bluuebunny/crossref_metadata_2025 huggingface dataset:
Aberrant Microtubule Organization and Wiskott-Aldrich Syndrome-like Defects in Platelets and Megakaryocytes of Profilin1-Deficient Mice
Authors: Stritt Simon , Bender Markus , Nurden Paquita , van Eeuwijk Judith , Zieger Barbara , Kentouche Karim , Schulze Harald , Morbach Henner , Stegner David , Heinze Katrin G , Dütting Sebastian , Gupta Shuchi , Witke Walter , Falet Hervé , Fischer Alain , Hartwig John H. , Nieswandt Bernhard
Published: October 2019

Wiskott-Aldrich syndrome (WAS) is a rare, X-chromosomal recessive disorder which is caused by mutations in the WAS gene and characterized by eczema, immunodeficiency and microthrombocytopenia (Thrasher and Burns, Nat Rev Immunol 2010). Interestingly, WAS protein (WASp)-deficient mice have normal-sized platelets and thus the molecular link between WAS mutations and its central hallmark microthrombocytopenia remains elusive.

Profilin1 (Pfn1) is a key actin-regulating protein that, besides actin, interacts with phosphoinositides and multiple proline-rich proteins including the WAS protein (WASp)/WASp-interacting protein (WIP) complex (Witke, Trends Cell Biol 2004; Ramesh et al., Proc Natl Acad Sci USA, 1997). Interestingly, similar to WAS patients, mice with Pfn1-null megakaryocytes/platelets suffered from microthrombocytopenia. We identified accelerated platelet clearance by macrophages and pre-mature platelet release into the bone marrow compartment as the major cause of the reduced platelet count in Pfn1-deficient mice.

Both, platelets from Pfn1-null mice and WAS patients contained abnormally organized and hyperstable microtubules. We next tested, if increased microtubule stability could account for the reduced size of Pfn1-deficient platelets. Treatment of control platelets with microtubule stabilizing toxins, such as the histone-deacetylase inhibitor trichostatin A (TSA) or taxol resulted in a decreased platelet size. This finding indicates that increased microtubule stability could account for the reduced platelet size in Pfn1-deficient mice but also in WAS patients. Based on these results we speculate that WASp might modulate Pfn1 function and dysregulation of this interaction leads to increased stability and altered organization of microtubules. In support of this, the subcellular localization of Pfn1 was altered in platelets of three WAS patients.

Together, these results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients.

Disclosures

No relevant conflicts of interest to declare.
The Expression of Functional Dopamine and Serotonin Receptors on Megakaryocytes
Authors: Mo Yang , Li Su-yi , Liang En-yu , Lian Qi-zhou , Meng Fan-yi
Published: October 2019

Monoamine dopamine (DA) and serotonin (5-HT) can affect the functions of many cells including platelets. Our previous studies have shown that 5-HT has potent effects on promoting megakaryopoiesis and platelet formation (Yang et al, Stem Cells, 2007; 2014). In this study, our hypothesis is that both DA and 5-HT receptors may have the development expression on platelets, megakaryocytes (MK) and hematopoietic stem cells (HSCs). The expression of DA receptors: DA1, DA2 and 5-HT receptors: 5-HT2A, 2B and 2C on these hematopoietic cells, and the effect of DA and 5-HT on the cell proliferation and apoptosis were investigated using RT-PCR, flow cytometry, immunofluorescence staining, annexin V, JC-1 and caspase-3 assay. Our results showed that dopamine DA1, DA2 receptors, 5-HT2A, 2B and 2C receptors were identified on human platelets, MK and HSCs using RT-PCR method. Using flow cytometry with monoclonal antibodies, these receptors were also identified on human platelets and MK. But these receptors expressed on human HSCs at a low levels. The expression of the five receptors were further confirmed on human bone marrow MK and HSC by using immunofluorescence staining. The functions of receptors were further studied in human MK and HSCs. DA at a low dose induced cell differention, but at a high dose induced cell apoptosis. This effect could be reduced by DA2 receptor blocker L-Sulpiride (L-SULP). In the other hand, 5-HT had an anti-apoptotic effect in MK cells. 5-HT also stimulated CFU-MK formation, this effect was reduced by 5-HT2R antagonist ketanserin. 5-HT also enhanced Akt phosphorylation, while addition of ketanserin compromised the 5-HT effect. 5-HT-driven MK effect was likely mediated via 5-HT2R and p-Akt. A higher number of proplatelet bearing MK was also detected in 5-HT-treated. Polymerization F-actin level was higher in 5-HT group. Adding ketanserin/PD98059, the intensity was reduced. Treated with 5-HT for 30 minutes ERK1/2 was activated. The adding of ketanserin/PD98059 to 5-HT cultures nullified the 5-HT effect. This effect was likely mediated via 5HT2R, p-ERK1/2, which leads to F-actin reorganization and proplatelet formation. This study demonstrated that the expression of DA and 5-HT functional receptors on human platelets, MKs and HSCs. DA and 5-HT may be involved in the regulation of thrombopoiesis.

