bluuebunny/crossref_metadata_2025 huggingface dataset:
Insights into Disturbed Hemostasis in the Quebec Platelet Disorder Using Thromboelastography, Blood Clots Formed at Low Shear, and Perfusion Studies of Fibrinolysis.
Authors: Diamandis Maria , Adam Frederic , Rivard Georges E. , Kahr Walter H.A. , Hayward Catherine P.M.
Published: October 2019

The Quebec Platelet Disorder (QPD) is an inherited disorder associated with delayed bleeding, increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets, normal to increased u-PA in plasma, consumption of platelet plasminogen activator inhibitor-1 (PAI-1), and increased plasmin generation in platelets with proteolysis of stored α-granule proteins, including factor V. Although accelerated fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood on the lysis of forming and preformed fibrin have not been evaluated. In this study, we modeled fibrinolysis in vitro using thromboelastography, biochemical evaluations of whole blood clots formed at low shear, and perfusion of blood over preformed fibrin. Thromboelastography (TEG) indicated that the clot formation and lysis phases were normal in QPD whole blood clots generated with tissue factor and observed for 180 min, which is the maximum time allowed by the TEG® software. However, when the TEG® were done with limiting amounts of tissue-type plasminogen activator (t-PA), QPD clots showed a shortened lysis phase. The addition of QPD platelets to normal blood hastened t-PA mediated lysis. In QPD whole blood clots, generated at low shear with exogenous thrombin (without t-PA), there was abnormal plasmin generation, with increased fibrinolysis unless fibrinolytic inhibitors were used to inhibit plasmin. These data excluded the defect in platelet factor V as the cause of abnormal lysis. Perfusion studies indicated that QPD platelets adhered to fibrin, although the amount of adhesion was reduced compared to control samples, likely because of the reduced platelet count in QPD blood. When QPD blood was perfused over labeled, preformed fibrin, without a stimulus for thrombin generation, there was accelerated loss of fibrin, and a corresponding increased generation of labeled fibrin degradation products. These results indicate that the QPD is associated with a “gain of function” abnormality that increases the lysis of forming or preformed clots, independent of thrombin generation. Our findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding and support the clinical observation that transfusion of normal platelets does not correct the defect in this bleeding disorder.
Afibrinogenaemia Resulting from a Novel Putative Splicing Mutation, and Evidence for Tissue Specific Expression of FGAα RNA.
Authors: Cutler Jacky , Bourdon Louise , Savidge Geoffrey F.
Published: October 2019

Congenital afibrinogenaemia is a rare autosomal recessive disorder characterised by the complete absence of fibrinogen in plasma. Fibrinogen is predominantly produced in hepatocytes from 3 homologous polypeptide chains (Aα, Bβ, γ) which assemble to form a hexameric structure. The genes that encode the 3 polypeptides (FGAα, FGBβ, FGγ) are clustered in an approximately 50kb region on chromosome 4q28-q31. Defects in each of the 3 genes have been implicated in afibrinogenaemia, and encompass missense and nonsense mutations, insertions, small and gross deletions and splicing defects.

We present a case of congenital afibrinogenaemia, diagnosed in a neonate, the only child of unrelated parents. Sequence analysis of the FGAα, FGBβ and FGγ genes identified a single, novel, homozygous nucleotide substitution (T→A) at IVS1 -19 of the FGAα gene. Both parents genotyped as heterozygous for this variant. Analysis of ~100 normal alleles failed to detect this change, thereby excluding it as a common polymorphism. Using splice prediction software, the IVS1 acceptor splice site is undetected, reflecting poor agreement with acceptor consensus sequences. The mutation presented here, results in the creation of a preferential acceptor splice site (Spliceview score of 77). It has been previously reported (Catia et al, 2003) that intron 1 is preferentially the last intron of the FGAα gene to be spliced, and it may be that the wild type exon 1-intron 1- exon 2- exon 3- exon 4 - exon 5 intermediate presents a better substrate to the spliceosome. If we accept this predicted splicing order the mutation described in this study will not affect expression. Instead, the resulting incorporation of 19 intronic bases will result in a premature termination sequence (TGA) and a truncated protein.

RNA work was undertaken to attempt to demonstrate these findings. However, RNA extracted from peripheral blood leukocytes from both parents failed to amplify for even the normal allele of FGAα (using primers located in exons 1and 5). Integrity of the samples was demonstrated by amplification of F8 RNA. To investigate this further, we tested a panel of commercially available RNAs from various tissues. RNA derived from liver, heart, spleen and kidney cells produced the expected product, with much higher yields being seen with liver cells, while RNA from brain cells and leukocytes failed to yield any product at all. Tissue specific expression has previously been described for FGγ RNA.

In summary: This case study has identified a novel splice site mutation in the FGAα gene which is predicted to result in the introduction of a premature termination sequence at the newly created codon -1. This could not be confirmed due to the previously unreported discovery that FGAα RNA is not expressed in peripheral leukocytes.
FVIII Recovery after a Single Infusion of Recalibrated ReFacto in 14 Severe Hemophilia A Patients.
Authors: Lambert Thierry , Guérois Claude , Gay Valerie , Stieltjes Natalie , Bertrand Marie-Anne , Derlon Annie , Sigaud Marianne , Hassoun Abel , Négrier Claude , Coatmelec Brigitte , Dreyfus Marie , Dubanchet Arnaud
Published: October 2019

Background: A field study among 6 reference laboratories in the EU using chromogenic substrate assays revealed in some laboratories a FVIII level lower than expected when measuring the potency of ReFacto, a B-domain deleted recombinant FVIII concentrate. In an attempt to resolve these discrepancies, the standard used for establishing the potency of ReFacto was recalibrated in 2003. Indeed after this recalibration, the amount of ReFacto protein in each International Unit (IU) has increased by approximately 20 percent without change in the labeled dosage strength.

