bluuebunny/crossref_metadata_2025_split huggingface dataset:
RAS and G-CSF Receptor Mutations Are Mutually Exclusive in Leukemogenesis in Severe Congenital Neutropenia.
Authors: Germeshausen Manuela , Kratz Chris , Ballmaier Matthias , Shannon Kevin M. , Welte Karl 
Published: April 2020

Abstract
Activating RAS mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) and were found in some patients with severe congenital neutropenia (CN) after transformation to MDS/AML (Kalra et al. Blood1995;86:4579). CN is a rare genetic disorder characterized by severe neutropenia leading to recurrent life-threatening bacterial infections due to the failure of myeloid cells to mature from promyelocytes and myelocytes to neutrophils. The risk for malignant transformation in CN is currently estimated at 14%. Based on earlier investigations one proposed model of the genetic events associated with progression of CN to MDS/leukemia starts with the acquirement of a somatic G-CSF Receptor gene (G-CSFR) mutation. In this model, RAS mutations and chromosomal aberrations such as monosomy 7 or trisomies represent late events in the transformation process. We have screened bone marrow specimens from 44 CN patients for mutations in the G-CSFR and in the KRAS2 and NRAS genes. Remarkably, we not only identified G-CSFR mutations in 10 out of 14 CN patients with MDS/AML, but also in 21 out of 30 patients without transformation. Only 1 out of 44 patients with CN harbored an activating RAS mutation (NRAS, c.37G&gt;C; G13R). In this patient, who developed secondary chronic myelomonocytic leukemia at the age of 18, the NRAS mutation was not detected in a bone marrow sample obtained at age 2. Results of cytogenetic investigations were available for 10 out of the 14 CN/MDS/leukemia patients. Chromosomal abnormalities were detected in all 10 patients, and included monosomy 7 (n=3), trisomies (n=3), deletions (n=2) and translocations (n=5). In addition to the cohort of 44 patients mentioned above, we analyzed 3 of the 5 CN patients with secondary myeloid leukemia in whom a NRAS mutation, c.35G&gt;A (G12D), was described previously (Kalra et al. Blood1995;86:4579). None of these patients carried a G-CSFR mutation. Combining our data with data available from the literature the frequency of RAS mutations in CN patients is now 6/55 (11%). Accordingly, RAS mutations are less common during leukemic progression in CN than suggested by earlier studies, which may reflect changes in demographics of the CN population and/or effects of early treatment with G-CSF. The sequential occurrence of G-CSFR and RAS mutations in the leukemogenesis is very rare. Instead, our genetic data suggest RAS and the G-CSFR mutations are largely mutually exclusive in CN and may deregulate common signaling pathways.
DOCK180 Is Induced with Differentiation from CD14 Positive Monocytes and It Regulates Phagocytosis in Both Macrophages and Dendritic Cells.
Authors: Fujimoto Katsuya , Nishio Mitsufumi , Nishihara Hiroshi , Sato Ikumi , Wang Lei , Tanaka Shinya , Koike Takao 
Published: April 2020

Abstract
A guanine nucleoside exchange factor (GEF), DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac. The expression of DOCK180 is mostly ubiquitous except in hematopoietic cells, while another CDM family protein, DOCK2 is strictly found in hematopoietic cells. Although a knockout study against DOCK2 demonstrated the essential role of DOCK2 regarding rodent T- and B-cell motility and chemotaxisis, no involvement of DOCK2 in the action of phagocytes, such as macrophages or dendritic cells (DCs), has yet been observed. We, therefore, investigated the possible role of DOCK180 in the phagocytosis of hematopoietic cells. CD14 positive monocytes, as well as T- or B lymphocytes, isolated from healthy human donors did not express DOCK180 observed by Western blotting. Consistent with this finding, a real time PCR study revealed near negligible levels of DOCK180 mRNA in monocytes. On the other hand, macrophages or DCs, differentiated from monocytes in vitro in the presence of M-CSF or IL-4 and GM-CSF for seven days, were found to express extremely high levels of DOCK180 protein and mRNA. Time course studies showed that DOCK180 mRNA rapidly increased by 6 hours after the induction of CD14 positive monocytes into both macrophages and DCs and its protein expression was clearly detected by Western blotting after Day 2. While the expression of DOCK180 on DCs gradually increased until it reached a plateu level on Day 4, the expression of macrophages showed a linear increase up to Day 7. In contrast to DOCK180, the expression of DOCK2 remained unchanged throughout this study. Furthermore, a phagocytosis assay using Zymosan A particles demonstrated an increase in the phagocyte activity accompanying the expression levels of DOCK180 during both types of differentiation. Protein knockdown assays against DOCK180 using siRNA also confirmed the significance of DOCK180 in the phagocytosis of differentiated macrophages and DCs. Finally, an immunohistochemical study with anti-DOCK180 monoclonal antibody showed that macrophages exhibit the phagocytosis in the front edge of inflammatory granulation tissue, and expressed a high level of DOCK180. Taken together, DOCK180 was thus found to play a role in the induction of phagocyte activity regarding the differentiation from monocytes into either macrophages or DCs, thereby demonstrating the biological significance of DOCK180 in the phagocytosis of human hematopoietic cells.
hDectin-1 Is Involved in the Uptake of Apoptotic and Infected Cells and Mediates Cross-Presentation of Engulfed Antigens.
Authors: Weck Markus M. , Grünebach Frank , Werth Daniela , Kanz Lothar , Sinzger Christian , Brossart Peter 
Published: April 2020

