bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allogeneic Hematopoietic Stem Cell Transplant Reverses The Phenotype Of DOCK8 Deficiency
Authors: Cuellar-Rodriguez Jennifer , Freeman Alexandra F , Gea-Banacloche Juan , Su Helen , Grossman Jennifer K. , Fry Terry J , Holland Steven M. , Hickstein Dennis D. 
Published: October 2019

Abstract

Background
DOCK8 deficiency— a combined immunodeficiency characterized by recurrent sinopulmonary infections and severe cutaneous DNA viral infections, eczema, food and drug allergies, and increased risk of viral driven malignancies— results from homozygous or compound heterozygous mutations in the dedicator of cytokinesis 8 protein. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a potential curative therapy for DOCK8 deficiency.

Methods
We treated 4 patients with DOCK8 deficiency with allogeneic HSCT using a reduced toxicity, myeloablative-conditioning regimen consisting of busulfan 3.2 mg/kg/day IV for 4 days and fludarabine 40 mg/m2/day for 4 days. Two patients received matched related donor allogeneic HSCT, and 2 received 10/10 HLA matched unrelated donor allogeneic HSCT. All patients received tacrolimus, and short methotrexate on days 1,3, 6, and 11, for graft-versus-host-disease (GVHD) prophylaxis. Indication for transplant was severe recurrent infections with end organ damage in three patients, and a refractory EBV-virus driven lymphoma at the time of unrelated donor PBSC transplant in the fourth patient.

Results
The ages of the patients at the time of transplant were 27, 25, 18, and 16. The median follow-up for patients was 7 months (range 5-18 months). All 4 patients engrafted at a median of 12 days, and all 4 had complete reconstitution of the CD3/CD4 and CD3/CD8 compartments with donor cells. Clinical correction of the DOCK8 clinical phenotype occurred within 3-6 months of engraftment and correlated with lymphocyte reconstitution. Mucositis was the major side effect of the transplant-conditioning regimen. All had transient worsening of their pre-transplant infections 1-3 months post-transplant. In the patient with an EBV-virus driven lymphoma that had been refractory to chemotherapy, there was complete resolution of the disease on PET scanning at 100 days post-transplant. One patient, who received unrelated donor bone marrow cells, had skin GVHD that responded to a short course of steroids. There was no chronic GVHD.

Conclusions
Allogeneic HSCT in DOCK8 deficiency results in reconstitution of the deficient lymphocyte compartments that are present pre-transplant and complete reversal of the infection susceptibility phenotype. There was minimal regimen-related toxicity, and the incidence of GVHD was low despite complete donor chimerism. With genetic testing for DOCK8 deficiency now more widely available, we anticipate that earlier diagnosis will enable patients to be transplanted earlier in their clinical course, before significant organ damage or the development of viral-driven malignancies

Disclosures:
No relevant conflicts of interest to declare.
Ferroportin Q248H Mutation Prevents Its Ubiquitination
Authors: Ammosova Tatiana , Ivanov Andrey , Nekhai Sergei A 
Published: October 2019

Abstract

Background
Ferroportin Q248H mutation is prevalent in African populations and leads to increased serum ferritin. Our recent study shows that ferroportin Q248H protein is resistant to physiologic hepcidin concentrations1. Also sickle cell disease patients with ferroportin Q248H heterozygote had lower serum ferritin concentration suggesting that the enhanced iron release by macrophages. Ferroportin glutamine 248 is located within the intracellular loop (residues 228-307), which is likely to be located in the cytoplasm. Recently ferroportin internalization was shown to be driven by ubiquitination of lysines lying within residues 229-269 including K229, K240, and K2472. The proximity of the K240 and especially to K247 to the Q248 residue suggests that a positively charged histidine in position 248 might change the overall negative charge of the 240eeetelkqlnlhk253sequence toward a more positive charge, which might affect ubiquitination and subsequent degradation of ferroportin. Here we analyzed and compared ubiquitination of WT and Q248H mutant ferroportin.

Results
WT ferroportin and Q248H mutant were expressed as EGFP-fusions in 293T cells and also combined with the expression of ubiquitin. Ferroportin was immunoprecipitated with anti-EGFP antibodies and analyzed by high resolution mass spectrometry using LTQ-Orbitrap. Phosphorylation and ubiquitination was determined using Proteome Discover and quantified using SIEVE 2.1 software.

Conclusions
WT ferroportin but not the Q248H mutant ferroportin was found to be ubquitinated on lysines 247 and 253 and also phosphorylated on Thr 144. Also WT ferroportin was found to associate with ubiquitine-conjugating enzyme E2 and ubiquitine protein ligase NEDD4. Thus hepcidin resistance of ferroportin Q248H could be due to its inability to undergo ubiquitination.

