Carboxyfluorescein Diacetate Succinimidyl Ester Research Articles

Telomerase reverse transcriptase induced thyroid carcinoma cell proliferation through PTEN/AKT signaling pathway

Thyroid carcinoma is the most common endocrine malignant tumor in the world, and so, there is a requirement to develop novel molecular targets for thyroid cancer diagnosis and treatment. Telomerase reverse transcriptase (TERT) was revealed to promote cell proliferation in a number of types of cell. To evaluate whether and how TERT functioned on papillary thyroid cancer (PTC) cell proliferation, the present study constructed TERT over-expression [recombined (r)TERT plasmid group] and interference [small interfering RNA (si)-TERT group] models by liposome transfection respectively to study the molecular mechanisms. The transfection efficiency was first detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting to analyze TERT levels compared with the negative control (NC) and control groups. Then MTT and carboxyfluorescein diacetate succinimidyl ester assays were performed to determine living cell proliferation and total cell proliferation respectively. Propidium iodide assay was used to detect alterations in cell cycle progression. RT-qPCR and western blotting were performed to detect associated factor variation. The results demonstrated that, following the generation of TERT overexpression or silencing PTC cells, the living cells and also total cell proliferation increased significantly in the rTERT group, and decreased significantly in siTERT group, when compared with the NC and control groups. The cell cycle was accelerated in the rTERT group, and blocked in the G1/S transition in the siTERT group. The mRNA and protein levels of P27, P53 and phosphatase and tensin homolog (PTEN) decreased significantly in the rTERP group and increased in the siTERP group, while cyclin dependent kinase 2 and Cyclin D1 increased significantly in the rTERP group and decreased in the siTERP group. The expression of cell division cycle 25A did not alter significantly. The protein levels of β-catenin and retinoblastoma were also unaltered. Protein kinase B (AKT) was detected once activated by TERT, and there were increased phosphorylated (p)-AKT protein levels in the rTERT group, and decreased p-AKT protein levels in the siTERT group. In conclusion, TERT could induce thyroid carcinoma cell proliferation mainly through the PTEN/AKT signaling pathway.

Overexpression of indoleamine 2, 3-dioxygenase contributes to the repair of human airway epithelial cells inhibited by dexamethasone via affecting the MAPK/ERK signaling pathway.

Indoleamine 2, 3-dioxygenase (IDO) catalyzes the degradation of trytophan, which serves a key role in immune suppression via regulating the production of several metabolites. The present study aimed to explore the effects and mechanisms of IDO in the repair of human airway epithelium suppressed by dexamethasone (DEX). Cell viability, proliferation and migration were evaluated using a Cell Counting Kit-8 (CCK-8), 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, and wound-healing assay, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and ELISA were performed to assess the levels of IDO, the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway-related factors and epidermal growth factor (EGF) expression, respectively. The results revealed that overexpression of IDO enhanced the cell viability, and promoted the proliferation and migration of 16HBE cells which repair was inhibited by DEX. Furthermore, it was indicated that overexpression of IDO affected the MAPK/ERK pathway. In conclusion, overexpression of IDO promoted the human airway epithelium repair inhibited by DEX through affecting MAPK/ERK pathway. The present study implied that IDO may be a potential genetic therapeutic agent and supported the utilization of IDO in asthma.

Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina.

To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells (EPCs) in a mouse model of laser-induced retinal injury. EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes: 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), 1,1'-dilinoleyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein (DiI-AcLDL), and green fluorescent protein (GFP). The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo. EPCs labeled with CFSE and DiI-AcLDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and DiI-AcLDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina. The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and DiI-AcLDL are suitable for short-term EPC-labeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

Effects of human umbilical cord-derived mesenchymal stem cells on hematologic malignancies.

