bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Marrow-Derived Mesenchymal Stem Cells Overexpressing Akt1 Protect Against ConA-Induced Liver Injury in Mice
Authors: Zhou Lukun , Liu Shuang , Lu Chuanyi M. , Yao Jianfeng , Shen Yuyan , Yang Xin , Feng Sizhou , Han Mingzhe
Published: October 2019

Liver injury associated with veno-occlusive disease and graft-versus-host disease (GVHD) is a frequent and severe complication of hematopoietic stem cell transplantation, and remains an important cause of transplant-related mortality. Bone marrow derived mesenchymal stem cells (MSCs) have been evaluated for the prevention and treatment of refractory GVHD. However, poor cell viability has limited the therapeutic capacity of mesenchymal stromal cell therapy in vivo. In this study, we genetically engineered C57BL/6 mouse bone marrow MSCs using ex vivo retroviral transduction to overexpress Akt1, a serine threonine kinase and pro-survival signal protein, and tested the hypothesis that Akt1-expressing MSCs (Akt1-MSCs) are more resistant to apoptosis and can ameliorate acute liver injury induced by concanavalin A (ConA) in BALB/c mice. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ (100 ng/mL) stimulation conditions, Akt1-GFP-MSCs had proliferation and survival (anti-apoptotic) advantages with down-regulated apoptosis pathways, compared to control GFP-MSCs. Twenty-four hours after receiving lethal dose of ConA (40 mg/kg, intravenous) (N=10 each group), no mouse survived, with or without 1x106 Akt1-MSCs or GFP-MSCs administration (intravenous); however, 3 and 1 survived in the 5×106 Akt1-MSCs group and 5×106 GFP-MSCs groups, respectively. In subsequent sub-lethal dose ConA (20 mg/kg) experiments, compared to GFP-MSCs, mice received Akt1-MSCs administration had significantly lower serum AST, ALT, TNF-α and IFN-γ levels and less histopathological abnormalities. In addition, Akt1-MSCs treated mice had significantly higher serum concentrations of IL-10, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). In vivo imaging showed that, hepatic fluorescence signal in sub-lethal ConA+Akt1-MSCs group was significantly stronger than ConA+GFP-MSCs group on day 0, and persisted up to 14 days, whereas the signal in ConA+GFP-MSCs, Akt1-MSCs and GFP-MSCs groups was negligible on both day 7 and day 14. Thus, bone marrow derived MSCs genetically enhanced with Akt1 had survival advantage in vitro and in vivo, and have the potential to be a potent therapy for prevention and amelioration of GVHD-associated liver impairment. Further translational pre-clinical studies are ongoing to further determine the efficacy, dosage and timing of administration of Akt1-MSCs in animal models.

Disclosures

No relevant conflicts of interest to declare.
5-Aza-2'-Deoxycytidine Promotes Cytotoxic Effect of CART-Cells on Leukaemia Cells
Authors: Dolnikov Alla , Rossi Swapna , Xu Ning , Klamer Guy , Shen Sylvie , Micklethwaite Kenneth P. , Lock Richard B , O'Brien Tracey
Published: October 2019

T cells modified to express CD19-specific chimeric antigen receptors (CAR) have shown anti-tumour efficacy in early phase clinical trials in patients with relapsed and refractory B-cell malignancies. In addition to direct cytotoxicity, chemotherapeutic drugs can have an immunomodulatory effect both through enhancing the tumour-specific immune response and increasing the tumour’s susceptibility to immune mediated destruction. Hence, combining immunomodulatory chemotherapy and CAR T-cells is an attractive approach for eliminating tumours, particularly in advanced stages. 5-aza-2'-deoxycytidine (5-AZA) is a hypomethylating agent that induces terminal differentiation, senescence or apoptosis in haematological malignancies. Here, we have explored a CAR-based immunotherapy combined with 5-AZA to maximise the effect of the CAR T-cells against CD19+ B-cell leukaemia. A second generation CAR including CD3zeta and the CD28 co-stimulatory domain was cloned into the PiggyBac-transposon vector and was used to generate CAR19 -T cells. Cord blood -derived mononuclear cells (MNC) were transfected with CAR19-transposon/transposase plasmids and expanded with CD3/28 beads for 2 weeks in the presence of 20ng/ml IL2 and 10ng/ml IL7. CAR19 T-cells efficiently induced cytolysis of CD19+ leukaemia cells in vitro and exhibited anti-tumour activity in vivo in a xenograft mouse model of leukaemia. Pre-treatment with 5-AZA produced greater leukaemia cell cytolysis in vitro and maximised anti-tumour activity of CAR19 T-cells in vivo against xenograft primary leukaemia compared to 5-AZA or CAR19 T-cells alone. In vitro analysis revealed that pre-treatment with 5-AZA up-regulates CD19 expression in leukaemia cells and improves CAR19 T-cell recognition of target cells increasing the formation of effector/ target cell conjugates and target cell cytolysis. Therefore using 5-AZA pre-treatment can be particularly useful for B-cell leukaemias with reduced expression of CD19. We have also demonstrated that pre-treatment of target cells with 5-AZA potentiates the effect of CAR19 T-cells used at low dose or low effector:target (E:T) suggesting that even small numbers of CAR19 T-cells can mediate a potent antitumor effect when combined with 5-AZA pre-treatment of target cells. This is particularly important for patients receiving limited numbers of CAR T-cells or for patients with large leukaemic burden. In addition, we speculate that the enhanced cellular cytotoxicity produced by 5-AZA-conditioning may allow the infusion of decreased numbers of CAR19 T-cells.

