ELISPOT检测人乳头瘤病毒特异性的记忆T细胞

应用ELISPOT方法检测人乳头瘤病毒感染后自发清除者外周血中抗原特异性的记忆T细胞。收集人乳头瘤病毒（HPV）感染后自发清除者外周血（病毒清除后74个月）,分离外周血单个核细胞（peripheralblood mononuclear cells,PBMC）。体外应用已鉴定的表位肽刺激PBMC,10 d后计数细胞,去除表位肽,继续培养。第11天,ELISPOT方法检测PBMC中HPV抗原特异性的记忆T细胞。PBMC经表位肽刺激10 d后,细胞数量有明显增加,由最初的4.1×105增加为4.2×106。第11天,细胞数量增加为4.65×106,为抗原刺激前细胞数的11.3倍。ELISPOT结果显示,PBMC中的记忆T细胞活化后,能够识别抗原递呈细胞递呈的抗原肽,并分泌IFN-γ。此HPV自发清除者外周血中抗原特异性的记忆T细胞的频数为0.007%。人乳头瘤病毒（HPV）感染后自发清除者外周血存在抗原特异性记忆T细胞,抗原肽可激活记忆T细胞,使之数量增加,分泌IFN-γ。ELISPOT可用于检测外周血中HPV特异性的记忆T细胞。     

Immobilized Cells

Three basic types of immobilization (i.e. without carrier, entrapment and immobilization on the carrier surface) of microbial cells, nonmicrobial cell populations and subcellular organelles are reviewed. These are further developed into a number of combined and less frequently used techniques of immobilization and application of cell biocatalysts for industrial biotransformations in pharmacy, food industry and agriculture, including novel approached and some unpublished authors’ results.     

固定化细胞合成生物表面活性剂鼠李糖脂的研究

从石油受污环境中分离筛选得到一株高产鼠李糖脂(rhamnolipid,RL)的假单胞菌(Pseudomonas sp.)B3。在游离细胞合成鼠李糖脂的基础上,应用包埋与交联相结合的复合固定化方法制备出性能优良的固定化细胞。以二次回归方程预测模型为基础,对发酵条件进行优化,得到B3固定化细胞合成RL的最优条件为:接种量15%,初始pH 7.0,合成温度38℃,120r·min-1振荡培养100h,RL的产量达到4 843.25mg·L-1,比游离细胞提高56.42%。制备的固定化细胞连续使用3个发酵周期,RL的产量均保持在4 517.75mg·L-1以上,说明B3固定化细胞具有用于连续发酵合成RL的可行性。     

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Specific Activation of Dendritic Cells Enhances Clearance of Bacillus anthracis following Infection

Dendritic cells are potent activators of the immune system and have a key role in linking innate and adaptive immune responses. In the current study we have used ex vivo pulsed bone marrow dendritic cells (BMDC) in a novel adoptive transfer strategy to protect against challenge with Bacillus anthracis, in a murine model. Pre-pulsing murine BMDC with either recombinant Protective Antigen (PA) or CpG significantly upregulated expression of the activation markers CD40, CD80, CD86 and MHC-II. Passive transfusion of mice with pulsed BMDC, concurrently with active immunisation with rPA in alum, significantly enhanced (p<0.001) PA-specific splenocyte responses seven days post-immunisation. Parallel studies using ex vivo DCs expanded from human peripheral blood and activated under the same conditions as the murine DC, demonstrated that human DCs had a PA dose-related significant increase in the markers CD40, CD80 and CCR7 and that the increases in CD40 and CD80 were maintained when the other activating components, CpG and HK B. anthracis were added to the rPA in culture. Mice vaccinated on a single occasion intra-muscularly with rPA and alum and concurrently transfused intra-dermally with pulsed BMDC, demonstrated 100% survival following lethal B. anthracis challenge and had significantly enhanced (p<0.05) bacterial clearance within 2 days, compared with mice vaccinated with rPA and alum alone.     

特异性抗体效价检测技术概述

特异性抗体效价是生物制品活性(浓度)的重要标志,通常采用生物学方法来测定,并以抗体与其相应抗原反应产生的、可观察到的标准免疫反应来表示。抗原抗体间除发生特异性反应外,还会产生交叉反应,从而使特异性抗体效价的检测出现偏差,这在实际应用中亟需避免。特异性抗体效价检测技术是疾病诊断、特异性免疫球蛋白制备和疫苗评价等领域的关键技术,在生物制品质量控制及医学临床实践中意义重大。本文对抗体效价检测的常规技术及其研究进展进行简要综述,并对这些技术的特异性、灵敏性、检测周期及应用情况等方面进行比较分析,以期对特异性抗体效价检测技术有一个系统全面的认知,有利于研究人员在实际应用中选择合适的技术,共同推动抗体检测技术的发展。     

特异性抗体效价检测技术概述

特异性抗体效价是生物制品活性（浓度）的重要标志,通常采用生物学方法来测定,并以抗体与其相应抗原反应产生的、可观察到的标准免疫反应来表示。抗原抗体间除发生特畀性反应外,还会产生交叉反应,从而使特异性抗体效价的检测出现偏差,这在实际应用中亟需避免。特异性抗体效价检测技术是疾病诊断、特异性免疫球蛋白制备和疫苗评价等领域的关键技术,在生物制品质量控制及医学临床实践中意义重大。本文对抗体效价检测的常规技术及其研究进展进行简要综述,并对这些技术的特异性、灵敏性、检测周期及应用情况等方面进行比较分析,以期对特异性抗体效价检测技术有一个系统全面的认知,有利于研究人员在实际应用中选择合适的技术,共同推动抗体检测技术的发展。     

