bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.