Abstract
Dectin-1 is a member of the c-type-lectin-like receptor family that was shown to be the major receptor for fungal beta-glucans and to play an important role in the cellular responses mediated by these carbohydrates. In the present study we identified the hDectin-1b splice variant as the major isoform expressed in in vitro generated mDC using a quantitative RT-PCR and western blot analysis. Interestingly, stimulation of immature DC with the toll like receptor ligands Poly I:C (TLR3) or LPS (TLR4) but not with TLR ligands 2 and 7 or TNF-a led to a dramatic down regulation of hDectin-1 protein expression. To further analyze the possible involvement of Dectin-1 in the phagocytosis of cellular material we recombinantly expressed the extracellular domain (ECD) of hDectin-1b and used it to stain tumor cells. The recombinant ECD showed a specific binding to several human tumor cell lines that could be increased by induction of apoptosis in malignant cells and inhibited by incubation of the extracellular domain with zymosan, a crude cell wall extract of saccharomyces cerevisiae. Furthermore, uptake of tumor cells by immature mDC was reduced by zymosan or the presence of the recombinant Dectin-1 in phagocytosis assays suggesting that Dectin-1 is involved in the engulfement of tumor cells by DC. In line with the expression analysis of hDectin-1, we found that phagocytosis of apoptotic cells was dramatically reduced upon stimulation of DCs with Poly I:C or LPS as compared to immature DC or DC activated with TLR2 or 7 ligands. We next analysed the role of Dectin-1 in the cross-presentation of cell derived antigens and used DC that were incubated with CMV infected fibroblasts and an autologous CTL line specific for the HLA-A2 binding pp65 peptide. Stimulation of CMV peptide specific CTL in ELIspot assays by DC that were incubated with CMV infected fibroblasts efficiently stimulated IFN-gamma secretion that could be inhibited by zymosan and the extracellular domain of Dectin-1. In line with the results from phagocytosis assays stimulation of CMV specific CTL was reduced by stimulation of DC with TLR2 and 4 ligands.
Our results identify hDectin-1 as a new receptor for endogenous ligands on mammalian cells and indicate an important role of this molecule in the clearing of apoptotic and infected cells and cross-presentation of cell derived antigens..
A Selective Small Molecule Factor VIIa Inhibitor, CRA-027483, Suppresses Acute Inflammation in Mice.
Authors: Dalrymple Stacie A. , San Pablo Florentino D. , Sukbuntherng Juthamas , Janc James W. , Young Wendy B. , Vijaykumar Dange , Scheerens Heleen 
Published: April 2020

