bluuebunny/crossref_metadata_2025_split huggingface dataset:
Iron-Overload Induces Apoptosis in Cardiomyocytes and Hepatocytes Via Mitochondrial/Caspase-3 Pathways.
Authors: Yang Mo , Chan Shing , Cheung Yiu Fai , Ha Shau Yin , Chan Godfrey ChiFung 
Published: April 2020

Abstract
Cardiomyopathy and liver damage due to iron-overload are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes and hepatic cells, and that TPO may exert protective effect on apoptosis of cardiomyocytes (Circulation, 2006). In this study, we demonstrated firstly that iron induced apoptosis in cardiomyocytes. Using H9C2 cells, we have shown that iron reduced cell viability in a dose-dependent manner (0.003–3 mM) (n=6). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM, 72 hrs) (n=6). The expression of active caspase-3 was significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (5, 10, 20, 50, 100 ng/mL, 72 hrs). The cell viability was significantly increased with the treatment of TPO at 50 ng/mL and 100 ng/mL (n=4). Dot-plot analysis of annexin V/PI staining demonstrated that TPO (50 ng/mL) significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis of H9C2 cells also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6). The population of phospho-Akt and Erk1/2 were also significantly increased after treatment by TPO (P&lt;0.05, n=4). Human liver cell line MIHA was also used as a cell model. We showed that iron-overload reduced cell viability in a dose-dependent manner (0.0375–0.6 mM) (n=7). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.15–0.6 mM) for 72 hrs (n=7). The expression of active caspase-3 was also significantly increased in iron-treated cells (n=5). We also found that TPO exerted proliferation effect on MIHA cell by activation of phospho-Akt. However, MIHA cells were cultured in the presence of iron (0.3 mM) with TPO (50 ng/mL, 72 hrs). The cell viability was not significantly increased with the treatment of TPO (n=5). Dot-plot analysis of annexin V/PI staining did not demonstrated that TPO reduced the population of apoptotic cells induced by iron-overload (n=5). Also, incubation with TPO did not decrease the iron-induced caspase-3 expression in these cells (n=5). Our findings suggest that iron-overload induces apoptosis in cardiomyocytes and hepatocytes via mitochondrial/caspase-3 pathways and that TPO might exert a protective effect on iron-overload induced apoptosis via the activation of Akt and Erk1/2 pathways in cardiomyocytes.
Growth Differentiation Factor 15 (GDF15) and Erythropoietin (EPO) Levels in Beta Talassemia Major Patients.
Authors: Salussoglia Ilaria , Volpe Gisella , Fracchia Silvia , Roggero Simona , Longo Filomena , Piga Antonio 
Published: April 2020

Abstract
Background: The serum level of GDF15 has been recently indicated as a possible marker of erythropoiesis (Tanno et al., Nature 2007) suggesting a role of its over-expression in contributing to iron overload in thalassemia syndromes by inhibiting hepcidin expression. The aim of present study has been to evaluate GDF15 serum levels in a homogeneous series of thalassemia patients and the relationship with transfusional parameters and iron status markers.
Methods: A group of consecutive patients with beta thalassemia major followed at our institution were included in the study. All patients were on regular transfusion and iron chelation treatment. Quantification of GDF15 on serum samples was performed with DuoSet ELISA for human GDF15 (R&D Systems) following the manufacturer’s protocol (Tanno et al., Nature 2007). Each patient had also a blood test for haemoglobin (Hb), serum iron, ferritin, transferrin, transferrin saturation and EPO levels. Liver Iron Concentration by SQUID and cardiac iron by MRI T2* have been assessed. The mean hemoglobin levels of the previous year (pre-transfusional, post-transfusional and mean) have been calculated for each individual. The presence of mild thalassemic mutations was used to classify mild or severe genotype. Clinical status has been assessed on the presence/absence of main complications (heart disease, liver disease, diabetes, hypothyroidism). Statistical analysis was performed using the software Statistica (StatSoft).
