bluuebunny/crossref_metadata_2025_split huggingface dataset:
Acute on Chronic Pulmonary Hypertension in Patients with Sickle Cell Disease.
Authors: Kato Gregory J. , Martyr Sabrina , Machado Roberto , Sachdev Vandana , Ernst Inez , Hunter Lori , Coles Wynona , Nichols James , Hunter Christian J. , Gladwin Mark T. 
Published: October 2019

Abstract
Secondary pulmonary hypertension (PHT), defined as a tricuspid regurgitant jet velocity (TRV) > 2.4 meters/second at rest, has been identified as an emerging complication in patients with sickle cell disease, with 32% prevalence. Patients with sickle cell pulmonary hypertension tend to have a higher mortality rate than patients with primary PHT at comparable pulmonary artery pressures (PAP). We analyzed prospectively obtained Doppler echocardiography measurements from 11 patients in steady-state and during vaso-occlusive crisis episodes or during exercise to determine whether pulmonary pressures became further elevated during stress. We found that tricuspid regurgitant jet velocity is significantly further elevated during vaso-occlusive crisis (2.61 ± 0.03 vs. 2.81 ± 0.06 m/sec (mean ± SEM), p = 0.01)(Fig. A). We assessed other laboratory parameters and found associated significant decreases in hemoglobin concentration (9.3 ± 0.3 vs. 8.4 ± 0.3 gm/dL, p = 0.02)(Fig. B), and significant elevations in lactate dehydrogenase (381 ± 39 vs. 442 ± 49 IU/L, p = 0.02)(Fig. C). These data are consistent with increased hemolysis during vaso-occlusive crisis, and the association of increased hemolysis with acute exacerbation of PHT in patients with SCD. In three patients at steady state, exercise echocardiograms were performed, showing acute marked rise in TRV during exercise (2.80 ± 0.12 vs. 3.37 ± 0.17 m/sec, p = 0.02)(Fig. D). These data bring to light several important points in the clinical assessment of tricuspid regurgitant jet velocity in patients with sickle cell disease. First, the relatively mild steady state PHT in patients with SCD is associated with previously unappreciated acute increases in PAP that may explain the high mortality rate in this population. This might be responsible for sudden death occurring during vaso-occlusive crisis or physical exertion. Second, this acute rise is associated with markers of increased hemolysis, further implicating hemolysis-associated derangement of nitric oxide homeostasis, endothelial function and PHT. Third, exercise measurements of TRV may be indicated in the clinical evaluation of patients with SCD and dyspnea on exertion, since it may unmask otherwise unappreciated PHT.
Figure Figure
Long Term Treatment with Hydroxyurea Does Not Prevent Development of Renal Dysfunction and Osteodystrophy in Patients with Sickle Cell Disease.
Authors: Voskaridou Ersi , Terpos Evangelos , Margeli Alexandra , Hantzi Eugenia , Stoupa Eleni , Loutradi Aphroditi , Papassotiriou Ioannis , Loukopoulos Dimitris 
Published: October 2019

Abstract
Hydroxyurea (HU) is presently considered as the main treatment for the reduction of sickle-cell crises; however, information regarding the potential of HU to inhibit progressive organ failure is scarce. The gradually failing renal function and renal osteodystrophy are well known complications of sickle cell disease (SCD). The pending question is whether administration of hydroxyurea over very long period of time may delay or prevent the appearance of these abnormalities. To this effect we evaluated the renal function and bone metabolism in 57 patients with HbS/β-thal (31M/26F; median age 32 years, range: 19–67 years) receiving hydroxyurea (usually 1 g/daily) continuously for 1 to 14 years (median 10.2 years). In addition to conventional renal biochemistry we measured the levels of serum and urinary β2-microglobulin (β2M), serum cystatin C (specific and sensitive index of glomerular filtration rate), and urine N-acetyl-b-D-glucosaminidase (NAG; reflecting the distal tubular cells function). The extent of renal osteodystrophy was evaluated by DEXA scans assessing bone mineral density (BMD), and by assaying various markers of (a) osteoclast function [soluble receptor activator of nuclear factor κB ligand (sRANKL), osteoprotegerin (OPG), and tartrate resistant acid phosphatase isoform 5b (TRACP-5b)], (b) bone resorption [C-telopeptide of collagen type I (CTX)], and (c) bone formation [bone-alkaline phosphatase (bALP) and osteocalcin (OC)]. The above parameters were also evaluated in 16 age- and gender-matched controls. Three patients (5.2%) had increased serum creatinine levels; 16 patients (28%) had more than 300 mg/day protein excretion in the urine, and 13 patients (22.8%) had microalbuminuria. Moreover, serum cystatin C was elevated in 16 patients (28%), NAG in 21 (36.8%), serum β2M in 34 (59.6%) and urinary β2M in one patient. In addition, 17 patients (29.8%) had osteoporosis/osteopenia in DEXA scans (comparable to HbS/β-thal patients who did not receive HU). Furthermore, all patients displayed significantly elevated levels of OPG (p=.001), sRANKL (p<.01), sRANKL/OPG ratio (p=.022), and CTX (p=.02), while significant correlations were found between serum cystatin C vs. both serum OPG and β2M levels as well as between cystatin C and the sRANKL/OPG ratio. Not only do these results suggest that HU does not prevent renal dysfunction in this cohort of patients but also highlight the role of RANKL/OPG pathway in the renal-induced bone disease of sickle cell syndromes. Furthermore, NAG, cystatin C and OPG may be useful as early biochemical markers for the assessment of renal impairment in SCD patients.
Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.
Authors: Miyake Noriko , Brun Ann C.M. , Magnusson Mattias , Scadden David T. , Karlsson Stefan 
Published: October 2019

