bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrinogen, Coagulation Times and Some Inflammation Markers In Patients with Different Stages of Sepsis
Authors: Mariscal Ignacio , Joanne Lim-Baga Grace
Published: October 2019

Abstract 5257

Fibrinogen, Coagulation Times and Some Inflammation Markers In Patients with Different Stages of Sepsis. Mariscal II MD, Lim-BagaGJ MD. Hospital Gómez Farias ISSSTE. Zapopan, México.

Inflammation and coagulation pathways, among others, are activated during sepsis (Sp). We tested a priori hypothesis that coagulation changes are related to clinical stages of Sp.

DESIGN.

Prospective non interventional study on patients with Sp, from November 2009–2010

SETTING.

Medicine hospitalization areas in a single third level hospital in western México

PATIENTS.

One hundred and eighty eight patients with Sp who were admitted to Medicine wards received standard critical care treatment.

INTERVENTION.

Patients were stratified into Sp, severe Sp and septic shock. Mortality was assessed at discharge and clotting test and inflammation markers were measured at admission and at discharge

MEASUREMENTS AND MAIN RESULTS.

Sp incidence was 9.6%. There were 114 patients with Sp, 57 severe Sp and 17 with septic shock; the mortality for the group was 23%; 6%, 34%, and 94% in Sp, severe Sp, and septic shock respectively. Initial fibrinogen (Fg) showed a trend to be higher as clinical stages of Sp advanced (346, 361,393 mg PNS), and final Fg decreased in Sp** and severe Sp* and shock (PNS). PT** and PTT** at discharge, were prolonged. Initial and final bands* were higher as the stages of Sp increased and decreased at discharge, except in those with septic shock. Comparing results by survivorship it was observed that initially and at discharge, non survivors had greater abnormalities of PT**, PTT**, and bands** than survivors. Fg**was lower in those who died

CONCLUSIONS.

The trend of the Initial Fg levels to increase as severity of Sp, its decrease at discharge, the prolongation of PT and PTT and the descend of platelets, suggest that the activation of the coagulation cascade increases as Sp progresses and, that this could be related to the severity of the inflammatory response and the vascular damage. The coagulopathy is suggestive of DIC andthat this participates in the prognosis. It would be important to set the clinical role of coagulation in the stages of Sp. A mayor numbers of patients must be studied and markers of DIC investigated.

Disclosures:

No relevant conflicts of interest to declare.
A Retrospective Review of the Management of Patients with Porphyria Cutanea Tarda At a Tertiary Care Centre
Authors: Yudin Jovana , Bacchus Farzana R. , Crowther Mark A.
Published: October 2019

Abstract 5290

Porphyria Cutanea Tarda (PCT) is caused by a deficiency in uroporphyrinogen decarboxylase, an enzyme in the heme biosynthesic pathway. This leads to an excess of porphyrins, which cause blistering and skin fragility on sun exposure. The goals of treatment are iron depletion and reduction of plasma porphyrins to ameliorate skin symptoms.

We reviewed the management of all PCT patients at St. Joseph's Hospital followed by the Hematology service, with the objective of describing their treatment course and its efficacy. The type of treatment received as well as the number and volume of phlebotomies were recorded. Outcomes of interest were skin symptom resolution (clinical remission) and biochemical remission, as measured by serum ferritin. Target ferritin was set at 30–50 μg/L.

A total of 18 patients with PCT met our inclusion criteria; 14 males and 4 females. Three patients had the familial subtype of PCT while 15 had the sporadic form. Precipitants included alcohol, hepatitis C, hemochromatosis and estrogens. All patients received phlebotomy and three received hydroxychloroquine. Most patients were referred and initially diagnosed by dermatologists. The total volume of phlebotomies per patient ranged from 750–9500 mL for resolution of cutaneous symptoms which was achieved in 75% of patients. At the end of the study period, 15/18 (83%) patients were in clinical remission and 12/18 (67%) were in biochemical remission.

Follow-up of PCT patients by a hospital hematology service allows for symptomatic control and biochemical remission in the vast majority of patients through the use of phlebotomy and occasionally hydroxychloroquine.

Disclosures:

No relevant conflicts of interest to declare.
Use of Biosimilar G-CSF Is Effective After Autologous Bone Marrow Transplantation
Authors: Kotwica Katarzyna , Cioch Maria B. , Wach Malgorzata , Manko Joanna , Jawniak Dariusz , Walter-Croneck Adam , Legiec Wojciech , Dmoszynska Anna
Published: October 2019

Abstract 5321

OBJECTIVES:

Granulocyte colony-stimulating factors (G-CSFs) are widely used to accelerate haematopoietic recovery after bone marrow transplantation (BMT). Regulatory approval of biosimilar G-CSFs in Europe has been on the basis of comparable efficacy, safety and quality as the originator product. However, data are not presently available for all G-CSF clinical settings. This is the first reported use of a biosimilar G-CSF for neutrophil recovery after BMT.

