Mean Fluorescence Research Articles

The roles of sub-micron and microscale roughness on shear-driven thrombosis on titanium alloy surfaces.

Continuous-flow ventricular assist devices (cfVADs) are implanted in patients with end-stage heart failure to assist with blood circulation. However, VAD implantation is associated with dangerous thrombotic complications. Our goal was to determine the impact of micron and sub-micron scale Ti6Al4V surface roughness on adherent platelet aggregate properties under clinically relevant shear rates. We used fluorescence microscopy to visualize platelets in real time as they adhered to Ti6Al4V coupons of varying degrees of roughness, including a smooth control, in microfluidic channels and quantified deposition using an image processing algorithm. We systematically characterized roughness using spatial frequencies to generalize results for more blood-biomaterial contact applications. We observed that on the control and sub-micron rough surfaces, at 1000 s-1 , platelets adhered uniformly on the surface. At 2000 s-1 , we observed small and stably adherent platelet aggregates. At 5500 s-1 , platelet aggregates were large, unstable and interconnected via fibrillar structures. On a surface with micron-scale roughness features, at all three shear rates, platelets deposited in the troughs of the roughened surface, and formed aggregates. Thrombus height at 2000 s-1 and 5500 s-1 was greatest on the roughest surface and lowest on the mirror-finished surface, as indicated by the mean fluorescence intensity. These results demonstrated that at high shear rates, thrombi form regardless of surface topography at the scales applied. At lower shear rates, micron-scale surface features cause thrombus formation, whereas submicron features result in innocuous platelet adhesion. These findings have implications for manufacturing costs and other considerations.

A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions.

The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.

Intraoperative near-infrared fluorescence imaging can identify pelvic nerves in patients with cervical cancer in real time during radical hysterectomy

PurposeRadical hysterectomy combined with pelvic lymphadenectomy is the standard treatment for early-stage cervical cancer, but unrecognized pelvic nerves are vulnerable to irreversible damage during surgery. This early clinical trial investigated the feasibility and safety of intraoperative near-infrared (NIR) fluorescence imaging (NIR-FI) with indocyanine green (ICG) for identifying pelvic nerves during radical hysterectomy for cervical cancer.MethodsSixty-six adults with cervical cancer were enrolled in this prospective, open-label, single-arm, single-center clinical trial. NIR-FI was performed in vivo to identify genitofemoral (GN), obturator (ON), and hypogastric (HN) nerves intraoperatively. The primary endpoint was the presence of fluorescence in pelvic nerves. Secondary endpoints were the ICG distribution in a nerve specimen and potential underlying causes of fluorescence emission in pelvic nerves.ResultsIn total, 63 patients were analyzed. The ON was visualized bilaterally in 100% (63/63) of patients, with a mean fluorescence signal-to-background ratio (SBR) of 5.3±2.1. The GN was identified bilaterally in 93.7% (59/63) of patients and unilaterally in the remaining 4 patients, with a mean SBR of 4.1±1.9. The HN was identified bilaterally in 81.0% (51/63) of patients and unilaterally in 7.9% (5/63) of patients, with a mean SBR of 3.5±1.3. ICG fluorescence was detected in frozen sections of a nerve specimen, and was mainly distributed in axons. No ICG-related complications were observed.ConclusionThis early clinical trial demonstrated the feasibility and safety of NIR-FI to visualize pelvic nerves intraoperatively. Thus, NIR-FI may help surgeons adjust surgical decision-making, avoid nerve damage, and improve surgical outcomes.Trial registrationClinicalTrials.gov NCT04224467

Sodium-glucose cotransporter type 2 inhibitors prevent ponatinib-induced endothelial senescence and disfunction: A potential rescue strategy

