bluuebunny/crossref_metadata_2025 huggingface dataset:
A Prospective Randomized Study of Rituximab and Dexamethasone vs Dexamethasone Alone in ITP.
Authors: Zaja Francesco , Mazza Patrizio , Vianelli Nicola , De Fina Marzia , Liso Vincenzo , Bonferroni Margherita , Usala Emilio , Cantoni Silvia , Ferrara Felicetto , Mirto Salvo , Visani Giuseppe , Fioritoni Giuseppe , Zaccaria Alfonso , Gugliotta Luigi , Mazzucconi Maria G. , Campagna Selenia , De Stefano Valerio , Veneri Dino , Carbone Cecilia , Semenzato Gianpietro , Soldano Franca , Gamba Enrica , Battista Marta , Baccarani Michele , Fanin Renato
Published: October 2019

Previous uncontrolled studies have highlighted the potential activity of Rituximab in patients with idiopathic thrombocytopenic purpura (ITP) relapsed or refractory to standard treatments. To better address this effect, a prospective randomized, multicenter, phase III study comparing treatment with Dexamethasone alone (arm A) vs Dexamethasone plus Rituximab (arm B) was started in July 2005 for adult patients with ITP according to the ASH guidelines. Main inclusion criteria were: age ≥ 18 years, untreated ITP, platelet (PLT) count ≤ 20 x109/L, HIV- HCV-HbsAg negativity, informed consent. Patients randomized to arm A received a single course of Dexamethasone 40 mg po on days +1, +2, +3, +4, while patients randomized to arm B received Dexamethasone (as in arm A) in association with Rituximab 375 mg/m2 iv on days +7, +14, +21, +28. Patients in arm A who failed to achieve a sustained response (SR) could be rescued with arm B treatment. The primary objective of the study was to compare SR, i.e. PLT ≥ 50 x 109/L at month + 6 of treatment. The secondary objectives were: the initial overall (OR= PLT ≥ 50 x109/L) and complete response (CR= PLT ≥ 100 x 109/L) by day 30 after starting treatment, respectively; the toxic profile. The statistical plan considered three interim analyses, after the first 50, 100 and 150 enrolled patients, with an estimated sample size of 198 patients (99 per arm). Table 1 summarizes the main demographic data and the results of efficacy and toxicity according to an intention to treat analysis of the first interim analysis. The toxic profile was characterized by only grade 3 adverse events (AE); no patient died during the study period. 16 patients of arm A were rescued with arm B. For this group SR was 81% and no patient experienced SAE or ≥ grade 3 AE. In accordance with the initial statistical plan of the study, which stated that patients’ recruitment would ceased if a ≥ 50% difference in sustained response was demonstrated, enrolment has been stopped in June 2007 with a total number of 103 randomized patients. This preliminary report indicates a significantly higher SR for arm B of treatment with no difference in toxicity profile. A final report will be prepared when the results on the entire study group will be available.

Table 1 Therapy Arm A Arm B Statistics Patients 24 26 Male/female 11/13 10/16 p = NS Age (median ± SD) 54.54 ± 18.78 48.65 ± 15.10 p = NS Initial OR 15 (62.5%) 18 (69%) p = NS Initial CR 10 (42%) 16 (61%) p = NS SR 7 (29%) 21 (81%) p = 0.0001 SAE or grade 3 AE 3 (12.5%) 2 (8%) p = NS
HOXA9 Represses Bim Expression in MLL Rearranged Leukemia: Implications for Drug Therapy.
Authors: Stubbs Matthew C. , Faber Joerg , Kung Andrew L. , Cameron Scott , Armstrong Scott A.
Published: October 2019

MLL translocations can be found in roughly 10% of acute leukemias, including over 70% of infant leukemias. These leukemias are often associated with a poor prognosis, making new therapeutics for this subtype of leukemias a priority. HoxA cluster genes have been shown to be upregulated by MLL fusion proteins, and make up a portion of a self-renewal associated gene expression signature found in murine models of MLL-AF9-expressing leukemia stem cells. The role of HoxA cluster genes in MLL-fusion mediated leukemia is not clearly understood, mainly due to lack of knowledge of HoxA downstream targets. One possible role for HoxA proteins in MLL-fusion induced leukemia is enhancement of growth and survival pathways. Evidence for this possibility can be found in C.elegans, where the pro-apoptotic BH3-only gene egl-1 is regulated by homeobox genes. Therefore we sought to determine whether a HoxA cluster gene could in fact regulate survival/apoptosis machinery. Here we demonstrate that HoxA9 serves as a repressor of the pro-apoptotic BH3-only family member Bim. Knockdown of HoxA9 using shRNA in the MLL-AF9 expressing AML cell line Molm14 leads to upregulation of Bim, and rapid induction of apoptosis. Interestingly, knockdown of Meis1 or Pbx2, known binding partners of HoxA9, showed only an intermediate effect, with slower growth, but without an increase in Bim levels. Other BH3-only family members Noxa and Puma showed no significant change in expression after HoxA9 suppression as determined by quantitative RT-PCR. Using a FLAG-HOXA9 expressing Molm14 cell line, we were able to determine by chromatin immunoprecipitation that HOXA9 is present at a DNA region containing a canonical HOX-PBX binding site (TGATTTAT) roughly 100bp downstream of the first exon-intron junction in the human Bim gene. As histone deacetylases (HDACs) are generally transcription repressors, it is plausible that HOXA9 recruits HDACs to the Bim gene, thus regulating its expression. Since HDAC inhibitors also activate BIM, we assessed BIM activation in Molm14 cells after treatment with an HDAC inhibitor, and noted similar kinetics of BIM activation, suggesting a possible link between HoxA9 mediated suppression of BIM and HDACs. We are now assessing HOXA9/HDAC interactions in MLL rearranged leukemia cells. These data suggest that HOXA9 ensures survival of leukemia cells by directly inhibiting transcription of the pro-apoptotic gene Bim, and that interfering with this mechanism may improve treatment for MLL-fusion bearing leukemias.
Circulating Angiopoietin-2 Predicts Time to Relapse after Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia.
Authors: Koenecke Christian , Kümpers Philipp , Lukasz Alexander , Hecker Hartmut , Dammann Elke , Verhagen Willem , David Sascha , Göhring Gudrun , Buchholz Stefanie , Weissinger Eva , Krauter Jürgen , Eder Matthias , Haller Hermann , Ganser Arnold
Published: October 2019