Objectives: The primary objective of this prospective study was to assess the FVIII recovery in severe hemophilia A patients receiving recalibrated ReFacto.

Methods: The study was conducted in 10 French Hemophilia Treatment Centers in Previously Treated Patients (> 150 exposure days to any FVIII concentrate). A series of 4 blood samples per patient were collected in a non-bleeding state, respectively before, 15mn, 30mn and 60mn after intravenous bolus infusion of a single dose of 50 ±5 IU/kg of ReFacto. Plasma FVIII activity was determined in a central lab using a chromogenic substrate assay (Coamatic FVIIITM).

Results: Fourteen severe hemophilia A patients (FVIII: C < 1%) were evaluable for intention to treat analysis. Median age was 25.5 years (range: 12–48). Median injected dose was 53.3 UI/kg (range 48.4–56.8). Maximal plasma FVIII activity level was obtained 15mn (n=10) or 30 mn (n=4) after the end of infusion. Mean incremental recovery (K value) was 2.20 ±0.27 UI/dL per UI/kg infused (range: 1.89–2.75) with a mean in vivo recovery of 105.2% (range: 87.6–133.8).

Conclusions: In most cases the peak of FVIII activity was obtained 15 mn after the end of infusion. Recovery of recalibrated ReFacto was similar to the expected recovery with full-length FVIII concentrates.
Treatment of Diffuse Alveolar Hemorrhage with Recombinant Factor VIIa in Patients with Hematologic Malignancies.
Authors: Jha Asit , Balasubramanian Manjula , Charania Roseleen , Sandilya Vijay , Topolsy David , Styler Michael
Published: October 2019

Diffuse alveolar hemorrhage (DAH) is a potentially life-threatening pulmonary complication in patients following hematopoietic stem-cell transplantation (HSCT) or acute leukemia induction therapy. This syndrome is associated with a high mortality rate. Current management strategies include high-dose steroids, platelet transfusions and mechanical ventilatory support to treat acute respiratory failure. Recombinant factor VIIa (rFVIIa) is approved for treatment of bleeding in patients with hemophilia A/B with inhibitors. A greater understanding of the mechanism by which rFVIIa restores hemostasis has become available; with the in-vitro evidence supporting that the thrombin burst achieved by rFVIIa is independent of the presence or binding to tissue factor. This insight has suggested a range of other potential clinical uses for the drug. We report our experience of using rFVIIa for the treatment of DAH in the setting of HSCT and acute leukemia induction therapy. We treated 5 episodes of DAH in 4 adult patients with hematologic malignancies. Two patients had undergone matched unrelated donor HSCT. Clinically, DAH was suspected because of progressive respiratory failure. DAH was confirmed by radiologic and bronchoscopic exams. All patients received high-dose steroids, broad-spectrum antibiotics and respiratory support, as indicated. Packed red cells and platelets were transfused to keep the hemoglobin above 8gm/dL and platelets above 50,000/μL. Patients received boluses of rFVIIa at 90mcg/kg, every 3-12 hrs. The dose of rFVIIa ranged from 7,200–28,000 mcg/day. All five episodes of DAH achieved clinicoradiologic improvement. No toxicity or adverse events were observed with rFVIIa use. Our experience indicates that rFVIIa might be an effective treatment option for patients with DAH, post-HSCT or acute leukemia induction therapy. The optimal dosing schedule in this clinical situation needs to be studied.

Patient demographics and outcomes Patient Diagnosis Pre-intervention rFVIIa used (microgms) PRBC/Platelet transfused (units) Clinical outcome OW ALL-MUD, BMT, day +75 8.8/12,000 24000/d X 4days 3/10 100 Non-rebreather to 6L nasal cannula O2 OW ALL-MUD, BMT, day +80 8/8,000 28000/d X 4 days 6/14 Ventilatory support to successful weaning CS Relapsed AML, S/P induction 8.8/19,000 8400/d X 2 days 2/4 O2 requirement decreased LW CML blast crisis, S/P induction 8.6/101,000 10800/d X 2 days 4/1 Clinically no bleeding, decreased O2 requirement RB Relapsed ALL 8.7/7,000 7200/d X 1 day 2/4 Ventilatory support to successful weaning
c-mpl Mutations in Congenital Amegakaryocytic Thrombocytopenia: Residual TPO Receptor Activity in Patients of Group CAMT II.
Authors: Ballmaier Matthias , Welte Karl , Germeshausen Manuela
Published: October 2019