Acknowledgments
This project was supported by NIH Research Grants 8G12MD007597 and P30HL107253.
References
1. Nekhai S, Xu M, Foster A, et al. Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin. Haematologica. 2013;98(3):455-463.
2. Qiao B, Sugianto P, Fung E, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Metab. 2012;15(6):918-924.

Disclosures:
No relevant conflicts of interest to declare.
Myocardial and Hepatic Iron Overload and Cardiac Function In Sickle/Thalassemia Patients Of Italian Origin
Authors: Meloni Antonella , Ruffo Giovan Battista , De Marchi Daniele , Cardinale Antonio , Pietrapertosa Anna , Facchini Elena , Toia Patrizia , De Franceschi Lucia , Positano Vincenzo , Lombardi Massimo , Pepe Alessia 
Published: October 2019

Abstract

Introduction
Sickle-thalassemia results from the combined heterozygosity for sickle-cell and β-thalassemia genes. This study evaluates myocardial and hepatic iron overload and cardiac function in Italian patients and explores their correlation with transfusions, age and sex.

Methods
Fifty-nine sickle-thalassemia patients (29 males, mean age 35.6±14.1 years), enrolled in the MIOT network underwent magnetic resonance imaging (MRI). T2* value for all 16 myocardial segments and global heart T2* value were calculated. Hepatic T2* value was converted into liver iron concentration (LIC). Cine images were acquired to quantify biventricular volumes and ejection fraction (EF).

Results
55 (93%) patients had all segmental T2* values normal (>20 ms). Of the 4 patients with abnormal segmental T2* values, all showed an heterogeneous myocardial iron overload (some segments with T2*>20 ms and other with T2*<20 ms) and only one had a global T2*<20 ms. The mean global heart T2* value was 34.4±6.2 ms. The mean LIC was 5.9±6.5 mg/g/dw and 30 patients (50.8%) had a pathological value (≥ 3 mg/g dw). There was a statistically significant positive correlation between global heart T2* and age but with poor linearity (R=0.368; P=0.004) and there was not a significant correlation between age and LIC.
Males and females had comparable global heart T2* values and LIC values.
Twenty patients were regularly transfused, 32 received sporadic transfusions while 7 were not transfused. The comparison among the three groups is shown in Table 1. We did not find significant differences in the global heart T2* value while patients regularly transfused had significantly higher LIC than sporadically transfused patients. Biventricular volumes indexed by body surface area and ejection fractions were comparable among the groups.

Conclusions
In respect of MIO, the sickle/thalassemia patients are similar to patients with homozygous SCD for which iron overloading is relatively rare. Hepatic iron overload may develop also in no regularly-transfused patients, maybe due to increased absorption of iron from the digestive tract, characteristic of both SCD and thalassemia intermedia patients. This finding underlines the importance to monitor by MRI also no regularly transfused sickle/thalassemia patients.

Disclosures:
No relevant conflicts of interest to declare.
Left and Right Ventricular Wall Motion Abnormalities In Thalassemia Major Patients
Authors: Meloni Antonella , Giuffrida Gaetano , Paci Cristina , Sardella Leonardo , Quota Alessandra , Bitti Pier Paolo , De Franceschi Lucia , Salvatori Cristina , Vinci Valentina , Positano Vincenzo , Lombardi Massimo , Pepe Alessia 
Published: October 2019

Abstract

Introduction
Left ventricular (LV) and right ventricular (RV) wall motion abnormalities can be detected through a qualitative analysis of cine magnetic resonance (MR) images. Moreover, MR is the gold standard technique for the evaluation of myocardial iron overload (MIO), biventricular global systolic function and myocardial fibrosis. We investigated the relationships between LV movement abnormalities and MIO, LV function and myocardial fibrosis as well as between RV motion and function in thalassemia major (TM) patients.

Methods
CMR was performed in 1092 TM patients (537 male; 30.6±8.5 years) enrolled in the Myocardial Iron Overload in Thalassemia Network. Cine images were acquired to evaluate wall motion and to quantify biventricular volumes and ejection fraction (EF). For MIO assessment, a T2* multislice approach was used. To detect myocardial fibrosis, late gadolinium enhanced (LGE) images were acquired. For the LV the 16-segment model of the AHA/ACC was taken into account during image analysis: wall motion, T2* value and presence/absence of enhancing area were evaluated for each segment.

