间接ELISA法用于细胞抗原特异的单克隆抗体筛选

在单克隆抗体（ｍＡｂ）制备过程中，间接ＥＬＩＳＡ方法用于可溶性蛋白为抗原的ｍＡｂ筛选。当得不到可溶性抗原的情况下，用细胞膜抗原或克隆抗原分子，再以痘苗病毒为载体转入真核细胞制备ｍＡｂ，可以用间接免疫荧光法进行筛选。由于此种方法费时，所需细胞数量大，影...

干细胞研究进展

干细胞包括胚胎干细胞与成体干细胞。介绍了近年来诱导胚胎干细胞向多种或某一种组织细胞分化的实验报道 ,并对成体干细胞“可塑性”

大脑皮质发育不良大鼠脑皮质和海马中的NeuN和GFAP表达研究

目的：探讨大脑皮质发育不良（DCD）大鼠大脑半球、海马的免疫组化病理学特点及其所致难治性癫痫（IE）的发生机制。方法：建立X-射线照射诱导的皮质下异位结节大鼠DCD模型，采用SP免疫组织化学方法检测神经元特异核蛋白抗原（NeuN）和星形胶质细胞特异抗原（GFAP）在大鼠脑皮质下和海马中异位结节的表达，阳性细胞计数，用SPSS10．0软件统计。结果：DCD组与正常对照或母鼠组白质比较，皮质下异位结节内NeuN免疫阳性神经元和GFAP免疫阳性细胞数密度分别增加30倍和16倍，差异有统计学意义（P〈0．05）。同龄而不同部位DCD病变组间脑皮质Ⅰ～Ⅲ层异位结节、皮质下异位结节和CA1区异位结节中NeuN、GFAP免疫阳性细胞数密度或阳性平均光密度值比较，差异无统计学意义。结论：DCD鼠脑NeuN及GFAP的分布证实了皮质下异位结节的内在属性，异位结节是灰质，主要是由神经元和星形胶质细胞构成。

New perspectives in occult hepatitis C virus infection

Occult hepatitis C virus (HCV) infection, defined as the presence of HCV RNA in liver and in peripheral blood mononuclear cells (PBMCs) in the absence of detectable viral RNA in serum by standard assays, can be found in anti-HCV positive patients with normal serum levels of liver enzymes and in anti-HCV negative patients with persistently elevated liver enzymes of unknown etiology. Occult HCV infection is distributed worldwide and all HCV genotypes seem to be involved in this infection. Occult hepatitis C has been found not only in anti-HCV positive subjects with normal values of liver enzymes or in chronic hepatitis of unknown origin but also in several groups at risk for HCV infection such as hemodialysis patients or family members of patients with occult HCV. This occult infection has been reported also in healthy populations without evidence of liver disease. Occult HCV infection seems to be less aggressive than chronic hepatitis C although patients affected by occult HCV may develop liver cirrhosis and even hepatocellular carcinoma. Thus, anti-HCV negative patients with occult HCV may benefit from antiviral therapy with pegylated-interferon plus ribavirin. The persistence of very low levels of HCV RNA in serum and in PBMCs, along with the maintenance of specific T-cell responses against HCV-antigens observed during a long-term follow-up of patients with occult hepatitis C, indicate that occult HCV is a persistent infection that is not spontaneously eradicated. This is an updated report on diagnosis, epidemiology and clinical implications of occult HCV with special emphasis on anti-HCV negative cases.

High doses of α-galactosylceramide potentiate experimental autoimmune encephalomyelitis by directly enhancing Th17 response

α-Galactosylceramide （u-GC） is widely known to activate invariant natural killer T （iNKT） cells to suppress my- elin antigen-specific Thl responses, protecting susceptible mice against experimental antoimmune encephalomyelitis 0EAE）. Here, we demonstrate an unexpected finding that high doses of α-GC exacerbated, rather than ameliorated, EAE. Similar results were observed when MOG35.ss-specific T cells treated with high-dose α-GC were transferred into naive syngeneic recipient mice. Further study showed that high doses of a-GC directly enhance the Thl7 and Thl re- sponse by activation of CD4＋CD44＋ memory T cells through phosphorylation of STAT3 and activation of NF-kB. Un- like the activation of iNKT cells by low doses of a-GC, high doses of a-GC directly interacted with CDld expressed on T ceils and activated Thl7 and Thl cells. Furthermore, antigen-presenting cells （APCs） predominantly express CDldl, whereas the majority of CD4~ T cells express CDld2. Knockdown of CDldl or CDld2 gene expression by RNAi interfered with the activation of iNKT or Thl7/Thl cells, respectively. Therefore, α-GC treatment could im- prove or worsen EAE by engaging either APCs or Thl7/Thl cells depending on the dose used.

