bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hematopoietic Stem Cells Can Give Rise to Satellite-Like Cells in Skeletal Muscles.
Authors: Yoshimoto Momoko , Heike Toshio , Shiota Mitsutaka , Kobayashi Hirohiko , Umeda Katsutsugu , Nakahata Tatsutoshi 
Published: October 2019

Abstract
Recent studies have indicated that bone marrow cells can regenerate damaged muscles, but also that they can adopt phenotype of other cells by cell fusion. It has also been reported that single hematopoietic stem cells (HSCs) can regenerate skeletal muscle although it is still controversial whether HSCs differentiate into satellite cells in muscle or not. In order to investigate the roles of HSCs in muscle regeneration and whether they can generate satellite cells or not, we purified and injected CD45+Lin−Sca-1+c-kit+(CD45+KSL) HSCs labeled by green fluorescent protein (GFP) into mice with or without irradiation. We examined time-course behavior of HSCs in recipient muscles with a fluorescent stereomicroscope and then immunohitochemical staining during the early and late phase after transplantation. Our direct visualization system gave evidence of massive GFP signals in all the muscles of only irradiated mice in early phase after transplantation. Transverse cryostat sections showed GFP+ Myosin+ muscle fibers along with numerous GFP+ hematopoietic cells in damaged muscle. We also found myogenin+GFP+ cells like myoblasts in very low number. These phenomena were temporal and GFP signals had dramatically reduced 30 days after transplantation. FISH analysis confirmed the GFP-DNAs in the nuclei of muscle fibers. These results suggested that most of GFP+HSCs fused with myofibers and participated in regeneration of damaged muscles, and a very few HSCs can differentiate into myoblast like cells expressing myogenin. After 6 months, GFP+ fibers could be hardly detected but GFP+c-Met+ mononuclear cells were located beneath the laminin+ basal lamina. Single fiber cultures from these mice showed proliferation of GFP+ fibers. These results suggested that HSC-derived cells settled beneath the basal lamina like satellite cells and might acquired the satellite cell activity.
Rhnull Syndrome: Identification of a Novel Mutation in RHce Gene.
Authors: Reid Marion E. , Rosa Karina A. , Powell Vivien I. , Lomas-Francis Christine , Costa Fernando F. , Stinghen Servio T. , Watanabe Alexandra M. , Carboni Edna K. , Baldon Joseani P. , Jucksch Maria Marta F. , Castilho Lilian 
Published: October 2019

Abstract
Background: Rhnull syndrome, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic mild hemolytic anemia. The suppression of Rh antigen expression for regulator types is attributed to mutations of the RHAG gene. The deficiency of Rh proteins on the red blood cells (RBCs) from the rare individuals of the Rhnull amorph type may be the result of homozygosity for a silent RHCE allele at the RH locus in which the RHD is absent. We studied a Brazilian family transmitting an amorph Rhnull disease gene on a consanguineous background and identified a novel mutation in RHce gene causing the loss of function phenotype.
Methods: RBCs from two Rhnull sisters (G1 and G2) of the amorph type and from family members were analyzed by serology and flow cytometry with monoclonal antibodies specific for Rh (D, C, c, E and e), and LW antigens, for RhAG and CD47 as well as alloantibodies directed against GPB (S, s and U) antigens. Genomic DNA samples and transcripts were tested by PCR and sequence analysis.
Results: RBCs from G1 and G2 did not react with the anti-Rh and anti-LW reagents and reacted weakly with anti-RhAG, anti-CD47, anti-s, and anti-U reagents. RBCs from G1 were S+ and from G2 S-. Molecular analyses showed a deletion of the nucleotide at position 963 in exon 7 of the RHce gene [GGG(Glu321→GG)] in addition to a deletion of the RHD gene. This nucleotide deletion in exon 7 introduced a frameshift after Glu321 and a premature stop codon, resulting in a shorter predicted protein with 359 amino acids, including a new C-terminal sequence that is likely to alter the protein conformation and impair the Rh complex assembly.
Conclusion: We describe a novel mutation of an amorph Rhnull disease gene leading to a loss of function phenotype. Our findings reinforce previous studies suggesting that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins plays a role in the stability of the Rh membrane complex.
Quantum Dots, Efficient Fluorescent Markers for Red Cells.
Authors: Castro Vagner , Farias Patrícia M.A. , Santos Beate S. , Menezes Frederico D. , Ferreira Ricardo C. , Lima Paulo R.M. , Fontes Adriana , Barjas-Castro Maria Lourdes , Cesar Carlos L. 
Published: October 2019