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome. Mutations in the gene for the thrombopoietin receptor c-mpl were defined as the molecular cause of CAMT. Extending our sequence analyses from 8 to a total of now 23 CAMT patients we could confirm our hypothesis of genotype-phenotype correlation in CAMT. Seven different nonsense, frame-shift or splice-site mutations, all predicted to lead to a complete loss of function of the thrombopoietin receptor were found in 13 patients belonging to group CAMT I with persistently low platelet counts and a fast progression into pancytopenia. Nine different missense mutations were detected in ten patients of group CAMT II, characterized by a transient increase in platelet counts over 50 nl−1 during the first years of life. Mutations spread nearly over the entire gene with a clear accumulation in exons 2 and 3. Except for two missense mutations in exon 8 and exon 12 coding for the second cytokine receptor homology domain and for the distal half of the intracytoplasmic domain, respectively, all mutations were found in exons 2–5 coding for the first cytokine receptor homology domain of the thrombopoietin receptor. The mutations found in group CAMT I patients were located in exons 2, 3 and in the splice sites between exons 1–3. Missense mutations found in patients of group CAMT II spreaded over exons 3, 4, 5, 8 and 12. Using in vitro assays with hematopoietic progenitors from patients of both patient groups we could demonstrate a residual activity of the thrombopoietin receptor in CAMT II patients: In samples from patients of group CAMT II the addition of TPO led to a significant higher number of total colonies. No difference was found in the number of megakaryocytic colonies. In contrast, TPO did show neither an effect on the total colony number nor on megakaryocytic colony number in samples from patients of group CAMT I. This is a first experimental confirmation of our hypothesis that the type of mutation has a strong influence on the course of the disease and that the transient increase of platelet counts and the delayed development of pancytopenia in CAMT II patients is due to a residual activity of the TPO receptor.
Rapid, Exponential Extra-Splenic Platelet Consumption in WASP Deficient Mice.
Authors: Strom Ted S. , Wan Jim Y. , Du Haiming , Jackson Carl W.
Published: October 2019

The thrombocytopenia seen in patients with the Wiskott-Aldrich Syndrome (WAS) is thought to be due primarily to rapid platelet consumption, and is markedly improved by splenectomy. While a murine model of WAS shows only mild thrombocytopenia, we have previously demonstrated rapid platelet turnover in this model; that splenectomy improves the platelet count in WASP-(C57Bl/6J) mice; and that the latter mice do not achieve the same platelet count found in splenectomized WT mice. Here we show that CMFDA-labeled WASP- platelets are consumed rapidly and exponentially in WT recipients, with an estimated lifespan of 18 hours (vs. 87 hours for WT platelets). WASP- platelets are consumed only slightly more slowly in splenectomized WT recipients (lifespan 28 hours, vs. 102 hr for WT platelets). On the C3H background, WASP- mice have normal platelet counts but show a similar rapid, exponential platelet consumption rate (lifespan 25 hours in either C3H or C57Bl/6J recipients, vs 85 hours for WT platelets). In vivo platelet biotinylation studies demonstrate less effective labeling of WASP- (C57Bl/6J) platelets than of WT platelets. After a plateau phase, in vivo labeled WASP- platelets show the same kind of rapid platelet turnover seen with ex vivo labeling (figure 1). Our results imply a three-fold increase in platelet production rate in WASP- mice on either background, consistent with the markedly increased splenic extramedullary hematopoiesis seen in WASP- mice. WASP- mice also show an increased number of bone marrow megkaryocytes, the ploidy distribution of which is normal. We conclude that WASP- mice demonstrate a significantly increased rate of extra-splenic platelet consumption that is largely (on the C57Bl/6 background) or completely (on the C3H background) compensated by increased platelet production. The ability of WASP- mice to compensate for their rapid platelet consumption, and the normal ploidy of their megakaryocytes, suggests that platelet production is not impaired by WASP deficiency in this model.

In vivo biotinylation of WASP-and WT platelets In vivo biotinylation of WASP-and WT platelets
Progressive Emergence of a Dominant Unit during Dual Unit Umbilical Cord Blood (UCB) Culture.
Authors: Liu Min , Reese Jane S. , Jaroscak Jennifer J. , Gerson Stanton L.
Published: October 2019