Mesenchymal stem cells (MSCs) have been used in hematopoietic stem cell transplantation for years. However, the safety of MSCs applied in various types of hematologic malignancy has not been comprehensively explored. In the present study, the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on six representative hematologic malignancy cell lines were explored, including leukemia, multiple myeloma and lymphoma cells. Direct and indirect co-culture models were established, and cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester staining. A cytometric bead array cytokine kit was used to quantify cytokines. The expression of interleukin (IL)-6 receptor elements on tumor cells was detected by reverse transcription-polymerase chain reaction and flow cytometry, and the effects of exogenous IL-6 on cell proliferation were determined using a Cell Counting kit-8 assay. The results demonstrated that hUC-MSCs inhibited the proliferation of most of the cell lines examined (THP-1, HL-60, K562 and RPMI-8226), but promoted the proliferation of Raji cells. In addition, hUC-MSCs secreted abundant IL-6, promoted the secretion of IL-10 by RPMI-8226 and Raji cells, and inhibited the secretion of tumor necrosis factor-α by THP-1 cells. These data indicate a varied effect of hUC-MSCs on various types of hematologic malignancy, including distinct mechanisms of cell-to-cell contact and cytokines. Researchers applying hUC-MSCs in lymphoma should be aware of a potential tumor growth-promoting effect.

Rosemary has immunosuppressant activity mediated through the STAT3 pathway

ObjectiveIn Europe extracts of Rosmarinus officinalis were traditionally used for the treatment of rheumatic diseases. We investigated the capacity of standardized aqueous extracts of Rosmarinus officinalis on human primary lymphocyte function in vitro, as activated lymphocytes are an important mediator of rheumatic diseases. MethodsLymphocyte proliferation was measured using membrane-permeable dye carboxyfluorescein diacetate succinimidyl ester (CFSE). Apoptosis was analysed by surface staining of phosphatidylserine (annexin V-assay) and necrosis was analysed by staining with propidium iodide. Modification of cell activity was detected by surface staining of CD69 and CD25. The activity of STAT3 in T-lymphocytes was determined by intracellular staining of STAT3 molecules. All endpoints were analyzed by using flow cytometry. The Rosmarinus officinalis extract was investigated at concentrations of 0.05–25 mg/mL. Analysis of the extract was performed using HPLC methods. ResultsRosmarinus officinalis inhibited proliferation of human lymphocytes and CD4+ T-cells in a dose-dependent manner (3.1–25 mg/mL) through induction of apoptosis. The intracellular signalling pathway STAT3 in T-cells, but not NF-kappaB and ERK1/2 in T- and B-cells was inhibited in a dose-dependent manner by Rosmarinus officinalis (0.2-6.2 mg/mL). Rosmanol, carnosolic acid, carnosol and trans-caffeic acid were tested in the same cellular models as the crude extract. From these, only trans-caffeic acid inhibited lymphocyte proliferation and STAT3 (30–100 μg/mL). Trans-caffeic acid was found in the extract in a concentration of 14.7 μg/mL. ConclusionsWe conclude that an immunosuppressive effect of Rosmarinus officinalis is mostly due to the effect of trans-caffeic acid. It results in inhibition of the activity of STAT3 causing induction of apoptosis and inhibition of proliferation of T-lymphocytes.

Extensive Proliferation of Cd4+ Lymphocyte by Both Phytohaemagglutinin A and Anti-Cd2/Cd3/ Cd28 Macsibeads

Abstract Background: Lymphocytes proliferate considerably following appropriate stimulation in vitro. Autologous T cells are obtained from whole blood or tissue sites in relatively limited amounts. We need a method to expand these cells efficiently, study their functions and manipulate them to create appropriate cells for transferring to the patient with infection and cancer. Objectives: The aim of this study is to determine proliferation ability of two different stimulators on CD4+ lymphocytes. Methods: Lymphocytes were isolated from blood samples of healthy donors after removing adherent cells (monocytes).The efficacy of MACSiBead™ coated with anti-CD2, anti-CD3, anti-CD28 (anti-CD2/CD3/CD28) was compared with Phytohaemagglutinin A (PHA) on CD4+ lymphocytes proliferation using carboxyfluorescein diacetate succinimidyl ester (CFSE) in cell culture media. The percentage of proliferating cells was analyzed using flow cytometry. Results: Both stimulators induced extensive proliferation of CD4+ lymphocytes but proliferation ability of PHA was higher compared to stimulation by anti-CD2/CD3/CD28 MACSiBead™. The proliferation rate of cells stimulated by PHA was 93.8% ± 3.37% whereas it was 85.2% ± 4.7% in cells stimulated by anti-CD2/CD3/CD28 MACSiBead™. Conclusions: Our results show that MACSiBead™ along with PHA can be used to obtain a large number of expanded CD4+ lymphocytes.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis

BackgroundSystemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development.MethodsTwenty-four patients with RA, 23 individuals positive for autoantibodies but without clinical disease (RA risk group) and 14 seronegative healthy control subjects underwent ultrasound-guided inguinal lymph node (LN) biopsy. Human LNSCs were isolated and expanded in vitro for functional analyses. In analogous co-cultures consisting of LNSCs and peripheral blood mononuclear cells, αCD3/αCD28-induced T-cell proliferation was measured using carboxyfluorescein diacetate succinimidyl ester dilution.ResultsFibroblast-like cells expanded from the LN biopsy comprised of fibroblastic reticular cells (gp38+CD31−) and double-negative (gp38−CD31−) cells. Cultured LNSCs stably expressed characteristic adhesion molecules and cytokines. Basal expression of C-X-C motif chemokine ligand 12 (CXCL12) was lower in LNSCs from RA risk individuals than in those from healthy control subjects. Key LN chemokines C-C motif chemokine ligand (CCL19), CCL21 and CXCL13 were induced in LNSCs upon stimulation with tumour necrosis factor-α and lymphotoxin α1β2, but to a lesser extent in LNSCs from patients with RA. The effect of human LNSCs on T-cell proliferation was ratio-dependent and altered in RA LNSCs.ConclusionsOverall, we developed an experimental model to facilitate research on the role of LNSCs during the earliest phases of RA. Using this innovative model, we show, for the first time to our knowledge, that the LN stromal environment is changed during the earliest phases of RA, probably contributing to deregulated immune responses early in disease pathogenesis.

CD18-mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles

Extracellular vesicles are submicron vesicles that upregulate the synthesis of proinflammatory mediators by lung epithelial cells.We investigated whether these structures adhere to lung epithelial cells, and whether adhesion is a prerequisite for their proinflammatory activity.Extracellular vesicles were generated by stimulation of normal human mononuclear cells with the calcium ionophore A23187, and labelled with carboxyfluorescein diacetate succinimidyl ester. Adhesion of vesicles to monolayers of immortalized bronchial epithelial (16HBE) and alveolar (A549) cells was analyzed by fluorescence microscopy. The role of candidate adhesion receptors was evaluated with inhibitory monoclonal antibodies and soluble peptides. The synthesis of proinflammatory mediators was assessed by ELISA.Transmission electron microscopy confirmed the generation of closed vesicles with an approximate size range between 50 and 600 nm. Adhesion of extracellular vesicles to epithelial cells was upregulated upon stimulation of the latter with tumor necrosis factor-α. Adhesion was blocked by an anti-CD18 antibody, by peptides containing the sequence RGD and, to a lesser extent, by an antibody to ICAM-1. The same molecules also blocked the upregulation of the synthesis of interleukin-8 and monocyte chemotactic protein-1 induced by extracellular vesicles.CD18-mediated adhesion of extracellular vesicles is a prerequisite for their proinflammatory activity.

Effect of the novel immunomodulator composed of muramyl dipeptide and anti-CD10 monoclonal antibody on dendritic cells in children with acute leukemia

