Mean Intensity Of Fluorescence Research Articles

Overexpression of KMT2E Is Associated with Higher Granulocytic Differentiation Rate in Response to ATRA-Based Treatment in Acute Promyelocytic Leukemia Cell Lines

Introduction: In the clinical setting, low KMT2E expression has been associated with poor prognosis in both acute myeloid leukemia (Damm et al. J Clin Oncol. 2011 Feb 20;29(6):682-9) and acute promyelocytic leukemia (APL) (Lucena-Araujo et al. Br J Haematol. 2014 Aug;166(4):540-9). Member of the Trithorax-group, the KMT2E is implicated in the positive transcriptional control of a specific set of genes related to hematopoiesis and differentiation processes in many cell types. Functional analyses showed that the encoded protein is involved in terminal myeloid differentiation and facilitate retinoic-acid induced granulopoiesis in human promyelocytes. Using lentiviral gene transfer, we hypothesized that the forced expression of KMT2E is implicated in the ATRA-induced granulocytic differentiation in APL. Aims: To investigate the effects of overexpression of KMT2E gene in cell viability and signaling in response to ATRA plus anthracycline or arsenic trioxide (ATO) treatment in APL cell lines. Methods: NB4 and NB4-R2 (resistant to ATRA treatment) cell lines were transduced with pMEG or pMEG- KMT2E lentiviral vectors and cultured with stable selection using puromycin. Before all functional experiments, cells were tested for KMT2E gene and protein expression by qPCR and confocal microscopy, respectively. The cells transduced with KMT2E and control cells were evaluated for cell viability (MTT assay), proliferation (Ki-67 staining), cell cycle (flow cytometry), mitochondrial reactive oxygen species (ROS) production (flow cytometry) in basal conditions and under treatment with ATRA (1 μM), ATO (0.187, 0.375, 0.75, 1.5 and 3 μM), and the association of ATO (1 μM) plus ATRA (1 μM) or cytarabine (150 nM). The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b expression (flow cytometry) plus morphological analysis (Leishman staining). The expression of the main target genes (p21, MDM2, and BMI1) encoding for proteins known to be involved in KMT2E function were evaluated by qPCR. Results: Overexpression of KMT2E was significantly associated with higher cell proliferation rates in basal or ATO and cytarabine treatment compared to vehicle treated cells (all P<.001). KMT2E overexpression significantly increase cell proliferation in NB4, but not in NB4-R2 cells (Ki-67 mean intensity of fluorescence (MFI) value - pMEG: 489±10 vs KMT2E : 1432±25; P<.001). The distribution of cells in the phases of the cell cycle was similar between groups. Using a nonlinear regression analysis, IC50 for ATO was of 2.96 and 6 μM for NB4 pMEG and KMT2E transduced cells, respectively. NB4- KMT2E cells showed lower levels of ROS generation upon ATO treatment (1.5 μM) compared to controls (ROS - MFI value - pMEG: 270.6±6 vs KMT2E : 84±11; P <.001). In the attempt to assess the role of KMT2E in granulocytic differentiation, NB4- KMT2E cells were treated with ATRA 1 μM. The cells containing an extra copy of KMT2E gene showed more differentiation rate in 72, 96 and 120 hours compared to empty vector transduced cells (all p<.05). Considering the expression of target genes of KMT2E, only p21 showed an increase in KMT2E cells treated with ATRA. Conclusion: In summary, the overexpression of KMT2E is associated with higher proliferation capacity and increased resistance to ATO treatment, associated with less ROS generation. DisclosuresNo relevant conflicts of interest to declare.

Inflammatory Signature, Oxidative Stress, and DNA Damage Response in DBA Pathogenesis