Umbilical cord blood (UCB) as a stem cell source for allogeneic transplantation is limited by the cell dose a single unit can provide to adult recipients. One approach to overcoming the cell dose limitation is the use of two UCB units. However, murine and human clinical studies have shown that after infusion of two UCB units, one unit dominates as early as 3 weeks post engraftment. To understand the biology that determines the engraftment of each donor, we investigated the effect of dual donor competition on hematopoietic progenitors from 3 unrelated units and 2 units from dizygotic twins during methylcellulose and LTC-IC co-culture of equal numbers of mononuclear cells from each unit pair. Donor chimerism was evaluated in primary CFU (t=0) and secondary CFU from LTC-IC cultures (weeks 4 and 5) by PCR of informative polymorphic short tandem repeats (STR analysis). In 10 units analyzed, the CFU frequency per 100,000 mononuclear cells was 152 ± 18 and the LTC-IC frequency was 1 in 20,000 ± 5.81. The mononuclear cell composition was CD34: 1.07 ±.29%; CD3: 56.3 ± 4.1%; CD33: 17.4 ± 3.4%; and CD19: 8.42 ± 1.1%. In 4 UCB pairs, the unit with the higher primary CFU frequency dominated by an average ratio of 2:1 based on STR analysis of the primary CFU co-culture. In secondary CFU derived from the LTC-IC cultures, the skewing progressively became more pronounced with the same unit identified in primary culture predominating by an average ratio of 7:1. In mixed cultures, the absolute number of primary CFU from the dominant units increased by 15 ± 2% over the number from individual units while CFU from the non-dominant units decreased by 37 ± 4%. Interestingly, there was no dominant unit in primary CFU cultures from one set of dizygotic twins and the CFU number from each unit increased by 30% in the mixed culture. This set of twins emerged with a dominant unit during LTC-IC culture, yielding a donor ratio of 2:1. We found no correlation between the predominating unit and the CD34 percentage or LTC-IC frequency. However, in all 5 dual UCB experiments, the unit with the higher CD3 percentage dominated in primary CFU cultures and was intensified during LTC-IC culture. These results demonstrate an early and progressive dominance of one donor based on CFU analysis during mixed UCB culture. The correlation with CD3 content suggests both an immediate and protracted T-cell effector mechanism even though the LTC-IC culture does not promote T-cell proliferation. Additionally, the increase in the absolute cell number of the dominant unit may suggest a graft vs. graft progenitor cell growth-stimulatory effect which may explain the observed more rapid engraftment of the dominant unit after dual UCB transplantation. Thus, immediate UCB competition predicts the dominant unit during in vitro culture and presumably mimics the process occurring after clinical dual UCB transplantation.
Quantitative Real Time PCR Analysis of MDR1 Transduced Rhesus Macaque Hematopoietic Stem Cells Shows No Indication for Proliferative Advantage after Long-Term Follow-Up.
Authors: Bozorgmehr Farastuk , Laufs Stephanie , Buss Eike C. , Nagy Katalina Z. , Sellers Stephanie E. , Zeller W. Jens , Dunbar Cynthia E. , Fruehauf Stefan
Published: October 2019

Introduction: It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral MDR1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analyses on the clonal level and a long-term follow-up in a model with more direct relevance to human HSC biology were lacking.

Methods: In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were transduced either with a MDR1 gene-retroviral vector (HaMDR1-vector) or a NeoR-retroviral vector (G1Na-vector). After autologous transplantation granulocytes and mononuclear cells of each animal were obtained at different time points and analysed by using a highly sensitive and specific ligation-mediated PCR (LM-PCR) and by quantitative real time PCR.

Results: In monkey M120 73 different cell populations (clones) were detected between 8 weeks and 4 years after transplantation; in monkey M038 49 clones could be identified between 16 weeks and 4 years after transplantation. Remarkably, 99 clones descend from the G1Na-vector, whereas only 23 clones descend from the HaMDR1-vector. Quantitative real time PCR analyses support these data: in both animals, the mean proportion of the G1Na-vector and the HaMDR1-vector, respectively, is 2,46%±0,014% and 0,455%±0,003%. Furthermore, single G1Na clones identified with LM-PCR 4 years after transplantation were quantified, showing a copy number between 1.43±0,52 and 7.1±0.34, according to 0.001%±0.0003% and 0.004%±0.0003% of the granulocytes fraction. In the MNC fraction, the G1Na clones were not detectable. At the same time point, 2 HaMDR1 clones were quantified, one in the granulocyte and one in the MNC fraction, respectively. Here, the copy numbers were 2.87±0.56 and 3.52±1.11, according to 0.002%±0.0003% and 0.002%±0.0008% of the granulocyte and MNC fraction, respectively.

Conclusions: We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.
Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.
Authors: Chunduri Sandeep , Mahmud Dolores , Abbasian Javaneh , Rondelli Damiano
Published: October 2019

Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.
Induction of Localized TNF Activity Against Tumors Using Bispecific Antibodies.
Authors: Krusch Matthias , Herrmann Tanja , Kanz Lothar , Jung Gundram , Salih Helmut R.
Published: October 2019

Tumor necrosis factor (TNF) is a highly effective cytokine with considerable anti tumor activity not only because of direct tumoricidal effects but also due to its strong inflammatory and anti-angiogenic properties. However, while impressive clinical responses were obtained by locoregional application, e.g. in isolated limb perfusion, systemic application of TNF causes intolerable side effects. Therefore, multiple attempts have aimed for targeted delivery of TNF to maximize therapeutic efficiency and sparing normal tissue from detrimental effects, e.g. by generating antibody-derived fusion proteins with human TNF replacing the IgG1 CH2/CH3 Fc domain. However, a highly selective and thus clinically applicable drug for stimulation of TNF-receptors (TNF-R) at the tumor site with minimal unspecific TNF-R stimulation is still not available. Here we report the generation of a bispecific EpCAM X TNFR1 Fab2 antibody (bsFab2). The Fab fragments of an agonistic antibody to mouse TNFR1 were chemically hybridized to anti-EpCAM Fab fragment which served as a targeting molecule on EpCAM transfected mouse melanoma cells. Bound to such cells the bsFab2 exerted strong TNF activity as measured by killing of TNF sensitive WEHI-164 cells at concentrations as low as 0.1ng/ml. In the presence of untransfected tumor cells weak killing was detectable only at antibody concentrations as high as 1000ng/ml. Even more, killing of WEHI-164 cells in the absence of tumor target cells was undetectable. This demonstrates that TNF activity is induced by the bsFab2 in a target cell restricted manner. Target cell restriction was also confirmed when caspase activation rather than cell death was measured. We also demonstrate that the bsFab2 is able to induce target antigen restricted cellular inflammatory responses like monocyte/macrophage cytokine production. Next we investigated the activity of the bsFab2 in the B16 melanoma mouse model with syngeneic, immunocompetent mice. C57Bl/6 mice were inoculated 2500 EpCAM transfected B16 melanoma cells and treated with 6x20μg of the bsFab2. While all of six treated mice survived without developing a tumor, in the control group five of six mice died within 10 weeks. No significant side effects were observed. Our data demonstrate that the TNF activity of the EpCAM X TNFR1-bsFab2 is strictly depending on the presence of EpCAM positive tumor cells, stimulates cellular inflammatory responses in immediate vicinity of target antigen expressing tumors and provide the rationale for further studies evaluating the clinical potential of selective TNFR activation with bispecific antibodies in humans.
IVIG Down-Regulates LPS-Induced TNFα Production Via Inhibition of IκBα Activation: A Possible Mechanism of Action in the Treatment of Bacterial Infections.
Authors: Su Brenda B. , Chan Howard H.W. , Branch Donald R. , Denomme Gregory A.
Published: October 2019

