bluuebunny/crossref_metadata_2025_split huggingface dataset:
Blood Induced Cartilage Changes in a Hemophilia A Murine Model.
Authors: Dunn Amy L. , Shehata Bahig M. 
Published: October 2019

Abstract
Background: Hemophilia A results from a congenital deficiency of clotting factor VIII. Patients with hemophila are unable to generate adequate thrombin during blood coagulation and therefore have a bleeding diathesis. Degenerative joint disease is the largest source of morbidity for hemophilic patients. The pathophysiology of this process is poorly understood but clinically, repeated bleeding into a joint leads to changes in the synovium, articular cartilage and underlying bone. It is not well known how quickly cartilage changes occur or if animal age has any impact on susceptibility to blood induced cartilage damage.
Materials and Methods: The E16 fVIII knockout mouse is a well-established model for hemophilia research. These mice contain a targeted disruption of the factor VIII gene at exon 16 (E16) in a C57BL/6 background. Despite having no detectable circulating fVIII activity they rarely suffer from spontaneous hemarthroses. Therefore hemarthroses was mimicked by injecting 5 μL of autologous whole blood into the hind knee. Six male mice per group in each of three ages; 12, 24 and 52 weeks were utilized. The animals were sacrificed 48 hours after injection. The injected and control contralateral knee joint from each animal was fixed in 10% formalin and then mounted in paraffin. After decalcification, five micrometer sections through the joint were obtained. Joint architecture was examined after hematoxylin and eosin staining. Proteoglycan content of articular cartilage was evaluated after alcian blue staining as part of the modified Mankin score. The pathologic specimens were also scored utilizing Valentino’s visual bleeding score. The Student’s t-test was utilized for significance testing.
Results: All mice showed evidence of blood remaining in the injected joint at 48 hours. The mean visual bleeding score was significantly higher for the injected knees versus the control knees of all age groups p<0.001. The mean visual bleeding score was significantly between the 12 and 24 week animals p=0.0112 and the 12 and 52 week animals p=0.0379, but not between the 24 and 52 week animals p=0.5423. The modified Mankin score was significantly higher between the injected and control knees of all age groups as seen in Figure 1 where the mean and standard deviation is shown for 6 animals in each group. Interestingly, one control knee in a 24 week old animal demonstrated synovial hyperplasia and decreased proteoglycan staining but had no evidence of blood in the joint. This likely represented a previous spontaneous hemorrhage. The modified Mankin score was highest for the 12 week old animals and lowest for the 52 week animals but did not achieve statistical significance p=0.5746.
Conclusions: Injection of autologous blood mimicked clinical hemarthrosis. Evidence of decreased proteoglycan staining of articular cartilage was evident within 48 hours of blood exposure. Additionally, there was a trend toward more prominent proteoglycan loss in the youngest animals.
Figure Figure
In-Situ Proliferation May Explain the Persistence of Host Derived Langerhans Cells Following Allogeneic Hematopoietic Stem Cell Transplantation with Reduced Intensity Conditioning.
Authors: Richards Julie , Pearce Laurence , Patten Piers E.M. , Buggins Andrea G.S. , Ho Aloyisius Y. , Pagliuca Anthony , Mufti Ghulam J. , Devereux Stephen 
Published: October 2019