METHODS:

A total of 23 consecutive patients (12 male, 11 female; mean ± SD age 47 ± 13 years) with haematological malignancy (multiple myeloma, n=12; Hodgkin’s lymphoma, n=6; non-Hodgkin’s lymphoma, n=4; acute myeloid leukaemia, n=1) were recruited at a single-centre. Nineteen patients were receiving their first BMT while it was the second autograft for 4 patients. Mobilisation chemotherapy consisted of high-dose (HD) VEP (n=14), ESHAP (n=4), HD Ara-C (n=3), HD cyclophosphamide (n=2) or ICE (n=1). Patients received biosimilar G-CSF (EP-2006, Sandoz Biopharmaceuticals) after myeloablative chemotherapy (primarily BEAM or melphalan 140/200 mg/m2 +/− bortozemib) with or without radiotherapy followed by autologous BMT. G-CSF therapy was started when absolute neutrophil count (ANC) was <0.5 × 109/l and was continued until ANC reached >1.5 × 109/l for 3 consecutive days. Response was evaluated after BMT using International Uniform Response Criteria.

RESULTS:

Mean ± SD number of CD34+ cells collected before transplantation was 10.1 ± 4.0 x106/kg/body weight. After BMT, one patient had a stringent complete response (CR), 7 patients had a CR, one patient a near CR, one patient a very good partial response (PR), 12 patients a PR, and one patient had progressive disease (overall response 96%). Mean recovery to ANC >0.5 × 109/l was 13.0 ± 4.0 days. Mean duration until platelet recovery >20 000/ml was 16.1 ± 4.4 days (not achieved in 5 patients at last available assessment). Mean duration of treatment with biosimilar G-CSF was 14.4 ± 5.1 days (range 6−23). Patients required antibiotics on a median of 4 days (range 1−6). Five patients (22%) experienced neutropenic events (neutropenic fever, n=4 and neutropenic enterocolitis and sepsis, n=1). Mean number of days in hospital was 28 ± 6.

CONCLUSION:

Biosimilar G-CSF appears to be effective in reducing the duration of neutropenia in patients undergoing myeloablative therapy followed by autologous BMT. The use of biosimilar G-CSFs may result in cost savings in cancer supportive care budgets.

Disclosures:

Dmoszynska: Mundipharma: Advisory Board; Roche: Honoraria.
Von Willebrand Factor Binds to the Endothelial Growth Factor Angiopoietin-2 Both within Endothelial Cells and Upon Release From Weibel Palade Bodies
Authors: Mckinnon Thomas A J , Starke Richard D , Ediriwickrema Kushani , Randi Anna Maria , Laffan Michael
Published: October 2019

Abstract 698

Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for homeostasis, also involved in inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and is either constitutively secreted or stored in Weibel-Palade bodies (WPB), ready to be released in response to endothelial stimulation. Several studies have shown that formation of WPB is dependent on the presence of VWF, and deletion of VWF in human umbilical vein EC (HUVEC) results in loss of WPB. Amongst the other proteins shown to co-localise to WPB is angiopoietin-2 (Ang2), a ligand of the receptor tyrosine kinase Tie-2. Ang2 regulates endothelial cell survival, vascular stability and maturation, by destabilizing quiescent endothelium and facilitating the response to inflammatory and angiogenic stimuli. VWF is required for storage of Ang2, and release of Ang-2 from EC is increased in VWF-deficient HUVEC. Recently, we have shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by Ang-2. In the present study we investigated the interaction between Ang2 and VWF. Binding analysis demonstrated that recombinant human Ang2 bound to purified plasma-derived VWF in a pH and calcium dependent manner, with optimal binding occurring at pH 6.5 and 10mM calcium, indicative of binding within the Golgi body. Generation of binding isotherms established that Ang2 bound to VWF with high affinity (KD∼3nM); furthermore binding affinity was not dependent on VWF conformation. Using an array of VWF constructs we determined that Ang2 bound predominantly to the VWF A1 domain, which also contains binding sites to the platelet receptor GPIb and extracellular matrix proteins. Co-immunoprecipitation experiments performed on TNFα- and ionomycin-stimulated HUVECs, to induce WPB exocytosis, confirmed that a portion of Ang2 remained bound to secreted VWF. Moreover, immunofluorescence staining of histamine-stimulated HUVECs to induce VWF release demonstrated the presence of Ang2 on VWF strings secreted from ECs. Finally we demonstrated that Ang2 bound to VWF was still able to interact with Tie-2. These data demonstrate that binding of Ang2 to VWF occurs within the cell; we propose that this is the mechanism mediating storage of Ang2 in WPB. Moreover, the finding that the Ang2-VWF interaction is preserved following secretion raises the intriguing possibility VWF may affect Ang2 function, possibly by localising Ang2 to the Tie 2 receptor under the shear forces experienced in flowing blood. Similarly, Ang-2 binding to VWF may modulate its interaction with receptors and extracellular matrix proteins, and ultimately influence the role of VWF in the angiogenic processes.

