bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum Ferritin Levels Are Associated with Vascular Damage in Patients with Nonalcoholic Fatty Liver Disease.
Authors: Valenti Luca , Swinkels Dorine W. , Burdick Larry , Dongiovanni Paola , Tjalsma Harold , Fargion Silvia , Fracanzani Anna Ludovica
Published: October 2019

Abstract 5098

Background and Aims

Increased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD.

Methods and Results

Vascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, HFE mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin (p=0.048). Carotid plaques were independently associated with age, ferritin (p=0.0004), glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and serum hepcidin-25 was independently associated with carotid plaques (p=0.05).

Conclusion

Ferritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin levels by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages.

Disclosures

No relevant conflicts of interest to declare.
Peculiar Hepatocellular Carcinoma Clinical Findings in patients with Thalassemia Syndromes.
Authors: Restivo Gaetano , Valenza Franco , D'Amato Fabio , Vitrano Angela , Rigano Paolo , Renda Disma , Cassarà Filippo , Maggio Aurelio
Published: October 2019

Abstract 5108

Background

Hepatocellular carcinoma (HCC) is the most common primary hepatic cancer, and the fifth and eighth most common malignancy worldwide in men and women, respectively. Hepatitis B and C, primary and secondary hemochromatosis and cirrhosis have been identified as major risk factors. Hepatitis C or B virus infection and secondary hemochromatosis are the major cause of morbidity and mortality. In the last years, HCC on cirrhosis is a common reported complication in patients with thalassemia syndromes1,2,3. In non thalassemia patients HCC is a late complication of cirrhosis and prognosis depends on suitability for treatment. In this group, the last is low (40%) considering that cirrhosis severe stage C, according to Child-Pugh score is generally observed (personal communication).

Aim of the study

describing, in a homogeneous cohort of thalassemia patients, the main clinical findings of HCC.

Patients and Methods

All subjects with risk factors for HCC as iron overloading, HCV or HBV chronic infection, histological evidence of cirrhosis were undergone every six months to an ultrasound evaluation associated with alphaphetoprotein determination. Nine new cases, since January 2000 until July 2009, were detected. Table I shows the main clinical findings. Mean value of serum ferritin levels, in the last 2 years before diagnosis, was 1049±721. All patients except one were anti-HCV positive (Table I). Seven of them were HCV-RNA positive (Table I). None of them had previous HBV infection.

Results

Only one patient had severe stage C, according to Child-Pugh score. None of the patients had significant high levels of alphaphetoprotein (AFP) at diagnosis. However, a slightly increase of AFP levels was shown, during the 6 months before diagnosis, in 50% of cases. Three patients had a delayed diagnosis because of atypical imaging for HCC at TC scan or a negative previous histology for HCC after liver biopsy. Five patients had multifocal HCC at the diagnosis (Table I). Four showed an unifocal HCC, developing two of them multifocal HCC during the follow-up (Table I). Four patients died during the follow-up for decompensated cirrhosis. Five patients are alive after treatment. Main used treatments are shown on Table I. The overall mean survival was 28±25 months (range 3-64).

Conclusions

1) almost all talassemia patients with HCC are suitable for treatment ; 2) baseline liver function is the most main factor conditioning survival and suitability for treatment ; 3) periodical serum AFP determnations can reveal a slight increase; 3) iron overloading alone can be related to the development of HCC in a liver with still good functional reserve, giving more opportunity for treatment 4) monitoring for HCC by liver ultrasound evaluation, every six months, in at risk patients with thalassemia syndromes should be mandatory.

Disclosures

No relevant conflicts of interest to declare.
Roles of Thromboxane A2 On High Salt Induced-Fetal Defect.
Authors: Pai Chen-Hsueh , Lin Shu-Wha , Yu I-Shing , Yen Ching-Tzu , Yang Yung-Li , Jou Shiann-Tarng , Lin Dong-Tsamn , Chen Jiann-Shiuh , Lin Shu-Rung
Published: October 2019

Abstract 5129

Introduction

Preeclampsia is a pregnancy-induced disorder. If a pregnant woman suffered from preeclampsia, her worse developed-placenta might result in growth retardation of the fetus, and in the most severe cases, it would cause fetal and maternal death. Several previous studies showed that circulating thromboxane A2 in preeclampsic women was higher than that of normal pregnant women, and higher thromboxane A2 level might induce thrombosis and hypertension in these patients. Thus, thromboxane A2 was thought to be an important metabolite relevant to the development of preeclampsia.

Materials and Methods

In order to clearly clarify the roles of thromboxane A2 on preeclampsia, wild type and thromboxane A2 synthase knockout pregnant mice were fed with drinking water containing high concentration of sodium chloride. The effect of thromboxane A2 on preeclampsia was observed by measuring blood pressure, urinary protein, systemic edema in pregnant mice, and placenta weight, fetal weight, fetal size and fetal sections were also measured and stained.

Results

In the groups of pregnant mice fed with high salt, maternal body weight, fetal size and fetal weight were severely decreased in wild type mice as compared to thromboxane A2 synthase knockout mice. Furthermore, abnormal cell degeneration was observed in fetus of wild type pregnant mice, but less in thromboxane A2 synthase knockout fetus.