Diamond-Blackfan anemia (DBA) is an inherited pure red cell aplasia caused mostly by pathogenic variants in genes encoding for ribosomal proteins (RPs). It was previously published that haploinsufficiency of RP leads to increased apoptosis of erythroid precursors and p53 upregulation in CD34+cells from DBA patients and in animal models. However, the exact mechanisms of p53 activation are still not completely understood. In our study, we examined the link between activation of p53 pathway, inflammation-evoked oxidative stress and DNA damage in DBA patients and cellular model.To investigate the pathogenesis of DBA, we prepared the RPL5- and RPS19-deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. The particular clones were heterozygous for different nucleotide size deletions in RPL5 or RPS19 genes resulting in decreased levels of these proteins to half when compared to control cells thus mimicking the RP haploinsuficiency in DBA patients. We examined the pathological consequences of RPL5 and RPS19 deficiency in both undifferentiated and differentiated cells; the differentiation was induced by either dimethyl sulfoxide - DMSO or hexamethylene bisacetamide - HMBA for at least 48 hours. Significantly decreased proliferation capacity and significantly increased apoptosis rate was detected for all undifferentiated RPL5- and RPS19-deficient clones in comparison with controlcells. Upon induction of erythroid differentiation, RPL5- and RPS19-deficient clones showed abnormal GATA1 expression (2-times lower than in control cells) and higher percentage of TUNEL+ apoptotic cells (RPL5-deficient cells 2.04%; RPS19-deficient cells 1.57%;controlcells 0.50%).The data from cellular models were consistent with an increased rate of apoptosis detected in the bone marrow of RPL5 and RPS19 mutant DBA patients by TUNEL assay (15-times more TUNEL+ cells in RPL5 and RPS19 mutant patients than in normal controls). These patients showed also induction of p53 in their bone marrow (p53 positive cells: 8.4% in RPL5-mutant patient and 7.3% in RPS19-mutant patient vs. 0.4% in normal controls). The activation of p53 was also confirmed in RPL5- and RPS19-deficient clones by detection of increased level of p53 phosphorylation.Because oxidative stress is known to induce p53, we examined oxidative DNA damage marker 8-oxoguanine (8-oxoG) in MEL-DBA clones. Cytospin slides of differentiated RPL5- and RPS19-deficient clones revealed elevated positivity for 8-oxoG in contrast to control cells. It is known that oxidative damage is caused by exposure of cells to inflammatory cytokines, which activate DNA damage response (DDR) through reactive oxygen species (ROS)-mediated DNA damage. We found upregulation of pro-inflammatory cytokines TNF-α and IL6 in RPL5- and RPS19-deficient cells and IL1b in RPS19-deficient cells. The increased expression of Map3k8 kinase or increased phosphorylation of p38 was detected in RPL5-deficient or RPS19-deficient cells; both of these molecules are known to play essential roles in the production of pro-inflammatory cytokines. We then analyzed the levels of pro-inflammatory cytokines in our DBA patients and observed that besides TNF-α, IL1b, and IL6 also other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF-γ, and GM-CSF) were upregulated in their serum. We also detected higher levels of ROS in differentiated MEL-DBA clones in comparison with controlcells. Increased ROS levels were also present in the serum of all DBA patients with elevated inflammatory cytokines in contrast to healthy controls. As a result of ROS production, DNA double-strand breaks can occur responding by phosphorylation of Ser-139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RPL5- and RPS19-deficient cells in comparison with control cells (mean intensity of fluorescence for RPL5-deficient cells: 2.63 ±0.34; RPS19-deficient cells: 3.29 ±0.41; and control cells: 1.02 ±0.22).Our results suggest that the defective ribosomal biogenesis is associated with the induction of inflammatory cytokine signature, oxidative stress, increased DNA damage and activation of DDR signaling. Further research is needed to elucidate the hierarchy of these processes.This study was supported by project GACR 15-13732S, AZV 16-32105A, IGA_LF_2017_015, and in part by LTAUSA17142 (BK). DisclosuresNo relevant conflicts of interest to declare.

Quantitative Assessment of Age-dependent Changes in Porphyrins from Fluorescence Images of Ultraviolet Photography by Image Processing

BackgroundSkin is the first line of defense against harmful external environmental factors. Skin flora living on the skin surface impact skin health and skin disease. Bacteria, form part of the unique and complex skin micro-ecological system. For example, Propionibacterium acnes (P. acnes) is a member of the anaerobic organisms and is involved in the induction of skin acne. It produces porphyrins that absorb ultraviolet light and emit red fluorescence in response. As a result, fluorescence surveillance of the skin can be important in both the diagnosis of skin acne and the evaluation of therapeutic effects. Many different measurement methods for single skin biophysical properties have been reported.. This study focused on the age-dependent changes in porphyrins for normal skin, and developed a novel algorithm to evaluate porphyrins using the fluorescence images by image processing quantitatively. Materials and methodsAn extraction algorithm was proposed for the segmentation of porphyrin fluorescence images in OpenCV. The algorithm consisted primarily of preprocessing, conversion from RGB color space to HSV color space, and classification of fluorescence. There are 3595 healthy Japanese aged 16–85 years enrolled in the study and fluorescence images were acquired from their cheek sites under 375 nm UV-LED excitation. Age-related fluorescence variation was conducted applying the algorithm implemented. ResultsA new extraction algorithm has been proposed with fluorescence image input and three indexes output, including the number of fluorescence, area of fluorescence, and mean intensity of fluorescence. Proposed algorithm was verified by three parameters, the accuracy, sensitivity, and precision, which refer to the ability of algorithm to detect the number of fluorescence correctly and repeatedly. The verification results were 71%, 72%, and 88% respectively, taking a validly fundamental step for skin health record and analysis. Furthermore, large-scale fluorescence image segmentation results revealed that similar trends were coming out for all three indexes in cheek as people get older. All the fluorescence number, area and mean intensity arrived at the highest at 30 years old and fell off since then. ConclusionThe number, area, and fluorescence intensity of porphyrins can be extracted well from fluorescence images with the proposed algorithm in the study, which has the potential to aid in thediagnosis of skin acne and predict skin conditions as an assisted tool. It is implicated that fluorescence status is influenced by age, which rises to the peak around 30 years old for normal cheek's skin.