Results: One hundred-forty patients (73 male, 67 females) were studied. The mean age was 27.9 ± 9.0 years (range: 3.5–42). One hundred (71%) were splenectomised. Betathalassemia major patients had elevated GDF15 serum levels (mean 6892 ± 6894 pg/mL; range 720–52521) in comparison with healthy volunteers (273 ± 104 pg/mL; range 129–401). GDF 15 levels were strongly related to EPO levels (r=0,81; p&lt;0,001). GDF15 levels were not related with age, gender, spleen, clinical status and iron markers. Patients with a severe genotype had higher GDF15 levels than mild genotype patients. GDF15 levels had a negative correlation with Hbs (p&lt;0,05 for actual Hb and pre-transfusional Hb; p&lt;0,001 for post-transfusional Hb and mean Hb). In thalassemia major patients with a severe genotype, GDF15 levels within thrice the normal range have been observed only in patients with pre-transfusional Hb above 9,6, post-transfusional Hb above 12,5 and a mean Hb above 11,3.
Conclusions: In beta thalassemia major patients on regular transfusion and iron chelation, serum GDF15 levels are high, inversely related to the haemoglobin levels maintained. Further studies of this marker may lead to a rethinking of the optimal transfusion therapy in these conditions.
Thrombin Inhibits NF-κb and RhoA Pathways in Cytokine-Stimulated Vascular Endothelial Cells When EPCR Is Occupied by Protein C.
Authors: Rezaie Alireza R. , Bae Jong-Sup 
Published: April 2020

Abstract
We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by protein C switches the PAR-1-dependent signaling specificity of thrombin from a permeability-enhancing to a barrier-protective response in endothelial cells. To determine whether the occupancy of EPCR by protein C renders thrombin a protective enzyme, thus up-regulating the expression of signaling molecules in the antiinflammatory pathways, we investigated the effects of thrombin and thrombin receptor agonist peptides (TRAP) on TNF-a-stimulated HUVECs in the absence and presence of the catalytically inactive protein C-S195A by monitoring the expression of cell surface adhesion molecules (VCAM- 1, ICAM-1 and E-selectin), adhesion of neutrophils to cytokine-stimulated endothelial cells, regulation of the Rho family of small GTPases and the activation of nuclear factorkB (NF-kB) pathway. Analysis of the results indicates that both thrombin and TRAP initiate proinflammatory responses in endothelial cells, thus neither thrombin nor TRAP influenced the proinflammatory effects of TNF-a in the absence of the protein C mutant. Interestingly, however, the occupancy of EPCR by the protein C mutant switched the PAR-1-dependent signaling specificity of thrombin and TRAP, thus leading to inhibition of the expression of all three adhesion molecules as well as the binding of neutrophils to TNFa-stimulated endothelial cells. Furthermore, similar to activated protein C, both thrombin and TRAP activated Rac1 and inhibited the activation of RhoA and NF-kB pathways in response to TNF-a in cells pretreated with protein C-S195A. Based on these results we conclude that when EPCR is bound by its natural ligand protein C, the cleavage of PAR-1 by thrombin initiates antiinflammatory responses in vascular endothelial cells.
Endothelial Dysfunction and Paradoxical Resistance to Thrombosis in a Transgenic Mouse Model of Severe Hyperhomocysteinemia.
Authors: Dayal Sanjana , Jensen Melissa J , Faraci Frank M , Kruger Warren D , Lentz Steven R. 