BackgroundPonatinib (PON), a third-generation tyrosine kinase inhibitor (TKI), has proven cardiovascular toxicity, with no known preventing agents usable to limit such side effect. Sodium-glucose cotransporter type 2 (SGLT2) inhibitors are a new class of glucose-lowering agents, featuring favorable cardiac and vascular effects. AimsWe assessed the effects of the SGLT2 inhibitors empagliflozin (EMPA) and dapagliflozin (DAPA) on human aortic endothelial cells (HAECs) and underlying vasculo-protective mechanisms in an in vitro model of PON-induced endothelial toxicity. Methods and resultsWe exposed HAECs to PON or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nmol/L) or dapagliflozin (DAPA) for 0–48 h exposure times. Compared with vehicle, incubations of HAECs with PON significantly reduced cell viability (0.56 ± 0.11 vs 0.23 ± 0.05 absorbance units, p < 0.01), increased the number of senescent cells at β-gal-assay (PON 9 ± 4 vs basal DMSO 3 ± 1 β-Gal+ cells/field, p < 0.01), decreased tubulization in Matrigel (PON PON: 6 ± 1 vs basal DMSO 12 ± 1 tubuli number/field, p < 0.05) with a non-statistically significant trend of PON to decrease the number of autophagic cells at immunofluorescence assay and flow cytometry. EMPA reverted the effects of PON on cell viability (E 500 + PON 0.24 ± 0.05 vs PON 0.56 ± 0.11 absorbance units, p < 0.01) and induced autophagy (E 500 7 ± 4.3 vs basal DMSO 2.6 ± 2.3 mean fluorescence vs PON 2.6 ± 2.4 mean fluorescence, p < 0.05). EMPA and DAPA also reversed the effects of PON on cell senescence (E 500 + PON 4 ± 1 and DAPA 100 4 ± 2 vs PON 9 ± 4 β-Gal+ cells/field, p < 0.01) and improved cell tubulization (E 500 + PON 21 ± 3 vs PON 6 ± 1 tubuli number/field, p < 0.05; DAPA 100 + PON 16 ± 2 vs PON 6 ± 1 tubuli number/field, p < 0.05). ConclusionEMPA and DAPA attenuate the vasculo-toxic effect exerted by PON by reverting endothelial cell senescence and dysfunction. These findings support the design of clinical studies exploring the vasculo-protective effects of EMPA or DAPA on PON-induced vascular toxicity.

Leveraging host-genetics and gut microbiota to determine immunocompetence in pigs

BackgroundThe gut microbiota influences host performance playing a relevant role in homeostasis and function of the immune system. The aim of the present work was to identify microbial signatures linked to immunity traits and to characterize the contribution of host-genome and gut microbiota to the immunocompetence in healthy pigs.ResultsTo achieve this goal, we undertook a combination of network, mixed model and microbial-wide association studies (MWAS) for 21 immunity traits and the relative abundance of gut bacterial communities in 389 pigs genotyped for 70K SNPs. The heritability (h2; proportion of phenotypic variance explained by the host genetics) and microbiability (m2; proportion of variance explained by the microbial composition) showed similar values for most of the analyzed immunity traits, except for both IgM and IgG in plasma that was dominated by the host genetics, and the haptoglobin in serum which was the trait with larger m2 (0.275) compared to h2 (0.138). Results from the MWAS suggested a polymicrobial nature of the immunocompetence in pigs and revealed associations between pigs gut microbiota composition and 15 of the analyzed traits. The lymphocytes phagocytic capacity (quantified as mean fluorescence) and the total number of monocytes in blood were the traits associated with the largest number of taxa (6 taxa). Among the associations identified by MWAS, 30% were confirmed by an information theory network approach. The strongest confirmed associations were between Fibrobacter and phagocytic capacity of lymphocytes (r = 0.37), followed by correlations between Streptococcus and the percentage of phagocytic lymphocytes (r = -0.34) and between Megasphaera and serum concentration of haptoglobin (r = 0.26). In the interaction network, Streptococcus and percentage of phagocytic lymphocytes were the keystone bacterial and immune-trait, respectively.ConclusionsOverall, our findings reveal an important connection between gut microbiota composition and immunity traits in pigs, and highlight the need to consider both sources of information, host genome and microbial levels, to accurately characterize immunocompetence in pigs.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Procoagulant Mechanisms in Patients with Myelodysplastic Syndrome

