bluuebunny/crossref_metadata_2025 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 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 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 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 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 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 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 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 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.
Association of Gene-Environment Interactions with Venous Thromboembolism (VTE): A Merged/Imputed Genome-Wide Scan/Candidate-Gene Case-Control Study
Authors: Heit John A. , Armasu Sebastian M. , Matsumoto Martha E. , Petterson Tanya M. , Rider David N. , Cunningham Julie M. , Asmann Yan W. , de Andrade Mariza
Published: October 2019

Abstract 2295

Background:

While interaction between Factor V Leiden and other VTE risk exposures compound VTE risk, whether other gene-environment interactions are associated with VTE is largely unknown.

Objective:

To test novel gene-environment interactions for an association with VTE.

Methods:

Genome-wide scan (Illumina 660Q [557,112 SNPs]) and candidate gene (12,551 SNPs in 764 genes within the anticoagulant, procoagulant, fibrinolytic and innate immunity pathways) genotypes from 1270 non-Hispanic adults of European ancestry with objectively-diagnosed VTE (cases; no cancer, venous catheter or antiphospholipid antibodies) and 1302 controls (frequency-matched on case age, gender, race, MI/stroke status) were merged and imputed to 2.5 million SNPs with MACH using HapMap Phase 2 (60 CEU). We tested pairwise interactions between these SNPS and each of 7 exposures (“stress” [defined as ever hospitalized, ever surgery, ever trauma, ever transfemoral procedure or ever leg paresis], ever surgery, ever neurosurgery, ever orthopedic surgery; and among women, ever pregnant, ≥3 pregnancies and ever oral contraceptives/hormone therapy), adjusted for age, gender, MI/stroke status and state of residence, using logistic regression.

Results:

The mean ± standard deviation case and control ages were 54.4 ± 16.2 and 55.6 ± 15.8 years, respectively and 51% were female. Among the 7 models, a significant interaction was found between stress and A2BP1 (rs735919, OR=0.2, p=2.8E-07), ever surgery and ABCG8 (rs6709904, OR=0.27, p=2.1E-07), orthopedic surgery and SLC24A3 (rs6081599, OR=0.53, p=2.8E-07), neurosurgery and TBC1D4 (rs17064485, OR=8.5, p=3.5E-07), every pregnant and MICALCL (rs11022321, OR=0.17, p=8.3E-07), >3 pregnancies and KLRB1 (rs2536929, OR=0.31, p=1.1E-07) and ever oral contraceptives/hormone therapy and LINGO2 (rs10969259, OR=2.02, p=4.9E-07). A2BP1 plays a role in RBC shape and size, ABCG8 is associated with severe hypercholesterolemia, SLC24A3 is important for intracellular calcium homeostasis, TBC1D4 participates in cell signaling in CD4+/CD45RO+ memory T-cells, MICALCL is an extracellular signal-regulated kinase 2-binding protein, KLRB1 regulates natural killer cell function, and LINGO2 encodes a leucine-rich repeat signaling protein. Our observed interaction of VTE risk factors with these genes to compound the risk of VTE requires replication in future studies. If confirmed, functional studies will be important to determine the roles of these gene products in the causal pathway to VTE.

Conclusion:

VTE risk factors and SNPs within several novel genes may interact to compound the risk of VTE.

Disclosures:

Heit: Daiichi Sankyo: Consultancy, Honoraria.
Physical Interaction of the Proto-Oncogene EVI1 with Hypermethylated in Cancer 1 (HIC1) Deregulates EVI1 Mediated Apoptosis in Cancer Cells
Authors: Pradhan Anjan Kumar , Chakraborty Soumen
Published: October 2019