Disclosures

No relevant conflicts of interest to declare.
The 1.7 Angstrom X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces
Authors: Brison Caileen M , Mullen Steven M , Podolsky Kira , Meeks Shannon L , Spiegel Paul Clinton
Published: October 2019

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity, however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 BU/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Lastly, differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with similar affinities, and the human K2227E mutant binds to phospholipid surfaces with a modestly decreased affinity.

Disclosures

No relevant conflicts of interest to declare.
p38α Is Required for Proper Proliferation of Hematopoietic Stem Cells during Stress
Authors: Karigane Daiki , Takubo Keiyo , Okamoto Shinichiro , Suda Toshio
Published: October 2019

Hematopoietic stem cells (HSCs) are capable of self-renewal and multilineage-differentiation during lifespan. HSCs are maintained in a quiescent state to avoid cellular senescence. Previous reports utilizing pharmacological inhibitors or shRNAs against p38MAPK suggest a pivotal role of p38MAPK-pRb-Ink4a signaling in induction of HSC senescence by hematological stress or chronological aging. However, no genetic evidence exists for p38MAPK-mediated cellular senescence in vivo. Here we report unexpected roles of the dominant isoform of p38MAPK family, p38α, in adult hematological system. p38MAPK has four isozymes, α, β, γ and δ. Among them, p38α isozyme was highly expressed in various bone marrow hematopoietic cells, and the expression level of p38α in HSCs was higher than differentiated cells (p<0.01). Phosphorylation of p38MAPK was mainly observed in multipotent progenitors but not in HSCs in steady-state hematopoiesis, in addition, physiological aging (1 year old mouse bone marrow) did not affect phosphorylation status of p38MAPK in steady state. In contrast, p38MAPK was phosphorylated in HSCs after transplantation or 5-FU treatment. Mean fluorescence index (MFI) of phosphorylation of p38MAPK in HSCs is significantly higher at day 3 post 5-FU treatment (250 mg/kg) than steady-state. MFI of phosphorylation of p38MAPK in HSCs was higher at day 1 post transplantation than steady state, and returned to normal at day 7 post transplantation. These results showed phosphorylation of p38α was immediately induced after hematopoietic demand.

p38α-deficient embryos die due to defective erythropoiesis in a non-cell-autonomous manner. Thus, we used a conditional knockout model; CAG-CreERT2:p38αfl/fl mouse to analyze the effects of p38α on adult hematopoiesis and HSCs. Expression level of p16Ink4a, one of the cellular senescence markers, was not significantly different between p38α-deficient mice and wild-type mice. Treatment of p38α-deficient mice with 5-FU exhibited defective recovery of hematopoiesis, and the survival rate were lower in p38α-deficient mice than wild-type (42.9%, N=7, p38α-deficient mice, vs 100%, wild-type, N=6, p<0.05). Loss of p38α in HSCs showed a defective transplantation capacity. Inducible loss of p38α in bone marrow chimera resulted in a gradual loss of peripheral blood chimerism of p38α-deficient cells. In addition, short-term BrdU incorporation assay showed that the cell cycle progression of p38α-deficient HSCs was suppressed (BrdU positive rate; 3.5±2.2%, N=9, p38α-deficient cells vs 6.5±2.6%, N=5, wild-type, p<0.05). Therefore, hematopoietic function was obviously lowered in p38α-deficient HSCs during hematopoietic stresses. These observations collectively support the requirement of p38α for proper proliferation of HSCs during stress hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.
CD43 but Not CD41 Marks the First Hematopoietic Stem Cells in the Human Embryo
Authors: Ivanovs Andrejs , Rybtsov Stanislav , Anderson Richard A. , Medvinsky Alexander
Published: October 2019

Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain multilineage hematopoiesis throughout the entire lifespan of the organism. Due to limited access to human embryonic tissues, early human HSC development remains a largely unexplored area of research. A better understanding of this process may be instrumental in creating novel protocols for in vitro generation of human HSCs from pluripotent stem cells to overcome the shortage of donor cells and increase the safety of HSC transplantations in the clinic.

We have recently described the spatio-temporal distribution of HSCs in the early human embryo and established the immunophenotype of the first human HSCs (Ivanovs et al., J. Exp. Med., 2011; Ivanovs et al., Stem Cell Reports, 2014). The first human HSCs emerge in the ventral domain of the dorsal aorta, possess high regenerative potential, and reside within the CD34+VE-cadherin+CD45+c-Kit+Thy-1+Endoglin+Runx1+CD38-/loCD45RA- cell population, which harbors all HSCs and committed hematopoietic progenitors in the human aorta-gonad-mesonephros (AGM) region. Here, we aimed to find a surface marker which would further enrich for the human AGM region HSCs. We found that CD41, which is the earliest surface marker of hematopoietic commitment in the mouse embryo (Ferkowicz et al., Development, 2003; Mikkola et al., Blood, 2003), does not mark the first human HSCs (3 independent experiments). Instead, the first HSCs in the human AGM region express CD43 (4 independent experiments). Of note, it has been shown previously that during human embryonic stem cell differentiation in vitro, CD43 but not CD41 marks multipotent hematopoietic progenitors (Vodyanik et al., Blood, 2006).