Predicting the risk of tumor progression in patients with early stages of adenocarcinoma and squamous cell lung carcinoma based on laboratory parameters

Non-small cell carcinoma (NSCLC) prevails in the structure of the incidence of lung cancer. In patients with I stage NSCLC, only 60-70% overcome the 5-year survival barrier, and at II stage it decreases to 35-40%. The reason for such a high mortality rate is almost always a relapse of the disease. The main histological forms of NSCLC - adenocarcinoma (AC) and squamous cell carcinoma (SCLC) - differ in the course, protocols and effectiveness of the treatment. Comparative survival data for AK and PCLC are controversial, and reliable biomarkers for determining the risk of tumor progression are lacking. In thus study we have investigated the possibility of using laboratory parameters characterizing the level of some blood proteins involved in carcinogenesis in patients with early stages of AC and SCLC to determine the risk of disease progression. We retrospectively analyzed the duration of the relapse-free period after surgical treatment for one year in 1250 patients (816 with stages I and II of adenocarcinoma, G1-3 and 434 with early stages of SCLC, G1-3). In 81 patients with AC and 36 - with SCLC (stages I-II, G1-3) the level of CYFRA 21-1 and SCC by electrochemiluminescent method, chemokines CXCL5, CXCL8, TPA, pyruvate kinase M2, HIF-1α and hyaluronic acid by enzyme immunoassay, receptors CXCR1, CXCR2, CD44v6 by flow cytometry were determined. Using the Kaplan-Meier graphical analysis, groups of low (stage I G1-2 + stage II G1) and high (stage I G3 + stage II G2-3) risk of tumor progression were identified. In the case of the one-year survival rate of patients with AC was higher than with SCLC. In patients with AC and a high risk of tumor recurrence, compared with a low one, the level of CYFRA 21-1, the mean intensity of fluorescence (MFI) of the CXCR1 receptor in granulocytes, and the relative content of the CXCR2 receptor in lymphocytes were higher. In the case of rapid progression of SCLC in patients, the relative content of the CXCR2 receptor in lymphocytes, the proportion of monocytes equipped with the CD44v6 receptor, and the SCC level were higher than with slow progression. Regression equations, including combinations of the above parameters (threshold value for AC - 0,512, for SCLC - 0,409, sensitivity - 91,9% and 90,0%, specificity - 90,0% and 87,5%, respectively), allow to predict the probability of tumor recurrence.

PF345 THE INVOLVEMENT OF OXIDATIVE STRESS, INFLAMMATION, AND DNA DAMAGE RESPONSE IN PATHOPHYSIOLOGY OF DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia mostly caused by different pathogenic variants in ribosomal protein (RP) genes. The ribosomal stress and subsequent activation of the p53 tumor suppressor pathway result in increased apoptosis of erythroid precursors and/or induction of cell cycle arrest.Aims:We studied the contribution of p53 pathway, oxidative stress, and inflammation to the DBA pathophysiology.Methods:We analyzed the bone marrow and peripheral blood samples from DBA patients listed in the Czech DBA Registry. We also prepared the RPL5‐ and RPS19‐deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. Activation of p53 pathway, oxidative DNA damage, and pro‐inflammatory cytokines were assessed in undifferentiated and differentiated RPL5‐ and RPS19‐deficient cells; the differentiation was induced by dimethyl sulfoxide (DMSO) for at least 48 hours.Results:Created RP‐deficient MEL cells presented the main phenotypic features of DBA patients’ erythroid cells: decreased levels of RPL5 or RPS19 proteins, decreased proliferation capacity, increased apoptosis, and abnormal GATA1 expression (2‐times lower than in control cells) upon differentiation. The increased p53 activation was also confirmed in RPL5‐ and RPS19‐deficient cells consistent with increased p53 expression in the bone marrow of DBA patients (p53+ cells: 8.4% in RPL5‐ and 7.3% in RPS19‐mutant patient vs. 0.4% in normal controls). One of the known inducer of p53 is oxidative stress causing oxidative DNA damage. Indeed, elevated positivity for 8‐oxoguanine (8‐oxoG), a marker of oxidative DNA damage, was detected in differentiated RP‐deficient MEL clones and in DBA patients’ samples in contrast to control samples. It is known, that reactive oxygen species (ROS) are produced under inflammation resulting in ROS‐mediated DNA damage response (DDR). The upregulation of pro‐inflammatory cytokines TNF‐α, IL6, and IL1b was found in RPL5‐ and RPS19‐deficient cells and also in the serum from DBA patients along with the upregulation of other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF‐γ, and GM‐CSF). Moreover, elevated pro‐inflammatory cytokines play a role in cellular senescence which was confirmed in RP‐deficient MEL clones by significantly elevated β‐galactosidase activity. In addition, increased ROS levels were detected in all differentiated RPL5‐ and RPS19‐deficient cells and in the serum of all DBA patients with elevated inflammatory cytokines. Finally, the DNA double‐strand breaks can occur as an outcome of ROS production responding by phosphorylation of Ser‐139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RP‐deficient MEL clones in comparison with control cells (mean intensity of fluorescence for RPL5‐deficient cells: 2.63 ± 0.34; RPS19‐deficient cells: 3.29 ± 0.41; control cells: 1.02 ± 0.22).Summary/Conclusion:The defective ribosomal biogenesis is associated with abnormal GATA1 expression and augmented apoptosis of erythroid cells. Our results show that the upregulation of inflammatory cytokines, senescence, oxidative stress, increased DNA damage, and activation of DDR signaling are also playing an important role in DBA pathogenesis. Our results also suggest the possibility of using the anti‐oxidative and anti‐inflammation therapy in DBA patients as the additive treatment for moderating DBA phenotype.Grants support: AZV 16–32105A, IGA_LF_2019_006, and by LTAUSA17142 (BK,LL).