Results
Abnormal motion of LV was found in 66 (6%) patients (60 hypokinetic and 6 dyskinetic). Table 1 shows the comparison between TM patients with normal and abnormal LV motion. Patients with abnormal LV motion were older and had significantly lower global T2* value and significantly higher number of segments with T2*<20 ms. Left volumes and mass indexed by body surface area were significantly higher in patients with abnormal LV motion while the EF was significantly lower. LGE areas were detected in 196 patients (18%) and were predominantly located in the mid-ventricular septum. There was a significant correlation between LGE and abnormal LV motion.
Abnormal motion of the RV was found in 35 (3.2%) patients (29 hypokinetic, 5 dyskinetic and 1 akynetic). Table 2 shows the comparison between TM patients with normal and abnormal RV motion. Patients with abnormal RV motion were older and they were more frequently males. Right volumes were significantly higher in patients with abnormal RV motion while the EF was significantly lower.
Abnormal LV motion was not correlated with abnormal RV motion. Seventeen patients showed movement abnormalities in both ventricles.

Conclusions
Movement abnormalities in the left ventricle were not really frequent in TM patients but were associated with age, MIO, LV dilation and dysfunction, and myocardial fibrosis. Movement abnormalities in the right ventricle were less frequent compared to the left ventricle, but were associated with age, sex , RV dilation and dysfunction.

Disclosures:
No relevant conflicts of interest to declare.
Genetic Mosaicism and Hemoglobinopathies; Uniparental Isodisomy
Authors: Harteveld Cornelis L , Phylipsen Marion , Giordano Piero C 
Published: October 2019

Abstract

Background
Recently we discovered three independent cases of “severe late onset beta-thalassemia”, all presenting with the mild phenotype of beta thalassemia minor up to adult age and developing a severe transfusion dependent phenotype in the third and fourth decade of life when a presumed homozygosity for the beta-thalassemia mutation is observed. We demonstrate that uniparental isodisomy (UPD) of part of chromosome 11p, a mechanism also seen in 20% of Beckwith-Wiedemann syndrome patients, accounts for the observed mosaicism in all three independent cases. Clonal selection for hematopoietic stem cells containing the UPD for the mutant beta-globin gene during life may account for the progressive development of the disease. Recently we discovered another case showing a similar mosaic UPD of 11p, presenting as a regular beta-thalassemia carrier.

Method
Direct sequencing of the beta-globin genes. Multiplex Ligation dependent Probe Amplification (MLPA) analysis of the HBB (beta-globin) gene cluster. Affymetrix GeneChip Human Mapping 262K NspI array (Santa Clara, CA, USA). Illumina OmniExpress 730K SNP Beads array (San Diego, CA, USA).

Results
In all cases molecular analysis shows sequences in which a near to complete homozygosity for the beta-gene mutation in DNA extracted from leucocytes is seen. Loss of heterozygosity due to allele drop-out or a deletion of one allele was excluded by direct sequencing and MLPA analysis. Affymetrix and/or Illumina SNP-array analysis revealed incomplete homozygosity for SNP’s along almost the entire short arm of chromosome 11 containing the beta-globin gene, indicating mosaicism for a partial uniparental isodisomy of chromosome 11p. Three patients were born asymptomatic as beta-thal carriers and developed a severe blood-transfusion dependent beta-thalassemia major at different ages and with different percentages of mosaicism. The fourth patient however did not develop the clinical severity despite of an almost 50% mosaicism determined from the DNA isolated from leucocytes.

Conclusion
We demonstrate that uniparental isodisomy of part of chromosome 11 accounts for the observed mosaicism in all four independent cases. The most probable mechanism seems clonal selection for hematopoietic stem cells containing the uniparental isodisomy for the mutant beta-globin gene during life, this may account for the progressive development of the disease. However, there seems to be no correlation between the percentage of mosaicism measured in the DNA isolated from the white cells and the severity of the clinical phenotype related to the expression in red cells, which strongly suggests hematopoietic tissue heterogeneity for the observed UPD containing cell lineages. This may have serious consequences for disease prediction and counseling, as this is largely dependent upon DNA isolated from leucocytes.

Disclosures:
No relevant conflicts of interest to declare.
A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition
Authors: Shen Bo , Zhao Xiaojuan , O'Brien Kelly A , Stojanovic-Terpo Aleksandra , Delaney Michael Keegan , Kim Kyungho , Cho Jaehyung , Lam Stephen C.-T , Du Xiaoping 
Published: October 2019

Abstract
Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding.

