Carboxyfluorescein Diacetate Succinimidyl Ester Research Articles

IgE recognition of the house dust mite allergen Der p 37 is associated with asthma

House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio=3.1) of asthma compared to Der p 37-negative patients. Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

Establishment of the reference intervals of whole blood neutrophil phagocytosis by flow cytometry.

ObjectiveTo investigate the reference intervals (RIs) of the whole blood neutrophil phagocytosis by flow cytometry (FCM) and to study the application value of neutrophil phagocytosis in infectious diseases.MethodsPathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) cultured for 18–24 h were labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA‐SE), and then incubated with whole blood at 37℃. The phagocytosis of pathogens by neutrophils was detected by flow cytometry, and a reference interval was established.ResultsIn the healthy adults, the reference interval for the neutrophil phagocytosis to Escherichia coli was 46.91%–83.09% and to Staphylococcus aureus was 33.92%–69.48%. This method showed good reproducibility. Neutrophil phagocytosis was negatively correlated with the neutrophil count, neutrophil percentage, and neutrophil‐to‐lymphocyte ratio (NLR, p < 0.05).ConclusionWe have successfully established the RIs of neutrophil phagocytosis in whole blood in healthy adults by flow cytometry (FCM), which might be of important clinical value in the diagnosis, treatment, and prognosis of infectious diseases.

Effect of IL-15 addition on asbestos-induced suppression of human cytotoxic T lymphocyte induction

BackgroundAsbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8+ T cells. Recently, we reported that asbestos-induced suppression of CTL induction is not due to insufficient levels of interleukin-2 (IL-2). In this study, we continue to investigate the mechanism responsible for the effect of asbestos fibers on the differentiation of CTLs and focus on interleukin-15 (IL-15) which is known to be a regulator of T lymphocyte proliferation.MethodsFor MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8+ T cells with fluorescence-labeled anti-CD3, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD25, and anti-granzyme B antibodies using flow cytometry. To examine the effect of IL-15 on the expression level of intracellular granzyme B in proliferating and non-proliferating CD8+ lymphocytes, PBMCs were stained using carboxyfluorescein diacetate succinimidyl ester (CFSE) and then washed and used for the MLR.ResultsIL-15 addition partially reversed the decrease in CD3+CD8+ cell numbers and facilitated complete recovery of granzyme B+ cell percentages. IL-15 completely reversed the asbestos-induced decrease in percentage of granzyme B+ cells in both non-proliferating CFSE-positive and proliferating CFSE-negative CD8+ cells. The asbestos-induced decrease in the percentage of CD25+ and CD45RO+ cells in CD8+ lymphocytes was not reversed by IL-15.ConclusionThese findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

Dendritic cells inclusion and cell-subset assessment improve flow-cytometry-based proliferation test in non-immediate drug hypersensitivity reactions.

Lymphocyte transformation test (LTT) has been widely used to evaluate non-immediate drug hypersensitivity reactions (NIDHRs). However, the lack of standardization and the low sensitivity have limited its routine diagnostic use. The drug presentation by dendritic cells (DCs) and the assessment of proliferation on effector cells have shown promising results. Flow-cytometry-based methods can help apply these improvements. We aimed to assess the added value of using drug-primed-DCs and the determination of the proliferative response of different lymphocyte subpopulations in NIDHRs. Patients with confirmed NIDHR were evaluated by both conventional (C-LTT) and with drug-primed-DCs LTT (dDC-LTT)analysing the proliferative response in T cells and other effector cell subpopulations by using the fluorescent molecule, carboxyfluorescein diacetate succinimidyl ester (CFSE). The C-LTT showed a significantly lower sensitivity (29.4%) compared with dDC-LTT (61.8%), which was confirmed analysing each particular clinical entity: SJS-TEN (62.5% vs 87.5%), MPE (15% vs 47.4%) and AGEP (33% vs 80%). When including the effector cell subpopulations involved in each clinical entity, CD3+ +CD4+ Th 1 or CD3+ +NK cells in SJS-TEN, CD3+ +CD4+ Th 1+NK cells in MPE and CD3+ +NK cells in AGEP, we could significantly increase the sensitivity of the in vitro test to 100%, 68.4% and 100%, respectively, with an overall sensitivity of 87% and 85% of specificity in NIDHR. The use of a flow-cytometry-based test, DCs as drug presenting cells, and focusing on effector cell subpopulations for each clinical entity significantly improved the drug-specific proliferative response in NIDHRs with a unique cellular in vitro test.