CK and PK contributed equally to this work. Angiopoietin-2 (Ang-2), a member of the Angiopoietin/Tie ligand-receptor system, represents one of the specific inducers required for angiogenesis. Recent data indicate that bone marrow neoangiogenesis plays an important pathogenic and prognostic role in malignancies of the hematopoietic system. We have recently shown that circulating Ang-2 is a predictor for relapse in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) in a cohort of high-risk (HR) myeloid malignancies (Kümpers, Koenecke et al.: BLOOD May 2008). The aim of this study was to validate our results in a larger cohort of patients. In contrast to our previous work we included different conditioning regimens as well as patients with Acute Myeloid Leukemia (AML) irrespective of their risk stratification according to cytogenetic (SWOG, ECOG criteria) and/ or mutational status (FLT3, NPM1). Circulating Ang-2 (in-house ELISA) was measured in serum of AML patients treated at our institution between 1991 and 2008 (n=191) immediately before standard (n=83) or reduced intensity conditioning (n= 104) for HSCT. Stem cell sources were BM (n=23), PBSC (n=163) or both (n=5) from MRD (n= 91), MMRD (n=7), MUD (n=67) and MMUD (n=26). The median age of the patients was 49 years (range 18–71). HSCT for AML (n=149) or secondary AML after MDS (n=42) was performed after primary induction failure (n=30) or for relapse (first relapse n=19, subsequent relapse n=5). 119 patients were transplanted in 1st CR; four in 2nd CR. 11 patients had no chemotherapy before HSCT. In this cohort, 114 of 159 evaluable patients suffered from HR-AML. Circulating pre-HSCT Ang-2 was elevated in patients (median (range): 1.7 (0.05–48.8) ng/ml) compared to healthy controls (0.92 (0.27–2.63) ng/ml; p<0.0001). Using univariate Cox regression analyses, patients’ age, conditioning regimen, remission status, cytogenetic/ mutational status, and Ang-2 were identified as prognostic variables. Multivariate Cox regression confirmed the independent prognostic impact of Ang-2 (hazard ratio 2.2, 95% CI 1.25–3.94, p = 0.007) and HR cytogenetics and/ or mutations (hazard ratio 1.5, 95% CI 1.09–2.11, p = 0.012) for Time to Relapse after HSCT. In this study we could show that Ang-2 is a valid and readily available biomarker to pre-estimate the risk of relapse in a large comprehensive HSCT cohort. Routine pre-HSCT measurement of Ang-2 may open new perspectives for riskadapted treatment of AML patients.
High-Sensitivity Monitoring of Hematopoietic Chimerism after Double Umbilical Cord Blood Transplantation by Real-Time Quantitative Polymerase Chain Reaction.
Authors: Marty-Grès Sophie , Moussiere Marielle , Avinens Odile , Andary Monique , Ramounau-Pigot Annie , Ceballos Patrice , Meniane Jean-Côme , Margueritte Geneviève , Fegueux Nathalie , Rossi Jean-Francois , Eliaou Jean-François
Published: October 2019

Double umbilical cord blood transplantation (DUCBT) has recently emerged as a treatment of hematological malignancies. Chimerism after DUCBT typically shows a single unit engrafting when it relies on polymerase chain reaction (PCR) fluorescent amplification of short tandem repeat-markers analyzed by capillary electrophoresis (STR-CE). As real-time quantitative PCR (RQ-PCR) of short insertion and deletion polymorphisms stands for a more sensitive method of detection especially for minor cell populations, the goal of our study was to assess by RQ-PCR the potential persistence of the unit not detected by STR-CE, defined as a nanochimerism. Between July 2005 and April 2008, 26 patients underwent DUCBT with a reduced-intensity regimen in our institution, as described by Barker JN et al. (fludarabine, cyclophosphamide, total body irradiation 2 Gy). Their median age was 54.5 years (range 11–67). Most patients had advanced hematological malignancies. They all received 4-6/6 HLA-matched cord units. Numbers of infused nucleated and CD34+ cells were higher than 2x107/kg and 1x105/kg, respectively. Monitoring of hematopoietic chimerism was routinely performed on DNA from total peripheral blood mononuclear cells (PBMC) and from blood T cells, weekly during the first month, then twice monthly for 2 months, and monthly, using STR-CE. All available patients with one cord unit chimerism were studied by RQ-PCR. The median follow-up time was 6.5 months (range 1–35). Twenty-three of 26 patients had a full donor chimerism from one cord unit by STR-CE, at day 14 (N=12), day 21 (N=4), day 30 (N=4), day 60 (N=1) or day 200 (N=2) on PBMC and on T cells. Among them, 15 patients were analyzed by RQ-PCR. Discrepancies between both methods appeared in 14 of 15 cases for PBMC and in 8 of 12 available DNAs from blood T cells. RQ-PCR could still detect the minor cord unit at least 21 days after its “disappearance” assessed by STR-CE on PBMC (N=7/14) and on T cells (N=4/8). These data suggested that evaluation of chimerism by RQ-PCR after DUCBT lowers the detection threshold (sensitivity 0.01%) and allows to quantify a nanochimerism that cannot be detected by STR-CE (sensitivity 1–5%). This is the first series ever published on the monitoring of chimerism after DUCBT using RQ-PCR. Clinical implications of this nanochimerism will have to be unraveled in larger series.
Low Dose Alemtuzumab Achieves Long-Term Engraftment with Low Level Mixed Chimerism in Related Haemopoietic Stem Cell Transplantation for Haemoglobinopathies
Authors: de la Fuente Josu , O’Boyle Farah , Killeen Natalie , Tso Allison , New Helen , Atra Ayad , Dokal Inderjeet , Vulliamy Tom , Davis John , Foroni Letizia , Szydlo Richard M. , Roberts Irene
Published: October 2019