Objective To study the effect of a new immunomodulator composed of muramyl dipeptide(MDP) and anti-CD10 monoclonal antibody (MDP-Ab) on the dendritic cells(DC) of children with acute leukemia. Methods DC was adopted to divide the children with acute lymphoblastic leukemia into 6 groups, including the control group, unconjugated anti-CD10 alone, unconjugated MDP alone, MDP-Ab alone, lipopolysaccharide (LPS) alone and MDP-Ab + LPS.The immunophenotypes, the endocytosis interleukin-12 (IL-12) were detected.The stimulation index of autologous lymphocytes was assayed by adopting 5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester(CFSE)-staining method.The supernatants of DC and autologous lymphocytes were used to detect the level of interferon-γ(IFN-γ) by using enzyme-linked immunosorbent assay. Results (1) DC immunophenotype: The expressions of human leukocyte antigen-DR(HLA-DR), mature molecule (CD83) and co-stimulatory molecules (CD80 and CD86) were increased significantly upon DC triggered with MDP-Ab, compared with the control group, unconjugated anti-CD10 group, and unconjugated MDP group, but lower than those in LPS and combination of MDP-Ab with LPS(F=629.62, P=0.000). (2) The level of IL-12: a significant increase in IL-12 level was detected in MDP-Ab group, LPS group, and combination of MDP-Ab with LPS group, compared with the control group, unconjugated anti-CD10 group, and unconjugated MDP group (F=857.87, P=0.000). There were significant differences among the first three groups. (3) Endocytosis assay: The uptake of DCs stimulated by unconjugated anti-CD10, unconjugated MDP, MDP-Ab immunoconjugate, LPS or combination was lower than that of immature DC in the control group which was (81.3±10.1)%. (4)Mixed lymphocyte reaction and IFN-γ level: DC, treated with MDP-Ab, LPS and combination, stimulated more CFSE positive cells and higher level of IFN-γ secretion than the control group and unconjugated anti-CD10 group, unconjugated MDP group. The most significance was observed in combination of MDP-Ab with LPS(F=393.36, P=0.000; F=2 497.18, P=0.000). Conclusion It is concluded that MDP-Ab could promote the proliferation and maturation of DC derived from blood of children with acute leukemia. Key words: Cancer vaccine; Anti-CD10 monoclonal antibody; Muramyl dipeptide; Dendritic cells; Leukemia; Child

The Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis in Intestinal Epithelial Cells Regulates Intestinal Barrier Function During Intestinal Epithelial Cells–CD4+T-Cell Interactions

Background/Aims: Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function. Methods: In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2–/– mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system. Results: The intestinal mucosal barrier damage in S1PR2–/– mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2–/– mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression. Conclusions: The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.

The Therapeutic Effect of ICAM-1-Overexpressing Mesenchymal Stem Cells on Acute Graft-Versus-Host Disease

Background/Aims: Mesenchymal stem cells (MSCs) do not readily migrate to appropriate sites, and this creates a major obstacle for their use in the treatment of graft-versus-host disease (GVHD). Intercellular adhesion molecule-1 (ICAM-1) can guide the homing of various immune cells to the proper anatomical location within secondary lymphoid organs (SLOs), which are the major niches for generating immune responses or tolerance. MSCs rarely migrate to SLOs after intravenous infusion, and are constitutively low expression of ICAM-1. So in our previous work, ICAM-1 was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (ICAM-1MSCs). Here, we hypothesized that ICAM-1^highMSCs may significantly improve their immunomodulatory effect. Methods: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate ICAM-1^highMSCs immunomodulatory effect on dendritic cells (DCs) and T cells in vitro and in vivo. MSCs were labeled with carboxyfluorescein diacetate succinimidylester (CFSE) to detect its distribution in mouse model. Results: Our in vitro analyses revealed ICAM-1 MSCs could suppress DCs maturation according to co-culture methods and suppress the T cell immune response according to the mixed lymphocyte response (MLR) and lymphoblast transformation test (LTT) tests. We found that infusion of ICAM-1^highMSCs potently prolonged the survival of GVHD mouse model. The infused ICAM-1^highMSCs migrate to SLOs in vivo, and suppressed DCs maturation, suppressed CD4+ T cell differentiation to Th1 cells, and increased the ratios of Treg cells. Conclusions: Taken together, these data demonstrate that ICAM-1^highMSCs had an enhanced immunosuppressive effect on DCs and T cells, which may help explain the protective effect in a GVHD model. This exciting therapeutic strategy may improve the clinical efficacy of MSC-based therapy for GVHD.