Up-Regulation of Donor Dendritic Cell PD-L1 Expression Reduced Recipient Lymphocyte Activation and Proliferation In Vitro

ObjectiveTo observe the effects of dendritic cells (DC) in donor C57BL/6 (H-2b) micetransfected with recombinant adenovirus vector Ad-PD-L1 on proliferation and activation of lymphocytes in recipient DBA/2 (H-2d) mice. MethodsThe pSport 1-mSD274 plasmid containing the full-length PD-L1 cDNA of the mouse was digested and subcloned to the shuttle plasmid pShuttle-GFP-CMV(-), and then the adenovirus skeleton plasmid pAdxsi-GFP-CMV-PD-L1 was constructed by enzymolysis and ligation, transformed into DH5α sensitive bacteria, and screened for positive clones. After enzyme digestion, sequencing, and identification, 293 cells were transfected with liposome after linearization for packaging and amplification, and the virus was purified by cesium chloride density gradient centrifugation. DC of donor C57BL/6 mice were isolated, cultured, and divided into the following 3 groups: group A, adenovirus vector Ad-PD-L1 transfection group; group B, empty vector transfection group; and group C, control group. Western blot was used to detect the expression of PD-L1 in each group of cells after transfection. Isolate lymphocytes from recipient DBA/2 mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and mixed with DC of donor C57BL/6 mice with lymphocytes of recipient DBA/2 mice. Flow cytometry was performed to observe the proliferation of lymphocytes. ResultsDigestion and sequencing confirmed that the recombinant adenovirus vector Ad-PD-L1 containing PD-L1 was successfully constructed. After transfection with DC of donor C57BL/6 mice, the expression of PD-L1 increased by 37% (P < .05), and the PD-L1 transfected DC and recipient DBA/2. Mouse lymphocytes were cocultured. Compared with the control group, the increased expression of PD-L1 significantly inhibited the proliferation and activation of lymphocytes. The lymphocyte proliferation of DBA/2 mice decreased by 41% (P < .01). ConclusionThe recombinant adenovirus vector Ad-PD-L1 containing the mouse PD-L1 gene was successfully constructed. After transfection with dendritic cells of donor C57BL/6 mice, PD-1/PD-L1 inhibited lymphocytes proliferation and activation of recipient DBA/2 mice through costimulatory pathway.

TSLP Produced by Aspergillus fumigatus-Stimulated DCs Promotes a Th17 Response Through the JAK/STAT Signaling Pathway in Fungal Keratitis.

PurposeThe purpose of this study was to explore the role of thymic stromal lymphopoietin (TSLP) secreted by Aspergillus fumigatus–stimulated dendritic cells (DCs) during the T helper 17 (Th17) immune response, and further clarify the mechanisms contributing to the Th17 immune response of fungal keratitis (FK).MethodsA carboxyfluorescein diacetate succinimidyl ester assay, PCR, and flow cytometry were performed to detect Th17 differentiation of CD4+ T cells; PCR, ELISA, and Western blot were used to detect the expression of TSLP and JAK/STAT–related proteins; Signaling pathways involved in Th17 response was evaluated using RNA sequence; C57BL/6 mice were infected with A. fumigatus and treated with ruxolitinib or BBI608. Slit-lamp examination, fluorescein staining, and clinical scores were used to assess the clinical manifestation.ResultsA. fumigatus–infected DCs could drive naïve CD4+ T-cell proliferation and promote the production of Th17 cytokines IL-17A, IL-17F, and IL-22. A. fumigatus stimulation increased the expression of TSLP in DCs. DC-derived TSLP contributed to a Th17-type inflammatory response via the JAK/STAT signaling pathway. TSLP small interfering RNA, TSLPR small interfering RNA, or JAK/STAT inhibitors inhibited the Th17 immune response induced by A. fumigatus–infected DCs. Moreover, TSLP promoted A. fumigatus keratitis disease progression in a mouse model. However, inhibition of the JAK/STAT signaling pathway using a specific inhibitor reversed the development of FK by A. fumigatus infection.ConclusionsTSLP secreted by A. fumigatus–stimulated DCs played a significant role in the Th17-dominant immune response of FK through its JAK/STAT activation. Our findings may contribute to the elucidation of the molecular mechanisms of FK and to the development of novel therapeutic approaches.