Abstract
Semiconductor quantum dots (QDs) are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. In comparison with organic dyes and fluorescent proteins, QDs are emerging as a new class of fluorescent labels with improved brightness, resistance against photobleaching, and multicolor fluorescence emission. These properties could improve the sensitivity of biological detection and imaging by at least 10 to 100 fold. Recent research has shown that QDs can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids and small molecules for use as fluorescent probes. The aim of the present study was to evaluate the application of a Cadmium Sulfide QDs method for the analysis of the expression and distribution of A antigen on living red blood cell surface. Highly luminescent and stable cadmium sulfide colloidal particles were obtained in the nanometer size range. Their surface was functionalized with commercially available monoclonal anti-A antibody via an one-pot glutaraldehyde cross linking procedure, followed by their conjugation to red cells. Samples were obtained from blood bank red cell units, of groups A (n=3), A2 (n=2) and O (n=3). Confocal microscopy (LSM 510 - Carl Zeiss, Jena, Germany) was used and for each cell type, the images were reproduced at least three times. The acquisition confocal image parameters, such as pinhole, filters, beam splitters and photomultiplier gain, were maintained constant to permit a comparative analysis of the luminescence intensity maps. Analyzing the intensity map of group A erythrocytes, a homogeneous emission profile was observed throughout the cell surface, suggesting a homogeneous distribution of A antigen. In contrast, the intensity map analysis of the confocal images showed a lower intensity pattern of the A2 group erythrocytes studied, suggesting different antigen expression on cell surface. The O group cells showed no emission. This result suggests that this new efficient, fast and simple labeling procedure may be employed as a quantitative tool to investigate the expression and distribution of molecules in red blood cells.
High Frequency of Unusual ABO Alleles in Patients with Leukemia.
Authors: Novaretti Marcia C.Z. , Pares Madalena M.N.S. , Domingues Alexandre E. , Dorlhiac-llacer Pedro E. , Chamone Dalton A.F. 
Published: October 2019

Abstract
Background: Mutations critical for ABO group phenotypes which encode the catalytic domain of ABO glycosyltransferases have been studied in healthy blood donors. Weakening of A and B transferases have been reported in patients with leukemia, particularly those in which the myeloid lineage is involved. The purpose of this study was to use sequence specific PCR (PCR-SSP) to perform ABO genotyping of leukemic patients.
Material and Methods: A total of 108 unrelated leukemic patients were initially classified serologically using tube test technique and gel test for ABO typing and subgroup detection. Genomic DNA was prepared from peripheral blood cells. All samples were then screened by 32 different PCR-SSP, each specific for a single nucleotide variation to screen 16 known polymorfic sites of exons 6 and 7 (modified from Seltsam, Transfusion 2003,43(4):428–39.
Results: The results of PCR screening were conclusive and consistent with the ABO phenotypes in 71 patients (65.07%). Common ABO alleles were found in only 15 (13.8%) of patients studied. Unusual ABO alleles were found in 26 of 28 blood group O patients (ABO*O05, ABO*O21, ABO*O30, ABO*O33, ABO*O35, ABO*O36, ABO*O43 and ABO*O45), in 20 of 43 blood group A patients (ABO*A103, ABO*A104, ABO*A106, ABO*A202, ABO*A203), in 8 of 10 blood group B patients (ABO*B104, ABO*B105) and in 2 of 4 AB blood group patients (ABO*Bx01, ABO*B103). In 37 patients (34.2%), PCR screening revealed allele combinations that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis.
Conclusion: This is the first report of using PCR-SSP for ABO genotyping of leukemic patients. The pathophysiologic role of unusual ABO allele combinations in patients with leukemia warrants further investigation.
Second Donation of Bone Marrow; Japan Marrow Donor Program (JMDP) Experience.
Authors: Akiyama Hideki , Hara Masamichi , Hino Masayuki , Sao Hiroshi , Hoshi Yasutaka , Ohto Hitoshi , Kai Shunro , Ochiai Ryoichi , Morita Kiyoshi , Nakao Yasuo 
Published: October 2019