Effects of Birch Polypore Mushroom, Piptoporus betulinus (Agaricomycetes), the "Iceman's Fungus", on Human Immune Cells.

Piptoporus betulinus, the mushroom that has been carried by Ötzi the "Iceman", has a long tradition of use in medicinal practice for its antiseptic, anticancer, and immune-enhancing properties. With this study, we aimed to investigate the immunomodulatory effects of P. betulinus on primary human immunocompetent cells. The influence of P. betulinus water and methanol extract on apoptosis and necrosis induction of T cells and monocytes was analyzed using annexin V/propidium iodide staining and proliferation of T cells by carboxyfluorescein diacetate succinimidyl ester staining using flow cytometry. The effects on T-cell activation (CD69/CD25) and dendritic cell maturation (CD83, CD86, and CD14) were assessed using flow cytometric analysis of distinct marker expression. Alterations of the secretion of effector mediators interferon (IFN)-γ by T cells and interleukin (IL)-8 by monocytes and dendritic cells were detected by enzyme-linked immunosorbent assay. None of the P. betulinus extracts had a significant influence on apoptosis and necrosis induction, T-cell proliferation, or T-cell activation status, but P. betulinus water extract caused a strong increase in IFN-γ release. The same extract was slightly protective against apoptosis of monocytes and further triggered IL-8 secretion by monocytes and dendritic cells. Moreover, P. betulinus water extract induced dendritic cell maturation. Our results demonstrate the immune-enhancing properties of P. betulinus.

Rapid flow cytometry-based assay for the evaluation of γδ T cell-mediated cytotoxicity.

The effector function of natural killer, lymphokine-activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium-release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell-mediated cytotoxicity. In the present study, target cells were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP-1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co-culture between γδ T cells and CFSE-labeled infected or uninfected THP-1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE-gated cell population was calculated by subtracting the value obtained for non-specific PI-positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two-color flow cytometric assay. In conclusion, the flow cytometric-based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.

Superior properties of CellTrace Yellow™ as a division tracking dye for human and murine lymphocytes.

The discovery of cell division tracking properties of 5‐(and‐6)‐carboxyfluorescein diacetate succinimidyl ester (CFSE) by Lyons and Parish in 1994 led to a broad range of new methods and numerous important biological discoveries. After labeling, CFSE is attached to free amine groups and intracellular proteins in the cytoplasm and nucleus of a cell, and halves in fluorescence intensity with each round of cell division, enabling enumeration of the number of divisions a cell has undergone. A range of popular division tracking dyes were subsequently developed, including CellTrace Violet (CTV), making available the green fluorescent channel previously occupied by CFSE. More recently, CellTrace Yellow (CTY) and CellTrace Far Red (CTFR), each with unique fluorescence properties, were introduced. In a comparison, we found that the fluorescence values of both dyes were well separated from autofluorescence, and enabled a greater number of divisions to be identified than CTV, before this limit was reached. These new dyes provided clear and well‐separated peaks for both murine and human B lymphocytes, and should find wide application. The range of excitation/emission spectra available for division tracking dyes now also facilitates multiplexing, that is, the labeling of cells with different combinations of dyes to give a unique fluorescence signature, allowing single cell in vitro and in vivo tracking. The combinatorial possibilities are significantly increased with these additional dyes.

