Carboxyfluorescein Diacetate Succinimidyl Ester Research Articles

210 Horse allogeneic mesenchymal stem cells perform homing and ameliorate endometrial inflammation after induced endometritis of mares

Post-mating induced endometritis (PMIE) is an acute inflammatory response of the endometrium to spermatozoa, linked to an incapability of some mares to drain out the fluids associated with inflammation. This is of pivotal importance for reproductive success in mares. Mesenchymal stem cells (MSCs) are potential candidates for anti-inflammatory uterine therapies. Here, we aimed to study inflammatory markers in the endometrium of healthy mares and of those with induced endometritis, before and after intrauterine inoculation of MSCs, and to characterise their homing potential invivo in an induced endometritis horse model. Nine mares during their ovulatory season were selected after gynaecologic examination (absence of free liquid in the uterus, no polymorphonuclear leucocytes (PMNs) at cytology, negative bacteriology, and grade I in Kenney's scale on uterine biopsies). Mares were infused in the uterine body with 2mL of 500×106 spermmL−1 previously killed by repeated frozen-thawing cycles. At 3h, uteri were flushed with 250mL of sterile saline and the inflammatory response was monitored in the lavages and biopsies. Parameters measured included cytology, protein expression of inflammatory markers (supernatant) after lavage centrifugation (800×g, 10min), ELISA, and immunostaining for interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). The mares were divided into three groups (3 mares each). Then, 24h after dead sperm challenge, group 1 received intrauterine infusion of 2×107 adipose MSC in 0.9% sterile saline; group 2, received the same amount of endometrial MSCs in the same vehicle; and group 3 received only saline. The volume of infusion in the uterine body was 20mL for all groups. Cells (passage 4) were previously labelled with 10μM Vybrant CFDA SE Cell Tracer Kit (ThermoFisher Scientific). After 48h, the same lavages, biopsies, and measurements as described above were performed. Additional biopsies were taken at Days 10 and 30 after intrauterine infusions. Biopsies were split in two, one for confocal microscopy and the other for quantitative PCR. Endometritis was induced in all mares, as judged by cytology and expression of protein markers of inflammation. After 48h, reduction in IL-6 and TNFα was detected by immunostaining of biopsies and confirmed by ELISA in the lavages, as well as by PCR. Homing was detected in all mares infused with MSC and it persisted at Days 10 and 30 after infusion. No homing was found in the control mares. As a result of these experiments, we conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells (adipose or endometrial). Both types of cells were nested in the endometrium at low quantities, although the number of cells actually detected at fixed time points was not quantified. Overall, we can propose that, given the number of homed cells detected and the marked decrease in inflammatory markers after inoculation of cells, MSCs exert their anti-inflammatory function preferentially by a paracrine mechanism and not necessarily by nesting and proliferation, although both events occur. Funding for this study was provided by Fondecyt 1150757.

Hapalindoles from the Cyanobacterium Hapalosiphon sp. Inhibit T Cell Proliferation.

Novel immunomodulating agents are currently sought after for the treatment of autoimmune diseases and cancers. In this context, a screening campaign of a collection of 575 cyanobacteria extracts for immunomodulatory effects has been conducted. The screening resulted in several active extracts. Here we report the results of subsequent studies on an extract from the cyanobacterium Hapalosiphon sp. CBT1235. We identified 5 hapalindoles as the compounds responsible for the observed immunomodulatory effect. These indole alkaloids are produced by several strains of the cyanobacterial family Hapalosiphonaceae. They are known for their anti-infective, cytotoxic, and other bioactivities. Modulation of the activity of human immune cells has not yet been described. The immunomodulatory activity of the hapalindoles was characterized in vitro using flow cytometry-based measurements of T cell proliferation after carboxyfluorescein diacetate succinimidyl ester staining, and apoptosis and necrosis induction after annexin V/propidium iodide staining. The most potent compound, hapalindole A, reduced T cell proliferation with an IC50 of 1.56 µM, while relevant levels of apoptosis were measurable only at 10-fold higher concentrations. Hapalindole A-formamide and hapalindole J-formamide, isolated for the first time from a natural source, had much lower activity than the nonformylated derivatives while, at the same time, being less selective for antiproliferative over apoptotic effects.