Abstract 2449

The ecotropic viral integration site I (EVII) gene has attracted considerable attention in recent years because of its reported involvement in chronic myeloid leukemia, high risk myelodysplastic syndrome and a very high percentage of acute myeloid leukemias. The most recent World Health Organization classification has designated AML with inv (3) or t(3;3) and associated RPN1/EVI1 fusion, as a distinct AML subgroup associated with an unfavorable prognosis. Studies also indicate that EVI1 may be overexpressed in a subset of human colon cancers, and that EVI1 might affect disease progression and/or sensitivity to chemotherapy. The presence of zinc finger domains that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, few target genes of EVI1 have been functionally identified. In the present study, we demonstrate by co-immunoprecipitation assay that the proximal zinc finger domain of EVI1 binds to HIC1, which abolishes the ability of EVI1 to induce expression of Bcl-xL. HIC1, is an epigenetically regulated transcriptional repressor that functionally cooperates with p53 to suppress age dependent development of cancer in mice. Inactivation of HIC1 results in up regulation of SIRT1, which deacetylate and inactivates p53; allowing cells to bypass apoptosis and DNA damage survival., Our study also demonstrates via indirect immuno fluorescence analysis that EVI1 and HIC1 proteins are colocalized in speckles in the nucleus. We recently reported that EVI1 not only binds the Bcl-xL promoter but also up regulates its transcriptional activity and protein expression. We now demonstrate by EMSA and luciferase assays that physical interaction between EVI1 and HIC1 releases the binding of EVI1 from the Bcl-xL promoter, and hence promotes apoptosis. Thus by directly binding to EVI1, HIC1 displaces EVI1 from its DNA consensus site leading to down regulation of Bcl-xL. Also we observed that posttranslational modification of EVI1, mainly acetylation plays a critical role in this EVI1-HIC1 protein-protein interaction. Further elucidation of the counteracting effects of HIC1 and EVI1 on Bcl-xL expression may provide new insights in EVI1 mediated disease progression.

Disclosures:

No relevant conflicts of interest to declare.
The Accuracy of PET in the Detection of Posttransplant Lymphoproliferative Disorder (PTLD)
Authors: Dierickx Daan , Verhoef Gregor , Gheysens Olivier , Tousseyn Thomas , Wolf-Peeters Christiane De , Wlodarska Iwona , Brepoels Lieselot
Published: October 2019

Abstract 2657

Introduction.

PET scan has emerged as a powerful tool in the diagnosis and staging of both Hodgkin and different subtypes of aggressive non-Hodgkin lymphoma. We investigated the accuracy of PET scan in 170 patients with suspected posttransplant lymphoproliferative disorder (PTLD).

Material and methods.

We retrospectively reviewed all patients in our center between 2003 and 2010 for whom a PET-scan was requested for the indication PTLD. 170 PET-scans in 150 patients were eligible for evaluation. In 45 cases, the patient had already a biopsy proven PTLD before PET-scanning and PET was considered a staging tool in these patients. In the remaining 125 PET-scans, PLTD was only suspected at the time PET was performed. In 73 (43%) and 97 (57%) cases PET scan without CT and combined PET/CT scan were ordered respectively. Because the aim of this study was to determine the accuracy of PET scan in diagnosis of PTLD, we evaluated only results of the PET scans. PET-scans were blindly scored on a four-point scale. Results were compared with biopsy as the gold standard.

Results.

The majority of PET scans were performed in kidney transplant recipients (35%), followed by liver (15%), lung (15%), heart (15%), hematopoietic stem cell (15%), combined (5%) and bowel (1%) transplantation. In 119 (70%) patients a biopsy was available. We found a sensitivity of PET of 89%, specificity of 89%, positive predictive value (PPV) of 91% and negative predictive value (NPV) of 87%. In a subanalysis of the 125 scans performed for differential diagnosis PTLD versus other diseases, sensitivity, specificity, PPV and NPV were 90%, 89%, 85% and 93% respectively. FDG-uptake in PTLD was generally high with a median mean and maximal standardized uptake value (SUV) of 9.0 and 17.4. False positive results were mainly due to infectious or inflammatory conditions, whereas false negative results were mainly reported in limited stage PTLD. Atypical extrandodal presentation was a frequent finding including diffuse pulmonary involvement and gastro-intestinal lesions.

Conclusions.

From these data, we can conclude that PET is highly sensitive for the detection of lesions of PTLD, and that PET has an excellent ability to differentiate PTLD from other diseases.