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay.

Can a discriminant threshold be determined for human sperm DNA oxidation? A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa; optimized consensus protocols are lacking and no thresholds of normality have been established. Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. This work was funded by institutional grants from the CNRS, INSERM and Université Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

Perfusion of the Rotator Cuff Tendon According to the Repair Configuration Using an Indocyanine Green Fluorescence Arthroscope: A Preliminary Report

Background: The disturbance of rotator cuff tendon perfusion has been connected with the suture-bridge configuration repair (SBCR) technique; however, in vivo assessments of the tendon blood supply have been problematic with other modalities. An evaluation of tissue perfusion by an indocyanine green (ICG) fluorescence arthroscope has been developed to counteract this difficulty. Purpose: To verify the hindrance of perfusion in SBCR, we used an ICG fluorescence camera to compare parallel-type transosseous repair (PTR) and SBCR in rabbits immediately and at 3 days after rotator cuff repair. Study Design: Controlled laboratory study. Methods: Acute rotator cuff repair was performed on the shoulders of 10 rabbits. Both shoulders were repaired using either PTR or SBCR. For PTR, simple repair was performed through 2 parallel transosseous tunnels created using a microdrill. For SBCR, 2 additional crisscross transosseous tunnels were added to mimic arthroscopic SBCR. Immediately after repair, ICG was injected through the ear vein, and images were recorded using an ICG fluorescence camera. Tendon perfusion was compared by measuring fluorescence intensity using ImageJ software in both methods. At 3 days after rotator cuff repair, a reassessment of ICG fluorescence was performed. In addition, as a subsidiary study, a comparison of each repair method and a healthy tendon was performed (PTR vs healthy tendon and SBCR vs healthy tendon). Six rabbits (3 for each comparison) were included. Results: Immediately after rotator cuff repair, the mean (±SD) grayscale intensity of ICG fluorescence was weaker in SBCR than PTR in 10 specimens (65.9 ± 47.6 vs 84.3 ± 53.4 per pixel, respectively; P = .003). At 3 days after repair, 8 specimens were included in the analysis because suture strands failed in 2 specimens in SBCR. The mean intensity of fluorescence was still weaker in SBCR compared with PTR (52.5 ± 13.7 vs 60.2 ± 22.7 per pixel, respectively; P = .077). The mean fluorescence intensity compared with a healthy tendon was 83.2% ± 9.5% in PTR and 63.2% ± 13.2% in SBCR. Conclusion: Our ICG fluorescence camera system was able to detect ICG fluorescence in an acute rabbit rotator cuff repair model. SBCR showed inferior tendon perfusion immediately after repair. At 3 days after repair, SBCR still showed inferior fluorescence intensity, although it did not reach statistical significance. Clinical Relevance: In this study, SBCR hindered perfusion at the tendon in the compressed area. This finding may affect rotator cuff tendon healing and failure mode.

Vascular Endothelial Growth Factor Expression in CD138+/CD19- and CD138+/CD19+ Plasma Cells in Monoclonal Gammopathies and Impact on Multiple Myeloma Prognosis

INTRODUCTIONVascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of extracellular matrix remodeling and inflammatory cytokine generation with an important role in the bone marrow microenvironment of multiple myeloma (MM). Angiogenesis is enhanced in the bone marrow of MM patients in parallel with tumor progression. Myeloma and stromal cells secrete angiogenic factors that include VEGF. Previous studies showed heterogeneity in the expression of VEGF between plasma cells (PCs) from the same MM patient. However, no clear association with expression levels, phenotypic subtypes of PCs and prognosis was demonstrated. MATHERIALS AND METHODSBone marrow PCs from 128 patients with monoclonal gammopathies, 60 patients with newly diagnosed symptomatic MM and 68 with monoclonal gammopathy of uncertain significance (MGUS) and also from 11 non-neoplastic controls (Ctr) were analysed between April 2010 and July 2013. We evaluated the expression of cytoplasmic VEGF with monoclonal antibodies by flow cytometry in the two populations of PCs, identified by gating CD138+/CD19- (clonal PCs) and CD138+/CD19+ (non-clonal PCs). The results are presented as percentage of PCs expressing VEGF and as expression levels of VEGF in mean intensity of fluorescence (MIF). The effects of VEGF expression on progression-free survival (PFS) and overall survival (OS) were analysed. For statistical analysis, software IBM SPSS Statistics v22 was used. Survival was estimated according to the Kaplan-Meier method. RESULTSIn our cohort of patients, median age was 70 (39-86) years, 52% were male. We found increased expression levels of VEGF in CD138+/CD19- PCs from MM (80 ± 7,5 MIF) compared to MGUS patients (61,7 ± 6,2 MIF) (p=0,011), as well as superior to CD138+/CD19+ PCs expression (39,92 ± 1,74 MIF) in both populations of patients (p<0,001 and p=0,02, respectively). No diferences were observed in the expression levels of VEGF in CD138+/CD19+ PCs from MM (39,92 ± 1,74 MIF), MGUS patients (41,18 ± 1,92 MIF) and controls (32,8±1,5 MIF). However, the percentage of CD138+/CD19+ expressing VEGF was significantly higher in MGUS (39,4±4%) and in MM patients (46,7±4,5%) compared to Ctr (13,5±0,5%)(p=0,019 and p=0,003, respectively). In MM patients, we also found an association between increased VEGF expression levels in CD138+/CD19- PCs (superior or equal to 175 MIF) and inferior PFS (p=0,002) and OS (p=0,003), irrespective of first line therapy (bortezomib-based regimens for fit patients or alkylating-based treatments for unfit patients). Interestingly, we also observed an incresed percentage of CD138+/CD19+ PCs (higher or equal to 21%) expressing VEGF in MM patients with a more favorable PFS (p= 0,04) and OS (p=0,008). CONCLUSIONSThe results of our investigation showed that CD138+/CD19- and CD138+/CD19+ PCs have diferences in what concerns VEGF expression, not only in MM patients, but also in MGUS patients. The increased expression of VEGF in clonal PCs from MM compared to MGUS patients evidences the relevance of VEGF in myelomagenesis. We also demonstrated a negative prognostic impact of an increased VEGF expression in CD138+/CD19- PCs, highlighting the role of VEGF in the survival and maintenance of clonal PCs and as a predictor of outcome in MM progression. The association between the percentage of CD138+/CD19+ PCs and survival supports the suggestion that these cells may not be neutral players in the complex pathogenesis of MM. The results of our study should be further investigated in larger series of patients. DisclosuresGeraldes:Celgene: Employment, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Characterization of an allotriploid strawberry Fragaria × bifera Duchesne (Rosaceae) from Europe