Background: MDS comprise a heterogeneous sort of disorders caused by cytogenetic changes, gene mutations, or both, responsible for altered multilineage morphological patterns that lead to a varying grade of peripheral blood cytopaenias and an increased risk of transformation to acute myeloid leukemia. Thrombocytopaenia is observed in aproximately 35% of MDS patients. Despite it is accepted that haemorrhagic events are more likely at platelet counts below 10,000/μl, in the case of MDS patients, whose platelets had a diminished ability to be activated, bleeding events are lower than expected. So, hemostasis in MDS patients is a complex issue where mechanisms additional to platelet count/function seem to be involved.Aim: This work aimed to identify procoagulant mechanisms in non-bleeders MDS patients that might protect them from bleeding.Methods: This is a prospective, observational and transversal study. Eighty four non-bleeders MDS patients (44% female, mean age of 77±25 years), were included. Sixty healthy subjects (45% female, mean age of 57±22 years) were recruited.Human peripheral blood samples were collected in 3.8% sodium citrate. Blood cell counts were performed with a Coulter Ac.T Diff cell counter (Beckman Coulter, Madrid, Spain). Platelet rich plasma (PRP) and platelet free plasma (PFP) were obtained by centrifugation.Surface exposure of phosphatidylserine (PS) and active caspase-3, -8 or -9 were assessed by flow cytometry measuring the binding of FITC-labeled Annexin V (BD Pharmingen) and with a specific kit for measuring these caspases (Millipore, Madrid, Spain).Prothrombinase complex binding to platelets was determined in quiescent washed platelets (1x108/mL) incubated with factor (F) Xa (5 nmol/L) and FVa (5nmol/L) obtained from Haemtech Inc (Cellsystems, Germany). Following fixation to cross-link platelet-bound FVa and FXa, specific or control monoclonal antibodies (0.1 μmol/L each, Haemtech Inc, Germany) were added. All samples were analized by flow cytometry.PS-associated and tissue factor (TF)-associated procoagulant activity of microparticles (MPs) was determined with the ZYMUPHEN MP-Activity kits (HYPHEN BioMed, Neuvillesur Oise, France).The identification of the MP's cellular origin was determined by flow cytometry, labeling MPs with Annexin-V-FITC and the following specific mAb conjugated with phycoerythrin: anti-CD41 mAb for platelets (Biocytex, Marseille, France); and anti-CD14 mAb for monocytes, anti-CD144 mAb for endothelial cells, anti-CD235 mAb for red cells and anti-CD45 mAb for leukocytes, all from BD Biosciences.The statistical analysis of the experimental data was using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA).Results: Platelets from patients with MDS presented higher levels of activated caspases-3,7, -8 and -9 and this situation was accompanied by a higher PS platelet surface exposure (TABLE 1)We evaluated if increased PS exposure was functional for anchoring prothrombinase complex. Binding of FVa and of FXa on platelet membrane surface was higher in quiescent platelets from MDS patients [FVa (Mean fluorescence), control: 49.7±8.5, MDS patients: 73.9±23.2, p<0.01; FXa (Mean fluorescence), control: 46.4±17.5 , MDS patients: 77.6±12.0, p<0.05)].A significantly higher procoagulant capacity of MPs associated either to PS (control: 8.0±2.5 nanomol/L; MDS: 17.8±3.5 nanomol/L, p<0.01) or TF (control: 0.4±2.5 nanomol/L; MDS: 7.5±2.5 nanomol/L, p<0.001) were observed in the MDS group.To determine the cellular source of MPs, flow cytometry analyses were performed and a significant increase was observed in the amount of monocyte- (events, control: 94±30, MDS patients: 220±29, p<0.05)and red cell-derived MPs (events, control: 142±55, MDS patients: 252±23, p<0.01) in the samples from the patients with MDS. In patients with MDS, TF-MP-associated thrombogenic ability correlated with monocyte percentage (Spearman r=0.5019, p<0.01).Conclusion: MDS patients, even those with normal platelet count, have a reduced platelet function with enhanced apoptosis but do not bleed in many cases. This fact might be due to the increase in tissue factor-containing microparticles and in the enhanced binding of prothrombinase complex to apoptotic platelets which, in turn, augmented procoagulant capacity of these patients.Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). [Display omitted] DisclosuresJimenez-Yuste:Roche: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding. Álvarez-Roman:Shire: Consultancy; Novo Nordisk: Consultancy.