Disclosures:

No relevant conflicts of interest to declare.
Activation of the Canonical Notch Signaling Pathway in the Hematopoietic Microenvironment Is Required to Maintain Normal Hematopoiesis
Authors: Wang Lin , Rodriguez-Rodriguez Sonia , Zhang Huajia , Mumaw Christen , Cardoso Angelo A , Carlesso Nadia
Published: October 2019

Abstract 723

Recent studies demonstrated that the causes of Myeloproliferative syndromes (MPS) is not limited to factors intrinsic to hematopoietic cells, but include also non-cell autonomous mechanisms. Here, we demonstrate that loss of CSL-dependent Notch signaling in the hematopoietic microenvironment leads to a lethal myeloproliferative disorder. Following pIpC induction, Mx1-Cre+/RBP-Jlox/lox mice showed a significantly increase in granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, spleen and liver and died by week 16–20. Survival at 5 months was 12% for the RBPJ KO mice compared to 100% for the control mice. Transplant studies revealed that the myeloproliferative disorder occurred rapidly when WT-hematopoietic cells were transplanted into MX1-Cre+/RBP-Jlox/lox-recipients, whereas WT-recipients of MX1-Cre+/RBP-Jlox/lox hematopoietic cells did not show onset of diseases at 6 months of follow-up. Furthermore, the disease was not transplantable in WT-recipients, indicating that abnormal Notch signaling in the microenvironment is critical for the development and sustainment of MPS. Transplants conducted with Lys/GFP HSC as WT donor cells, showed that transplant of myeloid progenitors (LK) are sufficient for the development of disease, as documented by rapid expansion of LK-Lys/GFP cells into MX1-Cre+/RBP-Jlox/lox recipients by intravital microscopy of the BM niche. Levels of hematopoietic cytokines were not significantly different in MX1-Cre+/RBP-Jlox/lox recipients prior to transplantation. Histological analysis of BM and spleen showed increased microvascular density and increased CD31+ EC by IHC. To dissect the mechanisms, we analyzed different cellular components of the microenvironment. Mx1Cre driven deletion of RBP-J occurred in osteoblasts, endothelial progenitors and mesenchymal stem cells. Co-culture experiments of HSC with these different cell types suggested cooperative effects in altering proliferation and differentiation of myeloid cells. Crosses of RBP-Jlox/lox mice with mice bearing cell type promoter specific Cre are ongoing to determine the cellular type(s) critical for the myeloid expansion and to identify downstream molecular mediators. These studies will provide further insights on the role of the microenvironment in contributing to myeloid proliferative diseases and leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Distinct Metabolic Dependency of Normal and Leukemic Cells in a Mouse Model
Authors: Yusuf Rushdia Z. , Acharya Sanket S. , Yu Vionnie , Saez Borja , Duvet Mildred , Scadden Elizabeth , Warraich Gohar , Clish Clary , Churchill Michael , Doench John , Logan David J. , Cook Colleen , Das Sudeshna , Brown Adam , Sykes Stephen M. , Chattopadhyay Shrikanta , Carpenter Anne E. , Heiden Mathew Vander , Armstrong Scott A. , Mukherjee Siddhartha , Ebert Benjamin L. , Scadden David T.
Published: October 2019

Abstract 759

We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice.

Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns.

L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified.

Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically.

Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally.

Disclosures:

No relevant conflicts of interest to declare.
The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) Is Essential for Survival and Proliferation of AML1-ETO Associated AML Cells
Authors: Shen Shuhong , Zheng Yi , Mulloy James C.
Published: October 2019

Abstract 923

AML1-ETO (AE), the product of translocation 8;21, is the most frequently observed fusion gene in acute myeloid leukemia (AML). Although AE is not sufficient to induce leukemia by itself, it endows a significant survival and growth advantage to hematopoietic progenitors. In a search of various AML microarray databases, we found that the Leukemia-Associated Rho Guanine nucleotide exchange factor (LARG) gene expression is consistently upregulated in samples from AE-positive AML patients compared to other types of AML. This observation was confirmed in an independent cohort of AML samples by qPCR and Western Blot analysis, and in a pre-leukemia cell model generated by AE expression in CD34+ human cord blood (CB) cells compared with CB cells transduced with the MLL-AF9 (MA9) fusion gene or control retroviral vector. ChIP-PCR assays further indicate that AE directly binds to the LARG regulatory region through cis-elements containing AML1 (Runx1) binding sites. LARG, a RhoA GEF, can activate the RhoA pathway which is important for interaction between cells and their environment. We found that the AE cell lines were more adhesive to fibronectin than the MA9 cell lines. When we used lentiviral vectors expressing shRNA to suppress LARG expression in AE+ pre-leukemic cells, the cells showed a significant decrease in adhesion to fibronectin. In addition, knockdown of LARG also significantly interfered with the growth of AE cells in that the shRNA transduced cells displayed a competitive disadvantage relative to non-transduced or control-transduced cells in a proliferation assay. Cell cycle analysis revealed that LARG knockdown induced cell cycle exit, while Annexin V staining showed that LARG suppression promoted apoptosis of AE cells. Because LARG is a well-known guanine nucleotide exchange factor (GEF) for RhoA GTPase, we examined the downstream signaling events of the LARG/Rho axis, including phosphorylation of MLC and AURORA, targets of Rho-associated protein kinase (ROCK). pMLC and pAURORA were significantly down-regulated upon knockdown of LARG. Phosphorylation of Stat3, another protein downstream of RhoA signaling important for the proliferation and survival of myeloid cells, was also decreased upon LARG knockdown. These findings suggest that LARG is a transcriptional target of the AML1-ETO fusion protein and the LARG-RhoA signaling pathway plays an essential role in the proliferation and survival of AE cells. The LARG/RhoA pathway may serve as a new therapeutic target in t(8;21) AML.

