bluuebunny/crossref_metadata_2025 huggingface dataset:
Feasibility Study Of a Randomized Control Trial To Evaluate An Internet-Based Self-Management Program For Adolescents With Hemophilia
Authors: Breakey Vicky R , Ignas Danial M , Warias Ashley , White Meghan , Blanchette Victor S. , Stinson Jennifer
Published: October 2019

Introduction

As adolescents with hemophilia approach adulthood, they must assume responsibility for their health and management of their disease. An online self-management program was developed to support adolescents during this transition.

Aim

To determine the feasibility of studying the online educational program using a non-blind, randomized control trial(RCT) design in terms of 1)Study accrual and attrition rates, 2) willingness to be randomized 3) compliance with the program and completion of the outcome measures, and 4) satisfaction with the program.

Methods

Adolescents, ages 13-18, were enrolled from three tertiary care centres in Canada in a pilot RCT (NCT01477437). After providing informed consent, adolescents were randomized to the intervention (8 week program with telephone coaching) or the control arm (no access, weekly telephone call as attention-strategy). Adolescents in both arms of the study were asked to complete pre- and post- outcome measures. Following completion of the program, quantitative analysis addressed the feasilibity measures and qualitative interviews were conducted to assess satisfaction.

Results

29 teens participated (intervention group, n=16, control group, n=13).Participants in the intervention arm spent an average of 50 minutes on the website per week completed the 8 modules in an average of 14 weeks (SD= 4.9). Among those randomized to the intervention, 2 participants dropped out of the study and 2 were lost to follow-up, resulting in an attrition rate of 25% (4/16). The control arm had a higher attrition rate of 54% (7/13) with 5 participants being lost to follow-up and 2 participants dropping out. For participants who completed the program, 17/18 (94%) completed the post-study outcome measures. Despite the study not being powered to assess efficacy, teens on the intervention arm showed significant improvement in disease-specific knowledge (p=0.004), self-efficacy (p=0.007) and transition preparedness (p=0.046) over time. There was a statistically significant improvement in the knowledge measure in the intervention group when compared to the control group (p=0.01). Overall, the teens found the website to be informative, comprehensive and easy to use and were satisfied with the program.

Conclusions

This feasibility study suggests benefit to the program and indicates a full scale RCT to be a reasonable next step with minor adjustments to the protocol. The higher than expected attrition rate in the control group, suggests the need to improve the strategies for maintaining participant engagement using a more active attention control group (static educational materials) in the design of this web-based intervention prior to a larger study to assess efficacy.

Disclosures:

Breakey: Baxter Bioscience: Research Funding.
Characteristics and Outcome Of Multiple Myeloma (MM) In African Americans (AA) Receiving Novel Agents and Autologous Stem Cell Transplantation (ASCT)
Authors: Bhatnagar Vishal , Wu Yin , Goloubeva Olga , Ruehle Kathleen , Milliron Todd , Yanovich Saul , Rapoport Aaron P. , Badros Ashraf
Published: October 2019

Background

African Americans (AAs) have a higher incidence of MM, about twice as much as Caucasians, and present at a younger age. Several studies have shown that presentation and outcomes of AA are similar to Caucasians treated with standard and high dose chemotherapy. The impact of induction with novel agents (IMIDs and proteosome inhibitors) and prognostic markers such as cytogenetics on different ethnic groups has not been fully evaluated.

Methods

We analyzed MM patients (AA=174 and Caucasians= 279) who underwent auto-SCT over fifteen years (1998-2013). Baseline characteristics are summarized in table below. Patients received induction therapy with standard chemotherapy (n=33) or dexamathesone (n=43) before 2001 or IMIDs or protesome inhibitors in addition to either chemotherapy (n=72) or dexamethasone (n=203) when treated after 2001. All patients received high-dose melphalan followed by auto-SCT. Response criteria were assessed according to the International Uniform Response criteria.

Results

AA were significantly younger but with no differences in disease stage or CRAB criteria at MM diagnosis with the exception of anemia. More AA women were referred and underwent auto-SCT relative to AA men. The subtype distribution was significantly different between AA and Caucasians (P <0.001). FISH and cytogenetics data for hyperdiploidy, hypodiploidy, deletions in chromosome 13q, 17p and 1p and IgH translocations were available for 271 patients. Approximately two-thirds of the patients had normal or favorable cytogenetics with more hyperdiploidy present in AA, although the population of patients with hyperdiploidy (43) is small for definitive conclusions. Response to induction was similar between AA and Caucasians, however response to novel agents plus dexamethasone was significantly better in Caucasians (p = 0.02). There was no difference in PFS between AA and Caucasians (median of 2.6 years; 95% CI: 2-3); In contrast, OS was significantly better in AA (median 7.7; 95% CI: 6.5-9.8) versus Caucasians (median 6.2; 95% CI: 5.5-7.5).