Interleukin-35 mitigates the function of murine transplanted islet cells via regulation of Treg/Th17 ratio.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes (T1D). Interleukin-35 (IL-35) is a recently discovered cytokine that exhibits potent immunosuppressive functions. However, the role of IL-35 in islet transplant rejection remains to be elucidated. In this study, we isolated islet cells of BALB/c mouse and purified CD4+ T cell subsets of a C57BL/6 mouse. The model for islet transplantation was established in vitro by co-culture of the islet cells and CD4+ T cells. IL-35 (20 ng/ml) was administered every other day. Following co-culture, the islet function and Treg/Th17 ratio were analyzed on days 1, 3, and 5. Furthermore, the Th17/Treg ratio was modulated (1:0–2), and the function of islet cells as well as proliferation of Th17 cells were analyzed. T cell sorting was performed using the magnetic bead sorting method; Treg and Th17 count using flow cytometry; cell proliferation detection using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method, and islet function test using the sugar stimulation test. Results showed that Th17 counts increased in the co-culture system. However, after administration of IL-35, the number of Treg cells increased significantly compared to that in the control group (50.7% of total CD4+ T cells on day 5 in IL-35 group vs. 9.5% in control group) whereas the proliferation rate of Th17 cells was significantly inhibited (0.3% in IL-35 group vs. 7.2% in control group on day 5). Reducing the Th17/Treg ratio significantly improved the function of transplanted islets. Treg inhibited Th17 proliferation and IL-35 enhanced this inhibitory effect. IL-35 mitigates the function of murine transplanted islet cells via regulation of the Treg/Th17 ratio. This might serve as a potential therapeutic strategy for in-vivo islet transplant rejection and T1D.

A Reliable and Non-destructive Method for Monitoring the Stromal pH in Isolated Chloroplasts Using a Fluorescent pH Probe.

The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpHenv), whether the concentration of ionophores used can effectively abolish the ΔpHenv is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpHenv can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r-square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpHenv can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin required to collapse the ΔpHenv was then studied. The establishment of a non-destructive method of monitoring the stromal pH will be valuable for studying the roles of the ΔpHenv in chloroplast physiology.

Regulatory effect of decitabine on human acute myeloid leukemia cell line HL-60 and its mechanism

Objective To investigate the effect of decitabine (DAC) on human acute myeloid leukemia (AML) cell line HL-60 and the regulating of natural killer (NK) cell activating receptor (NKG2D) ligands (NKG2DL), and to detect the molecular mechanism of JAK-STAT3-SOCS signaling pathway. Methods The effect of DAC on the proliferation of HL-60 was detected by using CCK-8 assay. The cell apoptosis was analyzed by using Annexin-V/PI double standard method. The expressions of receptor NKG2DL including MICA/B and ULBPs in HL-60 cells were detected by using flow cytometry (FCM). The killing activity of NK cells was analyzed by using carboxy fluorescein diacetate succinimidyl ester (CFSE). The expressions of JAK/STAT3 signaling pathway or molecules including STAT3, its upstream kinases JAK1, JAK2 and the negative regulator of STAT3, SOCS-1, SOCS-3 were examined by Western blot. Methylation level of the SOCS-1, SOCS-3 gene after the treatment of DAC was analyzed by using methylation-sensitive high resolution melting (MS-HRM). Results There was an obvious inhibitory effect of DAC on HL-60 cells. The cell viability of HL-60 treated with 0.2, 0.5, and 1.0 μmol/L DAC for 48 h was decreased by (25±11) %, (39±8) % and (50±7) % (P < 0.01) respectively compared with those cells without DAC treatment. The incidence of apoptosis was (24.77±7.50) %, (27.10±4.48) % and (30.53±3.93) % after DAC treatment for 48h respectively, which were higher than that of untreated cells [(3.11±0.50) %] (P < 0.01). DAC induced a significant up-regulation of MICA/B, ULBP-1, ULBP-3 in HL-60 cells, and enhanced the sensitivity of HL-60 cells to NK cytotoxicity. Western blot results showed that a down-regulating expression of STAT3 and JAK1, JAK2 protein was detected, in addition to the phosphor-STAT3 and phosphor-JAKs in HL-60 cells after DAC treatment, but the expressions of SOCS-1 and SOCS-3 protein were increased. HRM results showed that DAC could inhibit the methylation of SOCS-3 gene. Conclusion DAC can inhibit the proliferation of HL-60 cells, upregulate the expression of NKG2DL and enhance the cytotoxicity of NK targeted to HL-60 cells, which might be related to the activity regulation of intracellular JAK-STAT3-SOCS signaling pathway. Key words: Leukemia, myeloid, acute; Cell proliferation; Apoptosis; Molecular mechanisms of action; Decitabine

Chloroform extract of Hedyotis diffusa Willd inhibits viability of human colorectal cancer cells via suppression of AKT and ERK signaling pathways.

