ALBERT

Description: Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA–mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

Description: Background: Thrombin generation and other clotting assays suffer from a wide variation of pre-analytical variables. One of those pre-analytical variables is contact activation through blood withdrawal methods, different syringes, differences in blood coagulation tubes, blood transport and sample handling. It has been shown that the addition of contact activation inhibitors in low tissue factor activated thrombin generation leads to a correction of the, in these circumstances significant, increase in thrombin generation due to contact activation. We compare the novel 'thermostable inhibitor of contact activation' (TICA) to the current standard 'corn trypsin inhibitor' (CTI). Aim: Comparing the effectiveness of novel contact activation inhibitor TICA to the current standard CTI in low tissue factor-induced thrombin generation and recalcification in sodium citrate anticoagulated platelet poor plasma (PPP) and platelet rich plasma (PRP). Methods: We compared TICA, Corn trypsin inhibitor and plasma without contact activation inhibitors in low tissue factor PPP thrombin generation and in PRP recalcification thrombin generation, the latter the most sensitive condition for contact activation. In addition, we compared low tissue factor activated thrombin generation in plasma from severe hemophilia A patients with and without TICA during and after blood drawing. Thermostability - as a measure of shelf life - was measured and compared to CTI. Results: TICA is able to fully block contact activation in PRP recalcification experiments and is comparable to CTI in doing so. TICA significantly lowers low tissue factor induced thrombin generation by blocking contact activation. Pre-loading vacuum blood collection tubes with contact activation inhibitors is superior in inhibiting contact activation compared to addition of the inhibitor during the thrombin generation assay itself. TICA did not alter coagulation activity when added to FXIIa deficient plasma in thrombin generation. In contrast to CTI TICA is heat stable which will be of benefit to shelf life of pre-loaded blood drawing tubes. Conclusion: TICA is able to fully block contact activation and has several advantages over CTI. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Approximately 40% of children with a severe form of sickle cell disease (SCD) will develop cerebral white matter hyperintensities (WMHs), visible on magnetic resonance imaging (MRI). This may be associated with impaired neurocognitive functioning. It is unknown whether the volume of these WHMs is associated with the degree of neurocognitive dysfunction. Our objective was to investigate the association between volume of WMHs and neurocognitive functioning. Methods We prospectively included children with HbSS or HbS-beta(0)thalassemia aged 8-16 years. Exclusion criteria were prior stroke and chronic blood transfusion therapy. Volume of WMHs was calculated on MRI and patients were ranked by size of WMHs. Neurocognitive function was evaluated by testing intelligence (IQ, intelligence quotient), memory, visuo-motor functioning and executive functioning. Fatigue was measured using a validated questionnaire (Pediatric Quality of Life Inventory Multidimensional Fatigue Scale, PedsQL Fatigue) in which lower scores indicate more symptoms of fatigue. For each neurocognitive outcome, univariate linear regression was used to identify which variables (age, sex and hemoglobin level) were confounders. The independent association of volume of WMHs on neurocognitive outcomes was analyzed by multivariate linear regression, adjusted for these confounders when appropriate. The explained variance (R2) refers to the independently explained variance of volume of WMHs on the neurocognitive outcome and the presented p-value corresponds to the unique contribution of volume of WMHs on the outcome, both adjusted for confounders when appropriate. Results We included 38 children; mean age was 12.5 ± 2.7 years, WMHs were present in 50%. Mean full-scale, verbal IQ, performal IQ and Processing Speed Index were all between 85 and 90; this is significantly lower compared to the mean norm scores of 100. Our patients had significantly more symptoms of fatigue compared to Dutch reference values. A higher volume of WMHs was significantly associated with lower scores on full-scale IQ, verbal IQ and Processing Speed Index (see table). In addition, higher volume of WMHs was associated with higher scores of total and cognitive fatigue. Standardized beta coefficients ranged from -0.350 to -0.461, indicating a substantial negative effect of an increasing volume of WMHs on neurocognitive outcome. The volume of WMHs could explain between 12.1% and 21.2% of the variance of these outcomes. Conclusion Our findings suggest that the volume of WMHs is an independent predictor of full-scale IQ, verbal IQ, Processing Speed Index and fatigue in children with SCD. As WMHs are mostly found in the frontal lobe, this could explain the association with processing speed, which is an executive function and thought to be located in the frontal lobe. The association between WMHs and measures of fatigue has not been investigated before. Our results suggest the PedsQL Fatigue could be a promising screening tool for larger studies as it is easy and quick to administer and has a high validity. We suggest that future studies should consider taking the total volume of WMHs into account as an independent predictor of neurocognitive outcome, instead of only the presence or absence of WMHs. Taking the volume of WMHs into account is an important approach for individualized diagnostic and treatment strategies that could be further explored in a clinical setting. Table Prediction model of neurocognitive outcome by volume of white matter hyperintensities ß R2 p Full-scale IQ -0.382 0.146 0.018 Verbal IQ -0.460 0.212 0.004 Performal IQ -0.170 0.029 0.314 Processing Speed Index -0.461 0.212 0.005 PedsQL Fatigue, Total Score -0.350 0.121 0.025 PedsQL Fatigue, General Fatigue -0.289 0.083 0.071 PedsQL Fatigue, Cognitive Fatigue -0.352 0.123 0.026 IQ, Intelligence Quotient; PedsQL Fatigue, Pediatric Quality of Life Inventory Multidimensional Fatigue Scale. Disclosures No relevant conflicts of interest to declare.