Abstract
In Japan, JMDP, founded in 1991, had collected and transplanted more than 5,000 bone marrows by the end of 2003. Meanwhile, total of 137 donors donated their bone marrow twice. The efficiency and the safety of those donations and the efficacy of those cells in the engraftment were evaluated retrospectively. In JMDP, volunteers aged from 20 to 50 are enrolled as the donor. A year after the first donation, the donors are allowed to donate their marrow again, if they wish. Marrow was collected from the posterior iliac crest under general anesthesia and, generally, different patients received the marrow from the single donor. Peripheral blood stem cell harvest is not permitted by the JMDP. Their mean age was 36.6 and 36 of them were female. The volume of the marrow and the nucleated cell number collected in the first and the second donation were 760 ± 290 vs 857 ± 230 ml (p=0.001) and 15.3 ± 6.8 vs 15.6 ± 6.9 x 10E9 cell (p=0.674), respectively. Cell concentration of bone marrow solution and nucleated cell number per recipient’s weight were significantly lower in the second donation (2.2 ± 0.8 vs 1.8 ± 0.7X 10E7 cell/ml (p=0.0004) and 3.3 ± 1.1 vs 2.8 ± 1.1 x 10E8 cell/kg (p<0.0001), respectively) revealing decreased cell dose. The efficacy of those cells was also evaluated comparing the available data from 127 pairs of the patients who received the marrow. Within these 254 recipients, data of 8 patients (2 in the first and 6 in the second donation) are missing and another 19 (8 and 11) were excluded because of early death before day 28. There was no difference between the first and the second donated marrow in the date of neutrophil recovery (p=0.5214) and platelet recovery (p=0.4108). There were two cases each of slow engraftment (up to day 35) and late rejection in 1 and 5 patients respectively (p=0.2132). The adverse effects of those donations were also compared. The incidence of fever more than 38 degrees, the duration of the febrile period and other adverse effects such as urinary pain, sore throat, infectious disease, liver dysfunction and pain on the wound lasting more than 7 days were compared and no significant difference was observed. In conclusion, second donation can be performed safely and the functions of those cells were as good as the ones donated first. However, because the cell dose of the second collection could be lower, it might be necessary to collect more marrow than the expected.
Proteasome Inhibiters Up-Regulate TRAIL/Apo2L and Its Receptors Significantly Contributing to Proteasome Inhibitor-Induced Apoptosis in Primary Chronic Lymphocytic Leukemia (CLL) Cells.
Authors: Kabore Albert F. , McCrea Kristin , Johnston James B. , Gibson Spencer B. 
Published: October 2019

Abstract
The proteasome inhibitor, bortezomib has recently emerged as a new therapeutic treatment for refractory multiple myeloma and is presently being evaluated for other hematological malignancies either alone or in combination with other antitumor agents. Proteasome inhibitors cause the accumulation of many proteins but the precise mechanism responsible for their antitumor effect is unclear. In the present study, we have determined that cytotoxic effect the proteasome inhibitor MG-132 in primary chronic lymphocytic leukemia (CLL) cells is through the activation of the TRAIL (tumor necrosis factor-related apoptosis inducing ligand) apoptotic pathway. MG-132 induced apoptosis in approximately 70% of primary CLL cells as measured by annexin V staining. Addition of DR4:Fc that prevents TRAIL ligation with its receptors decreased the amount of MG-132 induced apoptosis by approximately 40% suggesting MG-132 caused activation of the TRAIL apoptotic pathway. MG-132 also up-regulated both the mRNA and protein levels of TRAIL and protein levels of TRAIL receptors DR4 and DR5. This upregulation correlated with activation of caspase 8 and cleavage of pro-apoptotic Bcl-2 family member Bid. Moreover, MG-132 treatment also induced a substantial reduction in the FLICE-like inhibitory protein (c-FLIP) protein levels. In contrast to CLL cells, proteasome inhibitors failed to activate the TRAIL apoptotic pathway in normal B-cells. This indicates that proteasome inhibitors are inducing apoptosis in primary CLL cells through activation of the TRAIL apoptotic signaling pathway through up-regulation of TRAIL and its cognate receptors and reduced FLIP expression. Thus, proteasome inhibitors may have a therapeutic role in CLL, either when used alone or in combination with TRAIL or antibodies against DR4/DR5.
Ribosomal Protein S19 and Diamond Blackfan Anemia.
Authors: Ellis Steven R. , Arce-Lara Carlos , Caffrey Jacqueline M. , Alvarez-Arias Diana A. 
Published: October 2019

