The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor

Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1-6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor. This interaction was also confirmed in mammalian cells. Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein. It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation. It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV.     

Fv structure of monoclonal antibody II-481 against herpes simplex virus Fc gamma-binding glycoprotein gE contains immunodominant complementarity determining region epitopes that react with human immunoglobulin M rheumatoid factors

Human immunoglobulin M (IgM) rheumatoid factors (RFs) show primary direct enzyme-linked immunosorbent assay (ELISA) reactivity with Fab rather than Fc fragments of monoclonal antibody (mAb) II-481 directed against the Fc gamma-binding site of herpes simplex virus glycoprotein gE. This preferential anti-Fab specificity suggests that RFs react with antigen-binding portions of mAb II-481 as anti-idiotypic antibodies directed at the combining site regions of mAb reacting with the Fc gamma-binding region of gE. Analysis of this idiotype-anti-idiotype reaction employed polymerase chain reaction amplification and sequencing of the variable heavy and light (VH and VL) regions of mAb II-481. When VH and VL regions of mAb II-481 were synthesized as overlapping 7-mer peptides on polypropylene pins, a panel of 10 polyclonal and 6 monoclonal human IgM RFs reacted primarily with epitopes within the three solvent-exposed mAb II-481 complementarity determining regions (CDRs). Preincubation of single CDR heptamer peptides with IgM RFs in free solution, resulted in 63-100% inhibition of RF binding to mAb II-481 on the ELISA plate, confirming the antigenic importance of linear CDR regions for RF reactivity. Combinations of two or three CDR peptides frequently produced 94-100% inhibition of RF binding to whole mAb II-481. Control peptides, singly or in combination, showed no inhibition. Computer modeling suggested that the RF-reactive mAb II-481 Fv region and a previously demonstrated RF-reactive CH3 epitope displayed considerable three-dimensional similarities in conformation. These studies may provide insight into limited shape homologies possibly involved in an RF anti-idiotypic reaction.     

Analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal antibodies to glycoprotein B.

The role of antibody in neonatal herpes simplex virus (HSV) infection remains controversial. A battery of well-characterized monoclonal antibodies to HSV glycoprotein B (gB), and polyclonal antibodies against synthetic peptides of predicted epitopes of HSV glycoprotein D (gD) were used to determine in vitro functional activity and association with protection against lethal infection in a murine model of neonatal HSV disease. Antiviral neutralization activity of HSV was not associated with antibody-dependent cellular cytotoxicity (ADCC) activity to HSV-infected cells in vitro. In a model of high dose challenge (10(4) PFU), protection was not afforded by any antibody alone, but was by antibody plus human mononuclear cells, and highly associated with ADCC functional activity (P less than 0.001). In a low dose challenge model, neutralizing activity of antibody alone was associated with protection in vivo (P less than 0.001). Of the nine neutralizing epitopes of gD in vitro, eight were predicted surface regions. Four of the five epitopic sites of gD (2-21, 267-276, 288-297, and 303-312) that were determined to be important targets of ADCC and in vivo protection were also predicted to be surface regions. The only exception was the antiserum to region 52-61 which was predicted to be buried and also showed these activities. ADCC as well as neutralizing antibody activity are important in protection against neonatal HSV infection.     

陕西省铜川市健康人群流行性乙型脑炎中和抗体水平监测结果分析

目的 了解陕西省铜川市健康人群流行性乙型脑炎(乙脑)抗体水平,为有效防控乙脑提供参考依据。 方法 采用整群随机抽样的方法,选择铜川市宜君县2岁、3~5岁、6~10岁、11~15岁、16~45岁、46~60岁和60岁组共216人,用微量中和试验检测乙脑病毒中和抗体。 结果 铜川市健康人群乙脑中和抗体阳性率为24.07%,抗体几何平均滴度(GMT)为1 ∶ 3.04;2岁组人群抗体阳性率及GMT 均最高,为63.33%和1 ∶ 11.06,其他年龄组的抗体阳性率为6.45%~31.03%,抗体GMT 为1 ∶ 1.78~1 ∶ 3.95。各年龄组抗体阳性率及GMT差异有统计学意义。 结论 铜川市健康人群乙脑抗体阳性率及GMT均较低,具有引发乙脑流行的风险,应加强乙脑疫苗免疫接种工作,提高人群免疫覆盖率。     

Detection of antibody to a new antigen induced by Epstein-Barr virus in rheumatoid arthritis.