Human embryonic tissues for our study were obtained after elective medical termination of pregnancy with informed written consent according to the Declaration of Helsinki. The study protocol was approved by the Lothian Research Ethics Committee. Employing magnetic cell separation and xenotransplantations, CD41- and CD43-positive and -negative cell populations were isolated from Carnegie stage 15–17 human AGM regions and individually transplanted into preconditioned adult NOD.Cg-PrkdcscidIl2rgtm1Wjl/Sz mice. Human hematopoietic contribution to the peripheral blood, bone marrow, spleen, and thymus of transplanted mice was assessed at 4 months after transplantation.

Our findings suggest that, although the process of HSC development is largely conserved between the mouse and the human, certain aspects vary substantially between the species, reflecting possible differences in the HSC specification program. We conclude that CD43 is a useful surface marker for the purification of the first human HSCs to enable further studies into molecular mechanisms underlying their development and high regenerative potential.

Disclosures

No relevant conflicts of interest to declare.
Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation
Authors: Swartz Kelsey , Qin Gangjian , Minella Alexander C
Published: October 2019

Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin ET74A T393A). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored.

We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation.

Disclosures

No relevant conflicts of interest to declare.
Role of Hepatocyte Growth Factor in Endothelial-Dependent Hematopoietic Stem Cell Regeneration
Authors: Zachman Derek K , Goldman Devorah C , Hamlin Kimberly L , Guha Chandan , Fleming William H.
Published: October 2019

Hematopoiesis is tightly regulated by the niche and highly sensitive to a variety of genotoxic stressors. The mechanisms responsible for the repair and regeneration of hematopoietic stem cells (HSCs) are poorly understood. Endothelial cells (ECs) are an essential component of the bone marrow microenvironment that provide regenerative signals for HSCs after injury; however, the identity of these signals remain largely unknown. We utilized RNA sequencing and a bioinformatics approach to identify hepatocyte growth factor (HGF) as an endothelial-derived mediator of HSC regeneration. HGF gene expression was 7.6-fold higher in arterial-derived ECs (HAEC) that regenerate functional long-term HSCs (CD150+LineageloSca1+Kit+ cells; CD150+LSK cells) compared to venous ECs (HUVEC) that support only hematopoietic progenitors. Furthermore, the HSC-supportive HAECs secreted up to 25 pg/mL HGF into conditioned media, whereas HGF was undetectable in media conditioned by HUVEC. To directly test the role of HGF in HSC regeneration, we supplemented co-cultures of irradiated bone marrow cells and primary ECs with recombinant HGF. Addition of HGF was sufficient to augment CD150+LSK cell regeneration up to 20-fold by HUVEC. This effect was abolished by treatment with the Met-specific small molecular inhibitor, PF04217903. Importantly, neither the addition of HGF alone to irradiated hematopoietic cells nor spiking HUVEC-conditioned media with HGF improved HSC regeneration in the absence of an EC monolayer. Together, these results indicate that HGF activates a signaling loop in ECs that supports HSC regeneration.

Disclosures

No relevant conflicts of interest to declare.
Histone Deacetylase 6 (HDAC6) Regulates Gene Transcription and Enucleation during Erythroid Differentiation
Authors: Li Xuehui , Yang Yurong , Lu Jianrong , McKinsey Timothy , Ji Peng , Qiu Yi
Published: October 2019

The deacetylation of histone and non-histone proteins by histone deacetylase (HDACs) plays a critical role in gene transcription and many other cellular processes in eukaryotic cells. Class II deacetylase HDAC6 is mainly localized in the cytoplasm and deacetylates tubulin and other cytoplasmic proteins. Earlier studies from our laboratory showed that HDAC6 can be transported to the nucleus, and can interact and deacetylate nuclear proteins, such as histones. In this study, we investigate the function of HDAC6 during erythroid differentiation. In proerythroblast cells, HDAC6 is detected in the nucleus and gradually migrates to the cytoplasm upon the induction of differentiation, indicating different HDAC6 functions during erythropoiesis. Inhibition of HDAC6 in mouse fetal liver erythroblasts impairs differentiation and enucleation. β-globin gene transcription is reduced by HDAC6 specific inhibitor, Tubastatin A, in cultured fetal erythroblasts. HDAC6 knockout mice also showed impaired globin gene transcription. HDAC6 specifically interacts with serine 2 phosphorylated Pol II and is recruited mainly at the transcribed region of β-globin, correlating with RNA Pol II recruitment. These results suggest that HDAC6 promotes erythroblast differentiation through the regulation of transcription elongation. HDAC6 also plays a role in regulating the enucleation process during the late stage of erythropoiesis. The formation of the contractile actin ring is disrupted and the enucleation process is blocked in cultured mouse fetal erythroblasts treated with the HDAC6 inhibitor. We further investigated the molecular mechanism of HDAC6 regulated enucleation. We found that HDAC6 interacts with and deacetylates mDia2, an effector of Rho GTPases that is required for erythroblast enucleation. Deacetylation of mDia2 is required for the formation of the contractile actin ring and subsequent enucleation. Altogether, our results identify the important role of HDAC6 in regulating transcription and enucleation during the different stages of mammalian erythropoiesis.