Description: Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized β-arrestin recruitment site. Similar to PAR2−/− mice, TF cytoplasmic domain–deleted (TFΔCT) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TFΔCT mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TFΔCT and TFΔCT/PAR2−/− mice, and increased tumor vessel diameters of TFΔCT mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.

Description: Key Points RNA interference of Serpinc1 and/or Proc allows for evaluation of the function of these genes, alone or in combination, in normal adult mice. RNA interference of Serpinc1 and Proc provides a novel, controlled mouse model for spontaneous venous thrombosis.

Description: Erythroid colonies from five patients with an early erythroblastic leukemia were obtained in “serum-free” cultures in the presence or absence of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and homogeneous native erythropoietin (Epo). Erythroid colonies with abnormal morphology and karyotype could be grown in different culture conditions. Their erythroid nature was ascertained by the presence of carbonic anhydrase I and glycophorin A. Leukemic erythroid progenitors strongly differed from normal progenitors in that spontaneous colonies were always obtained, sometimes with an extremely high plating efficiency (up to 5.7%). Colonies were found to be autonomous from exogenous hematopoietic growth factors because they were still obtained with a high plating efficiency at an average of one cell per culture in the absence of any added growth factor. No evidence for an autocrine secretion of Epo or GM-CSF emerged because Epo or GM- CSF could not be detected by biologic or radioimmunologic assays from the culture supernatant or cellular extracts of the leukemic cells and that Epo or GM-CSF antibodies did not block autonomous growth. In all cases, however, hematopoietic growth factors increased the plating efficiency of the abnormal erythroid progenitors. In the two “de novo” leukemias, leukemic erythroid progenitors responded primarily to Epo, whereas in the three other patients' (chronic myeloid leukemia) blast crisis they responded maximally to GM-CSF plus Epo. Recombinant erythroid-potentiating activity had no effect in any of these cases. These results suggest that the leukemic erythroid clonogenic cells arise from expansion of erythroid progenitors at different levels of differentiation (ie, CFU-E or BFU-E, depending upon the disease) and that autonomous growth is not related to a secretion of Epo or GM-CSF.