Haemopoietic stem cell transplantation (HSCT) is the only proven curative treatment available for haemoglobinopathies. To reduce transplant-related mortality and morbidity associated with graft-versus-host disease (GvHD) and facilitate donor engraftment we have used low-dose alemtuzumab in 43 consecutive patients (36 with thalassaemia and 7 with sickle cell disease; median age 6 years; range 2 to 16 years) transplanted since 2000. Donors were HLA-matched family donors in all cases. All patients were conditioned with oral busulfan 14 mg/kg (days -9 to -6), cyclophosphamide 200 mg/kg (days -5 to -2) and alemtuzumab 0.3 mg/kg (days -8 to -6) with post-HSCT methotrexate (10 mg/m2 days +4 and +7) and ciclosporin for six months. Hypertransfusion was initiated six weeks before conditioning and maintained until day +28 to suppress endogenous haemopoiesis and minimise graft rejection. Patients with thalassaemia were Pesaro class I or II. Patients with sickle cell disease were transplanted for stroke or recurrent vasooclusive crisis and/or acute chest syndrome not responding to hydroxycarbamide. The source of stem cells was bone marrow in all cases except one patient who received PBSC with a median cell dose of 3.08 x 108 TNC/kg (range 1.69 – 10.90 x 108 TNC/kg). Median follow up was 25.8 months (range 6.5 – 94.3 months). Chimerism studies were performed on peripheral blood genomic DNA using 8 microsatellites markers by genescanning. All patients are alive (43/43), 42 with long-term transfusion-independence and donor haematological values (5 years DFS 97.7%). One patient failed to engraft and was re-infused with autologous cells. Donor haemopoiesis was ≥ 95% on day +28 in all 42 patients who engrafted and was maintained at ≥ 95% in the majority of patients at 12 months post-HSCT. Eleven patients (11/42; 31%) have stable mixed chimerism at ≥ 12 months post-HSCT: donor haemopoiesis 90–94% (n=4); <90% (n=7; median 85%; range 45 – 89%). There have been no late relapses and no patients had falling donor haemopoiesis <90% after day +90. Acute GvHD ≥ grade 2 occurred in 5 patients (11.6%) and was steroid-responsive in all cases except the patient who received PBSC. Limited chronic GvHD was present in 2 patients (4.6%) >18 months post-HSCT (on no treatment) and Karnofsky score is 100% in all engrafted patients (42/42). In conclusion, addition of low-dose alemtuzumab (total dose 0.3 mg/kg) achieved long-term engraftment in almost all children undergoing HLA-matched family donor HSCT for haemoglobinopathies with minimal chronic GvHD and excellent DFS (97.7%).
Discovery of a New Molecular Marker, Prohibitin, and Development of Candidates for New Chemotherapeutic Agents Targeting Prohibitin in Primary AML Cells.
Authors: Kim Hye-Ran , Shin Myung-Geun , Lee Sun-Woo , Kim Hee-Nam , Lee Il-Kwon , Kim Hyeoung-Joon , Kook Hoon , Shin Jong- Hee , Suh Soon-Pal , Ryang Dong-Wook
Published: October 2019

In light of the recent studies that showed significant causal relationship between mitochondrial genetic mutation and apoptosis in pathology of various tumors and degenerative diseases, mitochondrial proteins have been interesting targets for the study of apoptosis in leukemia and other malignancies. This observation prompted us to analyze mitochondrial proteins and develop new anti-proliferative agents targeting them. Mitochondria were isolated from AML cells by density-gradient ultracentrifugation using swelling buffer and sucrose buffer. We identified 48 spots corresponding to 38 proteins in primary AML cells using 2-DE and mass spectrometry (MALDI-TOF/TOF technology), the expression of which were altered significantly compared to that of normal hematopoietic cells. Out of these deregulated proteins, 12 and 20 proteins were observed in up- or down-regulated spots, respectively. Interestingly, prohibitin (PHB) (gi4505773) was highly expressed in primary AML cells, which was confirmed by Western blot, immunohistochemistry and immunofluorecenct study in the primary AML bone marrow cells and sections. To assess the functional significance of aberrant prohibitin expression, we applied siRNA delivery for silencing of prohibitin. Transduction with a siRNA 11867 construct resulted in 75% decrease of AML cells as compared to the nonsilencing control construct after two days. Potent chemical substances that can alkylate PHB in AML cells, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU), were synthesized in our laboratory. Time and dose dependent manner of proliferation suppression when treated with CCEU and ICEU was observed in leukemic cell lines including THP-1, K-562 and Kasumi-1. Moreover, notable morphological transformation of leukemic cells was observed when treated with 50 – 200 umol of CCEU and ICEU for 24 hours. Cell cycle analysis of CCEU-and-ICEU-treated- THP-1 and Kasumi-1 cell lines showed a remarkable increase of the sub-G1 phase. Immunoblotting experiment revealed the change of cytoplasmic and nucleoplasmic PHB in K-562 cell line. Expression of cleaved caspase3 and poly ADP-ribose polymerases were also observed to have increased in primary AML cells and cell lines. By analyzing AML cell mitochondrial protein we discovered a new molecular marker, PHB, characteristically overexpressed in AML cells and developed new anti-cancer agents such as CCEU and ICEU that target against PHB in AML cells.
ChIP-Chip in Primary AML Blasts Identifies Peroxiredoxin-II as An Epigenetically Silenced Tumor Suppressor.
Authors: Agrawal-Singh Shuchi , Wiechmann Markus , Doths Sandra , Nolte Christina , Thoennissen Nils Heinrich , Klein Hans-Ulrich , Dugas Martin , Berdel Wolfgang E. , Koschmieder Steffen , Serve Hubert , Mueller-Tidow Carsten
Published: October 2019