Disclosures

No relevant conflicts of interest to declare.
Loss of mDia1 Mediates the Development of Del(5q) MDS through Upregulation of the Innate Immune Response and Induction of Neutropenia
Authors: Mei Yang , Zhao Baobing , Yang Jing , Ji Peng
Published: October 2019

mDia1, the diaphanous homolog 1 of Drosophila in mouse, is a formin protein involving in the linear actin polymerization. Recently, our group reported that patients with del(5q) myelodysplastic syndromes (MDS) showed a significantly decreased mDia1 expression. Mice with mDia1 deficiency developed age related hematologic features mimicking human MDS. In these mice, CD14 is aberrantly overexpressed on granulocytes, which sensitized the innate immune response upon the lipopolysaccharide(LPS) injection. Importantly, chronic LPS injection accelerated the development of MDS (Keerthivasan et al Blood 2014). Here we report that 1) the mDia1 knockout (KO) mice also have a hypersensitive innate immune response to damage-associated molecular pattern molecules (DAMPs), which can be partially blocked by CD14/Toll-like receptor 4(TLR4) signaling pathway inhibitors. This is significant since release of DAMPs from necrotic or senescent cells is a common age-related phenomenon. DAMPs are also detected in cancers including MDS. Thus our study may shed lights on how the deregulated innate immune responses get involved in the pathogenesis of MDS in a more pathophysiologically relevant etiology. 2) mDia1 KO mice have an increased Gr1/Mac1 expression on the granulocytes in peripheral blood. We demonstrate that the expression levels of Gr1/Mac1 were not changed in bone marrow granulocytes, suggesting a specific up-regulation of Gr1/Mac1 levels on circulating granulocytes in mDia1 knockout mice. Mac-1, as the predominant β2 integrin on granulocytes, plays key roles in the adhesion of leukocytes to the endothelium, and regulates the cell adhesion, migration, and chemotaxis. In this respect, the mDia1 knockout mice develop serve neutropenia, which could be due to the upregulation of Gr1/Mac1 and increased binding of the cells to intercellular adhesion molecule-1 (ICAM-1). Finally, we revealed the mechanism of Gr1/Mac1 upregulation by showing that loss of mDia1 significantly affected the endocytosis of Gr1 and Mac1 on granulocytes, however, the mRNA levels of Gr1 and Mac1 were not affected. Taken together, these studies reveal a significance of loss of mDia1 in the pathogenesis of del(5q) MDS through upregulation of innate immune response and accelerated granulocyte clearance.

Disclosures

No relevant conflicts of interest to declare.
Final Analysis of a Phase II Study of Intrapatient Dose-Escalation of Eltrombopag in Patients Receiving Azacitidine for Myelodysplasia/AML
Authors: Dickinson Michael J. , Herbert Kirsten , Sardjono Caroline , Le Thao , Zannino Diana , Wood Colin , Seymour John F , Kenealy Melita , Prince Miles
Published: October 2019

Background: Pre-existing thrombocytopenia in MDS/AML is worsened during the initial cycles of azacitidine (AZA) therapy, resulting in bleeding risk and possible platelet transfusion. Eltrombopag (EPG) is an oral TPO-receptor agonist. In vitro, it has anti-proliferative effects on AML blasts.

Aim:  To assess the safety of escalated doses of EPG in patients undergoing AZA for MDS/AML

Method An investigator-initiated phase-II, single arm, study of EPG with AZA. Inclusion: relapsed or de-novo MDS/CMML/AML (blasts 5-30%); or symptomatic cytopenia; or blasts 31-50% if >/=65 years or previously-treated disease; and platelets

Result Of 25 patients, 10 had prior therapy, which was chemo in 7; of these 6 had blasts >/=10%, 2 with blasts 20% or above. Of the 15 de-novo patients, 7 had blasts >/=20% (AML) and a further 2 had blasts between 10 and 19%. The median platelet count was 38x10^9/L (range 8-127). A median 11(2-24) cycles AZA and 6 cycles of EPG were delivered. One patient developed GrII EPG-related LFT abnormalities (resolved). Grade 3 fatigue was attributed to the combination in one patient. Thrombocytosis (>450 x109/L) resulting in EPG cessation occurred in 6 (at 50, 50, 150 and three at 200mg), without complications. 10 patients experienced reversible skin yellowing. Response/improvement was seen in 18 (72%): 7CR, 3CRm, 5HI-P,1 HI-N,2 with >50% blast reduction from >20% (8%). 5 patients had progression at first response. There were 19 events in total (17 progressions, 2 with worsening haematology). Median PFS was 12.0 months (95% CI 5.6 to 24.3 months). Median OS was 15.3 months (95% CI 11.9 to 31.7 months).