Abstract
Factor VIIa (FVIIa) is a well known member of the extrinsic coagulation cascade and therefore an attractive therapeutic target for thrombosis. CRA-027483 is a potent, selective, reversible small molecule inhibitor of human FVIIa that displays anti-thrombotic activities in vitro and in vivo. In addition to the well-characterized role of the FVIIa/tissue factor (TF) complex in coagulation, FVIIa/TF complex has more recently been implicated in inflammation via intracellular signaling initiated by the cleavage of protease activated receptors (PARs) by the complex. Therefore, we assessed the ability of CRA-027483 to inhibit acute inflammation using a mouse model of endotoxemia. The compound was delivered subcutaneously one hour prior to LPS challenge and serum or plasma was harvested at varying time points post-challenge. Soluble mediators of coagulation and inflammation induced by LPS were measured by ELISA. Additionally, prothrombin time (PT) and compound exposure were determined. CRA-027483 dose-dependently increased PT in mice as expected. Increases in PT correlated with increases in compound plasma concentrations. Coagulation markers plasminogen activator inhibitor-1 (PAI-1), D-dimer, and thrombin antithrombin (TAT) complex were dose-dependently inhibited by CRA-027483. In addition to increasing PT and inhibiting coagulation markers as expected, CRA-027483 also dose-dependently inhibited several pro-inflammatory mediators including cytokines, chemokines, and acute phase proteins. Inhibitory effects correlated with extension of PT, suggesting that the anti-inflammatory action of CRA-027483 was likely the direct result of inhibiting FVIIa/TF signaling rather than an off-target effect. Thus, we have demonstrated that selective inhibition of FVIIa with CRA-027483 results in suppression of acute inflammation in mice.
Inhibition of mTOR Reverses the Ability of c-MYC To Block the Differentiation of Murine Granulocytes.
Authors: Wall Meaghan , Poortinga Gretchen , Pearson Richard B. , Hannan Ross D. , McArthur Grant A. 
Published: April 2020

Abstract
The proto-oncogene c-MYC encodes a bHLH leucine-zipper transcription factor that acts as a potent differentiation inhibitor and as a promoter of cell cycle progression. Additionally, c-MYC and mTOR are widely acknowledged as major regulators of cell growth, the process by which cells accumulate biomass. Both c-MYC overexpression and constitutive activation of the PI 3-kinase/mTOR pathway are frequent events in acute myeloid leukaemia; a haemopoietic malignancy characterised by failure to undergo terminal differentiation. In this study we used the MPRO system, an in vitro murine myeloid terminal differentiation model, to study the relationship between cell growth effectors and differentiation in more detail. Enforced expression of c-MYC by the addition of 4-hydoxytamoxifen (4-OHT) to MPRO cells carrying the inducible mycER fusion protein prevents downregulation of growth and impairs retinoid-induced terminal myeloid differentiation. Because downregulation of mTOR limits growth we hypothesised that inhibition of mTOR may reverse the phenotypic effects of c-MYC. Strikingly, pharmacological inhibition of mTOR by rapamycin, but not growth limitation by other means such as amino acid deprivation or cycloheximide treatment, restores differentiation in granulocytes with enforced c-MYC expression (74.0±2.0% mature granulocytes in the rapamycin+4-OHT-treated mycER MPROs vs 4.5±1.0% in the cells treated with 4-OHT alone and 92.9±1.1% in vehicle-treated controls). Rapamycin restores cell growth parameters to levels equivalent to those of vehicle-treated control cells undergoing physiological granulocytic differentiation (2–3 fold reduction in 35S methionine incorporation in rapamycin+4-OHT-treated and vehicle-treated controls compared to levels in 4-OHT-treated cells) and facilitates cell cycle exit (26.0±7.14% of rapamycin+4-OHT-treated cells and 10.7±2.48% of vehicle-treated control cells in S phase vs 51.9±1.72% of tamoxifen-treated cells in S phase as determined by BrdU incorporation). Interestingly, rapamycin attenuates c-MYC’s ability to activate transcription of its target genes without impairing activation of RARα target genes. c-mycER protein levels are reduced while levels of retrovirus-driven c-mycER mRNA do not fall in rapamycin-treated MPRO mycER cells. Furthermore, c-mycER protein levels decrease in rapamycin-treated MPRO cells expressing the ER fusion of the mycT58A point mutation known to increase protein stability. Overall, this data suggests that rapamycin restores the differentiation potential of granulocytes in the MPRO mycER system by regulating c-MYC at the level of protein translation, rather than at the level of gene transcription or protein degradation. These findings suggest that mTOR could be targeted to influence terminal differentiation regulated by MYC in haemopoietic malignancies.
Capturing the Financial Impact of Heparin-Induced Thrombocytopenia.
Authors: Smythe Maureen A. , Koerber John M. , Mattson Joan C. 
Published: April 2020