Disclosures

No relevant conflicts of interest to declare.
What Can We Do for the Unrelated Donors of Hematopoietic Stem Cell to Lessen Their Agony?
Authors: Lee Mark , Na Jung Hwa , Jang Hyung In , Jang Jin Ho , Lee Jongchan , Lyu Chuhl Joo , Kim Sung-Yong
Published: October 2019

Objectives: The study purposes are as follows. What are the incidences of adverse events (AEs) after hematopoietic stem cell donations among the unrelated donors registered in Korea Marrow Donor Program (KMDP)? What donor factors have influence on their general discomfort and the limitation of daily activity at the time of stem cell collection? What prohibits the donors from donating stem cell repetitively as a Good Samaritan?

Methods: 1,666 consecutive unrelated donors were registered at KMDP from November 2007 to October 2013. General discomfort and pain were assessed using a visual analog scale (numerical measurement, 0 to 10) and other donor symptoms were assessed by making either yes or no reply to each item. The limitation of daily activity was assessed by numerical measurement (0 to 10 scales), 10 being the worst activity. The donor’s intention of repetitive donation was assessed by yes or no reply. The donor symptoms and the intention of repetitive donation were assessed within 48 hours after stem cell collection, and then at 1 week, 4 weeks, and 4 months thereafter.

Results: There were 1,292 males and 374 females. 1,525 donors underwent peripheral blood stem cells (PBSC) collection, 100 bone marrow (BM) harvest and 40 leukapheresis for donor leukocyte infusion (DLI). All of 1,525 PBSC donors received granulocyte-colony stimulating factor (G-CSF). 76 donors underwent central line insertion for PB cells collection. The degree of general discomfort and the limitation of daily activity were analyzed in variables of sex, stem cell source, G-CSF administration, collection method, collection period and the intention of repetitive donation. A multiple linear regression analysis showed that general discomfort at the time of stem cell collection was significantly influenced by sex and collection period (P<0.05). The limitation of daily activity at the time of stem cell collection was significantly influenced by sex, stem cell source, and collection method (P<0.05). A multiple logistic regression analysis showed that the intention of repetitive donation at the time of stem cell collection and thereafter was significantly influenced by sex only. At least 80% of stem cell donors had the intention of repetitive donation.

Compared to the donors using peripheral line, the donors using central line for PB cells collection suffered more frequently from various AEs: C-line site discomfort, hands and feet numbness, perioral numbness, dizziness, chills, nausea/vomiting and abdominal pain.

Conclusions: Stem cell collection via peripheral line is recommended to alleviate the physical stress and to improve daily activity and to encourage repetitive donation, and female donors are more sensitive to stem cell collection than male donors.

Disclosures

No relevant conflicts of interest to declare.
Cryostem: Establishment of a National Thematic Collection of Biological Samples Pre- and Post-Allogeneic Hematologic Stem Cell Transplantation for the Study of Graft-Versus-Host Disease
Authors: Peffault de Latour Regis , Fontenille Claire , Yakoub-Agha Ibrahim , Calmels Boris
Published: October 2019

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) is the only curative treatment for many patients with hematologic disease. Unfortunately, the transplanted immune system can reacts against the patient, inducing Graft-versus-Host Disease (GvHD). GvHD is the leading cause of treatment related mortality and morbidity following HSCT and is expected to increase in the forthcoming years. Pathophysiology and long-term determinants of GvHD are still largely unknown.

The French Society for Stem Cell Transplantation and Cell Therapy (SFGM-TC) launched the CryoStem project in 2011 with the goal of establishing a national biobank on GvHD. CryoStem has been selected and funded to the extent of 5,5 millions € over a period of 9 years by the French Government’s Investissements d’Avenir program. As of today, CryoStem brings together 33 HSCT Units and 23 Biological Resources Centers that actively contribute to a networked, prospective, longitudinal and standardized peripheral blood samples collection with concomitant well documented clinical data.

Patients sampling is scheduled before and after HSCT, depending on the occurrence or not of acute and/or chronic GvHD. Three types of pre-analytical products are processed and stored, according to standardized techniques: viable cells in DMSO, dried cell pellets and plasma. Samples and clinical data are anonymized and centralized in CryoStem-dedicated web-based database application.

Recruitment started in July 2012: currently, about 1,590 patients and 690 related donors have been included. So far, 3,820 blood samples have been processed, corresponding to 48,400 frozen pre-analytical samples. In order to guarantee the quality of frozen samples, an ISO9001 certification of the network is ongoing.

National projects are the only way to acquire the large number of samples needed for statistically significant results in a reasonable time. The collection is on embargo until further notice and the first call for scientific projects is expected to be launched in December 2014.

CryoStem is a crucial step toward progress in human GvHD research: it will allow collaborative studies between leading French and international research organizations and foster technology transfer with potential industrial players whenever possible to eventually improve the translational research on GvHD.

Disclosures

No relevant conflicts of interest to declare.
Rituximab in the Treatment of Epstein-Barr Virus Infection after hematopoietic stem cell Transplantation in Children
Authors: Peng Zhiyong , Li Chunfu , He Yuelin
Published: October 2019

OBJECTIVE: To investigate the efficacy and safety of Rituximab on Epstein-Barr virus infection disease after allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in children.