Intravenous IgG (IVIG) preparations, originally developed as a substitution therapy for patients with low plasma IgG, are used in the treatment of various immune and nonimmune disorders including hemolytic uremic syndrome, necrotizing fasciitis, and idiopathic thrombocytopenic purpura (ITP); an autoimmune pro-inflammatory condition. However, the pleotropic effects of IVIG on Fcγ receptor-bearing immune cells are not fully understood. Bacterial lipopolyssacharide (LPS) is a principal pro-inflammatory signaling molecule recognized by the innate immune system. LPS binds to monocyte/macrophage Toll-like receptor 4 (TLR4) to activate transcription factors such as NF-κB/IκBα. In turn, NF-κB activation results in cytokine secretion such as tumor necrosis factor alpha (TNFα); a key pro-inflammatory cytokine that leads to monocyte apoptosis. Since IVIG reduces pro-inflammatory responses, we hypothesized that IVIG may down-regulate the LPS-induced pro-inflammatory response in monocytes. Using the human monocyte-like cell line THP-1 that expresses FcγRs and TLR4, we examined LPS-induced intracellular signaling after in vitro treatment with IVIG. Enzyme-linked immunosorbent assay (ELISA) on culture supernatants showed that 30 minute pre-treatment of THP-1 cells with IVIG (5 mg/mL final) downregulated subsequent LPS (200 ng/mL)-induced secretion of TNFα. Western blotting showed that IVIG increased Akt phosphorylation and resulted in phosphorylation of S6 ribosomal protein and Erk1/2 signaling molecules. The activation of S6 ribosomal protein was blocked by the PI3K inhibitor LY294002. More importantly, IVIG treatment inhibited LPS-induced IκBα activation; the likely reason for the decrease in LPS-induced TNFα secretion. In conclusion, IVIG down-regulates LPS-induced signal transduction and proinflammatory cytokine secretion, an action that is likely responsible for its usefulness in the treatment of bacterial infections and in other diseases where down-regulation of NF-κB is desirable.
A Retrospective Study of Current Anticoagulant Use in Patients with Atrial Fibrillation: Results from a Managed Care Database.
Authors: Murray Miranda , Friedman Howard
Published: October 2019

The vitamin K antagonist warfarin is currently the mainstay of anticoagulant therapy for patients with atrial fibrillation (AF), in whom it has been shown to be effective in preventing stroke. However, it is underused because, despite the quality of clinical care, warfarin can be a difficult drug to manage well. When initiating therapy, patients may require several visits to a coagulation clinic to achieve an international normalized ratio (INR) within the safe and effective range (INR 2–3). Thereafter, frequent monitoring of patients receiving warfarin may still be required due to intrapatient variability. Despite the benefits, studies have shown that approximately 50% of patients who should receive warfarin are not prescribed it or do not receive it. This study was performed to investigate the use of warfarin in eligible patients with AF, for whom it was indicated. Using the US PharMetrics database from 51 managed care organizations, data entered between January 1997 and April 2004, from 5940 patients (>18 years of age with a diagnosis of AF [ICD 9] for whom warfarin was indicated), were analyzed. Patients were excluded if they were not in the database 30 days before and 120 days after their initial diagnosis of AF. Patients were divided into four distinct cohorts, based on treatment patterns: steady users (at least 3 months of warfarin use); discontinued users (used warfarin for only 3 months); non-users (warfarin use for <30 days); and intermittent users (used warfarin prior to first diagnosis of AF, or had less than two prescriptions). Analysis showed that only 31% (1854) of patients were steady users, whereas 59% (3521) of patients were non-users. Also, 4% (246) and 5% (319) of patients were discontinued users and intermittent users, respectively. In addition to a diagnosis of AF, many patients had significant risk factors for stroke: hypertension (48%); diabetes (23%); congestive heart failure (21%); age 75 or older (19%); history of stroke or transient ischemic attack (7%). However, the study showed that patients prescribed warfarin did not have more risk factors compared with long-term steady warfarin users and warfarin non-users (see table). Overall, this study shows that warfarin is underused, even in patients with significant risk factors for stroke, which highlights the need for new, simpler, oral anticoagulant therapy.