Abstract
Data evaluating the financial impact of heparin-induced thrombocytopenia (HIT) (a severe adverse drug reaction which can result in life threatening thrombosis) is lacking. The goal of this case-control study was to evaluate the financial impact of HIT. Case patients were those with a new diagnosis of HIT from April 2003 to March 2004 for whom matched controls were available. Controls for each case patient were matched for the DRG under which the hospital was reimbursed, the patient’s primary diagnosis code and their primary procedure code. Case patients required identification of &gt;1 control for inclusion. The hospital’s financial database was queried for length of stay (LOS), total cost, and reimbursement. For each case patient, the cost and reimbursement were compared to the cost and reimbursement for each group of matched controls. In an effort to eliminate the impact of variable reimbursement, a subset of only Medicare case and control patients was also evaluated. Of 72 new HIT patients, matched controls were identified for 31. The mean LOS for the case and control patients was 22.8 and 11.6 days respectively (p=0.006). The mean hospital cost of case and control patients was $55,440 and $26,505 respectively. From reimbursement minus cost calculations, our institution lost an average of $13,429 per HIT patient compared to an average of $393 per control patient (p=0.005). The mean LOS for Medicare cases (n=21) and matched Medicare controls was 26 and 14.6 days respectively (p=0.041). The mean hospital cost of Medicare case and control patients was $58,842 and $30,210 respectively. From reimbursement minus cost calculations for the Medicare subset, our institution lost an average of $20,229 per HIT case compared to $1844 per control patient (p&lt;0.0001). Assuming 72 new cases of HIT per year, our institution incurs a projected annual financial loss of $980,000 from HIT. The use of alternate anticoagulants, although having a higher acquisition cost, may offset this loss through HIT avoidance.
Cbl-Deficient Mice Display Erythropoietin Hypersensitivity and Enhanced Phosphorylation of Protein Kinase B/Akt.
Authors: Richmond Terri D. , Bailey Monica L. , Langdon Wallace Y. , Barber Dwayne L. 
Published: April 2020

Abstract
Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis, stimulating growth, preventing apoptosis, and promoting differentiation of red blood cell progenitors. The critical importance of EPO, EPO receptor (EPO-R) and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops in EPO, EPO-R or JAK2 knockout mice. Intracellular signal transduction pathways regulating growth, differentiation and cell survival downstream of the EPO-R and JAK2 are well documented. However, activation of the EPO-R is transient and down regulated by several negative regulators including tyrosine phosphatases, inositol phosphatases and ubiquitin ligases.
The negative regulator, Cbl, has been implicated as a tumour suppressor in murine sarcoma, B cell leukemia, and erythroleukemia. More recently, Cbl was found in a de novo form of acute myeloid leukemia and has been implicated in the formation of gastric tumours. Cbl is known to bind, ubiquitinate, and downregulate signaling from numerous activated hematopoietic and non-hematopoietic receptors. The discovery that Cbl is a target of EPO-dependent tyrosine phosphorylation, together with the finding that the EPO-R is ubiquitinated in vivo, led us to hypothesize that Cbl deficiency leads to altered murine erythropoiesis.
Resting C57Bl/6 Cbl-/- mice display normal hematologic parameters with the exception of an increased platelet count. However, Cbl deficient mice respond to phenylhydrazine-mediated anemia with increased reticulocyte production and hematocrit recovery. The hypersensitivity of Cbl deficient mice to anemia may be explained by a three-fold enhancement of splenic colony forming unit-erythroid (CFU-E) and an overall increase in burst forming unit-erythroid (BFU-E) and CFU-E. Furthermore, the elevated sensitivity of Cbl deficient mice to anemia is echoed by increased EPO-R and protein kinase B (PKB)/Akt phosphorylation in splenic erythroblasts at high levels of EPO stimulation.
Erythrocyte differentiation was examined by monitoring the expression of the erythroid markers CD71 (Transferrin Receptor) and Ter119. Cbl deficient mice do not have significantly more proerythroblasts than wild-type mice. Interestingly, Cbl deficient mice show impaired erythroid maturation with a 1.7 fold decrease in orthochromatophilic erythroblast levels, elevated erythroid apoptosis in the bone marrow, and no compensation by splenic erythroblast production.
These data (as well as earlier studies from our laboratory with STAT1-/- and SHIP-1-/- mice) illustrate the remarkable ability of the spleen to compensate for alterations in bone marrow erythropoiesis. It also suggests that Cbl regulates pathway(s) associated with regulation of erythroid cell survival through regulation of the PI 3 kinase, PKB/Akt signaling cascade.
GATA Motif on the Erythropoietin Gene Promoter Is Essential for Repression of Ectopic Constitutive Erythropoietin Production.
Authors: Obara Naoshi , Suzuki Norio , Ki-Bom Kim , Imagawa Shigehiko , Nagasawa Toshiro , Yamamoto Masayuki 
Published: April 2020

