bluuebunny/crossref_metadata_2025_split huggingface dataset:
mRNA and Protein Expression Levels of Secretory Leukocyte Protease Inhibitor (SLPI) Are Severely Reduced in Patients with Severe Congenital Neutropenia (CN)
Authors: Ellerbeck Wienke , Klimenkova Olga , Skokowa Julia , Welte Karl 
Published: October 2019

Abstract
Abstract 2165
Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-Neutrophil ELastase (NE), activities. Moreover, SLPI modulates intracellular signal transduction pathways such as NF-kB and Erk. The molecular interaction and the balance between NE and SLPI is tightly regulated. On the one side, NE upregulates the SLPI expression and at the other hand SLPI inhibits the NE-induced degradation of proteins.
We identified severe diminished levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN) harbouring either ELANE or HAX1 mutations, as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. SLPI protein levels in plasma of CN patients were also significantly reduced. We further analysed whether diminished levels of SLPI are associated with the „maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI using lentivirus-based transduction of the myeloid cell line NB4 with SLPI-specific shRNA and analysed ATRA-triggered myeloid differentiation. Indeed, myeloid differentiation was severely affected in NB4 cells transduced with SLPI-specific shRNA, as compared to control shRNA transduced cells.
Further, we analysed the mechanisms leading to SLPI downregulation. Previously, we identified severely reduced mRNA and protein levels of NE in myeloid cells and in plasma of CN patients with either ELANE or HAX1 mutations, as compared to healthy individuals. Knowing that NE induces SLPI expression, we assumed that diminished NE levels may be responsible for the low SLPI expression in CN patients. Indeed, inhibition of NE in the myeloid cell line NB4 using NE-specific shRNAs led to diminished expression of SLPI mRNA, as compared to ctrl shRNA transduced cells. At the same time, we also found that transduction of the myeloid cell line NB4 with wild type (WT) NE resulted in the increased expression of SLPI mRNA but mutated (MUT) forms of NE as found in CN patients were not able to induce SLPI mRNA, as compared to ctrl transduced cells. Taken together, both diminished NE levels and mutations in ELANE gene may cause downregulation of SLPI.
In summary, SLPI is severely downregulated in CN patients due to defective NE protein levels and ELANE mutations. As a consequence, the anti-microbial and antiinflammatory activities of SLPI are diminished in CN patients.

Disclosures:
No relevant conflicts of interest to declare.
Monocytic CCR2+ Myeloid Derived Suppressor Cells Promote Immune Escape by Limiting Activated CD8 T Cell Infiltration Into the Tumor Microenvironment
Authors: Lesokhin Alexander , Hohl Tobias , Merghoub Taha , Hirschhorn-cymerman Daniel , Pamer Eric G. , Houghton Alan N , Wolchok Jedd D 
Published: October 2019

Abstract
Abstract 2171
Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor progression in a process driven by soluble factors such as granulocyte-macrophage colony stimulating factor (GM-CSF). These cells contribute to the suppressive nature of the tumor microenvironment and interfere with the functions of cytotoxic anti-tumor T effector cells. To date, MDSC heterogeneity has presented a barrier to studying the properties of individual MDSC constituents in vivo.
Herein, we find that GM-CSF, a cytokine that promotes the numeric and functional development of monocytes, granulocytes and dendritic cells, and is frequently used as a vaccine adjuvant, is also critical for the expansion of a monocyte-derived MDSC population characterized by the expression of CD11b and the chemokine receptor CCR2. We demonstrate that these cells mediate T cell suppression in a contact dependent fashion and via the function of Arginase and inducible nitric oxide synthase, consistent with known MDSC functions. CD11b+CCR2 negative cells do not have suppressive capability despite also being expanded numerically by the actions of GM-CSF. Utilizing a toxin-mediated ablation strategy that targets CCR2-expressing cells, we demonstrate that monocytic MDSCs regulate activated CD8 T cell entry into the tumor site in vivo, thereby limiting the efficacy of immunotherapy.
Our results extend observations on the dual role of GM-CSF in both stimulation and suppression of tumor immunity and suggest therapeutic targeting of monocytic MDSC could enhance the outcomes of immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling
Authors: Krause Daniela S. , Fulzele Keertik , Barry Kevin , Lotinun Sutada , Baron Roland , Bonewald Lynda , Feng Jian Q , Chen Min , Weinstein Lee S , Bouxsein Mary , Scadden David T. , Divieti-Pajevic Paola 
Published: October 2019

Abstract
Abstract 219
Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated.
We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected.
In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls.
In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling.