Epigenetic silencing of tumor suppressors is a frequent event in leukemogenesis. We performed ChIP-Chip to identify genes with altered Histone H3 acetylation in primary AML specimens (n=115) and normal CD34+ progenitor cells (n=21). Interestingly, promoters of several members of the peroxiredoxin family (PrdxII and Prdx IV) were identified to be significantly Histone H3 hypoacetylated in primary AML blasts. Peroxiredoxins (Prdx) are antioxidant enzymes that regulate the amount of reactive oxygen species in the cell. Prdx scavenge H2O2 and protect cells from oxidative damage to cellular DNA, lipid and proteins. Confirmation by quantitative PCR revealed that the PrdxII promoter was hypoacetylated in AML, and mRNA expression was 10-fold induced in U937 cells upon exposure to the demethylating agent 5-Azadeoxycytidine (Aza). Demethylation of U937 cells by Aza resulted in increased Histone H3 acetylation at the PrdxII promoter. DNA hypermethylation was frequent in primary AML blasts (18/103) but not in control samples (0/40) as assessed by methylation-specific PCR. Hypoacetylation of Histone H3 and DNA hypermethylation was associated with repression of PrdxII mRNA and protein levels in primary AML samples. Decreased PrdxII protein expression as assessed by immunohistochemistry in a tissue microarray was associated with a poor prognosis in AML patients. On the functional level, PrdxII inhibited the growth of hematopoietic progenitor cells in colony assays and negatively influenced receptor tyrosine kinase signaling by inhibition of ERK and AKT activation upon overexpression. Conversely, PrdxII knock-down by shRNA in myeloid progenitor cell lines and in murine primary bone marrow cells led to enhanced growth and tyrosine kinase signaling. A decrease in PrdxII levels was associated with an increase in reactive oxygen species and enhanced phosphorylation of STAT5, AKT and ERK. Taken together, these findings suggest that chromatin modifications in AML suppress expression of PrdxII. Our data also revealed that PrdxII is a novel putative tumor suppressor in AML.
Rac GTPases Are Required for MLL-AF9-Induced Mixed Lineage Leukemia.
Authors: Wei Junping , Wunderlich Mark , Harris Chad , Mizukawa Benjamin , Zheng Yi , Williams David A. , Mulloy James C
Published: October 2019

The t(9;11) translocation fusion gene MLL-AF9 (MA9) is commonly found in acute myeloid and lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. It has recently been reported that MA9 leukemia cells express higher levels of the small GTPase protein Rac and CDC42 when compared to in vitro MA9 immortal cells in a murine model. To determine the importance of Rac GTPase signaling in MA9-induced transformation, we used an MA9 leukemia model we recently established involving MA9 fusion gene expression in human CD34+ cells. Treatment with the Rac specific inhibitor NSC23766, or transient knockdown of Rac expression by RNAi, induced rapid apoptosis in MA9 cells but not in normal cord blood or t(8;21) translocation fusion gene AML-ETO expressing cells. These data demonstrate that the Rac signaling pathway plays a critical role in the growth and survival of MA9 leukemia cells. To extend this work to an in vivo genetic model, we used mice deficient in Rac2 (Rac2-KO) or with floxed alleles of Rac1 in mice transgenic for Mx-Cre. Leukemia development was compared in mice transplanted with MA9-transduced wild type, Rac1 or Rac2 deficient low density bone marrow cells. Poly I:C injections were performed 2 weeks after transplantation to delete Rac1. Rac deletion was confirmed by PCR and western blot analysis. Mice that received either WT or Rac1−/ − MA9 expressing cells uniformly developed AML and died at 3 to 5 months. Mice transplanted with Rac2-KO cells expressing MA9 showed a decreased incidence and increased latency (6 to 10 months) of AML development despite the persistent engraftment of MA9-expressing cells. All three groups of mice maintained MA9 EGFP+ cells in peripheral blood over the entire experiment, and eventually gave rise to similar end stage AML with a Gr-1+/Mac-1+/Kit+/B220-/CD3- phenotype and myelomonocytic blast morphology. Combined with our observation in human CD34+ cord blood cells transduced with MA9, these in vitro and in vivo data indicate that MA9-mediated transformation and survival requires Rac and their downstream effectors. Rac2 signaling appears to be particularly important in the murine MA9 AML model. Therapeutic targeting of Rac could be a unique and important approach to treating MLL leukemia.
Epigenetic Inactivation of Notch Signaling Target Genes HES in B Cell Acute Lymphoblastic Leukemia.
Authors: Kuang Shaoqing , Zweidler-McKay Patrick , Yang Hui , Fang Zhi Hong , Tong Weigang , Garcia-Manero Guillermo
Published: October 2019

