Bone Marrow Proliferation Research Articles

Rehmannia Glutinosa Extract Activates Endothelial Progenitor Cells in a Rat Model of Myocardial Infarction through a SDF-1 α/CXCR4 Cascade

ObjectivesEndothelial progenitor cells (EPCs) can be used to repair tissues after myocardial infarction (MI) but EPC activators have adverse reactions. Rehmannia glutinosa is a herb used in traditional Chinese medicine, which can promote bone-marrow proliferation and protect the ischemic myocardium. We investigated the effects of Rehmannia glutinosa extract (RGE) on EPCs in a rat model of MI.MethodsA total of 120 male Wistar rats were randomized to 2 groups (n = 60 each) for treatment: high-dose RGE (1.5 g·kg−1·day−1 orally) for 8 weeks, then left anterior descending coronary artery ligation, mock surgery or no treatment, then RGE orally for 4 weeks; or normal saline (NS) as the above protocol. The infarct region of the left ventricle was assessed by serial sectioning and morphology. EPCs were evaluated by number and function. Protein and mRNA levels of CD133, vascular endothelial growth factor receptor 2 (VEGFR2), chemokine C-X-C motif receptor 4 (CXCR4), stromal cell–derived factor-1α (SDF-1α) were measured by immunohistochemistry, Western blot and quantitative PCR analysis.ResultsRGE significantly improved left ventricular function, decreased the ischemic area and the apoptotic index in the infarct myocardium, also decreased the concentration of serum cardiac troponin T and brain natriuretic peptide at the chronic stage after MI (from week 2 to week 4). RGE increased EPC number, proliferation, migration and tube-formation capacity. It was able to up-regulate the expression of angiogenesis-associated ligand/receptor, including CD133, VEGFR2 and SDF-1α/CXCR4. In vitro, the effect of RGE on SDF-1α/CXCR4 cascade was reversed by the CXCR4 specific antagonist AMD3100.ConclusionRGE may enhance the mobilization, migration and therapeutic angiogenesis of EPCs after MI by activating the SDF-1α/CXCR4 cascade.

Effects of human immunodeficiency virus on the erythrocyte and megakaryocyte lineages

Anaemia and thrombocytopenia are haematological disorders that can be detected in many human immunodeficiency virus (HIV)-positive patients during the development of HIV infection. The progressive decline of erythrocytes and platelets plays an important role both in HIV disease progression and in the clinical and therapeutic management of HIV-positive patients. HIV-dependent impairment of the megakaryocyte and erythrocyte lineages is multifactorial and particularly affects survival, proliferation and differentiation of bone marrow (BM) CD34+ haematopoietic progenitor cells, the activity of BM stromal cells and the regulation of cytokine networks. In this review, we analyse the major HIV-related mechanisms that are involved in the genesis and development of the anaemia and thrombocytopenia observed in HIV positive patients.

Prominent Reactive Plasmacytic Proliferation Presenting with Transient Pancytopenia: An Unusual Case

Reactive bone marrow plasmacytosis occurs in association with a variety of conditions. In such reactive cases, Plasma Cells (PCs) usually account for 10%-20% of cells in bone marrow trephine biopsy sections and rarely exceed 50%. Here in we report an unusual case of bone marrow aplasia associated with prominent atypical plasma cell proliferation and hypoplastic bone marrow.

“Blueberry muffin” rash and large right thigh mass: a unique presentation of Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a clonal proliferation of bone marrow derived antigen-presenting cells that can involve a spectrum of cutaneous findings, with or without internal organ involvement. Neonatal LCH almost always presents with skin findings, usually petechial papules and/or erosions in a seborrheic distribution, with or without extracutaneous involvement. Previously described as varying entities, LCH is now considered a single disease process demonstrating a spectrum of clinical findings. We report a unique case of neonatal LCH presenting with a "blueberry muffin" rash in conjunction with a large soft tissue tumor.

Abstract 19635: Glutathione-S-Transferase P Regulates Endothelial Progenitor Cell (EPC) Function in a c-Jun N-terminal kinase(JNK)-Dependent Manner and Enhances Neovascularization in Post-Infarction Heart Failure