Anti-CD123 CAR T-Cell Therapy for the Treatment of Myelodysplastic Syndrome

INTRODUCTIONMyelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis characterized by bone marrow dysplasia resulting in cytopenias. Currently, treatment options for MDS are limited to supportive care and hypomethylating agents with most patients eventually succumbing to the disease (anemia, infection or hemorrhage) or progressing to leukemia. Previously it have been demonstrated that CD123 is an important target for the treatment of MDS due to its aberrantly high expression in high-risk MDS cells compared to low-risk MDS cells and normal cells. As such, CD123-directed therapy may be useful in treating patients with high-risk MDS. METHODSChimeric antigen receptor (CAR) vector containing a CD123-specific single-chain variable fragment in combination with a CD28 costimulatory domain, CD3ζ signaling domain and tEGFR was expressed on healthy donor and patient derived T lymphocytes utilizing lentiviral vector delivery to target high-risk MDS cells. An anti-CD19 CAR was used as a negative control. CAR expressing or control T-cells were cocultured with MDS cell line (MDS-L) or primary MDS cells from patient bone marrow to examine in vitro anti-tumor function. Eradication of MDS cells was analyzed by flow cytometry 48hr after coculture. Intracellular TNF-α and cell surface CD107A were examined in effector cells 5 hours after cocultured with MDS cells. For in vivo analysis of CD123 CAR T cells, NSG-S mice were engrafted with 1.5x106 primary high-risk MDS cells for eight weeks, and were infused with autologous anti-CD123 CAR T-cells or control. Tumor burden was then analyzed on day 28. RESULTSHealthy donor derived anti-CD123 CAR T-cells effectively eliminated MDS cell line (MDS-L) (>99%), and primary bone marrow derived MDS cells (>70%) in vitro . The killing was associated with increased cytokine release and degranulation by anti-CD123 CAR T-cells, which did not occur with control anti-CD19 CAR T-cells. In addition, anti-CD123 CAR T-cells generated from patients with high-risk MDS showed 50~70% transduction efficiency. These cells successfully eradicated autologous CD123+ MDS cells at >99.9% in vitro compared to untransduced control T-cells (Figure 1A). The killing was also confirmed by increased intracellular TNF-α expression (Figure 1B). Furthermore, a patient derived xenograft system (PDX) was utilized to examine the killing ability of autologous anti-CD123 CAR T-cells in vivo . High-risk MDS bone marrow cells were engrafted in NSG-S mice. Eight weeks following engraftment, 7x104 autologous EGFRt+ anti-CD123 CAR T-cells or mock T-cells were infused via tail vein. On day 28 after autologous anti-CD123 CAR T-cell infusion, we observed significant decreases in both CD34+/CD38-/CD123+ MDS cells and MDS bulk (hCD45+/EGFRt-) while mice infused with mock CAR T-cells were unable to reduce tumor burden (Figure 1C and 1D). To extend the utility of our approach, we performed drug screening to identify the agents that increase expression of CD123 on MDS cells. We hypothesize that drugs inducing increased expression of CD123 will increase efficacy of anti-CD123 CAR T cell and avoid resistance arising from CD123 low/dim MDS progenitor cells. 134 various oncologic drugs were dosed at multiple concentrations (1nM, 100nM, 1uM, 0uM) and incubated with MDS-L cell line for 6hr, 16hr or 48hr. MDS-L cells were then analyzed for CD123 expression (Mean fluorescence) and cell viability (%live out of total) by flow cytometry. Eight drugs with low cytotoxicity significantly increased CD123 expression, including trifluridine (1uM), sunitinib (100nM), metoxantrone (1nM), dasatinib (10uM), imatinib (1uM), regorafenib (10uM), dabrafenib (100nM) and DEAB (100nM). These drug candidates will be examined further in primary MDS bone marrow samples and in combination with anti-CD123 CAR T-cells. CONCLUSIONThese results demonstrate that anti-CD123 CAR T-cells exhibit activity against high-risk MDS, and represent an effective therapeutic option for patients with high-risk MDS. [Display omitted] DisclosuresBudde:Mustang Therapeutics: Research Funding; Merck: Research Funding, Speakers Bureau; Amgen: Research Funding; KITE Pharmaceutical: Speakers Bureau; Precision Biosciences: Consultancy.

Analyzing Intracellular Gradients in Pollen Tubes.

Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.

Flow Cytometry Measurement of Reactive Oxygen Species Generation: Some Caveats and Their Solution

The redox state is very important in normal physiology and pathology. Yet, its measurement is not practiced within the clinic. The explanation is principally methodological—the inadequacy of the methods for application in the clinical laboratory. Flow cytometry is routinely utilized in hemato-oncology and immunology. Measuring of the cellular fluorescence by flow cytometry following loading with 2′7′-dichlorodihydrofluorescin diacetate (DCFDA) has become a standard experimental method for quantification of reactive oxygen species (ROS). However, the DCF fluorescence depends on the probe uptake and esterification as well as the presence of intracellular quenchers. Cells of various types, stages of differentiation or senescence, or under different conditions (e.g., pathological vs. normal) may differ in these respects. A one-time measurement does not take into consideration these differences. Herein, we describe a protocol that overcomes these caveats by determining the change in DCF fluorescence with time as a measure of the rate of ROS generation. Normal (n = 20) and thalassemia (n = 20) red blood cells (RBCs) samples were loaded with DCFDA, washed, and then incubated in a DCFDA-free medium. DCF fluorescence was measured at different times of incubation by flow cytometry and the mean fluorescence (MFC) calculated. The results showed that the rate of increase in the DCF fluorescence was linear in both types of RBCs, regardless of their basal level and hemoglobin content, with thalassemia RBCs demonstrating a 4.5-fold higher rate (265/h) compared with normal RBCs (58/h) (p < 0.001). In conclusion, this pulse-chase procedure may be used for flow cytometry clinical evaluation of general and cell type-specific oxidative stress under normal and pathological conditions.

