Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr‐1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr‐1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear‐punch injury in diabetic or non‐diabetic mice. The Gr‐1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr‐1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr‐1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF‐1 axis plays an important role in the homing of Gr‐1+CD11b+ myeloid cells to the site of injury. In addition, Gr‐1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr‐1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing. 相似文献
Flow cytometry provides a high throughput, multi‐dimensional analysis of cells flowing in suspension. In order to combine this feature with the ability to resolve detailed structures in 3D, we developed an optofluidic device that combines a microfluidic system with a dual beam trap. This allows for the rotation of single cells in a continuous flow, around an axis perpendicular to the imaging plane. The combination of both techniques enables the tomographic reconstruction of the 3D structure of the cell. In addition this method is capable to provide detailed 3D structural data for flow cytometry, as it improves the reconstructed z‐resolution of a standard microscopy system to produce images with isotropic resolution in all three axes.
Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction. In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs. Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells. The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma). In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha. To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin. These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression. Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37). 相似文献
Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule. 相似文献
A new type of high‐throughput imaging flow cytometer (>20 000 cells s‐1) based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. FACED imaging flow cytometers enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. It critically empowers largescale and deep characterization of single cells and their heterogeneity with high statistical power— an ability to become increasingly critical in single‐cell analysis adopted in a wide range of biomedical and life‐science applications. Further details can be found in the article by Wenwei Yan et al. ( e201700178 )
Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes. 相似文献
To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non‐self‐antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T‐cell repertoire was used. The response of EF4.1 mice to an I‐Ab‐associated epitope of the F‐MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg‐depleted EF4.1 mice were immunized, and the extent of the Vα2‐bearing, antigen‐specific TCR repertoire was characterized by high‐throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre‐immune repertoire. Injection of anti‐CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non‐self‐antigens. 相似文献
AbstractDendritic cells (DC) are antigen-presenting cells (APC) that are important for innate and acquired immune responses. Owing to their involvement in autoinflammation, autoimmunity and cancer, DC are useful cellular models for biomedical research. Appropriate DC production in vitro could aid the study of DC in many human diseases. We used fluorochrome-based flow cytometry assays to analyze the effects of culture period and maturation of monocyte-derived DC (MoDC) on their viability and necrosis, purity, CD11c expression and phagocytic capacity. The morphological changes that occur as purified monocytes become DC were assessed at 24 and 72 h, and 6 and 9 days in culture. The dynamics of certain cell surface markers of monocytes and mature MoDC (mMoDC) also were assessed using fluorescence-based assays. We found that day 6 of culture yielded the most functional immature MoDC (iMoDC) with maximal viability, purity, CD11c expression and appropriate phagocytic capacity. Mass production of viable MoDC could be useful for immunotherapy. 相似文献
Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8+ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3+PD‐1+ CD8+ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3?PD‐1+ cells. Tim‐3+PD‐1+ CD8+ T cells showed more evident properties associated with exhaustion than Tim‐3?PD‐1+ CD8+ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8+ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8+ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8+ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system. 相似文献
Natural Killer (NK) cells kill certain tumor cells and virus infected cells in an antigen-independent manner. Members of CD18 integrins such as CD11a, CD11b, and CD11c are expressed in all NK cells. CD18-blocking mAbs inhibit the killing activity of NK cells implying an essential role of these integrins in NK cell cytotoxicity. In this report we show that the pan CD18-activating mAb, 240Q, augments cytotoxicity of resting NK cells. Since activation of either CD11a or CD11c alone fails to augment the NK cell activity, we postulate that a functional synergy of the individual CD18 integrins is responsible for the observed stimulatory effect of pan CD18 activation on NK cell cytotoxicity. 相似文献
CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8+ T cells as a new subset of CD8+ regulatory T cells (Treg). Evidence exists that CD11c+CD8+ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c+ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c+CD8+ and CD11c+CD4+ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4+ T cells and CD8+ T cells in different tissues from three groups of male BALB/c mice—young, mature, and those with colorectal cancer (CRC). Analysis of CD3+CD11c+ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3+CD11c+ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4low and CD4high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8+PD-1+ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8+ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC. 相似文献
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