ALBERT

Description: Abstract 1542 Poster Board I-565 Introduction Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes. Materials and methods Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H2O2) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H2O2 stimulation. Results While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H2O2 stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD. Conclusion GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Mef2c and Mef2d are activated by the pre-B-cell receptor and are essential for pre-B-cell transition. Mef2c complexes with B-cell transcription factors to shut down the immediate early response and to initiate a new transcriptional network.

Description: Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor. Congenital defects in pre-B cell receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell receptor signaling represents a critical target for malignant transformation towards ALL. Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1-gene rearrangement are –like normal pre-B cells- highly selected for the expression of a functional pre-B cell receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell receptor resulted in a strong Ca2+ signal. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1, 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ-heavy chain locus encoding the central component of the pre-B cell receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ-chain was amplified, no pre-B cell receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell receptor signaling enables (E2A-PBX1; Type I) or suppresses (BCR-ABL1, MLL-AF4, Hyperdiploid; Type II) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1-induced Type I ALL cells (active pre-B cell receptor signaling) and MLL-AF4-induced Type II ALL cells (lack of pre-B cell receptor expression) with the BCR-ABL1 oncogene. Whereas growth of pre-B cell receptor-negative Type II ALL cells was accelerated by BCR-ABL1-transformation, pre-B cell receptor-positive Type I ALL cells rapidly eliminated by apoptosis within 9 days after BCR-ABL1-transduction. To identify factors that distinguish Type I (E2A-PBX1) and Type II (BCR-ABL1, MLLAF4, Hyperdiploid) ALL and that may explain the divergent role of pre-B cell receptor signaling in these groups, we performed a comparative gene expression including a metaanalysis of published microarray data and quantitative RT-PCR. Compared to E2A-PBX1 Type I ALL, MYC mRNA levels are on average 4-fold, 6-fold and 2.5-fold higher in BCR-ABL1, MLL-AF4 and Hyperdiploid ALL cells, respectively. To test whether high expression levels of MYC render leukemia cells non-permissive to pre-B cell receptor expression, we studied bone marrow B cell precursors from Rag2−/− mice that carry a tetracycline-inducible μ-chain transgene (Hess et al., 2001) as cell culture model for inducible pre-B cell receptor expression. When expression of the pre-B cell receptor was induced in normal IL7-dependent B cell precursors, the cells were induced to first proliferate and subsequently differentiate, hence mirroring normal stages of early B cell development. We then transformed Rag2−/− Tet-μ-chain B cell precursors by retroviral transduction with MYC. MYC-transformed cells rapidly outcompeted untransduced normal IL7-dependent B cell precursors in cell culture. Induction of pre-B cell receptor expression, however, completely reversed growth kinetics and within a few days, normal untransduced pre-B cell receptor-positive cells had a substantial growth advantage over MYC-transduced pre-B cell receptor-positive cells that were progressively lost in cell culture. Interestingly, this growth pattern was reversible by subsequent ablation of pre-B cell receptor expression: After Tet-mediated ablation of pre-B cell receptor expression, the initial growth kinetics were restored and MYC-transduced pre-B cell receptor-negative cells regained a substantial growth advantage. These findings demonstrate that different levels of MYC expression determine permissiveness of ALL cells for pre-B cell receptor signaling. Hence, the pre-B cell receptor suppresses outgrowth of Type II leukemia by censoring high levels of MYC expression.

Description: Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

Description: The atypical form of the kidney disease hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. In addition to mutations in complement regulators, factor H (FH)–specific autoantibodies have been reported for aHUS patients. The aim of the present study was to understand the role of these autoantibodies in aHUS. First, the binding sites of FH autoantibodies from 5 unrelated aHUS patients were mapped using recombinant FH fragments and competitor antibodies. For all 5 autoantibodies, the binding site was localized to the FH C-terminus. In a functional assay, isolated patient IgG inhibited FH binding to C3b. In addition, autoantibody-positive patients' plasma caused enhanced hemolysis of sheep erythrocytes, which was reversed by adding FH in excess. These results suggest that aHUS-associated FH autoantibodies mimic the effect of C-terminal FH mutations, as they inhibit the regulatory function of FH at cell surfaces by blocking its C-terminal recognition region.