A novel imaging flow cytometry method for the detection of histone H4 acetylation in myeloid cells

The histone deacetylase inhibitor (HDACI) valproic acid has been shown to inhibit the growth of multiple paediatric tumour types and is well tolerated in a childhood cancer setting. The current study was designed to develop a novel imaging flow cytometry method for the detection of histone H4 acetylation in white blood cells obtained from childhood cancer patients treated with valproic acid. HL-60 cells and whole blood samples from healthy volunteers were incubated with valproic acid (0-8mM) for 0-24hours, with additional blood samples collected from ependymoma patients receiving valproic acid on the SIOP Ependymoma II clinical trial. An imaging flow cytometry method was developed using an ImageStream®χ flow cytometer, collecting 100000 images per sample following excitation of PE tagged acH4 antibody and DAPI. The mean percentage of acH4-positive cells increased to a greater extent than increases in mean and median fluorescence intensity following incubation with valproic acid. Comparable results were observed for in vitro and ex vivo experiments, and the assay was shown to be appropriate for clinical sample analysis. Myeloid cells exhibited a smaller proportion of acH4-positive cells than the lymphoid population, but a greater fold increase above basal levels. The percentage of acH4-positive myeloid cells has the potential to be used as a robust pharmacodynamic biomarker for the measurement of acH4 for HDACIs. The developed assay is now being utilised in a clinical trial involving the treatment of childhood ependymoma patients with valproic acid.

Depth Profile Assessment of the Early Phase Deposition of Lysozyme on Soft Contact Lens Materials Using a Novel In Vitro Eye Model.

To characterize the location of fluorescently labeled lysozyme on commercial contact lenses (CLs) using an in vitro eye model that simulates tear volume, tear flow, air exposure, and mechanical wear. One commercially available conventional hydrogel CL material (etafilcon A) and three silicone hydrogel CL materials (balafilcon A, lotrafilcon B, and senofilcon A) were evaluated in this study. The CLs were mounted on the in vitro eye model and exposed to artificial tear fluid containing fluorescein isothiocyanate (FITC)-labeled lysozyme for 2 and 10 hrs. After these short incubation periods, circular discs were punched from the CLs at the center and periphery and were prepared for confocal laser scanning microscopy (CLSM). The CLSM captured a series of consecutive images spaced 5 μm apart, and the resulting images were rendered into two dimensional cross-sectional views of the CL. The mean fluorescence at each 5 μm slice was used to generate a histogram depicting the penetration of FITC-lysozyme into CLs. For both incubation periods, the CLSM images and histogram of etafilcon A showed that FITC-lysozyme is more concentrated at the lens surface, with a moderate amount of deposition in the lens matrix. For balafilcon A, FITC-lysozyme was evenly distributed throughout the lens. For lotrafilcon B, there was a greater amount of FITC-lysozyme deposition on the surfaces of the lens versus the matrix. Senofilcon A had differential FITC-lysozyme distribution profiles depending on the location of the lens. At the lens periphery, FITC-lysozyme primarily deposited on the surface, whereas FITC-lysozyme was uniformly distributed at the center of the lens. With the use of a sophisticated in vitro eye model, the study revealed a complex deposition pattern of FITC-labeled lysozyme on various CL materials after short periods of exposure. An understanding of the early deposition pattern of lysozyme on different CL material may elucidate new insights into the processes behind CL discomfort.

C-fos upregulates P-glycoprotein, contributing to the development of multidrug resistance in HEp-2 laryngeal cancer cells with VCR-induced resistance