Disclosures:

No relevant conflicts of interest to declare.
Posttransplant Lymphoproliferative Disorder in Kidney Transplant Patients: A Report of 68 Cases
Authors: Tsai Kathleen J , Bloom Jonathan H , Reshef Ran , Sawinski Deirdre , Bleicher Melissa , Goral Simin , Grossman Robert , Tsai Donald E , Bloom Roy D
Published: October 2019

Abstract 2662

Posttransplant Lymphoproliferative Disorder (PTLD) is a rare complication of kidney transplantation that often arises due to reactivation of the Epstein-Bar Virus (EBV). Given the rarity of this disease, a full understanding of its presentation and optimal therapies has yet to be determined. Out of the 3160 patients who received a kidney transplant at the Hospital of the University of Pennsylvania between October 1987 and December 2010, we provide a retrospective analysis of 68 patients who developed PTLD. These patients were 82% Caucasian, 62% male, and 9% combined kidney and pancreas transplant patients. The most common underlying diseases leading to transplantation were glomerulonephritis (26%) and diabetes mellitus (17%). Most patients received a kidney from an unrelated donor (74%) and experienced one or more rejection episodes prior to PTLD diagnosis (59%). The median age at PTLD diagnosis was 48 yrs (range 17–76 yrs), and the median time from transplantation to diagnosis was 3 yrs (range 6 days– 25 yrs). PTLD histology consisted of 48% monomorphic, 37% polymorphic, 7% plasmacytoma, 4% T cell, and 3% Hodgkin lymphoma. At diagnosis, patients were taking steroids (96%), cyclosporine (53%), tacrolimus (35%), azathioprine (52%), and/or mycophenolate mofetil (28%). Common initial PTLD symptoms include fever (48%), pain (42%), weight loss (33%), fatigue (33%), and mass (24%). The disease commonly presented in lymph nodes (38%), kidney allograft (29%), and/or GI tract (18%). Most PTLD cases were diagnosed at stage 1 (43%) or stage 4 (31%). Laboratory analyses reveal that 62% of patient tumors tested positive for EBV and 61% of patients had elevated LDH levels. Common PTLD therapies included reduction of immunosuppression (88%), treatment with rituximab (31%), surgery (29%), and/or radiation therapy (9%). Reduction of immunosuppression alone led to CR (50%), PR (8%), SD (4%), and PD (38%). Of the 68 patients, 21 (31%) ultimately died a PTLD related death.

Interestingly, PTLD patients under age 40 were more likely to have allograft involvement (p=0.007) and a polymorphic histology (p=0.007). Furthermore, allograft involvement tended to occur sooner after transplant (p=0.0001) and those with allograft involvement were more likely to present with allograft failure (p= 0.0001). Compared to monomorphic PTLD, polymorphic PTLD was more likely to be EBV positive (82% vs 51%, p=0.03) and have a lower chance of PTLD-related death (15% vs 40%, p=0.03). Finally, patients with allograft PTLD were more likely to have a polymorphic histology (80%, p<0.001). In summary, PTLD occurs in about 2% of kidney recipients and is typically EBV-associated. EBV positive PTLD is more common in younger recipients, frequently presenting with polymorphic histology in the allograft and appears to have a better prognosis.

Disclosures:

No relevant conflicts of interest to declare.
SF1126, a Pan-PI3K Inhibitor Has Superior Preclinical Activity to CAL-101 a PI3K Delta-Specific Inhibitor in Aggressive B-Cell Non-Hodgkin's Lymphoma
Authors: Mahadevan Daruka , Qi Wenqing , Stejskal Amy , Cooke Laurence , Garlich Joseph R
Published: October 2019

Abstract 2720

The PI3K pathway is constitutively active in B-cell non-Hodgkin lymphomas (B-NHL). PI3K pathway targeted therapies have focused on inhibiting mTORC1 (rapalogs) with a ∼20–48% response rate due to inactivation of mTORC1 resulting in G1 cell-cycle arrest or apoptosis. A mechanism of resistance to rapalogs is that mTORC2 is unaffected resulting in undesirable Akt activation. Strategies to block Akt up-regulation require novel agents that simultaneously block PI3K, mTORC1 and mTORC2. SF1126 is a novel pan-PI3K/mTORC1/mTORC2 inhibitor conjugated to an integrin targeted peptide RGD with potent anti-tumor activity in multiple solid tumor types. Here, we demonstrated SF1126 had potent anti-B-NHL activity and is superior to CAL-101 a PI3K delta-isoform specific inhibitor in a panel of aggressive B-NHL cell lines. Cells treated with SF1126 exhibited >90% decrease in pAkt and pGSK-3β confirming the mechanism of action of a pan-PI3K inhibitor. Moreover, SF1126 induced apoptosis in a dose and time dependent manner confirmed by flow cytometry, PARP cleavage and with an IC50 < 4μM. In contrast, CAL-101 was less active compared to SF1126 in inducing apoptosis (12% versus 25% in SUDHL-4 and 15% versus 23% in TMD-8) and cell proliferation (5.62μM versus 3.28μM SUDHL-4 and 5.31μM versus 1.47μM in TMD-8). SF1126 induced G1 cell cycle arrest at 2μM which contributes to suppression of cell proliferation. The cell cycle protein cyclin D1 is downstream of mTORC1, and the over-expression of cyclin D1 is a hallmark of mantle cell NHL (MCL). Consistent with this, cyclin D1 was significantly decreased by SF1126 compared to CAL-101. Lastly, the addition of Rituximab to SF1126 or CAL101 increased the apoptosis over single agent therapy in B-NHL cell lines. In conclusion, we demonstrate that SF1126 potently inhibits the constitutively activated PI3K/mTORC/Akt pathway in aggressive B-cell NHL cell lines with consequent suppressive effects on cell cycle progression, cell proliferation and induction of apoptosis. These findings provide a rationale for SF1126 in combination with rituximab as a novel therapeutic strategy for aggressive B-NHL and warrant early phase clinical trial evaluation [Funded by the Lymphoma SPORE 1 P50 CA 130805 01A1].

Disclosures:

Garlich: Semafore Pharmaceuticals: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
Slit2/Robo4 Signaling Modulates HIV-1 Gp120-Induced Lymphatic Hyperpermeability,
Authors: Zhang Xuefeng , Yu Jinlong , Kuzontkoski Paula M. , Li Dean Y. , Groopman Jerome E.
Published: October 2019

Abstract 3225

Dissemination of HIV in the host involves transit of the virus and virus-infected cells across the lymphatic endothelium. HIV may alter lymphatic endothelial permeability to foster dissemination, but the mechanism is largely unexplored. Using a primary human lymphatic endothelial cell model, we found that HIV-1 envelope protein gp120 induced lymphatic hyperpermeability by disturbing the normal function of Robo4, a novel regulator of endothelial permeability. HIV-1 gp120 induced fibronectin expression and integrin α5β1 phosphorylation, which led to the complexing of these three proteins, and their subsequent interaction with Robo4 through its fibronectin type III repeats. Moreover, pretreatment with an active N-terminus fragment of Slit2, a Robo4 agonist, protected lymphatic endothelial cells from HIV-1 gp120-induced hyperpermeability by inhibiting c-Src kinase activation. Our results indicate that targeting Slit2/Robo4 signaling may protect the integrity of the lymphatic barrier and limit the dissemination of HIV in the host.

Disclosures:

No relevant conflicts of interest to declare.
Integrin Alpha E (CD103) Beta 7 Expression Marks A Subset of Human Bone Marrow-Derived CD207+ Dendritic Cells Which Induce T Regulatory Cells Via Indoleamine 2,3-Dioxygenase,
Authors: Curti Antonio , Ocadlikova Darina , Trabanelli Sara , Evangelisti Cecilia , Ciciarello Marilena , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