Abstract
Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.
Genetic Evidence of a Novel Blood Differentiation Pathway from Lympho-Myeloid Hematopoietic Stem/Progenitor Cells, Independent of Common Myeloid Progenitors.
Authors: Hultquist Anne , Mansson Robert , Sigvardsson Mikael , Adolfsson Jorgen , Bryder David , Yang Liping , Thoren Lina , Jacobsen Sten E.W. 
Published: October 2019

Abstract
We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin−Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the cytokine tyrosine kinase receptor Flt3. Long-term HSCs (LT-HSCs) lack CD34 and Flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3−. A third LSK population is characterized by co-expression of CD34 and Flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocyte and monocyte) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential in vitro and in vivo. These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in further support of this new model through genetic profiling of these three HSC subpopulations, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to the pluripotent LSKCD34−Flt3− LT-HSCs and LSKCD34+Flt3− ST-HSCs, LSKCD34+Flt3+ cells downregulated or turned of genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor Gata-1. In contrast, the gene for Il-7rα, critically involved in early B and T cell development was upregulated in LSKFlt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and Pu.1 expression was sustained from LT-HSC through the LSKCD34+flt3+ stage Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+Flt3+ cells upregulating Il-7rα gene expression, sustained and co-expressed G-CSFR expression. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor (CMP). Our biological and gene expression data rather supports the existence of a pathway in which HSCs lose their lineage potentials one by one, starting with the Mk and E lineages.
Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.
Authors: Gupta Shiv K. , Gupta Mamta , Hoffman Barbara , Liebermann Dan A. 
Published: October 2019

Abstract
Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.
Normal Hematopoietic Stem Cell Functioning in p21−/− Mice.
Authors: Kamminga Leonie M. , van Pelt Kyrjon , Dontje Bert , de Haan Gerald 
Published: October 2019

Abstract
Recently, several studies have suggested that the family of cyclin-dependent kinase inhibitors plays a crucial role in regulating hematopoietic stem and progenitor pool size. However, due to a lack of appropriate transplantation models, competitive repopulation assays have not been performed. In the present study we have backcrossed a p21 null allele from mice with a mixed genetic background to inbred C57BL/6 mice. As expected, mouse embryonic fibroblasts (MEFs) derived from B6p21−/− mice failed to undergo senescence, whereas B6p21+/+ MEFs show a normal senescent phenotype. Moreover, B6p21−/− CFU-GM were more resistant to radiation compared to B6p21+/+. In contrast, homozygous deletion of the p21 allele did not affect the percentage of Lin− Sca-1+ c-kit+ cells in S-phase when measured by 7-AAD staining, and did not result in any alterations of in vitro cobblestone area forming cell activity. In a competitive repopulating assay different ratios of Ly5.2 BM cells from B6p21−/− or B6p21+/+ littermates were competed with 2 x 106 Ly5.1 B6 BM cells. Assuming similar repopulating capacity of both cell populations, expected chimerism was calculated. Surprisingly, observed and expected chimerism were identical, strongly suggesting that B6p21−/− stem cells had completely normal competitive repopulating activity for up to 1 year after transplant. Our data argue against an important role of p21 in maintaining stem cell function during steady-state hematopoiesis.
Human Cytomegalovirus Infection of Primitive Hematopoietic Progenitor Cells: A Model for Latency.
Authors: Goodrum Felicia D. , Jordan Craig T. , Terhune Scott S. , High Kevin P. , Shenk Thomas 
Published: October 2019