Successful Treatment of Chronic Myeloid Leukemia With Dasatinib After Kidney Transplantation: A Case Report

ObjectiveChronic myeloid leukemia (CML) is a rare malignancy in kidney transplant (KT) recipients. Although dasatinib is the first-line treatment for CML, it has inhibitory activity against CYP3A4; this might increase the blood concentration of tacrolimus (administered to KT patients for immune suppression). Furthermore, tacrolimus can also increase blood concentrations of dasatinib through P-glycoprotein inhibition. MethodsHere, we report a case of sustained molecular remission of CML with prolonged first-line dasatinib therapy in a KT recipient being treated with tacrolimus. A 61-year-old woman developed CML-chronic phase (CML-CP) 38 months post KT. Her maintenance immunosuppressive therapy consisted of tacrolimus, mycophenolate mofetil, and methylprednisolone. Considering the potential drug interaction with tacrolimus, dasatinib was administered at a low dose of 50 mg/day. Her immune status was evaluated regularly by assessing the mixed lymphocyte reaction (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique; immunosuppressive therapy was adjusted accordingly. ResultsThe patient achieved complete hematologic remission (CHR) after 1 month of dasatinib treatment. Six months after dasatinib treatment, she achieved a major molecular response. During the observation period, neither antibody-mediated nor acute cellular rejection were encountered in the patient. She remained in CHR with a major molecular response 12 months after the diagnosis of CML-CP. ConclusionData obtained from immune monitoring assays using CFSE-MLR helped us to successfully manage a KT recipient with CML-CP being treated with dasatinib. Drug-drug interactions are a key consideration while designing treatment regimens; such strategies would ensure that drug-drug interactions do not negatively affect the treatment outcomes.

Abstract 1421: Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells. METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo. CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC. Citation Format: Teruki Shimizu, Mako Tomogane, Yusuke Sano, Daiki Shimizu, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara. Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1421.

LncRNA KCNQ1OT1 delayed fracture healing through the Wnt/β-catenin pathway.

The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) on fracture healing and its possible mechanism. Abnormal lncRNAs were compared between patients with delayed fracture healing and those with normal fracture healing using gene expression profiling method. LncRNA expression in patients was verified by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Subsequently, the model of tibial fracture was successfully established in rabbits. The effect of lncRNA KCNQ1OT1 expression on tibial fracture healing in rabbits was explored. Meanwhile, the effects of lncRNA KCNQ1OT1 on cell proliferation and apoptosis were investigated by knockdown and overexpression experiments with HC-a as a cell model. Furthermore, Western blotting was used to explore the expressions of proteins in signaling pathway affected by lncRNA KCNQ1OT1. Gene expression profiling and qRT-PCR revealed that lncRNA KCNQ1OT1 was significantly down-regulated in bone tissues of patients with delayed fracture healing. Compared with the control group, knocking down lncRNA KCNQ1OT1 remarkably reduced the serum levels of alkaline phosphatase (ALP) and osteoprotegerin (OPG) in rabbits, and markedly decreased bone trabecular growth index (p<0.05). In HC-a cells, overexpression of lncRNA KCNQ1OT1 activated the Wnt/β-catenin signaling pathway, which could be suppressed by knocking down lncRNA KCNQ1OT1. Cell Counting Kit-8 (CCK-8) assay and 5(6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) results manifested that lncRNA KCNQ1OT1 remarkably promoted the proliferation and inhibited apoptosis of HC-a cells by activating the Wnt/β-catenin signaling pathway. LncRNA KCNQ1OT1 plays a vital role in delayed fracture healing. Moreover, it can induce cell proliferation and inhibit cell apoptosis by activating the Wnt/β-catenin signaling pathway. Therefore, KCNQ1OT1 may be used as a biomarker to predict the occurrence of delayed fracture healing.

Effects of intrinsic microbial stress factors on viability and physiological condition of yeasts isolated from spontaneously fermented cereal doughs