Published: April 2020

Abstract
Hyperhomocysteinemia induced by low folate or high methionine diets produces endothelial dysfunction and accelerated thrombosis in mice, but it is not known if these vascular effects are influenced by homocysteine-independent effects of dietary perturbation. To determine the vascular phenotype of severe hyperhomocysteinemia in the absence of dietary intervention, we utilized a recently developed transgenic mouse model of cystathionine β-synthase (CBS) deficiency. In this model, the early lethality of homozygous Cbs-deficient mice (Cbs-/-) is rescued by a zinc-inducible human CBSI278T (hCBS-I278T) transgene under the control of a metallotheinein promoter. The I278T mutation is a common CBS mutation found in human patients with homocystinuria. We bred Cbs+/− male to Cbs+/− hCBS-I278T female mice. The mothers were given drinking water supplemented with 25 mmol/L zinc sulfate. Pups were weaned at 4–5 weeks of age and did not receive supplemental zinc after weaning. At 20 weeks of age, plasma total homocysteine was significantly higher in Cbs-/- hCBS-I278T mice (350±17 μmol/L) compared with Cbs+/+ hCBS-I278T (8.0±0.6 μmol/L) or wild-type Cbs+/+ (7.2±0.8 μmol/L) littermates (P&lt;0.001). Maximal dilatation of pial arterioles to the endotheliumdependent dilator, acetylcholine, was decreased significantly in Cbs-/- CBS-I278T mice (10±2 %) compared with Cbs+/+ hCBS-I278T (25±2 %) mice (P&lt;0.001). Dilatation responses to the endothelium-independent vasodilator, nitroprusside, were similar in both groups. Surprisingly, the time to stable occlusion after photochemical injury (rose bengal and green laser) of the carotid artery was significantly delayed in Cbs-/- hCBSI278T mice (71±10 minutes) compared with Cbs+/+ hCBS-I278T (30±10 minutes) or Cbs+/+ mice (33±8 minutes) (P&lt;0.05). The tail bleeding time was comparable in all 3 groups. We conclude that severe hyperhomocysteinemia due to genetic deficiency of Cbs produces endothelial dysfunction but not accelerated thrombosis in the absence of dietary intervention in mice.
The Multikinase Inhibitor Sorafenib Inhibts Proliferation in B- and T-Lymphoblastic Cell Lines Via the PI3K/Akt Pathway.
Authors: Schult Catrin , Dahlhaus Meike , Boldt Sonja , Glass Änne , Ruck Sabine , Sawitzky Mandy , Lange Sandra , Amoroso Francesca , Freund Mathias , Neri Luca Maria , Junghanss Christian 
Published: April 2020

Abstract
Background: The phosphatidylinositol 3 Kinase (PI3K)/Akt signalling pathway is involved in the regulation of proliferation, apoptosis and angiogenesis. Dysregulation of PI3K/Akt has been described for several solid and hematological tumors. Its role in acute lymphoblastic leukemogenesis (ALL) is unkown. Here, we investigated whether the inhibition of PI3K/Akt kinases influences apoptosis, necrosis and cell proliferation.
Patients and Methods: ALL and high grade NHL cell lines with different cytogenetics and phenotypes were used (SEM, RS4;11, REH, Jurkat, DOGKIT). Phosphorylationstatus of Akt (Ser473, Thr308) and FOXO3A (Thr32) were determined by western blot (WB). Cells were incubated for 96h with LY29004 (5μM, 12.5μM, 25μM; PI3K inhibitor) or sorafenib (0.73μM, 7.3μM). In order to detect synergistic effects with other cytotoxic drugs cells were treated with combinations of sorafenib and doxorubicine, cytosin-arabinoside, and mTOR inhibitor (RAD001), respecitvely. Cell number, apoptosis, necrosis, protein phosphorylation and metabolic activity were determined at 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, WB and WST-1 testing. Whole genome Affymetrix gene expression arrays (U133 Plus 2.0) were performed in order to detect differential gene expression compared to controls treated with DMSO (dimethylsulfoxid) only.
Results: In all ALL cell lines phosphorylated Akt (pAKT) was detected. Levels of pAkt and pFOXO3A differed between cell lines significantly, with Jurkat and SEM demonstrating high activation levels. PI3K inhibition by LY29004 led to decreased proliferation in SEM cells with only slight increases in apoptosis and moderate increase in necrosis rates (20%). Sorafenib inhibited the proliferation of SEM, Jurkat and RS4;11 significantly, with most pronounced effects at 96h. Maximal apoptosis and necrosis rates increased and ranged from 11 to 39% and 53 to 84%, respectively. Metabolic activity decreased significantly already after 24h. As early as 0.5h after treatment complete disappearance (SEM, RS4;11) or marked decrease (Jurkat) in levels of pAkt and pFOXO occurred. Combination of sorafenib with conventional cytotoxic drugs failed to demonstrate synergistic effects. Sorafenib treatment induced differential gene expression in several genes e.g. genes involved in apoptosis such as BIMBAM, caspases and CDKs.
