Is the C-C Motif Ligand 2–C-C Chemokine Receptor 2 Axis a Promising Target for Cancer Therapy and Diagnosis?

C-C motif ligand 2 (CCL2) was originally reported as a chemical mediator attracting mononuclear cells to inflammatory tissue. Many studies have reported that CCL2 can directly activate cancer cells through a variety of mechanisms. CCL2 can also promote cancer progression indirectly through increasing the recruitment of tumor-associated macrophages into the tumor microenvironment. The role of CCL2 in cancer progression has gradually been understood, and various preclinical cancer models elucidate that CCL2 and its receptor C-C chemokine receptor 2 (CCR2) are attractive targets for intervention in cancer development. However, clinically available drugs that regulate the CCL2–CCR2 axis as anticancer agents are not available at this time. The complete elucidation of not only the oncological but also the physiological functions of the CCL2–CCR2 axis is required for achieving a satisfactory effect of the CCL2–CCR2 axis-targeted therapy.     

DNA‐Directed Assembly of Antibody–Fluorophore Conjugates for Quantitative Multiparametric Flow Cytometry

Multiparametric flow cytometry offers a powerful approach to single‐cell analysis with broad applications in research and diagnostics. Despite advances in instrumentation, progress in methodology has lagged. Currently there is no simple and efficient method for antibody labeling or quantifying the number of antibodies bound per cell. Herein, we describe a DNA‐directed assembly approach to fluorescent labeling that overcomes these barriers. Oligonucleotide‐tagged antibodies and microparticles can be annealed to complementary oligonucleotides bearing fluorophores to create assay‐specific labeling probes and controls, respectively. The ratio of the fluorescence intensity of labeled cells to the control particles allows direct conversion of qualitative data to quantitative units of antibody binding per cell. Importantly, a single antibody can be labeled with any fluorophore by using a simple mix‐and‐match labeling strategy. Thus, any antibody can provide a quantitative probe in any fluorescent channel, thus overcoming major barriers to the use of flow cytometry as a technique for systems biology and clinical diagnostics.     

Disrupted Homeostatic Cytokines Expression in Secondary Lymph Organs during HIV Infection

Research has firmly established that infection by human immunodeficiency virus (HIV) leads to structural disruption in secondary lymph organs (SLOs) and that IL-7 expression by SLOs is downregulated in simian immunodeficiency virus (SIV)-infected rhesus macaques. However, the foregoing has not been demonstrated in HIV-infected patients. As well, SLO-produced chemokines and cytokines, other than IL-7, have not been tested. In this study, SLOs in HIV-infected patients exhibit decreased levels of lymphoid cytokines, such as IL-7 and C–C motif chemokine ligand 21 (CCL21), due to lower expression of lymphotoxin (LT)-β. Previous research has shown that LT-β is produced mainly by CD4⁺T cells in rhesus macaques, while our study found the same level of LT-β expressed by CD4⁺T and CD8⁺T cells in humans. CD8⁺T cells substitute for depleted CD4⁺T cells LT-β production. Only the total number of CD3⁺T cells can account for the majority of LT-β in human SLOs. This study indicates a possible mechanism and a potential target for improvement of SLO function in HIV-infected patients, a novel adjuvant therapy for AIDS.     

Potential Use of Quantum Dots in Flow Cytometry

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.     

A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes

The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.     

Novel and Selective Fluorescent σ2‐Receptor Ligand with a 3,4‐Dihydroisoquinolin‐1‐one Scaffold: A Tool to Study σ2 Receptors in Living Cells

Although sigma‐2 (σ₂) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ₂‐selective fluorescent tracer (compound 5 ) as a specific tool to study σ₂ receptors. By using flow cytometry with 5 , we performed competition binding studies on three different cell lines where we also detected the content of the σ₂ receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ₁/σ₂ fluorescent tracer ( 1 ) also allowed for the detection of σ₁ receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ₁, and Scatchard analysis for σ₂ receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology.     

Role of SDF1/CXCR4 Interaction in Experimental Hemiplegic Models with Neural Cell Transplantation

Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES) cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1) in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.     

基于微流体数字化技术的流式细胞术的设计

针对流式细胞术的核心之一——单细胞的液流驱动系统,提出了基于微流体数字化驱动-控制技术实现单细胞排列及输出的方法,分析了单细胞流的驱动机理,通过对流动室施加数字化可编码的高频驱动脉冲,细胞从连续的鞘液流中分割成单细胞流输出。设计了一种流式细胞仪核心部分石英微流动室,并采用石英玻璃精细加工而成。进行了人体外周血淋巴细胞的驱动实验研究,可通过荧光显微镜实时观测微通道内的单细胞流动情况,方法简单,操作方便。