bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfer of Immunity Using Vaccinate and Boost Strategy with Bispecific Antibody Armed T Cells (BATs) in Metastatic Breast Cancer Patients
Authors: Thakur Archana , Uberti Joseph P. , Ratanatharathorn Voravit , Lum Lawrence G. 
Published: October 2019

Abstract
Background. In our recent phase I immunotherapy (IT) trial in 23 women with metastatic breast cancer (MBC), 8 infusions of activated T cells (ATC) armed with anti-CD3 x anti-HER2 bispecific antibody (HER2Bi) given in combination with interleukin-2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) induced specific anti-breast cancer immunity. This study investigated whether specific cellular and humoral anti-breast cancer immunity induced by infusions of HER2 bispecific antibody armed T cells (BATs) could be transferred after high dose chemotherapy (HDC) and stem cell transplant (SCT) in MBC patients. Methods. T cell were activated with OKT3 and expanded in IL-2. ATC were harvested, armed with HER2Bi, and cryopreserved in 8 doses for twice weekly infusions for 4 weeks along with IL-2 and GM-CSF. Seven to 14 days after the last infusion of BATs, the patient was leukapheresed again to obtain and expand immune T cells. Multiple infusions of immune ATC after the HDC and SCT were given to boost the transferred cellular and humoral immune responses which were monitored up to 24 months. Results. Six of 8 MBC patients enrolled were evaluable in the protocol, no dose-limiting toxicity, delays in engraftment, or life-threatening infections were observed. Five of 6 evaluable patients exhibited increased anti- breast cancer cytotoxicity and IFN-γ Elispots after vaccination with BATs and up to 18 months post SCT. Serum and culture supernatants from in vitro antibody synthesis assay showed gradual increases in anti-SK-BR-3 IgG levels after SCT. Serum cytokine profile showed increases in IL-12 and Th1 cytokines. One out of 6 evaluable patients who rapidly progressed showed poor immune response, had high serum levels of Th2 cytokines and no evidence of transfer of immunity. Flow cytometry analysis of Vβ repertoire pattern in PBMC collected post IT and post SCT indicate transfer of the major Vβ clones post SCT that produced IFN-γ upon stimulation with breast cancer cells (Fig. 1). A significant correlation (r=1.0; p<0.002) between immune ATC cytotoxicity directed at breast cancer cells and time to progression (TTP) suggests that more robust vaccinations with a Th1 shift in cytokine profiles can lead to clinical benefit (Fig. 2). Conclusions. Our pilot study suggests that cellular and humoral immunity was transferred and boosted using immune T cells after SCT. There was robust reconstitution of T and B cell functions early after SCT as evidenced by CTL and NK activity, IFN-γ EliSpots, in vivo/in vitro antibody synthesis, and Th1 cytokine responses.BATs induced endogenous anti- breast cancer cellular, humoral and innate immunity that could be detected after SCT and may have provided clinically meaningful anti-tumor immunity.

Disclosures
No relevant conflicts of interest to declare.
Gene Editing of the Human Pklr Gene in Human Hematopoietic Progenitors to Correct Pyruvate Kinase Deficiency
Authors: Quintana-Bustamante Oscar , Fañanas Sara , Orman Israel , Gouble Agnès , Galetto Roman , Poirot Laurent , Bueren Juan A. , Segovia Jose C. 
Published: October 2019

Abstract
Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. This gene encodes the erythroid specific Pyruvate Kinase (RPK) enzyme, implying that this defective enzyme fails to produce normal levels of ATP and consequently, erythrocytes from PKD patients show an energetic imbalance. Hematopoietic stem cell gene therapy using gene editing will be the safest approach to treat PKD patients. However, its direct application in hematopoietic progenitor cells (HPCs) is challenging. Different gene editing approaches are being explored to correct PKD, either by Knock-In of an optimized cDNA sequence in the first introns of the gene or by site-specific correction using ssODN. While Knock-In strategy will allow the treatment of most PKD mutations, an ssODN-mediated gene editing will correct specific PKD patient-mutations. In our Knock-In system, a recombination matrix carrying an exon 3-11 partial codon optimized version of RPK cDNA and a puromycin selection cassette is inserted in the second intron of the PKLR gene assisted by TALEN® nucleases. We have previously shown that this approach was effective to correct PKD phenotype in PKDiPSC lines. Here, we have tested the feasibility of our approach in hematopoietic progenitors. Thus, the therapeutic matrix together with specific TALEN® for the second intron of PKLR was electroporated in purified cord blood CD34+ from healthy donors. Cells were then expanded and puromycin selected to enrich the population for gene edited ones. Up to 96% of the colony forming units that survived the puromycin selection showed the specific integration of the donor cassette. After transplantation into immunodeficient NSG mice, a low though detectable percentage of gene edited cells was . Additionally, CRISPR-Cas9 site-specific correction is being developed by combining guide RNAs directed against PKD patient's mutation and precise ssODN to restore RPK protein. Overall, our data show that gene editing in engraftable HPCs is feasible, although further improvements are needed to increase efficiency of this gene therapy appraoch prior to consider its therapeutic application in PKD patients.

Disclosures
Gouble: Cellectis: Employment. Galetto:Cellectis SA: Employment. Poirot:Cellectis: Employment.
Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium
Authors: Sugimura Ryohichi , Han Areum , Jha Deepak , Lu Yi-Fen , Goettel Jeremy A , Serrao Erik , Rowe Robert Grant , Wong Irene , Sousa Patricia , Ditadi Andrea , Keller Gordon , Engelman Alan , Snapper Scott , Doulatov Sergei , Daley George Q. 
Published: October 2019

Abstract
A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Disclosures
No relevant conflicts of interest to declare.
Thrombin Generation: Plasma Composition Imbalance and Mortality in HIV
Authors: Brummel-Ziedins Kathleen E. , Baker Jason V , Gissel Matthew , Neuhaus Jacqueline , Neaton James D , Tracy Russell P. 
Published: October 2019

Abstract
Introduction: Effective treatment of HIV infection with antiretroviral therapy (ART) has prolonged survival and shifted causes of death to non-AIDS defining illnesses such as cardiovascular disease (CVD). Increased thrombin generation is evident from increased D-dimer levels in HIV; previously we have shown that inflammation and viral replication associate with imbalances in pro and anticoagulant factors resulting in increased thrombin generation profiles when mathematically modeled. We explore for the first time the hypothesis that factor compositional imbalance increases simulated thrombin generation and predicts mortality in HIV.
Methods: In a nested case-control study of the SMART and ESPRIT participants randomized to continuous ART, we evaluated cases of all-cause mortality and controls (1:3) matched on enrollment date, age (± 5yrs), study, and country. Thrombin generation in response to a 5 pM tissue factor initiator for each individual was calculated by a mathematical model incorporating levels of factors II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor and protein C. Thrombin generation metrics included time to clot, maximum rate (MaxR), maximum level (MaxL) and area under the curve (AUC). Conditional logistic regression was used to compare parameters between cases and controls, adjusted for age, gender, and race/ethnicity.
Results: Among cases (n=129) and controls (n=369), respectively, median CD4 count was 451 and 496 cells/mm3, and 65 and 77% had HIV viral suppression (<400 copies/mL). Levels of antithrombin (median: 125% vs. 128%, p=0.084) and protein C (median: 109% vs. 117%, p=0.005) were decreased and factor V (median: 112% vs 98%, p=0.014), FVIII (median: 145% vs 129%, p=0.006) and TFPI (median: 27 ng/mL vs. 26 ng/mL, p=0.073) were higher in cases versus controls. The overall effect is a more procoagulant simulated phenotype. In mortality cases versus controls, MaxR (median: 0.44 nM/s vs. 0.35 nM/s, p=0.064), MaxL (68 nM vs. 56 nM, p=0.037) and AUC (18 µM*s vs. 14 µM*s, p=0.016) were all more procoagulant, although clot time was unaffected. Among mortality cases thrombin generation (MaxL) was greater among those in the highest versus lowest quartile of IL-6 (p=0.006). In controls thrombin generation (MaxL) did not vary by IL-6 level (p=0.93).
Conclusions: Antithrombin, FV and FVIII, and PC were seen as the major contributors to the increased simulated thrombin generation among HIV+ mortality cases. Our results are consistent with the hypothesis that HIV is associated with an increase in thrombin generation via altered balance of pro and anticoagulant factors, and the resulting coagulopathy is associated with increased mortality.

Disclosures
No relevant conflicts of interest to declare.
Ribosomal Protein Rpl22 Regulates Development and Transformation Via Altering RNA Processing
Authors: Wang Minshi , Ser Zheng , Rao Shuyun , Fahl Shawn , Zhang Yong , Peri Suraj , Kentsis Alex , Wiest David L 
Published: October 2019

Abstract
Although it has long been reported that mutations in ribosome proteins (RP) are associated with increased cancer risk in humans, the molecular basis why which RP mutations do so remains unclear. Nevertheless, the prevailing view is that RP mutations, such as Rps19, are thought to alter transformation potential through general impairment of ribosome biogenesis or function. Importantly, recent observations are beginning to challenge this notion as too simplistic. We have determined that the RP, Rpl22, is not essential for ribosome biogenesis or global protein synthesis; however, its inactivation impairs the development of normal T lymphocytes and increases their transformation potential. Indeed, RPL22 is inactivated in human T acute lymphoblastic leukemia (T-ALL) and this is associated with reduced survival. Moreover, Rpl22-deficiency accelerates development of leukemia in a myristylated Akt2 transgenic (MyrAkt2 Tg) mouse model of T-ALL. To gain insight into how Rpl22 inactivation facilitates development of leukemia, we are performing unbiased transcriptomic and proteomic analysis on Rpl22+/+ and Rpl22-/- thymic lymphomas arising in the MyrAkt2 Tg model, and in an Rpl22-/- lymphoma reconstituted with Rpl22. Interestingly, relatively few changes in mRNA transcript read depth were observed; however, substantial differences in the proteome were observed. Pathway analysis revealed that the loss of Rpl22 altered the expression of proteins regulating RNA-processing, in particular RNA-splicing. Interestingly, interrogation of the transrciptome data for alternative splicing revealed that alterations in exon usage. The ability of Rpl22 to influence splicing appears to be conserved across species as alternative splicing was also observed in zebrafish embryos in which Rpl22 was knocked down using morpholino oligonucleotides. Consequently, we hypothesize that Rpl22 regulates biological events through its ability of binding to RNA targets, and controlling the expression of their protein products at least in part through altering mRNA splicing. How Rpl22 changes mRNA splicing pattern is currently under investigation.

Disclosures
No relevant conflicts of interest to declare.
Measurement of Quality of Life and Depression of Spouse Caregivers for Survivors after Hematopoietic Stem Cell Transplantation
Authors: Park Silvia , Cho Juhee , Jang Jun Ho , Jung Chul Won , Heo Mi Hwa 
Published: October 2019

Abstract
Introduction: Hematopoietic stem cell transplantation (HSCT) is a quite exhausting process, which encompasses multitude of chemotherapy before HSCT and long term care and surveillance in post transplant period. Caregivers are also exposed to this whole process, and are often the sole provider of physical and emotional support for the patients.
Methods: This was a cross-sectional, HSCT survivor-spouse caregiver matching study to determine the caregiver's quality of life (QoL) and depression. From September 2013 to March 2015, a total of 103 HSCT survivor-partner pairs were answered to a set of questionnaire for the assessment of QOL and depression, which were measured by the World Health Organization Quality of Life (WHOQOL)- BREF 26 and Patient Health Questionnaire-9 (PHQ9), respectively.
Results: Among a total of 103 pairs, the data from 97 married couples were finally used for the analysis. Mean age of the caregiver participants was 49.7 years (range 30-73) and majority of caregivers were female (n=65, 67%). More than 70% of pairs were assessed with a monthly income of
$2,000-4,000 or more, and 55.2% of caregivers responded to have co-morbidities. Mean duration since transplant was 3.2 years, and allogeneic HSCT comprised 68.7% of cases. As a result, overall QoL, psychological health, social relationships and environmental health were not significantly different between survivors and caregivers (p=0.345, 0.424, 0.415, and 0.253) whereas physical QoL was reported to be better in caregivers compared to survivors (p=0.011). Mean depression scale of depression was comparable between survivors and caregivers with mean score of 5.3 and 5.1 (p=0.812) and there was no difference in the proportion of severe depression between the two groups (15.6% in survivors, 13.7% in caregivers) (p=0.270). It was revealed that family income had significantly correlation with caregiver's overall QoL and environmental health (p=0.013, and 0.023), and female gender, co-morbidites and family income were the important factors associated with caregiver's depression (p=0.007, 0.017 and 0.049).
Conclusion: The present study identified that there was no significant difference in QoL and the level of depression between HSCT survivors and spouse caregivers. Family income, gender and co-morbidites showed significant association with caregiver's distress. We suggest that psychosocial intervention starting from a clinical attention to both survivors and caregivers is needed, and that social support to families who are financially challenged has to be considered.

Disclosures
Jang: Kyowa Hakko Kirin Co., Ltd.: Research Funding.
Wnt Signaling in Red Blood Cells
Authors: Rosin-Arbesfeld Rina , Caspi Michal , Siman-Tov Ronen , Levi Yakir , Perry Chava 
Published: October 2019

Abstract
Introduction : Red blood cells (RBCs) are the most common cell type in the human body.
These cells deliver oxygen to the body's tissues and are composed of a cytoplasm that is rich in hemoglobin and is surrounded by a membrane that is essential for the cells function by providing properties such as stability and deformability. The membrane is composed of a lipid bilayer, transmembrane proteins, and a filamentous meshwork of proteins such as actin and adducin that forms the cells cytoskeleton along the entire cytoplasmic surface of the membrane. RBCs lack a nucleus and other cellular organelles that enable gene expression. Thus, it has been assumed, that signaling cascades such as the Wnt signaling pathway are not active in RBCs.
The Wnt pathways are fundamental for normal development and homeostasis and regulate, among other, cell growth, motility and differentiation. Although there is no functional data connecting between Wnt signaling and RBCs, previous proteomic studies have shown that some of the non-canonical Wnt signaling components, such as specific small GTPases and kinases are present in RBCs. In this study, we show for the first time that Wnt ligands activate signaling cascades in RBCs.
Methods : RBCs were collect from healthy donors and treated with different Wnt ligands. A large number of methods were used to evaluate the RBCs morphological properties, life span, vitality, flexibility and protein expression patterns.
Results : Our results clearly show that different Wnt ligands can dramatically increase the live span of RBCs by affecting the cells membrane cytoskeleton. Some of the Wnts affects include activation of GTPases such as Rac, JNK and RhoA, which lead to actin modification. These changes in the actin cytoskeleton result in increased membrane stability. In parallel, treating RBCs with Wnts leads to activation of PKC and RhoA resulting in phosphorylation of adducin which in turn increases the cells membrane flexibility and improves the vitality of the cell.
Summary : Our novel findings indicate that the non-canonical Wnt signaling pathway in RBCs is active and can stabilize the RBCs cytoskeleton enhancing the vitality, deformability and life span of the cell.
These novel findings may help in the development of new therapeutic strategies for people suffering from different hemolytic disease that effect the RBCs cytoskeleton and provide new insights into the improvement of stored RBC units.

Disclosures
No relevant conflicts of interest to declare.
Clinical Characteristics and Factors of the Impact on the Survival in 133 De Novo FLT3-ITD Acute Myeloid Leukemia with Intermediate Chromosome Karyoptye from a Single Center of China
Authors: Qiu Shaowei , Lin Dong , Wei Hui , Zhang Guangji , Zhou Chunlin , Liu Kaiqi , Wei Shuning , Wang Ying , Liu Bingcheng , Gong Benfa , Gong Xiaoyuan , Liu Yuntao , Mi Yingchang , Wang Jianxiang 
Published: October 2019

Abstract
Objects: Investigate the clinical characteristics and factors of the impact on the survival of FLT-ITD mutation in the acute myeloid leukemia(AML) with intermediate chromosome karyoptye.
Methods: We retrospectively collected and analyzed information regarding clinical characteristics and survival data from 133 patients with newly diagnosed AML with FLT3-ITD mutation and intermediate chromosome karyoptye from 2001-2015.The median follow-up time was 13.02 months.
Results: 1. The median age was 37, median WBC was 50×109/L , the incidence between male and female was similar, the most common FAB subtype was M5(60.2%), the hepatosplenomegaly incidence was 8.4% .
2.The complete remission (CR) was 64.5% after first induction chemotherapy. CR could reached 74.4% or 57.1% when induction regimen included the medium dose cytarabine(100mg/m2 d1-4;1g/m2 q12h d5-7) or standard dose cytarabine(Ara-C)(100mg/m2d1-7) respectively(p=0.063).
3. Totally 131 patients received chemotherapy, and subsequently 22 patients received transplantation after chemotherapy. When the patients only received chemotherapy, 3-year OS was 39.9%, 3-year DFS was 44.8% and while in the patients who received transplantation, 3-year OS was 73.5%, 3-year DFS was 77.5%, the outcome was better (p<0.05).
4. Univariable analysis showed that OS was related with age, hepatosplenomegaly, transplantation, WBC, the remission of induction chemotherapy. DFS was related with transplantation, courses for CR. Cox regression showed that transplantation and remission of induction chemotherapy were the independent prognosis factor. When the patient only received chemotherapy, FACS-MRD persisted negative was the independent prognosis factor.
Conclusions: The FLT3-ITD de novo AML patients with intermediate chromosome karyoptye had unique characteristics. The patients who attained CR after induction chemotherapy and received transplantation would get better outcome, while in the patients who only received chemotherapy, FACS-MRD persisted negative implied better outcome. The induction regimen that contained medium Ara-C could reach higher CR , that may translated into better survival, therefore it may be the appropriate choice for FLT3-ITD AML patients.

Disclosures
No relevant conflicts of interest to declare.