Abstract
Langerhans cells (LCs) are antigen presenting cells found in the epidermis. They are thought to originate from bone marrow precursors although studies in mice suggest they have the ability to self renew in situ. Murine models have also shown that recipient LCs are required for the development of acute graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Engraftment of LCs following human allo-HSCT has previously been assessed using migration from isolated epidermal sheets, which may be inaccurate because of the potential for differential migration of donor and recipient cells. We therefore studied LC engraftment using fluorescence immunophenotyping and simultaneous in-situ hybridisation for X/Y chromosomes in sex mismatched transplants. Skin biopsies were performed at days 28, 56 and 100, 6 months and 1 year post transplant on 8 patients receiving alemtuzumab based RIC allo-HSCT regimens. Ten micron cryosections were taken, LCs labelled with anti-CD1a and X/Y chromosomes detected with the CEP X/Y probe kit. Slides were examined on a Zeiss LSM510 Meta confocal microscope and Z-stack images were collected to cover a volume of 100μm3. Chimerism of purified CD15+ and CD3+ peripheral blood cells was determined in parallel by polymerase chain reaction amplification of short tandem repeat sequences. Results are tabulated below and show that compared to CD15+ myeloid blood cells, donor LC engraftment is markedly delayed (day 28, 56 and 100 paired t-tests give P<0.0001, P=0.001 and P=0.0302 respectively). Of the 8 patients studied, 1 went on to develop biopsy proven GVHD. In all cases studied, recipient LCs could still be detected albeit in small numbers at the 1-year time point. Since these findings suggested that epidermal LCs are either very persistent or replicate in-situ, we next stained samples of normal and post transplant GVHD skin with both CD1a and the proliferation marker Ki67. In both cases a proportion of CD1a+ cells also expressed Ki67 (13.5% of normals, n=8; 18.6% of GVHD n=9 p=0.33). Both recipient and donor derived LCs appear to be capable of proliferating since both male and female Ki67+ CD1a+ cells were identified in 2 cases for which appropriate samples were available. In conclusion, our results provide independent confirmation that recipient LCs may persist for a prolonged period following allo-HSCT and show that at least in part, this may be due to self renewal within the epidermis.
Post transplant chimerism analysis Cell Type Day 28 Day 56 Day 100 6 Months 1 Year Median % donor chimerism at specific time points following allo-HSCT. LC 18.5 (n=8) 73 (n=7) 90 (n=7) 93.5 (n=6) 97 (n=3) CD3+ 99 (n=8) 80.5 (n=6) 66.5 (n=6) 79.5 (n=4) 100 (n=3) CD15+ 100 (n=8) 100 (n=6) 100 (n=6) 100 (n=4) 100 (n=2)
GMP-Conform Generation and Cultivation of Unrestricted Somatic Stems Cells (USSC) from Cord Blood Using the SEPAX©-Separation Method a Closed Culture System Applying Cell Stacks.
Authors: Radke Teja F. , Buchheiser Anja , Lefort Aurélie , Maleki Mahtab , Wernet Peter , Kögler Gesine 
Published: October 2019

Abstract
Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.
C1q Complement Cascade Protein as a Novel Modulator of the SDF-1-CXCR4 Axis and Hematopoietic Stem/Progenitor Cell Trafficking.
Authors: Jalili Ali , Shirvaikar Neeta , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna 
Published: October 2019

Abstract
The first component of the complement (C) system C1q has multiple immune functions including acting as a chemoattractant for mature blood cells, and it has recently been reported that hematopoietic stem/progenitor cells (HSPC) express the C1q receptor, C1qRp. We have reported that the complement components C3 and C5 play an important role in the mobilization of HSPC and that G-CSF mobilization activates C by the classical Ig-dependent pathway (Blood2004;103:2071; Stem Cells 2007, in press). While C3 plays an important role in the retention of HSPC in BM, C5 is pivotal for their egress into peripheral blood. The aim of this study was to examine a possible role for C1q in the mobilization/homing of HSPC. We found (using RT-PCR and Western blotting) that the C1q gene and protein are strongly expressed in human mobilized peripheral blood (mPB) CD34+ cells, but not in steady-state bone marrow (BM) CD34+ cells. Importantly, we found that G-CSF stimulation of BM cells induces the expression of C1q in CD34+ cells, as detected by flow cytometry. Moreover, we demonstrated the presence of the receptor for C1q, C1qRp, in BM, mPB and cord blood (CB) CD34+ cells as well as in myeloid, megakaryocytic and erythroid progenitors ex vivo-expanded from CD34+ cells. The stromal derived factor (SDF)-1-CXCR4 axis plays a critical role in HSPC homing to BM and we showed using confocal microscopy that pre-incubation (for 1 h) of CB CD34+ cells on C1q-coated slides increased incorporation of the SDF-1 receptor, CXCR4, into membrane lipid rafts while C1q-coated filters increased chemoattraction (compared to bare filters) to an SDF-1 gradient. Although C1q itself is not a chemoattractant for HSPC, it primed the chemotactic response of CD34+ cells to a low SDF-1 gradient (10 ng/mL) to the extent that it approximated response to a high SDF-1 gradient (200 ng/mL), as well as the chemoinvasion of CB CD34+ cells across reconstituted basement membrane (Matrigel) to a low gradient of SDF-1, which was inhibited by the monoclonal anti-C1q antibody. In addition, C1q stimulated matrix-degrading metalloproteinase-9 secretion by CB CD34+ cells. In conclusion, we demonstrated for the first time that G-CSF mobilization strongly upregulates C1q in human HSPC which express the functional C1q receptor and that the first component of the complement cascade is involved in HSPC trafficking. This supports our previous data that C as an important modulator of the SDF-1-CXCR4 axis plays a pivotal role in the mobilization of HSPC.
Autologous Implantation of Bone Marrow for Non-Unions and Avascular Necrosis of the Bone.
Authors: Nedelcu Elena , Vierria Dawn , Taylor Douglas , Dwyre Denis , Jamali Amir 
Published: October 2019

