bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of the Btk Inhibitor Ibrutinib on Monocyte Responses to Antibodies
Authors: Ren Li , Campbell Amanda , Gautam Shalini , Fang Huiqing , Fatehchand Kavin , Stiff Andrew , Mehta Payal , Mo Xiaokui , Fang Xuexun , Byrd John C. , Carson William , Butchar Jonathan P. , Tridandapani Susheela 
Published: October 2019

Abstract
The novel and irreversible tyrosine kinase inhibitor, Ibrutinib, has shown significant activities across a variety of B-cell tumors such as Chronic Lymphocytic Leukemia (CLL) and B-cell Non-Hodgkin lymphoma, either alone or in combination with antibody therapy. Fcγ receptors (FcγR) on effector immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses. Here we tested the effect of Ibrutinib on FcγR-mediated activities in monocytes.
Our results indicated that Ibrutinib does not affect monocyte FcγR-mediated phagocytosis even at concentrations higher than those achieved physiologically, but suppresses FcγR-mediated cytokine production. This phenomenon was also seen in macrophages from Xid mice, in which there is a decrease in FcγR-mediated cytokine production but not phagocytosis.
One major signaling event downstream of Btk that is blocked by Ibrutinib is calcium flux. Thus, we next tested whether phagocytosis was independent of calcium flux in order to help explain the lack of effect that Ibrutinib had on monocyte phagocytic ability. Results showed that intracellular calcium flux is required for FcγR-mediated cytokine production but not for phagocytosis. In order to verify this finding, we then measured the activation of the GTPase Rac, which is responsible for actin polymerization needed for phagocytosis. Results showed that Ibrutinib did not inhibit Rac activation, nor did inhibition of calcium flux by BAPTA-AM.
We next asked whether the effect of Ibrutinib on cytokine production could be reversed by IFNγ priming since NK cells produce significant levels of IFNγ in response to antibody therapy. Treatment of monocytes with IFNγ prior to Ibrutinib treatment resulted in a lack of inhibition of cytokine production, suggesting that IFNγ signaling can overcome Ibrutinib effects. We are currently in the process of examining the mechanisms by which IFNγ can protect from Ibrutinib effects on monocytes.
These results suggest that combing Ibrutinib with monoclonal antibodies can enhance B-CLL killing while not affecting macrophage effector function.

Disclosures
Byrd: Acerta Pharma BV: Research Funding.
Circadian Clock Protein CRY Controls B-Cell Intrinsic Tolerance
Authors: Cao Qi , Zhao Xuan , Gery Sigal , Chen Zhengshan , Deng Ruishu , Sun Haibo , Lin De-chen , Zhao Zhiqiang , Said Jonathan W , Li Quan-zhen , Muschen Markus , Evans Ronald M. , Koeffler H. Phillip 
Published: October 2019

Abstract
The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice.
The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control.

Disclosures
No relevant conflicts of interest to declare.
Tumorantigen-Specific CD40B Cells: Combining Enhanced Antigen-Presentation and Antibody-Secretion for Tumor Targeting
Authors: Wennhold Kerstin , Thelen Martin , gracia Marquez Maria , Scheid Christof , Shimabukuro-Vornhagen Alexander , von Bergwelt-Baildon Michael 
Published: October 2019

Abstract
Efficient antigen presentation is a prerequisite for the development of a T-cell-mediated immune response. Dendritic cells (DCs) are the most prominent professional antigen-presenting cells (APCs). However, they have several disadvantages as cellular adjuvant in cancer immunotherapy. Therefore, an alternative approach was developed, in which polyclonal B cells can serve as potent APCs by treatment with the CD40 ligand. We demonstrated that CD40-activation dramatically improves antigen presentation by B cells, efficiently inducing naive and memory CD4+ and CD8+ T-cell responses. Moreover, these CD40-activated (CD40) B cells home to secondary lymphoid organs. However, antigen presentation by antigen-specific B cells is more effective compared to polyclonal B cells. Therefore, we use tumorantigen-specific B cells to improve the antigen-presenting function of CD40B cells. Purified human and murine tumorantigen-specific B cells highly upregulate activation markers upon CD40-stimulation, which results in an enhanced CD4+ and CD8+ T cell response in vitro and in vivo. This response is significantly lower in polyclonal CD40B cells and comparable to the stimulation induced by mature dendritic cells. Moreover, antigen-specific B cells could be stimulated in vitro to differentiate into antibody-secreting plasma cells. Treatment of E.G7 lymphoma-bearing mice with a combination of antigen-specific CD40B cells and plasma cells results in inhibition of tumor growth and prolonged survival. Moreover, antigen-specific B cells home to the tumor site and the spleen, where they encounter T cells. These results provide new insights into the role of activated antigen-specific B cells as APCs and their use for cancer immunotherapy.