Conclusion

MM is heterogeneous disease. AA disease characteristics are different from Caucasians (subtype and cytogenetics). AA have inferior responses to novel agents, despite comparable and probably better survival to Caucasians, suggesting a favorable disease biology. Future studies focusing on AA patients are urgently needed to validate these observations prospectively.

Disclosures:

No relevant conflicts of interest to declare.
Peripherally Inserted Central Catheters Are a Good Option For Venous Access In The Patients With Hematologic Diseases
Authors: Takaiwa Naoko , Seki Masanori , Kurita Naoki , Yokoyama Yasuhisa , Sakata-Yanagimoto Mamiko , Obara Naoshi , Suzukawa Kazumi , Hasegawa Yuichi , Chiba Shigeru
Published: October 2019

Background

Central venous catheters are widely used for treatments of hematologic diseases. Conventional central venous catheters (CVC), however, often cause complications such as pneumothorax, hematoma, catheter-related blood stream infection (CR-BSI), and so on. Peripherally inserted central venous Catheters (PICC) are recently expected to reduce these complications. The aim of this study was to compare the frequencies of and risk factors for complications with PICC and CVC in patients with hematologic diseases.

Patients and Methods

We retrospectively reviewed all 363 patients who had inserted PICC or CVC for treatment of hematologic diseases in January 2011 through July 2013 at the University of Tsukuba Hospital. Overall and device-specific frequencies of infectious and non-infectious complications were evaluated and potential risk factors were captured.

Results

The PICC group (N = 215) and CVC group (N = 148) were similar in terms of clinical backgrounds, types of complications during catheter insertion, and total time of catheter-inserted periods. The CVC group had significantly higher proportions of patients who received hematopoietic cell transplantation (PICC 17.7% vs. CVC 49.3%, P<0.001). The significant intergroup differences were found in frequencies of CR-BSI (PICC, 1.4/1000 catheter days and CVC, 5.9/1000 catheter days; P<0.001) and local infection at the insertion site (PICC, 0.3/1000 catheter days and CVC, 2.3/1000 catheter days; P=0.002). The cumulative incidence of catheter removal for infective infectious complications was higher in the CVC group than the PICC group (PICC 8.7% vs. CVC 37.3%, P<0.001). Multivariate analysis showed that PICC significantly reduced the incidence of CR-BSI (odds ratio (OR), 0.15; 95% confidence interval (CI), 0.06-0.33; P<0.001). The incidence of CR-BSI was also significant higher in patients who received hematopoietic cell transplantation (OR, 2.4; 95%CI, 1.22-4.69; P=0.01). Phlebitis and deep vein thrombosis was noted in no patient in PICC group and 1 patient in CVC group.

Conclusion

Our date suggest that PICC is superior to CVC in terms of infectious complications. Low incidence of thrombophlebitis, observed in this study, is a key to a major change of practice from CVC to PICC.

Disclosures:

No relevant conflicts of interest to declare.
Determinants Of Cost Of Care For Persons With Severe Hemophilia
Authors: Evans Michael W. , Eyster M. Elaine , Hollenbeak Christopher S.
Published: October 2019

Background

The ability of a person with hemophilia to adhere to treatment recommendations aimed at preventing and controlling bleeding likely impacts clinical outcomes and resource utilization. Annual cost of replacement factor for persons with severe hemophilia on prophylaxis is estimated to be as high as $300,000 per year (Johnson KA and Zhou ZY. ASH Annual Meeting Educational Program. 2011; 413-418). Hemophilia care is optimized when delivered by a highly specialized hemophilia treatment center (HTC) (Soucie et al. Blood. 2000; 96: 437-442). Consolidation of care for persons with hemophilia from a large geographic region within an HTC results in demographic heterogeneity that may impact an individual's ability to adhere to treatment recommendations. Little is known regarding the impact of socio-demographic factors on treatment outcomes for persons with hemophilia. The purpose of this study was to identify disease-related, treatment-related and demographic variables that have a significant impact on morbidity and resource utilization.

Methods

We identified 69 persons with severe hemophilia treated at our HTC between June 2009 and June 2012. We collected data for the variables listed below in table 1. To assess morbidity and resource utilization, we collected data for total factor use, total outpatient cost, total inpatient cost, frequency of inpatient and outpatient encounters and length of stay when hospitalized. Risk factors were identified using linear regression. A subset analysis was performed to evaluate the impact of prophylaxis on patients without inhibitors. All statistical analyses were performed using STATA (version 12.0 College Station, TX). Statistical significance was defined as P < 0.05.

Results

Age and inhibitor status were the only variables to significantly impact cost in both inpatient and outpatient settings (Table 1). The statistically significant beneficial impact of prophylaxis was confirmed by subgroup analysis of patients without inhibitors with respect to number of hospitalizations and length of stay, but not total cost.

Conclusions

Identifying and addressing potential barriers to care will likely reduce morbidity and cost for persons with hemophilia. Caregiver status and age are demographic variables that may contribute to poor medical adherence resulting in increased morbidity and cost of care for persons with severe hemophilia.