Abstract
Introduction: Autologous implantation of bone marrow (BM) stem cells for avascular necrosis of the bone has been previously demonstrated to be feasible and safe procedure. The benefit of using autologous stem cells for bone regeneration consists of avoidance of graft rejection, graft-versus-host disease, and the toxic and infectious complications associated with immunosuppressive regimens.
Materials and Methods: We developed an efficient protocol to process autologous BM for volume reduction and mononuclear cell concentration that can be immediately used for autologous implantation. The end-product is a concentrated BM cell suspension that was defined by several parameters as follows: volume, mononuclear cell count, CD34-positive cell count. A total of five patients under general anesthesia underwent BM aspiration, followed by immediate intraoperative BM concentration and implantation at the injured site. Four patients had avascular necrosis of the bone and one had tibia non-union. The patients were followed for one year and monitored clinically and by imaging studies.
Results: All five patients treated with autologous bone marrow implantation at the avascular necrosis or non-union site tolerated the procedure well. One year follow-up of these patients reveals improved clinical and functional status. The imaging performed pre and one-year-post procedure revealed no evidence of bone collapse and preserved joint space.
Conclusions: Autologous implantation of concentrated bone marrow is a safe and promising treatment for non-unions and avascular necrosis of the bone. Additional studies for further characterization of the concentrated BM used for implantation are in progress.
GATA-1 Represses PU.1/Sfpi-1 Gene Transcription in Erythro-Megakaryocytic Progenitors.
Authors: Chou Stella T. , Bailey Charles L. , Hardison Ross C. , Blobel Gerd A. , Vakoc Christopher R. , Weiss Mitchell J. 
Published: October 2019

Abstract
Commitment of multipotential hematopoietic progenitors to unique cell fates is determined by the induction and interplay of specific nuclear factors that reinforce unilineage transcriptional programs. Previously, we reported that loss of transcription factor GATA-1 promotes the expansion of bipotential megakaryocyte erythroid progenitors (MEPs) from embryonic stem cell or fetal liver derived hematopoietic progenitors. These mutant cells, termed G1ME (for Gata-1− Megakaryocyte-Erythroid), proliferate in culture and differentiate into committed megakaryocytes and erythroblasts when GATA-1 activity is restored. To evaluate gene regulation at the MEP stage of hematopoiesis, we performed microarray analysis of G1ME cells before and after GATA-1-induced differentiation. Expression of GATA-1 in G1ME cells induced numerous erythroid and megakaryocytic target genes. In addition, undifferentiated G1ME cells expressed numerous granulocyte and macrophage genes, suggesting that loss of GATA-1 derepresses a myeloid program in MEPs. Myeloid genes were rapidly inhibited upon expression of GATA-1. In particular, mRNA encoding the myeloid transcription factor PU.1 and more than thirty of its downstream targets were repressed by GATA-1. Chromatin immunoprecipitation (ChIP) showed that GATA-1 bound directly to the PU.1 gene (Sfpi1) at the promoter and at a −18kb upstream region coincident with repression. At the same regions, GATA-1 triggered release of both GATA-2, a related transcription factor expressed in multipotential progenitors, and PU.1, a positive autoregulator of its own gene. While GATA-1 is known to inhibit PU.1 functions through direct protein interactions, this work shows that antagonism also occurs at the level of Sfpi1 gene transcription. Together, our findings indicate that GATA-1 not only positively regulates an erythro-megakaryocytic program of gene expression, but also actively restrains myelopoiesis by inhibiting Sfpi1 expression directly. More generally, our work reveals a new mechanism through which cross-antagonism between transcription factors reinforces lineage commitment decisions in hematopoiesis.
GATA-1 Interferes with Monopoiesis by Blocking a Positive Regulatory Circuit Involving VDR and PU.1 - Results from a Retroviral Dominant Effector Screen.
Authors: Taschner Sabine , Gobel Florian , Mag Mario Kumerz , Jorgl Almut , Richter Susanne , Strobl Herbert 
Published: October 2019