A new antigen which is different from Epstein-Barr virion antigen was detected in NC-37 cells infected with Epstein-Barr virus (EBV). A significant elevation of the titer of antibody to this new antigen was observed in the sera of the patients with rheumatoid arthritis (RA), but not in the sera of controls. From the standpoint of the etiologic role of EBV in RA, it is interesting that the antibody to the new antigen is not detected in the healthy persons in contrast to other viral antibodies. Therefore, it differs from any other viral antibodies so far reported.     

Identification of overlapping HLA class I-restricted cytotoxic T cell epitopes in a conserved region of the human immunodeficiency virus type 1 envelope glycoprotein: definition of minimum epitopes and analysis of the effects of sequence variation.

Although the immunologic basis of protective immunity in human immunodeficiency virus type 1 (HIV-1) infection has not yet been defined, virus-specific cytotoxic T lymphocytes (CTL) are likely to be an important host defense and may be a critical feature of an effective vaccine. These observations, along with the inclusion of the HIV-1 envelope in the majority of vaccine candidates presently in clinical trials, underscore the importance of the precise characterization of the cellular immune responses to this protein. Although humoral immune responses to the envelope protein have been extensively characterized, relatively little information is available regarding the envelope epitopes recognized by virus-specific CTL and the effects of sequence variation within these epitopes. Here we report the identification of two overlapping CTL epitopes in a highly conserved region of the HIV-1 transmembrane envelope protein, gp41, using CTL clones derived from two seropositive subjects. An eight-amino acid peptide was defined as the minimum epitope recognized by HLA-B8-restricted CTL derived from one subject, and in a second subject, an overlapping nine-amino acid peptide was identified as the minimal epitope for HLA-B14-restricted CTL clones. Selected single amino acid substitutions representing those found in naturally occurring HIV-1 isolates resulted in partial to complete loss of recognition of these epitopes. These data indicate the presence of a highly conserved region in the HIV-1 envelope glycoprotein that is immunogenic for CTL responses. In addition, they suggest that natural sequence variation may lead to escape from immune detection by HIV-1-specific CTL. Since the region containing these epitopes has been previously shown to contain an immunodominant B cell epitope and also overlaps with a major histocompatibility complex class II T cell epitope recognized by CD4+ CTL from HIV-1 rgp160 vaccine recipients, it may be particularly important for HIV-1 vaccine development. Finally, the identification of minimal CTL epitopes presented by class I HLA molecules should facilitate the definition of allele-specific motifs.     

抗CCP抗体与类风湿关节炎的相关性研究

目的研究抗环瓜氨酸肽抗体（CCP）（抗CCP抗体）与类风湿关节炎（HA）的相关性。方法利用酶联免疫吸附法（ELISA）测定患者抗CCP抗体在RA中的含量。结果抗CCP抗体的敏感性比类风湿因子（RF）的敏感性低。但抗CCP抗体的特异性远高于RF,RA患者的RF的阳性率在抗CCP抗体阳性组和阴性组有显著性差异,但抗CCP抗体与年龄、红细胞沉降率、C反应蛋白及病程均无显著性差异。结论研究证明抗CCP抗体含量与疾病活动度（红细胞沉降率、C反应蛋白）无相关性,而和RF存在正相关,并且抗CCP抗体阳性组和阴性组红细胞沉降率、C反应蛋白比较无显著性差异。     

TRIM34分子B细胞抗原表位的生物信息学筛选及抗体制备

通过生物信息学软件预测TRIM34蛋白的B细胞抗原表位,制备兔源性抗TRIM34多肽抗体并验证其特异性。音先运用VectorNTI11.5和Lasergene7.1软件,分析TRIM34蛋白的氨基酸序列,筛选出B细胞抗原表位序列。人工合成TRIM34多肽抗原,并在多肽氨基端与钥孔血蓝蛋白(KLH)偶联。免疫新西兰大白兔,制备抗TRIM34多肽抗体。通过酶联免疫吸附实验(ELISA)检测抗TRIM34多肽抗体的效价,并通过蛋白印迹法和激光共聚焦法检测抗体对TRIM34蛋白的特异性识别能力。经ELISA检测,抗TRIM34多肽抗体的效价为1:8000。经蛋白印迹检测,抗TRIM34多肽抗体能特异性识别外源性和内源性TRIM34蛋白,同时该抗TRIM34多肽抗体不能识别TRIM 34旁系同源分子TRIM6、TRIM5和TRIM22。经间接免疫荧光染色并通过激光共聚焦检测,发现抗TRIM34多肽抗体可以用来显示TRIM34蛋白的亚细胞定位情况。本研究利用生物信息学分析,筛选并合成TRIM34蛋白抗原多肽,成功制备特异识别TRIM34蛋白的兔源多肽抗体。     

A combined indirect elisa and immunoblotting for the detection of intrathecal herpes simplex virus igg antibody synthesis in patients with herpes simplex virus encephalitis

A combined indirect ELISA and immuno-blotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.     