Disclosures:
No elevant conflicts of interest to declare.
Aberrant Type-2-Lactosaminoglycan Synthesis Severely Impairs Thrombopoiesis
Authors: Giannini Silvia , Begonja Antonija Jurak , Adelmann Max , Hoffmeister Karin M. 
Published: October 2019

Abstract
Platelet recovery following myelosuppressive/myeloablative chemotherapy is crucial to avoid bleeding complications of cancer treatment. Platelets are produced by bone marrow megakaryocytes (MKs), which develop and mature from hematopoietic stem cells (HSC). Mature MKs interact with sinusoidal bone marrow endothelial cells to form transendothelial pseudopods called proplatelets from which platelets are released into the bloodstream. Platelet survival is dependent on correct glycan expression. We here investigate the role of Type-2-Lactosaminoglycans (Type-2-LacNAc) in platelet production. beta1,4Galactosyltransferase 1 (beta4GalT1) is a major enzyme involved in Type-2-LacNAc synthesis which adds Galactose (Gal) to terminal N-Acetylglucosamine (GlcNAc) to form beta1,4Gal-GlcNAc (Type-2-LacNAc).beta4GalT1 deficient mice die in uthero between E15.5 and E16.5. A small percentage of beta4GalT1-/- mice survive until adulthood and they have severe macrothrombocytopenia but normal platelet clearance. Lethally irradiated wild type mice transplanted with beta4GalT1 deficient fetal liver cells failed to produce circulating beta4GalT1 deficient platelets, in marked contrast to beta4GalT1 deficient white blood cells, despite beta4GalT1 deficient MKs have been detected in the bone marrow of transplanted mice. beta4GalT1 deficient fetal liver MKs poorly produce proplatelets in vitro, following their normal maturation and differentiation, as judged by number, morphology, ploidy and expression of main surface glycoproteins. Our data strongly support the notion that glycosylation mediated by beta4GalT1 is crucial for platelet production in vitro and in vivo and demonstrate for the first time a role for post-translational glycan modification in platelet production.

Disclosures:
No relevant conflicts of interest to declare.
IL-1β Plays An Important Role On Thrombocytosis In An Immune Vasculitis Model Via Promoting Megakaryopoiesis
Authors: Yang Mo , Zhou Min , Li Su yi , Chong Beng , Li Xiao jing 
Published: October 2019

Abstract
Thrombocytosis and inflammation cytokines may be involved in the pathogenesis of vasculitis. Our previous study have showed that major inflammation cytokine IL-1β play an important role on in-vitro megakaryopoiesis (Yang M et al, Br J Haematol 2000). In this study, we investigated the changes of IL-1β and megakaryopoiesis and the effect of aspirin in an immune vasculitis model. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin. In this model, platelet number and function of periphery blood, megakaryocyte number and the CFU-MK formation of the bone marrow, and serum levels of inflammatory cytokines were investigated. After treatment with BSA for 7 days, the platelet count, platelet aggregation and the expression of AnnexinⅤ were significantly increased in this vasculitis model group compared with normal control group (n=6). The serum levels of inflammatory cytokine IL-1β was also significantly higher in vasculitis model. There were positive correlations between platelet count and IL-1β levels (R=0.55), platelet aggregation and IL-1β levels (R=0.603). Treatment with aspirin (100 mg/kg/d) significantly decreased all these parameters, showing aspirin had anti-platelets and anti-inflammation effects. Our results also demonstrated that megakaryocyte number and the formation of CFU-MK were significantly increased in vasculitis group as compared to those in normal group. Treatment with aspirin significantly reduced the number of megakaryocytes and the formations of CFU-MK in bone marrow in this immune vasculitis model. Our study further demonstrated that IL-1β alone or in combination with TPO induced in-vitro CFU-MK formation. Using RT-PCR techniques, the mRNA of of IL-1 type I and type II receptors (IL-1 RI and RII) were detected in cultured CD61+ CD41+ cells and four megakaryocytic cell lines. The expression of IL-1 RI and RII was also confirmed by flow cytometry and immunofluorescence staining in bone marrow megakaryocytes. Moreover, the IL-1R bloker can reduced IL-1β induced megakaryopoiesis. This sudy showed that IL-1β may play an important role in the pathogenesis of immune vasculitis. Aspirin has anti-inflammation effects in this model which may be mediated via inhibiting megakaryopoiesis and platelet formation.

Disclosures:
No relevant conflicts of interest to declare.
Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription
Authors: Nanan Kyster , LeBrun David P 
Published: October 2019

Abstract
E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV.
ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house.
Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis.
In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented.

Disclosures:
No relevant conflicts of interest to declare.