Abstract 3233

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in tryptophan catabolism along the kynurenine pathway. IDO expressed by different cell subsets inhibits T-cell activation, proliferation and survival and induces regulatory T cells (Tregs), thus mediating immunological tolerance. Although human monocyte-derived dendritic cells (DCs) have been shown to express IDO, little is known about its expression in other subsets of human DCs, including those generated from CD34+ hematopoietic progenitors (CD34+-derived DCs). In particular, no data are currently available for IDO expression in CD34+-derived DC subsets. CD34+-derived DCs were generated from healthy donors from purified CD34+ cells after 7 days of culture with GM-CSF and TNF-a. Then, DCs were separated into CD1a−CD14+ and CD1a+CD14− cells. DCs subsets were analyzed for IDO expression by real-time PCR and western immunoblot, kynurenine production, inhibition of allogeneic proliferation and Tregs induction. CD34+ cells did not express IDO mRNA expression regardless of the progenitor cell sources (cord blood, mobilized peripheral blood, bone marrow). During DC differentiation, IDO expression and function, evaluated by enzymatic and immunological tests, was markedly induced at day 7. Interestingly, the expression of IDO was shown to be 10 times higher in the CD1a+ compartment as compared to CD1a- cell fraction. IDO expression resulted in increased production of kynurenine and in reduced allostimulatory capacity of T-cell proliferation. Moreover, CD1a+ cells were shown to induce a population of CD4+CD25+Foxp3+ which acted as Tregs by inhibiting allogeneic T cell proliferation. This effect was abrogated by the addition of the IDO inhibitor 1-methyl tryptophan. Phenotypically, IDO-expressing CD1a+ cells expressed typical marker of Langerhans cells such as CD207, CD11b, CD1c, as well as CD103, which has been recently identified as a marker for tolerogenic DCs. Importantly, IDO expression was mainly detected in the CD103+ CD207+ fraction, which induces Tregs through an IDO-dependent manner. In conclusion, DC differentiation of CD34+ cells results in the expression of a functionally active IDO protein in CD103-expressing DCs. Given the role of IDO in regulating immune tolerance, a subset of bone marrow-derived DCs, expressing CD103, may be intrinsically committed to function as regulatory DCs. These data point toward IDO expression as part of a tolerogenic signature during DC development.

Disclosures:

No relevant conflicts of interest to declare.
Desialylated Platelets Are Cleared From the Circulation by the Hepatic Asialoglycoprotein Receptor,
Authors: Grozovsky Renata , Giannini Silvia , Hoffmeister Karin M.
Published: October 2019

Abstract 3257

The regulatory mechanisms of platelet homeostasis remain elusive. We investigated here the role of hepatic asialoglycoprotein receptor (a.k.a. Ashwell-Morell receptor) in platelet clearance. Mice lacking the hepatic asialoglycoprotein receptor Asgpr2 subunit had increased platelet survivals (T1/2 = 49.5±2h) when compared to wild type (WT, T1/2 = 31±4h) mice. Consequently, Asgpr2−/− mice had platelet counts increased by ∼20%, compared to WT, with increased terminal galactose exposure, as demonstrated using the galactose specific lectin RCA1. Bone marrow and spleen megakaryocyte numbers were reduced by ∼15% and ∼20% in Asgpr2−/− mice, compared to WT mice. Sialidase (NA, Clostidium perfringens, 50mU/mice) maximally desialylated circulating platelets when injected intravenously, as evidenced by increased RCA1 binding. Sialidase injection resulted in a ∼60% depletion of circulating platelets after 24h in Asgpr2−/− mice, compared to >90% in WT mice, indicating that desialylated platelets were partially removed by Asgpr1/2. In contrast to platelets, red blood cell counts were unaffected by sialidase treatment. Sialidase injection for 72h resulted in a 2.3-fold and 1.2-fold increase in megakaryocyte numbers in the spleen and bone marrow of WT mice, respectively, but not in Asgpr2−/− mice. In contrast to sialidase treatment, injections of rabbit anti-mouse platelet serum (RAMPS) depleted >95% of circulating platelets and increased by 70% bone marrow, but not spleen MK numbers in both WT and Asgpr2−/− mice. The data shows that removal of desialylated, i.e, senescent, platelets by the hepatic Ashwell-Morell receptor differs to that of antibody-mediated platelet clearance.