Analysis of Same Selected Immunomodulatory Properties of Chorionic Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a population of adherent cells that can be isolated from multiple adult tissues. MSCs have immunomodulatory capacity and the ability to differentiate into many cell lines. Research study examines the immunomodulatory properties of MSCs isolated from chorion (CMSCs). Following the stimulation process, it was found that MSCs are capable of immunomodulatory action via the release of bioactive molecules as well as through direct contact with the immune cells. Immunomodulatory potential of the CMSCs was analyzed by modifying proliferative capacity of mitogen-activated lymphocytes. CMSCs and lymphocytes were tested in cell-to-cell contact. Lymphocytes were stained with carboxyfluorescein diacetate succinimidyl ester. Inhibition of the proliferation of activated lymphocytes was observed. Following the co-cultivation, the expression of markers involved in the immune response modulation was assessed. Afterwards, an increase in CMSCs expression of IL-10 was detected. Following the co-cultivation with activated lymphocyte, adhesion molecules CD54 and CD44 in the CMSCs increased. An increase of CD54 expression was observed. The properties of CMSCs, adherence and differentiation ability, were confirmed. The phenotype of CMSCs CD105+, CD90+, CD73+, CD44+, CD29+, CD45−, CD34−, CD54+ was characterized. It was demonstrated that chorion-derived MSCs have important immunomodulatory effects.

Depletion of SNRNP200 inhibits the osteo\u2212/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla

BackgroundTissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs’ function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research.MethodsThe alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo−/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins.ResultsDepletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo−/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2.ConclusionsDepletion of SNRNP200 repressed osteo−/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

Ethyl Pyruvate-Derived Transdifferentiation of Astrocytes to Oligodendrogenesis in Cuprizone-Induced Demyelinating Model.

Astrocytes redifferentiate into oligodendrogenesis, raising the possibility that astrocytes may be a potential target in the treatment of adult demyelinated lesion. Upon the basis of the improvement of behavior abnormality and demyelination by ethyl pyruvate (EP) treatment, we further explored whether EP affects the function of astrocytes, especially the transdifferentiation of astrocytes into oligodendrogenesis. The results showed that EP treatment increased the accumulation of astrocytes in myelin sheath and promoted the phagocytosis of myelin debris by astrocytes in vivo and in vitro. At the same time, EP treatment induced astrocytes to upregulate the expression of CNTF and BDNF in the corpus callosum and striatum as well as cultured astrocytes, accompanied by increased expression of nestin, Sox2, and β-catenin and decreased expression of Notch1 by astrocytes. As a result, EP treatment effectively promoted the generation of NG2+ and PDGF-Ra+ oligodendrocyte precursor cells (OPCs) that, in part, express astrocyte marker GFAP. Further confirmation was performed by intracerebral injection of primary astrocytes labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). As expected, NG2+ OPCs expressing CFSE and Sox2 were elevated in the corpus callosum of mice treated with EP following transplantation, revealing that EP can convert astrocytes into myelinating cells. Our results indicate the possibility that EP lead to effective myelin repair in patients suffering from myelination deficit.Graphical Abstract The diagram of EP action for promoting myelin regeneration in CPZ model. EP promoted migration and enrichment of astrocytes to demyelinated tissue and induced astrocytes to express neurotrophic CNTF and BDNF as well as translation factor nestin, Sox2, and β-catenin, which should contribute to astrocytes to differentiate of oligodendrogenesis. At the same time, EP promoted astrocytes to phagocytized myelin debris for removing the harmful substances of myelin regeneration.