Abstract
In multipotent hematopoietic progenitors the co-expression of several lineage affiliated transcription factors occurs prior to commitment. The mechanism underlying the repression of alternative lineage pathways is poorly understood. We screened a complex retroviral fetal liver cDNA library for regulatory proteins that repress monopoiesis in a phenotype-based differentiation assay system. This functional genetic “dominant effector” strategy revealed a positive circuit regulating monopoiesis that is established by PU.1 and vitamin D receptor (VDR) as well as a novel repressor function of GATA-1. Increasing levels of PU.1 sensitize progenitors to VDR-dependent monopoiesis, and VDR/retinoic X receptor (RXR) signalling directly transactivates PU.1 gene expression. This positive regulatory loop is repressed by GATA-1 via the VDR. Specifically, we show that the GATA-1 N-terminal region, which is required for the inhibition of monocytic cell proliferation, represses and physically interacts with VDR. Additionally, PU.1 binding via the GATA-1 C-terminus is sufficient to inhibit monocyte differentiation without impairing myeloid cell proliferation. Thus, loss of GATA-1 N-terminal function allows for the expansion of immature myeloid progenitors. We propose that GATA-1 represses monopoiesis in multipotent myeloid progenitors or precursors by interacting with both VDR and PU.1, ultimately enabling alternative lineage programs.
ZFP143 Activates C/EBPα Transcription in Myeloid Cells.
Authors: Bartholdy Boris , Gonzalez David , Tenen Daniel G. 
Published: October 2019

Abstract
C/EBPα is a key transcription factor involved in myeloid differentiation and frequently mutated or deregulated in human acute myeloid leukemias (AML) as well as in blast crisis of chronic myelogenous leukemia (CML). Disruption of its function contributes to the differentiation block observed in these diseases and thus to leukemogenesis. Here, we have identified a conserved region in the C/EBPα promoter that is important for activation of C/EBPα transcription in myeloid cells and narrowed it down to a conserved site of approximately 25 bp that contains a consensus binding site for ZFP143, a seven-zinc finger transcription factor. In gel retardation assays, this region bound a factor of approximately 100 kDa that we biochemically purified and identified by mass spectrometry as being indeed ZFP143. In vivo binding of ZFP143 to its bona fide binding site in the C/EBPα promoter was also detected by chromatin immunoprecipitation assays. We are now studying the in vivo effect of ZFP143 on C/EBPα transcription in mice that carry an inducible gene trap in the ZFP143 locus. While mice homozygous for the active gene trap - that prevents ZFP143 transcription - die at an early embryonic stage, preliminary data from heterozygous mice shows indeed a reduction of C/EBPα mRNA levels, suggesting a role for ZFP143 in C/EBPα transcription activation. Ongoing experiments aim at selectively inactivating ZFP143 in the hematopoietic system using the Mx1-Cre / loxP system.
Evi-1 Promotes Para-Aortic Splanchnopleural Hematopoiesis through Up-Regulation of GATA-2 and Repression of TGF-β Signaling.
Authors: Sato Tomohiko , Goyama Susumu , Kurokawa Mineo 
Published: October 2019

Abstract
The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in myeloid tumors in mice. Evi-1 is highly expressed in cases with human acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) as a consequence of chromosomal rearrangements involving 3q26, where Evi-1 is mapped. Mice deficient in Evi-1 die during embryogenesis with widespread hypocellularity, hemorrhaging, and disruption in the development of the heart, somite, and neural crest-derived cells. It was recently reported that Evi-1 is expressed at a high level in the para-aortic splanchnopleural (P-Sp) region, from which definitive hematopoiesis originates. Hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are decreased in number with defective proliferation capacity, and this defective proliferation of Evi-1-deficient P-Sp cells is successfully rescued in vitro by retroviral transfer of GATA-2. However, detailed molecular mechanisms underlying Evi-1-mediated hematopoiesis remain to be elucidated. In the present study, we used a coculture system of cells derived from the P-Sp region with a layer of a stromal cell line OP9, on which hematopoietic cell development is efficiently induced. In this culture system, Evi-1-deficient P-Sp-derived cells showed severely decreased colony forming capacity. This defect was overcome by reactivating Evi-1 retrovirally. Using this assay, we examined a hematopoietic potential of a series of Evi-1 mutants and found that the first zinc finger domain and the acidic domain are required for the hematopoietic rescue of Evi-1-deficient P-Sp cells. These two domains were also related to some extent for GATA-2 up-regulation, suggesting that GATA-2 is one of the key molecules in Evi-1-mediated hematopoiesis. Furthermore, we found that blocking of TGF-β signaling is also able to recover the hematopoietic defect of Evi-1-deficient P-Sp cells. These findings suggest that Evi-1 promotes hematopoietic stem/progenitor expansion during embryogenesis through at least two pathways: up-regulation of GATA-2 and inhibition of TGF-β signaling.