Conclusions

These results demonstrate that knockout pregnant mice were more resistant to high salt treatment, implying the roles of thromboxane A2 on the development of maternal and fetal defects during high salt treatment.

Disclosures

No relevant conflicts of interest to declare.
Quality of Life in Adolescents and Adults with Thalassemia: A Report of the Thalassemia Longitudinal Cohort.
Authors: Sobota Amy , Yamashita Robert , Xu Yan , Trachtenberg Felicia , Kleinert Dorothy A. , Giardina Patricia J , Kwiatkowski Janet L , Foote Dru , Thayalasuthan Vivekanandan , Porter John B , Thompson Alexis A. , Schilling Leann , Quinn Charles T. , Neufeld Ellis J
Published: October 2019

Abstract 556

Background:

Advances in the treatment of thalassemia have led to increased life expectancy, making outcomes such as health related quality of life (HRQOL) especially important in assessing interventions. However little has been published about the HRQOL of patients living with thalassemia. We report the baseline data from the Thalassemia Clinical Research Network's (TCRNs) Thalassemia Longitudinal Cohort (TLC) study.

Patients and Methods:

The TCRN is an NIH sponsored network of 16 major thalassemia centers in the US, Canada and London. This study is part of the TCRN's longitudinal cohort (TLC) study. HRQOL was measured by self-report with the Medical Outcomes Study 36-Item Short Form Health Survey version 2 (SF36v2), a generic measure of functional health status and well being with results reported as 8 subscale and two summary measures. It has been validated in numerous populations and diseases, and is considered the gold standard for measuring HRQOL. We report here the results from 245 patients over the age of 14 who completed baseline assessments. Comparisons within the study population and with US norms using t-test and linear regression were performed.

Results:

There were 245 patients [47% male; mean age 29 years (range 14-58)]. When compared to US norms, TLC patients had statistically significant (p<0.05) worse HRQOL on 5 of the 8 SF36v2 subscales (physical functioning, role-physical, general health, social functioning and role-emotional) and on both summary scales (physical component summary and mental component summary). As is also seen in the US population, women had worse HRQOL than men on all subscales except vitality, and on both component summary scores. HRQOL decreased with age in all scales, more so than expected compared to US norms.

Conclusions:

In general, adolescents and adults with thalassemia have worse HRQOL than the US population, despite contemporary therapy. Further investigation is needed to determine predictors of low HRQOL so that these may be better addressed by a multidisciplinary team.

Disclosures:

No relevant conflicts of interest to declare.
Signal Transducer and Activator of Transcription (Stat)-3 Activates the Receptor Tyrosine Kinase-Like Orphan (ROR)-1 Gene in Chronic Lymphocytic Leukemia (CLL).
Authors: Ping Li , Harris David , Liu Zhiming , Keating Michael , Estrov Zeev
Published: October 2019

Abstract 57

ROR1, an embryonic protein involved in organogenesis and Wnt signaling, is expressed in B-cell CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors ψ-interferon activation sequence (GAS)-like elements typically activated by Stat3, we sought to determine whether Stat3 activates ROR1. In MM1 cells interleukin (IL)-6 induced Stat3 phosphorylation and upregulated ROR1 whereas STAT3-siRNA downregulated both Stat3 and ROR1 protein levels, suggesting that Stat3 transcribes ROR1. Therefore, we cloned the human ROR1 promoter, generated a series of truncated promoter constructs and assessed their activity by using the luciferase assay. We found that IL-6 augmented the luciferase activity of ROR1 -195, ROR1 -666, ROR1 -834, and co-transfection with Stat3-siRNA significantly attenuated it, suggesting that IL-6 enhanced ROR1 expression by activating Stat3. Furthermore, we established that a region, located between bp -122 and -134, harbors a GAS-like element and activates the ROR1 promoter upon exposure to IL-6. Binding of Stat3 to that region in IL-6-stimulated MM1 cells was confirmed by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether Stat3 transcribes ROR1 in CLL, we obtained fresh CLL cells and by using the same GAS-like element-containing probe we performed EMSA. CLL cell nuclear protein bound this probe and anti-Stat3 and -phsophoserine Stat3 antibodies induced a super-shift. CLL cell ChIP confirmed that Stat3 binds to the promoter of ROR1 as well as the promoters of the Stat3-regulated genes STAT3, c-Myc and P21, but not that of the control gene RPL30. Finally, using qRT-PCR and western immunoblotting we determined that STAT3-shRNA downregulated ROR1, STAT3 and STAT3-regulated gene mRNA by 4-6 fold, and Stat3 and ROR1 protein levels by 50%. Taken together, these data suggest that constitutively activated Stat3 binds to the ROR1 promoter, activates transcription, and induces production of ROR1 protein in CLL cells.

Disclosures:

No relevant conflicts of interest to declare.
Hemolysis-Associated Elevation in Tricuspid Regurgitation Velocity Predicts Reduction in Six-Minute Walk Distance After Two Years of Follow up in Children and Adolescents with Sickle Cell Disease.
Authors: Gordeuk Victor R. , Luchtman-Jones Lori , Campbell Andrew D. , Rana Sohail R , Nouraie Mehdi , Sable Craig , Dham Niti , Ensing Gregory , Minniti Caterina , Kato Gregory J , Gladwin Mark , Castro Oswaldo L
Published: October 2019

Abstract 574

Background.