Disclosures:

No relevant conflicts of interest to declare.
Clopidogrel, a P2Y12 Receptor Antagonist, Potentiates the Inflammatory Response in a Rat Model of Peptidoglycan Polysaccharide - Induced Arthritis,
Authors: Garcia Analia E , Mada Sripal R , Rico Mario C , De La Cadena Raul A. , Kunapuli Satya P.
Published: October 2019

Abstract 3287

Clopidogrel is a widely used anti-thrombotic drug that prevents platelet activation by irreversibly antagonizing P2Y12 receptor. This drug could be co-administrated in patients afflicted with inflammatory conditions, such as rheumatoid arthritis and lupus. Hence it is of importance to determine if blocking P2Y12 receptors has any therapeutic benefit during the inflammatory response. Therefore, we evaluated the effect of clopidogrel using an animal model of chronic inflammation. Genetically susceptible Lewis rats were injected intraperitoneally with peptidoglycan polysaccharide (PGPS) to develop arthritis and followed for 21 days. Interestingly, we noticed an increased level of inflammation in induced arthritis animals treated with clopidogrel compared with induced arthritis animals alone, including a) increased in joint diameter between day 15 and 21 (p<0.0001) with augmented clinical manifestations of inflammation, b) an elevated neutrophil blood count and an increased circulating platelet count (926 ± 195 × 103 vs. 561.25 ± 97 × 103 mL; p<0.05). Furthermore, microscopic differences were observed at the site of joint inflammation namely: a significant increase in leukocyte infiltration, pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in induced arthritis animals. Plasma levels of pro-inflammatory cytokines, namely IL-1 beta, interferon (IFN) gamma, and IL-6, were significantly increased in the induced arthritis animals treated with clopidogrel when compared to induced arthritis animals alone (p<0.05). In contrast, plasma levels of IL-10 were significantly lower when animals were treated with clopidogrel compared to induced arthritis animals alone (p<0.05). Plasma levels of platelet factor 4 (PF4) were increased in all animals treated with PGPS, however PF4 levels show not difference with clopidogrel treatment. This last finding suggests that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. In summary, according to clinical observations, histopathology measurements and cytokine results, the antagonism of the P2Y12 receptor has a pro-inflammatory effect in this model of erosive arthritis, which might not be mediated only by platelets. These data provide new insights into the role that thienopyridine metabolites play during chronic inflammation distant from their action on platelets and with a detrimental outcome in this model of erosive arthritis

Disclosures:

No relevant conflicts of interest to declare.
Dose-Finding Study: Rational Proceeding in Patients with VWD,
Authors: Halimeh Susan , Rott Hannelore , Nowak-Gottl Ulrike
Published: October 2019

Abstract 3326

In patients with von Willebrand's disease (VWD) replacement therapy with factor VIII/VWF concentrates is increasingly applied as prophylactic regimen. Since 2000, 82 consecutively enrolled patients with clinically relevant bleeding episodes were diagnosed with VWD. In all patients, decision for initiating prophylaxis was based on a bleeding score >2 prior to diagnosis, concomitant with recurrent bleedings associated with anemia in patients with on-demand therapy. We report results on secondary prophylactic VWF replacemment therapy applied 13 children, 7 adolescents and 4 adults (n=24) with VWD type 1 (n=4), 2 (n=12) and the severe type 3 (n=8).

Based on individual 24h VWF:RCo recoveries [aim: 0.01 to 0.02 IU/ml > baseline] median dose was 40 [min. 20- max. 47] IU/kg, 15 patients were give substitution therapy semiweekly, 7 patients thrice weekly and 2 children four times per week. Within a 12-months-period hemoglobin conentrations returned to normal values. Median duration of prophylaxis was 3 years. Recurrent bleeding episodes stopped in 23/24 patients. One female type 3 patient additionally needed locally applied tranexamic acid [tooth bruising]. Following a 12-months observation period the monthly bleeding frequency and the bleeding score was significantly reduced [3 vs. 0.07; 3 vs. 0: p< 0.001], compared to the pre-prophylaxis/ pre-diagnostic values.

Conclusion:

The use of secondary prophylactic VWF replacement therapy with FVIII/VWF concentrate is an effective tolerated treatment modality, highly benefical for patients with VWD, who present recurrent bleeding events during an on-demand therapy.

Disclosures:

No relevant conflicts of interest to declare.