Conclusions: Inhibition of the PI3K/Akt pathway seems to be a potential therapeutic target in ALL and high grade lymphoma cells. Sorafenib as a multikinase inhibitor approved for clincial application in solid tumors displayes significant antileukemic activity in vitro and might be a potential drug for a targed therapy approach in ALL.
Approach to Establish Long Term Anti-Acute Myeloid Leukemia (AML) Immune Responses by Inducing Central Memory AML Specific CD4 T Cells.
Authors: Zhong Ruikun , Lane Thomas A. , Ball Edward D 
Published: April 2020

Abstract
The efficacy of adoptive cell therapy of cancer and leukemia is often limited by the failure of cultured T cells, particularly cloned CD8+ T cells, to persist in vivo, and insight into the basis for the poor survival of the transferred cells is lacking. We previously reported a novel culture method that induces AML dendritic cell differentiation and primes in situ AML-reactive T cells (AMLDC culture) (Zhong et al. Exp Hematol2008, 36:486). Highly reactive anti-AML T-cell lines were generated. These T-cell lines caused specific lysis of autologous AML cells, but not autologous LCL or allogeneic AML cells, and they depleted autologous AML colony-forming cells (CFC), but not normal CFC. The culture procedure has been further optimized in this study. We found that by the addition of the TLR-4 agonist LPS,(1–100 ng/ml) in the last 24 hours of AMLDC culture (day 6), CD80, CD86, CD53, CD83 or HLA-Dr expression of AML cells pre-induced by cytokine combination GM-CSF/IL-4 or GM-CSF/IL4/IL2/IL7/IL12 could be significantly enhanced (n=6, P&lt;0.005–0.04; n=8, P&lt;0.0001–0.005 respectively). Addition of LPS increased the IFN-gamma secretion by T cells generated from AMLDC culture in response to autologous AML cells 4–10 fold (3 of 3; P&lt;0.001). Addition of TNF-alpha, (10–20 ng/ml) in the last 24 hours of culture could also significantly enhance the expression of above surface molecules. However, LPS induced significantly higher expression of CD80 and CD86 on AMLDC compared with TNF-alpha. T cells generated from AMLDC culture with TNF-alpha led to differential effects on autologous AML reactivity. IFN-gamma secretion was enhanced in 4 of 7 studies and suppressed in 3 of 7. The significantly enhanced AML cell reactivity of autologous T cells generated from AMLDC culture by adding LPS was also demonstrated in limiting dilution AMLDC culture in 96 well plates. The majority of T cell lines selected and expanded from AMLDC culture with LPS were CD3+CD4+ (12 of 18), and 18 T cell lines tested expressed moderate to high level of CD62L, implying the central memory phenotype.
Conclusions: Timely exposure of AMLDC culture to TLR-4 agonists, followed by T cell expansion, may promote the generation of AML-reactive T cells and differentiation toward the central memory phenotype. Theoretically, this should promote long-term maintenance and potential of regulation of both humoral and cellular immune responses against AML upon infusion of AML reactive autologous T cells derived from such cultures and may therefore enhance the therapeutic efficacy of these cells.
Donor Characteristics Affecting Graft Failure and Survival after Unrelated Donor Transplantation with Reduced Intensity Conditioning Regimens (RIC) for Hematologic Malignancies.