Elevated tricuspid regurgitation velocity (TRV) as determined by echocardiography correlates with elevated systolic pulmonary artery pressure and is associated with increased morbidity and mortality in adults with sickle cell disease. The importance of elevated TRV in children and adolescents with sickle cell disease is not known. The Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH) study is an ongoing, longitudinal and observational multicenter study of children with sickle cell disease.

Methods.

Baseline echocardiography and six-minute walk test were performed prospectively in 361 children and adolescents with sickle cell disease at steady state and then repeat studies were performed in 209 after a median of 22 months of follow up (range 10 months to 36 months), also at steady state. A hemolytic component was derived by principal component analysis of baseline values for reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and total bilirubin.

Results.

TRV or six-minute walk test were measured at both baseline and follow-up in 193 patients. Twenty-one of these 193 patients had elevated TRV of 2.60 m/sec or higher at baseline. Elevated baseline TRV was associated with high hemolytic rate in 15 patients, defined as hemolytic component above the median for the population studied, and with lower hemolytic rate in six patients. Elevated baseline TRV with high hemolytic rate predicted elevated TRV at follow up (odds ratio 7.7; 95% confidence interval [CI] 2.5 to 24.2; P <0.001) but elevated baseline TRV with lower hemolytic rate did not (odds ratio 1.6; 95% CI 0.2 to 14.1; P = 0.7). Elevated baseline TRV with high hemolytic rate also predicted a decline in the six-minute walk distance by 10% or more at follow-up (hazard ratio 3.9; 95% CI 1.4 to 10.7; P = 0.009). In contrast, higher cardiac output as measured by the left ventricular end diastolic dimension z-score was associated with reduced risk for a decline in the walk distance (hazard ratio 0.7; 95%CI 0.6 to 0.9; P = 0.006).

Conclusion.

Steady-state TRV elevation in association with a high hemolytic rate occurs on screening in about 8% of children and adolescents with sickle cell disease and is predictive of elevated TRV and reduced six-minute walk distance after approximately two years of follow. Such children may be at risk for adverse clinical consequences of pulmonary hypertension as young adults. Further studies are indicated to identify the molecular mechanisms and to develop appropriate medical management for children and adolescents with this complication.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Phosphodiesterase Type 5 Inhibitor Therapy Reduces Priapic Activity in Transgenic Sickle Cell Mice: Restoring Nitric Oxide/Redox Balance in the Penis.
Authors: Burnett Arthur L. , Musicki Biljana , Champion Hunter C. , Bivalacqua Trinity J.
Published: October 2019

Abstract 576

Introduction:

Sickle cell disease (SCD)-associated priapism is characterized by aberrant nitric oxide (NO) regulatory pathway signaling and reduced endothelial NO bioavailability resulting in phosphodiesterase type 5 (PDE5) dysregulation in the penis. Our objective was to determine the effect of chronic PDE5 inhibitor therapy on in vivo erectile responses as well as on NO signaling and redox actions in the penis in an animal model of SCD in order to identify mechanisms associated with this therapeutic effect. Our animal model comprised transgenic sickle cell (SS) mice, expressing human sickle hemoglobin.

Methods:

Five groups of mice were used: 1) wild type (WT), 2) sickle cell heterozygotes (Hemi), 3) sickle cell homozygotes (Sickle), 4) WT + sildenafil (100 mg/kg per day), and 5) Sickle + sildenafil (100 mg/kg per day). All experimental groups underwent cavernous nerve electrical stimulation (CNS; 0.5 - 6 V) to assess erectile function (intracavernous pressure; ICP), and their responses were compared to WT. The frequency of erectile responses (erections/hr) pre- and post-stimulation was also determined. Analyses of penes were: total endothelial NOS (eNOS) and its phosphorylated/activated form (p-eNOS, Ser-1177), and PDE5 expressions; eNOS uncoupling (ratio of active eNOS dimer vs. inactive monomer); reactive oxygen species (ROS) generation; cGMP, NOS and PDE5 activities; and electron microscopy.

Results:

Erectile responses to CNS were significantly enhanced (P<0.05) in the Sickle when compared to Hemi and WT at all voltage settings. Sickle mice treated with sildenafil had a reduction (P<0.05) in spontaneous erections and reduced erectile responses to CNS, while sildenafil therapy had no effect on erectile responses in WT. EM showed endothelial cell interruption with loss of normal endothelial architecture and increased smooth muscle cell hyperplasia in Sickle mice penes. Sickle mice penes had reduced eNOS activity, p-eNOS expression, cGMP level, and PDE5 activity and increased eNOS uncoupling and ROS generation. Sildenafil therapy restored cGMP, NOS and PDE5 activities, improved p-eNOS expression, prevented eNOS uncoupling, and reduced ROS generation in Sickle mice penes compared to WT.

