic evidence is emerging, revealing its inappropriate expression and secretion in a wide range of disease states including atherosclerosis and tumourigenesis. This review covers the known role and consequences of cathepsin S activity in these pathological disorders, highlighting various studies that have demonstrated its utility as a therapeutic target. This review also examines challenges that exist towards the development of agents that specifically target this protease and discusses the studies to date that have applied cathepsin S inhibitors in disease models.     

Co-occurrence of albumin complexes with IgE and IgG in the serum of an IgE myeloma patient

The serum of a patient with a clinically and immunologically identified multiple myeloma of the IgE class was found to contain both IgE-albumin and IgG-albumin complexes. These complexes were partially purified and some of their properties studied by biochemical and immunochemical methods. The IgG-albumin interaction was dissociated by 5.0 M guanidine hydrochloride, while the IgE-albumin interaction was dissociated upon reduction by mercaptoethanol, suggesting that the proteins were linked by intermolecular disulfide bonds. Complex formation between pathological or normal immunoglobulins with albumin have been reported for IgG, IgM and IgA but not for IgD or IgE. The present observation seems to be the first in which an IgE myeloma protein was involved.     

Relationship between IgE and IgG antibodies in type I allergy.

Serum samples of human patients with immediate type allergy were examined for both IgE (by radioallergosorbent test) and IgG (by enzyme immunoassay) antibodies to several of the common inhalant and food allergens. The results show a statistically significant correlation between the titers of anti-allergen antibodies of both isotypes. The data indicate that immune stimulation in atopic individuals is not restricted to the IgE isotype, but equally affects the IgG-producing antibody systems. The statistical relationship observed may either be due to common pathways in the production of both antibody classes in atopic people, or may be explained by preferential binding of allergens to circulating IgE-IgG immune complexes.     

Hemostasis of Tiger Prawn Penaeus monodon Affected by Vibrio harveyi,Extracellular Products,and a Toxic Cysteine Protease

ABSTRACT: The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied. The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease or PBS (control) was observed for 2 h at 25 C. Of these, only the control group was clottable while all the other groups were unclottable. A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (Rα coagulogen) to neutralize the clotting ability of normal prawn hemolymph. The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels. The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using Rα coagulogen. In addition, the apparent disappearance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using Rα coagulogen or by reducing the time for pre-incubation from 2 h to 30 min. Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph. These complex events may significantly contribute to the pathogenicity of V. harveyi in the prawn.     

犬蛔虫眼病病人血清中特异性IgG和IgE抗体水平的测定(英文)

目的研究犬蛔虫眼病病人血清中特异性IgG和IgE的抗体的水平。方法应用ELISA和Western-blotting。从东京医科齿科大学国际环境寄生虫学分野实验室储存的脉络膜炎的眼病病人血清中随意抽取105份血清,检测犬蛔虫抗体。结果通过ELISA法,105份血清中有82份IgG和IgE均为阴性(78 .1 %) ,12例血清仅IgG阳性(11 .4 %) ,3例血清仅IgE阳性(2 .9 %) ,8例血清IgG和IgE均为阳性。所有的IgG和IgE阳性的血清,进一步进行Western-blot。IgG的反应带分布在幼虫分泌排泄抗原分子重量(97 .2 -14 .3kDa)的整个范围内,IgE的反应带分布在幼虫分泌排泄抗原分子重量(97 .2—14 .3kDa)的相对狭窄的范围内(29 -45kDa)。结论在这个研究中,我们清楚的说明了一些脉络膜炎病例血清中IgE抗体比IgG抗体具有特异性,因此IgE在眼犬蛔虫病的诊断中具有特异性,IgE在眼犬蛔虫病诊断中的价值还要进行进一步的研究。     

IgE and IgG4 antibody responses to Aedes saliva in African children

Aedes mosquitoes are the major vectors of (re)-emerging infections including arboviruses (dengue, Chikungunya, yellow fever) in developing countries. Moreover, the emergence of Aedes-borne diseases in the developed world is currently a source of concern. Evaluation of human immune responses to Aedes bites could be a useful immuno-epidemiological tool for evaluating exposure to Aedes-borne diseases and thus predicting the risk of such emerging diseases. Specific IgE and IgG4 antibody (Ab) responses to Aedes aegypti saliva were evaluated in young Senegalese children living in an area of exposure to the Aedes vector. Specific IgE and IgG4 responses increased during rainy season of high exposure to Aedes bites. In addition, the evolution of anti-saliva isotype levels during the rainy season presented spatial heterogeneity between the studied villages. These preliminaries results support the potential approach of using anti-saliva Ab responses for evaluating exposure to Aedes vectors and risks of emerging arbovirus infections.     

Activity of specific IgG, IgM and IgE antibodies in human trichinellosis.

The activity of IgG, IgM and IgE antibodies to somatic antigen of Trichinella spiralis in the sera of patients with trichinellosis at various intervals after infection was examined by means of ELISA. Mathematical analysis of the dose-response curves was used. Elevated level of IgG and IgM antibodies of relatively high avidity and of rather low IgE avidity was documented. Amount or avidity of IgG antibodies was found to be most useful for the diagnosis of trichinellosis (85% positive results in patients' sera). The isotype of IgM avidity constitutes a better diagnostic value than the amount of it (60% and 35% of positive results, respectively).     