Allopolyploidy has played an important role in the plant evolution. To assess its role in speciation, it is necessary to examine fertility and crossability of hybrids. A hybrid clone of the genus Fragaria with different and complex morphology compared to F. vesca, F. viridis and F. moschata, was detected in Germany (in Bayreuth, Bavaria). The genome size of these plants was measured using flow cytometry and their fertility was tested in experimental crossing. The parental origin of the hybrid was revealed using RAPD approach. From the mean intensity of fluorescence emitted by PI-stained nuclei for F. moschata, F. vesca, F. viridis and the hybrid, triploidy of the hybrid could be indicated. The hybrid shared an 1800bp and 880bp long species-specific RAPDs bands with F. viridis and F. vesca, respectively, indicating them as the parental species of the hybrid. The hybrid did not produce any fruit in selfing, open pollination and when crossed by pollen of F. vesca and F. viridis, all showing female sterility of the hybrid. The hybrid had 78% pollen sterility, however, pollinating F. vesca by pollen of the hybrid produced viable seed and F1 plants, indicating its male fertility. This work shows allopolyploidy role in the evolution and speciation of Fragaria, and may suggest the study site as potential new centre of Fragaria speciation.

Erythroid Adhesion Molecule Expression Profile in Sickle Cell Anemia Infants

Introduction: In normal conditions, circulating red blood cells (RBCs) are not supposed to adhere. Some erythroid adhesion molecules expressed on young reticulocytes are therefore lost upon maturation. Conversely, abnormal RBC adhesiveness in sickle cell anemia (SCA) through activation, sustained or increased expression of adhesion molecules are considered central in vaso occlusive crisis (VOC), the hallmark of SCA.SCA is seldom symptomatic in the first six months of life. One main explanation lies in the sustained level of fetal hemoglobin (HbF) and F cells during this period preventing HbS polymerization. However, infra clinical vaso occlusion, particularly in the spleen, has been demonstrated to be present and the absolute reticulocyte count is paradoxically elevated in the first semester of life, evidencing the very early onset of hemolysis.Our objectives were to analyze the profile of erythroid adhesion molecules in SCA and non-SCA infants in combination with HbF level, in order to evidence significant differences specifically attributable to SCA, informative on very early pathophysiology and disease severity.Patients and methods: Infants were enrolled in a longitudinal multi center prospective study on prognostic factors in SCA between September 2010 and March 2013. Blood sampling was performed at enrolment (3-6 months) at steady state, in asymptomatic infants. In parallel, blood samples from infants with no hemoglobinopathy were collected. Flow cytometer analysis (BD FACSCanto II) was performed using murine monoclonal antibodies against CD36, CD44, CD47, CD49d, CD58, CD99, CD147, CD239 and CD242. Statistical analysis was performed with Graphpad software, Prism 6. Differences in the percentage of positive cells and the level of expression of molecules between groups were calculated with Mann Whitney test. Results were considered statistically significant at an a-risk level of 5%.Results:Fifty-four SCA infants were included and compared to 17 non-SCA infants. Median age in the two groups was not statistically different (144 days, range 81-196 versus 128, range 68-621, p=0,84). Mean HbF level was, as previously described, statistically increased in SCA compared to non-SCA infants (41.2% +/-11.2 versus 10,4 % +/- 1,8; p<0,0001). Median reticulocyte count tended to be more elevated in SCA infants (2.6 % range 0,5-10) compared to non-SCA infants (2 range 1-3,1) without reaching statistical significance (p=0,052).Reticulocytes from SCA infants display a distinct surface molecule expression profile, with a statistically increased percentage of reticulocytes expressing the following adhesion molecules: CD239 (Lu/BCAM), CD242 (ICAM-4), CD47, CD99, CD58, CD147 and CD44 (Figure 1). Furthermore the mean intensity of fluorescence is statistically increased concerning CD239, CD58 and CD242 (data not shown). Surprisingly, known adhesion markers demonstrated to play an important role in SCA pathogeny i.e. CD36 and CD49d (α4β1/Very Late Antigen-4) are not significantly increased in SCA infants (Figure 1)No significant difference of expression profile was found on mature or total RBCs between SCA and non-SCA infants (data not shown). [Display omitted] Discussion: Recent publications have pointed to the significance of reticulocytosis associated with an increased risk of death or stroke during childhood. Our results further demonstrate that this early rise of reticulocyte count in SCA infants, despite elevated HbF level, include reticulocytes with a specific adhesion molecule expression profile. This numerically small subpopulation of red cells may play in fact a major role in this complex disease. An interesting hypothesis may be that the spleen, as long as its function is preserved, prevents VOC by trapping this subpopulation in very young infants. As spleen function decreases, proadhesive reticulocytes remain in the circulation, which may, in combination with the decline of HbF, subsequently favor extra splenic VOC.Ongoing analysis will confirm if this idiosyncratic erythroid adhesion molecule profile in infants indeed correlates with HbF level and, importantly, correlates with functional activation, in order to serve as a readily available biomarker of disease severity. DisclosuresDe Montalembert:Novartis: Speakers Bureau. Le Van Kim:SHIRE: Research Funding.