Hedyotis diffusa Willd (HDW) is a widely used traditional Chinese medicine in clinical therapy to treat various types of cancer, including colorectal cancer (CRC), but its effective polar fractions and functional mechanisms remain unclear. The aim of the present study was to determine the most effective extract of HDW and to investigate its effects on the regulation of CRC cell proliferation and apoptosis, as well as to investigate the underlying molecular mechanisms. The results demonstrated that the chloroform extract of HDW (CEHDW) exhibited the most anticancer ability. Furthermore, results of the MTT assay, colony formation, carboxyfluorescein diacetate succinimidyl ester assay and annexin V/propidium iodide staining suggested that CEHDW significantly inhibits proliferation and promotes apoptosis in the SW620 CRC cell line. Additionally, reverse transcription-polymerase chain reaction and western blot analysis demonstrated that CEHDW treatment downregulated the expression of Survivin, proliferating cell nuclear antigen, Cyclin D1, cyclin-dependent kinase 4 and B-cell lymphoma 2 (Bcl-2), and upregulated the expression of Bcl-2-associated X protein at the mRNA and protein levels. CEHDW also decreased the phosphorylation of protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK), which indicated that the suppression of the AKT and ERK signaling pathways may be one of the underlying molecular mechanisms by which CEHDW exhibited its anticancer effect. Thus, CEHDW may be a promising agent for anticancer therapy.

Migration of normal stem CD34 + cells to the brain in experimental models of ischemic stroke and glioblastoma

Background: Glyoblastoma is one of the most common brain tumors, which is characterized by high aggressiveness and unfavorable prognosis. One of the leading causes of the therapeutic resistance of glioblastoma is associated with tumor stem cells (TSC). One of the promising ways to solve this problem is the creation of biomedical products based on stem and progenitor cells of the human bone marrow. We have established, in vitro, that the stem CD34 + cells of the bone marrow are able to migrate toward the cells of different types. In vivo, stem cells of bone marrow CD 34+ actively migrate to the tumor tissue, where they accumulate in the area of invasion and penetrate into the hypoxic zones of the tumor. We suggested that the appearance of CD34 + cells in the neoplastic tissue is associated with the processes of neoplastic angiogenesis, and the role of hypoxia in this case is secondary. The study of migration processes and the homing of stem cells in the pathological focus in the brain on experimental models of acute ischemia and glioblastoma. Methods: The work was performed on 60 mature Wistar female rats divided into 2 groups (N = 30). The animals of the first group underwent stereotactic implantation of C6 glioma cells in the brain, tumor development was confirmed by magnetic resonance imaging (MRI) data; the second group - the ligation of the common and internal carotid artery on the left was performed surgically. The development of the stroke was confirmed by MRI data. The haploidentical CD34 + stem cells stained with the fluorescent vital dye Vybrant® CFDA SE (V12883, Molecular Probes, USA) were injected into the tail vein. The animals were removed from the experiment on days 4 and 8, the material was subjected to morphological and immunohistochemical analysis. Results: Four days after the infusion of cells on the glioblastoma model, the fluorescent label was detected at the periphery of the tumor site, which was associated with localized blood vessels in this region. On day 8, the area of fluorescing objects was 12.7 ± 9.2% at the tumor boundary, 47.6 ± 9.3% in the parenchyma of the tumor tissue. In the experimental model of ischemia, fluorescent substances in the spectrum of characteristic CFDA on days 4 and 8 were not detected. Conclusions: Hypoxic brain damage is not enough to ensure the effective homing of cells in the tumor. With a glioblastome formed a specific type of microglia, this circumstance allows one to assume the possibility of increasing the life expectancy of patients with glioblastoma. Legal entity responsible for the study: School of Biomedicine, Far Eastern Federal University, Russian Federation Funding: Russian Scientific Fund (14-15-00084) Disclosure: All authors have declared no conflicts of interest.