Improvements in Flow Cytometry-Based Cytotoxicity Assay.

The flow cytometry-based assay has been increasingly used to assess the cell-mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive 51 Cr release assay (CRA), such as higher sensitivity at the single-cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross-staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross-staining occurred between the effector and target cells after 4h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector-target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry-based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost-effective way. © 2020 International Society for Advancement of Cytometry.

Both quiescent and proliferating cells circulate in the blood of the invasive apple snail Pomacea canaliculata

Gastropod hematopoiesis occurs at specialized tissues in some species, but the evidence also suggests that hemocyte generation is maybe widespread in the connective tissues or the blood system in others. In Ampullariidae (Caenogastropoda), both the kidney and the lung contain putative hematopoietic cells, which react to immune challenges. In the current study, we wanted to explore if hematopoiesis occurs in the blood of Pomacea canaliculata. Thus, we obtained circulating hemocytes from donor animals and tested their ability to proliferate in the blood of conspecific recipients. We tracked cell proliferation by labeling the donors' hemocytes with the fluorescent cell proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE). Transferred CFSE-labeled hemocytes survived and proliferated into the recipients’ circulation for at least 17 days. We also determined the cell cycle status of circulating hemocytes by using the propidium iodide (PI) and acridine orange (AO) staining methods. Flow cytometry analyses showed that most PI-stained hemocytes were in the G1 phase (~96%), while a lower proportion of cells were through the G2/S-M transition (~4%). When we instead used AO-staining, we further distinguished a subpopulation of cells (~5%) of low size, complexity-granularity, and RNA content. We regarded this subpopulation as quiescent cells. In separate experimental sets, we complemented these findings by assessing in circulating hemocytes two evolutionary conserved features of quiescent, undifferentiated cells. First, we used JC-1 staining to determine the mitochondrial membrane potential (Ψm) of circulating hemocytes, which is expected to be low in quiescent cells. Most hemocytes (~87%) showed high aggregation of JC-1, which indicates a high Ψm. Besides that, a small hemocyte subpopulation (~11%) showed low aggregation of the dye, thus indicating a low Ψm. It is known that the transition from a quiescent to a proliferating state associates with an increase of the Ψm. The specificity of these changes was here controlled by membrane depolarization with the Ψm disruptor CCCP. Second, we stained hemocytes with Hoechst33342 dye to determine the efflux activity of ABC transporters, which participate in the multixenobiotic resistance system characteristic of undifferentiated cells. Most hemocytes (>99%) showed a low dye-efflux activity, but a small proportion of cells (0.06–0.12%) showed a high dye-efflux activity, which was significantly inhibited by 100 and 500 μM verapamil, and thus is indicative of an undifferentiated subpopulation of circulating hemocytes. Taken together, our results suggest that, among circulating hemocytes, there are cells with the ability to proliferate or to stay in a quiescent state and behave as progenitor cells later, either in the circulation or the hematopoietic tissues/organs.