Description: Abstract 391 Introduction Traditionally, the focus of VTE diagnostic is on deep vein thrombosis (DVT) of the leg and pulmonary embolism. Until recently, upper extremity DVT (UEDVT) was regarded as an uncommon and relatively benign presentation of venous thromboembolism; however, the more widespread use of central venous catheters has caused a significant increase in its incidence. Moreover, recent data indicate that 10–25% of these patients may have pulmonary embolism. Therefore, effective and safe diagnostic strategies are needed. The use of an algorithm combining a clinical decision score, D-dimer and ultrasonography is well-established for suspected lower limb DVT, but has not been evaluated in suspected UEDVT. This diagnostic management study assessed the safety and feasibility of a new diagnostic algorithm in patients with clinically suspected UEDVT. Methods In- and outpatients with suspected UEDVT were recruited from January 2010 until July 2012 in 17 hospitals in Europe and the United States. To confirm an acceptable failure rate of excluding UEDVT (upper 95% confidence interval below 3%), approximately 400 patients needed to be included. Main exclusion criteria were previous UEDVT and the use of therapeutic doses of anticoagulants. Informed consent was obtained from all participants. The algorithm consisted of the sequential application of the Constans' clinical decision score (Constans et al, Thromb Haemost 2008), D-dimer testing and compression ultrasonography. Patients were first categorized as UEDVT likely or unlikely by the Constans' score. In the patients with an unlikely score and a normal D-dimer, UEDVT was considered excluded and no further testing was done. All other patients underwent compression ultrasonography, which first assessed the deep veins for the presence of UEDVT and then the superficial veins for the presence of superficial vein thrombosis (SVT). Ultrasonography was repeated in case of an indeterminate ultrasonography result, or in patients with a high probability score, abnormal D-dimer and a normal ultrasonography. The primary outcome was the 3-month incidence of symptomatic UEDVT and pulmonary embolism in patients with a diagnostic work-up excluding both UEDVT and SVT. Results The study population comprised of 356 consecutive patients with suspected UEDVT. The algorithm was feasible and completed in 96% (Figure). Of the 356 patients, 181 had a low probability score and D-dimer was measured. In 78 patients (22%) a normal D-dimer combined with a low probability score excluded UEDVT without any imaging, and these patients all had an uneventful 3 month follow up. An abnormal D-dimer test result was found in 100 patients, who underwent ultrasonography. In 3 patients, D-dimer measurement was not done, and these patients received ultrasonography right away. Of all patients with a low probability score, 15 patients had UEDVT, 24 patients had SVT, while thrombosis (both deep and superficial) was excluded in 141 patients. One remaining patient died of progressive cancer before ultrasonography could be done. All 175 patients with a high probability score underwent compression ultrasonography right away, which was repeated if indicated. Of these patients with a high probability score, 82 had UEDVT and 22 had SVT. In 71 patients, thrombosis was excluded, including 11 patients in whom the protocol was not followed completely. To summarize, of the 356 included patients, 97 patients had UEDVT (27%), 46 had SVT (13%) and in 212 patients the algorithm excluded UEDVT and SVT (60%). Of all patients in whom the algorithm excluded UEDVT and SVT, one patient developed UEDVT during follow-up for an overall failure rate of 0.47% (95%CI: 0.0–2.6%). Final results of the definitive study population of 407 patients will be presented. Conclusions A new diagnostic algorithm which combines a clinical decision score, D-dimer and ultrasonography can safely and effectively exclude venous thrombosis of the upper extremity. This approach is attractive as it is simple, quick and non-invasive, and very similar to the well established algorithm for suspected DVT of the leg which could facilitate its implementation in clinical practice. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 821 Introduction: Sickle cell disease (SCD) is characterized by recurrent acute vaso-occlusive painful crisis frequently leading to SCD related complications such as acute chest syndrome, stroke, multi-organ failure and even sudden death. The complex pathophysiology of the vaso-occlusive painful crisis is mediated by activation of endothelial cells, adhesion of sickled erythrocytes and neutrophils, oxidative stress, coagulation activation and increased release of inflammatory mediators, resulting in ischemic organ damage. Recently, neutrophils have been demonstrated to form neutrophil extracellular traps (NETs) upon activation. Nucleosomes and histones exposed together with neutrophil proteases, such as elastase on these NETs have been shown to kill efficiently bacteria. NET formation has been shown to propagate coagulation in sepsis and in deep venous thrombosis. In addition, nucleosomes and histones exposed on NETs have been shown to be strongly cytotoxic to endothelial cells. Beside the exposure on NETs, nucleosomes can be actively released into the circulation from dead cells. Circulating nucleosomes detected in sepsis have been reported to correlate with severity of inflammation, organ dysfunction and mortality. However, no studies are available yet on the dynamics of nucleosomes and NETs in sickle cell patients suffering from painful crisis. The aim of this case-control study was to assess plasma levels of circulating nucleosomes and human neutrophil elastase–α1-antitrypsin (EA) complexes as measure of systemic neutrophil activation, in sickle cell patients during steady state and painful crisis. Methods: Plasma levels of nucleosomes and EA as a measure of neutrophil activation were measured in 74 patients in asymptomatic state (49 HbSS/HbSβ0-thalassemia, and 25 HbSC/HbSβ+-thalassemia), 70 painful crises (53 HbSS/HbSβ°-thalassemia and 17 HbSC/HbSβ+-thalassemia) in 49 patients and in 24 HbAA healthy controls using Enzyme-Linked Immunosorbent Assay (ELISA). Results: Plasma levels of nucleosomes in both HbSS/HbSβ°-thalassemia and HbSC/HbSβ+-thalassemia patients were significantly higher during painful crisis (median; interquartile range, 20.2; 8.9 – 129.0 U/ml, P 〈 0.0001 and 11.7; 5.1 – 67.7 U/ml, P = 0.045 respectively) as compared to patients in steady state (6.0; 3.0 – 9.8 U/ml and 7.1; 4.6 – 9.6 U/ml respectively). Nucleosomes levels in healthy controls were just above the detection limit of the assay (5.0; 5.0 – 6.5) U/ml). Plasma levels of EA in HbSS/HbSβ°-thalassemia patients were significantly increased during painful crisis as compared to steady state (75.1; 56.5 – 102.4 vs. 45.7; 34.7 – 59.7 ng/ml, P 〈 0.0001). Also in HbSC/HbSβ+-thalassemia patients, EA levels were higher during painful crisis than in steady state, though the difference did not reach statistical significance (62.0; 48.0 – 96.7 vs. 50.2; 33.3 – 67.7, P = 0.051). Plasma levels of EA in healthy controls (39.9; 31.5 – 62.2 ng/ml) were comparable with those in steady state patients. In a paired analysis of 36 patients, included both during steady state and painful crisis, significant increments were observed during painful crisis in levels of both nucleosomes (from 5.0; 3.0 – 10.8 to 20.2; 6.8 – 94.3 U/ml, P 〈 0.0001) and EA (from 47.9; 36.0 – 67.6 to 70.6; 55.9 – 101.4 ng/ml, P 〈 0.0001), as compared to steady state. During painful crisis, EA levels were strongly correlated with levels of nucleosomes in both HbSS/HbSβ°-thalassemia (Spearman's rank (Sr)=0.55, P

Description: Abstract 2572 Poster Board II-549 Introduction: Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls. Methods: Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC). Results: PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis. Conclusion: In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed. Disclosures: No relevant conflicts of interest to declare.