Abstract 9919: PH-low Insertion Peptide (pHLIP) Targets Ischemic Myocardium

pHLIP molecule (∼3 kD) inserts into a lipid bilayer when ambient pH is low. Originally, this property was used for targeted delivery of small molecules, liposomes and nanoparticles conjugated with pHLIP to visualize and treat tumors. Since cardiac ischemia causes a decrease in tissue pH we hypothesized that pHLIP can selectively bind to ischemic myocardium. METHODS. We studied pH-sensitive (Var7) and a pH-insensitive (kVar7) variants of pHLIP conjugated with fluorescent dye Alexa488 in three models. (1) Local myocardial ischemia was induced by repetitively occluding (10 min) and reperfusing (5 s) left coronary artery of anesthetized mice for 2 hr; 100 μL of 80 μM pHLIP were administered IV 3 min prior to the first occlusion. In the end, hearts were excised and retrogradely perfused with Evans Blue to delineate the area at risk. Hearts were frozen and sectioned to measure mean intensity of fluorescence (F). (2) Low flow (5% of normal flow rate) global ischemia was induced in Langendorff-perfused mouse hearts paced at 5 Hz and perfused with 1 μM pHLIP for 15 min. (3) Effects of 10 μM pHLIP on transmembrane potential and developed force were studied in isolated preparations of paced (4 Hz) murine left atrial appendages (AA) at normal and low (6.5) pH. RESULTS. In the model of local ischemia, when pH-sensitive Var7 was used, area of bright fluorescence coincided with the area at risk. F in the area at risk normalized to F in intact myocardium was higher with Var7 (3.89±.43, n=4) than with pH-insensitive kVar7 (1.11±.09, n=5, p.05). In global ischemia, F was higher in the hearts perfused with Var7 vs either kVar7 or Var7 in the absence of ischemia (Var7: 76±8, n=5; kVar7: 25±5, n=4; Var7 + no ischemia: 12±4, n=3, p&lt;.05). In AA exposed to pH 6.5 (but not at pH 7.4), F was higher with Var7 than with kVar7 (76±9 vs 28±3, n=5; p&lt;.05). Neither at the normal or low pH, pHLIP variants affected parameters of transmembrane potential or developed force. CONCLUSIONS. pH-sensitive pHLIP selectively binds to ischemic myocardium without affecting either ectrical or mechanical activity. Based on analogy with applications in oncology, pHLIP can be used for targeted delivery of contrasting agents and drugs to ischemic regions of the heart.

Phagocytic and Oxidative Burst Activity of Neutrophils in Patients With Spinal Cord Injury

ObjectiveTo evaluate phagocytic activity and neutrophil oxidative burst functions in patients with spinal cord injury (SCI) because alterations in neutrophil metabolic activity can be one of the causes of immune mechanism damage contributing to repeated bacterial infections. DesignA controlled and cross-sectional study. SettingDepartments of physical medicine and rehabilitation and immunology. ParticipantsPatients with SCI (N=34) and 28 healthy controls. InterventionsPhagocytosis and oxidative burst in whole-blood neutrophils were assessed by flow cytometry. The percentage of phagocytizing cells after in vitro incubation with Escherichia coli, phagocytic activity (mean intensity of fluorescence [MIF]) and the percentage of neutrophiloxidative burst, and the MIF value of the production of reactive oxygen intermediates (ROIs) were analyzed. In addition, clinical assessment including the level of injury, American Spinal Injury Association scores, and functional status were carried out. Main Outcome MeasuresNot applicable. ResultsAlthough the percentage of E. coli phagocytizing neutrophils was not different between groups, the MIF value of absorbed E. coli was significantly lower in patients with SCI than in controls (P<.05). The MIF value of ROI production by neutrophils with both stimulator of phorbol 12-myristate 13-acetate and E. coli was significantly higher in patients with SCI (P<.05). ConclusionsIn patients with SCI, decreased phagocytic activity of neutrophils may be a result of a regulatory mechanism to minimize the deleterious effects of increased neutrophil burst activity.