Conclusions:

The priapic activity in the Sickle mouse is associated with a reduction in the functions of NOS and PDE5 and an NO/redox imbalance in the penis, and chronic PDE5 inhibitor therapy reverses this phenotype by restoring normal NO-mediated penile vascular homeostasis. These data support the use of PDE5 inhibitors for the treatment of priapism associated with hematologic dyscrasias.

Disclosures:

Burnett: Pfizer Inc: Research Funding; Lilly LLC: Research Funding. Off Label Use: Sildenafil and tadalafil are FDA approved for treating erectile dysfunction.
Tyrosine Phosphorylation of Runx1 In Megakaryocytes by Src Family Kinases
Authors: Huang Hui , Waldon Zachary , Chan Gordon , Zhu Helen , Steen Hanno , Feng Gen-Sheng , Neel Benjamin , Cantor Alan
Published: October 2019

Abstract 742

Runx1 and its cofactor, CBF-beta, are the most frequent targets of chromosomal translocations in human leukemias. Point mutations in Runx-1 also occur in some cases of myelodysplastic syndrome and undifferentiated leukemia. During normal hematopoiesis, Runx1 is required for the ontogeny of all definitive hematopoietic stem cells and for the proper maturation of megakaryocytes (Mks) and lymphocytes. Despite these critical roles, the regulation of Runx1 activity via cell signaling pathways remains incompletely understood. Here, we report that Runx-1 is tyrosine phosphorylated in Mks. This occurs on multiple residues and is mediated by src-family tyrosine kinases (SFKs). Loss of Runx1 tyrosine phosphorylation correlates with phorbol ester induced differentiation of L8057 megakaryoblastic cells, suggesting a negative regulatory function. Consistent with this model, retroviral expression of a tyrosine non-phosphorylatable mutant Runx1 molecule increases primary murine fetal liver Mk maturation and Runx1 target gene expression to a greater extent than wild type Runx1. Moreover, treatment of wild type primary Mks with SFK inhibitors markedly enhances Mk maturation, as previously reported (Lannutti BJ, et al 2005 Blood;105:3875-3878). Treatment of L8057 cells with the pan-tyrosine phosphatase inhibitor Na3VO4, significantly increases Runx1 tyrosine phosphorylation levels, suggesting that tyrosine phosphorylation of Runx1 is dynamically regulated under steady-state conditions. Using a proteomic approach, we found that Runx1 physically interacts with the non-receptor tyrosine phosphatase SHP-2 (Ptpn11). We validated this interaction and showed that it occurs via direct interactions involving the Runx1 runt domain. ShRNA mediated knock down of SHP-2 in L8057 cells increases Runx1 tyrosine phosphorylation levels. Conditional knockout of SHP-2 in Mks using SHP-2fl/fl, PF4-Cre mice leads to reduced peripheral blood platelet counts and delayed platelet recovery following transient anti-GPIb antibody induced immune thrombocytopenia. Lastly, we show that treatment of TPA-induced L8057 cells with Na3VO4 markedly diminishes binding between Runx1 and the key Mk transcription factor GATA-1. Taken together, our data suggest that tyrosine phosphorylation of Runx1 via SFKs inhibits Runx1 function. Dephosphorylation, at least in part via SHP-2, relieves this inhibition and promotes Mk maturation. These effects are likely mediated through altered protein-protein interactions.

Disclosures:

No relevant conflicts of interest to declare.
Patterns of Use of FDG-PET for the Initial Staging of Follicular Lymphoma (FL) and Its Impact on Initial Treatment Strategy and Outcomes In the National Comprehensive Cancer Network (NCCN) Lymphoma Database
Authors: Abou-Nassar Karim E , Vanderplas Ann , Friedberg Jonathan W. , Abel Gregory , Niland Joyce , Rodriguez Maria A. , Czuczman Myron S. , Millenson Michael , Crosby Allison , Gordon Leo I , Zelenetz Andrew D , LaCasce Ann
Published: October 2019

Abstract 81

Background:

The management of follicular lymphoma (FL) can vary according to stage at presentation. The use of 18-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) for initial staging of FL may result in upstaging and/or changes in treatment strategy. In this study, we describe the patterns of use of FDG-PET for the initial staging of patients with newly diagnosed FL and its potential impact on treatment.

Methods:

Data were obtained from the NCCN Non-Hodgkin's Lymphoma Outcomes Database, a prospective cohort study collecting comprehensive clinical, treatment, and outcome data for patients treated at 7 participating NCCN centers. Patients who presented between January 1, 2001 and September 30, 2009 with newly diagnosed low grade FL (grade 1, 2), received staging imaging within 6 weeks of diagnosis, and with at least 6 months of follow-up were included. We compared baseline and clinical characteristics as well as treatment among patients by receipt of FDG-PET for initial staging.

Results:

We identified 953 eligible FL patients with a mean age of 56 years; 532 (56%) underwent FDG-PET as part of initial staging. Use of FDG-PET varied significantly across NCCN centers (p<0.0001). Among patients who underwent FDG-PET for initial staging, 438 (82%) received early treatment (defined as treatment within 180 days of diagnosis) compared to 259 (61.5%) who were staged with conventional CT scanning only (p<0.0001).