Fc Receptor is Involved in Nk Cell Functional Anergy Induced by Miapaca2 Tumor Cell Line

ABSTRACT Impaired NK cytotoxicity has been linked to poor cancer prognosis, but its mechanisms are not clearly established. Increasing data demonstrate that NK cells lose cytotoxicity after interaction with NK cell-sensitive tumor cells. In this paper, we provide evidence that the human adenocarcinoma cell line MiaPaCa2 and TNFα and TGFβ-treated MiaPaCa2 cultures (MiaPaCa2-TT) induced functional anergy of NK cells via FGL2 protein. MiaPaCa2-TT cultures decreased expression of IFNγ, CD107a, DNAM-1, and stimulated expression of PD1 by NK cells, as well as inhibited their cytotoxic activity in a greater manner compared to the parental culture. More importantly, we found that co-cultivation with anergized NK cells decreased expression of IFNγ and CD107a by naïve NK cells, which supports the hypothesis of NK cell functional anergy transmission. The obtained results suggest a mechanism by which tumor cells may inhibit cytotoxic functions of tumor-infiltrating and circulating NK cells in cancer. Abbreviations: CFSE: Carboxyfluorescein diacetate succinimidyl ester; CSCs: Cancer stem cells; FGL2: Fibrinogen-like protein 2; mAbs: Monoclonal antibodies; MiaPaCa2: Human adenocarcinoma cell line; MiaPaCa2-ТТ: Adenocarcinoma cell line MiaPaCa2 cells stimulated with TNFα and TGFβ-1; PI: Propidium iodide; TGFβ: Transforming growth factor beta; TME: Tumor microenvironment; TNFα: Tumor necrosis factor alfa

The ATF6-EGF Pathway Mediates the Awakening of Slow-Cycling Chemoresistant Cells and Tumor Recurrence by Stimulating Tumor Angiogenesis.

Slow-cycling cancer cells (SCCs) with a quiescence-like phenotype are believed to perpetrate cancer relapse and progression. However, the mechanisms that mediate SCC-derived tumor recurrence are poorly understood. Here, we investigated the mechanisms underlying cancer recurrence after chemotherapy, focusing on the interplay between SCCs and the tumor microenvironment. We established a preclinical model of SCCs by exposing non-small-cell lung cancer (NSCLC) cells to either the proliferation-dependent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) or chemotherapeutic drugs. An RNA sequencing analysis revealed that the established SCCs exhibited the upregulation of a group of genes, especially epidermal growth factor (EGF). Increases in the number of vascular endothelial growth factor receptor (VEGFR)-positive vascular endothelial cells and epidermal growth factor receptor (EGFR) activation were found in NSCLC cell line- and patient-derived xenograft tumors that progressed upon chemotherapy. EGFR tyrosine kinase inhibitors effectively suppressed the migration and tube formation of vascular endothelial cells. Furthermore, activating transcription factor 6 (ATF6) induced the upregulation of EGF, and its antagonism effectively suppressed these SCC-mediated events and inhibited tumor recurrence after chemotherapy. These results suggest that the ATF6-EGF signaling axis in SCCs functions to trigger the angiogenesis switch in residual tumors after chemotherapy and is thus a driving force for the switch from SCCs to actively cycling cancer cells, leading to tumor recurrence.

Mesenchymal stem cells activate Notch signaling to induce regulatory dendritic cells in LPS-induced acute lung injury