Disclosures:
No relevant conflicts of interest to declare.
Disaggregation Signals Initiated by Ligand Binding and Platelet Aggregation
Authors: Gao Cunji , Fang Juan , Zhi Huiying , Wilcox David A. , Newman Peter J. 
Published: October 2019

Abstract
Abstract 2196
Accumulating evidence suggests that activation of the major platelet integrin, αIIbβ3, and subsequent platelet aggregation are intrinsically dynamic and reversible processes, especially when platelets are activated by low-dose agonists like ADP. When aggregation is not robust enough to activate feed-forward autocrine amplification pathways, platelet integrins revert to their inactivated state and fibrinogen dissociates, resulting in platelet disaggregation. Failure to disaggregate following low-dose agonist stimulation has been known for many years to correlate with occlusive arterial disease and diabetes mellitus, however the mechanisms by which integrins become inactivated leading to dissociation of fibrinogen is not well understood. Because phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling is known to be required to maintain integrins in an activated state and to support the formation of stable platelet aggregates, we examined whether the PI3K/Akt signaling might be actively reversed when platelets aggregate in response to ADP. We found that Akt becomes completely dephosphorylated in an aggregation-dependent manner. SHIP-1, a negative regulator of PI3K signaling, also becomes phosphorylated and activated during the early stages of platelet aggregation. These results suggest that fibrinogen binding and platelet aggregation initiate negative feedback through the PI3K/Akt signaling pathway to control unnecessary platelet aggregation responses. To gain further insight into the mechanism of platelet disaggregation, we examined the dynamic interaction of αIIbβ3 with talin – a cytosolic protein whose association with the integrin β3 cytoplasmic domain is required for integrin activation. We found that talin translocates to αIIbβ3 upon ADP stimulation, but dissociates from it during the process of platelet disaggregation. Blocking ADP-induced platelet aggregation with RGDW inhibited dissociation of talin from the integrin, again supporting the notion that that platelet aggregation initiates negative, as well as positive, signals to regulate the extent of agonist-induced platelet aggregation. Finally, we found that platelets missing PLCγ2 or FcγRIIa, each of which plays a positive role downstream of αIIbβ3-mediated outside-in signaling, are less prone to disaggregate following low-dose agonist stimulation than are their wild-type counterparts, although neither of them plays a role in ADP-induced platelet aggregation. Taken together, these data demonstrate that platelet aggregation and integrin αIIbβ3-mediated outside-in signaling not only initiates signals that amplify integrin activation and platelet aggregation, but also guards against inadvertent activation by shifting activated integrin αIIbβ3 back to its resting state, triggering platelet contraction and leading to dissociation of platelet aggregates.

Disclosures:
No relevant conflicts of interest to declare.
Flow Analysis of Von Willebrand Factor Collagen Binding Mutants
Authors: McKinnon Thomas A J , Nowak Agata Anna , Hua Alina , Millar Carolyn , Laffan Michael 
Published: October 2019

Abstract
Abstract 2213
Von Willebrand Factor (VWF) binds to exposed sub-endothelial collagen at sites of vessel injury principally via its A3 domain, although some evidence suggests that the A1 domain can compensate for the A3 domain under flow conditions if the A3 domain is absent or non-functional. Recently, several naturally occurring Von Willebrand disease-causing mutations have been indentified in the A3 domain; S1731T, W1745C, S1783, H1786D and most recently M1761K, as well as one mutation in the A1 domain (I1343V) all of which have defective collagen binding. While the collagen binding function of these mutations has been assessed under static conditions it remains to be established if these affect collagen binding under shear stress. In the present study the collagen binding mutants were expressed in HEK293T cells and collagen binding function determined using an in vitro flow assay. All of the mutations were expressed at similar levels to wild type (wt) VWF and demonstrated normal multimeric patterns and binding to GPIbα under static conditions. As expected, collagen binding analysis under static conditions confirmed the collagen binding defect of all the mutants, with reduced or abolished binding to both collagens type I and III for all the mutants except S1731T which demonstrated normal binding to collagen type III and slightly reduced binding to collagen type I. Analysis of platelet capture under flow conditions confirmed that all the mutants were able to capture platelets similarly to wtVWF. Analysis of VWF mediated platelet capture to a collagen surface under flow conditions confirmed the phenotype of the collagen binding mutants. With the exception of S1731T, which demonstrated normal platelet capture on both collagens, none of the mutants were able to bind to collagen type I or III under flow conditions, or mediate platelet capture at high shear stress. The collagen binding function of these mutants under flow was partially restored when co-expressed with wtVWF. Interestingly, in contrast to a previous study, a VWF variant lacking the A3 domain (VWF-ΔA3) failed to bind to collagen under shear stress and was not able to mediate platelet capture to collagen. Together these data confirm that the major collagen binding site in VWF is located in the A3 domain and demonstrate that collagen binding mutations affect VWF mediated platelet capture under shear stress.