Among 189 patients with early stage FL (ESFL), 120 (63.5%) underwent FDG-PET for initial staging. Significant differences in use were observed across NCCN centers (p<0.0001). Of all ESFL, 55 (29.1%) were treated with radiotherapy (RT) alone, 68 (36%) with chemotherapy/immunotherapy (CIT) alone, 28 (14.8%) with combined RT and CIT and 38 (20.1%) were observed. Initial treatment strategy for ESFL varied significantly according to NCCN centers (p<0.001). Choice of initial treatment strategy for ESFL did not vary significantly by use of FDG-PET (p=0.09).

Conclusions:

The use of FDG-PET for staging of FL varies by center and is widespread despite a paucity of evidence supporting its use in this disease. Patients who undergo FDG-PET scan for initial staging appear more likely to receive early therapy although this may not be directly attributed to FDG-PET results. The management of ESFL is surprisingly heterogeneous and varies across NCCN centers but is not influenced by the use of FDG-PET scan at staging. Given the widespread use and high cost of FDG-PET, its clinical utility in ESFL should be further evaluated.

Disclosures:

No relevant conflicts of interest to declare.
S1P1 Agonists for Use as Adjunct Mobilizing Agents
Authors: Harun Nadia , Thien Marilyn , Juarez Julius G , Bradstock Kenneth Francis , Bendall Linda J.
Published: October 2019

Abstract 826

Harvesting hematopoietic stem cells (HSC) mobilised into peripheral blood (PB) for transplantation is mediated through bone marrow (BM) retentive and egress factors. Factors that retain hematopoietic stem cells (HSC) in the BM are well defined, with CXCL12 and VCAM1 playing major roles. However, the factors involved in the egress of HSC from the BM into the peripheral blood (PB) are currently uncharacterised. Sphingosine-1-Phosphate (S1P) is a lymphoid organ egress factor for lymphocytes, mediated through the S1P1 receptor, which is also expressed on HSC. We hypothesised that S1P mediates the egress of HSC out of the BM and into the PB.

Our laboratory used a number of different mouse models with various S1P levels or S1P receptor expression to elucidate the role of the S1P gradient between the BM and PB. Sphingosine kinase-1 knock-out (SK1KO) mice were utilized for their reduced PB S1P levels. A sphingosine lyase inhibitor 4′deoxypyridoxine (DOP) was used to increase BM S1P levels. Mice treated with FTY720 for 14h had suppressed S1P1 expression and an S1P1 conditional knock-out mouse was also generated by our group. Animals were also treated with S1P receptor agonists such as SEW2871. Mobilisation experiments, competitive repopulation assays and chemotaxis assays were performed utilizing the various models.

Plasma from SK1KO mice had a reduced capacity to induce migration in haematopoietic progenitor cells (HPC), confirming the chemokine activity of S1P. Consistent with this, AMD3100 induced mobilization was inhibited in SK1KO mice and DOP treated mice, demonstrating the role of an S1P gradient in HPC mobilization. Mice treated with FTY720 significantly inhibited AMD3100, although not G-CSF, mediated mobilisation of HPC in mice. No HPC accumulation was detected in secondary lymphoid organs such as lymph nodes or spleen. Most importantly, FTY720 treatment reduced the number of transplantable HSC in the blood following AMD3100-mediated mobilisation using a competitive repopulation assay. Our laboratory also generated an S1P1 conditional knock-out mouse. When mobilised with AMD3100, these S1P1 knock-out animals displayed a marked reduction in HPC mobilisation compared to wild-type animals. Finally, the S1P1 agonist SEW2871 increased HPC mobilisation synergistically, by approximately 2 fold when combined with AMD3100, but not G-CSF.

S1P supports the egress of HSC from the BM into the PB following inhibition of the CXCL12/CXCR4 axis. S1P1 conditional knock-out mice display a significantly reduced mobilising capacity. S1P receptor agonist, SEW2871, acts synergistically with AMD3100 to increase HPC mobilisation in vivo, raising the possibility that such a combination may increase the efficiency of HSC collection for transplantation purposes.

Disclosures:

No relevant conflicts of interest to declare.
Ror1 Targeting Monoclonal Antibodies Induced Apoptosis of Chronic Lymphocytic Leukemia Cells– A Potential Novel Therapeutic Approach
Authors: Rabbani Hodjattallah , Daneshmanesh Amir Hossein , Khan Abdul Salam , Hojjat-Farsangi Mohammad , Jeddi-Tehrani Mahmood , Akhondi Mohammad Mehdi , Österborg Anders , Shokri Fazel , Mellstedt Håakan
Published: October 2019

Abstract 916

Methods:

A peptide-based mouse monoclonal antibody generation method was used for producing MAbs against the three extracellular domains of Ror1. Twenty CLL patients (10 with progressive and 10 with non-progressive disease) were enrolled in this study. Flow cytometry was used for surface staining of Ror1. Annexin V and propidium iodide (flow cytometry) as well as PARP cleavage (Western blot) was used for detection of apoptosis.

Results:

Six monoclonal antibodies of different isotypes (IgG, IgM) were produced against Ror1. The frequency of Ror1 positive cells were in the range of 24–89%. Patients with progressive disease had a significantly higher number of Ror1 positive cells as compared to those with non-progressive disease as well as in patients with unmutated compared to mutated IgVH genes. All six antibodies alone induced apoptosis (Annexin V and PARP cleavage) of CLL cells. A higher frequency of apoptotic CLL cells was induced by the antibodies against the CRD region (ligand binding site for Wnt proteins) as well as one antibody against the kringle domain (also a binding region for regulatory proteins) . Apoptosis induced by these three antibodies alone was significantly higher than that induced by Rituximab (p<0.001). Apoptosis induction by all antibodies was statistically significantly augmented by crosslinking of the Ror1 antibodies with anti-Fc F(ab’)2 fragment antibodies. None of the antibodies induced apoptosis of PBMC of healthy individuals and normal PBMC did not express Ror1.

Conclusion:

Monoclonal antibodies alone against the RTK, Ror1, were shown to selectively kill CLL cells. Development of MAb targeting Ror1 might be a novel therapeutic approach complementary to existing therapies.

Disclosures:

No relevant conflicts of interest to declare.
The Function of Thymic Innate TCRαß+ CD8+ T Cells Is Regulated by Constitutive Expression of B7-H1
Authors: Rafei Moutih , Hardy Marie-Pierre , Perreault Claude
Published: October 2019

Abstract 955

Innate T cells display features of Ag-experienced T cells (e.g., high levels of CD44) before any encounter with non-self antigens. They include NKT cells, CD8α/α T cells, as well as an undefined (and presumably very small) proportion of naïve CD4 and CD8 α/β T cells. We used the RAG2-GFP transgenic mouse model to differentiate naïve innate single-positive thymocytes (GFP+CD44high) from re-circulating effector/memory T cells (GFP-CD44high) T cells and naïve conventional (GFP+CD44low) thymocytes. We found that innate T cells represent about 10% of TCRαβ+ single-positive GFP+ thymocytes. To evaluate their proliferative ability under lymphopenic conditions, conventional and innate T cells were sorted and injected in TCRβKO mice. Four weeks later, expansion of conventional T cells was 2.3-fold greater than that of innate T cells. Comprehensive phenotypic analyses of GFP+CD8+CD44low vs GFP+CD8+CD44hi thymocytes revealed that B7-H1 was absent on conventional thymocytes but constitutively expressed on innate CD8+ thymocytes. Moreover, in vitro stimulation of innate T cells with anti-CD3 and anti-CD28 antibodies induced a strong proliferation and secretion of inflammatory mediators such as interferon (IFN)-gamma. The addition of anti-B7-H1 neutralizing antibody, however, inhibited proliferation and IFN-gamma production in a dose-dependent manner. To see whether B7-H1 ligation influenced the viability of CD8+ thymocytes, GFP+CD8+CD44low and GFP+CD8+CD44hi thymocytes were treated for 48 hrs with anti-B7-H1 antibody in vitro then stained for annexin-V and propidium iodine. Under these conditions, 70% of innate GFP+CD8+CD44hi T cells underwent apoptosis as opposed to 30% with conventional thymocytes. To test their bystander properties, we added conventional or innate T cells as third-party cells to an in vitro antigen stimulation assay. Briefly, B6 peritoneal macrophages pulsed with recombinant chicken ovalbumin (rOVA) presented OVA-derived peptides in the context of MHCI, and served as antigen-presenting cells (APCs). For responder cells, we added naive T cells from T cell receptor-transgenic OTI mice that recognize OVA. Interestingly, innate thymocytes were capable of promoting the activation of responding T cells; an effect that was lost upon treatment of innate CD8 T-cells with anti-B7-H1 neutralizing antibody. The sum of these data suggests a role for B7-H1 in the development, survival and proliferation of innate α/β T cells.

Acknowledgments

Moutih Rafei holds a Fellowship from the CIHR. Claude Perreault holds a Canada Research Chair in Immunobiology. This work was supported by grant # 42384 from the CIHR.

Disclosures:

No relevant conflicts of interest to declare.
JAK2 Inhibition with TG101348 Inhibits Monosomy 7 Myelodysplastic Syndromes (MDS) Bone Marrow Cells In Vitro: a Potential Targeted Therapy for Monosomy 7 MDS
Authors: Olnes Matthew J. , Poon Andrea , Tucker Zachary , Young Neal S. , Sloand Elaine M
Published: October 2019