BackgroundMesenchymal stem cells (MSCs) have been shown to alleviate acute lung injury (ALI) and induce the production of regulatory dendritic cells (DCregs), but the potential link between these two cell types remains unclear. The goal of this study was to investigate the effect and mechanism of MSC-induced regulatory dendritic cells in ALI mice.Material/methodsIn vivo experiments, C57BL/6 wild-type male mice were sacrificed at different times after intratracheal injection of LPS to observe changes in lung DC maturation and pathological damage. MSCs, DCregs or/and carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled DCs were administered to the mice by tail vein, and flow cytometry was performed to measure the phenotype of lung DCs and T cells. Lung injury was estimated by the lung wet weight/body weight ratio and histopathological analysis. In vitro, Western blotting or flow cytometry was used to detect the expression of Notch ligand or receptor in MSCs or DCs after coculture or LPS stimulation. Finally, in vivo and in vitro, we used the Notch signaling inhibitor DAPT to verify the effect of the Notch pathway on MSC-induced DCregs and their pulmonary protection.ResultsWe showed significant accumulation and maturation of lung DCs 2 h after intratracheal injection of LPS, which were positively correlated with the lung pathological injury score. MSC treatment alleviated ALI lung injury, accompanied by a decrease in the number and maturity of classical DCs in the lungs. CFSE-labeled DCs migrated to the lungs of ALI mice more than those of the normal group, and the elimination of CFSE-labeled DCs in the blood was slower. MSCs inhibited the migration of CFSE-labeled DCs to the lung and promoted their elimination in the blood. DCregs, which are obtained by contact coculture of mDCs with MSCs, expressed reduced levels of MHCII, CD86, CD40 and increased levels of PD-L1, and had a reduced ability to stimulate lymphocyte proliferation and activation (expression of CD44 and CD69). mDCs expressing Notch2 significantly increased after coculture with MSCs or rhJagged1, and MSCs expressed more Jagged1 after LPS stimulation. After stimulation of mDCs with recombinant Jagged1, DCs with low expression of MHCII, CD86 and CD40 were also induced, and the effects of both rhJagged1 and MSCs on DCs were blocked by the Notch inhibitor DAPT. Intra-airway DAPT reversed the inhibitory effect of mesenchymal stem cells on DC recruitment to the lungs and its maturation.ConclusionsOur results suggested that the recruitment and maturation of lung DCs is an important process in early ALI, MSCs attenuate LPS-induced ALI by inducing the production of DCregs by activating Notch signaling.

Assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis

Horse mesenchymal stem cells (MSC) are potential anti-inflammatory tools for post-breeding induced endometritis (PBIE). In this research MSCs isolated from the endometrium or subcutaneous fat of the same donors were infused iu into mares with PBIE for assessment of their anti-inflammatory action and engraftment. PBIE was induced in nine gynecologically healthy mares by iu infusion of 500 million dead sperm in saline. Inflammatory markers were analyzed in uterine lavages and biopsies immediately before (phase I) and 3 h after infusion of sperm (phase II). Measurements: polymorph nuclear cells (PMN), proteins IL-6 and TNFα (ELISA in the lavages) and immunostaining in biopsies, transcripts of IL-1α, 6, 8, 10, TNFα and COX2 (qPCR of pelleted lavages). At 24 h after sperm deposition (phase III), mares were instilled iu with 20 ml of saline containing 2 × 107 adipose MSCs (n = 3, group 1) or endometrial MSCs (n = 3, group 2). Cells were labeled previously with carboxyfluorescein diacetate succinimidyl ester (CFDA SE). A third group (n = 3) received 20 mL of sterile saline alone. After 48 h another biopsy/lavage were done and the same parameters analyzed. For engraftment, additional biopsies were taken at days 10 and 30 of sperm infusion and analyzed by confocal microscopy. Dead sperm in saline markedly increased PMNs counts, IL-6 and TNFα expression in the ELISA (p < 0.05) and immunostaining. In phase III a significant reduction (p < 0.0001) of PMN was found in all samples, including control mares. A decrease (p < 0.05) of IL-6 and TNF-α was detected by ELISA, in the groups that received MSC, but not in control group. In the aMSC-treated group, a significant decrease was found in the expression of (IL1α, p = 0.0003; IL-6 p 0.04; IL-8, p = 0.006, TNFα p = 0.004). Expression of IL-10 and COX2 remained unchanged (p = 0.08). In the mares that received eMSC, IL-6 and 8 decreased significantly (p = 0.01), IL-10 increased (p = 0.009), while TNFα, COX2 and IL1α did not significantly change their expression. In the engraftment experiment CFDA label was found sparingly in all the samples analyzed until day 30, mainly at the stromal compartment of the endometrium. No differences in the engraftment pattern was found among cell origins. We conclude that inoculation of MSCs significantly reduced inflammation independently of the origin of the cells and that cells perform limited engraftment detectable after one month of infusion. These findings can be of help for the design of new anti-inflammatory therapies of uterine diseases in mares.

Systematic Evaluation of PKH Labelling on Extracellular Vesicle Size by Nanoparticle Tracking Analysis

Extracellular vesicles (EVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.