Disclosures:

No relevant conflicts of interest to declare.
Exosome Mediated Wnt-Signaling Augments Side Population In Aggressive Lymphoma
Authors: Koch Raphael , Demant Martin , Aung Thiha , Guentsch Annemarie , Diering Nina , Chapuy Bjoern , Truemper Lorenz , Wulf Gerald
Published: October 2019

Introduction

Patients with aggressive B-cell lymphoma are treated in curative intention. However, some patients experience fatal relapse, originating from refractory lymphoma cells with the capacity for clonogenic regrowth. We here addressed repopulation capacity of lymphoma cell subpopulations and the mechanisms regulating the populational composition in the growing tumor.

Material & Methods

We identified side population (SP) cells in diffuse large B-cell lymphoma cell lines and patient samples with the DNA-binding dye Hoechst33342, analyzed clonogenicity in vitro and in vivo and screened for differentially expressed genes and DNA-methylation patterns. A GFP-containing lentiviral vector construct was used to keep track of side population cells cultured among mixed cultures of SP and nonSP cells. Manipulation of canonical wnt-signaling was performed by lentiviral sh-RNA constructs as well as pharmacological tankyrase-inhibition by XAV-939. In vitro data were supported by in vivo experiments using a chorioallantoic membrane-assay.

Results

Colony assays and suspension cultures of sorted SP and nonSP cells revealed restriction of clonogenic potential to the SP cell population as well as resurgence of nonSP cells from purified SP cell progenitors, while mixed culture assays using a GFP-vector construct tracing the SP vs. nonSP-population revealed homeostasis between the two populations, showing both SP and nonSP cells contributing to either cell compartment. SP cells show enhanced canonical wnt-signaling and increased exosomal secretion of wnt3a. Suppression of canonical wnt-signaling resulted in reduced clonogenicity. Exosome stimulation of DLBCL cell lines resulted in increased clonogenicity, stabilization of beta catenin and enhanced TOP/FOP activity.

Conclusion

Here we show that tumor cells reversibly switch between states of autonomous and non-autonomous clonogenicity, and that such transitions are regulated by exosome-mediated wnt signaling.

Disclosures:

No relevant conflicts of interest to declare.
Detection Of MYD88 L265P In Peripheral Blood Of Patients With Waldenström's Macroglobulinemia and IgM Monoclonal Gammopathy Of Undetermined Significance
Authors: Xu Lian , Hunter Zachary , Yang Guang , Zhou Yangsheng , Liu Xia , Cao Yang , Manning Robert , Patterson Christopher , Tripsas Christina K , Kanan Sandra , Chen Jiaji G , Lindeman Neil I , Treon Steven Peter
Published: October 2019

Waldenström's macroglobulinemia (WM) is characterized by bone marrow (BM) infiltration with lymphoplasmacytic cells and production of an IgM paraprotein. Lymphocytosis is uncommon in WM, and many patients exhibit peripheral B-cell lymphopenia. Recently, by whole genome sequencing, we identified a highly recurrent somatic mutation (L265P) in MYD88. Using real-time allele-specific polymerase chain reaction (AS-PCR), we demonstrated the presence of MYD88 L265P in 93% and 54% of patients with WM and IgM monoclonal gammopathy of undetermined significance (MGUS), respectively (Xu et al, Blood 2013).

To clarify the feasibility of using real-time AS-PCR assay to detect MYD88 L265P in peripheral blood, we first examined unselected mononuclear cells from peripheral blood (PB) samples of 88 patients with untreated WM. 81/88 (92%) of these patients were MYD88 L265P positive by bone marrow (BM) examination of CD19 selected B-cells; Of the 81 BM-MYD88 L265P positive patients, 32 (40%) demonstrated MYD88 L265P by AS-PCR in unselected PB mononuclear cells (PBMC) obtained at the time of BM examination. To enhance the ability to detect MYD88 L265P in PB samples, we next used AS-PCR in CD19 selected PBMC obtained from 198 WM patients which included untreated and previously treated patients. Among untreated patients, 106/118 (89.8%) were positive for MYD88 L265P in CD19 selected PB, whereas among previously treated patients, 55/80 (68.8%) were positive for MYD88 L265P (p=0.0002 vs. untreated). Simultaneously analyzed PB and BM samples from 65 untreated WM patients demonstrated positivity for MYD88 L265P in 58/65 (89.2%) and 55/65 (84.6%) of CD19 selected BM and PB samples, respectively. These findings yielded a sensitivity of 94.8%, specificity of 100%, positive and negative predictive values of 100% and 70%, respectively for AS-PCR testing of PB CD19+ cells for MYD88 L265P in untreated WM patients. In addition, we analyzed 12 untreated IgM MGUS patients with paired sampling of CD19 selected PB and BM cells. 6/12 (50%) IgM MGUS patients were positive for MYD88 L265P in BM CD19+ cells, with 5/6 (83%) of these positive patients demonstrating MYD88 L265P by AS-PCR in CD19 selected PB samples.