BackgroundLaryngeal cancer tends to have a very poor prognosis due to the unsatisfactory efficacy of chemotherapy for this cancer. Multidrug resistance (MDR) is the main cause of chemotherapy failure. The proto-oncogene c-fos has been shown to be involved in the development of MDR in several tumor types, but few studies have evaluated the relationship between c-fos and MDR in laryngeal cancer. We investigated the role of c-fos in MDR development in laryngeal cancer cells (cell line: human epithelial type 2, HEp-2) using the chemotherapeutic vincristine (VCR).MethodsHEp-2/VCR drug resistance was established by selection against an increasing drug concentration gradient. The expressions of c-fos and multidrug resistance 1 (mdr1) were measured using qPCR and western blot. C-fos overexpression or knockdown was performed in various cells. The intracellular rhodamine-123 (Rh-123) accumulation assay was used to detect the transport capacity of P-glycoprotein (P-gp, which is encoded by the mdr1 gene).ResultsHEp-2 cells with VCR-induced resistance (HEp-2/VCR cells) were not only resistant to VCR but also evolved cross-resistance to other chemotherapeutic drugs. The expressions of the c-fos and mdr1genes were significantly higher in the HEp-2/VCR cells than in control cells. C-fos overexpression in HEp-2 cells (c-fos WT) resulted in increased P-gp expression and increased the IC50 for 5-FU. C-fos knockdown in the HEp-2/VCR cells (c-fos shRNA) resulted in decreased P-gp expression and decreased IC50 for 5-FU. An intracellular Rh-123 accumulation assay showed that the mean intracellular fluorescence intensity (MFI) was lower in the HEp-2/VCR cells than in HEp-2 cells. C-fos WT cells also showed lower MFI. By contrast, c-fos shRNA cells exhibited a higher MFI than the control group.ConclusionC-fos increased the expression of P-gp and mdr1 in the HEp-2/VCR cells, and enhanced the efflux function of the cells, thereby contributing to the development of MDR.

Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have.Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation.Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP).To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer.Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP.Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions.To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses.Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p<0.01; ADP-induced PAC1 binding: 63±14 % in controls and 50±23 % in ITP, p<0.05). Diminished responses to activation were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients.Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p<0.05].Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p <0.02; MF for TRAP-induced FVa binding: 44.1±11.4 in controls, 81.4±38 in ITP, p<0.001; MF for basal FXa binding: 45.7±18.4 in controls, 58.1±24 in ITP, p <0.005; MF for TRAP-induced FXa binding: 46.1±16.4 in controls, 72.0±24 in ITP, p<0.05). The lower the platelet count the higher increase in PS exposure (Spearman r =-0,518, p <0.001) and the union of FVa (Spearman r = -0.8571, p <0.001) and FXa (Spearman r = -0.7455, p<0.05).Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP.This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457 DisclosuresNo relevant conflicts of interest to declare.

Temporal patterns in innate immunity parameters in reef‐building corals and linkages with local climatic conditions

AbstractExtremes in seasonal environmental conditions can significantly impact the health and physiological functioning of reef corals, underscoring the need for knowledge of seasonally specific baselines from which to monitor and forecast impending stress. Increases above summertime means in seawater temperature, sunlight intensity, turbidity, or sedimentation may reduce coral immunocompetency and increase disease and bleaching susceptibility. We analyzed temporal patterns in innate immunity parameters over nine time points throughout one year to establish baseline levels from which anomalies might be detected for representative species from three major reef‐building coral families (Acroporidae, Faviidae, and Poritidae). Temporal patterns in both phenoloxidase activity and expression of green fluorescent protein‐like proteins varied among the three families, as did overall constitutive levels. For example, Porites cylindrica had 2.8‐fold higher yearly average levels of phenoloxidase activity than Acropora millepora, which had the lowest levels. In contrast, mean fluorescence was lowest in Acropora millepora and highest in Echinopora mammiformis. Relationships between the potential physical drivers (seasonal variation in seawater temperature, rainfall, salinity) and temporal patterns in these parameters also differed among the three species. For example, phenoloxidase activity was positively correlated with seawater temperature in A. millepora, but negatively correlated in both E. mammiformis and P. cylindrica. Distinctions in constitutive levels and temporal patterns in these parameters among species suggest that corals from these three families have evolved different strategies for investing resources into innate immune parameters. Such differences highlight the need for species‐specific baselines and long‐term assessments to accurately predict coral reef trajectories in rapidly changing environments.

Assessment of myeloid and monocytic dysplasia by flow cytometry in de novo AML helps define an AML with myelodysplasia-related changes category

AimsIn recent years, multiparameter flow cytometry has been increasingly recognised as an important tool in diagnosis of myelodysplastic syndrome and acute myeloid leukaemia (AML). Assessment of myeloid and monocytic ‘immunophenotypic’...