METHODS: A retrospective analysis was performed based on clinical data of 25 children with median age of 6 years, (3~14 years) diagnosed as EBV infection and received Rituximab from Jan. 2012 to Jun. 2014 in our center. Of them, 3 patients were diagnosed as post-transplant lymphoproliferative disorders (PTLD). All patients received Rituximab at dose of 375 mg/m2 once a week for 4 sequence weeks or discontinued therapy when EBV viral loads was <500 copies/mL two times consecutively. The complete remission (CR) was defined as EBV viral loads <500 copies/mL consecutively at least two time and without any related clinical symptoms. Side effects during infusion were evaluated by Common Terminology Criteria for Adverse Events.

RESULTS: The median EBV-infection time was on day 70 (18~200) after HSCT. Each of patients received a median of 2 (1~4) infusions. There were no severe side effects during the infusion of Rituximab. The cumulative complete remission (CR) rate were 88% in total. The CR rate was 56.0±9.9 %, 80.0±8.0%, 84.0±7.3%, and 88.0±6.5%, respectively, in 1st, 2nd, 3rd, and 4th week. Two patients died, one due to infections and the other died of hepatitis while receiving therapy. The CR rate was 100% in patients with PTLD (3/3).

CONCLUSIONS: These data suggest Rituximab is safe and effective when treating children with EBV infection disease after allo-HSCT.

Disclosures

No relevant conflicts of interest to declare.
Effect of Severe Hypoalbuminemia on Myelosuppression and Other Toxicities of High Dose Melphalan and Autologous Stem Cell Transplantation in AL Amyloidosis Patients
Authors: Meehan Robert , Seldin David , Sloan John Mark , Quillen Karen , Brauneis Dina , Shelton Anthony C , Sanchorawala Vaishali
Published: October 2019

Background: Treatment of AL amyloidosis with high dose intravenous melphalan followed by autologous stem cell transplant (HDM/SCT) is effective in inducing hematologic and clinical remissions associated with prolonged survival. The major toxicities are myelosuppression and GI side effects. Studies have shown that ~75% of melphalan in the blood is bound to plasma proteins, with ~25% free. We hypothesized that AL patients with severe nephrotic syndrome and profound hypoalbuminemia might have a higher fraction of free melphalan, a higher effective dose, and greater toxicity of treatment.

Methods: Patients with AL amyloidosis and severe hypoalbuminemia, defined as serum albumin level of < 2 g/dL, treated from 2011 to 2013, were studied retrospectively. The stem cell transplant database was queried for dose of HDM, treatment-related complications, and days of neutrophil and platelet engraftment after SCT.

Results: Of 71 patients with AL amyloidosis who underwent HDM/SCT between Jan 2011 and Dec 2013, 12 patients had severe hypoalbuminemia. Of these, 5 received full HDM at 200 mg/m2 and 7 received modified HDM at 140 mg/m2. All patients received GCSF mobilized peripheral blood stem cells following HDM, with a median stem cell dose of CD34+ cells 8.1 x 106/kg (range, 4.0 to 12.2). The median time to engraftment of neutrophils was 11 days, and not statistically different based upon melphalan dose. The median time to platelet engraftment was 13 days, and also did not differ significantly by dose. These times were similar to controls without severe hypoalbuminemia. Grade 4 toxicities were observed in 2 of 7 patients with modified HDM/SCT and 1 of 5 patients with full HDM/SCT.

Conclusions: These data suggest that patients with severe hypoalbuminemia do not have more prolonged myelosuppression or increased non-hematologic toxicities compared to other patients. In this retrospective study, we did not measure free melphalan concentrations in the blood. However, these data suggest that patients with severe hypoalbuminemia do not require adjustment of melphalan dosing.

Disclosures

No relevant conflicts of interest to declare.
Successful Unrelated Peripheral Blood Stem Cell Transplantation from Unrelated Donor for a Concomitant Paroxysmal Nocturnal Hemoglobinuria and Heterozygous α-Thalassemia Patient
Authors: Wu Xuedong , Wen Jianyun , Liu Xuan , Wang Yanhua , Lu Linglong , Li Na , Li Chunfu
Published: October 2019

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell (HSC) disorder that manifests as hemolytic anemia, venous thrombosis, and deficient hematopoiesis. PNH concomitant with inherited hemolytic anemia has been rarely reported. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the only curative therapeutic measure. Here, We present our first allo-HSCT form unrelated donor for a 23-year-old patient with PNH and heterozygous α-thalassemia.

Case report: Flow cytometry of the patient's peripheral blood demonstrated that 7.9% and 11.9% of the erythrocytes were CD59 and CD55 deficient, respectively. Thalassemia gene analysis revealed heterozygous α-thalassemia. The patient was diagnosed with PNH and heterozygous α-thalassemia, and became transfusion dependent 1 year later and was admitted to our hospital in March 2011. Conditioning was consisited of Cyclophosphamide (d-10 to d-9), Fludarabine(d-8 to d-4), Busulfan (d-7 to d-5), Thiotepa (d-4) and ATG-F (d-3 to d-1). Stem cell source was peripheral blood stem cells (PBSC) harvested on the 16th October 2011 from a HLA-matched (8/8) unrelated donor. The infused mononuclear cell (MNC) and CD34+cell does were 8×08/kg and 1.38×106/kg body weight of the recipient, respecitively.The time to platelets engraftment and leukocyte engraftment was 21 and 25 days,respecitively.The patient did not accoured acute graft versus host disease (aGVHD). Out-patient were followed up for 2 years, the patient's liver and kidney function, urine which showed normal, as well as reviewed the implant remains for independent plantentry.