The overall results demonstrate a high concordance of MYD88 L265P status between PB and BM CD19 selected samples, particularly in untreated WM patients. The detection of MYD88 L265P by CD19 selected PB AS-PCR examination provides a convenient and less invasive method to support the diagnosis of WM and IgM MGUS.

Disclosures:

No relevant conflicts of interest to declare.
Epigenetic Silencing Of Mir-137 Contributes To The Proliferation Of Multiple Myeloma By Targeting AURKA
Authors: Qin Yu , Li Fei , An Gang , Hao Mu , Zang Meirong , Qiu Lugui
Published: October 2019

Background

MicroRNAs are non-coding small RNAs that modulate protein expression and are implicated in the pathogenesis of much kind of cancers. miR-137 was reported to act as a tumor suppressor in different cancers. In the present study, we describe the epigenetic regulation of miR-137 and its contribution in Multiple Myeloma (MM).

Methods and Results

Real-time RT-PCR was used to screen the expression levels of miR-137 in MM cell lines and CD138+ cell sorted from MM patients, which confirmed the downregulation of miR-137 in MM cell lines and patients, and the expression of cell lines increasing treated with epigenetic drugs 5-aza-dC. The methylation status of miR-137 CpG island was determined by bisulfite pyrosequencing and methylation specific polymerase chain reaction (MSP). Methylation of the miR-137 CpG island was frequently observed in MM cell lines and patients but not in healthy donor and MGUS. Cck-8 assay showed transfection of miR-137 precursor in MM cells significantly inhibited cell proliferation and increased cell drug sensitivity. Importantly, Ectopic expression of miR-137 in MM cells inhibited phosphorylation of mitogen-activated protein kinase (MAPK/ERK). To further identify miR-137 targets, we used bioinformatics analysis and confirmed using a luciferase reporter assay. To validate AURKA as miR-137 target, we cloned the 3' UTR sequence of human AURKA into the luciferase-expressing vector psiCHECK. 293Tcells were transiently transfected with this construct in the presence of pre-miR-137 or a scrambled oligonucleotide acting as a negative control. As reported in luciferase plate reader, miR-137 significantly reduced luciferase activity compared with the scrambled control miRNA. This indicated that miR-137 binds to the 3'UTR of AURKA and impairs its mRNA translation. Furthermore, NCI-H929 cells were transfected with AURKA shRNA vector psiHIV-mH1-AURK and westernblot showed phosphorylation of ERK was also significantly decreased.

Conclusion

miR-137 was epigenetic Silenced and targeted AURKA expression to contribute to the proliferation through MAPK/ERK pathway in MM.

Disclosures:

No relevant conflicts of interest to declare.
ANKHD1 Silencing Reduces In Vitro Clonogenicity and In Vivo Tumorogenicity Of Multiple Myeloma Cells
Authors: Dhyani Anamika , Machado-Neto João Agostinho , Favaro Patricia , Olalla Saad Sara Teresinha
Published: October 2019

Introduction

ANKHD1 is a multiple ankyrin repeats containing protein, highly expressed in cancers, such as acute leukemia. Earlier studies showed that ANKHD1 is highly expressed and plays important role in proliferation and cell cycle progression of multiple myeloma (MM) cells. It was also observed that ANKHD1 downregulation modulates cell cycle gene expression and upregulates p21 irresepective of TP53 mutational status of MM cell lines.

Objective

The present study aimed to study the effect ofANKHD1 silencing on MM growth both in vitro (clonogenicity, migration) and in vivo (xenograft tumor mice model). The purpose was to investigate the feasibility of ANKHD1 gene therapy for MM.

Methods

In the present study, ANKHD1 expression was silenced using short hairpin RNA (shRNA)-lentiviral delivery vector in MM cell lines (U266 and MM1S). For control MM cells were tranduced by lentiviral shRNA against LacZ. Downregulation of ANKHD1 expression was confirmed by qPCR and Western blot. Colony formation capacity and migration of control and ANKHD1 silenced MM cells was determined by methylcellulose and transwell migration assays, respectively. For in vivo MM growth, NOD-SCID mice were divided in two groups injected with control and ANKHD1 silenced cells, separately. Mice were observed daily for tumor growth. Once the tumor size reached 1 mm3, mice in both groups were sacrificed and tumor was excised to measure tumor volume and weight.