Platelet improvement was seen in 54% (13/24) of patients with baseline platelets <100 at median (range) at 46d (7-107) following commencement of AZA. Only one patient (4%) had an improvement in platelets following the monotherapy phase.

Conclusion Eltrombopag could be safely delivered at these doses. A phase III international study has commenced to better define the role of this combination as supportive care for patients undergoing treatment with azacitidine. Table:Characteristics and best responses observed.MDS/AML CharacteristicsBest ResponseMDS <20% Blasts, De Novo (N=7)5CR, 1SD, 1 HI-NAML >/=20%, De Novo (N=8)1CR, 1PR, 3 Hi-P, 1SD, 2 PDPrior therapy for MDS/AMLBlasts <20% (N=7)1CR, 3CRm, 1SD, 1PD, 1Hi-PPrior therapy for MDS/AMLBlasts >/=20% (N=3)2PD, 1 Hi-P

Figure: Overall survival of the trial population. Figure:. Overall survival of the trial population.

Disclosures

Dickinson: GSK: Consultancy; Celgene: Honoraria. Off Label Use: Eltrombopag for MDS. Sardjono:GSK: Employment. Seymour:Celgene: Consultancy, Honoraria, Speakers Bureau. Kenealy:Celgene: Honoraria, Research Funding. Prince:Celgene: Consultancy, Honoraria, Research Funding.
Clinical Presentation and Treatment Responses in IgM AL Amyloidosis, a Series of 106 Patients
Authors: Sloan John Mark , Sissoko Moussa , Seldin David C , Sworder Brian , Angelino Kennneth , Broce Mike , Berk John L , Sanchorawala Vaishali
Published: October 2019

Objective: AL amyloidosis is a multiorgan disease due to deposition of misfolded monoclonal immunoglobulin light chains. AL amyloidosis with IgM paraproteinemia is a rare variant of this disease; accounting for approximately 6% of AL amyloidosis cases. The clonal cell of origin may be a plasma cell or lymphoplasmacytic cell, and treatments targeting each have been employed. This study describes clinical and laboratory features of IgM AL amyloidosis patients and their response to different therapeutic regimens.

Methods: 106 patients with IgM-related AL amyloidosis were evaluated at the BUMC Amyloidosis Center during 1996-2012. Treatment and response information was available on 46 treated after 2003, during the era of new agents for plasma cell diseases. In this cohort, there were 5 treatment groups: high-dose melphalan/stem cell transplant (HDM/SCT); bortezomib-based regimens; standard doses of the alkylating agents melphalan or cyclophosphamide; immunomodulatory agents (IMiDs); and rituximab-based regimens. Treatment regimens were assigned by bone marrow pathology and patient-specific factors.

Results: For the 106 patients, the median age at diagnosis was 67 years (range: 38-89 years) with 52 (56%) males. The kidney was the most commonly involved organ (51%) followed by heart (41%) and GI tract (23%). Median IgM was 590 g/dL (range: 29-9130). The clonal population had lambda light chain restriction in 69% of patients. For the 46 patients treated after 2003, overall response rates and rates of very good partial or complete responses were were 100% and 80% respectively, with HDM/SCT; 82% and 30% with bortezomib-containing regimens; 80% and 25% with regimens that included rituximab; 75% and 0% with regimens in which an IMiD was included; and 63% and 20% with other alkylator regimens. There was no association between treatment response and organ involvement, sex, or age.

Disclosures

Sloan: Millenium: Consultancy.
Lovastatin Induces Platelet Apoptosis
Authors: Zhao Qing , Chen Mengxing , Zhou Ling , Zhao Lili , Yan Rong , Dai Kesheng
Published: October 2019

Introduction

Lovastatin, the inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, is widely used in treatments of coronary artery diseases. Lovastatin induces apoptosis in nucleate cells, such as macrophages, cancer cells, and skeletal muscle cells. Lovastatin has side effects of thrombocytopenia and hemorrhage, however, the possible pathogenesis of the side effects have still remained unknown.

Methods and Results

In this study, we found that collagen and thrombin-induced platelet aggregation was obviously reduced in platelets treated by lovastatin, while platelet activation (expression of P-selectin or PAC-1 binding on platelet surface) was not detected. The apoptosis markers in both intrinsic mitochondrial pathway (ΔΨm changes, PS exposure, Bax/Bak and Bcl-XL expression, and caspase9/3 activation) and extrinsic pathway (caspase 8) were detected in platelets treated with lovastatin. Apoptosis decreased in platelets after being pretreated by tetrapeptide arginine-glycine-aspartic acid-serine (RGDS) peptide (the GPIIb/IIIa antagonist). Lovastatin was

intraperitoneally injected into C57 mice at a dose of 5 mg/kg for four hours. The circulating platelets were significantly decreased by lovastatin compared with that of vehicle control.