Glutathione-S-transferase P (GSTP), via non-catalytic inhibition of JNK, modulates proliferation of bone marrow (BM) myeloid progenitors; however, its effects on EPCs are unknown. We hypothesized that GSTP supports normal EPC function via JNK suppression in BM-derived cells, and thereby promotes neovascularization in heart failure (HF). EPCs (CD34+VEGFR2+) and activated monocytes (F4/80+CD11b+) in BM and peripheral blood (PB) were determined by flow cytometry in wild type (WT) and GSTP-/- mice. Compared with WT, GSTP-/- mice exhibited significantly (p<0.01) reduced EPC number in PB (0.13±0.03 vs 0.21±0.07%) and BM (1.0±0.2 vs 1.7±0.6%), and increased activated monocytes, despite augmented numbers of CD34+CD45+ BM hematopoietic stem cells (HSCs; 6.7±0.8 vs 5.8±0.44%). PB EPCs in GSTP-/- mice were restored (p<0.05) with 5 days treatment in vivo with the specific JNK inhibitor, SP600125 (0.26±0.05% vs 0.15±0.04% before SP60025), whereas circulating activated monocytes were significantly reduced. Compared with WT, GSTP-/- BM-derived mononuclear cells cultured in endothelial growth medium for 10 d demonstrated reduced EPC colonies, less EPC migration in Transwell plates (VEGF gradient), and diminished vascular tube formation, indicating that GSTP deficiency decreased EPC differentiation from BM progenitors and EPC functional characteristics. All of these parameters were restored upon concomitant treatment with SP600125, and JNK phosphorylation, which was augmented in GSTP-/- BM, was significantly decreased by SP600125, suggesting JNK dependence of the GSTP-mediated effects on EPCs. Furthermore, EPCs, monocytes, and cardiac remodeling were examined WT and GSTP-/- mice with HF induced by left coronary ligation. As compared with WT, GSTP-/- HF mice exhibited (p<0.05): 1) fewer circulating PB EPCs and increased activated monocytes; 2) increased BM-HSCs but reduced BM EPCs; and 3) exaggerated pathological remodeling, augmented inflammation, and reduced capillary density and neovascularization in failing hearts. We conclude that GSTP promotes EPC function, mediated by inhibition of JNK in BM-derived progenitor cells, and also suppresses inflammation, and thereby imparts cardioprotective benefits in the post-infarct failing heart.

Pharmacodynamic Activity and Preclinical Safety of CSL362, a Novel Humanised, Affinity Matured Monoclonal Antibody Against Human Interleukin 3 Receptor

AbstractAbstract 1524CSL362, a novel humanized, affinity matured monoclonal antibody (mAB), is currently under development at CSL Limited. CSL362 neutralizes Interleukin-3 (IL-3) by binding to the human IL-3 receptor alpha chain (IL-3Ra/CD123). Furthermore, CSL362 has been engineered to bind to FcγRIIIa receptor (CD16) with high affinity and engages with effector cells of the innate immune system to elicit antibody-dependent cellular cytotoxicity (ADCC) against target cells expressing IL-3Ra such as AML blasts and leukemic stem cells.A non-clinical program was conducted to investigate the general safety and toxicity profile as well as the pharmacokinetic and pharmacodynamic properties of CSL362 in non-human primates. Cynomolgus monkeys (n=6 per dose cohort) were administered weekly intravenous infusions of CSL362 at doses of either 0, 3, 10, 30 or 100 mg/kg for 5 weeks with a 6 week recovery period. The potential of CSL362 to induce hypersensitivity or allergic reactions, its impact on safety pharmacology variables as well as local tolerance were assessed. Furthermore, the possible adverse effects of CSL362 on the proliferation, differentiation and maturation of bone marrow derived hematopoetic progenitors were explored. The potential for immunotoxicity of CSL362 was assessed by means of peripheral blood immunophenotyping, T cell-dependent antibody response measurements after keyhole limpet hemocyanin-immunization, measurements of natural killer cell (NK), macrophage and granulocyte function, and quantification of serum cytokine release. The ability to ablate cells expressing IL-3Ra was also determined by evaluating the numbers of basophils, plasmacytoid dendritic cells (pDCs) and monocytes in the peripheral blood. Plasma concentrations of CSL362 were determined to evaluate the pharmacokinetic characteristics. In parallel, the specific immune response against CSL362 was monitored throughout the 11 week study duration.Plasma level determination of CSL362 revealed adequate exposure and a PK profile similar to other humanized mABs. Specific anti-CSL362 antibody formation was observed in 18 % of the study animals. Treatment with CSL362 did not result in any anaphylactic or allergic reaction and had no adverse effect on systemic toxicity parameters or safety pharmacology variables measured. As predicted from in vitro and ex vivo studies, CSL362 produced a marked, dose-dependent and sustained depletion of peripheral blood basophils and pDCs which was associated with a transient decrease in NK cell numbers. No adverse effects of CSL362 on peripheral blood monocytes or pluripotent progenitor cells in the bone marrow was observed, furthermore the function of normal peripheral blood cells expressing IL-3Ra remained unaffected by CSL362. Intriguingly, a clear dose-dependent trend to NK cell recovery was observed following the final dose at week 5 during the subsequent recovery period which preceded the reappearance of basophils and pDC's. Together with the outcome of a tissue-cross reactivity study this proof of specific pharmacodynamic activity confirmed the Cynomolgus monkey as a relevant species. Overall, treatment with CSL362 was very well tolerated and 100 mg/kg, the highest tested dose, was designated the no-observed-adverse-effect-level.In conclusion, the results from this non-clinical safety program demonstrate an excellent tolerance and favorable safety profile of CSL362. This study supports the further clinical development of this mAB in the remission maintenance of patients with CD123-positive acute myeloid leukemia. Disclosures:Herzog:CSL Behring GmbH: Employment. Busfield:CSL Ltd.: Employment. Biondo:CSL Ltd.: Employment. Vairo:CSL Ltd.: Employment. DeWitte:CSL Behring: Employment. Pragst:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment. Nash:CSL Ltd.: Employment. Zollner:CSL Behring GmbH: Employment.