Disclosures
Scheid: Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.
The Second CGHC Motif of Protein Disulfide Isomerase Mediates Thrombosis
Authors: Zhou Junsong , Wu Yi , Wang Lu , Rauova Lubica , Hayes Vincent M , Poncz Mortimer , Essex David W 
Published: October 2019

Abstract
Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation.

Disclosures
No relevant conflicts of interest to declare.
Increased Levels of Histone in Human Plasma in Septic Patients with DIC
Authors: Kawasugi Kazuo , Yamamoto Tadashi , Shirafuji Naoki , Yamada Shingo , Takashi Ito , Maruyama Ikuro 
Published: October 2019

Abstract
<Background and Methods>
Sepsis is a life-threatening condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome, SIRS). Histones are essential for packing DNA into the cell nucleus and also play role in regulating gene expression but the new study (Jun Xu et al, Nature Medicine 15. 1318, 2009) have shown histones released into blood stream at the onset of sepsis can have devastating effects (endothelial damage and organ failure etc). However, plasma levels of histone are unclear in septic patients with DIC. Therefore, We developed a sandwich ELISA to quantify serum or plasma histone H3 levels and measured plasma levels of histone H3 in septic patients with DIC (n=30).
<Results>
DIC was diagnosed on the Japanese Ministry of Health and Welfare (JMHW) DIC diagnostic criteria. The thrombin antithrombin complexes (TAT) levels were higher in septic patients with DIC as reported by others. The working range of this ELISA was 3-1000 ng/ml. In healthy volunteers, plasma levels of histone (H3) were not detectable. We detected high levels of extracellular hisotone (H3) in the plasma of all septic patients with DIC by sandwich ELISA. Mean plasma levels of hisotone (H3) were 70.5 ± 63.2 (12.9-336.5) ng/ml in septic patients with DIC. However, plasma levels of extracellular histone (H3) were negative in acute promyelocytic leukemia (APL)-induced DIC (n=5). Also, Plasma levels of histone were 10 ng/ml or less by sandwich ELISA in septic patients without DIC (n=8).
<Conclusion>
These results suggest that assess of extracellular hisotones in the plasma may be helpful in the making the diagnosis in septic patients with DIC. Also, it appears that extracellular hisotones in the plasma may contribute to develop DIC in septic patients.

Disclosures
No relevant conflicts of interest to declare.
Hematopoietic Stem Cells Develop in the Absence of Endothelial Cadherin 5 Expression
Authors: Anderson Heidi , Patch Taylor , Reddy Pavan , Hagedorn Elliott , Tamplin Owen J. , Bauer Daniel E. , Paw Barry H. , Vestweber Dietmar , Zon Leonard I. , Orkin Stuart H. , Daley George Q. , Shah Dhvanit I. 
Published: October 2019

Abstract
Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from Cadherin 5 (Cdh5, VE-cadherin)+ endothelial precursors, and isolated populations of Cdh5+ cells from mouse embryos and embryonic stem (ES) cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Since Cdh5-/- mice embryos die before the first HSCs emerge, it is unknown if Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlbbw306), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Utilizing time-lapse imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5-/- GFP+/+ cells inchimeric mice, we demonstrated that Cdh5-/- GFP+/+ HSCs emerging from E10.5 and E11.5 AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multi-lineage adult blood. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.

Disclosures
Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder. Orkin:Editas Medicine: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Pfizer: Research Funding; Sangamo Biosciences: Consultancy.
Thrombopoietin (TPO) Induces Hematopoietic Differentiation of Mouse ES Cells Via HIF-1 Alpha-Dependent Activation of a BMP4 Autoregulatory Loop
Authors: Zahabi Azadeh , Morishima Tatsuya , Pramono Andri , Lan Dan , Kanz Lothar , Welte Karl , Skokowa Julia 
Published: October 2019

Abstract
Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the optimal conditions for the production of hematopoietic cells as a source for transplantation or experimental use. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-mpl. This knowledge, together with observations in vitro and in animal models in vivo, suggests that TPO/c-mpl signaling promotes early hematopoiesis. However, the downstream mechanisms underlying TPO signaling are not fully elucidated. Here, we describe for the first time a direct connection between the TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in the hematopoietic fate decision of ES cells. BMP4 is a classical morphogen and a well-known inducer of early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 by ES cells with concomitant upregulation of the BMP receptor, BMPR1A, phosphorylation of Smad1, 5, and 8 and activation of the specific target genes, Id1, 2, and 3, and Msx1 and 2. This was mediated by TPO-dependent binding of the HIF-1α transcription factor to the BMP4 gene promoter, resulting in further activation of the BMP4-autoregulatory loop. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cell. Taken together, our findings contribute to the understanding the mechanisms of hematopoietic differentiaiton of ES cells and might help to establish new methods for the efficient production of hematopoietic stem cells in vitro.