Fermented cereal doughs constitute a predominant part of West African diets. The environment of fermented doughs can be hostile for microbial survival due to high levels of microbial metabolites such as weak carboxylic organic acids and ethanol. In order to get a better understanding of the intrinsic factors affecting the microbial successions of yeasts during dough fermentation, survival and physiological responses of the yeasts associated with West African fermented cereal doughs were investigated at exposure to relevant concentrations of microbial inhibitory compounds. Three strains each of the predominant species, i.e. Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kudriavzevii as well as the opportunistic pathogen Candida glabrata were studied. The strains were exposed to individual stress factors of cereal doughs, i.e. (i) pH 3.4, (ii) 3% (v/v) ethanol (EtOHpH3.4), (iii) 285 mM lactic acid (LApH3.4) and (iv) 150 mM acetic acid (AApH3.4) as well as to combinations of these stress factors, i.e. (v) (LA + AA)pH 3.4 and (vi) (LA + AA+EtOH)pH 3.4. Growth and single cell viability were studied by flow cytometry using combined SYTO 13 and propidium iodide (PI) staining. Intracellular pH (pHi), plasma membrane integrity and micro-colony development of stressed cells were studied by fluorescence microscopy using PI and carboxyfluorescein diacetate succinimidyl ester (CFDA-se).Viability of the yeast strains was not affected by pH 3.4 and 3% (v/v) ethanol (EtOHpH3.4). 285 mM lactic acid (LApH3.4) reduced the specific growth rate (μmax) from 0.27-0.41 h−1 to 0.11–0.26 h−1 and the viability from 100% to 2.6–41.7% at 72 h of exposure in most yeast strains, except for two strains of C. glabrata. 150 mM acetic acid (AApH3.4) as well as the combinations (LA + AA)pH 3.4 and (LA + AA+EtOH)pH 3.4 reduced μmax to 0.0 h−1 and induced significant cell death for all the yeast strains. Exposed to (LA + AA+EtOH)pH 3.4, the most resistant yeast strains belonged to S. cerevisiae followed by P. kudriavzevii, whereas C. glabrata and K. marxianus were more sensitive. Strain variations were observed within all four species. When transferred to non-stress conditions, i.e. MYGP, pH 5.6, after exposure to (LA + AA+EtOH)pH 3.4 for 6 h, 45% of the single cells of the most resistant S. cerevisiae strain kept their plasma membrane integrity, recovered their pHi to near physiological range (pHi = 6.1–7.4) and resumed proliferation after 3–24 h of lag phase. The results obtained are valuable in order to change processing conditions of the dough to favor the survival of preferable yeast species, i.e. S. cerevisiae and K. marxianus and inhibit opportunistic pathogen yeast species as C. glabrata.

Flavonoids kaempferide and 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells

ObjectiveKaempferide and 4,2′-dihydroxy-4′,5′,6′-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odorata Linn. leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated. MethodsConfluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of differentiation. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins α were assessed using AdipoRed, Oil red O and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively. ResultsKaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis. ConclusionKaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.

Solamargine inhibits proliferation and promotes apoptosis of CM-319 human chordoma cells through suppression of notch pathway.

BackgroundSolamargine (SM), which represents a natural steroid alkaloid glycoside compound and a cytotoxic agent, has been proved to enhance the sensitivity of lung cancer cells to tumor necrosis factors (TNFs). In this study, we aimed to investigate the roles and mechanisms of SM in chordoma.MethodsCell viability, proliferation, apoptosis and cell cycle were measured by cell counting Kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry (FCM), respectively. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed to detect the expressions of related mRNAs and proteins.ResultsThe results revealed that SM distinctly suppressed the proliferation of CM-319 cells. SM significantly induced the CM-319 cells apoptosis through up-regulating the expression levels of Caspase-3/8/9. The cell cycle of CM-319 cells was blocked by SM in G1 phase. Moreover, SM could significantly suppress the Notch pathway in CM-319 cells.ConclusionsIn conclusion, SM suppressed the proliferation and enhanced the apoptosis ability of CM-319 cells via suppressing the Notch pathway. The results suggested that SM might be a novel therapeutic agent and supported the utilization of SM in chordoma.

Solamargine inhibits proliferation and promotes apoptosis of CM-319 human chordoma cells through suppression of notch pathway

Background: Solamargine (SM), which represents a natural steroid alkaloid glycoside compound and a cytotoxic agent, has been proved to enhance the sensitivity of lung cancer cells to tumor necrosis factors (TNFs). In this study, we aimed to investigate the roles and mechanisms of SM in chordoma. Methods: Cell viability, proliferation, apoptosis and cell cycle were measured by cell counting Kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and flow cytometry (FCM), respectively. Western blot and quantitative real-time reverse transcription PCR (qRT-PCR) assays were performed to detect the expressions of related mRNAs and proteins. Results: The results revealed that SM distinctly suppressed the proliferation of CM-319 cells. SM significantly induced the CM-319 cells apoptosis through up-regulating the expression levels of Caspase-3/8/9. The cell cycle of CM-319 cells was blocked by SM in G1 phase. Moreover, SM could significantly suppress the Notch pathway in CM-319 cells. Conclusions: In conclusion, SM suppressed the proliferation and enhanced the apoptosis ability of CM-319 cells via suppressing the Notch pathway. The results suggested that SM might be a novel therapeutic agent and supported the utilization of SM in chordoma.