Efficacy of the investigational mTOR kinase inhibitor MLN0128/INK128 in models of B-cell acute lymphoblastic leukemia

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

The Proliferation Index of Specific Bone Marrow Cell Compartments from Myelodysplastic Syndromes Is Associated with the Diagnostic and Patient Outcome

Myelodysplastic syndromes (MDS) are clonal stem cell disorders which frequently show a hypercellular dysplastic bone marrow (BM) associated with inefficient hematopoiesis and peripheral cytopenias due to increased apoptosis and maturation blockades. Currently, little is known about the role of cell proliferation in compensating for the BM failure syndrome and in determining patient outcome. Here, we analyzed the proliferation index (PI) of different compartments of BM hematopoietic cells in 106 MDS patients compared to both normal/reactive BM (n = 94) and acute myeloid leukemia (AML; n = 30 cases) using multiparameter flow cytometry. Our results show abnormally increased overall BM proliferation profiles in MDS which significantly differ between early/low-risk and advanced/high-risk cases. Early/low-risk patients showed increased proliferation of non-lymphoid CD34+ precursors, maturing neutrophils and nucleated red blood cells (NRBC), while the PI of these compartments of BM precursors progressively fell below normal values towards AML levels in advanced/high-risk MDS. Decreased proliferation of non-lymphoid CD34+ and NRBC precursors was significantly associated with adverse disease features, shorter overall survival (OS) and transformation to AML, both in the whole series and when low- and high-risk MDS patients were separately considered, the PI of NRBC emerging as the most powerful independent predictor for OS and progression to AML. In conclusion, assessment of the PI of NRBC, and potentially also of other compartments of BM precursors (e.g.: myeloid CD34+ HPC), could significantly contribute to a better management of MDS.

A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition

Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies.

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.

Adult and umbilical cord blood-derived platelet-rich plasma for mesenchymal stem cell proliferation, chemotaxis, and cryo-preservation

Platelet-rich plasma (PRP) was prepared from human adult peripheral blood and from human umbilical cord (uc) blood and the properties were compared in a series of in vitro bioassays. Quantification of growth factors in PRP and platelet-poor plasma (PPP) fractions revealed increased levels of mitogenic growth factors PDGF-AB, PDGF-BB, and FGF-2, the angiogenic agent VEGF and the chemokine RANTES in ucPRP compared to adult PRP (aPRP) and PPP. To compare the ability of the various PRP products to stimulate proliferation of human bone marrow (BM), rat BM and compact bone (CB)-derived mesenchymal stem cells (MSC), cells were cultured in serum-free media for 4 and 7 days with varying concentrations of PRP, PPP, or combinations of recombinant mitogens. It was found that while all forms of PRP and PPP were more mitogenic than fetal bovine serum, ucPRP resulted in significantly higher proliferation by 7 days than adult PRP and PPP. We observed that addition of as little as 0.1% ucPRP caused greater proliferation of MSC effects than the most potent combination of recombinant growth factors tested, namely PDGF-AB + PDGF-BB + FGF-2, each at 10 ng/mL. Similarly, in chemotaxis assays, ucPRP showed greater potency than adult PRP, PPP from either source, or indeed than combinations of either recombinant growth factors (PDGF, FGF, and TGF-β1) or chemokines previously shown to stimulate chemotactic migration of MSC. Lastly, we successfully demonstrated that PRP and PPP represented a viable alternative to FBS containing media for the cryo-preservation of MSC from human and rat BM.