Disclosures
No relevant conflicts of interest to declare.
Interferon Alpha Mediated Activation of the Bone Marrow Stem Cell Vascular Niche
Authors: Prendergast Áine M. , Kuck Andrea , Von Essen Mieke , Essers Marieke A. G. 
Published: October 2019

Abstract
Endothelial cells (ECs) significantly influence the response of an organism to inflammation and infection. In the bone marrow, these cells form a major part of the bone marrow vascular niche, which regulates stem cell function and influences stem cell fate. The primary response to infection involves synthesis of immune-modulatory cytokines, such as interferon alpha (IFNα). We, and others, have shown that in contrast to the anti-proliferative effect of IFNα on hematopoietic stem cells (HSCs) in vitro, in vivo, IFNα induces cell cycle entry of quiescent HSCs (Essers et al. 2009). Given the contrasting outcome of in vitro and in vivo exposure of HSCs to IFNα, it is probable that niche cells and molecular maintenance signals from the niche are required for IFNα-induced activation of quiescent HSCs. Here, we now show that although interferon signaling itself in niche cells is not required for HSC activation, niche components do respond to IFNα stimulation. Of these, bone marrow ECs are rapidly and indirectly stimulated following IFNα treatment in vivo. The vascular system, lined by ECs, is a central primary responder following inflammatory insult with a multifaceted role, including transport of immune cells and promotion of a rapid return to homeostasis. We have found that IFNα stimulation in vivo results in an increased bone marrow vascularity, visualized by fluorescence imaging of cryo-sectioned murine femurs immunostained with the vascular basement membrane protein, laminin. IFNα-mediated activation of ECs involves the expression of key inflammatory and endothelial-stimulatory markers, including VE-cadherin and ESAM, and also an increased vascular leakiness in the bone marrow, demonstrated by the Evans blue assay. In accordance with this finding of vascular activation, we confirmed that VEGF, which is an established regulator of vascular dynamics, is rapidly up regulated in the bone marrow supernatant of treated mice. Furthermore, we can also demonstrate a key role for VEGF in our observed IFNα-mediated stimulation of ECs by abrogation of this activation using co-treatment with Avastin (bevacizumab) in vivo. We are now investigating the feedback from this activation of ECs on the hematopoietic system itself. In summary, these data indicate a rapid and indirect stimulation of the bone marrow vascular niche in an inflammatory setting. In addition, they support a previously undescribed cellular instruction from an activated hematopoietic system to a niche component.

Disclosures
No relevant conflicts of interest to declare.
5-Fluorouracil Treatment Leads to Activation of Stem Cell Niche By Reconstructing Mesenchymal Stromal Cells and Exert a Distinct Microenvironmental Impact on Normal and Leukemic Cells
Authors: Jeong Seon-Yeong , Kim Jin-A , Oh Il-Hoan 
Published: October 2019