The Notch signaling pathway has been implicated in multiple functions during normal hemato-lymphoid development. It also plays critical roles in T-cell leukemogenesis through influencing T-cell proliferation, differentiation and survival. In contrast, we have previously reported a tumor suppressor role in B-cell leukemias, where Notch signaling leads to growth inhibition and apoptosis. The Notch target genes Hairy/Enhancer of Split (HES1-7) encode transcriptional repressors with basic helix-loop-helix (bHLH) domains. Functional and phenotypic analyses of some of the HES family members have been reported, however, expression and epigenetic regulation of the HES family in leukemia is largely unknown. Using Methylated CpG Island Amplification (MCA) / DNA promoter microarray, we identified several HES family genes as hypermethylated in B cell acute lymphoblastic leukemia (B ALL). We further investigated the comprehensive methylation profiles of HES family genes in a panel of leukemia cell lines and ALL patient samples by bisulfite pyrosequencing. Aberrant DNA methylation of HES2, HES4, HES5 and HES6 was detected in most B ALL cell lines including B-JAB, RS4:11, REH, Raji and Ramos but not in normal B cell controls. In contrast, in T cell leukemia cell lines such as Molt4, PEER, T-ALL1 and J-TAG, these genes were generally unmethylated. In B ALL patient samples, the frequencies of DNA methylation in the promoter regions of these genes were 25% for HES2, 50% for HES4, 76% for HES5 and 71% for HES6. Expression analysis of HES4, HES5 and HES6 in leukemia cell lines by real-time PCR further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with DNA methyltransferase inhibitor 5’-aza-2’-deoxycytidine resulted in HES gene re-expression. Finally, forced re-expression of HES5 and HES6 in methylation silenced Rs4 and REH cell lines inhibited cell growth. These results suggest that the Notch/HES signaling pathway is epigenetically-inactivated in B ALL. These data support the role of the HES family as tumor suppressors in pre-B ALL and establish epigenetic modulation as a novel mechanism of Notch pathway regulation. We anticipate that therapies capable of activating Notch/HES signaling may have therapeutic potential in B cell leukemias.
The Anti Von Willebrand Factor Aptamer ARC1779 Prevents the Desmopressin-Induced Thrombocytopenia in VWD Type 2B.
Authors: Paulinska Petra , Jilma Bernd , Firbas Christa , Schaub Robert G , Gilbert James C. , Knoebl Paul
Published: October 2019

Introduction: Von Willebrand Disease (VWD) Type 2B is characterized by increased affinity of VWF for the platelet glycoprotein receptor Ib (GPIb). Consequently, infusion of desmopressin, a standard of treatment for other forms of VWD, causes transient thrombocytopenia in patients with VWD-2B. The aim of the study was to test whether the novel anti-VWF aptamer ARC1779, which inhibits the interaction of VWF with GPIb, could abrogate the transient desmopressin-induced thrombocytopenia in VWD-2B.

Patients and Methods: This was part of a clinical trial using a double-blind, randomized, placebo-controlled, crossover design. A patient with VWD 2B received desmopressin (0.4 mcg/kg) alone, or desmopressin after pre-treatment with ARC1779 targeted to a plasma concentration of 10 mcg/mL. Multiple blood samples were obtained over 24 hours to measure von Willebrand Factor Antigen, Ristocetin Cofactor (RiCO), FVIII activity and multimers.

Results: Desmopressin alone (open circles) increased VWF:Ag by 38% (not shown), and VWF:RiCo 3.4-fold within 30 min, and profoundly decreased platelet counts by 87%. In striking contrast, ARC1779 (solid triangles) completely prevented the desmopressin induced fall in platelet counts (Figure), although the increase in vWF:Ag and vWF:RiCO was greater (2.7-fold and 12-fold, respectively).

Conclusions: This randomized, double-blind, placebo-controlled experiment shows that ARC1779 effectively prevents consumption of VWF and platelets in response to desmopressin in VWD Type 2B. It provides in vivo proof of concept that ARC1779 is a potent inhibitor of the VWF A1 domain interaction with GPIb.

Figure Figure
Subclinical Deep Venous Thrombosis Affects 10 % of Haemophiliacs Undergoing Major Orthopaedic Surgery.
Authors: Hermans Cedric R. , Lambert Catherine
Published: October 2019

The management of venous thromboembolism (VTE) in patients with haemophilia undergoing major orthopaedic surgery requiring intensive replacement therapy remains controversial. Information about the incidence of proven VTE in this setting is lacking. Despite the absence of data, half of haemophilia comprehensive centers in Europe recently reported the use of pharmacological antithrombotic prophylaxis in this setting.

We conducted a prospective study to evaluate the occurrence of deep venous thrombosis (DVT) of the lower limbs in all consecutive patients with haemophilia referred for major orthopaedic surgery at the Cliniques universitaires Saint-Luc, Brussels, Belgium. Screening for DVT by unilateral (13) or bilateral (16) compression ultrasonography (US) of the lower limbs was performed 5 to 12 days after 29 major orthopaedic surgeries (total hip replacement (5), total knee replacement (TKR) (15), ankle arthrodesis (4), decompressive laminectomy for lumbar stenosis (1), femoral osteosynthesis (4)) in 22 patients with severe (16), moderate (4) or mild (2) haemophilia A (20) or B (2), intensively treated with continuous infusion of clotting factor concentrate. None of the patients received pharmacological anti-thrombotic prophylaxis with LMWH. There was no case of clinical DVT or pulmonary embolism during the 3-months post-operative clinical surveillance. However distal DVT involving a single peroneal vein without proximal extension was identified in two patients with severe HA after TKR and in one patient with mild HB after decompressive laminectomy. The latter was efficiently treated with a short course of LMWH. The two others resolved spontaneously without antithrombotic therapy.