Conclusion

Lovastatin induced platelet apoptosis by both intrinsic mitochondrial and extrinsic pathways, which might be the possible pathogenesis of thrombocytopenia or hemorrhage in patients treated with lovastatin.

Disclosures

No relevant conflicts of interest to declare.
Do Sexual Hormones Influence the Coagulation Parameters during the Normal Menstrual Cycle
Authors: Halimeh Susan , Rott Hannelore , Osseiran Ihsan , Kappert Guenther , Siebert Manuela
Published: October 2019

Introduction:

The blood coagulation can be influenced by different variables like mental stress, physical activity or oral anticoagulants. During the normal menstrual cycle changes have been observed on different time points. A study of Chaireti et al (1) found an association of progesterone and factor II, factor VII and factor X.

We investigated the possible influence of the sexual hormones on the coagulation parameters.

Samples and methods:

We conducted a laboratory work group in 35 women sent to our lab. The following tests were conducted: Blood count, VWF:RCo, VWF:Ag, VWF:CB, Fibrinogen (Clauss), activities of FII, FV, FVII, FVIII (clotting and chromogenic), FIX, FX, FXI, FXII, FXIII, FSH, LH, Progesterone and Oestradiole during the menstrual cycle on predefined time points (day 1-6, day 7-11, day 12-18, day 19-23, day 24-28).

Results:

We found slight variations in nearly all coagulation parameters but without significance. The box-plots of the Von Willebrand activity shows that the means are relative close together. Clear variations could not be found. The Friedman-Test showed no significance. As expected the changes of the sexual hormones during the menstrual cycle showed highest significance.

Conclusion:

The influence of sexual hormones on coagulation parameters was not significant. The slight fluctuations may be caused by imprecision of the measurement and intra individual changes from day to day. Changes during the menstrual cycle could not be verified.

Nevertheless it is advisable to investigate unclear negative results, when a conspicuous medical history or a corresponding clinic occurs. It is not necessary to regard the time point in the menstrual cycle for the blood collection.

Literature: Chaireti R, Gustafsson KM, Byström B, Bremme K, Lindahl TL, Endogenous thromboin potential is higher during the luteal phase than during the follicular phase of a normal menstrual cycle, Hum Repro, 2013 Jul, 28 (7) 1846-52

Disclosures

No relevant conflicts of interest to declare.
Since the Process of Transfusion-Induced Alloimmunization Is Not Harmless, Can We Mitigate It?
Authors: Szymanski Irma O. , Orton Sharyn L
Published: October 2019

Transfusion protocols have remained unchanged for decades. Accordingly, it is necessary to avoid transfusion reactions by giving patients ABO- and irregular antibody-compatible red blood cells (RBC). Prevention of immunization to the Rh antigen D is also required since anti-D may cause a serious hemolytic disease of fetus and newborn. Prevention of transfusion-induced immunization to other blood group antigens is neither required nor feasible for the general transfused population.

We consider the process of alloimmunization to be harmful due to the following untoward effects: 1) Destruction of transfused RBC negates transfusion's intended therapeutic benefits and may cause transfusion reactions. 2) Some alloantibodies become undetectable after a relatively short period (e.g., anti-Jka) so that in the future such seemingly compatible transfusions cause severe, sometimes fatal delayed transfusion reactions. 3) RBC autoimmunity may follow alloimmunization and cause hemolytic anemia in the recipient.

To take the first step toward preventing transfusion-induced alloimmunization we calculated, using our antibody database, the immunogenicity of RBC antigens in a predominantly white female and male populations according to the methods of *Giblett (Transfusion 1961;1(4): 233) and **Tormey and Stack. (Blood 2009;114: 4279). The latter method corrects immunogenicities for antibody evanescence. The Table below shows that the three highly immunogenic antigens against which clinically significant antibodies were frequently formed were E, Jka and K, both in females and males. Immunogenicity of Rh C antigen was not calculated, since anti-C occurs most often with anti-D.

Figure 1 Figure 1.

In summary, we suggest a first step toward improving immunological safety of RBC transfusions by preventing immunization to E, Jka and K antigens. Automated typing or DNA analysis would facilitate mass typing of both donors' and recipients' RBC antigens.

Disclosures

No relevant conflicts of interest to declare.
miRNA Expression Profiling in Mesenchymal Stromal/Stem Cells Derived from G-CSF Primed Bone Marrows
Authors: Gunel-Ozcan Aysen , Cagnan Ilgin , Kansu Emin , Uckan Duygu
Published: October 2019

Regulatory microRNAs (miRNAs) are involved in the molecular control of hematopoiesis and diseases that result from aberrations during this process. Bone marrow (BM) transplantation is a commonly used therapy for patients with certain hematopoietic diseases. Prior to BM transplantation, donors are occasionally treated with granulocyte colony-stimulating factor (G-CSF) to increase the number of hematopoietic stem or progenitor cells in the collected stem cell product. Here we aimed to assess the effects of G-CSF priming on miRNA expression of BM derived mesenchymal stromal/stem cells (BM-MSCs) from healthy donors.