Disclosures:
No relevant conflicts of interest to declare.
The Role of Factor VIII C Domains in Sorting to Weibel-Palade Bodies
Authors: Bouwens Eveline , van den Biggelaar Maartje , Voorberg Jan , Mertens Koen 
Published: October 2019

Abstract
Abstract 2241
Recent studies have shown that factor VIII (FVIII) expressed in endothelial cells sorts with von Willebrand factor (VWF) to secretory Weibel-Palade bodies (WPBs). The sorting mechanism remains controversial although VWF is thought to be essential. However, mutations that lead to impaired FVIII-VWF complex assembly do not reduce the sorting efficiency of FVIII. As factor V (FV) and FVIII are highly homologous in structure, we addressed the possibility that FV sorts to WPBs as well. Our study was designed to identify domains in FVIII that are needed for sorting to WPBs by means of domain deletions and FVIII-FV domain exchange. As the C domains of FVIII contain membrane and VWF binding sites, we particularly focused on comparing the C domains of FVIII and FV. Blood outgrowth endothelial cells (BOECs) were transduced with lentiviral vectors encoding FV, FVIII deletion mutants, or FVIII-FV chimeras. We found by confocal microscopy and subcellular fractionations that FV displays a strong reduction in sorting efficiency (2% sorting efficiency) compared to FVIII (20% sorting efficiency). This indicates that sorting to WPBs is mediated by FVIII-specific structural elements. As the C domains of FVIII are implicated in membrane and VWF binding, these domains could drive sorting to WPBs. Therefore, we constructed FVIII variants lacking C domains to establish their role in WPB sorting. Quantitative determination of the sorting efficiencies demonstrates that the C1 domain is not of major importance for sorting to WPBs (10% sorting efficiency), whereas the C2 domain is (not detectable in WPB fractions). Moreover, exchanging the FVIII C domains for corresponding domains of FV also suggests that the C2 domain drives WPB sorting (3% sorting efficiency). This leads to the conclusion that FVIII sorting to WPBs is driven by FVIII-specific structural elements in both C domains, but in particular the C2 domain.

Disclosures:
No relevant conflicts of interest to declare.
Consequences of Switching From Prophylactic Treatment to On-Demand Treatment in Late Teens and Early Adults with Severe Hemophilia A. the TEEN/TWEN Study
Authors: Manco-Johnson Marilyn J. , Sanders Joann , Ewing Nadia , Humphries Thomas J. 
Published: October 2019

Abstract
Abstract 2288
The TEEN/TWEN study is an observational, prospective, case-controlled, multicenter study to evaluate long-term prophylaxis patients and to determine the consequences of switching from prophylaxis to on-demand treatment. Thirty-eight males, aged 14–29, with severe hemophilia A were enrolled and followed for 18 months. Group 1 (n=22) remained on prophylaxis, Group 2 (n=5) switched to on-demand treatment ≤12 months prior to entry, and Group 3 (n=11) switched to on demand ≥13 months prior to entry. The primary and main secondary efficacy criteria were presented at ISTH, 2011 in Kyoto, Japan, and showed clear benefits of continuing prophylaxis in decreased bleeding and improved quality of life. Here, we present the secondary and exploratory variables examined in the study, including the Gilbert score, target joints, Hemophilia Activities List (HAL), Godin Leisure Time, treatment satisfaction, and State Trait Anxiety Inventory (STAI).

Results:
Gilbert score changes were small, and limited to the ankles, knees, and elbows. Group 2 had the highest overall bleeding score. Of the exploratory variables, Group 3 usually had numerically worse values than the other groups. For the HAL, Group 2 had a higher increase overall than Group 1. The STAI showed an increase in trait anxiety for Groups 1 and 2 during the study, and increase in state anxiety for group 1 but a decrease for Group 2. No other distinguishing findings emerged.