Disclosures:

No relevant conflicts of interest to declare.
Differential Prognostic Effect of IDH1 Versus IDH2 Mutations in Myelodysplastic Syndromes: A Mayo Clinic Study of 277 Patients
Authors: Patnaik Mrinal M. , Hanson Curtis A. , Hodnefield Janice M , Lasho Terra L , Finke Christy , Knudson Ryan A , Ketterling Rhett P , Pardanani Animesh , Tefferi Ayalew
Published: October 2019

Abstract 971

Unlike the case in acute myeloid leukemia, there is limited information on the prognostic impact of IDH mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (six R132S and two R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts (RARS), 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1, and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (p=0.0004; HR 4.0, 95% CI 1.9–8.8), revised International Prognostic Scoring System (IPSS-R) risk category (p<0.0001), and red cell transfusion need (p=0.002) as independent predictors of inferior survival (Figure 1). In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (p=0.001; HR 7.0, 95% CI 2.3–20.8). The presence of IDH2 R140Q did not affect overall (p=0.54) or leukemia-free (p=0.81) survival (Figure 2). The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS.

Disclosures:

No relevant conflicts of interest to declare.
Inhibition of RARα2 or Its Downstream Signaling Pathways Decreases Drug Resistance in Myeloma
Authors: Tricot Guido J. , Yang Ye , Xiao Fang , Zangari Maurizio , Xu Hongwei , Zhan Fenghuang
Published: October 2019

Abstract 989

Background:

We have previously reported that the 30% of newly diagnosed myeloma (MM) patients expressing RARα2, had a significantly inferior outcome. RARα2 expression was also significantly increased in rapidly relapsing myelomas compared to paired baseline samples, indicating the existence at diagnosis of a RARα2 subclone, which is drug-resistant. We further demonstrated that RARα2 expression was significantly higher in MM cell line-derived and primary MM stem cells (MMSC) than in CD138+ bulk MM cells. In this study, we further explore the role of RARα2 in myeloma drug resistance.

Materials and methods:

RARα2 related drug resistance was evaluated by clonogenic formation assays, using 20,000 MM cells from the RARα2 high-expressing ARK and KMS11 MM cell lines, treated with all-trans retinoic acid (ATRA) (1nM, 10nM), Wnt inhibitor CAY10404 (1 nM, 10nM), Hedgehog inhibitor cyclopamine (1nM, 10nM), bortezomib (1nM, 10nM), as well as doxorubicin (50nM, 100nM), etoposide (50nM, 100nM), and verapamil (50nM). To determine whether inhibition of RARa2 decreased drug resistance, 1.0 × 106 KMS11 cells, made resistant to bortezomib, were transfected with RARα2 shRNA and injected subcutaneously into 20 NOD/SCID mice. The 5TGM1 myeloma mice were used to determine whether targeting RARa2 or its signaling pathways could eliminate MMSC.

Results:

After serial replating for 6 weeks, MMSCs (CD138- fraction) exhibited greater clonogenic expansion than the control CD138+ fraction, while ATRA, an inhibitor of RARα2, induced potent clonogenic inhibition on MMSC. We also showed in vitro that over-expression of RARα2 in low-expressing MM cell lines, ARP1 and OCI-MY5 resulted in increased clonogenic potential and drug-resistance. In a xenograft myeloma mouse model, knockdown of RARα2 in the KSM11bortezomib-resistant cells decreased resistance to bortezomib. We further identified that RARα2 induced drug resistance by activating the drug efflux pump gene ABCC3 through Wnt and Hedgehog signaling. Inhibition of Wnt (CAY10404) signaling or the ABC transporter by verapamil overcame the drug-resistance in ARP1 and OCI-MY5 cells caused by RARα2 over-expression. Finally, targeting RARa2 or its pathways using ATRA, CAY10404 and cyclopamine significantly reduced the tumor burden as determined by idiotype IgG2 protein levels and increased survival compared to untreated controls (P < 0.05) in the 5TGM1 mice after injection of 5TGM1 MMSC.

Conclusion:

Over-expression of RARa2 induces drug resistance by activating the drug efflux pump gene ABCC3 through activation of the Wnt and Hedgehog pathways, while inhibition of RARα2 decreases drug resistance. We also provide a possible strategy to eliminate MMSC by targeting RARa2 and/or its downstream targets, such as the Wnt and Hedgehog pathways.