Previously isolated and characterized BM-MSCs from 3 days G-CSF (10 μg/kg/d) treated (n=5) and untreated (n=8) healthy donors were used for miRNA expression profiling with Affymetrix GeneChip 2.0 Array. Following Robust Multi-array Average (RMA) normalization, data analysis was carried out in Partek Genomics Suite software package (Partek Incorporated). Differentially expressed miRNAs between G-CSF primed and unprimed groups were determined (p<0.05, fold change ±1.5).

A total of 2156 miRNAs were upregulated whereas 2436 miRNAs were downregulated after in-vivo G-CSF priming. The expression of hp_hsa-mir-1238 and has-miR-3180-3p non-coding RNAs was significantly increased in G-CSF primed BM-MSCs (p<0.001). Modular enrichment analysis of hsa-miR-3180-3p predicted target genes by GeneCodis 3 software revealed that some may play a role in different types of cancer such as small cell lung cancer (Adjusted p-value: 8.47338e-7, 0.000382029). Our data indicate that G-CSF treatment changes miRNA expression of bone marrow stromal cells (MSCs) and this change might be important to evaluate for long-term biosafety.

Disclosures

No relevant conflicts of interest to declare.
Bone Marrow Contains High Levels of Microparticles
Authors: Rank Andreas , Nieuwland Rienk , Koehler Anton , Kolb Hans Jochem , Toth Bettina
Published: October 2019

Background: Microparticles (MP) are shed in humans from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. Alternations in plasma levels have been found in a variety of diseases, mostly associated with thrombosis and inflammation. However, little is known about tissue levels of MP. The aim of this pilot study was to investigate the bone marrow compartment as a possible source of circulating MP.

Material and Methods: MP were analysed in bone marrow blood and peripheral blood in 12 healthy stem cell donors. MPs were isolated and measured by flow-cytometric double-staining (FACScalibur, BD) with Anexin V and cell-specific antibodies for hematopoietic cells, platelets, white blood cells, red blood cells and endothelial cells. Statistical analysis was performed with SPSS for Windows 20.0.

Results: Total MP levels differed between bone marrow and peripheral blood: 14.8 x109/l [8.5–19.3] vs. 9.2 x109/l [3.8–14.8] (median value [25-75 percentile]), p=0.060. Pattern of MP´s origin varied likewise: in bone marrow blood (bmb), main cell source of MP were CD235a positive red blood cells/erythropoetic cells (6.4x109/l [3.7–13.7], 43.2% of total bmb MP), CD61 positive platelets/megacaryocytes (4.1x109/l [1.7–10.1], 27.7% of total bmb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (3.8x109/l [2.7-4.9], 25.7% of total bmb MP) and CD62e positive endothelial cells (0.3x109/l [0.1-1.0], 2.0% of total bmb MP). In contrast, MP in peripheral blood (pb) mainly derived from CD61 positive platelets/megacaryocytes (8.5x109/l [2.4–14.4], 92.0% of total pb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (0.5x109/l [0.2–1.1], 4.5% of total pb MP), CD235a positive red blood cells/erythropoetic cells (0.2x109/l [0.1–0.6], 1.8% of total pb MP), and CD62e positive endothelial cells (0.1x109/l [0.0–0.1], 0.9% of total pb MP). Mean levels of tissue factor bearing MP were low in bone marrow blood as well as in peripheral blood (0.19 x109/l [0.16 – 0.29] and 0.08 x109/l [0.05 – 0.12], respectively, p=0.004).

Conclusion: Within our study we were able to detect MP in bone marrow blood. When comparing MP from bone marrow and peripheral blood, major differences with regard to cell source and concentration was present. Our data suggest that the blood – bone marrow barrier is not only existentant for cells, but also for circulating MP.

Disclosures

No relevant conflicts of interest to declare.
A New Approach of PNH Clone Detection
Authors: Galtseva Irina V. , Fidarova Zalina T. , Mikhailova Elena A. , Borisov Vyacheslav I. , Troitskaia Vera V. , Parovichnikova Elena N. , Savchenko Valeri G.
Published: October 2019

Introduction

The presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube.

Objectives: improvement of PNH cells detection technique.

Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group.

The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed.

Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1).

Figure 1 Figure 1.

In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations.

Table 1. Average value of PNH clone on leukocytes in groups of patients both techniques PNH clone Gr, % (mean) Mon, % (mean) CD24-/FLAER- CD157-/FLAER- CD14-/FLAER- CD157-/FLAER- 0%-0,99% 0,178 0,349 0,323 0,378 1%-9,99% 3,98 3,76 9,54 9,98 >10% 71,52 71,52 71,85 71,34

Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone.