Prediction of HLA-A 2.1-restricted CTL epitopes from IGFBP7 antigen of lung carcinoma

Objective： With the development of peptide-based cancer specific immunotherapy, the prediction of CTL epitopes from insulin-like growth factor-binding protein 7 （IGFBP7） is very important for some research about tumor metastasis. Because HLA-A2.1-expressing individuals cover 〉50% in the population of China, we aimed at identifying IGFBPT-encoded peptide presented by HLA-A2.1. Methods： In our study, a HLA-A2.1 restricted CTL epitope was identified by using the following two-step procedure： （a） computer-based epitope prediction from the amino acid sequence of IGFBP7 antigen; （b） Validation with epitope molecular modeling. Results： We obtained four epitopes with high immunogenicity scores by all of the three algorithms, i.e., BIMAS, SYFPEITH1 and IMTECH. Each of the four candidates satisfied the criteria of the HLA-A2.1- restricted CTL epitopes in molecular modeling analysis. Conclusion： The combination of BIMAS, SYFPEITHI and IMTECH method can improve the prediction efficiency and accuracy. Due to this research herein, this four epitopes have potential value for further studied, also have potential application in peptide-mediated immunotherapy. These epitopes may be useful in the design of therapeutic peptide vaccine for lung carcinoma and as immunotherapeutic strategies against lung carcinoma after identified by immunology experiment.

IL-13Ra2-and glioma stem cell-pulsed dendritic cells induce glioma cell death in vitro

Objective Gliomas are the most common malignant tumors in the central nervous system.Despite multiple therapies including surgery,chemotherapy,and radiotherapy,the prognosis of patients remains poor.Immunotherapy is an alternative method of treating glioma,and the use of dendritic cell vaccines is one of the promising treatment options.However,there is no specific tumor cell antigen that can trigger dendritic cells(DCs).IL-13Ra2 is a specific antigen expressed in glioma cells;in the current study,we have attempted to explore whether IL-13Ra2 could be the antigen that triggers DCs and to envisage its application as potential therapy for glioma.Methods The expression of IL-13Ra2 was detected in U251 glioma cell lines and primary glioma tissues using different methods.DCs from human blood were isolated and pulsed with recombinant IL-13Ra2,following which the cytotoxicity of these DCs on glioma cells was detected and analyzed.Results About 55.9% human glioma tissue cells expressed IL-13Ra2,while normal brain tissue cells did not show any expression.DC vaccines loaded with IL-13Ra2,glioma cell antigen,and brain tumor stem cell(BTSC) antigen could significantly stimulate the proliferation of T lymphocytes and induce cell death in the glioma tissue.Compared to other groups,DC vaccines loaded with BTSC antigen showed the strongest ability to activate cytotoxic T lymphocytes(CTLs),while the glioma cell antigen group showed no significant difference.Conclusion IL-13Ra2,which is expressed in gliomas and by glioma stem cells,as well as IL-13Ra2 could prove to be potential antigens for DC vaccine-based immunotherapy.

Development of HBsAg-Binding Aptamers that bind HepG2.2.15 cells via HBV surface antigen

Hepatitis B virus surface antigen （HBsAg）, a specific antigen on the membrane of Hepatitis B virus （HBV）-infected cells, provides a perfect target for therapeutic drugs. The development of reagents with high affinity and specificity to the HBsAg is of great significance to the early-stage diagnosis and treatment of HBV infection. Herein, we report the selection of RNA aptamers that can specifically bind to HBsAg protein and HBsAg-positive hepatocytes. One high affinity aptamer, HBs-A22, was isolated from an initial 115 met library of -1.1 ×10^15 random-sequence RNA molecules using the SELEX procedure. The selected aptamer HBs-A22 bound specifically to hepatoma cell line HepG2.2.15 that expresses HBsAg but did not bind to HBsAg-devoid HepG2 cells. This is the first reported RNA aptamer which could bind to a HBV specific antigen. This newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at HBV-infected cells.