A Study of IgE in Immunoglobulin Preparations for Intravenous Administration

We have studied the IgE content of different lots of commercial intravenous IgG preparations used in our day hospital as replacement therapy for patients with agammaglobulinemia. Two enzymatic methods were used: Phadezym and FAST. The average amounts of IgE detected in Endobulin and Sandoglobulin lots were very high with respect to serum values in the general population. In some lots we found low titers of specific IgE (RAST class 1) to house dust mite, rye grass and cow's milk. We also found in most preparations the presence of IgG anti-IgE which are usually present in sera from atopic patients. Despite a low post-infusion increase of serum IgE and the absence of sensitization or adverse reactions observed, a careful selection of donors with normal IgE levels may need to be recommended to manufacturers in the future.     

Comparisons of serum total IgE, IgG, and IgG1 levels in patients with and without echinococcosis-induced anaphylactic shock

We investigated serum total immunoglobulin E (IgE), IgG, and IgG1 levels in patients with and without echinococcosis-induced anaphylactic shock. This was a case-control study of 11 patients with echinococcosis-induced anaphylactic shock and 22 echinococcosis patients with cyst rupture but without anaphylactic shock. Blood was collected before surgery (T0), at the time of cyst rupture (T1), and shock (Tx), 1 h (T2), 1 day (T3), and 1 week (T4) after cyst rupture. Serum IgE, IgG, and IgG1 were determined by enzyme-linked immunosorbent assay. Serum IgE, IgG, and IgG1 levels were significantly higher in patients who developed anaphylactic shock at all time points. Increased pre-surgical IgG and IgG1 levels were identified to be a significant risk factors for developing anaphylactic shock. The results showed that a serum IgG concentration of 312.25 μg/mL could be used as a cut-off point to predict whether an echinococcosis patient would develop anaphylactic shock.     

食物抗原特异性IgG和IgE在功能性胃肠病发病中的作用

背景：许多功能性胃肠病患者于进食某些食物后出现症状加重。目的：测定肠易激综合征（IBS）和功能性消化不良（FD）患者的血清食物抗原特异性IgG和IgE，探讨食物过敏在功能性胃肠病发病中的作用。方法：符合罗马Ⅱ标准的37例IBS患者、28例FD患者以及20例健康志愿者纳入本研究。以酶联免疫吸附测定（ELISA）检测受试者血清中14种食物抗原特异性IgG和IgE，同时测定血清总IgE水平。通过问卷调查行症状评分，分析血清食物抗原特异性IgG水平与症状总评分的相关性。结果：与健康对照组相比，IBS组血清中螃蟹、鸡蛋、河虾、大豆和小麦抗原特异性IgG水平显著升高（P〈0．05），FD组血清中鸡蛋和大豆抗原特异性IgG水平显著升高（P〈0．05）。三组间血清总IgE水平和食物抗原特异性IgE阳性率差异无统计学意义。IBS组和FD组血清食物抗原特异性IgG水平与症状总评分之间无明显相关性。结论：IBS和FD患者血清中部分食物抗原特异性IgG水平显著升高．提示IgG介导的食物过敏可能是功能性胃肠病潜在的发病机制之一。     

Contrasting functions of IgG and IgE antimalarial antibodies in uncomplicated and severe Plasmodium falciparum malaria

Plasmodial infection results in a significant elevation of the blood concentrations of immunoglobulins including IgE. Two well-characterized groups of adult Thai patients with either uncomplicated or severe Plasmodium falciparum malaria were studied over a period of four weeks. The mean parasitemias were approximately three-fold higher in patients with severe malaria than in those with uncomplicated disease. The mean concentrations of both total IgG and IgG antiplasmodial antibodies tended to be highest in the group with uncomplicated disease while total IgE and IgE antibodies were higher in the group with severe disease. The IgE antibodies detected in approximately 65% of the patients were positively correlated to parasitemia. These results suggest that antiplasmodial IgG antibodies are involved in reducing the severity of P. falciparum malaria, while IgE antibodies may contribute to the pathogenesis of this infection.     

乙型肝炎病毒基因疫苗诱导小鼠产生免疫应答的效应

目的 :构建编码乙型肝炎病毒 (HBV)表面蛋白S的重组质粒pCR 3 .1 S,将之直接肌肉注射balb/c小鼠 ,观察小鼠HBV特异的免疫应答。方法 :以ELISA法检测小鼠血清 ,3H TdR掺入法测定淋巴细胞增殖 ,51 Cr 4h释放法检测淋巴细胞杀伤功能。结果 :与空载体对照组相比较 ,基因疫苗诱发小鼠产生良好的抗HBs反应及HBV特异的细胞免疫应答 (P <0 .0 5)。结论 :基因疫苗pCR 3 .1 S可诱导balb/c小鼠产生全面的免疫应答