Conclusions: The present case suggests that allo-HSCT can be used to cure patient with PNH and heterozygous α-thalassemia. Allo-HCT from unrelated donors is a new and potentially curative option for patients with PNH and inherited hemolytic anemia.

Disclosures

No relevant conflicts of interest to declare.
Human C1q Deficiency and Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Olsson Richard , Ringden Olle , Truedsson Lennart , Åhlin Anders
Published: October 2019

Human C1q deficiency is a rare disorder associated with systemic lupus erythematosus (SLE) and increased susceptibility to severe bacterial infections with mainly encapsulated organisms. Among patients with C1q deficiency there is a great variation in the severity of symptoms, however, the majority of patients requires comprehensive medical therapy and some develop treatment resistant disease. Since C1q is produced by monocytes it has been speculated that allogeneic hematopoietic stem cell transplantation (allo-HSCT) may cure these patients. At our center, we have so far treated five patients with C1q deficiency. In three cases, SLE symptoms remained relatively mild after the initiation of medical therapy, but two patients developed treatment resistant SLE and we decided to pursue with allo-HSCT. Here, we report an unrelated allo-HSCT in a 9-year-old boy who suffered from refractory SLE of the central nervous system and a related allo-HSCT in a 12-year-old girl with refractory SLE. Both patients restored C1q production post-transplant, and the severity of their SLE was attenuated. The boy developed post-transplant lymphoproliferative disease, which resolved after rituximab and donor lymphocyte infusions (DLI). Unfortunately, the DLIs induced cortisone-resistant severe gastrointestinal graft-versus-host disease, and he died from multiple organ failure four months post-transplant. The girl is doing well 21 months post-transplant, and clinically all signs of SLE have resolved. We believe that allo-HSCT is a promising treatment in patients with C1q deficiency and severe SLE, which needs further investigation.

Disclosures

No relevant conflicts of interest to declare.
High Glucose/Thrombin-Induced Endothelial Inflammation Via Microrna-146a and Nox4 Regulation
Authors: Peng Ching-Tien , Lo Wan Yu , Wang Huang Joe
Published: October 2019

Diabetes is associated with hyperglycemia and increased thrombin generation. It is unknown whether high glucose (HG)/thrombin can modulate the expression of NAPDH oxidase (Nox) subtypes in human aortic endothelial cells (HAECs). Besides, we investigate whether miR-146a is involved in endothelial cell inflammation. We observed that HG (25 mmol/l) exerted a synergistic effect with thrombin (2 U/ml) for induction of Nox4 mRNA level in HAECs. The increased Nox4 mRNA was associated with increased Nox4 protein and ROS production. We also demonstrated that HG/thrombin treatment increased interleukin-8 and interleukin-6 protein levels. Besides, HG/thrombin treatment caused an 11.43-fold increase of THP-1 adhesion to HAECs. In Silico analysis identified homology between miR-146a and the 3’-UTR of the human Nox4 mRNA, suggesting a potential regulation of Nox4 by miR-146a. Furthermore, HG/thrombin treatment decreased miR146a expression to 58% of the control, indicating an impaired feedback restrain of HG/thrombin-induced endothelial inflammation. MiR-146a mimic transfection prevented HG/thrombin-induced upregulation of Nox4 mRNA, Nox4 protein, and ROS generation. In addition, inflammatory phenotypes were attenuated in miR-146a mimic-transfected HAECs. In conclusion, miR-146a is involved in the regulation of endothelial inflammation via modulation of Nox4 in an in-vitro milieu mimicking diabetic atherothrombosis.

Disclosures

No relevant conflicts of interest to declare.
Evidence of Defibrotide Internalization and Its Protective Effect in a Hepatic Endothelial in Vitro model
Authors: Diaz-Ricart Maribel , Palomo Marta , Mir Enric , Rovira Montserrat , Escolar Gines , Carreras Enric
Published: October 2019

INTRODUCTION: Defibrotide (DF) interferes with several steps of the coagulation-inflammation cycle. Last October (2013), DF received EMA authorization for its use in the treatment of severe hepatic veno-occlusive disease (VOD) after hematopoietic cell transplantation (HCT). Since endothelial dysfunction has been associated with the development of VOD, among other HCT complications, we decided to explore the mechanisms of action of DF in a hepatic endothelial in vitromodel.

OBJECTIVES: To investigate the interaction of DF with a hepatic endothelial cell line from human origin (SK-HEP-1), the mechanisms involved in its traffic and its potential protective effect on fludarabine induced apoptotic mechanisms.

METHODS: SK-HEP-1 cells were exposed to DF (20µg/ml), previously labelled with ULYSIS® Nucleic Acid Labeling Kit, for up to 24 hours. Confocal microscopy (CM, Leica TCS SP5) was used to localize DF into the different cell compartments. Additional experiments were performed to assess colocalization of DF with different endocytosis pathways, by using specific antibodies. MTT assay to measure cell viability was performed in SK-HEP-1 cells after incubation with DF at different concentrations (10 to 200µg/mL, for 24h) followed by fludarabine exposure (at 20µg/mL, for 24h).