In vitro and in vivo analyses of a genetically-restricted antigen specific factor from mixed cell cultures of macrophage, T and B lymphocytes

An immunostimulatory factor was identified to be secreted by antigen-pulsed maorophages. This factor was able to induce the generation of antigen specific T helper lymphocytes in vitro as well as in vivo. Further in vitro experiments testing for the genetic restriction of this factor indicated that it is a genetically-restricted antigen specific factor (ASF). The Cunningham plaque assay was used to quantify the generation of T helper lymphocytes by measuring the number of plaque forming cells after sequential incubations of antigen-pulsed maorophages with T lymphocytes, and then spleen cells, and finally the TNP-coated sheep red blood cells.

Construction of HLA/Peptide Tetramer with Peptide-Linked β_2 Microglobulin

Analysis of the frequency of antigen-specific cytotoxic T lymphocytes (CTLs) ex vivo is largely dependent on the use of MHC/peptide tetramers. However, the latter reagents have not been widely available, most likely because of their costly and time-consuming production. In this report we utilized an economic strategy to construct HLA/peptide tetramers with recombinant peptide-linked β2 microglobulin (β2m). The HLA-A2-restricted, melanoma antigen MARTI-derived pep- tide MART127-35( AAGIGILTV) was fused to the N terminus of human β2m through a 15-amino acid (aa)-long linker before being refolded with the recombinant biotinylated HLA-A2 heavy chain ectodomain. The resulted 2-component (2C) monomer was then tetramerized with phycoerythin-labeled streptavidin. The experimental result showed that the 2C HLA-A2/ MART127-35 monomer was shown to bind to the HLA class complex-specific monoclonal antibody W6/32 and the HLA-A2/ MART127-35 complex-specific single chain antibody fragment (scFv) 8.3, suggesting the correctness of its specificity. Fur- thermore, the 2C HLA-A2/MART127-35 tetramer detected a specific CD8+ T cell population in HLA-A2-restricted melanoma infiltrating lymphocytes as the conventional 3C HLA-A2/MART127-35 tetramer. The yield of 2C HLA-A2/MART127-35 monomer was 2. 5 times more than that of the conventional 3C monomer. Taken together, these data indicate that the HLA-A2/ MART127-35 tetramer can be generated conveniently through the use of MART127-35 peptide-β2 m fusion proteins, which can fa- cilitate the monitoring of HLA-A2-restricted, MART1-specific CTL responses in patients with melanoma.

Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity Factors

Background： Plasmodium falciparum malaria remains a major life-threatening disease. Recently, the Plasmodium apoptosis-linked pathogenicity factors （PALPF） have been identified. These antigens PALPF are expressed only by P falciparum-infected erythrocytes triggering endothelial cell apoptosis （apoptogenic）. Methods： We designed ten synthetic peptides （PI to P10） from PALPF： PF07 0032, PF10_0226, PFI0130c, PFD0875c and MAL13P1.206, and analyzed their antigenicity with an ELISA method using plasma samples from subjects living in Dienga, Gabon. Results： Four peptides showed good reactivity with human antibodies. The prevalence rate of specific IgG was 61%, 51%, 44% and 34% for P5, P6, P4 and P2, respectively. The median optical density of total IgG anti-P2 was higher than that directed against P4 and P6 （P = 0.009; P = 0.012 respectively）. The prevalence rate oflgG subclasses determined with plasma samples recognizing peptide 5 for IgGl, 2, 3 and 4 isotypes was 69%, 45%, 76% and 62%, respectively. All the subjects had at least one immunoglobulin subclass, while 13 （44%） had both IgG1 and IgG3 antibodies. There was no significant difference in the prevalence rate of anti-P5 IgG1, IgG3 and IgG4. Conclusion： These results warrant further immunogenicity studies of peptides 2, 4, 5 and 6 with a view of a tentative to antimalarial vaccine development.

四聚体技术的新进展

四聚体技术是一种可直接对抗原特异的T淋巴细胞进行标记的新方法。多种MHC分子和多种来源的多肽分子被用于制备四聚体 ,研究抗原特异的T细胞在抗病毒感染、自身免疫病和抗肿瘤中的作用。另外 。