Results

Corroborating the results obtained in our earlier studies, in the present study also inhibition of ANKHD1 expression suppressed growth of MM cells in vitro. MM cell lines tranduced with ANKHD1 shRNA showed significantly low number of colonies ten days after plating in methylcellulose medium as compared to control (p<0.05). Similarly, in transwell migration assay, cell lines transduced with ANKHD1 showed significantly less migration as in response to 10% FBS at lower chamber as compared to control group (p<0.05) in both the cell lines analyzed. Further in xenograft MM mice model, the growth of tumor was visibly suppressed in mice injected with ANKHD1 silenced cells compared to control group. There was significant difference in tumor size (volume) between these 2 groups (P< 0.006). The tumor weight of the inhibition group was 0.71 ±0.2 g, significantly lighter than those of the control group (1.211 ± 0.5 g, P =0.02)

Conclusion

Our data indicates ANKHD1 downregulation significantly inhibits colony-forming ability and migration of both glucocorticoid resistant (U266) and sensitive (MM1S) MM cells. Further, gene silencing of ANKHD1 also resulted in reduced in vivo tumor growth in NOD/SCID mice. Collectively, the result obtained indicates that ANKHD1 may be a target for gene therapy in MM.

Disclosures:

No relevant conflicts of interest to declare.
Srf Is Required For Neutrophil Migration In Response To Inflammation
Authors: Taylor Ashley , Tang Wenwen , Bruscia Emanuela , Zhang Ping-Xia , Lin Aiping , Gaines Peter , Vu Linh , Wu Dianqing , Halene Stephanie
Published: October 2019

Serum Response Factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown, that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of loss of SRF in neutrophils, we crossed Srffl/fl mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in mice in whom Srf is selectively deleted in the hematopoietic system (Srf KO). Srf KO neutrophils fail to migrate to sites of inflammation in vivo. In a model mimicking lung infection by nebulization of lipopolysaccharide (LPS), significantly fewer neutrophils are recruited to the inflamed lungs 4 and 24 hours after LPS administration as evident by cell numbers retrieved in the bronchoalveolar lavage fluid (BAL, total cells: WT 0.568 ± 0.093x106 vs. KO 0.128 ± 0.024x106 at 4 hours and WT 1.337 ± 0.369x106 vs. KO 0.347 ± 0.045x106 at 24 hours; p <0.005 at 4 hours, p < 0.05 at 24 hours). Similarly, in an in vivo peritonitis model, where all other immune cells normally express Srf, significantly fewer Srf KO than WT neutrophils are recruited to the inflamed peritoneal space resulting in significantly reduced Srf KO neutrophil numbers in the peritoneal lavage fluid. To directly assess neutrophil migration and chemotaxis we performed in vitro transwell assays and assessed neutrophil migration in the Dunn chamber assay. Srf KO neutrophils show a significant migration defect in response to fMLP and KC. We next assessed actin polymerization in Srf WT and KO neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to fMLP. Neutrophil polarization in response to cytokine stimuli is markedly decreased. In addition, Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in neutrophil adhesion and migration. Srf KO neutrophils show normal expression of the integrin LFA1 (CD11a/CD18), however, Mac1 (CD11b/CD18) expression is markedly increased on the cell surface as assessed by flow cytometry and Immunofluorescent staining, despite reduced mRNA expression. We find that trafficking of Mac1, essential for directed cell migration, is disrupted in Srf KO neutrophils, likely contributing to the migratory defect. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, thus underscoring an essential function for SRF and its pathway in health and disease.

Disclosures:

No relevant conflicts of interest to declare.
MiR-155 Regulates Acute GvHD By Reducing NLRP3 Inflammasome Activity Via Targeting SOCS1 In Antigen-Presenting Cells
Authors: Chen Sophia , Iype Joseena , Grundmann Sebastian , Idzko Marco , Schmitt-Gräff Annette , Dierbach Heide , Beck Yvonne , Finke Jürgen , Duyster Justus , Zeiser Robert
Published: October 2019

Allogeneic hematopoietic cell transplantation (alloHCT) is limited by acute graft-versus-host disease (GvHD) which occurs in 30-40 % of patients. Despite numerous clinical studies, the standard immunosuppressive regimens for prevention of acute GvHD have changed little in the last two decades. A better understanding of the pathophysiology of GvHD may help to improve the outcome after alloHCT.

Acute GvHD is mediated by donor T cells that are activated by recipient antigen-presenting cells (APCs). Since microRNA-155 (miR-155) was shown to regulate the activation of different innate immune cell subsets, we aimed to determine its function for APCs during an allogeneic immune response.

We observed upregulation of miR-155 in recipient APCs residing in secondary lymphoid organs and in the intestinal tract when GvHD developed. By using gene targeted mice, we observed that miR-155 deficiency of the recipient led to improved survival (p=0.0011), lower GvHD histopathology scores (small bowel: p=0.0003, large bowel: p=0.0209, liver: p=0.0035) and reduced serum levels of proinflammatory cytokines (IFNγ: p=0.0305, IL-12: p=0.0274, MCP-1: p=0.0016). Using miR-155-/- bone marrow chimeric mice lacking miR-155 in the hematopoietic system and adoptive transfer of miR-155+/+ versus miR-155-/- APCs, we determined that this phenotype was dependent on miR-155 deficiency in the APC compartment. Mechanistically, miR-155-/- APCs showed reduced ERK phosphorylation in response to lipopolysaccharides (LPS) and adenosine-5'-triphosphate (ATP) as compared to miR-155+/+ APCs. Cleaved caspase-1, the indicator of an active NLRP3 inflammasome, was also reduced in miR-155-/- APCs. Conversely, suppressor of cytokine signaling 1 (SOCS1), a known target of miR-155, was increased in miR-155-/- APCs as compared to miR-155+/+ APCs. SOCS1 leads to proteasomal degradation of the p65 subunit of NF-κB and thereby may interfere with NLRP3 inflammasome activation.