Risk factors Total population [n (%)] Steady warfarin (%) Discontinued (%) Non-warfarin (%) Intermittent (%) NB Cumulative numbers of risk factors were constructed by assigning one point for each of the following: congestive heart failure; hypertension; age >75; diabetes; with two points added for prior stroke or transient ischemic attack 0 2036 (32) 32 29 36 34 1 1774 (30) 30 27 30 30 2 1197 (20) 22 23 19 23 3 623 (10) 11 13 10 8 4 204 (3) 3 4 4 4 5/6 106 (2) 2 3 1 1
Molecular and Phenotypic Analysis of Philadelphia Chromosome-Positive Bilineage Leukemia: Possibility of a Lineage Switch from T-Lymphoid Leukemia Progenitor to Myeloid Cells Via PU.1 Expression.
Authors: Nishii Kazuhiro , Monma Fumihiko , Lorenzo Felipe , Katayama Naoyuki , Shiku Hiroshi
Published: October 2019

The occurrence of acute bilineage leukemia is thought to be the malignant transformation of a myeloid or lymphoid leukemic progenitor with the potential to differentiate into the other lineages; however, the mechanisms of this lineage switch are not well understood. Here, we show the extremely rare case of adult Philadelphia chromosome positive acute bilineage leukemia, characterized by T-cell acute lymphoblastic leukemia, CD7+CD5+CD14−, and acute myelomonocytic leukemia, CD7−CD5−CD14+. Chromosome analysis showed 46,XY,del(7)(p11.2),t(9;22)(q34;q11.2) in all metaphase and leukemic cells expressed a minor BCR/ABL chimeric gene. When the CD5+CD14− and CD5−CD14+ cells were sorted, a fusion gene of BCR/ABL and a same clonal rearranged band of a T-cell receptor (TCR) gene were detected in both populations. Nucleotide sequencing of the TCRg gene revealed the clonal rearrangement of the V8-JGT2 complex in both populations. Over-expression of PU.1, which plays a fundamental role in myelomonocyte development was found in the sorted CD34+CD7+ and CD5−CD14+, but not CD5+CD14− cells. These results suggest that leukemic progenitor cells in the T-lineage with del(7),t(9;22) chromosome have the potential to differentiate into myeloid lineage and enforced PU.1 expression may contribute in part of this phenomenon. Studies of bilineage leukemia will be important for the understanding of lineage commitment and switch in hematopoietic cells.
Chromosome 21 Encoded RUNX1 and ETS-2 Overexpression in Regenerating Hematopoiesis in Children with Down Syndrome - Implications for Leukemiogenesis?.
Authors: Reinhardt Dirk , Wortmann Kristin , Kolar Miriam , Klusmann Jan H. , Puhlmann Ulrike , Bourquin Jean P. , Creutzig Ursula , Langebrake Claudia
Published: October 2019

Children with Down Syndrome (DS) are at an 150 fold increased risk to develop acute megakaryoblastic leukemia (AMKL) within the first 4 years of life. About 10% of newborns with trisomy 21 showed transient myeloproliferative disorder (TMD). Although mutations of the transcriptional factor GATA1, resulting in the shortened GATA1s have been shown in almost all blasts in DS-AMKL and TMD the predisposition to leukemiogenesis related to trisomy 21 is not clear. TMD occurred during embryonic stress hematopoiesis leading to the hepatic proliferation of the GATA1s positive blasts. Typically blasts disappeared within the first 3 month of live, however after a median time of 1.3years (0.6 to 3.7 years) 20% of the children suffered AMKL and required intensive cytostatic treatment.

The expression of chromosome 21 encoded hematological transcription factors (TFs) such RUNX1, ETS-2 and ERG were analysed in leukemic blasts from DS- TMD(n=7), DS-AMKL (n=25), DS without hematological disorder (n=10), AMKL (n=10) and healthy controls (n=7) by qRT-PCR.

Results: No increase of RUNX1, ETS-2 and ERG expression could be shown. By contrast, ERG was decrease in all leukemias and in DS without hematological disorder (p Anova.<0.002). GATA1s was significantly overexpressed in TMD and DS-AMKL (pAnova <0.02), whereas GATA1 expression in AMKL and controls was not changed. GATA2 was elevated (pAnova <0.01) in all megakaryoblastic leukemias, with or without DS (pAnova <0.0001). PU.1, typically associated with early lymphatic differentiation and granulopoiesis was down regulated in all megakaryoblastic leukemias and, surprisingly, in DS without hematological disorder. This confirmed previously reported results by gene-array analysis1.

To get further insight in the predisposition caused by trisomy 21 we analysed regenerating hematopoiesis in DS (n=14) partly resembling embryonic stress hematopoiesis. Correlated to the amount of bone marrow activation (CD38 positivity) a myeloid cell population (CD13/CD33 positive); with the co-expression of CD56 (NCAM) and CD36 (thrombospondin-receptor) could be detected by immunophenotyping (median percentage all nucleated bone marrow cells: 73±10%). In children without DS but regenerating hematopoiesis (n=41) a similar population of 4.6±1.8% (p<0.00001) could be detected.

For further analysis the CD33/CD56 positive cells were sorted (FACSVantage). The cells showed normal myeloid morphology and differentiation, lack of GATA1s mutation, but an aberrant TF expression pattern. RUNX1 was 10-fold and ETS-2 5-fold higher expressed compared to controls (p<0.012).