Disclosures

No relevant conflicts of interest to declare.
IgG Subclasses Abnormal in Immune-Related Pancytopenia
Authors: Shao Zonghong , Shao Yuanyuan , Fu Rong
Published: October 2019

In recent years, we found some patients with idiopathic cytopenia of undetermined significance (ICUS)responded well to corticosteroid and/or high-dose intravenous immunoglobulin treatment. We had detected both IgG and IgM antibodies by bone marrow mononuclear-cell-Coombs test, flow cytometric and immunofluorescence analysis on the various bone marrow cell membrane. We also detected IgG in the BM supernatant of these ICUS patients, which can target several antigens on hematopoietic cells membranes by western blot. Then We termed this group of ICUS cases with autoantibodies as immuno-related hemocytopenia(IRP). IgG include IgGl, IgG2, IgG3, IgG4. IgG subclass defects, increased or imbalance in autoimmune diseases, infectious diseases, cancer and other diseases is an indication of the immune response, and IgG subclass in the development of the disease process also plays a different effect. Analysis of IgG subclass is beneficial for the disease etiology, pathogenesis and prognosis. 46 IRP patients and 20 normal controls were enrolled in this study. 58.69% patients’ hematopoietic cell antibody IgG were positive tested by flow cytometric. Hematopoietic cell antibody IgG-positive patients’ bone marrow supernatant were detected and analyzed, IgG1 and IgG3 were increased in the bone marrow supernatant of the patients with IRP, positively related to CD5+ B lymphocytes, and negative correlated with hematopoietic function. Further studies are needed to confirm which IgG subclass plays an important role in the pathogenesis of IRP.

Disclosures

No relevant conflicts of interest to declare.
Proteomics Profiles of CD34+ Cells in Bone Marrow of Patients with Severe Aplastic Anemia
Authors: Shao Zonghong , Qi Weiwei , Fu Rong
Published: October 2019

Objective

Application of isobaric tags for relative and absolute quantification (iTRAQ) technology, the proteins of CD34+ cells in bone marrow for patients with severe aplastic anemia (SAA) and normal controls were analyzed. To obtain differential proteomics profiles of CD34+ cells in bone marrow for SAA specificity. To preliminary screen the autoantigen which may cause immune system hyperfunction in SAA, providing a theoretical basis for further elucidating the immune pathogenesis of SAA.

Methods

Untreated SAA patients, remission SAA patients, as well as normal controls in Tianjin Medical University General Hospital from 2013.1 to 2014.2 were enrolled in this study. CD34+ cells in bone marrow for 29 cases of recovery SAA patients and 10 normal human were purified by immunomagnetic cell sorting, total protein were extracted and were processed enzymolysis, labeled sample by iTRAQ reagent, then analyzed the protein of sample by multidimensional liquid chromatography tandem mass spectrometry of Q Exactive, identified proteins by application of database, compared the different expression of proteins between SAA patients and normal controls, to preliminary screen the autoantigen which may cause immune system hyperfunction in SAA.

Results

Expression of differential proteomic profiles for CD34+ cells in SAA bone marrow were obtained. The protein confidence > 95% and identification of peptide protein false positive rate < 1% were limited, 15120 peptides and 3208 protein were identified. The Ratio of different proteins (SAA group/ normal group) greater than 2.794 and less than 0.323 and the confidence limit greater than 95% were limited , 157 different proteins with high confidence were identified, of which 54 up-regulated proteins, 103 down regulated proteins. The physiological function of proteins involved cell apoptosis, cell cycle, RNA splicing and metabolism, protein modification and transfer, the ubiquitin proteasome mediated protein degradation.

Conclusions

The proteome profiles of CD34+ cells in SAA bone marrow were obtained successfully, and it was proved that iTRAQ marker tandem mass spectrometry for proteome of bone marrow cells for patients with SAA was an effective method. It was found that 157 abnormal expression proteins of CD34+ cells in SAA bone marrow, the function of proteins mainly involved apoptosis, cell cycle, protein modification and transport, RNA processing and splicing, the ubiquitin proteasome system. We preliminary predicted autoantigen which may cause SAA immune "waterfall" activation.

Disclosures

No relevant conflicts of interest to declare.
Telomere Length Changes of Lymphocytes in Aplastic Anemia
Authors: Fu Rong , Zhang Jiangbo , Shao Zonghong
Published: October 2019

Aplastic anemia (AA) is a primary disorder of severe bone marrow failure characterized by pancytopenia. The pathogenesis of AA is closely related to T cell hyperfunction. Abnormal telomere shortening of bone marrow mononuclear cells which has been reported in AA not only lead to genomic instability and apoptosis but also regulate T cells immunity to antigen. Interestingly,lymphocytes with shorter telomere length have undergone apoptosis escape in autoimmune disease. In our study, the relative telomere length (RTLs) of CD3+, CD3+CD4+, CD3+CD8+, CD19+ and CD34+ cells were investigated in 14 untreated AA patients and 32 controls. The RTLs of CD3+,CD3+CD4+ and CD3+CD8+T lymphocytes were shorter than those of the controls. Meanwhile, no differences in CD19+B and CD34+ cells were found between AA and controls. A change in telomere length may be involved in the pathogenesis of AA and could be considered as a predictive biomarker for the diagnose of AA.

Disclosures

No relevant conflicts of interest to declare.