Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).     

利用流式细胞术测定CTL活性方法在EIAV免疫学中的应用

利用PKH 26和CFSE两种荧光染料对靶细胞染色,建立了一种通过流式细胞术进行马传染性贫血症病毒(Equine infectious anemia virus,EIAV)抗原特异性细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)反应的新方法,避免了经典的Cr51释放法对检测人员的放射线威胁,降低了本底释放,提高了检测的灵敏度.将该检测方法用于检测EIAV疫苗毒接种马和嵌合克隆接种马的细胞免疫反应变化趋势,数据显示细胞免疫反应在接种后3个月达到成熟阶段而后保持在较高的反应水平.该方法的成功建立和应用为研究EIAV减毒疫苗的免疫机制提供了好的研究手段,也为其他病毒的免疫学研究提供了新的参考方法.     

利用流式细胞术测定CTL活性方法在EIAV免疫学中的应用

利用PKH-26和CFSE两种荧光染料对靶细胞染色,建立了一种通过流式细胞术进行马传染性贫血症病毒 (Equine infectious anemia virus,EIAV)抗原特异性细胞毒性T淋巴细胞(Cytotoxic T lymphocytes,CTL)反应的 新方法,避免了经典的Cr51释放法对检测人员的放射线威胁,降低了本底释放,提高了检测的灵敏度。将该检测方 法用于检测EIAV疫苗毒接种马和嵌合克隆接种马的细胞免疫反应变化趋势,数据显示细胞免疫反应在接种后3 个月达到成熟阶段而后保持在较高的反应水平。该方法的成功建立和应用为研究EIAV减毒疫苗的免疫机制提 供了好的研究手段,也为其他病毒的免疫学研究提供了新的参考方法。     

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.     

A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.     

Mechanism-Based Inhibitors: Development of a High Throughput Coupled Enzyme Assay to Screen for Novel Antimalarials

Identifying potent enzyme inhibitors through a robust HTS assay is currently thought to be the most efficient way of searching for lead molecules. We have developed a HTS assay that mimics a crucial step in an essential metabolic pathway, the purine salvage pathway of the malarial parasite Plasmodium falciparum. In this assay we have used purified recombinant enzymes: hypoxanthine guanine phosphoribosyl transferase (HGPRT) and inosine monophosphate dehydrogenase (IMPDH) from the malarial parasite and the human host, respectively. These two enzymes, which work in tandem, are used to set up a coupled assay that is robust enough to meet the stringent criteria of an HTS assay. In the first phase of our screen we seem to have identified novel inhibitors that kill the parasite by inhibiting the salvage pathway of the parasite.     

Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69⁺ T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 10³-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.     

Circulating Tumor Cells: Clinically Relevant Molecular Access Based on a Novel CTC Flow Cell

Background

Contemporary cancer diagnostics are becoming increasing reliant upon sophisticated new molecular methods for analyzing genetic information. Limiting the scope of these new technologies is the lack of adequate solid tumor tissue samples. Patients may present with tumors that are not accessible to biopsy or adequate for longitudinal monitoring. One attractive alternate source is cancer cells in the peripheral blood. These rare circulating tumor cells (CTC) require enrichment and isolation before molecular analysis can be performed. Current CTC platforms lack either the throughput or reliability to use in a clinical setting or they provide CTC samples at purities that restrict molecular access by limiting the molecular tools available.

Methodology/Principal Findings

Recent advances in magetophoresis and microfluidics have been employed to produce an automated platform called LiquidBiopsy®. This platform uses high throughput sheath flow microfluidics for the positive selection of CTC populations. Furthermore the platform quantitatively isolates cells useful for molecular methods such as detection of mutations. CTC recovery was characterized and validated with an accuracy (<20% error) and a precision (CV<25%) down to at least 9 CTC/ml. Using anti-EpCAM antibodies as the capture agent, the platform recovers 78% of MCF7 cells within the linear range. Non specific recovery of background cells is independent of target cell density and averages 55 cells/mL. 10% purity can be achieved with as low as 6 CTCs/mL and better than 1% purity can be achieved with 1 CTC/mL.

Conclusions/Significance

The LiquidBiopsy platform is an automated validated platform that provides high throughput molecular access to the CTC population. It can be validated and integrated into the lab flow enabling CTC enumeration as well as recovery of consistently high purity samples for molecular analysis such as quantitative PCR and Next Generation Sequencing. This tool opens the way for clinically relevant genetic profiling of CTCs.     

分子信标检测限制性内切酶活性的新型荧光分析方法

利用分子信标被切割后荧光信号的变化.发展了一种简便的检测限制性内切酶活性的新型荧光分析方法.以限制性内切酶Xsp I为例.将其识别位点嵌入具有茎环结构的分子信标探针(Molecular Beacon,MB)的环状部分,利用这种分子信标在溶液中形成的瞬时二聚体结构,实现了限制性内切酶对分子信标的切割.在优化的条件下,反应初速度与酶的浓度成正比,线性范围为0.05～50 U/mL,检测限为0.05 U/mL.通过改变分子信标环部的识别位点的序列.还可以检测其他限制性内切酶如Alu I的活性.     

A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity

Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10^–12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.     