Summarized, (1) DS-AMKL and TMD leukemic blasts showed no general gene-dosage effect. However, (2) in stimulated bone marrow (stress hematopoiesis) trisomy 21 led to an overexpression of chromosome 21 encoded TFs, which might contribute to leukemiogenesis.
Human BCL11B Is Expressed in Normal T Cells and Differentially Expressed in T-Cell Malignancies.
Authors: Pulford Karen , Lyne Linden , Roncador Giovanna , Kominami Ryo , Banham Alison H.
Published: October 2019

B-cell lymphomaleukaemia 11B (BCL11B), the human homologue of murine Bcl11b/Rit-1/CTIP2, was originally identified as a novel tumor suppressor gene in a study of gamma-radiation induced thymic lymphomas in mice. Later studies, however, showed Bcl11b to have a vital role in T cell development and survival, with BCL11B translocations involving a variety of partner genes being reported in both T-cell and myeloid leukemias. The human BCL11B gene, located at 14q32.2, encodes an 832aa Kruppel C2H2 zinc finger protein that is functionally uncharacterized but likely to act as a transcriptional regulator. Analysis of publically available normal tissue Affymetrix microarray expression data indicates an expression pattern restricted to hematopoietic cells, with high levels of BCL11B transcripts being present only in peripheral blood T cells, NK cells, thymus and tonsil.

We have used two rabbit polyclonal anti-BCL11B antibodies to study the distribution of BCL11B protein in both normal and neoplastic human cells. These reagents, raised against two distinct regions of the murine Bcl11b protein (zinc finger and C-terminus), recognized the human BCL11B protein. While neither antibody stained B-cells in tonsil, one was crossreactive with the highly homologous BCL11AXL protein by Western blotting. In normal tissues, BCL11B protein expression was confined to the nuclei of the vast majority of T cells in thymus (foetal and adult) and tonsil. High levels of BCL11B were detected in T-cell lines, including the Molt-4, CCRF-CEM and Jurkat T-cell acute lymphoblastic leukaemia (T-ALL) derived cell lines. No expression was detected in any B-cell derived (pre-B to plasma cell stage) or myeloid cell lines studied. These results are consistent with the microarray gene expression data. In T-cell malignancies, BCL11B protein was only detected in a proportion of tumors, including 5/6 T-ALLs (one being weakly stained) and 2/8 peripheral T-cell lymphomas (weak cytoplasmic staining only). Interestingly, no expression was detected in ALK-positive anaplastic large cell lymphoma lines or tumors. Further studies of a larger series of T-cell malignancies are in progress. All other tumors studied, including B-ALL, chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, myeloma and Hodgkin’s lymphoma, were unlabelled.

In conclusion, the distribution pattern of the BCL11B protein in a wide range of both normal and neoplastic tissues is described for the first time. The study of BCL11B expression is an invaluable first step towards elucidating the role of this protein in T-cell biology and the significance of its differential expression in T-cell malignancies.
G1 Cell-Cycle Arrest and Apoptosis by Histone Deacetylase Inhibition in MLL-AF9 Acute Myeloid Leukemia Cells Is MLL-AF9 Independent.
Authors: Tonelli Roberto , Sartini Roberta , Fronza Raffaele , Freccero Francesca , Franzoni Monica , Dongiovanni Danilo , Ballarini Marco , Ferrari Simona , D’Apolito Maria , Di Cola Gabriella , Capranico Giovanni , Khobta Andriy , Campanini Renato , Paolucci Paolo , Minucci Saverio , Pession Andrea
Published: October 2019

Acute myeloid leukemia (AML) with MLL rearrangements (MLLmut), found mainly in M5 or M4 FAB subtypes, is frequent in infants and secondary leukemias. The most common MLL translocation gives rise to MLL-AF9. MLL protein interacts with histone deacetylases (HDACs) -1 and -2 through the MLL repression domain.

We report the effects of HDAC inhibition by valproic acid (VPA) in MLL-AF9 AML-M5 cells (THP-1, MM6 and MOLM-13) and MLLmut AML-M5 blasts. VPA led to histone hyper-acetylation, strong cell-growth inhibition, G1 cell-cycle arrest and apoptosis. Combined treatment with all-trans-retinoic-acid (ATRA) did not substantially improve these effects. VPA increased MLL-AF9 transcription, indicating that VPA overcomes the cell-growth promoting activity and resistance to apoptosis conferred by MLL-AF9 in AML-M5 cells, even with increased MLL-AF9. A small number of genes were significantly affected by VPA in p53-absent THP-1 cells (GeneChip analysis), and the majority of these were up-regulated. VPA potently induced p21 and cyclin G2 (CG2) expression. p21 and CG2 targeted inhibition demonstrated that p21 acts as a key regulator in the VPA-inducted G1 cell-cycle arrest, while induction of CG2 has no effect.

These data suggest that these poor prognosis patients may benefit from HDAC inhibitor therapy.
In Acute Leukemia, the Polymorphism −211C>T in the Promoter Region of the Multidrug Resistance-Associated Protein 3 (MRP3) Is Not Associated with the Expression Level of the Gene.
Authors: Dorfel Claus , Sauerbrey Axel , Gruhn Bernd , Zintl Felix , Steinbach Daniel
Published: October 2019

We could recently show that the expression of the multidrug resistance-associated protein 3 (MRP3; ABCC3) is an important prognostic factor in ALL and in AML (Blood2003; 102:4493–8 and Clin Cancer Res2003; 9:1083–6).