Overall, our data indicate that miR-155 is required in the recipient APC compartment for NLRP3 inflammasome activation in response to LPS and ATP. These findings could help to reduce tissue damage related proinflammatory effects induced by LPS and ATP which activate the intestinal immune system following cytotoxic therapy by antagonizing miR-155 function with antagomir treatment.

Disclosures:

No relevant conflicts of interest to declare.
Phase 1 Trial Of Bone Marrow Stromal Cells (Bone Marrow-derived MSCS) To Treat Tissue Damage In Allogeneic Stem Cell Transplant Recipients: Biological Markers Correlate With Clinical Responses and Survival
Authors: Battiwalla Minoo , Yin Fang , Ito Sawa , Feng Xingmin , Chinian Fariba , Melenhorst J. Joseph , Koklanaris Eleftheria K , Sabatino Marianna , Stroncek David , Samsel Leigh , Klotz Jeffrey K. , Hensel Nancy F. , Robey Pamela , Barrett A. John
Published: October 2019

Bone marrow stromal cells (BMSC, also known as bone marrow-derived “mesenchymal stem cells”) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage, BMSC for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following BMSC infusion. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were monitored for plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and after BMSC infusion. BMSCs were prepared from marrow aspirates from healthy volunteers with the expansion of 3 passages. Ten subjects were infused a fixed dose of 2 x 106 BMSCs /kg weekly for 3 doses. There was no treatment related toxicity (primary endpoint). Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first BMSC infusion (fig1). Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for tissue injury. The GVHD complete responders survived significantly longer (>300 days vs a median of 33 days), had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts but there was no clear difference in natural or induced Tregs. Cytokine changes also segregated with survival. These results confirm that BMSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. However BMSC were ineffective in patients with more aggressive GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSC, requires a relatively intact immune system. Early detection and BMSC treatment appear important in patients with refractory GVHD.

Disclosures:

No relevant conflicts of interest to declare.
BALB/c- Specific L29V Polymorphism In SIRPA Determines The Efficient Engraftment Of Human Hematopoiesis In Xenogeneic Model
Authors: Iwamoto Chika , Takenaka Katsuto , Urata Shingo , Shima Takahiro , Yamauchi Takuji , Kuriyama Takuro , Daitoku Shinya , Saito Yasuyuki , Itoh Katsuhiko , Kishimoto Junji , Kohda Daisuke , Matozaki Takashi , Akashi Koichi
Published: October 2019

It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the non-obese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant “don’t eat me” signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. We then developed the C57BL/6.Rag2nullIl2rgnull strain harboring NOD-specific Sirpa, named the BRGS mouse. The efficiency of human cell engraftment in BRGS mice was equal to that of NOD.Rag2nullIl2rgnull mice. These findings clearly show that in addition to depletion of lymphocytes, inactivation of phagocytosis via the CD47-SIRPA interaction is one of the critical determinants to establish an efficient xenogeneic transplantation system. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. DNA sequencing of the SIRPA IgV domain located in the CD47 binding site in each strain was performed. We identified 2 SNPs unique to BALB/c mice, and 3 SNPs unique to both BALB/c and NOD mice. Among these 5 SNPs, BALB/c strain has a Val residue at the 29th amino acid, whereas all other mouse strains have a Leu residue. Introduction of C57BL/6 SIRPA carrying the L29V mutation into C57BL/6 conferred the binding affinity to human CD47 equivalent to that of BALB/c SIRPA, indicating that L29V mutation at least contributes to the enhanced binding of BALB/c SIRPA to human CD47. Thus, BALB/c-specific L29V polymorphism in SIRPA renders to be able to recognize human CD47. Our data strongly suggest that the mouse strain effect on xenogeneic engraftment might be ascribed mainly to “macrophage tolerance” based on the binding affinity of strain-specific polymorphic murine SIRPA with human CD47. This information should be useful to develop a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells.