Novel Method for Evaluating In Vitro Activity of Anidulafungin in Combination with Amphotericin B or Azoles

A combination of drugs possessing different targets has been used as salvage therapy, although without scientific support. In vitro studies validating such combinations are scarce, and the methodology is very laborious and time-consuming. This study proposes a flow cytometric (FC) protocol as an alternative to evaluate the effect of the combination of anidulafungin (AND) with amphotericin B (AMB) and azoles (fluconazole and voriconazole), tested upon 39 and 36 Candida strains, respectively. The concentration assayed in the combination was 0.5× MIC of each drug. The membrane potential marker DiBAC(4)(3) [Bis-(1,3-dibutylbarbituric acid) trimethine oxonol] was used for AND-AMB, and the metabolic marker FUN-1 was used for AND-azoles. Drug interaction was determined by calculating a staining index (SI): the sum of the percentage of depolarized cells (DC) after treatment with drug combinations divided by the DC of the drug alone, and the sum of the mean intensity of fluorescence (MIF) displayed by cells treated with drug combinations divided by the MIF of the drug alone for FUN-1. An SI of <1 means antagonism, an SI between 1 and 4 means no interaction, and an SI of >4 means synergism. The combination of AND and AMB by FC and checkerboard was synergistic for 46 and 43% of isolates and antagonistic for 5 and 8%, respectively. For the combination of AND and azoles, it was synergistic for 36% and antagonistic for 3% by FC and synergistic for 44% and antagonistic for 3% by checkerboard. When the FC method was compared to the gold standard checkerboard method, the agreement was 0.91 (95% confidence interval [95% CI] of 0.88 to 0.94), sensitivity was 0.88 (95% CI of 0.73 to 0.95), and specificity was 0.95 (95% CI of 0.84 to 1). Thus, FC is a rapid and reliable method (<2 h) to assess the effect of antifungal combinations.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.

全血法によるFlow Cytometryを用いた抗好中球細胞質抗体(ANCA)の検出法について

It has been reported that anti-neutrophil cytoplasmic antibody (ANCA) is a specific serological marker for systemic necrotizing vasculitis, such as Wegener's granulomatosis, microscopic polyarteritis and idiopathic crescentic glomerulonephritis. Although the standard method for ANCA detection enplays an indirect immunofluorescence technique (IIF), the method is not objective. We attempted to detect ANCA by a more objective method using flow cytometry. Thirty ANCA positive sera (cytoplasmic in 4, perinuclear in 26) and 20 ANCA negative sera including 2 sera with anti-nuclear antibody (ANA) detected by standard IIF were used for this study. For flow cytometric analysis, heparinized blood was taken from normal healthy volunteers. Neutrophils in the heparinized blood was incubated with human recombinant tumor necrosis factor alpha (rTNF alpha) in order to facilitate reaction between the antigens of ANCA and ANCA. Primed neutrophils were incubated with sera. After washing with phosphate buffered saline, immunofluorescence-labeled antibody against human IgG was added and incubated. The mean intensity of fluorescence (MIF) of the neutrophils was measured by flow cytometry (FACS, Becton-Dickinson CO., U.S.A.) for each sample. ANCA was considered positive by flow cytometric analysis if MIF of the sample was over the mean +2SD for normal human sera. By flow cytometric analysis, ANCA was detected in all ANCA positive samples by IIF and in 3 ANCA negative samples by IIF, of which 2 had ANA. However, it was possible to discriminate serum with positive ANCA from serum with positive ANA by flow cytograms because ANA reacted with the nuclei of both lymphocytes and neutrophils showing two peaks on the histogram.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined Posttransplant Prophylactic IVIg/Anti-CD 20/Plasmapheresis in Kidney Recipients With Preformed Donor-Specific Antibodies: A Pilot Study

This study assesses the immunologic, functional, and histologic course of kidney recipients with preformed donor-specific alloantibodies (DSA) receiving deceased donor kidneys according to two prophylactic strategies that have been sequentially applied posttransplant. The first strategy combined posttransplant quadritherapy/intravenous immunoglobulin (group 1, n=36) and the second added to the above protocol anti-CD20/plasmapheresis (group 2, n=18). All patients had a concomitant evaluation of glomerular filtration rate, protocol biopsies, and DSA mean intensity of fluorescence (MFI) at 3 month and 1 year posttransplant. Peak and day-0 class-I or II DSAmax-MFI were similar in both groups. The rate of acute antibody-mediated rejection (AMR) was similar in both groups (19.6% vs. 16.6%, respectively). At 1 year posttransplant, group 2 was characterized by lower microcirculation inflammation lesions (glomerulitis+capilaritis score of 1.8+/-0.2 vs. 2.7+/-0.2, respectively, P=0.03), a lower rate of transplant glomerulopathy (7% vs. 38%, P=0.02), and a lower rate of chronic AMR (41.3% vs. 13.3%, respectively, P=0.03). The decline in DSA-MFI from day 0 to 1 year was 44%+/-13% in group 1 compared with 80%+/-8% in group 2 (P=0.02). Finally, the 1-year glomerular filtration rate was 43+/-16 vs. 54+/-16 mL/min/1.73 m(2) in groups 1 and 2, respectively (P=0.04). This study raises the possibility that a more intensive day 0 prophylactic immunosuppressive strategy combining intravenous immunoglobulin/anti-CD20/plasmapheresis in this high-risk population, despite similar rates of early acute clinical humoral rejection, is associated with significant differences in long-term function and chronic AMR rate. Future prospective randomized studies are needed to assess the best strategies to be applied in light of the pretransplant immunologic risk stratification.