RESULTS: CM analysis revealed a significant interaction of DF with endothelial cell membranes followed by internalization and redistribution to the cytoplasm. Mechanisms involved seem to be dependent on endosomes and cytoskeletal assembly. DF did not reach the cell nucleus even after 24h of exposure. In addition, MTT assay demonstrated the protective effect of DF (at concentrations >20µg/mL) on the fludarabine damage on endothelial cells.

CONCLUSIONS: Our studies show that DF interacts with endothelial cell membranes, becoming internalized and redistributed into the endothelial cell cytoplasm without evidence in the nucleus. Moreover, DF protects hepatic endothelial cells from the toxic effect of fludarabine. Our findings may contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathological situations.

Partially supported by grant SAF2011-28214 (Spanish MINECO and FEDER).

Disclosures

Diaz-Ricart: Gentium / Jazz Pharmaceuticals: Research Funding. Palomo:Gentium / Jazz Pharmaceuticals: Research Funding. Mir:Gentium / Jazz Pharmaceuticals: Research Funding. Carreras:Gentium / Jazz Pharmaceuticals: Research Funding.
Base Excision Repair Glycosylase Activity Is Impaired in a Subgroup of Acute Myeloid Leukemia Resulting in Increased Levels of Oxidative Base Lesions
Authors: Olipitz Werner , Lind Karin , Monsberger Nicole , Katschnig Anna , Mangerich Aswin , Hofer Sybille , Schulz Eduard , Quehenberger Franz , Schlembach Dietmar , Robier Christoph , Woelfler Albert , Zebisch Armin , Sill Heinz
Published: October 2019

Base excision repair (BER) is the primary DNA repair mechanism dealing with oxidative base lesions. Oxidative DNA base lesions are the predominant type of DNA damage in mammalian cells. Deficiencies in glycosylases, the BER initiating enzymes, have been associated with increased genomic instability and increased frequencies of cancer. Here we investigated the role of oxidative BER in acute myeloid leukemia (AML).

We determined oxidative BER activity in 99 primary AML blast cell samples, 34 CD34+ umbilical cord blood cell samples and 27 AML cell lines using the alkaline comet assay. Oxidative base lesion levels were determined in 10 AML cell lines using a modified version of the Comet assay with the bacterial enzymes Fpg and Endo III as well as using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Using nuclear protein extracts in an oligonucleotide incision assay we tested the enzymatic activity of oxidative glycosylases. Finally, mutational analysis, gene expression analysis and protein expression of oxidative glycosylases was used using Sanger sequencing, real time PCR and western blot of nuclear extracts, respectively.

We found DNA strand incision of oxidatively damaged bases significantly impaired in primary AML cells as compared to UCB cells (p= 0.003) suggesting a deficiency in BER glycosylases. In addition, 5/27 AML cell lines showed impaired DNA strand incision activity. We hypothesized that BER deficient cells harbor an increased number of oxidative base lesions compared to BER proficient cells. Using a modified comet assay and LC-MS/MS we were able to show that increased numbers of unrepaired oxidative base lesions were indeed present in glycosylase deficient AML cells (comet assay: p= 0.0001; mass spec: p= 0.03). We then evaluated the activity of the predominant oxidative DNA glycosylase, OGG1, and found significantly decreased DNA strand incision activity in BER deficient cells as compared to proficient cells (p= 0.002) further supporting the fact that glycosylases are impaired in BER deficient cells. Determining causes of BER deficiency preliminary experiments showed significantly decreased expression of nuclear OGG1 protein in BER deficient cells but did not reveal novel non-synonymous mutations or a difference in gene expression.

Taken together we found impaired BER glycosylases in a substantial number of primary AML samples and AML cell lines resulting in increased levels of potentially mutagenic oxidative DNA base lesions

Disclosures

No relevant conflicts of interest to declare.
Genetic and Metabolic Determinants of Methotrexate Induced Mucositis in Pediatric Acute Lymphoblastic Leukemia
Authors: den Hoed Marissa , Lopez-Lopez E. , Te Winkel Mariël L. , Tissing Wim , de Rooij Jasmijn , Gutierrez-Camino A. , Garcia-Orad A. , den Boer E. , Pieters Rob , Pluijm S.M.F. , de Jonge R. , van den Heuvel-Eibrink Marry M.
Published: October 2019

Introduction: Methotrexate (MTX) is an important and effective chemotherapeutic drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). However MTX can induce toxicity, which can lead to amendments of treatment and subsequent impaired survival. The aim of this study was to identify metabolic and genetic determinants of MTX toxicity.

Patients and Methods: In this prospective study, 134 Dutch pediatric ALL patients were treated with four high dosages MTX (HD-MTX: 5 g/m2) every other week according to the DCOG-ALL10 protocol. Toxicity was prospectively scored and a National Cancer Institute (NCI) grade ≥3 was considered clinically relevant toxicity. Plasma MTX levels were measured at 24 and 48 hours after each HD-MTX infusion. Erythrocyte folate, plasma folate and plasma homocysteine levels were determined at the start of protocol M. Seventeen single nucleotide polymorphisms (SNPs) in 7 candidate genes in the MTX pathway were analyzed.