Disclosures:

No relevant conflicts of interest to declare.
Periostin Acts As An Important Cell Cycle Regulator Of Adult Hematopoietic Stem Cells Via Binding To Integrin-αvβ3
Authors: Khurana Satish , Verfaillie Catherine M.
Published: October 2019

Osteoblasts are one of the important cellular components of the niche for hematopoietic stem cells (HSCs) in mammalian bone marrow (BM). Integrin receptors not only play a key role in HSC adhesion within the BM niche but also transfer regulatory signals from the microenvironment to HSCs. Periostin (Postn or osteoblast specific factor-1; OSF-1) is expressed in osteoblasts in addition to many other tissues, and acts as a ligand for Integrin-αvβ3 (ITGAV-B3). We identified POSTN as an important regulator of the cell cycle in adult murine HSCs. POSTN inhibited culture induced proliferation of HSCs thereby decreasing the total number of cells following 2-5 day culture of primitive HSCs, identified as CD150+CD48-Lin-Sca-1+c-kit+ (CD150 KLS) cells with SCF and TPO, while increasing the proportion of long-term (LT-) HSCs. Culture for 5 days with POSTN decreased the short-term (ST-) engraftment of progeny of 200 CD150 KLS cells, while significantly increasing LT- engraftment of the donor derived cells. A significant fraction of CD150 KLS cells expressed ITGAV as well as ITGB3. POSTN did not affect proliferation of HSCs in vitro following blocking of ITGAV with neutralizing antibodies. Among the important cell cycle regulators, we found an increase in p27kip1 expression in HSCs. Preliminary studies on possible signaling mechanisms involved, showed that POSTN inhibits Akt phosphorylation, known to mediate inhibition of both expression and activation of p27Kip1. Intravenous infusion of recombinant POSTN protein significantly decreased proliferation of hematopoietic progenitors as shown by Brdu incorporation and Hoechst/Pyronin staining. Interestingly, POSTN infusion also led to an increase in the number of KLS as well as CD150 KLS cells in the BM. Studies on characterization of the hematopoietic system of Postn-/- mice are underway. To further determine the role of ITGAV in HSCs, we used blocking antibodies against ITGAV and performed homing and engraftment studies. No effect on either homing potential or engraftment of ST- and LT- engraftment was seen. However, the competitive repopulation of ITGAV- CD150 KLS cells was significantly lower that that of ITGAV+ CD150 KLS cells (isolated using non-blocking antibodies). Therefore, our studies confirm the importance of ITGAV expression on primitive HSCs as well as presents POSTN as an important cell cycle regulator in the hematopoietic system.

Disclosures:

No relevant conflicts of interest to declare.
Prospective Isolation Of Human Erythroid Lineage-Committed Progenitors
Authors: Mori Yasuo , Seita Jun , Pluvinage John V. , Chen James Y. , Weissman Irving L.
Published: October 2019

Anemia is one of the findings most frequently seen in patients with myelodysplastic syndrome (MDS); however, in our hands, significant loss of megakaryocyte/erythrocyte progenitors (MEP) is not observed among these patients (PNAS, 2013, 110(8):3011-6). Unipotent erythroid-committed progenitors (EPs), estimated to exist downstream of bipotent MEPs, may be involved in the pathogenesis of refractory anemia among MDS patients. Previously, we identified mouse (m) EPs (mEPs) in bone marrow. The expression of endoglin (CD105) was a key marker to isolate mEPs: mEPs were the Lineage(lin)-Sca-1-c-kit+CD16/32-CD150+CD105+CD41- population in murine bone marrow (Cell Stem Cell, 2007, 1(4):428-42). Here we show that the human (h) counterpart (hEPs) is prospectively isolatable in human bone marrow. We analyzed the expression of hCD105 in addition to hCD71 (known to be an early erythroid marker) in human stem and progenitor populations, and found a fraction (32.9±13.6%, n = 5) of common myeloid progenitors (CMPs; lin-CD34+CD38+CD45RA-IL-3Ra+) and a major part (85.1±5.5%) of MEPs (lin-CD34+CD38+CD45RA-IL-3Ra-) expressed hCD71. A portion of CD71+ MEPs co-expressed CD105 (CD105+MEPs; 32.3±6.5% of MEPs). Neither hCD105 nor hCD71 was detectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-CD45RA-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), or granulocyte/monocyte progenitors (GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS. In vitro, CD71+ cells within the CMP fraction showed differentiation potential skewed toward MegE lineage, representing the transitional stage to MEPs. CD71+MEPs mostly generated BFU-E, although they still retained some MegK potential, while output of CD105+MEPs was completely restricted to erythroid lineage, and colonies from this fraction contained a small number of mature (enucleated) erythrocytes (CFU-E type). Thus we termed CD71+MEPs and CD105+MEPs as human erythroid-biased MEPs (E-MEPs) and hEPs, respectively. In short-term liquid culture, hE-MEPs gave rise to hEPs in the presence of SCF, TPO and EPO, whereas hEPs could not generate hE-MEPs, suggesting a hierarchical progression from E-MEPs to EPs. These newly classified populations might be a very useful tool for understanding the molecular mechanisms of human erythroid development, and should be analyzed in patients with erythroid-related disorders (e.g., MDS, polycythemia vera, or aplastic anemia).