Lang et al. showed that the single nucleotide polymorphism −211C>T (NCBI SNP ID: rs4793665) in the promoter region of the MRP3 gene is associated with the expression of the gene in liver cells (Pharmacogenetics2004;14:155–64). They found that individuals who were homozygous for cytosine in position 211 showed higher levels of MRP3 mRNA and protein. Lang et al. also provided evidence that the polymorphism is located at a binding site for transcription factors and that the polymorphism affected the binding of such factors. This could explain the influence of the genotype on the transcription level of the gene.

Because of these findings, we hypothesized that the SNP −211C>T might also determine the expression of MRP3 in acute leukemia and therefore be an inborn determinant of drug resistance. In order to prove this hypothesis we analyzed this SNP in our previously described groups of ALL and AML patients.

The genotype was determined using a ready to use TaqMan® SNP Genotyping Assay and the ABI PrismTM 7700 Sequence Detector (Assay ID: C_27829307_10; Applied Biosystems, Foster City, CA, USA).

Samples from 139 patients were analyzed. The frequency of the genotypes was: TT = 32% (n=44), CC = 22% (n=31), and CT = 46% (n=64). We did not find any correlation between the genotype and the expression of MRP3 (p=0.44). The result was the same, when patients with AML, T-lineage ALL, and precursor B-lineage ALL were analyzed separately. We also did not find a correlation between the genotype and response to therapy or the chance of survival.

In conclusion, the SNP −211C>T in the promoter region of MRP3 does not have a major impact on the level of MRP3 gene expression in acute leukemia and it does not determine the response to therapy. Possibly, the putative transcription regulator which binds at this part of the MRP3 gene is not active in acute leukemia.
Arsenic Trioxide Induces Regulated, Death Receptor- and Caspase-Independent Cell Death through the Mitochondrial Death Pathway.
Authors: Scholz Christian , Richter Antje , Lehmann Mario , Schulze-Osthoff Klaus , Dörken Bernd , Daniel Peter T.
Published: October 2019

Arsenic trioxide (As2O3, arsenite) efficiently kills cells from various hematologic malignancies and has successfully been employed especially for the treatment of acute promyelocytic leukemia. There, and in lymphoid cells we demonstrated that arsenite induces cell death in a caspase-2 and -9-independent fashion. Here, we address a potential role of death receptor signaling through the FADD/caspase-8 death inducing signaling complex in arsenite-induced cell death. In detail, we demonstrate that arsenite induces cell death independently of caspase-8 or FADD and cannot be blocked by disruption of CD95/Fas receptor ligand interaction. Unlike in death receptor ligation-induced apoptosis, As2O3-induced cell death was not blocked by the broad spectrum caspase inhibitor z-VAD-fmk or the caspase-8-specific inhibitor z-IETD-fmk. Nevertheless, arsenite-induced cell death occurred in a regulated manner and was abrogated upon Bcl-2 overexpression. In contrast, arsenite-induced cell demise was neither blocked by the caspase-9 inhibitor z-LEHD-fmk nor substantially inhibited through the expression of a dominant negative caspase-9 mutant. Altogether our data demonstrate that As2O3-induced cell death occurs independently of the extrinsic death receptor pathway of apoptosis. Cell death proceeds entirely via an intrinsic, Bcl-2 controlled mitochondrial pathway that does, however, not rely on caspase-9.
Apoptotic Cross-Talk between Organelles Induced by Fenretinide in Hematopoietic Malignancy.
Authors: Wang Kankan , Xiao Dakai , Du Yanzhi , Fan Huiyong , Chen Yulong , Chen Saijuan , Chen Zhu , Zhang Ji
Published: October 2019

Fenretinide (N-4-hydroxyphenyl retinamide, 4-HPR), a synthetic analogue of all trans retinoic acid (ATRA), has shown promising potentials in chemopreventive/therapeutic treatment of human malignancies due to its minimal toxicity, ability to control tumor growth in animal models, and synergism with a number of other anti-cancer agents. Clinical trials of fenretinide, as a preventive agent, have proven its efficacy in a number of tumor types including breast cancer, ovary carcinoma, prostate cancer, neuroblastoma and leukemia. In contrast to ATRA which induces cell differentiaion, fenretinide induces cell apoptosis in a variety of tumor cell lines. Although intensive studies have revealed that fenretinide may trigger apoptosis via induction of different signal molecules such as reactive oxygen species (ROS) and ceramide, mechanisms underlying fenretinide induced apoptosis are far from clear, which is largely due to the fact that a multitude of responses occur in cells upon fenretinide treatment. Accordingly, we have applied an approach integrated with cDNA microarray, advanced technology of data and literature mining, and methods of protein biochemistry to study the process of fenretinide-induced apoptosis in leukemia cell line NB4 in detail. Numerous novel features with the temporospatial relationship were revealed, indicating the coordinated regulation of molecular network from various aspects of cellular activities to program cell death. These features include changes in transcriptional regulation, translational regulation, signal transduction, metabolism and stress response. In consequence, cells treated with fenretinide appear to enter programmed cell death through complex cross-talk between organelles such as mitochondria and endoplsmic reticulum. This study may therefore have substantial impact on the development of more sophisticated protocols in leukemia and probably other malignancies as well.