Description: Background: Urgent surgery or life-threatening bleeding requires prompt reversal of the anticoagulant effects of the direct thrombin inhibitor dabigatran. As no specific reversal agent in situations of life-threatening bleeding with dabigatran is currently available, this study assessed the ability of a four-factor prothrombin complex concentrate (PCC) as mono-therapy as well as PCC in combination with tranexamic acid (TX) and fibrinogen in an experimental polytrauma pig model. Methods: After ethical approval dabigatran etexilate (30 mg/kg p.o. twice daily, n=24) was administered to male pigs for 3 days. On day 4, the pigs were anesthetized and given a 90 min infusion of active dabigatran to achieve consistent, supratherapeutic plasma levels. A standardized polytrauma including bilateral femur fractures and a blunt liver injury was induced. Animals underwent hemorrhagic shock and were resuscitated using Ringer’s solution (1 L). Twelve min after polytrauma, animals received either placebo (control group, n=6); TX, 20 mg/kg plus fibrinogen (80 mg/kg; TX+F group, n=6); PCC alone (PCC group, 50 U/kg, n=6); or TX (20 mg/kg) plus fibrinogen (80 mg/kg) plus PCC (50 U/kg; TX+F+PCC group, n=6) according to randomized group allocation. Coagulation was assessed by thromboelastometry, coagulation parameters and diluted TT. Blood loss (BL) was measured over the observation period of 240 min. Data were analyzed by ANOVA (± SD). Results: Dabigatran levels were 504 ± 171 ng/mL prior to injury and plasma levels remained significantly elevated in all animals throughout the observation period. The degree of injury was similar among animals with comparable BL of 803 ± 46 mL 12 min post injury. Anticoagulation with dabigatran without intervention resulted in a total BL of 3521 ± 600 mL, with 100% mortality and mean survival time of 101 ± 34 min (range: 67 - 148 min; p

Description: E. coli. L-Asparaginase repeated injections induce immunization. Anti-Asparaginase antibodies can provoke clinical hypersensitivity reactions and/or silently inactivate enzyme activity. Consequently, L-Asparaginase clearance is increased, implying a lack of L-asparagine deamination. Firstly, we developed an assay able to detect the presence of neutralizing factors including anti-Asparaginase antibodies. Next we investigated in a mouse model if loading L-Asparaginase into red blood cells (RBC) may be a way to protect its activity against neutralizing factors. A rabbit was immunized injecting 0.5 mg of L-Asparaginase (167 IU) mixed with Freund’s adjuvant every 3 weeks for 4-fold. The animal was euthanized and the final serum collected. Part of this final serum was immuno-adsorbed onto protein A for IgG antibodies purification. L-Asparaginase activity was measured by monitoring the kinetics of ammonia generation from the hydrolysis of asparagine. This assay was adapted to a biochemistry automated analyzer. When mixed with undiluted serum from the immunized rabbit, L-Asparaginase activity (0.8 to 100 IU/ml) was totally inhibited for all the concentration range within 15 min at 37°C. In the other hand, up to 1/128 serial dilutions of serum totally inhibited 2 IU/ml L-Asparaginase. As a control, undiluted pre-immunization serum from the same animal did not significantly affect L-Asparaginase activity. To identify the neutralizing factors, IgG from serum were purified by protein-A. As performed with serum, successive dilutions of IgG were mixed with 1.25 IU/ml L-Asparaginase. The IgG inhibited enzyme activity at the 1/128 dilution by 97%, thus proving their neutralizing effect on L-Asparaginase. To simulate the presence of neutralizing antibodies in the patient, we injected 7.5 μg of rabbit IgG into OF1 mice. Control mice were injected with phosphate buffered saline (PBS). Twenty minutes later mice either received 80 IU/kg of native E. coli L-Asparaginase or the same dose entrapped into OF1 mouse RBC. L-Asparaginase was loaded into murine RBC by reversible hypotonic dialysis, followed by a resealing step. The RBC thus acts as a bioreactor where plasmatic asparagine enters and is cleaved by the entrapped L-Asparaginase inside the erythrocyte. L-Asparaginase activity inside the erythrocyte was quantified at 68 IU per ml of erythrocytes, and the extracellular enzyme activity was less to 9% of total enzyme activity. Mice were sacrificed 6 hours after the administration of native or encapsulated L-Asparaginase. Free L-Asparaginase was totally inactivated in plasma of anti-Asparaginase IgG pre-treated mice: 0.002 ±0.002 IU/ml vs 0.417 ±0.103 IU/ml in PBS pre-treated mice. In addition, when L-Asparaginase is loaded inside RBC the activity is maintained irrespective of the presence of antibodies (0.798 ±0.126 IU/ml with IgG vs 0.879 ±0.146 IU/ml without). Moreover asparagine was not deaminated in IgG pre-treated mice who received free L-Asparaginase (27 ±1.6 μmol/L), while below 2 μmol/L in all the other groups. In conclusion, this newly developed assay can predict in vivo L-Asparaginase inefficacy. In addition, L-Asparaginase loaded into RBC is protected against neutralizing antibodies and its efficacy is maintained.