In conclusion, sub-clinical DVT affects up to 10 % of patients with haemophilia undergoing major orthopedic surgery. All thrombotic events were distal and resolved spontaneously or with a short course of LMWH without complications. This study suggests that routine screening for DVT by compressive US is indicated in patients with haemophilia undergoing major orthopaedic surgery and not receiving pharmacological thromboprophylaxis. Further studies are needed to determine if systematic pharmacological prevention of DVT is required in this setting as in patients without haemophilia.
Monocyte-Derived Microparticles Improve Hemostasis in Factor VIIIDeficient Mice: Exploring the Role of PSGL-1/P-Selectin.
Authors: Gross Peter L. , Vaezzadeh Nima , Ivicic Lori , Ni Ran , Esposito Bruno , Bai Xufang , Akram Ali
Published: October 2019

Introduction: Microparticles derived from leukocytes contribute to fibrin formation at thrombi in vivo and factor VIII-deficient (FVIII) mice treated with an agent that elevates their microparticles have decreased bleeding. A novel therapy for hemophilia patients with inhibitors is needed. We evaluated whether microparticles generated in vitro could improve hemostasis in FVIII mice.

Methods: Mouse CD11b positive monocytes, isolated by MACS, or cultured monocytic cells (WEHI274.1) were treated with the calcium ionophore A23187. The resulting microparticles (isolated by differential centrifugation, and defined as CD18 positive events less than 1 μm diameter) or PIPES buffer were infused intravenously into FVIII-deficient mice (B6.129S4-F8tm1Kaz) or control wild type B6.129 mice prior to evaluation. The amount of platelets in laser-generated thrombi in cremaster muscle arterioles was evaluated using high-speed intravital fluorescence microscopy. The amount of hemoglobin shed from a 2 mm tail tip amputation measured blood loss.

Results: Infusion of MPs at doses above 1000/g resulted in the death of wild type mice; FVIII-deficient mice tolerated MPs at doses up to 4000/g. Blood loss after tail clip in FVIII-deficient mice was 6-fold higher than blood loss from wild type mice. Blood loss after tail clip in FVIII-deficient mice was reduced to normal after the infusion of MPs at concentrations as low as 400/g. MPs, at 400/g, from CD11b positive cells isolated from wild type, FVIII-deficient mice or PSGL-1-deficient mice all similarly reduced blood loss after tail clip in FVIII-deficient mice. The biological half life of MP effect on tail-bleeding was 3 hours.

Platelet accumulation in cremaster arteriolar thrombi was impaired in FVIII-deficient mice. Infusion of MPs at a concentration of 1000/g normalized platelet accumulation, but infusion of MPs at a lower concentration (400/g) did not.

Conclusion: Abnormal hemostasis in FVIII-deficient mice can be temporarily reversed by the infusion of in vitro generated monocyte-derived MPs, including MPs derived from monocytes from FVIII-deficient or PSGL-1-deficient mice. The dose whereby MPs normalize FVIII-deficient mice is different between the hemostasis and thrombosis models. To explore whether P-selectin at injuries is required for the effect of MPs, we have generated by cross-breeding FVIII/P-selectin double deficient mice. These mice are born at expected mendelian frequency. Two of 20 male FVIII/P-selectin double deficient mice had spontaneous bleeding at 8 weeks of age, one in the thigh and one from the ear. FVIII/P-selectin double deficient mice also have prolonged tail bleeding times, which will serve as a model for testing the P-selectin targeting of microparticles.
Primary Prophylaxis Prevents the Onset of Arthropathy: Longitudinal Results of 603 Boys with Severe Factor VIII Deficiency Analyzed through the Centers for Disease Control Universal Data Collection (CDC UDC) Study.
Authors: Manco-Johnson Marilyn J. , Wang Chengbin , Konkle Barbara A. , Ingram-Rich Robina , Humes Steven , Soucie J. Michael
Published: October 2019

BACKGROUND: Arthropathy is the most common complication of severe factor VIII deficiency and causes chronic pain, functional disability and increased cost of care. Early institution of primary prophylaxis (PP) was found to prevent hemophilic arthropathy at age 6 years in a prospective randomized clinical trial including 65 boys (NEJM2007;357:535–44). The current study was conducted to determine the prevalence and efficacy of primary prophylaxis to prevent joint disease in the larger population of young children enrolled in the CDC UDC data base.

METHODS: Eligibility for this analytic project included boys with factor VIII < 1% entered onto the UDC study at or before 36 months of age. Treatment was clinically determined and defined as prophylactic which could be primary (PP, initiated at or before 36 months), secondary (SP, initiated at or following 42 months), or indeterminate (IP, initiated between 36 and 42 months); or episodic (E, without routine prophylaxis). A target joint was defined as one with four or more bleeding events within 6 months. Kaplan-Meier curves were used to evaluate survival without target joint development over time; Cox modeling was employed to assess risk factors for target joint development. Univariate and multivariable analyses were used to model risk factors for bleeding.

RESULTS: Of 603 eligible boys, treatment included: PP in 252 (42%), SP in 73 (12%), E in 240 (40%). 38 boys (6%) with IP were excluded from the analysis. In comparison to children treated with E, those on PP were, on average: older (2.17 vs 1.87 years, p < 0.0001); more likely to have had a bleeding event (96 vs 91%, p=0.002); more likely to have received home infusions (90 vs 49%, p < 0.0001); and more likely to have suffered an intracranial hemorrhage (11.3 vs 4.1%, p=0.02). Despite their increased experience with joint and other bleeding events, during the period of prospective observation, children on PP had significantly less joint bleeding (OR 0.51, CI 0.39–0.66, p < 0.0001) and showed improved survival time without onset of a target joint (p = 0.002 overall, and 0.004 between PP and E, figure 1). Results for muscle or any bleeding were similar. In multivariable analysis PP exerted the greatest protective effect on risk of target joint development (hazard ratio 0.38, 95% CI 0.22–0.66, P = 0.001). Factors associated with E therapy included non-white race (p < 0.0001) and insurance coverage other than commercial (p = 0.004), although these factors were not associated with poor joint outcomes.

