Preparation and Application of Modified VEGFR-2 Cell Membrane Chromatographic Separation System

The vascular endothelial growth factor receptor-2 (VEGFR-2) in some tumor cells is a significant target for drug discovery. In this work, a modified model of VEGFR-2 cell membrane stationary phase (CMSP) was prepared by immobilizing U251 cell membrane onto the surface of chitosan-silica (CTS-SiO₂) hybrid carrier. The surface and chromatographic characteristics of VEGFR-2 CMSP were studied. We have developed modified VEGFR-2 cell membrane chromatography for screening drugs and sunitinib malate was used as a positive control. The interaction between the new compounds and membrane receptor was determined by the capacity factors (kʹ). The in vitro cytotoxicity of 10 new compounds on U251 cell viability was determined by MTT test separately to verify the potential pharmacological activity. The modified VEGFR-2 cell membrane chromatographic system demonstrated fast and effective characteristics for screening leading compounds.

     

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

Summary A new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.     

Prostate Cell Membrane Chromatography�CLC/MS Method for Screening ��1A-Adrenoceptor Antagonists from Lotus Plumule

The ??_1A-adrenoceptor (??_1A-AR) plays an important role in drug discovery and development. An online analytical method coupling prostate cell membrane chromatography (CMC) with high-performance liquid chromatography/mass spectrometry (LC/MS) was established to screen ??_1A-AR antagonists in traditional Chinese medicines (TCMs). The prostate cell membrane stationary phase (CMSP) was prepared by immobilizing the prostate cell membrane onto the surface of the silica carrier. The surface and chromatographic characteristics of the prostate CMSP were studied using tamsulosin as a model molecule. The retained fractions from the prostate CMC were analyzed by transferring them to an LC/MS system through a 10-port switch valve. The active component, which could act on the prostate cell membrane and receptor on it (such as ??_1A-AR), was determined using a displacement experiment. The results indicated that liensinine, isoliensine, and neferine from Lotus Plumule exhibited similar retention characteristics to the control drug tamsulosin when utilizing the prostate CMC model. This new prostate CMC?CLC/MS method is applicable for screening ??_1A-AR antagonists from TCMs such as Lotus Plumule and could be employed as a drug discovery tool for natural medicinal herbs.     

Real-time monitoring of cell migration,phagocytosis and cell surface receptor dynamics using a novel,live-cell opto-microfluidic technique

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic “Lab-in-a-Trench” (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell–cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective–diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.     

Cell Membrane Chromatography Correlated with Functional Assay for Ligand–β-Adrenergic Receptor Affinities

This study was performed to investigate whether the retention factor (k) from cell membrane chromatography (CMC) can be used to assess the affinity of ligands to β-adrenergic receptor (β-AR) and the correlationship between the factor and pharmacoligical effects. The cell membrane of guinea pig myocardium membrane was immobilized on the surface of the silica carrier as the cell membrane stationary phase (CMSP) for the rapid on-line chromatographic evaluation of ligand binding affinity to β-AR. The affinity was also evaluated by functional assay using the same tissues. Correlation analysis was used to assess the correlationship of these two methods. The retention factors in guinea pig myocardium CMSP were: (?)-propranolol (33.9) > (+)-propranolol (27.0) > metopranolol (23.2) > esmolol (17.7) > practolol (13.2) > sotalol (9.56). Compared to the affinity rank orders obtained from functional assay in the same myocardium, there was a positive correlation (r ² = 0.9729, n = 18, p < 0.0001) between both data sets. These results showed that CMC can be used to evaluate drug–receptor affinities of drug candidates as the functional assays.     

Activation of B cells using Schneider 2 cells expressing CD40 ligand for the enhancement of antigen presentation in vitro

CD40 ligand (CD40L) expressed by activated CD4+ T cells is a family member of membrane bound TNF family ligand and its interaction with CD40 expressed in APC has been shown to contribute in enhancing immune response. Exogenous stimulation through CD40 has been performed using soluble trimeric CD40L, anti-CD40 monoclonal antibody and cells expressing CD40L. Schneider 2 (S2) cells, a cell line derived from Drosophila melanogaster, was transfected with a plasmid vector, pAc5.1/V5-HisA, for the constitutive expression of CD40L (S2-CD40L). Upon incubation of S2-CD40L with B-lymphocytes for 6 days, activated B cells were examined by counting B cell numbers and for activation markers including CD86 and HLA Class II molecules. The activated B cells were tested for its efficient APC function by mixed lymphocyte reactions (MLR) and enzyme-linked Immunospot (ELISPOT) assay. S2-CD40L was cultured for a year and maintained CD40L expression (>90%). S2-CD40L induced B cell activation as demonstrated by increment of total B cells and up-regulation of CD86 and MHC Class II molecules. Activated B cells pulsed with peptide from human cytomegalovirus pp65 antigen efficiently induced both proliferation and IFN-gamma secretion of T cells. Our result suggests that S2-CD40L can efficiently and conveniently generate B cells as a functional APC and represents a potential role for B-cell mediated cancer immunotherapy.     