Proliferation, Differentiation and Immunoregulatory Capacities of Brown and White Adipose-Derived Stem Cells from Young and Aged Mice.

Background and ObjectivesAdipose tissue is a source of mesenchymal stem cells, which have the potential to differentiate into various types of cells. Adipose-derived stem cells (ADSCs) are now recognized as an accessible, abundant, and reliable stem cells suitable for tissue engineering and regenerative medicine applications. However, few literatures gave a comprehensive report on the capacities of ADSCs harvested from different sites. Especially, the capacities of ADSCs from aged mice remained unclear. In this study, we investigated several main capacities of brown adipose derived stem cells (B-ADSCs) and white adipose derived stem cells (W-ADSCs) from both young and aged mice.Methods and ResultsWhen isolated from young mice, B-ADSCs showed a stronger proliferation rate and higher osteogenic, adipogenic and myocardial differentiation ability than W-ADSCs. Carboxy fluorescein diacetate succinimidyl ester (CFSE) labeling test suggested no significant difference in immunosuppression capacity between B-ADSCs and W-ADSCs. Similarly, no difference between these two were found in several immune related molecules, such as programmed death-ligand 1 (PD-L1), intercellular cell adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), interleukin 10 (IL10), and suppressor of cytokine signaling 1 (socs1). When isolated from aged mice, B-ADSCs also showed a stronger proliferation rate and higher osteogenic, adipogenic and myocardial differentiation ability than W-ADSCs; however, it demonstrated an attenuated immunosuppression capacity compared to W-ADSCs.ConclusionsIn summary, our data showed that ADSCs’ characteristics were tissue source dependent and changed with age. It provided evidence for choosing the right tissue-specific ADSCs for clinical application and fundamental research.

IL-2 enhances ex vivo–expanded regulatory T-cell persistence after adoptive transfer

AbstractAs regulatory T cell (Treg) adoptive therapy continues to develop clinically, there is a need to determine which immunomodulatory agents pair most compatibly with Tregs to enable persistence and stabilize suppressor function. Prior work has shown that mechanistic target of rapamycin inhibition can increase the stability of thymic Tregs. In this study, we investigated the transcriptomic signatures of ex vivo–expanded Tregs after adoptive transfer in the setting of clinically relevant immunosuppression using a nonhuman primate (NHP) model as a prelude to future transplant studies. Here, we found that adding interleukin-2 (IL-2) to rapamycin in vivo supported a logarithmic increase in the half-life of adoptively transferred carboxyfluorescein diacetate succinimidyl ester–labeled, autologous NHP Tregs, effectively doubling the number of cells in the peripheral blood Treg compartment compared with Treg infusion when rapamycin was given alone. Using single-cell transcriptomics, we found that transferred ex vivo–expanded Tregs initially exhibit a gene expression signature consistent with an activated state. Moreover, those cells with the highest levels of activation also expressed genes associated with p53-mediated apoptosis. In contrast, transferred Tregs interrogated at day +20 posttransfer demonstrated a gene signature more similar to published profiles of resting Tregs. Together, these preclinical data further support combining IL-2 and rapamycin in vivo as adjunctive therapy for ex vivo–expanded adoptively transferred Tregs and suggest that the activation status of ex vivo–expanded Tregs is critical to their persistence.

Doxorubicin selectively induces apoptosis through the inhibition of a novel isoform of Bcl‑2 in acute myeloid leukaemia MOLM‑13 cells with reduced Beclin 1 expression