In vivo fluorescent cercariae reveal the entry portals of Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Strigeidae) into the gilthead seabream Sparus aurata L.

BackgroundDespite their complex life-cycles involving various types of hosts and free-living stages, digenean trematodes are becoming recurrent model systems. The infection and penetration strategy of the larval stages, i.e. cercariae, into the fish host is poorly understood and information regarding their entry portals is not well-known for most species. Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Digenea, Strigeidae) uses the gilthead seabream (Sparus aurata L.), an important marine fish in Mediterranean aquaculture, as a second intermediate host, where they encyst in the brain as metacercariae. Labelling the cercariae with in vivo fluorescent dyes helped us to track their entry into the fish, revealing the penetration pattern that C. longicollis uses to infect S. aurata.MethodsTwo different fluorescent dyes were used: carboxyfluorescein diacetate succinimidyl ester (CFSE) and Hoechst 33342 (NB). Three ascending concentrations of each dye were tested to detect any effect on labelled cercarial performance, by recording their survival for the first 5 h post-labelling (hpl) and 24 hpl, as well as their activity for 5 hpl. Labelled cercariae were used to track the penetration points into fish, and cercarial infectivity and later encystment were analysed by recording brain-encysted metacercariae in fish infected with labelled and control cercariae after 20 days of infection.ResultsAlthough the different dye concentrations showed diverse effects on both survival and activity, intermediate doses of CFSE did not show any short-term effect on survival, permitting a brighter and longer recognition of cercariae on the host body surface. Therefore, CFSE helped to determine the penetration points of C. longicollis into the fish, denoting their aggregation on the head, eye and gills region, as well as on the dorsal fin and the lower side. Only CFSE-labelled cercariae showed a decreased number of encysted metacercariae when compared to control.ConclusionsOur study suggests that CFSE is an adequate labelling method for short-term in vivo studies, whereas NB would better suit in vivo studies on long-term performance. Cardiocephaloides longicollis cercariae seem to be attracted to areas near to the brain or those that are likely to be connected to migration routes to neuronal canals.

A Potent CD1d-binding Glycolipid for iNKT-Cell-based Therapy Against Human Breast Cancer.

Invariant natural killer T-cells (iNKT) stimulated by CD1d-binding glycolipids have been shown to exert antitumor effects by a number of studies in a mouse model. Breast cancer is a devastating disease, with different types of breast cancer recurring locally or distant as metastatic/advanced disease following initial treatment. The aim of this study was to examine the tumoricidal effect of a CD1d-binding glycolipid, called 7DW8-5, against a highly invasive human breast cancer cell line both in vitro and in vivo. Parental MDA-MB-231 cells and MDA-MB-231 cells transduced with human CD1d were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), followed by loading with glycolipids. After co-culturing with human iNKT cells, the cells were permeabilized and stained with Alexa Flour 647-conjugated antibody to active caspase-3, and analyzed using a BD LSR II. For the in vivo tumoricidal effect, MDA-MB-231 cells transduced with human CD1d and luciferase genes were injected into the mammary fat pad of female NOD/SCID/IL2rγnull (NSG) mice, followed by the injection of human iNKT cells with or without 7DW8-5, and the levels of luminescence were analyzed with whole-body imaging. Human iNKT cells could kill CD1d-expressing human breast cancer cells in vitro in the presence of 7DW8-5, but not α-GalCer. As for in vivo, the adoptive transfer of human iNKT cells into tumor-challenged NSG mice significantly inhibited the growth of CD1d+ MDA-MB-231 human breast cancer cells in the presence of 7DW8-5. CD1d-binding, glycolipid-based iNKT-cell therapy is suggested as a potent and effective treatment against breast cancer in humans.

The prognostic value of Tiam1 correlates with its roles in epithelial-mesenchymal transition progression and angiogenesis in lung adenocarcinoma.