A new A431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A₄₃₁ cells) are an important target for drug discovery. We have developed a new A₄₃₁/cell membrane chromatography (A₄₃₁/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A₄₃₁ cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A₄₃₁/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A₄₃₁ cell growth in vitro. This new A₄₃₁/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

EGFR/cell membrane chromatography-online-high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Radix Angelicae Pubescentis

The intracellular kinase domains of the epidermal growth factor receptor (EGFR) in some tumor cells are significant targets for drug discovery. We have developed a new EGFR cell membrane chromatography (EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis. In this study, the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase (CMSP) in the EGFR/CMC model. The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online. As a result, osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. This new EGFR/CMConline-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs, for instance, Radix Angelicae Pubescentis. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Comparison of Determination of Drug–Muscarinic Receptor Affinity by Cell-Membrane Chromatography and by Radioligand-Binding Assay with the Cerebrum Membrane of the Rat

This study was performed to determine whether cell-membrane chromatography (CMC) can be used to reflect the selectivity and specificity of interactions between drugs and the muscarinic acetylcholine receptor (mAChR). A cell membrane stationary phase (CMSP) was prepared by immobilizing rat cerebrum cell membrane on the surface of a silica carrier and used for rapid on-line chromatographic evaluation of ligand binding affinity to mAChR. Comparison of the results with affinity rank orders obtained from radioligand-binding assays using the same cerebrum membrane indicated a positive correlation (r²=0.8640, n=18, P < 0.0001) between the data sets and proved that CMC can be used to evaluate drug–receptor affinity for drug candidates.Revised: 19 July and 15 November 2004     

Cell Membrane Chromatography Correlated with Functional Assay for Ligand–β-Adrenergic Receptor Affinities

This study was performed to investigate whether the retention factor (k) from cell membrane chromatography (CMC) can be used to assess the affinity of ligands to β-adrenergic receptor (β-AR) and the correlationship between the factor and pharmacoligical effects. The cell membrane of guinea pig myocardium membrane was immobilized on the surface of the silica carrier as the cell membrane stationary phase (CMSP) for the rapid on-line chromatographic evaluation of ligand binding affinity to β-AR. The affinity was also evaluated by functional assay using the same tissues. Correlation analysis was used to assess the correlationship of these two methods. The retention factors in guinea pig myocardium CMSP were: (−)-propranolol (33.9) > (+)-propranolol (27.0) > metopranolol (23.2) > esmolol (17.7) > practolol (13.2) > sotalol (9.56). Compared to the affinity rank orders obtained from functional assay in the same myocardium, there was a positive correlation (r ² = 0.9729, n = 18, p < 0.0001) between both data sets. These results showed that CMC can be used to evaluate drug–receptor affinities of drug candidates as the functional assays.

     

Development of a Stationary Phase of Vascular Smooth Muscle Cell Membrane Chromatography and Its Chromatographic Affinity Characteristics

A novel stationary phase of vascular smooth muscle cell membrane chromatography (VSM-CMC) was developed by immobilizing the vascular smooth muscle cell membrane onto the surface of silica and was presented for bioaffinity chromatography. The protein level and Na⁺, K⁺-ATP enzymatic activity of the vascular smooth muscle cell membrane stationary phase (VSM-CMSP) were detected. The surface characteristics of the VSM-CMSP were tested using scanning electron microscopy and surface energy spectrometry. The retention characteristics of four dihydropyridines (amlodipine besylate, nicardipine hydrochloride, nitrendipine and nifedipine) were investigated using a VSM-CMSP column (10 mm × 2 mm, I.D.) packed with VSM-CMSP. The logarithm of the capacity factor (logk??) was taken as a measure of the affinities of the calcium antagonists toward the vascular smooth muscle cell membrane and receptors. The surface characteristics of the VSM-CMSP were very different from that of the normal and reversed stationary phase, and the VSM-CMSP was found to have cell membrane activity and chromatographic separation. Moreover, there was a significant correlation between the affinity in the VSM-CMC system and the effect in vitro with respect to the pharmacological effect. It is concluded that the VSM-CMC system can serve as a type of bioaffinity chromatography for studying the interaction between drug molecules and target sites (e.g., receptors) in cell membranes, and for screening active compounds from complex agents.     