Polycythemia of Mice with Human Gain-of-Function EPOR (mtHEPOR) Is Transiently Corrected in Perinatal Life in Association with Low Epo and Increased Erythrocyte Phosphatidylserine Exposure

Gain-of-function mutations of erythropoietin receptor (EPOR) resulting from truncation of the cytoplasmic domain of EPOR are associated with primary familial and congenital polycythemia (PFCP). We generated and previously reported a PFCP mouse model where the murine EpoR gene (mEpoR) was replaced with wild-type human EPOR (wtHEPOR) or mutant human EPOR (mtHEPOR) genes. We showed that mtHEPOR mice become polycythemic at 3-6 weeks of age, but not at birth, similar to the polycythemia phenotype of affected humans, while the wtHEPOR mice were anemic (Divoky et al, Proc Natl Acad Sci U S A 2001;98:986). In addition, we previously reported (Yoon et al, ASH Annual Meeting Abstracts 2005 106: Abstract 567) that mtHEPOR fetuses are polycythemic at the mid- and late-gestation stages (embryonic days, E12.4-18.5), while wtHEPOR fetuses are anemic, similar to what we observed in adult mice. The mtHEPOR fetal liver cells (FLCs) revealed augmented and sustained activation of Stat5, while wtHEPOR FLCs had significantly reduced Stat5 activation. We also previously presented (Yoon and Prchal, ASH Annual Meeting Abstracts 2007 110: Abstract 3664) that the fetal polycythemia of mtHEPOR mice is associated with a delayed switch from primitive to definitive erythropoiesis, and that polycythemia in mtHEPOR mice is transiently corrected in perinatal life and reappears at 3 weeks after birth. Here, we hypothesized that the change of oxygen tension at delivery (from the uterus to ambient atmosphere) and differences in Epo levels may account for the inexplicable perinatal absence of polycythemia in mtHEPOR mice.Epo levels in adult mice were substantially elevated in wtHEPOR (1271±240 pg/mL) and significantly reduced in mtHEPOR (98±51 pg/mL) when compared to mEpoR mice (265±62 pg/mL). We then proposed that destruction of young red blood cells (RBCs), termed neocytolysis (a mechanism that overcorrects increased RBC mass generated during chronic hypoxemia after restoration of normoxic conditions) contributes to the anemia of newborns (Song et al, J Mol Med 2015;93:857). When adult mice were challenged by rapid conversion from 10 days of hypoxia (12% of O2) to normoxia, the mtHEPOR mice had greater changes of hematocrit (drop of 16.9%) than other genotypes (drop of 14.8% for mEpoR and13.8% for wtHEPOR). Similarly, the most pronounced decrease of hematocrit after birth, at perinatal day 7, (PN7) was detected in mtHEPOR mice (drop of 56%) followed by mEpoR (drop of 48%) and only a relatively moderate decrease of hematocrit in wtHEPOR neonates (drop of 24%). In parallel, PN7 mtHEPOR newborns had the lowest levels of Epo (310±102 pg/mL), while PN7 wtHEPOR neonates had Epo levels markedly increased (2757±912 pg/mL); the Epo levels of control mEpoR at PN7 littermates were 494±102 pg/mL. This suggests that Epo protects from neocytolysis. These changes corresponded to exposure of phosphatidylserine; a stimulus for RBC removal from circulation. There was a gradual increase of phosphatidylserine-positive RBCs in mtHEPOR neonates and, to a lower extent, also in mEpoR between PN0 and PN7 with a maximum positivity at PN7 (mean fluorescence: 6.5±2.8 and 6.2±1.7 for mtHEPOR and mEpoR, respectively). This correlated with a maximal decrease of hematocrit and lowest Epo levels at PN7 in mtHEPOR. The proportion of phosphatidylserine-positive RBCs then declined with age (mean fluorescence of 0.6 in adult mtHEPOR mice). In contrast, wtHEPOR mice had the lowest numbers of phosphatidylserine-positive RBCs, which were comparable at all analyzed time-points (mean fluorescence at PN0=0.7±0.1; PN7=1.3±0.3; in adults=0.6±0.04), consistent with the lowest reduction in perinatal hematocrit and very high Epo levels in these mice.In conclusion, our study describes that transient correction of polycythemia of the mtHEPOR PFCP mouse model in the perinatal period is associated with low Epo levels and increased erythrocyte phosphatidylserine exposure. Increased phosphatidylserine exposure on the membrane of mtHEPOR RBCs is consistent with accelerated destruction of these cells by macrophages, thus contributing to the decrease of hematocrit in mtHEPOR neonatal mice.Authorship: JS and BK: equal credit as first authorsAcknowledgment: VD and MH were supported by the Czech Science Foundation (project P301/12/1503) and by the Ministry of Health Czech Republic (project NT13587). DisclosuresNo relevant conflicts of interest to declare.

Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface. Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys539 with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using XaAlexa488 as donor and VaAlexa532 as acceptor. XaAlexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of VaAlexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of VaAlexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of VaAlexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of XaAlexa488 in the absence of Va. A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell. DisclosuresNo relevant conflicts of interest to declare.