Abstract
In response to anemia erythropoietin ( Epo) gene transcription is markedly induced in the kidney and liver. This regulation has been ascribed for the promoter GATA motif and 3′ enhancer. To elucidate how the Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of renal cortex and hepatocytes surrounding the central vein. Surprisingly, the renal Epo-producing cells were identified with neuron-like morphology and expression of neuronal markers. We also examined the regulatory mechanism of Epo gene using the transgenes in which mutations had been introduced in the GATA promoter motif, since we have reported the motif as a negative regulatory element for the inducible Epo gene expression. A single nucleotide mutation in the motif resulted in constitutive ectopic expression of GFP in the renal distal tubules, collecting ducts and certain epithelial cells of other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in the distal tubular cells, these factors are likely to constitutively repress ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell-type specific expression of the Epo gene.
The Sickle Cell-Pulmonary Hypertension Screening Study: ECHO Findings at Two-Years of Follow Up.
Authors: Castro Oswaldo , Kato Gregory , Sachdev Vandana , Machado Roberto , Ernst Inez , Coles Wynona A. , Nichols James S. , Hunter Lori A. , Gladwin Mark T. 
Published: April 2020

Abstract
In a prospective study started in 2001 echocardiography (ECHO) was used to screen 195 unselected adult sickle cell patients for pulmonary hypertension (PHTN). The prevalence of PHTN [tricuspid regurgitant jet velocity (TRV) of 2.5 m/sec or higher, corresponding to a pulmonary artery systolic pressure (PAs) of at least 35 mm Hg] was 32 %. Markers of hemolysis (low Hb, and high reticulocytes and LDH) were associated with PHTN risk. Although the pulmonary pressures in sickle-related PHTN were not as high as in other forms of PHTN, they were predictive of short survival (NEJM2004;350:886).
We now report the TRV results in 113 patients who had repeat ECHO exams at 2.1 years (range 1.2–4.1 y) after study enrollment. Their mean age was 36.2 ± 10.3 y and 59 % were female. Their hemoglobin types were: SS, 77.7 %; SC, 14.3 %; Sickle-thal.+, 6.3 %; and Sickle-thal.0, 1.8 %. At baseline, 40 patients had PHTN (mean TRV 2.75 m/sec) and their mean TRV at the 2 y follow up was 2.71 m/sec (p=0.45). In 6 subjects the TRV decreased to normal values from baselines of 2.5 – 3 m/sec. Nevertheless, the mortality of the patients diagnosed with PHTN at baseline currently stands at 40% at 40 months of follow-up. Seventy-three patients did not have PHTN at enrollment (mean TRV 2.0 m/sec) and as a group their TRV at the 2 y follow up had not changed significantly (mean 2.1, p=0.1). However, 11 of the 73 patients (15.1%) with initially normal TRV developed PHTN at 2 years (mean TRV 2.70, range 2.5–3.5 m/sec). The table shows that age, sex, Hb type, Hb F %, and hydroxyurea treatment of the patients who developed PHTN at the 2 y follow up were not significantly different from those who did not.
However, patients who developed PHTN were significantly more anemic, had higher reticulocyte counts, and higher serum ferritin values. A trend toward an increased bilirubin and LDH was observed. Hence, hemolysis probably played a pathogenetic role in these patients’ increased pulmonary pressures, as is also postulated for sickle patients who had PHTN at baseline.
These preliminary results suggest that the incidence of PHTN in adult sickle patients could be as high as 7 % per year and that ECHO screening of these patients every two years is probably indicated.
No PHTN PHTN P value N 62 11 Mean age, y (±SD) 34.1 ± 8.9 34.6 ± 13.4 0.85 Sex (% female) 56 64 0.75 Non-SS (%) 27 18 0.5 On hydroxyurea (%) 41 25 0.5 Hb (g/dl, mean±SD) 9.98 ± 1.74 8.68 ± 1.91 0.04 Hb F (%,mean±SD) 7.9 ± 6.8 7.5 ± 7.4 0.869 Abs. Retics (x1000/mm3, mean±SD) 205 ± 107 288 ± 151 0.045 LDH (U/L, mean±SD) 286 ± 107 359 ± 179 0.09 Bilirubin (mg/dl, mean±SD) 2.5 ± 1.88 3.7 ± 2.58 0.10 Ferritin (mg/L, mean±SD) 632 ± 925 1439 ± 1849 0.046 Creatinine (mg/dl, mean±SD) 0.76 ± 0.23 0.69 ± 0.25 0.36