Disclosures:

No relevant conflicts of interest to declare.
Notch Signaling in Graft-Versus-Host Disease
Authors: Maillard Ivan
Published: October 2019

Abstract SCI-14

Notch is a highly conserved signaling pathway with multiple functions in health and disease. In the hematopoietic system, Notch was first described for its essential role at early stages of T cell development in the thymus and for its involvement in T cell acute lymphoblastic leukemia. In addition, Notch is being increasingly recognized as a potent regulator of antigen-driven mature T cell responses with context-dependent effects. These effects are influenced by the regulated expression of Jagged and Delta-like Notch ligands in antigen-presenting cells under the control of Toll-like receptors and other innate signals. We are investigating the importance of the Notch pathway in T cell alloimmunity in the setting of allogeneic bone marrow or peripheral blood stem cell transplantation (allo-BMT). After allo-BMT, alloimmune T cell responses mediate beneficial graft-versus-tumor (GVT) activity as well as detrimental graft-versus-host disease (GVHD). Using the pan-Notch inhibitor DNMAML and other genetic models of Notch inhibition, we have discovered an essential function for Notch signaling in donor-derived alloreactive T cells. In multiple mouse models of allo-BMT, infusion of Notch-deficient T cells as compared to wild-type T cells led to dramatically reduced GVHD severity and lethality. This effect was apparent for both CD4+ and CD8+ T cells and did not involve global immunosuppression, since Notch-deficient T cells proliferated normally and expanded in host lymphohematopoietic organs. However, Notch-deficient donor T cells failed to accumulate in the gut, a key GVHD target organ, and produced markedly decreased amounts of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. In parallel, Notch inhibition was associated with increased accumulation of FoxP3+CD4+ T cells. Decreased cytokine production could not be explained by a classical T helper differentiation defect. In contrast to their defective induction of GVHD, Notch-deficient T cells remained capable of mediating cytotoxic and anti-tumor responses both in vitro and in vivo, leading to preserved GVT activity even against large numbers of tumor cells. We are currently investigating the molecular and cellular mechanisms of Notch action in alloreactive T cells. We are also exploring the role of individual Notch receptors and ligands at the alloimmune synapse in vivo. Altogether, our findings identify Notch inhibition in donor T cells as a novel strategy to induce beneficial immunomodulation rather than global immunosuppression after allo-BMT.

Disclosures:

No relevant conflicts of interest to declare.
Modulation of HIF-2 Function with Small Molecules: Did Nature Beat Us to It?
Authors: Bruick Richard K. , Gardner Kevin H , MacMillan John , Tambar Uttam
Published: October 2019

Abstract SCI-39

Hypoxia inducible factors (HIFs) govern a mammalian cellular hypoxic response, regulating the transcription of hundreds of genes in response to low oxygen levels. Though this pathway performs critical functions under physiological conditions, increased levels of HIFs are also highly correlated with several disease states. As such, the development of artificial compounds that directly and selectively regulate HIF function is of interest. We have used a combination of biophysical, biochemical, and chemical approaches to study the structure and function of the Per-ARNT-Sim (PAS) domains of human HIFs, which serve as critical protein/protein interaction modules in assembling the functional HIF heterodimer. These studies have revealed the presence of a putative ligand-binding site within one of the HIF-2α PAS domains. A series of biased and unbiased approaches were undertaken to identify compounds that bind at this site and trigger conformational changes within the domain, leading to dissociation of the HIF-2 heterodimer in vitro and in living cells. Consequently, the expression of many hypoxia-inducible genes can be coordinately downregulated by a single agent. Despite its high amino acid identity, HIF-1α lacks a comparable ligand-binding site, indicating that these agents will be useful in deciphering differences in the biological roles of the HIF-1 and HIF-2 isoforms. The presence of a preformed cavity within HIF-2α is particularly as exciting as it suggests the potential for direct physiological regulation of HIF-2 by endogenous metabolite(s). Together, these studies offer further insight into HIF function and regulation while providing chemical tools that advance therapeutic applications.

Disclosures:

No relevant conflicts of interest to declare.
The Yin and Yang of Interleukin-21 in Allergy, Autoimmunity and Cancer
Authors: Leonard Warren J.
Published: October 2019