Inverted Erythrocyte Membranes Are a Novel Platform for Expressing Phosphatidylserine and Investigating the Antiphospholipid Syndrome

Background: The antiphospholipid (aPL) syndrome (APS) is an acquired thrombophilic disorder in which autoantibodies target proteins bound to anionic phospholipids; the mechanisms involved have not yet been clearly established. Anionic phospholipids are normally present in the internal leaflets of cytoplasmic membranes and become expressed after cell injury or death. Current diagnosis of APS is based on a panel of non-mechanistic "criteria" tests, which consist of the paradoxical lupus anticoagulant (LA) and immunoassays for autoantibodies binding to anionic phospholipids (cardiolipin) or β2glycoprotein I. Also, there has been difficulty in identifying biologically relevant and robust phospholipid platforms for analyzing aPL-membrane interactions. We therefore decided upon a novel path by: 1) developing a cell-based model consisting of inverted erythrocyte membranes (iEMs) and 2) developing novel ways of detecting aPL-iEM interactions.Materials & Methods: iEMs were prepared from erythrocytes using an established protocol, suspended in 20 mOsm Tris buffer and used fresh or stored at -80°C. iEMs were incubated with normal or patient plasmas for a minimum of 30 minutes and washed by centrifugation at 15,000g. Binding of IgG to iEMs was investigated by goat anti-human IgG-Alexa 633 (GαH IgG-Alexa 633) in flow cytometry. PS exposure on membranes was confirmed by microscopy with annexin A5 (A5)-Oregon green and A5-Alexa 647 binding in flow cytometry. Bright field images of aggregated membranes were obtained to visually assess aggregation. The potential of iEMs to serve as phospholipids necessary for coagulation was assessed through calibrated automated thrombinography.Results: Incubation of iEMs with normal and aPL plasmas resulted in increased IgG binding to the iEMs, as determined by flow cytometry (figure 1A). The IgG staining was more pronounced with aPL compared to normal plasmas (mean fluorescence ± standard deviation; aPL (n = 3): 10004 ± 5416 versus healthy (n = 3): 4055 ± 1680). When studying these samples with bright field microscopy aggregate formation of iEMs was observed. The aggregates induced by aPL plasma were more numerous and larger than those forming in plasma of healthy donors (figure 2A-B). Amplified agglutination was observed in the presence of broad spectrum anti-hIgG/anti-hC3d reagent (figure 2C-D).Fluorescence microscopy of iEMs incubated with A5-OG confirmed that a population of iEMs was exposing PS. Thrombin generation (TG) studies established that iEMs contribute to coagulation reactions by providing a source of anionic phospholipids. TG increased dose-dependently with higher levels of iEMs until a plateau concentration was reached. Additionally, A5 resistance was investigated by means of flow cytometry. The aPL plasmas induced a decrease in A5 binding to iEMs compared to normal plasma and the control conditions (figure 1B), consistent with the functional A5 resistance assay.Discussion: We have developed a novel model for a more physiological approach to phospholipid based assays in APS using iEMs as a source of anionic phospholipids. They are easily prepared by hypotonic lysis of erythrocytes and can be frozen for storage until use.We have thus far demonstrated that a higher level of iEM aggregation is induced by aPL samples compared to healthy plasmas. Moreover, iEMs expose PS and dose-dependently accelerate TG by functioning as a platform for coagulation. Previous findings on A5 resistance induced by aPL plasma are confirmed with this iEM model.Conclusion: IEMs offer a novel and robust biologically-derived platform; they express PS, promote coagulation, bind aPL antibodies and open different approaches to investigate APS. [Display omitted] [Display omitted] DisclosuresDe Laat:Synapse bv: Employment.

The Effect of Time on Bone Fluorescence: Implications for Using Alternate Light Sources to Search for Skeletal Remains.

Bones fluoresce when exposed to certain wavelengths of shortwave light, and this property can be useful in locating and sorting skeletal remains in forensic contexts. The proteins in bone collagen are largely responsible for its fluorescent properties, but these proteins degrade and denature over time. This study examined the fluorescence of bones from four temporal groups (recent, semi-recent, ancient, and historic) ranging from 0 to 1064 years before present. Specimens were photographed under 490 nm wavelength light, and fluorescence was quantified by converting intensity to a gray scale value based on the RGB color model using ImageJ(®) software. Significant (p < 0.05) differences were found in mean fluorescence between all four temporal groups, and a 0.324 coefficient of correlation indicates a significant (inverse) relationship between fluorescence and time. Bone fluorescence decreases with time, but some fluorescence is retained even in older samples. Fluorescence can therefore be reliably used in many modern skeletal remains searches.