Differential cellular and microbial responses to nano-/micron-scale titanium surface roughness induced by hydrogen peroxide treatment

This study investigated the effects of combined titanium nano-/micron-scale roughness, induced by hydrogen peroxide pre-treatments, on bone marrow stromal cell responses and Porphyromonas gingivalis adherence invitro. Untreated surfaces exhibited nano-scale features, while hydrogen peroxide treatments promoted increased nano-/micron-scale roughness. Bone marrow stromal cell attachment and proliferation were maintained with 6 h and 24 h treatments, but significantly decreased on 1-week and 4-week-treated surfaces. Bone marrow stromal cells on 6 h-4 week-treated titanium demonstrated enhanced osteogenic differentiation versus untreated surfaces. P. gingivalis adherence was significantly increased on 24 h-4 week surfaces. Results suggest that 6 h but less than 24 h treatments maintain or promote bone marrow stromal cell responses while minimizing microbial adherence, potentially enhancing titanium surface bio-activation for osseointegration.

Commensal bacteria-derived signals regulate basophil hematopoiesis and allergic inflammation.

Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (TH2) cytokine–dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we identify that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum immunoglobulin E (IgE) levels, increased steady–state circulating basophil populations, and exaggerated basophil–mediated TH2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell–intrinsic expression of MyD88 was required to limit serum IgE levels and circulating basophil populations in mice. Commensal–derived signals were found to influence basophil development by limiting proliferation of bone marrow–resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal–derived signals influence basophil hematopoiesis and susceptibility to TH2 cytokine–dependent inflammation and allergic disease.

Serum concentrations and clinical significance of soluble CD40 ligand in patients with multiple myeloma

Multiple myeloma (MM) is a disease of plasma cells that express the CD40 receptor. Binding of the CD40 by its natural ligand, CD40 ligand (CD40L), produces growth arrest and/or apoptosis in MM. To evaluate serum levels of soluble CD40L (sCD40L) in MM patients and to correlate them with markers of disease activity and angiogenesis, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-6 (IL-6), proliferation marker Ki-67 proliferation index (Ki-67 PI) and bone marrow plasma cell infiltration, fifty-eight MM patients were studied in diagnosis and 43 of them after completion of treatment. Serum levels of sCD40L, VEGF, HGF and IL-6 were measured by ELISA, whereas Ki-67 PI and bone marrow plasma cell infiltration were measured by immunohistochemistry. Pre-treatment levels of sCD40L in MM patients were higher compared to controls and to their levels after effective treatment. Treatment regimen did not affect the degree of reduction of sCD40L levels, whereas patient in partial remission had increased levels compared to those with better response. Significant differences were found among disease stages. There were also positive correlations between CD40L with HGF, VEGF, IL-6 and Ki-67 PI. Elevated serum sCD40L is found in patients with advanced MM stage and can be reduced after effective treatment. Increased levels of this mediator are correlated with angiogenic cytokines, providing evidences that CD40L/CD40 interactions play a significant role in the mechanisms of angiogenesis in MM patients.

Wnt signalling: a mediator of the heart-bone marrow axis after myocardial injury?

Wnt signalling: a mediator of the heart-bone marrow axis after myocardial injury?

The Proliferation and Differentiation Capacity of Bone Marrow Derived- Human Mesenchymal Stem Cells in Early and Late Doubling

The Proliferation and Differentiation Capacity of Bone Marrow Derived- Human Mesenchymal Stem Cells in Early and Late Doubling

Constitutively active PTH/PTHrP receptor specifically expressed in osteoblasts enhances bone formation induced by bone marrow ablation

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1-constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady-state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild-type mice. Whereas de novo bone formation was restricted to the ablated area in wild-type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase-positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt-signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation-induced recruitment, proliferation, and differentiation of osteoprogenitors.

Efficacy and Safety of Eltrombopag in Elderly Patients with Chronic Immune Thrombocytopenia: Analysis of Five Clinical Trials,