基于肿瘤超早期免疫应答信息放大指标naCTL,TCR/BCR/HLA有效多样性等免疫信息学多维指标的中医(肿瘤)治未病测知、评价模型(体系)构建

肿瘤细胞在形成瘤体前(超早期)是干预最佳时期,但限于该时期肿瘤细胞数量,当前主要针对其本身的检测(如CTC,ct DNA等)难以实现肿瘤细胞与免疫力的量化,这不仅无法实现该时期精准预防,也影响中医(肿瘤)治未病深入测知、干预、评价。鉴于当前检测局限性,该评价体系转而检测与肿瘤细胞直接相关的、经其激活、增殖至高数量级的肿瘤新生抗原特异性细胞毒性T淋巴细胞(naCTL),免疫组有效性多样性(TCR/BCR/HLA在递呈、识别肿瘤新生抗原上的有效多样性),并引入免疫检查点(check point)、中医病证(治未病)信息等,运用多种数据分析及相关算法,建立免疫正常化多维统计模型。该体系充分放大肿瘤超早期信息,同时,深度融合中医整体观、平衡观思想与扶正祛邪基本治则,为机体免疫正常化及中医(肿瘤)治未病提供量化测知、评价新方法,也为中医证候科学内涵与中药复方药效评价提供新思路,新方法。

Establishing guidelines for CAR-T cells： challenges and considerations

T cells, genetically modified by chimeric antigen receptors(CAR-T), are endowed with specificity to a desired antigen and are cytotoxic to cells expressing the targeted antigen. CAR-T-based cancer immunotherapy is a promising therapy for curing hematological malignancy, such as acute lymphoid leukemia, and is promising for extending their efficacy to defeat solid tumors. To date, dozens of different CAR-T cells have been evaluated in clinical trials to treat tumors; this necessitates the establishment of guidelines for the production and application of CAR-T cells. However, it is challenging to standardize CAR-T cancer therapy because it involves a combination of gene therapy and cell therapy. In this review, we compare the existing guidelines for CAR-T cells and discuss the challenges and considerations for establishing guidance for CAR-T-based cancer immunotherapy.

Armed T cells with CAR for cancer immunotherapy

The development of genetic engineering has enabled the modification of stem cells and somatic cells.T cells exert immune responses against cancer cells.Efforts to redirect T cell specificity of a chimeric antigen receptor(CAR)to a desired antigen began in the 1990s(Gross et al.,1989;Kuwana et al.,1987).In 2006,the first clinical trial using carbonic anhydrase IX CAR-T cells to fight renal cancer was conducted(Lamers et al.,2006).Until 2011,Porter et al.exploited CD19 CAR-T to treat refractory/relapsed chronic lymphoid leukemia(Porter et al.,2011).Subsequently。

Application of surface plasmon resonance imaging technique for i PSCs identification

Induced pluripotent stem cells （iPSCs） are an attractive cell source for regenerative medicine through cell therapy or drug screening. But application of iPSCs in regenerative medicine requires rapid and accurate charac- terization of iPSCs. Here, we demonstrate the detection of multiple antigens present in iPSC lysate using rapid, label- free surface plasmon resonance imaging （SPRi） assay. Validation of pluripotency is an important aspect of iPSC research. In this study, we fabricated antibody array against pluripotency biomarkers and found that our array suc- cessfully detect corresponding antigens in stem cell lysate. Each antibody recognized its specific antigens presented in iPSC lysates and a certain degree of variability was observed in comparison with other cell lysates. The results suggested that SPRi is a versatile technology feasible for the detection of multiple antigens presented in iPSC lysate. Further extension of this method may be applied in the characterization and high-throughput biomarker profiling of iPSCs.

预防性丙型肝炎病毒疫苗研究新策略

全球有超过7 000万名丙型肝炎病毒(Hepatitis C virus,HCV)慢性感染患者,给社会带来严重的公共卫生安全问题。直接抗病毒药物(Direct antiviral drugs,DAA)能够有效地治愈慢性丙型肝炎病毒感染,但治愈后患者仍存在再感染的可能,预防性疫苗(Prophylactic vaccine)是彻底切断HCV传播的最有效途径。目前HCV预防性疫苗的研发已取得一定进展,能够诱导极少数受试者产生广谱中和抗体(Broadly neutralizing antibodies),如何在绝大多数个体中诱导保护性广谱中和抗体则是当前HCV预防性疫苗研究所面临的最大挑战。本文总结了当前疫苗免疫学的研究进展:包括抗原特异的B细胞前体(Precursor B cell)、滤泡辅助性T细胞(Follicular T helper cell,Tfh)、滤泡调节性T细胞(Follicular T regulatory cell,Tfr)、抗体重链基因1-69(VH1-69)编码抗体成熟特点;基于胚系靶向(Germline-targeting)的免疫原设计与序贯免疫策略(Sequential immunization)等,期望能为HCV疫苗研究提供新的思路。