Specific Kilting of Human Melanoma Cells With an Efficient 10B-Compound on Monoclonal Antibodies

We previously established methods which have enabled us to target a sufficient number of ¹⁰B atoms on human melanoma cells to destroy them by thermal neutron irradiation. Monoclonal antibodies were here used as vector of ¹⁰B atoms on the target cell. Thermal neutrons require at least 10⁹¹⁰B atoms to destroy the cell. In order to accumulate an adequate number of ¹⁰B atoms on target cells, our first approach was to make an effective compound that contains 12 atoms of ¹⁰B in a molecule. The second step was to conjugate the compound with an avidin molecule (¹⁰B₁₂-avidin). One molecule of the ¹⁰B₁₂-avidin carries about 30 atoms of ¹⁰B. This ¹⁰B₁₂-avidin can be specifically targeted on human melanoma cells by biotinated monoclonal antibodies specific for the cells. Furthermore, the number of ¹⁰B atoms on target cells can be augmented by a hapten-antihapten monoclonal antibody system. The cultured human melanoma cells treated with these methods were damaged by thermal neutron irradiation. This is the first study that indicates thermal neutrons do injure target cells boronated by monoclonal antibodies.     

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice

As most infections by the helminth parasite elicit the recruitment of CD4⁺CD25⁺Foxp3⁺ T (T_reg) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T_reg cells, we compared the expression levels of T_reg cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T_reg cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T_reg cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T_reg cells in the muscle tissue.     

Production of L-Phenylacetyl Carbinol by Immobilized Cells of Saccharomyces Cerevisiae

Conversion of benzaldehyde to L-phenylacetyl carbinol (L-PAC) was achieved with immobilized, growing cells of Saccharomyces cerevisiae in different reactors. Product formation increased (31%) with the subsequent initial reuses of the entrapped cells. Biomass production and PAC formation depleted (40 and 57%, respectively) after 4-5 continuous growth and biotransformation cycles. With the regeneration of the biocatalysts, catalytic activity of the cells was resumed. The highest yields were in a stirred tank reactor (29 g PAC) from 77 g benzeldehyde with 14 repeated uses of entrapped cells.     

Immobilized Cells in Meat Fermentation

Abstract

The immobilization of microbial cells can contribute to fermented meat technology at two basic levels. First, the solid/semisolid nature (low available water) of the substrate restricts the mobility of cells and results in spatial organizations based on “natural immobilization” within the fermentation matrix. The microniches formed influence the fermentation biochemistry through mass transfer limitations and the subsequent development and activity of the microflora. This form of immobilization controls the nature of competition between subpopu-lations within the microflora and ultimately exerts an effect on the ecological competence (ability to survive and compete) of the various cultures present. Second, immobilized cell technology (ICT) can be used to enhance the ecological competence of starter cultures added to initiate the fermentation. Immobilization matrices such as alginate can provide microniches or microenvironments that protect the culture during freezing or lyophilization, during subsequent rehydration, and when in competition with indigenous microflora. The regulated release of cells from the microenvironments can also contribute to competitive ability. The regulation of both immobilization processes can result in enhanced fermentation activity.     

Specific Inhibition of Tumor Cells by Oncogenic EGFR Specific Silencing by RNA interference

Anticancer agents that have minimal effects on normal cells and tissues are ideal cancer drugs. Here, we show specific inhibition of human cancer cells carrying oncogenic mutations in the epidermal growth factor receptor (EGFR) gene by means of oncogenic allele-specific RNA interference (RNAi), both in vivo and in vitro. The allele-specific RNAi (ASP-RNAi) treatment did not affect normal cells or tissues that had no target oncogenic allele, whereas the suppression of a normal EGFR allele by a conventional in vivo RNAi caused adverse effects, i.e., normal EGFR is vital. Taken together, our current findings suggest that specific inhibition of oncogenic EGFR alleles without affecting the normal EGFR allele may provide a safe treatment approach for cancer patients and that ASP-RNAi treatment may be capable of becoming a safe and effective, anticancer treatment method.     

Activation of Thymus Cells by Histocompatibility Antigens

“Educated” or “activated” thymus cells have been shown, by the use of “strong” histocompatibility antigens, to be immunocompetent only against the antigens by which they were originally activated.     

固定化细胞应用进展

固定化细胞技术是酶工程的核心技术之一,它将酶工程提高到一个新水平。该技术简化了工业分离纯化的步骤,并使酶反应的连续生产成为现实。目前,该技术已经广泛应用于食品、发酵、三废处理等行业,经济效益显著。首先分析了固定化细胞的优缺点,介绍了近年来在食品、发酵和三废处理行业的应用,最后对其应用进行了展望。     

Contribution of Herpesvirus Specific CD8 T Cells to Anti-Viral T Cell Response in Humans

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2_low) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-γ during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.