Abstract SCI-6

Interleukin-21 (IL-21) is a four-α-helical bundle type 1 cytokine. Its receptor consists of IL-21R and the common cytokine receptor γ chain, γc, which is shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is encoded by the gene that is mutated in humans with X-linked severe combined immunodeficiency (XSCID). Each of these cytokines activates JAK1, which associates with the more distinctive receptor component, and JAK3, which associates with γc. IL-21 signals in part via the activation of STAT proteins, with STAT3 being induced more potently than STAT1 or STAT5 proteins. Interestingly, we have demonstrated that STAT3 globally cooperates with IRF4 in the induction of IL-21-regulated genes in T cells. IL-21 is produced by populations of CD4+ T cells, including Th17 cells, T follicular helper cells, and NKT cells. It is a pleiotropic cytokine that exerts a broad and complex array of biological actions on multiple lineages. IL-21 is pro-apoptotic for incompletely activated B cells, thereby serving a role for B cells similar to that of IL-2 related to activation-induced cell death in T cells. However, IL-21 also promotes the expansion and differentiation of activated B cells to plasma cells via its induction of Prdm1, which encodes BLIMP1, and the differentiation of cells into T follicular helper cells via its induction of Bcl6. IL-21 also promotes immunoglobulin class switch, and defective signaling by IL-21 and IL-4 explains the defective B cell function and panhypogammaglobulinemia that is observed in humans with XSCID. In addition, IL-21 has potent actions on CD8+ T cells, cooperating with IL-15 and IL-7 in the expansion of these cells. Conversely, IL-21 has inhibitory effects on antigen presentation by dendritic cells. IL-21 is implicated in multiple forms of autoimmunity, with IL21 and IL21R loci being associated with a range of human autoimmune diseases. Moreover, IL-21 is required for the development of systemic lupus erythematosus, type 1 diabetes mellitus, and experimental autoimmune uveitis in mouse model systems. In contrast to its promotion of autoimmune disease, IL-21 has potential anti-tumor actions, with elimination of established solid tumors in mouse models. In addition to these actions, IL-21 has also been demonstrated to be a potent inducer of IL-10 and thereby to exhibit immunosuppressive effects. In summary, IL-21 has a range of biological actions on multiple lineages, with varied effected related to apoptosis and differentiation, as well as both promoting autoimmunity yet limiting tumor growth. Regulation the actions of IL-21 in vivo therefore has potential clinical utility for a range of diseases.

Disclosures:

Leonard: NIH: I am an inventor on NIH patents and patent applications related to IL-21.
Acute Lymphocytic Leukemia and Down Syndrome
Authors: Izraeli Shai
Published: October 2019

Abstract SCI-8

Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis.

Disclosures:

No relevant conflicts of interest to declare.
EBV-Induced Lymphoproliferation
Authors: Rooney Cliona M.
Published: October 2019

Abstract SCI-9

The similar genome structures, methods of entry, replication, and egress of herpesviruses suggest a common evolutionary origin dating back about 200 million years. EBV, a gammaherpesvirus, has co-evolved with its human host alone for over 80 million years and has developed a relationship that is seldom detrimental to its host and may even be beneficial. For example, EBV-specific T cells with heterologous specificity may protect against other pathogens. Unlike RNA viruses, the sequence of EBV, including its strong immunogenic epitopes, is remarkably conserved even across geographically separated populations. This underscores the comfortable relationship that EBV has developed with the host immune response, which tightly controls but never eliminates the virus. Rarely, this relationship breaks down resulting in diverse diseases and malignancies with underlying causes that are poorly understood. Is mononucleosis restricted to adolescents because of a too active or an inadequate immune response? Why is post-transplant lymphoma (PTLD) not more common in T cell depleted stem cell recipients? How can EBV-positive malignancies develop in patients who have circulating T cells specific for the viral tumor antigens expressed in those malignancies and who control their general EBV infection? Patients with nasopharyngeal carcinoma (NPC) or EBV-positive lymphoma seldom display an elevated virus load in peripheral blood lymphocytes. To prevent EBV-associated diseases we must understand their root causes, which, in turn, require an understanding of the virus’ complex life cycle that comprises involve four types of latency in B cells and replication in both B cells and mucosal epithelium. Each phase of the life cycle is represented both in normal infected cells and in one or more diseases. For example, virus replication is observed in tonsillar epithelium and in the oral hairy leukoplakias of HIV patients. Virus replication is also indirectly associated with NPC; its development is preceded by elevated antibody titers to the viral lytic cycle antigens despite the fact that NPC cells express only latency proteins. There are four patterns of viral latency and three are observed in tumor cells. Type 1 latency (expression of only two of over 90 viral proteins) is observed in normal circulating B cells, Burkitt’s lymphoma and gastric carcinoma. Type 2 latency is shared by the T, B, and NK lymphomas that occur in immunocompetent individuals. Type 3 latency, the most immunogenic form of latency in which nine latency-associated proteins are expressed, is seen in tonsils, in lymphomas of the immunosuppressed and elderly, and in leiomyosarcoma. Therapies may specifically target the function or immunogenicity of the viral proteins, while prophylaxis, usually by vaccination, aims to prevent them. We will discuss the types of latency observed in EBV-associated lymphoproliferations in the context of immunotherapies that might be used to control them.