免疫球蛋白重链可变段和T细胞受体可变段的分子进化研究

为阐明免疫球蛋白(Ig)和T细胞受体(TCR)在抗体多样性产生机理上的异同,作者比较了Ig重链可变段(Ig V_H)和TCR可变段(TCR V)的密码子替代率和协同进化,并分析异同的原因。共搜集8种鼠和3种人的TCR α链可变段(V_α),11种鼠和1种人的TCRβ链可变段(V_β),以及2种鼠和4种人的T细胞γ链可变段;同时搜集11种鼠、3种人、3种南美鳄鱼和1种鲨鱼的Ig V_(H_(o))研究结果揭示:(1)对编码蛋白质的密码子来说,TCR V(包括V_α和V_β)的核苷酸替代率为Ig V_H的2.4倍,说明前者有更高的替代率。(2)以协同进化而言,TCR V和Ig V_H的基因重复率分别为1.7×10~(-6)和1.6×10~(-6)/基因年。两者几乎相同,均系低速保持者。TCR V的数目(V_α为100,V_β为30)远少于Ig V_H(数目为300),原因是前者受到主要组织相容性复合体的制约,即受到负选择,这与中性学说观点相一致。文章还讨论了体细胞突变和DNA重排对两类抗体多样性产生上的作用,并探讨了IgV_H和TCR V的假基因问题。     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Abstract

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post‐translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR α and β chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR α and β chains was accomplished using site‐directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL‐2 in response to substrate‐immobilized antibody to TCR. However, mutation of serine‐88 in the protein kinase A phosphorylation site of the TCR β chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR‐associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine‐88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three‐dimensional structure of the complex of SEB and TCR, are consistent with the view that serine‐88 is important for the contact of the TCR β chain with SEB.     

A Theoretical and Experimental Study of Competition Between Solution and Surface Receptors for Ligand in a Biacore Flow Cell

Rate constants that characterize the kinetics of binding and dissociation between biomolecules carry fundamental information about the biological processes these molecules are involved in. An instrument that is widely used to determine these rate constants is the Biacore. In a Biacore experiment, one of the reactants, which we will call the receptor, is immobilized on a sensor chip. During the binding phase of the experiment the other reactant flows past the chip. After binding, buffer alone is introduced into the flow cell and dissociation is monitored. Often surface-based binding assays are influenced by the transport of the reactant in solution, complicating the determination of the chemical rate constants from the observed binding kinetics. We propose a new way to determine the dissociation rate constant by adding soluble receptor during dissociation. The method is tested first on simulated data and then on Biacore experiments where the lac repressor protein binds and dissociates from a stretch of double stranded DNA containing the lac repressor binding site. With this method we find a dissociation rate constant k_d=0.075 ± 0.005s^-1, a value that is faster than previously obtained from Biacore experiments. In developing our method to analyze these experiments we obtain an expression for the transport limited rate constant for a Biacore experiment when soluble receptor is present during dissociation.     

一种新的非病毒基因转移载体及其体外细胞转染活性研究

目的：用低分子量的聚乙亚胺(PEI)有效地进行基因转染,为基因转染在基因治疗中的应用提供一种可靠、廉价、高效的方法。方法：将带有绿色荧光蛋白(GFP)报告基因的真核表达质粒与阳离子聚合物聚乙亚胺(PEI)结合,用肝癌细胞系SMMC7721实验,研究PEI分子量与转染活性以及可能引起的细胞毒性之间的关系;进一步研究在血清存在的情况下,PEG(聚乙二醇8000)、叶酸等物质对PEI介导的转染效率的影响。结果：分子量为600Da的PEI在pH值为6．0时与质粒DNA以1：1的质量比混合,细胞转染效率最高为43．6 /-7．3％,随着分子量的增大,转染活性略有下降;进一步研究发现,在血清存在下,20μM的叶酸和15％的PEG能有效地改善PEI介导的转染活性,使其转染活性提高了13％;用光学相差显微镜检测了PEI潜在的细胞毒性,结果发现分子量为600Da的PEI没有使细胞形态改变或死亡,但是随着分子量的增大,PEI潜在的细胞毒性也相应增大。结论：PEI是一种高效、有用的非病毒载体,能够在培养的哺乳动物细胞中进行基因转移,这对疾病的基因治疗具有潜在的应用价值。     

Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain

Abstract: A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4-dihy-droxyphenylacetic acid, homovanillic acid, 3-methoxytyra-mine (3-MT), noradrenaline, 3-methoxy-4-hydroxyphenyl-ethyleneglycol (MOPEG), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40%; wt/wt) and graphite powder (60%; wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24–67%; (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20–60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8–16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed-phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G-10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HC1O₄, and the excess of HC1O₄ was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer. Adjustment of the pH was necessary so that elution of 5-HT from the Sephadex G-10 columns in the same fraction with 3-MT was avoided. If these compounds were in the same solution, their peaks would overlap on HPLC. MOPEG sulfate was purified by diethylaminoethyl-Sephadex A-25 (anion exchange resin) from the first fraction collected from the Sephadex G-10 columns. The contents of the compounds under investigation in nine brain parts agreed with those found by other investigators.