Conclusions:
Although the primary and main secondary efficacy variables showed clear benefits of continuing prophylaxis, the remaining secondary variables and the exploratory variables did not clearly distinguish between Groups 1 and 2, although most showed worse results for Group 3. For these variables the changes in Groups 1and 2 were usually small, occurred in a similar pattern in both groups, or showed inconsistent patterns. The changes in Group 3 suggest that quality of life decreases with length of time off prophylaxis. Small cell numbers, outliers, being in a clinical trial and questionnaire fatigue may be contributing factors.

Disclosures:
Manco-Johnson: Octapharma AG: Consultancy; Bayer: Research Funding. Ewing:CSL Behring: Consultancy, Honoraria, Research Funding, Travel funds; Baxter: Consultancy, Honoraria, Research Funding, Travel funds; Bayer: Consultancy, Honoraria, Research Funding, Travel funds; Biogen Idec: Consultancy, Honoraria, Research Funding, Travel funds; Grifols: Consultancy, Honoraria, Research Funding, Travel funds; Inspiration: Consultancy, Honoraria, Research Funding, Travel funds; NovoNordisk: Consultancy, Honoraria, Research Funding, Travel funds; Octapharma: Consultancy, Honoraria, Research Funding, Travel funds; Pfizer: Consultancy, Honoraria, Research Funding, Travel funds; Talecris: Consultancy, Honoraria, Research Funding, Travel funds. Humphries:Bayer: Employment.
A Recombinant Thrombomodulin Improves Haemostatic Disturbance and Inflammation in Setpic Patients with DIC
Authors: Yada Noritaka , Nishio Kenji , Fukushima Hidetada , Seki Tadahiko , Urizono Yasuyuki , Hata Michiaki , Asai Hideki , Doi Masaaki , Matsui Hideto , Sugimoto Mitsuhiko , Okuchi Kazuo 
Published: October 2019

Abstract
Abstract 2293

Background:
Recently, a recombinant human soluble thrombomodulin (rTM) has become commercially available for patients with disseminated intravascular coagulation (DIC) in Japan, which is composed of the active, extracellular domain of thrombomodulin. However, its anti-thromobotic and anti-inflammatory effect during the clinical course in patients with DIC has not been reported. Aim: To investigate the effect of rTM on mortality, haemostatic disturbance, and inflammation in septic patients with DIC. Methods: The patients with sepsis who met the Japanese diagnostic criteria for acute DIC and showed a level of antithrombin (AT) lower than 70% were recruited and divided into two groups, one group (Control group) treated with AT products and Gabexate mesilate (GM), and the other group (TM group) treated with rTM (0.06 mg kg(-1) for 30 min, once daily) in addition to AT and GM. The effect of rTM during the clinical course were investigated from the differences between TM and Control groups in mortality, DIC scoring, haemostatic and inflammatory markers on days 0, 3, 5, 7 (day 0 is just before initiation of therapy). Results & Discussion: Eighteen patients were included in the TM group, and 16 patients in the Control group. There were no differences in APACHEII score at day 0, DIC score at day 0, or mortality at day 28 between the groups. The effects of the rTM were as follows; 1) Patients in the TM group showed earlier DIC resolution at day 5 than Control at day 7. 2) Hypercoagulable state expressed by the increased levels of soluble fibrin monomer (SF) or thrombin-antithrombin complex (TAT) was improved more quickly in TM group compared with Control group, suggesting that rTM decreased thrombin generation. 3) AT was increased much more at day 3 after AT product administration in TM group than in Control group, which also can be explained by suppression of thrombin generation by rTM. 4) Increased levels of the complex of α2PI/plasmin, D-dimer, and fibrin/fibrinogen degradation product were also improved earlier in TM group than Control group. 5) Plasmin-alfa2 plasmin inhibitor complex (PIC) was significantly lower at day 7 in TM group than Control group, suggesting anti-fibrinolytic effect of rTM. 6) Decreased level of ADAMTS13 increased more quickly in TM group. 5) TNF-α, IL-6, HMGB1 were decreased significantly at day 3 compared with day 1 only in TM group. These anti-inflammatory effect can be evoked through direct sequestration of HMGB1 by rTM or decreased thrombin generation by rTM, because thrombin is a strong pro-inflammatory molecule through PAR1. Conclusion: rTM may be beneficial in improving septic DIC via its anti-thrombotic and anti-inflammatory properties.

Disclosures:
No relevant conflicts of interest to declare.