Results: Grade ≥3 mucositis occurred in 20% of the patients, skin toxicity in 7%, diarrhea in 3%, and neurotoxicity in 3%. Mucositis occurred especially after the first course compared to the other courses (p=0.006). Mucositis was not associated with plasma MTX, plasma folate or plasma homocysteine levels. Patients with mucositis had higher baseline levels of erythrocyte folate (median 1.2 μmol/L vs. 1.4 μmol/L, p<0.008). Wildtype rs7317112 in the ABCC4 gene was the only SNP associated with a higher frequency of mucositis (AA (39%) vs. AG/GG (15%), p=0.016).

Conclusion: Mucositis is the most frequent occurring toxicity during the HD-MTX phase in pediatric ALL treatment, and occurs especially after the first MTX course. Only a higher baseline erythrocyte folate, which reflects the accumulation of MTX polyglutamates in mucosal cells, and the wild-type variant of rs7317112 SNP in ABCC4 were determinants of mucositis in pediatric ALL during MTX-HD treatment.

Disclosures

No relevant conflicts of interest to declare.
miRNAs, Immune Signaling, and Myelodysplastic Syndromes Pathogenesis
Authors: Karsan Aly
Published: October 2019

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell hematologic malignancies characterized by cytopenias as a result of ineffective hematopoiesis and a propensity to progress to acute myeloid leukemia. In low-risk MDS, a characteristic finding in the bone marrow is that of increased apoptosis. The most common structural genomic aberration observed in MDS is the interstitial deletion of the long arm of chromosome 5. MDS with isolated del(5q) is a subtype of MDS characterized by severe anemia and variable neutropenia, but normal or high platelet counts with dysplastic megakaryocytes. MicroRNAs (miRNAs) are short noncoding RNAs capable of exerting their effects by postranscriptionally regulating numerous mRNA targets. We have shown that deletion of chromosome 5q correlates with loss miR-145 and miR-146a that are abundant in hematopoietic stem/progenitor cells (HSPC). Genes involved in innate immune signaling are significantly overrepresented when predicted targets of these two miRNas are surveyed. Specifically, Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) are targets of miR-145 and miR-16a, respectively. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6, to activate innate immune signaling in mouse HSPC, results in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow, in order to aberrantly activate innate immune signaling, progress either to marrow failure or acute myeloid leukemia. Loss of these miRNAs and consequent inappropriate immune signaling results in suppression of HSPC with a relatively greater effect on normal HSPC. Thus, inappropriate activation of innate immune signaling in HSPC phenocopies several general clinical features of low-risk MDS and of del(5q) MDS in particular. Recent work from our group defines additional cytokine pathways that are dysregulated by loss of miR-143 and miR-145. The impact of cytokine dysregulation on HSPC and the marrow microenvironment will be discussed.

Disclosures

Karsan: Celgene: Research Funding.
Lgr5 Stem Cells in Self-Renewal and Cancer
Authors: Clevers Hans
Published: October 2019

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost “generic” marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear, tongue, and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so-called “mini-guts” in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D culture systems have been developed for the Lgr5+ve stem cells of stomach, liver, pancreas and kidney. Using CRISPR/Cas9 technology, the cystic fibrosis transmembrane conductance regulator (CFTR) locus has been corrected in intestinal organoids of cystic fibrosis patients.

Disclosures

No relevant conflicts of interest to declare.
New Approach in Assessing the Pharmacokinetic/Pharmacodynamic Relationship in Patients with Severe Hemophilia A Using Data from BAY 81-8973 Studies
Authors: Garmann Dirk , Frede Matthias , Shah Anita , Ploeger Bart
Published: October 2019

Background: The relationship between factor VIII (FVIII) level or time spent below a certain FVIII level and bleeding rate has previously been analyzed using different regression analysis methods (Collins PW, et al. J Thromb Haemost. 2009;7[3]:413-420; Ahnström J, et al. Haemophilia. 2004;10[6]:689-697; den Uijl IE, et al. Haemophilia. 2011;17[1]:41-44). Although a relationship could be generally confirmed, results were not fully consistent. Nevertheless pharmacokinetic (PK)-guided dosing that targets a certain trough FVIII level, usually 1 IU/dL, is used in clinical practice.

Objectives: To establish a PK/pharmacodynamic (PD) relationship between FVIII replacement and the occurrence of repeated bleeds and to investigate the effect of PK-guided dosing by simulations

Patients/methods: Data from 121 patients aged 12-61 years with severe hemophilia A (FVIII <1 IU/dL) who were treated prophylactically with a new full-length recombinant FVIII (BAY 81-8973) and who had PK information available were included in the analysis. The PK/PD relationship was analyzed with a repeated time-to-event method, using nonlinear mixed-effect modeling.

Results: A relationship between FVIII concentrations over time and bleeding hazard could be identified. In addition, a time-dependent effect was significant in the analysis, resulting in a decrease in bleeding risk when patients were treated prophylactically with BAY 81-8973. Documented bleeds in the year before the study gave insight into the different bleeding risks of patients. Simulation results show that a fixed dose of FVIII on a study level might be as effective as PK-guided dosing.

Conclusion: The patient's documented bleeding history (phenotype) should be considered when dosing decisions for replacement therapy are being made.