Abstract 973

The myelodysplastic syndromes (MDS) are bone marrow disorders characterized by cytopenias and a variable risk of progression to acute myeloid leukemia (AML). Monosomy 7 is the second most common cytogenetic abnormality in MDS, and the most frequent karyotypic aberration occurring in aplastic anemia patients following immunosuppressive therapy. Monosomy 7 MDS carries a particularly poor prognosis, with patients manifesting severe cytopenias and a high propensity to develop treatment-refractory AML. There are currently no targeted therapies for this disorder. We previously reported that monosomy 7 bone marrow mononuclear cells (BMMNCs) express high levels of a differentiation-defective granulocyte colony stimulating factor (G-CSF) receptor isoform (IV), an alternative splice variant that exhibits constitutive signaling through the JAK-2 and STAT-1 pathway, while levels of STAT-3 and -5 are unchanged (Sloand et al, PNAS, 2006, 103:14483). As a result, the cell's ability to differentiate is limited, while its ability to proliferate remains intact. Here we examine the effects of the highly selective JAK2 inhibitor TG101348 on monosomy 7 aneuploidy in BMMNCs, as well as the activity of this compound on CD34+ stem cells and CD13+ myeloid cells in culture, and on the JAK-2 signaling apparatus. Incubation of BMMNCs with TG101348 for 5 days significantly decreased absolute numbers of monosomy 7 aneuploid cells in a concentration dependent manner versus vehicle- treated controls (0.187 × 106 vs 1.08 × 106, P=0.007), while diploid cell numbers remained stable (0.338 × 106 vs 0.213 × 106, P=0.50). Flow cytometry experiments demonstrated that incubation with increasing concentrations of TG101348 decreased the absolute number of CD34+CD13- stem cells, and increased numbers of more differentiated CD34-CD13+ myeloid cells, with median CD34+/CD13+ ratios of 6.547 and 2.216 for cells treated with vehicle and 100 nM TG101348, respectively. By immunoblot, STAT-1 protein expression in monosomy 7 BMMNCs treated with 1uM TG101348 was decreased relative to vehicle- treated controls, while there was no difference in STAT-3 and STAT-5 levels. Thus TG101348 decreases monosomy 7 MDS blasts in vitro through inhibition of JAK-2/STAT-1 signaling, a finding that warrants further study of this agent in clinical trials for patients with monosomy 7 MDS and AML.

Disclosures:

No relevant conflicts of interest to declare.
Genomic Variants Associated with Childhood Acute Lymphoblastic Leukemia
Authors: Relling Mary V.
Published: October 2019

Abstract SCI-11

Genome-wide interrogations have a role in addressing both the etiology and the responsiveness of childhood acute lymphoblastic leukemia (ALL). Recent work by our own1 and other groups identified common polymorphisms in ARID5B, and to a lesser extent in IKZF1, as predisposing to the development of childhood ALL. Polymorphisms in these two genes can account for ∼ 40% of the population attributable risk of ALL, and differences in the frequency of the minor allele among different ancestral groups can account for a large portion of the differences in the incidence of ALL among different race groups. The association of these inherited polymorphisms with specific ALL subtypes (e.g. ARID5B with risk of hyperdiploid ALL) indicates that germline polymorphisms affect not only risk of ALL but may also affect or confound association analyses between germline variants and the probability of cure of ALL. The biological basis for the prognostic differences that exist among ALL subtypes remain largely unknown, and it is plausible that inherited polymorphisms may affect both susceptibility to subtypes of ALL as well as to drug responsiveness. Genome-wide analyses have also identified polymorphisms associated with eradication of MRD across multiple treatment protocols.2 Polymorphisms in IL15 indicate variants that likely affect the inherent pharmacodynamic responsiveness of ALL to drug-induced apoptosis. Approximately 20% of the polymorphisms associated with MRD were also associated with rapid drug clearance in the host, even though pharmacokinetic data were only available for 2 of the 4–8 medications used during induction. These findings lead us to suggest that perhaps half of the polymorphisms associated with eradication of ALL are related to effects on host drug clearance, and about half have penetrant effects on the inherent responsiveness of the ALL cells themselves. A genome-wide analysis for the determinants of clearance of one such drug, methotrexate, identified a strong effect of polymorphisms in the SLCO1B1 transporter.3 Genome-wide approaches have identified the importance of genes that decades of candidate gene approaches did not reveal, illustrating the utility of an agnostic approach to genotype-phenotype association studies in childhood ALL.

1. Treviño LR, Yang W, French D, et al. Germline genomic variations associated with childhood acute lymphoblastic leukemia. Nat Genet 41:1001–5, 2009.

2. Yang J, Cheng C, Yang W, et al. Genome-wide interrogation of germline genetic variation associated with treatment response in childhood acute lymphoblastic leukemia. JAMA 301:393–403, 2009.

3. Treviño LR, Shimasaki N, Yang W, et al. Germline genetic variation in an organic anion transporter polypeptide associated with methotrexate pharmacokinetics and clinical effects. J Clin Oncol 27:5972–8, 2009.

Disclosures:

Relling: St. Jude Children's Research Hospital: Employment, Patents & Royalties; Enzon Pharmaceuticals: Research Funding.
Heme, Iron, and the Kidney
Authors: Nath Karl A.
Published: October 2019