CONCLUSION: In a large population of children with severe hemophilia A on the CDC UDC registry, PP prevents joint and other bleeding and delays target joint development. Factors limiting application of PP must be explored and addressed.

Figure Figure
Alteration of Circulating Regulatory T Cells in Japanese Adult Patients with Immune Thrombocytopenic Purpura after Eradication of Helicobacter Pylori.
Authors: Miyazaki Koji , Danbara Mikio , Ohsaka Manabu , Suzuki Yuhko , Horie Ryouichi , Higashihara Masaaki
Published: October 2019

Immune thrombocytopenic purpura (ITP) is an autoimmune disorder, and eradication of Helicobacter pylori (HP) has been demonstrated to be an effective and tolerable firstline treatment for the patients infected with HP in Japan. However the mechanism has still remained to be uncovered. Recently CD4+CD25high+Foxp3+ regulatory T cells (Tregs), which regulate autoreactive T cells, have been identified, and suggested to play an important role in pathogenesis of ITP, as well as other autoimmune disorders. It has been shown that Tregs are reduced and suppressed in the ITP patients. And a recent report has demonstrated that the defective Tregs are restored upon the rituximab treatment. However, the effects of HP eradication therapy on Tregs have not been determined. The aim of this study is to investigate the circulating Tregs in ITP patients treated with HP eradication. And we also attempted to elucidate the mechanisms of the treatment. The peripheral blood CD4+CD25high+ Tregs were measured by flow cytometry before and after the treatment in 21 Japanese adults with HP-positive chronic ITP. We also confirmed the expression of Foxp3 in this cellular population with the permeabilized mononuclear cells. Among 21 patients, the platelet counts increased in 13 cases (responders), but not in 9 cases (non-responders). In responders the numbers of Tregs have been restored, but not in non-responders after the treatment. It is interesting here that the amounts of Tregs were still transiently elevated in an early phase (2–3weeks) after the treatment in some non-responders without recovery of the platelet counts. Furthermore, in three cases, who failed in pylorus elimination, Tregs were also transiently increased in number associated with brief recovery of the platelet counts, and reduced to the initial level in about two months. After the successful re-eradication, the numbers of Tregs and platelets have been restored. From these results it is shown that HP eradication can modulate Tregs to increase the platelet counts for ITP patients. We also demonstrate that the increase of Tregs by HP eradication was more rapid than that by rituximab, which required about three months. Further, it might be suggested that there are two phases of the therapy with HP eradication for ITP. In an initial stage, the therapy itself could have an effect modulating the immune systems to potentiate Tregs. Some drugs, such as macrolide antibiotics including clarithromycin have been demonstrated to be a potent immunomodulator. However, this phase is not sufficient for the successful treatment, as shown in the cases of failure in HP elimination. In a retentive stage, extermination of HP is also necessary for sustainment of restored Tregs.
The Uroporphomethene Inhibitor Causitive for Porphyria Cutanea Tarda (PCT) Is Generated by Oxidation of Hydroxymethylbilane (HMB)
Authors: Phillips John D , Bergonia Hector A , Reilly Christopher A , Franklin Michael R , Kushner James P
Published: October 2019

Porphyria cutanea tarda (PCT) is caused by a uroporphomethene (UPN) inhibitor of uroporphyrinogen decarboxylase (Phillips, JD et al, Proc Natl Acad Sci U S A. 2007 104(12):5079–84). In the absence of the inhibitor very little uroporphyrin (URO) is secreted in the urine and the excreted URO is predominantly the isomer-III (URO-III). In contrast, in patients with PCT and mice with experimental PCT the predominant porphyrin excreted in urine is uroporphyrin-I (URO-I). Direct measurements of uroporphyrinogen III synthase activity (UIIIS) in mice with PCT have shown no reduction in activity indicating that an alternate mechanism is responsible. We hypothesized that oxidation of a single bridge carbon of HMB, the product of the porphobilinogen deaminase reaction, is the initial event leading to the porphomethene. Our structural data indicate that oxidized HMB cannot serve as a substrate for UIIIS (Schubert, HL et al, Biochem J. 2008 47(33):8648–55) but can non-enzymatically cyclize to form UPN I. UPN I would then serve as the inhibitor of uroporphyrinogen decarboxylase but and then be fully oxidized to yield URO-I. To test this hypothesis we generated authentic UPN’s I and III. URO’s I and III were reduced with palladium on carbon to yield uroporphyrinogens (Uro’gen) I and III. UPN’s I and III were then generated by controlled photo-oxidation of the corresponding Uro’gens. We then utilized LC-MS to separate and characterize the synthesized UPN’s. Retention times of the two compounds differed by approximately 1 min (UPN III elutes prior to UPN I) but the profile of the daughter ions derived from the UPN’s (molecular mass 835 Da) was identical. The same approach was applied to clarified liver cytosol from mice with experimental PCT. The retention time of the 835 Da UPN identified in mouse liver cytosol corresponded to that of the synthesized UPN I and yielded identical daughter ions. These data support the hypothesis that oxidation of HMB leads to formation of UPN I in vivo. This is concordant with our view that UPN I serves both as the inhibitor of URO-D and as the source of the excess URO-I found in PCT.
Human SIRPA Polymorphism Modulates Macrophage-Mediated Suppression of Human Hematopoiesis
Authors: Kuriyama Takuro , Takenaka Katsuto , Kumano Takashi , Oku Seido , Urata Shingo , Nagafuji Koji , Dick John E. , Danska Jayne S , Akashi Koichi
Published: October 2019