BackgroundTiam1 has been identified as an oncogene and acts as an activator of GTPase Rac. Tiam1 was reported to be a promoter of cancer progression in various cancer types, while in lung adenocarcinoma, its mechanism of action is poorly understood.Materials and MethodsImmunohistochemistry staining and Western blot assay were used to determine Tiam1 expression in lung adenocarcinoma tissues, and its association with prognosis was determined by statistical analysis. We depleted Tiam1 in both A549 and H1975 cancer cell lines. Carboxyfluorescein diacetate succinimidyl ester staining and colony formation assays were used to evaluate its impact on cell proliferation ability after depletion. Transwell migration assay and wound healing assays were performed to determine its impact on migration ability of both cell lines. Western blot assay and immunofluorescence staining were used to analyze the association between Tiam1 and epithelial–mesenchymal transition (EMT) progression. Tube formation assay and vasculogenic mimicry assay were used to show the impact of Tiam1 depletion on cancer angiogenesis.ResultsIn this study, we demonstrated that Tiam1 overexpression in lung adenocarcinoma was significantly associated with advanced tumor grade and poor prognosis. In vitro assays indicated that Tiam1 depletion significantly inhibited cell proliferation, colony formation, and migration capacities in A549 and H1975 cells. Further investigations revealed that Tiam1 plays an important role in EMT program enhancement, angiogenesis, and accelerated tumor progression. Notably, Tiam1 depletion in cancer cells strongly inhibited human umbilical vein endothelial cell angiogenesis and vasculogenic mimicry capacities of both cancer cell lines.ConclusionTiam1 overexpression is associated with lung adenocarcinoma progression and may indicate poor prognosis. Tiam1 accelerated tumor progression due to EMT and angiogenesis enhancement. Our data may provide a novel therapeutic target for lung adenocarcinoma.

CFSE: A New Method for Identifying Human Limbal Stem Cells and Following Their Migration in Human Cornea.

To develop a method capable of identifying human corneal limbal stem cells (LSCs) and follow their proliferation and migration in the epithelium. Ten fresh matched pairs of cadaveric normal human corneas were obtained from donors. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used to target LSCs. The distribution of CFSE-positive cell clusters was analyzed by fluorescence microscopy by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Fluorescence was digitally recorded for seven days, and the rate of cell movement was determined. CFSE-labeled cells were tracked in corneas. Analysis of time sequences revealed that they moved centripetally. Daily average CFSE-labeled LSC movement was 0.073±0.01 cm (±SD). CFSE allowed us to identify LSCs and to track their centripetal migration from the limbal basal layer to the anterior ocular surface. This experimental system appears to be a valuable tool for further studies on corneal epithelial cell migration and proliferation.

Antigen-specific CD4+ and CD8+ T-cell responses in PBMC from pony mares immunized with either native or recombinant zona pellucida vaccines

Few studies have investigated the cell mediated immune response during zona pellucida-based immunocontraception, despite hypothesized cytotoxic T-cell involvement in ovarian dysfunction associated with these vaccines. This study aimed to investigate antigen-specific anamnestic responses of helper (CD4+) and cytotoxic (CD8+) T-lymphocytes in peripheral blood mononuclear cells (PBMC) isolated from pony mares before and after their treatment with native porcine zona pellucida (pZP), recombinant pZP3 and pZP4 antigens (reZP) or adjuvanted saline. Mares were randomly assigned to pZP, reZP and control groups (n = 7 per group). Treatments consisted of a primary vaccination or saline (V1; Day 0) incorporating Freund's modified complete adjuvant, followed by a single booster (V2; Day 35) incorporating Freund's incomplete adjuvant. Peripheral blood mononuclear cells were isolated and cryopreserved immediately prior to V1 (Day 0) and five weeks post V2 (Day 70). Relative proliferation of T-lymphocytes in response to pZP antigen was assessed using carboxyfluorescein diacetate succinimidyl ester dilution with immunophenotyping, analysed via flow cytometry. Significant pZP-specific CD4+ and CD8+ T-lymphocyte responses were detected in PBMC isolated from mares treated with either pZP or reZP, in comparison to pre-treatment samples. In the pZP group, but not the reZP group, CD8+ T-cell proliferation showed significant negative correlations to circulating progesterone, oestradiol and anti-Müllerian hormone levels. Results suggest that antigen-specific CD8+ T-cells may play a role in ovarian suppression observed during pZP immunocontraception in this species.