Abstract
Diamond Blackfan Anemia (DBA) is one of several bone marrow failures that have been linked to defects in ribosome synthesis. 25% of DBA cases are linked to mutations in ribosomal protein S19 (Rps19). The etiology of the remaining cases is unknown. To gain a better understanding of the function of the Rps19 family of proteins we have characterized members of this protein family in the yeast, Saccharomyces cerevisiae. In yeast, Rps19 is encoded by duplicated genes, RPS19A and RPS19B. Yeast cells lacking both RPS19 genes are not viable, whereas those lacking a single gene are viable but have growth defects. These latter strains are defective in a specific step in rRNA processing that preferentially affects the maturation of 40S ribosomal subunits. We scanned other yeast strains with mutations in genes for 40S subunit proteins for processing phenotypes similar to RPS19 mutants. Several have phenotypes that overlap with RPS19 mutants, but only RPS18 stands out as being virtually identical to RPS19 mutants. The human RPS18 gene is therefore a candidate locus for pathogenic mutations in DBA patients with normal RPS19. We are currently developing strategies to sequence RPS18 genes from DBA patients with normal RPS19 to determine if mutations in RPS18 are associated with DBA. We have also developed a yeast system for the functional testing of mutant alleles of RPS19 found in DBA patients. In general, a mutation is considered pathogenic if it is not found in unaffected family members and in the general population. We have found, however, that several missense mutations classified as pathogenic in DBA patients do not affect Rps19 function in the yeast system. The failure of these mutations to affect Rps19 function in yeast points to a need for functional testing of RPS19 mutant alleles in human cells.
Generation of Dendritic Cells in a Closed System Using GMP Guidelines.
Authors: Giulivi Antonio , Halpenny Mike , Birch Paul , Yang Lyn , Martin Lisa 
Published: October 2019

Abstract
Objective: The application of a dendritic cell (DC) based immunotherapy requires the ability to consistently produce a clinically acceptable product using Good Manufacturing Practice (GMP). We describe our experience using the Nexell SteriCell container to generate monocyte derived DC in a closed system using GMP guidelines.
Design/Materials and Methods: Leukapheresis product was used as source material to first isolate monocytes by adherence to culture bag and then generate DC from monocytes using a serum free media containing GM-CSF and IL-4 and culturing for 7 days at 37°C /5% CO2. DC generation was characterized by phenotyping cultured cells for decreased expression of CD14 and increased appearance of CD80, CD86 and DR. In addition to phenotyping, cultures were tested pre and post culturing for viability and microbial contamination.
Results: Cultured cells expressed a decreased expression of CD14 (mean % decrease 58.8 /range 11.2–94.9) and increased expression of CD 80/86 (mean fold increase 23.6X /range 5.7 – 36.2), and DR (mean fold increase 9.3X /range 3.2 – 16). All microbial cultures were negative and viability ranged from 54% to 90% post incubation.
Conclusions: The Canadian Blood Services cell processing laboratory is a FACT (Foundation for the Accreditation of Cellular Therapy) accredited facility following GMP guidelines. The results obtained from our study demonstrate a system easily adaptable to larger scale production of DC for possible future immunotherapy clinical application.
Upregulation of MT1-MMP Expression by Hyaluronic Acid Enhances Homing-Related Responses of Hematopoietic CD34+ Cells to an SDF-1 Gradient.
Authors: Shirvaikar Neeta , Montano Jencet , Turner Andrew R. , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna 
Published: October 2019

Abstract
Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.
Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy.
Authors: Konno Akihiro , Jagadeesh G. Jayashree , Moratto Daniele , Bosticardo Marita , Holt Ingeborg , Muul Linda M. , Schurman Shepherd H. , Wolfsberg Tyra , Candotti Fabio 
Published: October 2019

Abstract
Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking.
We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001).
These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.