Signal regulatory protein-α (SIRPA) is an immunoglobulin superfamily transmembrane protein with intracellular docking sites for two Src homology domain containing tyrosine phosphatases, and most abundantly expressed in neurons and myeloid cells. SIRPA is a critical immune inhibitory receptor on macrophages. CD47 is a ligand for SIRPA, and CD47 interaction with SIRPA serves as a ‘self-recognition’ that prevents phagocytosis of the cells expressing CD47. Recently, we (Nature immunology 2007) identified polymorphism in murine Sirpa as a critical modulator of engraftment in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation, and non-obese diabetic (NOD) mouse Sirpa polymorphism has far greater reactivity with human CD47 than that of the respective alleles of other strains resulting in effective engraftment of human hematopoietic cells. In addition, as well as the mouse, human SIRPA is highly polymorphic in the IgV domain. Then, we examined whether polymorphism in SIRPA induced the difference for suppressive effect of human macrophage on hematopoiesis. First, we examined sequence alignment of SIRPA IgV domain (exon 3 of SIRPA) of healthy control 18 people. We identified two major variants; 6 people with variant1 (V1) and 12 people with variant2 (V2). The SIRPA amino acid sequences of V1 and V2 are different in 13 residues, and its residues in orthologous position between species that is polymorphic between NOD and other strains as well as between V2 and V1. Next, we examined that whether there is a difference in the effect on hematopoiesis between V1 macrophage and V2 macrophage by long term culture-initiating cells (LTC-IC) assay on MS-5 stromal cells. For macrophage preparation, peripheral mononuclear cells from healthy donors were purified by positive selection using MACS CD14 Micro Beads (Miltenyi Biotec), and they were incubated with M-CSF for 3 days. For LTC-IC assay, 5×102 differentiated macrophages were seeded onto established MS-5 stroma in 96-well tissue culture plates. The next day, human hematopoietic CD34+ cells were seeded at doses of 102 to 5×103 cells per well and cultured for 4 weeks. At the end of the culture, cells were detached and plated into methylcellulose progenitor assays. Macrophage had a suppressive effect on the number of LTC-IC in all cultures. There was tendency that V1 macrophage had a greater suppression compared to V2 macrophage, however the differences between V1 and V2 were marginal (p=0.03). These data suggests that human macrophages have suppressive effect on hematopoiesis, and human SIRPA polymorphism modulates macrophage-mediated suppression of hematopoiesis in allogenic model, likewise in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation. Moreover, SIRPA polymorphism might be related to graft failure in the allogenic hematopoietic stem cell transplantation.
The Potent Deacetylase Inhibitor Trichostatin a (TSA) Increases CXCR4 Expression in Hematopoietic Stem/Progenitor Cells by Chromatin Remodelling
Authors: Gul Hilal , Marquez-Curtis Leah A. , Lo Jennifer , Jahroudi Nadia , Turner A. Robert , Larratt Loree M. , Janowska-Wieczorek Anna
Published: October 2019

Stromal-cell derived factor (SDF)-1α/CXCL12 and its cognate receptor, CXCR4, play a crucial role in the trafficking of normal hematopoietic stem/progenitor cells (HSPC) and their homing/retention in bone marrow. Consequently, modulation of CXCR4 expression in HSPC could be applied therapeutically to improve the efficiency of HSPC transplantation. It is known that gene expression can be regulated by chromatin remodelling. Two groups of histone modifying enzymes, histone acetyltransferase (HAT) and histone deacetylase (HDAC) participate in the regulation of chromatin structure, and hence gene expression. Disruption of normal HAT or HDAC activities has been found in many human cancers. Recently, several structurally diverse and highly specific HDAC inhibitors (HDI) have been reported. They act as strong modulators of growth, differentiation and apoptosis in several types of cancer, particularly acute myeloid leukemia (AML). However, very little is known regarding the effects of HDI on HSPC. We have previously shown that a specific short-chain fatty acid HDI, valproic acid (VPA), enhances CXCR4 expression and function in normal HSPC (Blood2007: 110; 425a). In order to determine whether other structurally diverse classes of HDI are able to influence CXCR4 expression in HSPC through chromatin remodelling, we investigated the effect of potent hydroxamic acid HDI Trichostatin A (TSA) on CXCR4 in normal HSPC. We examined the effect of TSA on CXCR4 expression (by FACS and real-time RT-PCR), modulation of CXCR4 transcription (chromatin immunoprecipitation (X-ChIP) analysis) and on functional response towards an SDF-1α gradient (by chemotaxis assay) of HSPC (CD34+ cells from cord blood (CB) and the models of immature hematopoietic cells expressing CD34 antigen, namely AML cell lines KG-1a and KG-1). Cells were incubated for 24 h in IMDM supplemented with 20% FCS in the presence of TSA (0.1 μM). We found that TSA increases the percentage of CXCR4-expressing CB CD34+, KG-1a, KG-1 cells (2.5-, 8- and 3-fold, respectively). This effect was also confirmed at the mRNA level in CB CD34+, KG-1a and KG-1 cells (by about 2.5-, 5- and 2.5-fold up-regulation, respectively). Moreover, X-ChIP analysis showed a significant increase in association of acetyl-histone H4 binding to the CXCR4 promoter in CB CD34+ and KG-1 cells (2- and 1.7-fold, respectively). TSA was also shown to significantly increase the chemotaxis of KG-1a cells towards SDF-1α (20 ng/mL), which was inhibited by AMD3100, a potent antagonist of CXCR4. We conclude that other HDI such as TSA regulate CXCR4 expression in HSPC by chromatin remodelling and we suggest that priming of HSPC with HDI may improve their homing and engraftment into bone marrow, especially in CB transplantation.