Aptamer-containing surfaces for selective capture of CD4 expressing cells

Aptamers have recently emerged as an excellent alternative to antibodies because of their inherent stability and ease of modification. In this paper, we describe the development of an aptamer-based surface for capture of cells expressing CD4 antigen. The glass or silicon surfaces were modified with amine-terminated silanes and then modified with thiolated RNA aptamer against CD4. Modification of the surface was first characterized by ellipsometry to demonstrate assembly of biointerface components and to show specific capture of recombinant CD4 protein. Subsequently, surfaces were challenged with model lymphocytes (cell lines) that were either positive or negative for CD4 antigen. Our experiments show that aptamer-functionalized surfaces have similar capture efficiency to substrates containing anti-CD4 antibody. To mimick capture of specific T-cells from a complex cell mixture, aptamer-modified surfaces were exposed to binary mixtures containing Molt-3 cells (CD4+) spiked into Daudi B cells (CD4-). 94% purity of CD4 cells was observed on aptamer-containing surfaces from an initial fraction of 15% of CD4. Given the importance of CD4 cell enumeration in HIV/AIDS diagnosis and monitoring, aptamer-based devices may offer an opportunity for novel cell detection strategies and may yield more robust and less expensive blood analysis devices in the future.     

Role of the Immune System in Mediating the Antitumor Effect of Benzophenothiazine Photodynamic Therapy

The role of the host immune system in contributing to tumor regression following benzophenothiazine photodynamic therapy (PDT) was examined. Photodynamic therapy with 2-iodo-5-ethylamino-9-diethylaminobenzo[a]-phenothiazinium chloride (2I-EtNBS) eradicated EMT-6 mammary fibrosarcomas in 75-100% of treated mice. In contrast, PDT failed to inhibit tumor growth in T-cell-deficient nude mice. Furthermore, T-cell depletion studies with anti-CD8 antibody revealed that the CD8+ T-cell population was critical for an effective PDT response (tumor volume 14 days post-PDT: 262 mm3 vs 59 mm3 in controls; P < 0.01). Because anti-CD4 antibody inhibited tumor growth in the absence of PDT, the role of CD4+ T cells remains unclear. Depletion of natural killer (NK) cells in vivo with anti-asialo-GM1 antibody significantly reduced a suboptimal PDT effect relative to vehicle controls (tumor volume 13 days post-PDT: 513 mm3 vs 85 mm3, respectively; P < 0.001). However, splenic NK cells obtained from PDT-treated tumor-bearing mice were not cytotoxic in vitro against EMT-6 cells, suggesting that NK cells contribute to the PDT effect in vivo by an indirect mechanism. In addition, when mice with complete tumor regression following PDT were rechallenged 28 days later with 5 x 10(5) EMT-6 cells, tumor growth was significantly inhibited as compared to controls (tumor volume 40 days postrechallenge: 137 mm3 vs 833 mm3 in controls; P < 0.03; percent animals without tumor in five experiments: 67% vs 8% in controls). Collectively, these results demonstrate that CD8+ T cells are required to prevent tumor regrowth after 2I-EtNBS-PDT, NK cells contribute to this response and such PDT can elicit protective antitumor immunity.     

Study on molecular interactions between proteins on live cell membranes using quantum dot-based fluorescence resonance energy transfer

Mouse anti-human CD71 monoclonal antibody (anti-CD71) was conjugated with red quantum dots (QDs; 5.3 nm, emission wavelength λ _em = 614 nm) and used to label HeLa cells successfully. Then green QD-labeled goat anti-mouse immunoglobulin G (IgG; the size of the green QDs was 2.2 nm; λ _em = 544 nm) was added to bind the red-QD-conjugated anti-CD71 on the cell surface by immunoreactions. Such interaction between anti-CD71 and IgG lasted 4 min and was observed from the fluorescence spectra: the fluorescence intensity of the “red” peak at 614 nm increased by 32%; meanwhile that of the “green” one at 544 nm decreased by 55%. The ratio of the fluorescence intensities (I _544 nm/I _614 nm) decreased from 0.5 to 0.2. The fluorescence spectra as well as cell imaging showed that fluorescence resonance energy transfer took place between these two kinds of QDs on the HeLa cells through interactions between the primary antibody and the secondary antibody.     

Detecting interferon-gamma release from human CD4 T-cells using surface plasmon resonance

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-γ – an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4⁺ T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-γ Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset.     

Analysis of leukocyte membrane protein interactions using protein microarrays

Background  

Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.     

Attenuation of airway inflammation by simvastatin and the implications for asthma treatment: is the jury still out?