Abstract
Human cytomegalovirus (CMV) is beta-herpesvirus that infects the majority of the population worldwide and establishes a lifelong relationship with its host by way of a latent infection. Although, CMV is of little consequence to healthy individuals, reactivation of CMV from latency can cause life-threatening disease in immunocompromised individuals such as bone marrow transplant recipients and AIDS patients. CMV establishes a latent infection in cells of the hematopoietic system. The focus of our work is to elucidate the mechanisms governing a latent infection and reactivation from latency. It is likely that both viral and cellular mechanisms govern the latent program. We have developed a novel in vitro model for CMV latency using primary human hematopoietic progenitors. Using this model to compare CMV infection in CD34+ hematopoietic subpopulations, the outcome of CMV infection was found to depend on the nature of the subpopulation of cells infected. Only primitive CD34+/CD38− progenitor cells harboured an infection with the hallmarks of latency. This subpopulation transiently expressed a unique subset of CMV genes in the absence of viral replication. Importantly, viral replication could be reactivated from this subpopulation. Other primitive (CD34+/c-kit+) and more mature (CD34+/CD38+ and CD34+/c-kit−) subpopulations resulted in an apparently abortive infection. Strikingly, a CD34+ subpopulation expressing a stem cell phenotype supported a productive infection. These results indicate that cellular determinants contribute to the outcome of HCMV infection in hematopoietic cells. Further, we have also demonstrated that viral determinants play a role in establishing a latent infection using our in vitro model. Comparing the clinical Toledo and FIX strains and the laboratory-adapted AD169 strain of CMV, only the clinical strains were able to establish an infection consistent with latency. AD169, by contrast, replicated productively. Genetic sequences unique to clinical strains have been identified and may contribute to its ability to establish a latent infection in vitro. These studies indicate that both viral and cellular determinants contribute to the establishment of CMV latency. Furthermore, these determinants may serve as targets for CMV disease prophylaxis following bone marrow transplantation or in AIDS patients.
Platelet Release Occurs in the Bone Marrow Sinusoids and Spleen but Not in the Lungs Following Hematopoietic Stem Cell Transplant in the Mouse.
Authors: Kreinest Deanna , Sola Martha , Li Xiao-Miao , Sanders Ronald , Jorgensen Marda , Scott Edward , Slayton William B. 
Published: October 2019

Abstract
The steps that lead to platelet production are poorly understood. Current theories suggest that megakaryocytes mature under the influence of contact with sinusoidal endothelium, and release platelets either in the sinusoids or in the lungs. We hypothesized that platelet release would be accentuated following hematopoietic stem cell transplant, and that sites of platelet release would be apparent during the period of platelet recovery. We transplanted highly purified hematopoietic stem cells based on lack of expression of markers for mature lineages (Linneg) and expression of Sca-1, c-kit, and Thy-1.1 (KTSL cells), and subfractionated these cells based on low expression of Rhodamine 1-2-3, into lethally irradiated hosts expressing an allelic version of glucose phosphate isomerase to identify donor and host-derived platelets. We collected bones, lungs, livers and spleens on day 7, 14, 21, and 28 post-tranplant, and stained formalin/fixed tissue with anti-Von Willebrand Factor antibody to identify megakaryocytes (5–10 animals per cohort, 2 separate experiments). We scored megakaryocytes based on their location relative to endothelial cells, and whether they were releasing platelets based on extension of proplatelet processes into the vascular spaces. Almost every megakaryocyte was associated with the endothelium during the period of platelet recovery, and we did not identify megakaryocytes that were migrating to the endothelium. We saw numerous megakaryocyte releasing platelets in both the bone marrow and the spleen during the time of platelet recovery, which occurred on days 13–28 following transplant of purified stem cells. Some of these megakaryocytes had disrupted the endothelium and were incorporated into the sinusoidal wall. Others were completely within the sinusoidal spaces. Between 30 and 50% of megakaryocytes were releasing platelets in the spleen and bone marrow at any given time following transplant, and platelet release did not correlate with the platelet counts. These levels were similar to levels of platelet release seen in healthy control mice. In contrast, we saw no identifiable megakaryocytes in the liver and lung during the period of platelet recovery. Our results suggest that in the mouse, the bone marrow and spleen, and not the lung, are major sites of platelet release following stem cell transplant.
Profiling Retroviral Integration Sites in Donor T-Lymphocytes for Suicide Gene Therapy.
Authors: Giordano Frank A. , Laufs Stephanie , Nagy Katalin Z. , Fehse Boris , Hotz-Wagenblatt Agnes , Lauterborn Daniel , Fellenberg Kurt , Kuehlcke Klaus , Naundorf Sonja , Zander Axel R. , Zeller Jens W. , Fruehauf Stefan 
Published: October 2019

Abstract
Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR).
41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31).
Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.