The overexpression of anti-apoptotic Bcl-2 in acute myeloid leukaemia (AML) may contribute to difficulties in eradicating these cells during chemotherapy. In the present study, doxorubicin (Dox) was evaluated for its potential to induce selective apoptotic cell death in AML MOLM-13 cells and to modulate autophagy through Bcl-2 and Beclin 1 protein expression. Annexin V/propidium iodide and 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometric analyses were conducted to determine the effects of Dox on cell death and cell proliferation, respectively, following 48 h of co-incubation with AML MOLM-13 or U-937 monocytic cells. The protein expression levels of Bcl-2 and Beclin 1 in untreated and treated cells were quantified by western blot analysis. Dox reduced the viability of MOLM-13 cells partly by inhibiting cell division and inducing cell apoptosis. Dox demonstrated a level of selectivity in its cytotoxicity against MOLM-13 compared to U-937 cells (P<0.05). Dox induced a significant decrease in Beclin 1 protein levels in MOLM-13 cells without significantly affecting the protein levels in U-937 monocytes. A novel Bcl-2 15-20 kDa (p15-20-Bcl-2) isoform was found to be selectively expressed in AML MOLM-13 cells (but absent in the leukaemic cell lines tested, OCI-AML2, CML K562 and U-937). Dox induced a highly significant inhibition of p15-20-Bcl-2 at concentrations of 0.5, 0.75 and 1 µM (P<0.01). However, the usual 26 kDa Bcl-2 (p26-Bcl-2-α) isoform protein expression was not affected by the drug in either the MOLM-13 or U-937 cells. It was thus postulated that Dox exhibited some selectivity by targeting the p15-20-Bcl-2 isoform in MOLM-13 cells and activating Beclin 1 to induce cell death.

Immune function of myeloid-derived suppressor cells and its mechanism in obstructive sleep apnea syndrome

Objective: To investigate the immune function of myeloid-derived suppressor cells (MDSC) and its mechanism in obstructive sleep apnea syndrome (OSAS). Methods: Twenty OSAS patients who were diagnosed by polysomnography (Apnea Hyponea Index>30 events/h) from Sleep Disorders Center at First Affiliated Hospital between January 2015 and December 2016 were selected. The percent of CD14(+) low expression or lack of human leukocyte antigen DR (HLA-DR(-/low)) MDSC in the CD14(+) monocyte from both OSAS patients and healthy people were analyzed by flow cytometry. In vitro assay, MDSC from OSAS patients and health people were sorted by flow cytometry and T cells were sorted with negative isolation kit. For T-cell proliferation assays, the carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells were respectively incubated with autologous MDSC. CFSE fluorescence intensity of T cells was detected by flow cytometry. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used to evaluate the concentrations of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10, transforming growth factor-β(1) (TGF-β(1)), positive rate of programmed death ligand-L1 (PD-L1), relative transcript level of Arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), hypoxic inducible factor-1α (HIF-1α) expressed by MDSC. Results: Comparing to healthy people, the percentage of CD14(+)HLA-DR(-/low) MDSC in CD14(+) monocyte was significantly elevated [(12.5±1.5)% vs (3.5±0.4)%, P<0.05]. In vitro, OSAS patient-derived MDSC exhibited a stronger suppressive effect on T-cell proliferation [(23.2±1.1)% vs (53.7±3.2)%, P<0.05]. Further analysis revealed that OSAS patient-derived MDSC secreted much higher concentrations of IL-6, TNF-α, IL-10 and TGF-β(1) [(1 316±163) vs (642±72) ng/L, (316±35) vs (167±18) ng/L, (385±42) vs (108±26) ng/L and (44 276±4 589) vs (9 557±1 124) ng/L] (all P<0.05). The percentage of membrane molecule PD-L1-positive cells in OSAS patient-derived MDSC was obviously higher than that in healthy people-derived MDSC [(75.6±7.9)% vs (30.6±2.5)%, P<0.05]. Compared with healthy people-derived MDSC, the relative transcript level of Arg1, iNOS and HIF-1α in OSAS patient-derived MDSC was also increased by (4.6±0.5), (2.8±0.3) and (4.3±0.4) fold, respectively (all P<0.05). Conclusions: OSAS may be capable of inducing the proliferation of MDSC and its expression of immunosuppressive molecules by activating HIF-1α signal, thereby enhancing the immunosuppressive ability of MDSC.