Disclosures:

No relevant conflicts of interest to declare.
Ruxolitinib Inhibits Dendritic Cell Function By Interfering With The MAP-Kinase/NF-Kb Signalling Pathways
Authors: Held Stefanie AE , Heine Annkristin , Wolf Dominik , Brossart Peter
Published: October 2019

The recently approved Jak-Inhibitor ruxolitinib improves clinical symptoms in patients with myelofibrosis and autoimmune diseases such as rheumatoid arthritis. It has been shown in myelofibrosis that the clinical benefit occurs independent of the Jak mutational status, indicating that not only diseased, but also healthy cells like immune cells might be affected by the treatment. In a previous study we investigated the effects of ruxolitinib on dendritic cells (DCs) as the most professional antigen presenting immune cells. We were able to show that ruxolitinib markedly impairs human and murine DC phenotype, DC activation and migration and the induction of allogeneic and antigen-specific T cell responses in vitro and in vivo. In regard to good clinical responses in myelofibrosis patients lacking the Jak-2-mutation, it seems rather unlikely that Jak-inhibition is the only responsible for the observed in vivo and in vitro effects. In the present study, we therefore aimed to investigate whether other signalling mechanisms might be additionally involved. To exclude significant effects on Jaks other than Jak-2, we first analyzed the more selective Jak-2 inhibitor TG101348 in our in vitro models. Here, we were able to show that both inhibitors impair DC phenotype and function to a similar extend. We next investigated the role of the MAP-kinase and the NF-kB signalling pathways. Therefore, human monocyte-derived immature and mature DCs were treated with different concentrations of ruxolitinib for distinct durations, followed by preparation of whole cell lysates and nuclear extracts to perform Western blot analyses. We found that ruxolitinib treatment resulted in a reduced phosphorylation and expression of the MAP-kinases ERK and p38, while the housekeeping gene GAPDH was not affected. This effect was independent of apoptosis induction as the expression of caspase 3 was constant. Moreover, the expression of the NF-kB family members RelA and cRel were markedly reduced.

Taken together, we have shown that the Jak-inhibitior ruxolitinib significantly impairs DC phenotype and function resulting in a markedly reduced induction of allogeneic and antigen-specific T cell responses. To analyze potentially involved underlying signalling mechanisms, we were able to show that exposure of DCs to ruxolitinib leads to a distinct downregulation of the MAP-kinase ERK and p38 as well as a diminished expression of NF-kB family members downstream in the MAP-kinase signalling pathway. This may indicate that not only the Jak/Stat signalling pathway might be involved in the immunological effects of ruxolitinib in non-mutated cells, but also the MAP-kinase pathway downstream of Jak2. Our work might add a piece of information for a deeper understanding of the clinical efficacy of ruxolitinib in diseases characterized by an exaggerated inflammation, such as myelofibrosis or autoimmune disorders.

Disclosures:

Wolf: Novartis: Honoraria, Research Funding.
Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation
Authors: Yi Wenxiu , Li Wei , Ren Lijie , Mao Xinliang , Zhu Li
Published: October 2019

The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu)

Disclosures:

No relevant conflicts of interest to declare.
Megakaryocyte Abnormalities Occur In The Absence Of Cell-Mediated Immunity In a Murine Model Of Passive Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Hunt Kristin , Hou Yu , Aslam Rukhsana , Speck Edwin R. , Ni Heyu , Lazarus Alan H
Published: October 2019

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to humoral mediated autoimmunity it is now evident that T cells can also play a contributing role to the thrombocytopenia. In fact, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the elements of both the humoral and cell mediated immune systems still remain incompletely defined. In murine passive models of ITP, the direct administration of anti-platelet antibodies can result in severe thrombocytopenia which is evident within 24 hours of injection. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the anti-platelet antibody also has an effect on megakaryocytes. To unequivocally determine if antiplatelet antibodies have an effect on megakaryocytes in an in vivo model, BALB/c mice were intravenously administered 2 ug of an anti-GPIIbIIIa antibody (MReg30) or 50 uL of a high tittered anti-GPIIIa (anti-β3) serum from BALB/c GPIIIa (CD61) knockout mice immunized with wild type platelets. Platelet counts were assessed over time and the bone marrow and spleens were harvested for histological examination of megakaryocytes. Both preparations of antiplatelet antibodies significantly reduced platelet numbers within 1 day of antibody or serum administration. This thrombocytopenia could be rescued by administration of 2 g/kg of IVIg ip. Compared with naïve control mice, histological (H&E staining) examination of the bone marrow and spleens revealed that megakaryocytes were significantly increased in number and all exhibited abnormalities consistent with apoptosis e.g. pyknotic nuclei. IVIg administration completely prevented these megakaryocyte abnormalities. These results show that passively administered anti-platelet antibodies not only affect platelet counts but also significantly affect megakaryocyte physiology in the absence of cell mediated immunity.

Disclosures:

No relevant conflicts of interest to declare.