A Novel PAX5-KIDINS220 Fusion Protein Activates Multiple Signals Associated with Therapeutic Resistance

Background: It is well known that PAX5 related fusion proteins are mainly associated with leukemogenesis of B cell precursor acute lymphoblastic leukemia (B-ALL) through dominant negative effect against normal PAX5, resulting in impairment of B cell differentiation. However, different biological property of PAX5 fusion protein which is related to B-ALL has not been fully investigated. Here, we performed functional analysis of novel fusion protein, PAX5-KIDINS220 (P-K220) which was identified in pediatric Ph-like ALL patient. Methods: Full length of P-K220 fusion gene was cloned into pRetroX-Tight-Pur retroviral vector. We also established the PAX5-N construct that contained only PAX5 region (1-306 aa) of P-K220 so that we could analyze the importance of KIDINS220 in P-K220. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing P-K220 or PAX5-N under doxycycline (DOX) dependent manner. P-K220 was also cloned into pAcGFP1-C1 vector and pMSCVneo vector to perform localization assay and luciferase reporter assay. Gene expression analysis was performed using Mouse Genome 430 2.0 Array. In cytotoxic assay using co-culture system with murine mesenchymal stromal cell, MS-5, Ba/F3 cells that were pre-stained with Carboxyfluorescein diacetate succinimidyl ester (CFSE) were seeded on MS-5 stromal cell, then 10 nM vincristine (VCR) was applied. 72 hours later, flow cytometric analysis was performed to determine the fraction and proliferation ability of viable Ba/F3 cells. The proliferation ability was estimated by CFSE fluorescence that attenuates depending on cell proliferation. Results and discussions: P-K220 and PAX5-N were successfully expressed in Ba/F3 cells, confirmed by western blotting. Localization assay revealed that GFP tagged P-K220 was localized in a nucleus of HEK293-T cells under confocal microscopy, suggesting that P-K220 acted as a transcriptional factor. Luciferase reporter assay revealed that P-K220 protein inhibited PAX5 transcriptional activity in dominant negative fashion. Although P-K220 was identified in Ph-like ALL patient and activated JAK2-STAT5 pathway through reduction of Socs5 expression, Ba/F3 cells expressing P-K220 protein did not acquire IL-3 independency. P-K220 attenuated proliferation of Ba/F3 cells (p=0.02), which was in contrast to PAX5-JAK2. To reveal which pathways were affected by P-K220, gene expression analysis was performed. Gene set enrichment analysis revealed that multiple pathways related to chemotaxis, migration, such as IL-15 pathway, were activated in P-K220 expressing Ba/F3 cells. RQ-PCR and western blotting confirmed P-K220 induced expression of IL-15 in RNA (p=0.018) and protein level, suggesting that P-K220 might be associated with infiltration of leukemic cells into extramedullary site. Next, we examined whether P-K220 was associated with sensitivity of chemotherapeutic agents, such as vincristine (VCR), cytarabine, prednisolone, methotrexate, and 6-mercaptoprine. P-K220 expressing Ba/F3 cells showed higher VCR IC50 (0.4 nM vs 2.5 nM) and decreased Annexin V positive fraction under the condition of 10 nM VCR (39.4 % vs 23.8 %, p=0.04). In co-culture analysis with MS-5 stromal cell under the 10 nM VCR, the fraction of viable Ba/F3 cells that adhered to MS-5 was significantly increased with expression of P-K220 than PAX5-N or Dox (-) (11.6% vs 7.3% vs 6.8%, p<0.001), and proliferation ability was also significantly increased with expression of P-K220, that was estimated by mean CFSE fluorescence (479.6 vs 990.0 vs 909.7, p<0.001), suggesting P-K220 was associated with VCR resistance. Gene expression analysis and RQ-PCR revealed that P-K220 induced expression of Abcc5 which was reported to be associated with VCR resistance (p=0.003). Conclusion: P-K220 did not induce high proliferation of Ba/F3 cells even though identified in Ph-like ALL patient. However, P-K220 might be associated with treatment resistance through the infiltration of leukemic cells into extramedullary sites partly via activation of IL-15 pathway and resistance to VCR via induction of Abcc5. DisclosuresNo relevant conflicts of interest to declare.

Theaflavins attenuate ethanol‑induced oxidative stress and cell apoptosis in gastric mucosa epithelial cells via downregulation of the mitogen‑activated protein kinase pathway.

Ethanol-induced diseases of the gastric mucosa are the most common and refractory diseases of gastrointestinal system in clinic, and are mediated by oxidative stress and apoptosis pathways. Theaflavins (TFs) are considered to be antioxidants. The present study aimed to determine the molecular mechanism underlying the ability of TFs to attenuate ethanol-induced oxidative stress and apoptosis in GES-1 gastric mucosa epithelial cells. A Cell Counting Kit-8 (CCK-8) assay was performed to investigate the cell viability of GES-1 cells following administration of ethanol (0.5 mol/l) and subsequent treatment with TFs (20, 40 and 80 µg/ml) for specific time intervals. A carboxyfluorescein diacetate succinimidyl ester assay was used to measure proliferation and further investigate the results of the CCK-8 assay. Flow cytometry was performed to measure reactive oxygen species (ROS) levels and the apoptosis rates of GES-1 cells. Furthermore, levels of oxidative stress-associated factors, including malondialdehyde, superoxide dismutase and glutathione, were investigated using commercial kits. Reverse transcription-quantitative polymerase chain reaction and western blot assays were performed to determine the expression levels of apoptosis-associated factors, as well as the phosphorylation levels of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). The results of the present study demonstrated that treatment with ethanol inhibited GES-1 cell proliferation, and enhanced ROS levels and apoptosis rates, potentially via downregulation of B-cell lymphoma-2 (Bcl-2) expression and upregulation of Bcl-2-associated X and caspase-3 expression levels, as well as enhancing the phosphorylation levels of ERK, JNK and p38. However, treatment with TFs was revealed to attenuate the effects of ethanol administration on GES-1 cells in a dose-dependent manner. In conclusion, TFs may attenuate ethanol-induced oxidative stress and apoptosis in gastric mucosa epithelial cells via downregulation of various mitogen-activated protein kinase pathways.

Abstract 4747: Neutrophils in gastric cancer tissue inhibit the proliferation of CD4+T cells and contribute to immunosuppression

Abstract [Background] Neutrophils are essential effector cells in the host's defence against invasive pathogens, On the other hand, neutrophils in the tumor-microenvironment are known to have potential to induce angiogenesis, lymphangiogenesis, and be an important factor in tumor progression. We have reported that neutrophils infiltrating in gastric cancer tissue (Tumor-Associated Neutrophil: TAN) correlated with lymph node metastasis, systemic inflammatory response and be a poor prognostic factor. So we hypothesized that neutrophils in gastric cancer tissue suppressed anti-tumor immune response, but their details are still unclear. [Aim] The aim of this study was to investigate the immunosuppressive ability of neutrophils in gastric cancer tissue and to explore the influence of neutrophils on the proliferation of CD4 (+) T cells. [Materials and Methods] We isolated neutrophils from blood samples taken from healthy donors and added tumor tissue culture supernatants (TTCS) purified from human scirrhous gastric cancer cell line (OCUM12) or purified supernatants from patients who underwent gastrectomy at our department. Then, we examined the expression of apoptotic cells, PDL-1, and HLA-DR on neutrophils with TTCS. We also observed the expression of PD-1, CD25 on T cells co-cultured with neutrophils added TTCS. Furthermore, CD4 T cell labeled Carboxyfluorescein diacetate succinimidyl ester (CFSE) were co-cultured with allogenic dendritic cells and neutrophils with TTCS, and the proliferation of CD4 (+) T cells were examined by flow cytometry. We also performed immunohistochemistry (IHC) staining using anti CD15 and anti PD-1 antibodies for 117 patients who underwent gastrectomy in our department from 2007 to 2013 to explore the positional relationship. [Results] Neutrophils with TTCS were upregulated the expression of PDL-1 than those without TTCS. We also found that the apoptotic cell ratio and the expression of HLA-DR on neutrophils with TTCS was decreasing. CD4 (+) T cells co-cultured with neutrophils added TTCS were suppressed their proliferation, and upregulated the expression of PD-1 and downregulated the expression of CD25. IHC showed that PD-1 positive cells formed clusters and TAN was infiltrating around the clusters. And a positive correlation was found between the number of TAN and PD-1 positive cells in the scatter plot. [Conclusion] Our findings suggested that neutrophils in gastric cancer tissue inhibited the proliferation of CD4 T cells and formed a local immunosuppressive environment through the PD1-PDL1 pathway. Citation Format: Soichiro Hiramatsu, Hiroaki Tanaka, Yoshihito Yamakoshi, Junya Nishimura, Tatsuro Tamura, Takahiro Toyokawa, Kazuya Muguruma, Masaichi Ohira. Neutrophils in gastric cancer tissue inhibit the proliferation of CD4+T cells and contribute to immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4747.

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.