Disclosures:

No relevant conflicts of interest to declare.
CD59 Deficiency Is Associated with Chronic Hemolysis and Childhood Chronic Immune Mediated Neuropathy
Authors: Mevorach Dror , Elpeleg Orly
Published: October 2019

Abstract 3187

CD59 deficiency is a common finding in red and white blood cells in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria (PNH) where acquired mutation in the PIGA gene leads to membrane loss of GPI-anchored membrane proteins, including CD59. The current study objective was the elucidation of the molecular basis of childhood familial chronic Coombs' negative hemolysis and relapsing polyneuropathy, mimicking chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from four unrelated families. A founder mutation was searched using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55 and CD14 was examined in blood cells by flow cytometry followed by western blot of the CD59 protein. A homozygous missense mutation in CD59, was identified in all the patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jews of North-African origin. The mutated protein was present in the patients' cells in normal amount but was undetectable on the membrane surface.

Conclusion: This is the first clinical and molecular characterization of a novel clinical syndrome. CD59 mutation is associated with a failure of proper localization of CD59 protein in the cell surface. This is clinically manifested in infancy by chronic hemolysis and relapsing peripheral demyelinating disease.

Disclosures:

No relevant conflicts of interest to declare.
Monocyte Chemotactic Protein-1 Is Associated with Microvascular Abnormalities and Serum Ferritin Concentrations in Sickle Cell Disease Patients
Authors: Green Ralph , Miller Joshua W , Samarron Sandra L , Lin Xin , Cheung Anthony T , Wun Ted
Published: October 2019

Abstract 3255

Introduction:

Microvascular abnormalities underlie much of the morbidity observed in sickle cell disease (SCD). Adherence of sickle erythrocytes and leukocytes to the microvascular endothelium leads to vaso-occlusion and end organ ischemia/reperfusion injury. Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the recruitment of monocytes to sites of endothelial activation or injury and thus contributes to the pathophysiology of vascular disease. In SCD patients, circulating MCP-1 concentrations are elevated during both steady state and vaso-occlusive crisis. However, the relationship between MCP-1 and microvascular abnormalities in SCD patients has not been assessed. In the present study, we measured a panel of circulating cytokines including serum MCP-1 and hematological biomarkers including serum ferritin in SCD patients who were being monitored by computer-assisted intravital microscopy (CAIM) to assess severity of microvasculopathy.

Methods:

The study included 31 steady state SCD patients (22 females, 9 males) ranging in age from 4–61 years. MCP-1 concentrations were measured by Luminex multiple analyte profiling. Ferritin concentrations were measured by chemiluminometric immunossay. The conjunctival microvasculature was assessed using CAIM. A severity index on a scale of 0–15 was calculated as described previously (Cheung et al, Am J Hematol 85:899, 2010) to quantify the degree of microvasculopathy observed among the patients.

Results:

The mean ± SD MCP-1 concentration was 445 ± 253 pg/ml (range: 122–1180 pg/ml), mean ± SD ferritin concentration was 1675 ± 1883 ng/ml (range 18–5825 ng/ml), and mean ± SD severity index was 5.0 ± 2.5 (range: 0–9). By multiple regression analysis, with controlling for age and gender, MCP-1 was directly correlated with severity index (p = 0.046), and serum ferritin was directly correlated with MCP-1 (p = 0.019).

Conclusions:

Monocyte recruitment to the site of endothelial injury involves chemotactic signaling mediated in part by MCP-1, levels of which rise in SCD. Our results indicate that MCP-1 is associated with and may directly contribute to the microvasculopathy observed in SCD patients. Furthermore, we find that MCP-1 levels are correlated with ferritin concentrations. Ferritin reflects body iron stores, and an increase of iron stores in SCD resulting from chronic blood transfusion as well as increased iron absorption associated with hemolysis may thus lead to monocyte/macrophage recruitment to the vascular endothelium and contribute to vasculopathy. Measurements of MCP-1 and ferritin may also serve as surrogate biomarkers of microvasculopathy severity and inflammation.

Disclosures:

Green: Emisphere - Consultancy : Consultancy; Teva Pharmaceuticals - expert testimony: Consultancy.
Interleukin-21-Induced Granzyme B-Expressing B Lymphocytes Infiltrate Tumors and Regulate T Cells
Authors: Lindner Stefanie , Dahlke Karen , Sontheimer Kai , Hagn Magdalena , Kaltenmeier Christof , Barth Thomas F.E. , Beyer Thamara , Reister Frank , Fabricius Dorit , Lotfi Ramin , Lunov Oleg , Nienhaus Ulrich , Simmet Thomas , Kreienberg Rolf , Moeller Peter , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: October 2019

Abstract 3278

The role of B cells in tumor infiltrations is controversial. Different studies suggest that certain tumor-infiltrating B cell populations exhibit regulatory potential. Here, we demonstrate that the microenvironment of various solid tumors contains granzyme B (GrB)-expressing B cells adjacent to IL-21-providing T cells. GrB-mediated effector T cell modulation is already known from regulatory T cells (Treg) and plasmacytoid dendritic cells. We now show that IL-21 induces B cells to express high levels of GrB and to modulate T cell proliferation by GrB-dependent degradation of the T cell receptor z-chain. Detailed characterization of IL-21-induced GrB+ B cells reveals a CD19+CD38+CD1d+CD147+ phenotype and expression of additional regulatory molecules including IL-10, IDO and CD25. Of note, GrB induction is accompanied by both BCR- and TLR-mediated signals and GrB expression levels are influenced by B cell expression of CD5. In summary, we demonstrate that IL-21 induces GrB-expressing regulatory B cells, which are detected in tumor infiltrations, and which may contribute to the modulation of cellular adaptive immune responses by Treg-like mechanisms. Our findings may stimulate the development of novel diagnostic and cell therapeutic approaches to the management of malignant, autoimmune and graft-versus-host pathologies.