Description: A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, a group of 27 patients with HIV-1 infection was studied. In all patients, a marked reduction of in vitro colony formation by erythroid, granulomacrophagic, and megakaryocytic bone marrow progenitors was observed in comparison to normal donors. HIV-1 infection of marrow progenitors was investigated in studying individual colonies with the polymerase chain reaction (PCR) technique. No HIV-1 DNA could be detected in these colonies, suggesting either that marrow progenitors were not infected or that infected progenitors were not able to generate colonies in vitro. The addition of antisense oligonucleotides directed against HIV tat or nef sequences in the culture medium led to a significant increase in colony formation, suggesting that HIV replication in hematopoietic progenitors could be responsible for their defective growth. However, no HIV-1-infected colonies could be detected by PCR after the antisense treatment, indicating that the increase in colony number was not due to the proliferation and differentiation of infected progenitors but to an inhibition of HIV replication in an accessory cell. This last hypothesis was further confirmed by the absence of effects of antisense oligomers on the plating efficiency of hematopoietic progenitors grown from CD34+ cells. These data indicate that hematologic abnormalities of HIV-infected patients cannot be explained by a direct infection of hematopoietic progenitor cells and suggest that a defective modulation of progenitor cell growth by HIV replication outside these cells might play a role in these abnormalities.

Description: Background: For many years the ALFA Group has used high dose DNR, i.e 80 mg/m2/day for 3d as part of the induction regimen for untreated adult AML pts. As the equivalence of DNR and Ida is not known we conducted a phase III study to compare high dose DNR to Ida. We also tested the effect of IL2 for maintenance. Methods: Newly diagnosed AML, aged 50 to 70 years, were randomized to receive AraC 200 mg/m2/day IV x 7 d with either DNR: 80 mg/m2/d x 3 d (arm 1) or Ida: 12 mg/m2/d x 3 d (arm 2) or x 4 d (arm 3). Pts who failed could receive a salvage course with Mitoxantrone x 2 d and AraC1g/m2 12 hrs x 4d. Response was assessed after induction+/− salvage chemotherapy. CR pts received 2 consolidation courses according to the induction arm with DNR: 80 mg/m2 or Ida:12 mg/m2 for 1 day (first course) or 2 days (2nd course) and AraC:1g/m2/12 hrs x 4 days. Pts in CCR after the two consolidation courses were randomized to receive or not a maintenance immunotherapy with IL2: 5.106/m2 for 5 days monthly for12 months. Results: From 2001 to 2006, 468 pts were included (median age: 60 years). The 3 treatment arms were well matched for pretreatment characteristics. CR was achieved in 360/468 pts (77%): 109 (70%) pts arm 1, 129 (83%) arm 2, 122 (78%) arm 3 (p=0.02).70 pts, (45%) pts in the DNR arm reached CR after 1 course vs 97 (62%) and 90 (57%) in Ida arms (p= 0.006). Pts in Ida arms experienced more grade 3 or 4 mucositis (p=0.004) but no differences were observed between the 3 arms for duration of hospitalization, time to PMN or plts recovery, incidence of grade 3 or 4 infectious episodes and treatment related mortality. 3 year cumulative incidence of relapse was 69%, 63%, 62% resp in arms 1,2,3 (p=NS). 3 year EFS was 19%, 30%, 26% resp (p=0.06 for arm 1 vs arms 2 and 3). 3 year OS was 31%, 40% 41% resp in arms 1,2,3 (p=NS). Age (〈 or 〉60 years), sex, initial WBC counts, initial LDH (nl or 〉nl), DNR or Ida arms, need for a salvage course were not predictive for relapse, while 2yCIR was 43%, 64%, 77% among respectively fav, intermediate and unfav cytogenetic risk groups resp (p=0.0046). Of the 219 pts alive in CR after consolidations, 161 (73%) were randomized for maintenance. Only 22 of the 77 pts randomized for IL2 completed the 1 year treatment. 32 and 23 pts stopped IL2 resp because of relapse or intolerance. There were no differences in relapse or OS in both maintenance arms. Conclusion: Ida treatment even when compared to high doses of DNR yields higher CR rate and more CR after one course. This effect translated in slightly better EFS but not better CIR or OS. IL2 maintenance treatment at least as scheduled in this trial was difficult to apply and showed no impact on disease course.