Association among Fas expression in leucocytes, serum Fas and Fas-ligand concentrations and hepatic inflammation and fibrosis in chronic hepatitis C

Replication of the hepatitis C virus (HCV) in peripheral blood mononuclear cells (PBMC) may impair immune functions and establish persistent infection. The aim of this study was to assess the influence of HCV on PBMC and their susceptibility to apoptosis in relation to liver inflammation and fibrosis. Eighty-one patients with chronic hepatitis C (CHC) were enrolled in this study. Flow cytometry was used to determine the amount of T cells (CD4(+), CD8(+)), B cells (CD19(+)), monocytes (CD14(+)) and natural killer cells (CD16(+)) in the peripheral blood and the expression of CD95(+) (CD95/APO-1) in each subset. Serum concentrations of sFas and sFasL were assessed by the enzyme-linked immunosorbent assay method. An increased expression of Fas was observed in CD4(+) and CD8(+) cells in CHC. There was a more prominent expression of Fas on CD4(+) cells in HCV genotype 1b in contrast to 3a. Increased Fas expression on CD4(+) cells was seen in advanced stages of liver disease. Fas expression on monocytes was lower in advanced stages of liver inflammation and fibrosis. Serum sFas concentration was higher in CHC compared with the control group. There was an association between sFasL concentration and inflammatory activity in the liver. Serum sFasL concentration correlated positively with the mean intensity of fluorescence of the Fas receptor in CD4(+) and CD8(+) cells, granulocytes and monocytes. These findings indicate that there is an increased susceptibility of PBMC to apoptosis, which can be attributed to the constant contact of leucocytes with the inflamed liver tissue, or from direct HCV influence.

Antithymocyte Globulins Bind to Human Umbilical Vein Endothelial Cells

Polyclonal antithymocyte globulins (ATGs) are immunosuppressive agents used for the treatment of organ rejection after transplantation. ATGs induce complement-mediated cell death of T lymphocytes and may decrease leukocyte adhesion. However, little is known about their effects on endothelial cells (ECs). Our aim was to study whether they bind to human umbilical vein endothelial cells (HUVECs). HUVECs obtained from umbilical cords were cultured and incubated with ATGs. A group incubated without ATG served as controls. All groups were coincubated with an anti-rabbit-IgG. Binding of ATGs to HUVECs was investigated by means of flow cytometry. Statistical analysis was performed using one-way analysis of variance (ANOVA). ATGs were bound to HUVECs with or without prior stimulation by tumor necrosis factor-alpha (TNF-alpha). Binding of ATGs to HUVECs was >99% in all treated groups. The mean intensity of fluorescence was constant in the ATG-treated groups. Our results demonstrated that ATGs bind to HUVECs before and after stimulation with TNF-alpha. Binding of ATGs to HUVECs suggests an independent endothelial mechanism of ATG action.

Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.

Comparison of Combination Plasmapheresis/IVIg/ Anti-CD20 Versus High-Dose IVIg in the Treatment of Antibody-Mediated Rejection

Different strategies appear to improve the success in treatment of antibody-mediated rejection (AMR), although no one best method has yet emerged. The objective of this study was to compare the efficacy of the combination of Plasmapheresis/intravenous immunoglobulin (IVIg)/anti-CD20-based regimes versus high-dose IVIg alone in the treatment of AMR. Group A (12 patients) was treated with high-dose IVIg between January 2000 and December 2003; group B (12 patients) was treated by Plasmapheresis/IVIg/anti-CD20 between January 2004 and December 2005. Graft survival at 36 months was 91.7% in group B versus 50% in group A (p = 0.02). Donor-specific human leukocyte antigens (DSA) levels detected by Luminex single antigen (Luminex SA) and ELISA, 3 months postrejection are significantly lower in group B than in group A: DSA ELISA class 2 score 6-8 (p = 0.02), DSA mean intensity of fluorescence (MFI) max (p = 0.009) and DSA mean MFI (p = 0.0004). The persistence of elevated DSA levels posttreatment is more frequent in patients with graft loss as compared to those with preserved renal function: score 6-8 on ELISA (p = 0.04); mean MFI (p = 0.00009) and MFImax (p = 0.018). We conclude that: (1) high dose IVIg alone is inferior to Plasmapheresis/IVIg/anti-CD20 as therapy for AMR and (2)DSA postrejection can be quantified using solid phase assays, showing that 3 months after AMR, DSA levels are higher in patients with graft loss.