Abstract
Reactivation of endogenous hematopoietic stem cells (HSCs) are initiated by stimulation of bone marrow niche triggered by various injury signals. Here, we show that treatment with 5-fluorouracil (5-FU) leads to reconstruction of bone marrow (BM) microenvironment to establish an activated niche stimulating hematopoietic stem cells (HSCs). First, we show that pre-treatment with 5-FU leads to engraftment of donor cells in non-irradiated recipient mice without affecting the homing efficiency of HSCs into BM. The HSC activation effects were reproduced in-vitro by co-culturing hematopoietic cells with CD45-Ter119- stromal cells derived from 5-FU treated BM, but not by co-culture with CD45+ cells or stromal cells obtained from enzymatic digestion of bone from the same mice. Examination of BM mesenchymal cells after 5-FU treatment revealed a rapid emergence of high-proliferating mesenchymal progenitors exhibiting large size colony (CFU-F) and higher self-renewal of colonogenic cells 3-5 days after 5-FU treatment, which was concomitantly associated with regeneration of CD34+Lin-Sca-1+c-kit+ (LSK) cells in the same BM. The cellular changes in mesenchymal stroma was associated with rapid emergence of characteristic mesenchymal cell populations (PDGFR-a+/Leptin receptor+/SSEA-3+: PLS) with 650-folds increase of the PLS cells in BM in 3 days after 5-FU treatment. However, the increase of these PLS mesenchymal cells were not associated with increase in mitotic activity of mesenchymal cells (<5% BrdU+ cells), indicating phenotypic conversion of subpopulation in BM. Moreover, cellular changes in mesenchymal niche were associated with rapid increase of mesenchymal cells expressing cross-talk molecules such as CXCL-12 (20-folds), Jagged-1 (13-folds) and DLL-1 (15-folds). Furthermore, in-vivo administration of chemicals blocking CXCL-12 and notch signaling during the recovery from the 5-FU treatment led to the significant loss of LSK-SLAM cells in the regenerated BM. Interestingly, the BM niche activated by 5-FU exerted a distinct effect on normal and leukemic cells in a manner that it provide higher support on the primitive state of normal HSCs than for MN-1 induced leukemia cells. Thus, leukemic mice engrafted with MN-1 cells exhibited a decrease in primitive leukemic cell (Lin-c-kit+) and higher survival by 5-FU treatment than those treated by radiation. Taken together, our study reveals the cellular reconstruction of mesenchymal niche in BM during stimulus-induced niche activation and provides an insight on the selective niche targeting as a novel therapeutic strategies for hematological diseases.

Disclosures
No relevant conflicts of interest to declare.
A Human Bone Marrow Failure Syndrome Caused By a Homozygous Mutation in MYSM1
Authors: Bahrami Ehsan , Racek Tomas , Witzel Maximilian , Puchalka Jacek , Greif-Kohistani Naschla , Kotlarz Daniel , Orth Michael , Lauber Kirsten , Walz Christoph , Herz Waleed Al , Horny Hans-Peter , Schmidt Heinrich , Albert Michael H. , Klein Christoph 
Published: October 2019

Abstract
Inherited bone marrow failure syndromes comprise a heterogeneous group of genetic disorders characterized by dysfunction of hematopoietic stem or progenitor cells. We present a consanguineous pedigree with two siblings with early-onset, progressive bone marrow failure (neutropenia, anemia, thrombocytopenia) associated with trigonocephaply, hyperplastic gingiva, cataract, accessory mammilla, dental changes and mild neurocognitive developmental delay. Pancytopenia at birth as well as non-compaction cardiomyopathy was observed in one of the patients.
Bone marrow histology showed marked hypocellularity with decrease of all hematopoietic cell lines but without cellular atypia, monocytosis, blast excess or fibrosis. Fanconi anemia was excluded by mitomycin C induced chromosomal breakage studies.
Immunophenotyping of peripheral blood cells showed a reduction of mature B cells, reduced switched memory B cells, whereas the level of activated B-cells and plasma blasts were increased. T cell proliferation upon PHA and specific antigens was normal.
Using whole exome sequencing in patients and both parents we could identify a single potentially disease causing homozygous stop codon mutation (NM_001085487: c.1168G>T: p.E390*) in MYSM1 (Myb-Like, SWIRM And MPN Domain-Containing Protein 1). A family with two affected patients carrying the same mutation has been reported previously (Alsultan A et al, Blood 122:3844, 2013). MYSM1 is a Histone H2A deubiquitinase that has previously been implicated in controlling hematopoietic stem cells, progenitor B cells, and NK cells in mice (Nijnik A et al, Blood 119:1370, 2012).
MYSM1 protein was absent in patients' EBV-transformed B cell lines (EBV-LCLs). MYSM1-deficient EBV-LCLs had elevated γ-H2AX levels, indicative of increased genomic instability. Upon exposure to UV light, we observed a sustained expression of p53 and phospho-p53 as well as p38 MAPK and phospho-p38 MAPK in patients' fibroblasts in comparison to fibroblasts from healthy individuals.
In view of the progressive pancytopenia, both siblings underwent allogeneic hematopoietic stem cell transplantation from 10/10 HLA-matched family donors after reduced intensity conditioning with alemtuzumab, fludarabine and treosulfan at 2 and 4 years of age, respectively. The procedure was well tolerated, and both siblings exhibit full donor hematopoietic reconstitution at 21 and 26 months after transplant.
In summary, we here identify a novel human bone marrow failure syndrome caused by MYSM1-deficiency that can be cured by allogeneic hematopoietic stem cell transplantation.

Disclosures
No relevant conflicts of interest to declare.