Disclosures

Garmann: Bayer Pharma AG: Employment. Frede:Bayer Pharma AG: Employment. Shah:Bayer HealthCare: Employment. Ploeger:Bayer Pharma AG: Employment.
Head Trauma Is the Major Risk Factor for Cerebral Sinus-Vein Thrombosis
Authors: Giladi Ornit , Steinberg David , Peleg Kobi , Tanne David , Givon Adi , Grossman Ehud , Klein Yoram , Avigdori Shirli , Grinberg Gal , Katz Rachel , Shalev Varda , Salomon Ophira
Published: October 2019

Cerebral sinus vein thrombosis (CSVT) is a rare disease with significant neurological sequellae and high mortality rate. Incidence of CSVT diagnosis in the western world has increased despite the reduced occurrence of infectious sinus thrombosis related to otitis media and mastoiditis. The objective of this study was to identify risk factors that may explain the predisposition to the site specific thrombosis based on patients from a single tertiary medical center.

The study included 90 consecutive patients aged 15 and up that were diagnosed with acute CSVT from January 2002 to September 2014 at the Sheba Medical Center. As a control group we used the data extracted from the national trauma registry for the years 2012 and 2013 and from Maccabi Healthcare Services, the second largest health care maintenance organization (HMO) in Israel.

Trauma history up to one month prior to diagnosis of CVST was found in 13 (14%) patients (10 men and 3 women). Six patients had skull fractures, the others had blunt trauma. Data from the national trauma registry were used to compute annual age and gender specific head trauma rates. The overall SMR was 941 (p < 0.0001); the separate results for men and women were 1206 and 543, respectively. Another important risk factor was infections confined to the head and neck in 7% of the cases and brain tumor in 8%. At the time of CVST, 23 of 50 (46%) women had a hormonal risk factor. The SMR for OC use was 1.63 (p=0.0298). Prothrombotic polymorphisms were detected in 16 of 63 (25.4%) patients who were tested for factor V Leiden and prothrombin G20210A mutation (OR=3.47, p=0.002) in comparison to 49% in DVT patients (OR=9.95, p<0.0001). In 29 of 90 patients at least one of the risk factors for atherosclerosis (hypertension, diabetes or hypercholesterolemia) was discerned but this was very close to the expected number adjusted for sex and age and SMR was 0.98. None of the risk factors correlated with severity of disease and outcome.

These data suggest that search for CVST in patients with recent trauma and headache even after intact head CT is required. The other risk factors, such as hormone related and prothrombotic polymorphisms, were not specific just for CVST and the latter play a lesser role in CVST than in DVT.

Disclosures

No relevant conflicts of interest to declare.
Dickkopf 1 (Dkk1) Regulates Hematopoietic Stem Cell Regeneration
Authors: Himburg Heather A , Doan Phuong L , Yan Xiao , Quarmyne Mamle , Zhao Liman , Tran Evelyn , Chao Nelson J. , Harris Jeffrey , Chute John P
Published: October 2019

Bone marrow endothelial cells (BM ECs) have been shown to regulate HSC regeneration following myelosuppression. The role of osteolineage cells in regulating HSC regeneration remains less well understood. Here, we show that deletion of the pro-apoptotic genes, Bak and Bax, in osterix (Osx)-expressing osteoprogenitor cells promoted HSC regeneration and hematopoietic radioprotection of mice following total body irradiation (TBI). We identified Dkk1 to be enriched in the BM of radioprotected OsxCre;Bak1-/-;BaxFL/- mice and found that Bak /Bax-deficient osteolineage cells expressed increased levels of Dkk1 compared to Bax-expressing osteolineage cells (p=0.003). Treatment of irradiated BM HSCs with DKK1 in vitro significantly increased the recovery of phenotypic HSCs (p=0.0002), colony forming cells (CFCs)(p=0.003) and long-term repopulating HSCs compared to control cultures (p=0.009). Systemic administration of Dkk1 to lethally irradiated C57Bl6 mice accelerated the recovery of mature blood counts (p=0.008), BM HSCs (p=0.008) and progenitor cells (p=0.007). Furthermore, survival after lethal irradiation was markedly increased in Dkk1 treated mice (93%) compared to saline controls (27%; p=0.0004). Conversely, systemic administration of anti-Dkk1 antibody significantly delayed recovery of BM HSCs (p=0.002), peripheral white blood cells (p=0.0004), neutrophils (p<0.0001) and lymphocytes (p=0.002) in irradiated mice compared to irradiated, control mice. Dkk1 promoted HSC regeneration via suppression of reactive oxygen species (ROS) and inhibition of caspase activation in HSCs following irradiation. Dkk1 represents a novel, osteoprogenitor cell-derived paracrine factor which is necessary for normal hematopoietic regeneration following irradiation and can be therapeutically delivered to accelerate hematopoietic reconstitution.