Disclosures:

No relevant conflicts of interest to declare.
IL-35 Suppresses Lipopolysaccharide-Induced Endothelial Cell Activation Through Downregulation of MAPK Signaling-Mediated Upregulation of Adhesion Molecule VCAM-1
Authors: Sha Xiaojin , Meng Shu , Li Xinyuan , Pastrana Jahaira Lopez , Wang Hong , Yang Xiao-Feng
Published: October 2019

Abstract 3310

Our previous reports showed that survival/apoptosis of CD4+CD25+Foxp3+ regulatory T cells (Tregs) modulates vascular inflammation even though the mode of Tregs inhibition was unknown. Interleukin-35 (IL-35), consisting of two subunits Epstein-Barr virus–induced gene 3 (EBI3) and p35, is a novel anti-inflammatory cytokine, which is a member of the interleukin-12 (IL-12) cytokine family. IL-35 is produced by Tregs. It has been shown that IL-35 suppresses chronic inflammatory diseases such as asthma and inflammatory bowel diseases. However, an important question of whether IL-35 can carry out Tregs suppression and inhibit endothelial cell (EC) activation in acute inflammation remained unknown. Here we found that IL-35 significantly inhibits lung neutrophil infiltration into the surrounding areas of bronchioles and alveolar space when induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild type mice and EBI3-deficient mice. Furthermore, cremaster microvasculature study using intravital microscopy showed IL-35 significantly suppresses leukocyte adhesion to the vascular wall as well, suggesting IL-35 inhibition of endothelial activation. Mechanistically, IL-35 inhibited LPS-induced upregulation of adhesion molecules on human aortic endothelial cells, a marker of endothelial activation, including vascular cell adhesion molecule 1 (VCAM-1). IL-35 acted through new IL-35 dimeric receptors gp130 and IL-12Rβ2, and inhibited VCAM-1 promoter transcription in mitogen-activated protein kinase (MAPK)-mediated pathway. These results provide a novel insight on Tregs and IL-35 inhibition of vascular inflammation.

Disclosures:

No relevant conflicts of interest to declare.
Rituximab Does Not Prevent Relapse in Patients with Thrombotic Thrombocytopenic Purpura
Authors: Jatinder Goyal , Lima Jose L. O. , Adamski Jill , Marques Marisa
Published: October 2019

Abstract 3318

Objective:

In the last decade, rituximab has been added to therapeutic plasma exchange (TPE) to treat patients with thrombotic thrombocytopenic purpura (TTP) who appear resistant to TPE. We sought to determine first if rituximab prevented TTP relapse. In addition, if relapse has occurred, we compared the rates of relapse of patients treated with TPE alone with those treated with a combination of TPE and rituximab.

Methods:

We retrospectively reviewed the medical records of all adult patients treated for TTP between 2003 and 2008 at our institution. TTP was defined as thrombocytopenia, hemolytic anemia and ADAMTS13 activity less than 10% due to an inhibitor. None of the patients had congenital TTP. Patient demographics, laboratory data, treatment characteristics and follow up details were collected from their electronic and apheresis' medical records. Kaplan-Meier curves were drawn for survival and Cox proportional hazards models were applied to look for independent predictors of relapse-free survival (RFS).

Results:

A total of 20 patients underwent TPE only (Group 1) as compared to 18 patients who also received rituximab during admission with TTP (Group 2). Table 1 shows that both groups were balanced at baseline for demographic and laboratory data. However, patients in group 2 had longer duration of hospital stay (p<0.0001), underwent more TPE procedures (p<0.0001) and took longer to achieve remission (p<0.0001). The mean follow up in group 1 was 77.5 (±22.4) months and in group 2 was 68.6 (±28.5) months. At follow-up, 5 patients from group 1 relapsed (25%) as compared with 6 patients from group 2 (35%) (p=0.50). The 1-year, 3-year and 5-year RFS rates were 95%, 85% and 74% for group 1, and 94%, 76% and 71%, respectively, for group 2 (p=0.53 using log rank test). On univariate analysis, only age at the time of treatment (p=0.05) and duration of follow-up after treatment (p=0.03) were predictors of relapse. However, on multivariate analysis, no independent predictors of relapse were identified.

Conclusion:

Rituximab does not prevent or reduce rates of relapse when used with TPE in patients with TTP. Since rituximab was added to patients later in their TPE course due to delayed response to treatment, it may yet have a role in decreasing the number of TPEs needed to achieve a response if it were started earlier during hospitalization for TTP.

Disclosures:

No relevant conflicts of interest to declare.