Abstract SCI-26

The kidney is often the target of injury by heme proteins and states of iron overload. Exogenous heme proteins, such as hemoglobin delivered to the kidney in hemolytic states, or myoglobin imposed upon the kidney following rhabdomyolysis, can injure the kidney via pathways that include vasoconstriction, direct cytotoxicity, and tubular cast formation. The former two pathways critically involve the heme prosthetic group: heme can bind vasodilator gases such as nitric oxide and carbon monoxide, while free heme can be cytotoxic because it is lipophilic, lipid membrane-transmissible, lipid membrane-destabilizing, prooxidant, and proinflammatory. Renal injury can also arise from endogenous heme proteins, such as cytochrome P450 enzymes, which can be destabilized by ischemic and nephrotoxic insults; such destabilization leads to the release of heme and incurs heme-dependent injury. The toxicity of heme may also reflect, at least in part, the release of iron and the attendant increase in labile cellular iron, the latter representing a potent catalyst for oxidative stress; increased levels of cellular iron may also originate from intracellular nonheme sources. Heme-iron can not only induce acute kidney injury, but also can provoke chronic kidney disease by virtue of its proinflammatory and profibrotic effects. Mechanisms that protect against heme-dependent and iron-dependent toxicity include heme oxygenase (HO), the rate-limiting enzyme in heme degradation, and increased synthesis of ferritin. Induction of HO-1, the inducible HO isoform, is protective against renal injury because of the following mechanisms: 1) the prevention of acute elevation in cellular heme concentrations otherwise incurred by cell injury; 2) the safe sequestration of iron in iron-binding proteins such as ferritin, or the cellular extrusion of iron by iron-exporting proteins; 3) the generation of antioxidant, anti-inflammatory metabolites such as bile pigments; and 4) the generation of carbon monoxide which is an antiapoptotic, anti-inflammatory, and vasorelaxant gas. Carbon monoxide can also be cytoprotective by binding cytochrome P450 enzymes, and thereby preventing their destabilization and the release of heme that subsequently occurs. In addition to these areas, this presentation discusses the pathobiologic and clinical significance of the siderophore-binding protein, NGAL (Neutrophil gelatinase-associated lipocalin): NGAL protects against renal ischemic injury through mechanisms that require the induction of HO-1; NGAL is increasingly utilized as a biomarker of acute kidney injury. The presentation concludes by discussing the use of iron supplementation in the treatment of anemia of chronic kidney disease and therapeutic strategies that may be designed from understanding endogenous and adaptive mechanisms that protect against the toxicity of heme-iron.

Disclosures:

No relevant conflicts of interest to declare.
Functional Genomics Approaches to Platelet Signaling
Authors: Ouwehand Willem Hendrik , Soranzo Nicole
Published: October 2019

Abstract SCI-37

The count, volume and function of platelets varies in the population and there is ample evidence that all 3 quantitative traits (QT) are highly heritable. The control of platelet function is complex with a large number of forward and reverse regulatory pathways that promote and inhibit thrombus formation, respectively. We used 4 parallel genomics approaches with the aim to identify novel regulators of platelet function. First we established a genome-wide expression (GWE) HaemAtlas of all 8 haematological elements, including erythroblasts and megakaryocytes (MKs)(1). Mining of this data identified 279 transcripts that were relatively over-expressed in MKs if compared with the results obtained with the 7 remaining blood cell types. Of these, 75 transcripts encode membrane proteins, many with known and several with a hitherto unknown function (2). Second we determined the platelet response to ADP and a collagen mimetic in 500 healthy subjects of the Platelet Function Cohort (PFC)(3). WGE studies of platelet RNA samples from 37 PFC subjects, selected to be representative of the observed variation, identified 63 transcripts that were correlated with function. Third the 500 PFC samples were typed for 1536 SNPs tagging 108 candidate genes for sequence variation and this identified 19 associations (p-value ≤ 0.005) (3). All 19 QT loci (QTLs), but the GP6 gene, were novel. Based on the above 9 genes were selected for functional studies with platelets from genotyped healthy subjects and by morpholino-based gene knockdown in a model of laser-induced thrombus formation in Danio rerio (2,3). This identified BAMBI, COMMD7, LRRC32 and LRRFIP1 playing a role in the promotion and DCBLD2, ESAM, G6B and GTF2A2 in the inhibition of thrombus formation. Finally 2 of the 15 QTLs for platelet volume and count identified in about 15,000 healthy subjects with genome-wide typing information showed an effect on platelet function demonstrating that sequence variants that modify platelet volume may also exert an effect on function (4). (1) Macaulay, I. C. et al., Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Blood 109 (8), 3260 (2007); Watkins, N. A. et al., A HaemAtlas: characterizing gene expression in differentiated human blood cells. Blood 113 (19), e1 (2009); (2) O'Connor, M. N. et al., Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins. Blood 113 (19), 4754 (2009).; (3) Jones, C. I. et al., A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways. Blood 114 (7), 1405 (2009); (4) Soranzo, N. et al., A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium. Nat Genet 41 (11), 1182 (2009).

Disclosures:

No relevant conflicts of interest to declare.
Angioblast Behavior in Arterial-Venous Segregation
Authors: Herbert Shane P. , Stainier Didier
Published: October 2019

Abstract SCI-43

Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from pre-existing vessels (angiogenesis). Using high resolution imaging of zebrafish vascular development we discovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated in part by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial/venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

Disclosures:

No relevant conflicts of interest to declare.
Complementary Costimulation of Human T Cell Subpopulations by CD28 and CD81
Authors: Levy Shoshana , Sagi Yael
Published: October 2019

Abstract 1124

CD81 is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells - primary human naïve T cells are better costimulated by CD81, while the memory T cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared to that by CD28. We found that IL-6 played a role in the activation of the naïve T cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.

Disclosures:

No relevant conflicts of interest to declare.