Disclosures

Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.
Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML
Authors: Mahmud Hasan , Scherpen Frank JG , Meeuwsen-de Boer Tiny , Lourens Harm Jan , de Bont Eveline S.
Published: October 2019

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Still about 30% of patients with t(8;21) AML struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to identify new potential druggable targets. The aim of this study was to perform kinome profiling on 29 AML patients (cytogenetically normal, CN-AML, n=17 and t(8;21), n=12) and 4 normal bone marrow (NBM) samples to identify new potential druggable targets relevant to t(8;21) AML. We defined 130 peptides differentially phosphorylated between CN-AML, t(8;21) and NBM (p<0.05). With this technique, phospholipase C gamma 1 (PLCγ1_Y783) was identified as one of the highest phosphorylated peptide in t(8;21) AML. The knowledge about the role of PLCγ1 in leukemia progression is unknown. Therefore, we selected PLCγ1 to study its functional role in t(8;21) AML cell line (Kasumi-1) and also to test its efficacy as a potential target and provide proof of principle for this approach. PLCγ1 peptide phosphorylation was significantly higher in all t(8;21) AML samples when compared to NBM or to CN-AML (p<0.001 and p<0.05 respectively). mRNA expression of PLCγ1 in a publicly available paediatric AML database (http://r2.amc.nl) also showed significantly higher expression in t(8;21) AML compared to other AML karyotypes (p<0.001). PLCγ1 silencing with the specific short hairpin RNAs (two constructs, shRNAs PLCγ1-A and shRNAs PLCγ1-B), showed 50% and 80% decrease in PLCγ1 mRNA level compared with the control (scramble shRNA). These results were confirmed by PLCγ1 protein level analysis by western blotting. The shRNA-mediated silencing of PLCγ1 leads to suppression of the Kasumi-1 cell proliferation and viability at day 8 after transduction (p<0.05). The percentage of apoptosis in PLCγ1 supressing kasumi-1 cells at day 4 was two-fold higher than in scramble (p<0.01). To understand mechanistic insights of inhibited cell proliferation of PLCγ1 knock down cells, human phosphokinase microarray was performed and higher phosphorylation of Chk2_T68 and AMPK-α1_T183 were found in these cells. Higher Chk2 phosphorylation may responsible for cell cycle arrest and induced cell death of PLCγ1 supressing kasumi-1 cells. Similarly, higher phosphorylation of AMPK-α1 may inhibited cell proliferation of PLCγ1 knockdown cells. In conclusion kinome profiling is an elegant approach to identify interesting potential druggable targets. The results from proof of principle experiments strongly support the notion that suppression of PLCγ1 inhibited cell growth and induced apoptosis in Kasumi-1 cells.

Disclosures

No relevant conflicts of interest to declare.
Impact of Erythropoietin Modulation Using Hyperbaric Oxygen on Umbilical Cord Blood CD34+ Cell Homing
Authors: Aljitawi Omar S. , Lin Tara L. , Vielhauer George , Cheung Aaron , Capitano Maegan L. , Li Dandan , Wise Amanda , Lipe Brea C. , Paul Soumen , Laughlin Mary , Broxmeyer Hal E.
Published: October 2019

Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates EPO which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined EPO receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate EPO effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum EPO levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to EPO during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum EPO blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p <0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood EPO and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation.

Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient.

Disclosures

No relevant conflicts of interest to declare.
Extrafollicular CD4+ T and B Interaction Induces Chronic Gvhd in the Absence of Germinal Center Formation
Authors: Deng Ruishu , Hurtz Christian , Yue Chanyu , Xiao Gang , Yu Hua , Muschen Markus , Forman Stephen J. , Martin Paul J. , Zeng Defu
Published: October 2019

Chronic graft-versus-host disease (GVHD) in patients with allogeneic hematopoietic cell transplantation (HCT) is an immune-mediated syndrome. Both CD4+ T and B cells are required for disease induction, but details of this interaction have not been fully defined. Other groups have reported that germinal centers are enlarged in mice with chronic GVHD and that GC formation is required for induction of the disease. On the other hand, patients with chronic GVHD have a predominance of IgG1 in the serum with little evidence of IgG somatic hypermutation by one year after HCT, suggesting a lack of GC formation (Suzuki et al: Blood. 1996;87:1873-1880; Glas et al: Blood. 2000; 96:1064-1069). Consistently, we observed that GCs were not detectable in mice with chronic GVHD, including MHC-matched C57BL/6 (H-2b) recipients given LP/J (H-2b) grafts and BALB/c (H-2d) recipients given DBA/2 (H-2d) or B10D2 (H-2d) grafts, and MHC-mismatched BALB/c (H-2d) recipients given C57BL/6 (H-2b) grafts. Furthermore, BALB/c recipients given grafts from BCL6-deficient (BCL6 fl/fl Mb1-Cre) C57BL/6 (H-2b) donors lacking the ability to form GC developed chronic GVHD with similar severity to recipients given control BCL6-sufficient grafts. Consistent with lack of GC formation, recipients of BCL6-deficient grafts had profound reduction of serum IgG2b and IgG2c but moderate reduction of IgG1 as compared to recipients given control BCL6-sufficient grafts. The presence of IgG1 indicates extrafollicular CD4+ T and B interaction, which is Stat-3 dependent. Further experiments showed BALB/c recipients given grafts from Stat3-deficient (Stat-3 fl/fl CD4-Cre) C57BL/6 donors did not develop chronic GVHD. Taken together, these results indicate that GC formation is not required for development of chronic GVHD and that extrafollicular CD4+ T and B interaction can induce chronic GVHD. This work is supported by NIH R01 AI066008 and The Beckman Research Institute Excellence Award (to D. Zeng).

Disclosures

Forman: Mustang: Research Funding; Amgen: Consultancy. Martin:Neovii: Research Funding; RegImmune: Research Funding; Enlivex: Consultancy; Janssen: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy.