bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Adenylate Kinase 2 Deficiency Inhibits Hematopoietic Cell Differentiation towards Neutrophil and T Lymphoid Lineages.
Authors: Six Emmanuelle M. , Lagresle-Peyrou Chantal , Demerens-deChappedelaine Corinne , Rieux-Laucat Frederic , Didati Andrea , Stolzenberg Marie-Claude , Cassinat Bruno , Chomienne Christine , Fischer Alain , Cavazzana-Calvo Marina 
Published: October 2019

Abstract
Abstract 78
Reticular dysgenesis (RD), an autosomal recessive form of human Severe Combined Immunodeficiency is characterized by the absence of blood neutrophils and T lymphocytes. This pathology is due to biallelic mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of AK2 protein expression. AK2 is a mitochondrial protein which regulates the homeostasis of cellular adenine nucleotides by converting ADP into ATP and AMP. In order to understand the precise role of AK2 in hematopoiesis, we have developed a RNA interference strategy through lentiviral-mediated gene transfer of AK2 short hairpin RNAs (shAK2). The knock-down of AK2 in human or murine hematopoietic stem cells (HSC) inhibits their capacity to form granulocyte colonies in methycellulose assays and prevents them to generate mature polynucleated cells in liquid culture in the presence of G-CSF. We also determine the ability of shAK2-transduced HSC to differentiate along the T lymphoid lineage after co-culture on a OP9Delta1 stroma cell line. Our data demonstrated that the apparition of CD4+CD8+ cells was profoundly reduced in the presence of shAK2. To delineate the mechanism involved in this defect, we also studied the neutrophil differentiation of the HL60 promyelocytic cell line, following retinoic acid treatment. In this system, the absence of AK2 expression led to an arrest of neutrophil differentiation process, increased cell apoptosis and disrupt the mitochondrial membrane potential. All these data suggest a novel mechanism regulating hematopoietic cell differentiation, and involved in one of the most severe human immunodeficiency syndromes.

Disclosures:
No relevant conflicts of interest to declare.
Longitudinal Analysis of Antibody Response to Immunization in Pediatric Survivors After Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Inaba Hiroto , Posner Meredith , Hartford Christine , Yang Jie , Pei Deqing , Hayden Randall , Srinivasan Ashok , Triplett Brandon , Pui Ching-Hon , Leung Wing 
Published: October 2019

Abstract
Abstract 795
Re-immunization is a common practice after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the optimal vaccine schedule and long-term response have not been established. We prospectively and longitudinally evaluated the antibody response to childhood vaccines in 210 survivors after allo-HSCT. Positive titer lasting for 5 years or more after immunization was observed more often for diphtheria (98.1%), tetanus (96.1%), rubella (92.9%), and polio vaccines (96.2% for type 1; 98.1% for type 2 and 3), but less often for pertussis (25.0%), measles (65.4%), mumps (64.3%), and hepatitis B (75.0%). As shown in the table below, vaccine failure was associated with patients who were older at the time of re-immunization, received a T cell depleted graft, had a high-risk hematological malignancy, lower IgG levels, higher IgM levels, chronic GvHD, positive recipient CMV status, and negative titers prior to immunization. These clinical factors are useful to formulate immunization and monitoring strategies for survivors after allo-HSCT. Patients at risk for vaccine failure should be monitored closely during long-term follow-up, and booster immunizations should be considered when there is no seroconversion.Table.Factors associated with negative titers after re-immunizationVaccineFactorOdds ratio95% confidence intervalp value DiphteriaT cell depleted graft5.521.14-26.820.034 TetanusOlder age at immunization1.541.05-2.220.026 Lower IgG level*1.031.00-1.050.033 MeaslesLower IgG level*1.011.00-1.020.022 MumpsHigh risk vs. standard risk2.631.02-6.670.045 RubellaHigher IgM level*1.121.03-1.230.012 High risk vs. standard risk16.71.56-100.000.02 PoliovirusType 1Older age at immunization1.431.05-1.920.021 Positive recipeint CMV titer3.181.58-6.410.001 Higher IgM level*1.081.01-1.150.027 Negative titer before immunization8.332.17-33.330.002 Type 2Older age at immunization1.411.04-1.920.027 Positive recipeint CMV titer2.651.36-5.200.004 Higher IgM level*1.061.00-1.140.04 Negative titer before immunization14.34.55-50.00<0.001 Type 3Older age at immunization1.411.04-1.890.027 Positive recipient CMV titer3.111.67-5.80<0.001 Higher IgM level*1.061.00-1.120.035 Chronic GVHD2.71.05-6.950.04 Negative titer before immunization5.881.92-20.000.002*Total IgG and IgM, not disease specific Ig levels

Disclosures:
No relevant conflicts of interest to declare.
Adoptive Transfer of Hexon-Specific T-Cells as a Treatment of Adenovirus Reactivation Following Allogeneic Stem Cell Transplantation.
Authors: Opherk Kathrin , Schuster Friedhelm R , Bethge Wolfgang Andreas , Bader Peter , Greil Johann , Stuhler Gernot , Albert Michael H , Sauer Martin G. , Handgretinger Rupert , Lang Peter , Feuchtinger Tobias 
Published: October 2019

Abstract
Abstract 796
In pediatric patients human adenovirus (HAdV) was identified as a common viral pathogen responsible for significant morbidity and mortality post allo SCT. Antiviral chemotherapy is often insufficient. Given that T-cell immunity is crucial for protection against adenoviral infection/reactivation, cellular immunotherapy is a promising therapeutic option. The capsid protein Hexon has been shown to contain immunodominant T-cell epitopes, with T-cell responses in the majority of the healthy population. Therefore a prospective phase I/II clinical study was performed analysing safety and feasibility of adoptive Hexon-specific T-cell transfer in patients after allogeneic SCT and HAdV infection refractory to Cidofovir treatment. Hexon-specific T-cells were isolated from the SCT-donor by using the IFNγ secretion system and small T-cell populations were immediately infused, without in vitro expansion steps. Fourty pediatric and adult patients with a mean age of 15 years were treated according to the study protocol after haploidentical, matched unrelated and matched sibling donor SCT between day 11 and 327 post SCT. The T-cell dose varied from 300-25000 T-cells/kg. No acute toxicitiy was observed. In two patients GvHD °I-°II of the skin occured within two weeks after administration of specific T-cells, one patient also developed GvHD of the gut. In vivo T-cell responses were absent in all patients before adoptive T-cell transfer and detectable in 70% of evaluable patients within the first weeks after adoptive transfer, associated with a clinical and/or virological response to the adoptive T-cell transfer. However, in patients with adenoviral disease response rate was lower and 6 of 14 evaluable patients succumbed with the infection within few days, in spite of adoptive immunotherapy. This lead to the assumption, that adoptive treatment in patients with severe infection related morbidity was to late during the course of infection. In conclusion we could show that adoptive immunotherapy is safe, feasible and a promising therapeutic option in patients with HAdV infection. Infusion of small IFNγ producing Hexon-specific T-cells populations resulted in an in vivo expansion of specific T-cells in the majority of cases. Emergence of in vivo T-cell responses was closely associated with a clearance or reduction of the viral load.

Disclosures:
No relevant conflicts of interest to declare.
Identification of Single Human Hematopoietic Stem Cells Capable of Long-Term Multilineage Engraftment and Self-Renewal.
Authors: Notta Faiyaz , Doulatov Sergei , Dick John E. 
Published: October 2019

Abstract
Abstract 816
A fundamental tenet that has guided our insight into the biology of hematopoietic stem cells (HSCs) over the past 50 years is the principle that an HSC can only be assayed by functional repopulation of an irradiated host1. In its strictest definition, only a HSC can provide long-term reconstitution of all the major lineages following single cell transplantation. However, the existing strategies for human HSC isolation lack quantitation and do not submit to this rigorous standard, thus precluding further biological analysis. Here, we report the prospective and quantitative analysis of human cord blood (CB) HSCs transplanted into female NOD/SCID/IL-2Rgcnull mice. We identify integrin a6 (CD49f) as a novel marker of cord blood (CB) HSCs and report that single Lin-CD34+CD38-CD90+CD45RA-RholoCD49fhi cells can reconstitute myeloid, B-, and T-cell lineages for 18 weeks. 5 of 29 mice transplanted with single cells gave rise to human cells indicating that approximately 20% of cells in this fraction are HSCs. This advance finally enables utilization of near-homogeneous populations of human HSCs to gain insight into their biology and to harness them for stem cell-based therapeutics.

Disclosures:
No relevant conflicts of interest to declare.
HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.
Authors: Chapman Michael A , Brunet Jean-Philippe , Keats Jonathan J , Baker Angela , Adli Mazhar , Schinzel Anna C. , Ahmann Gregory , Christina Harview , Moore Anna , Shanmugam Vijayalakshmi , Perkins Louise M. , Auclair Daniel , Hahn William C. , Bernstein Bradley E. , Bergsagel Leif , Fonseca Rafael , Carpten John , Trent Jeffrey , Golub Todd R. 
Published: October 2019

Abstract
Abstract 832
We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM.

Disclosures:
No relevant conflicts of interest to declare.
Autologous Hematopoietic Stem Cell Transplantation (autoHSCT) in CLL: First Results of An EBMT Randomized Trial Comparing Autotransplant Versus Wait and Watch.
Authors: Michallet Mauricette , Dreger Peter , Sutton Laurent , Brand Ronald , Richards Sue , van Biezen Anja , Sobh Mohamad , Gratwohl Alois , Kolbe Karin , Dearden Claire E. , Hallek Michael , de Witte T. M. , Niederwieser Dietger W. , Leporrier Michel , Milligan Donald 
Published: October 2019

Abstract
Abstract 877
This phase-III randomized EBMT-intergroup trial studied the impact of a consolidating autoHSCT vs no consolidation for patients with CLL in Binet stage A progressive, B or C , in CR, nodular PR or VGPR after first or second line therapy. The primary objective was to show that autoHSCT increased the 5-year progression-free survival (PFS) by 30%. Although it had been calculated that 270 patients were to be randomized, the study was terminated by the steering committee in July 2007 due to poor accrual. Here we present a first analysis based on 69% of expected follow-up forms.

Results:
Between November 2001 and July 2007, 223 patients were enrolled (SFGM-TC/FCLLG n=98, MRC n=62, GCLLSG n=32, SAKK n=10, other EBMT centers n=17). There were 74% males and 26% females. Binet stages were progressive A 13%, B 67%, C 20%; 59% were in CR, and 41% in very good or nodular PR. Of note, SFGM-TC/FCLLG included only patients in CR. 82% of the patients were enrolled in 1st, and 18% in 2nd remission. Patients were randomized between group 1 (autoHSCT n=112) and group 2 (observation n=111) after an induction treatment which was left at the discretion of the investigators. Median PFS was 43 months in the observation group but not reached in the autoHSCT group; 5-year PFS was 48% and 65%, respectively (p=0.005). Accordingly, autoHSCT halved the relapse risk (5-year relapse incidence 25% vs. 51%; HR 0.4 [0.23-0.71], p=0.002). Cox modeling for randomization arm, Binet stage, disease status, line of treatment, contributing group (country), and the interaction between randomization arm and contributing group confirmed that autoHSCT significantly improved PFS (HR 0.41 [0.23-0.75] p=0.004). The beneficial effect of autoHSCT was stable over all contributing groups although patients accrued by SFGM-TC/FCLLG overall had a significantly better PFS than patients from other countries (HR 0.2 [0.08-0.55], p=0.001). At 5 years, the probability of OS was 92% and 91% for autoHSCT and observation, respectively. Significant differences in terms of non-relapse death were not observed. At the last follow up, among 205 evaluable patients, 186 are alive (147CR, 39 relapse), 19 died (14 from relapse and 5 from non-relapse causes) . In conclusion, in patients with CLL in first or second remission, consolidating autoHSCT reduces the risk of progression (PFS) by more than 50%, but has no effect on overall survival.

Disclosures:
No relevant conflicts of interest to declare.
Detrimental Role of Excess Adenosine-Mediated 2,3-Diphosphoglycerate Induction in Erythrocyte Sickling and Novel Mechanism-Based Therapies.
Authors: Zhang Yujin , Dai Yingbo , Wen Jiaming , Kellems Rodney E. , Blackburn Michael R , Juneja Harinder S. , Xia Yang 
Published: October 2019

Abstract
Abstract 906

Introduction:
Sickle cell disease (SCD) is a devastating inherited hemolytic disorder due to a single missense mutation in the β-globin gene resulting in the production of hemoglobin S (HbS). Despite our knowledge of the molecular defect in HbS, we remain unable to control HbS polymerization and erythrocyte sickling under hypoxic conditions, which are central to the pathophysiology of the disease.

Methods and Results:
Here we report that adenosine, a cellular metabolite that is well known to be induced under hypoxic/ischemic conditions, is increased in the blood circulation of patients with SCD and in SCD transgenic (Tg) mice. To our surprise, we found that adenosine contributes to hypoxia-induced sickling of cultured erythrocytes from humans with SCD. Next, to evaluate the pathogenic role of increased adenosine in erythrocyte signaling in vivo, we took advantage of SCD transgenic (Tg) mice, a well accepted animal model of SCD. Intriguingly, treatment of SCD Tg mice with polyethylene glycol-modified adenosine deaminase (PEG-ADA) enzyme therapy to lower circulating adenosine levels reduced sickling and attenuated multiple tissue damage (including lung, kidney, liver and spleen) seen in SCD Tg mice. These findings revealed a previously unrecognized role for excess adenosine in sickling and the progression to multiple life-threatening complications. Next, by screening erythrocytes for small metabolites that are induced by adenosine and may contribute to sickling, we determined that 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific metabolite known to promote O2 release from Hb, is elevated in erythrocytes of both SCD Tg mice and humans. Using both pharmacological and genetic approaches, we demonstrated that adenosine-mediated 2,3-DPG induction is through A2B receptor signaling and that this signaling pathway is a major contributor to hypoxia-induced erythrocyte sickling.

Conclusion and Significance:
Overall, we have identified for the first time that elevated adenosine induces 2,3-DPG via A2BR signaling and contributes to sickling in both human and mouse with SCD. Thus, our studies provide strong support for the new concept that adenosine-mediated 2,3-DPG induction is beneficial for normal erythrocytes by promoting O2 release from Hb to hypoxic tissues. However, for SCD patients, the beneficial effect of adenosine-mediated 2,3-DPG induction becomes detrimental by inducing erythrocyte sickling due to 2,3-DPG mediated increased O2 release and increased deoxyHbS polymerization. Our findings reveal a novel therapeutic possibility to treat and prevent sickling and progression to multiple life-threatening complications by targeting on the adenosine signaling pathway.

Disclosures:
No relevant conflicts of interest to declare.
Crosstalk Between the Bone and Immune Systems: Osteoclasts Function as Antigen-Presenting Cells and Activate CD4+ and CD8+ T Cells.
Authors: Li Haiyan , Hong Sungyoul , Qian Jianfei , Yang Jing , Zhang Liang , Yi Qing 
Published: October 2019

Abstract
Abstract 918
Bone is a dynamic tissue that is constantly being remodeled by bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OB). Bone destruction, observed in patients with autoimmune disease rheumatoid arthritis and malignancies such as multiple myeloma, is believed to be caused by hyperactivation of OCs. OCs differentiate from hematopoietic monocytic precursors under stimulation by the cytokines RANKL (receptor activator of nuclear factor κB ligand) and M-CSF (macrophage colony-stimulating factor), which are produced primarily by bone marrow stromal cells, OBs, and activated T cells. As OCs are derived from monocytes/macrophages lineage similar to dendritic cells (DCs), we hypothesized that OCs could serve as antigen-presenting cells (APCs) to activate T cells. In this study, OCs were generated from human monocytes with the stimulation of RANKL and M-CSF in vitro. DCs derived from monocytes with the stimulation of GM-CSF and IL-4 were used as positive controls. First, we examined the phenotype of OCs by flow cytometry, and OCs were detached by non-enzymatic cell dissociation solution. Results showed that OCs expressed MHC class I and II molecules and costimulatory molecules important for APCs such as CD80, CD86 and CD40. The expression of these molecules could be upregulated by LPS and IFN-γ. Second, we showed by PCR that OCs expressed IL-10, TGF-β, IL-6 and TNF-α, but not IL-12p35 and p40. Third, we examined the ability of OCs to present alloantigens and activate alloreactive T cells in a mixed lymphocyte reaction assay. OCs could present allogeneic antigens and activate both CD4+ and CD8+ alloreactive T cells to proliferate as detected by [3H]thymidine incorporation and CFSE dilution assay. The activation was restricted by MHC class I and II molecules. Finally, we recruited tetanus toxoid (TT)-immunized healthy donors to test whether OCs could uptake exogenous soluble antigens and present them to CD4+ T cells. OCs pulsed with TT could activate autologous specific CD4+ T cells, which was MHC II molecule restricted. These findings indicate that OCs could function as APCs and activate both CD4+ and CD8+ T cells. Thus, our study provides new insight to the effect of OCs on the immune system especially T cells. This is not only important for a better understanding of the crosstalk between the bone and immune system but also may help develop novel strategies for treating diseases such as rheumatoid arthritis and multiple myeloma, which affect both the bone and immune systems.

Disclosures:
No relevant conflicts of interest to declare.
Coordinate Loss of a MicroRNA Mir 145 and a Protein-Coding Gene RPS14 Cooperate in the Pathogenesis of 5q- Syndrome.
Authors: Kumar Madhu , Narla Anupama , Nonami Atsushi , Ball Brian , Chin Christine , Chen Cindy , Kutok Jeffery Lorne , Galili Naomi , Raza Azra , Attar Eyal C. , GIlliland D. Gary , Jacks Tyler , Ebert Benjamin L. 
Published: October 2019

Abstract
Abstract 947
Large hemizygous chromosomal deletions are among the most common molecular abnormalities in cancer, but the identification of critical haploinsufficiency disease genes within the deleted regions has been difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a well-studied chromosomal deletion syndrome characterized by a consistent clinical phenotype with macrocytic anemia and thrombocytosis. We have previously shown that while hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not account for the thrombocytosis. Evaluation of the effects of all protein coding genes in the CDR on hematopoietic differentiation showed no genes other than RPS14 altered the ratio of megakaryocytic to erythroid cells, either alone or in combination with RPS14. We therefore examined the 5q- syndrome CDR for non-coding RNAs and identified a microRNA, miR-145, which targets Fli-1, a transcriptional factor that regulates megakaryocyte development. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 in CD34+ cells decreases megakaryocyte production, while inhibition of miR-145 or overexpression of Fli-1 has the reciprocal effect. These findings have been validated in vivo using transgenic mice. Moreover, the combined loss of miR-145 and RPS14 cooperate to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate for the first time that coordinate deletion of a microRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome.

Disclosures:
No relevant conflicts of interest to declare.