Although some studies have explained the immunomodulatory effects of statins, the exact mechanisms and the therapeutic significance of these molecules remain to be elucidated. This study not only evaluated the therapeutic potential and inhibitory mechanism of simvastatin in an ovalbumin (OVA)-specific asthma model in mice but also sought to clarify the future directions indicated by previous studies through a thorough review of the literature. BALB/c mice were sensitized to OVA and then administered three OVA challenges. On each challenge day, 40 mg kg⁻¹ simvastatin was injected before the challenge. The airway responsiveness, inflammatory cell composition, and cytokine levels in bronchoalveolar lavage (BAL) fluid were assessed after the final challenge, and the T cell composition and adhesion molecule expression in lung homogenates were determined. The administration of simvastatin decreased the airway responsiveness, the number of airway inflammatory cells, and the interleukin (IL)-4, IL-5 and IL-13 concentrations in BAL fluid compared with vehicle-treated mice (P<0.05). Histologically, the number of inflammatory cells and mucus-containing goblet cells in lung tissues also decreased in the simvastatin-treated mice. Flow cytometry showed that simvastatin treatment significantly reduced the percentage of pulmonary CD4⁺ cells and the CD4⁺/CD8⁺ T-cell ratio (P<0.05). Simvastatin treatment also decreased the expression of the vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 proteins, as measured in homogenized lung tissues (P<0.05) and human epithelial cells. The reduction in the T cell influx as a result of the decreased expression of cell adhesion molecules is one of the mechanisms by which simvastatin attenuates airway responsiveness and allergic inflammation. Rigorous review of the literature together with our findings suggested that simvastatin should be further developed as a potential therapeutic strategy for allergic asthma.     

Selective capturing and detection of Salmonella typhi on polycarbonate membrane using bioconjugated quantum dots

Present work demonstrates the utilization of surface modified polycarbonate (PC) membrane as solid phase and antibody conjugated CdSe/ZnS quantum dots (QDs) as fluorescent label for the sensitive and selective detection of Salmonella typhi (S. typhi) in water in a period of 2.5 h. PC membrane was surface modified with glycine and activated by EDC/NHS for immobilization of S. typhi specific IgG. Antibody immobilized porous PC membrane was incubated with bacteria contaminated water for immunocapturing of S. typhi. Antibody conjugated QDs were also prepared by using carbodiimide chemistry. Both modified PC membrane and quantum dots were characterized by using various modern analytical tools. It was estimated that 1.95 molecules of QDs were successfully bio-conjugated per unit of IgG. PC membrane with captured bacteria was incubated with prepared IgG conjugated QDs for the formation of sandwich complex. Analysis of the regions of interest (ROI) in fluorescent micrographs showed that newly developed method based on PC and fluorescent QDs has 100 times higher detection sensitivity (100 cells/mL) as compared with detection using conventional dye (FITC) based methods.     

细胞膜色谱法研究5-羟色胺受体与藁本内酯的亲和作用

建立了大鼠纹状体细胞膜色谱前沿分析法,研究5-羟色胺(5-HT)受体5-HT_1D与藁本内酯的亲和作用。通过大鼠纹状体组织制备得到色谱固定相,利用酶联免疫吸附剂测定法(ELISA)分别测定硅胶吸附前后细胞膜悬液中5-HT的量,求得细胞膜固定相上5-HT受体含量为每克硅胶(40.5±2.3) pg。利用细胞膜色谱与液相色谱的离线联用,特异性地识别混合对照品中的舒马普坦和藁本内酯;以不同浓度(24.2~242 nmol/L)的5-HT_1D受体激动剂舒马普坦为模型药物,连续通过细胞膜色谱柱,记录舒马普坦的突破曲线,测得舒马普坦与受体作用的平衡解离常数(K_D)为389 nmol/L;并将舒马普坦通过不同浓度(37.0~370 nmol/L)的藁本内酯饱和后的细胞膜色谱柱,记录色谱柱饱和前后舒马普坦突破曲线的变化,测得藁本内酯与受体作用的K_D值为4.21 μmol/L。该方法快速、有效,适用于求解存在竞争结合时药物与受体作用的平衡解离常数。     

Inhibitory Effect of Anti-HER-2 Anti-CD3 Bi-specific Antibody on the Growth of Gastric Carcinoma

Introduction Gastriccancerisoneofthemostcommonlyen counteredmalignantdiseasesworldwide,especiallyin AsiaandAfrica[1].Thecombinationofoperation,chemotherapyandradiotherapyisusedfortreatinggas triccarcinoma.However,the5yearsurvivalrateof patientsofgastricca…