Abstract 3294▪▪This icon denotes a clinically relevant abstract Introduction:Patients (pts) with immune thrombocytopenia (ITP) with platelet counts persistently <30,000/μL are at risk for severe and even fatal bleeding events; this risk increases with age. Risk for major non-fatal hemorrhage has been estimated at 3% per year for pts <40 years and 71% per year for pts >60. The 5-year risk of fatal hemorrhage has been estimated at 2% for pts <40 years and 48% for pts >60 (Cohen 2000). Eltrombopag, an oral, nonpeptide thrombopoietin receptor agonist approved for the treatment of chronic ITP, stimulates bone marrow progenitor cells, promoting differentiation and proliferation of megakaryocytes and platelet production. No data have been reported on the safety and efficacy of eltrombopag specifically in elderly pts. Aim: To assess efficacy and safety of eltrombopag in pts ≥65 years. Methods: A retrospective analysis by age was completed for 446 adult chronic ITP pts receiving eltrombopag in 5 clinical trials: two 6-week and one 6-month placebo-controlled studies (TRA100773A/B, RAISE); an open-label study with pts treated intermittently in 3 cycles of up to 6 weeks (REPEAT); and an ongoing extension study (EXTEND) of 299 pts who completed a prior eltrombopag trial. Bleeding was assessed prospectively using the WHO Bleeding Scale and using the NCI Common Terminology Criteria for Adverse Events (CTCAE) v3.0. Results: Pts receiving eltrombopag in each trial aged 18–49, 50–64, and ≥65 are shown in the Table. In the 6-week trials, response was defined as platelets ≥50,000/μL at day 43. In TRA100773A (eltrombopag 50 mg), response was achieved by 67% of pts aged 18–49 and 50–64, and 100% in pts ≥65. In TRA100773B, response was achieved by 50% of pts 18–49, 64% of pts 50–64, and 74% of pts ≥65. In RAISE, at any given visit, 40–53% of pts aged 18–49, 40–61% aged 50–64, and 42–75% ≥65 responded (platelets >50,000/μL and <400,000/μL). In REPEAT, response (platelets ≥50,000/μL and ≥2x baseline) in all 3 cycles was achieved by 67%, 72%, and 83% of pts who responded in Cycle 1, in the 18–49, 50–64, and ≥65 groups. In EXTEND, 84%, 94%, and 86% of pts aged 18–49, 50–64, and ≥65 had a response (platelets ≥50,000/μL); 15, 18, and 6 of these pts had platelets ≥50,000/μL at baseline. In EXTEND, 66%, 73%, and 78% of pts aged 18–49, 50–64, and ≥65 responded for >50% of assessments and 43%, 48%, and 53% for >75% of assessments. Across all 5 studies, pts aged 18–49 (n=220), 50–64 (n=148), and ≥65 (n=78) were treated with eltrombopag for a median (range) of 399 (5–1255), 484.5 (5–1302), and 244.5 (2–1267) days; median daily dose was 53 mg for pts 18–49 and 50 mg each for pts 50–64 and ≥65. The most common adverse events (AEs) (headache, nasopharyngitis, upper respiratory tract infection, and diarrhea) across all 5 studies were reported in similar proportions of pts across age groups. Fatigue, arthralgia, constipation, and cataracts were more common in elderly pts (11%, 10%, 5%, and 4% for pts 18–49 vs 19% each for pts ≥65). Thromboembolic events were reported in 4 (2%), 5 (3%), and 7 (9%) pts aged 18–49, 50–64, and ≥65. No age specific trend toward arterial vs venous events was observed. Proportions of liver enzymes elevation and bone marrow reticulin grade ≥2 were similar across age groups. Bleeding serious AEs (SAEs) were reported in 7% of pts aged 18–49 and 50–64, and 3% of pts ≥65. The most common bleeding SAEs were GI and CNS events, with no apparent difference in proportion of pts among age groups. Two pts experienced a fatal bleeding event (GI and CNS hemorrhages) 55 and 107 days after stopping eltrombopag; both were 18–49 and never responded to eltrombopag. Conclusion: No significant difference in the safety or efficacy profile of eltrombopag was observed for elderly versus younger pts, although elderly pts seemed to exhibit slightly more robust responses and slightly more nonhemorrhagic AEs (including thrombosis), which are not unexpected in an elderly population.TableNumber of Pts Evaluable for Efficacy by Age Group for Each StudyStudyAge Group18–4950–64≥65TRA100773A, na1593TRA100773B, na401519RAISE, n754416REPEAT, nb25207EXTEND, nc14810249aPts received ≥1 dose of 50 mg eltrombopag/day and had baseline platelets <30,000/μL.bIncludes only pts who responded in Cycle 1.cAll pts completed a prior eltrombopag study. Disclosures:Olney:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees. Pabinger:GlaxoSmithKline: Research Funding, Speakers Bureau. Mayer:GlaxoSmithKline: Employment, Equity Ownership. Bakshi:GlaxoSmithKline: Employment. Bailey:GlaxoSmithKline: Employment, Equity Ownership. Brainsky:GlaxoSmithKline: Employment, Equity Ownership.

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?

Enhanced biological effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant, on HL60 cells

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.