A comparison of pyrophosphatase and peroxidase as markers in the immunoenzyme analysis of the tick-borne encephalitis virus and antibodies to it]

The authors compare the sensitivities of pyrophosphatase and peroxidase conjugates with immunoglobulins in assays of tick-borne encephalitis (TBE) virus antigens and anti-TBE antibodies in human sera. Pyrophosphatase conjugates are similar to peroxidase ones in their enzymic and immunochemical characteristics and are not inferior to them in sensitivity in titration of antibodies and superior in assays with antigens. A number of advantages of pyrophosphatase conjugates, i.e. a simple preparation procedure, stable substrate and enzymic reaction product, color range convenient for visual recording, etc., make these conjugates preferable vs. the peroxidase ones and recommend them for wide use at diagnostic laboratories.     

Rapid induction of specific cytotoxic T lymphocytes against melanoma-associated antigens by a recombinant vaccinia virus vector expressing multiple immunodominant epitopes and costimulatory molecules in vivo

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.     

山西和河南省部分地区人群流行性乙型脑炎病毒中和抗体水平调查

目的调查山西和河南省部分地区健康人群血清中流行性乙型脑炎(乙脑)病毒中和抗体水平。方法对采集的血清标本按1:10,1:20,1:40,1:80四个稀释度稀释后进行乙脑病毒中和试验,结果判定采用细胞病变观察方法并结合ELISA测定结果进行综合判定。结果调查结果显示,所测标本中乙脑病毒中和抗体保护率以临猗县最高(87.6%),其次是栾川县(58.2%)和万荣县(51.4%)。经χ2检验,临猗县与万荣县中和抗体保护率差异有统计学意义(χ2=42.812,P=0.000),临猗县的中和抗体保护率高于万荣县,栾川县与万荣县的中和抗体保护率差异无统计学意义(χ2=1.466,P=0.226)。两个年龄组(≤40岁和40岁)中和抗体保护率比较显示,临猗县差异无统计学意义(χ2=0.057,P=0.811),万荣县差异有统计学意义(χ2=5.505,P=0.019),栾川县差异无统计学意义(χ2=2.129,P=0.145)。结论三个县健康人群乙脑病毒中和抗体保护率不同,这可能与当地乙脑的流行状况以及疫苗的接种情况有关。     

新型多酸化合物(POM-2)抗乙型肝炎

目的 研究新型多酸化合物(FOM-2)体外抗乙型肝炎病毒(HBV)的活性和体外毒性.方法 采用实时荧光定量PCR分析受试多酸化合物对HepG2.2.15细胞外HBV DNA的抑制率,计算其50%抑制浓度(IC50)值和90%抑制浓度(IC90)值;采用乙型肝炎病毒e(s)抗原诊断试剂盒测定细胞培养上清液中HBeAg、HBsAg的含量,测定受试多酸化合物对HBeAg、HBsAg分泌的抑制率;采用MIT法测定受试多酸化合物对HepG2.2.15细胞的毒性,计算其50%致死浓度(CC50)值.结果 新型多酸化合物对HepG2.2.15细胞外HBV DNA的50%抑制浓度(IC50)和90%抑制浓度(IC90)分别为13.42 mg·L-1、59.47 mg·L-1.各浓度实验组对HBeAg、HBsAg分泌的抑制率均高于对照组(P＜0.05).受试多酸化合物对HepG2.2.15细胞的50%致死浓度(CC50)值为2899.21 mg·L-1.结论 新型多酸化合物(FOM-2)体外毒性低,对HepG2.2.15细胞外HBV DNA及HbeAg、HBsAg的分泌具有较好的抑制作用.     

Structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of CBA/Ca mice to influenza hemagglutinin

Information on the antigenic structure of influenza hemagglutinin (HA) has been deduced previously from sequence analyses of laboratory mutant viruses selected, in vitro, with neutralizing monoclonal antibody (mAb) established exclusively from BALB/c (H-2d) mice; and there has been no attempt to investigate the influence of host genetic background, or natural route of infection, on the protective antibody repertoire. CBA/Ca mice are extremely sensitive to X31 virus infection, and in the present study a structural analysis was made of the antibody repertoire, by direct sequencing of the HA genes of laboratory mutant viruses selected, in ovo with mAb from CBA/Ca mice primed by natural infection with X31 virus at two different infectious doses. Single nucleotide substitutions in the HA genes of mutant viruses identified both novel and immunodominant antigenic sites on the HA1 subunit: a majority of mAbs, from different donors, were of the IgG2a isotype and were specific for HA1 158 Gly. In addition, novel laboratory mutants were obtained containing substitutions in the HA1 subunit that had not been reported previously for H3 subtype viruses, either natural variants or laboratory mutants, at residues: HA1 62 Ile----Arg; HA1 165 Asn----Ser (resulting in the loss of a N-glycosylation site); and HA1 273 Pro----Leu. Our findings suggest that host genetic background and/or a natural route of infection may be significant factors in the selection of different and distinct neutralizing antibody responses to influenza HA and therefore be of some relevance in our further understanding of the immune pressure for antigenic drift, and the immunogenic features of a protective antigen.     

In vitro susceptibility of Bacillus anthracis to various antibacterial agents and their time-kill activity

OBJECTIVES: To investigate the in vitro acquisition of resistance to antibiotics by Bacillus anthracis. METHODS: The in vitro activities of 18 antibacterial agents against two strains of B. anthracis, the Sterne strain and the Russian anthrax vaccine strain ST-1, were tested by determining the MICs and by measuring the rates of antibiotic kill at 5x and 10x MIC. RESULTS: The fluoroquinolones ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, the beta-lactams penicillin G and amoxicillin, the macrolide clarithromycin, the ketolide telithromycin, as well as clindamycin, rifampicin and quinupristin/dalfopristin had MICs in the range of 0.03-0.25 mg/L. Minocycline had an MIC of 0.03 mg/L, as did penicillin, against the ST-1 strain. Ciprofloxacin had an MIC of 0.03 mg/L against both strains. Erythromycin, vancomycin and the oxazolidinone linezolid were less active (MIC 0.5-2.5 mg/L). Ceftriaxone was the least active, having an MIC of 8.0 mg/L. Chloramphenicol was inactive (MIC > 256 mg/L). Quinupristin/dalfopristin, rifampicin and moxifloxacin showed the most rapid bacterial killing, achieving a complete eradication of detectable organisms (2 log(10) reduction within 0.5-3 h and 4 log(10) reduction within 0.5-4 h for both strains at concentrations of 5x and 10x the MIC). The beta-lactams and vancomycin demonstrated a 2-4 log(10) reduction within 5-15 h. Ceftriaxone had a similar effect to penicillin and amoxicillin against the ST-1 strain, but a slower effect than these two beta-lactams against the Sterne strain. The macrolides, tetracyclines and linezolid demonstrated a lower kill rate, while chloramphenicol did not kill at all. CONCLUSIONS: These data expand on the spectrum of agents recommended for the treatment of anthrax (ciprofloxacin, penicillin G and tetracyclines) and add new options, such as other fluoroquinolones, amoxicillin, rifampicin and quinupristin/dalfopristin, as potential therapeutic agents.     

Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus

Tenofovir is an acyclic nucleotide analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Tenofovir disoproxil fumarate (tenofovir DF), a bis-alkoxyester prodrug of tenofovir, is approved for the treatment of HIV and is currently being developed to treat chronic hepatitis B. In this report, we further characterize the in vitro activity of tenofovir against HBV as well as its metabolism in hepatic cells. We show that tenofovir is efficiently phosphorylated to tenofovir diphosphate (TFV-DP) in both HepG2 cells and primary human hepatocytes. TFV-DP has a long intracellular half-life (95 h) and is a potent and competitive inhibitor of HBV polymerase (Ki = 0.18 microM). Tenofovir has a 50% effective concentration of 1.1 microM against HBV in cell-based assays, and potency is improved > 50-fold by the addition of bis-isoproxil progroups. Tenofovir has previously demonstrated full activity against lamivudine-resistant HBV in vitro and clinically. Here we show that the major adefovir resistance mutation, rtN236T, confers three- to fourfold-reduced susceptibility to tenofovir in cell culture; the clinical significance of this susceptibility shift has not yet been determined. The rtA194T HBV polymerase mutation recently identified in tenofovir DF-treated HIV/HBV-coinfected patients did not confer in vitro resistance to tenofovir as a single mutation or in a lamivudine-resistant viral background. Overall, the antiviral and metabolic profile of tenofovir supports its development for the treatment of chronic hepatitis B.