Authors: Passweg Jakob R. , Kan Fangyu , Zhang Mei-Jie , Rocha Vanderson , Isola Luis M. , Gee Adrian P. , Gibson John , Lazarus Hillard M. , Laughlin Mary J. , Loren Alison , Marks David , Gratwohl Alois , Eapen Mary 
Published: April 2020

Abstract
Impact of donor characteristics is well described for standard intensity unrelated donor and matched sibling donor transplants but may differ in recipients of unrelated donor RIC transplants. Less immunosuppressive regimens at transplantation may lead to higher graft failure rates. We examined risk factors affecting graft failure, acute and chronic graft-versus-host disease (GVHD) and survival after RIC unrelated donor transplants in 715 patients with acute (n=394) and chronic leukemia (n=74), myelodysplastic syndrome (n=70) and non-Hodgkin lymphoma (n=177). Graft failure was defined as &lt;5% donor chimerism within 3 months after transplantation. 159 patients received bone marrow (BM) and 556 peripheral blood (PB) grafts. All transplantations occurred in 1999–2006 in the US. Median follow-up of surviving patients was 36 months (range 6–92). All donors and recipients were typed for HLA A, B, C and DRB1 using high resolution molecular methods. Mismatches at low resolution (antigen) and high resolution (allele) were considered together and are described collectively as mismatches. The day-28 incidence of neutrophil recovery (≥ 0.5 x 109/L) was 96%. After initial neutrophil recovery most patients (n=506) had &gt;95% donor chimerism. 63 patients had &lt;5% donor cells and the remaining 146 patients, 5–95% by 3-months post-transplant. In multivariate analysis, the only factor associated with graft failure was transplantation of BM vs. PB grafts (odds ratio 2.36, p=0.002). We specifically looked for an effect of donor-recipient sex match on graft failure and female donor parity on GvHD and found none. As expected risks of acute graft-versus-host disease (GVHD) were higher after mismatched transplants (p=0.015). No donor characteristic was associated with chronic GVHD. The only donor characteristic affecting overall survival was donor-recipient HLA disparity: 3-year overall survival rates were significantly lower at 23% after mismatched transplants compared to 42% after HLA-matched transplants (p=0.008). Additionally, mortality rates were significantly higher in patients older than 50 years (p=0.005), performance score &lt;90 (p=0.002) and when transplantation occurred with active disease (p&lt;0.001). As seen in recipients of myeloablative conditioning regimens, the only donor characteristic associated with survival is donor-recipient HLA disparity. Donor age, donor-recipient sex match, donor parity and donor cytomegalovirus serostatus were not associated graft failure or survival after unrelated donor RIC transplants.
Potential Use of miRNAs as Platelet Biomarkers of Storage.
Authors: Kannan Meganathan , Mohan Ketha V. K. , Kulkarni Sandhya , Atreya Chintamani D 
Published: April 2020

Abstract
MicroRNAs (miRNAs) are a class of small endogenous noncoding regulatory RNAs, which control cellular gene expression in eukaryotes at post-transcriptional level. MicroRNAs have been shown to play a role in cell differentiation and apoptosis by down regulating the translation of their target mRNAs. Differential expression profiles of miRNAs often help in identifying specific miRNAs as biomarkers of interest to a variety of cellular states, such as cancer. One of the key cellular components of blood that is of interest to transfusion medicine to control bleeding is ‘platelet concentrates (PCs)’. Platelets are stored at room temperature (22 0C) for up to 5 days for transfusion and during storage platelets undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo following transfusion. Therefore identifying markers that truly reflect the cellular status at any given time during storage would help in developing efficacy biomarkers for stored platelets. Since mature platelets do not have a nucleus, but they do contain numerous mRNAs and undergo translational regulation, we hypothesized that miRNAs as translational regulators could play a crucial role in platelet biology and therefore may serve as biomarkers of platelets during storage. In this study, we explored differential miRNA profiling of 52-apoptosis-associated miRNAs of platelets using PCs obtained from the National Institutes of Health (NIH) blood bank and were stored at room temperature (22 0C) for up to 9 days. Samples were collected at day 0, day 2, and day 9. Out of 52 miRNAs analyzed by microarray, we observed that while two miRNAs levels had increased, another two miRNA levels decreased drastically from day 0 to day 9 (Table 1).
miRNA Day-0 Day-2 Day-9 Let-7b 1.12 0.015 15.1 miR-16 0.045 0.26 23.5 miR-145 35.09 10.18 5.415 miR-7 24.435 9.145 1.51
The differential microarray analysis presented here clearly suggests that miRNAs certainly have the potential to be useful as biomarkers of platelets during storage. Further analysis of the targets of these miRNAs in platelets will help understand the underlying mechanisms of platelet lesions.
The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
Generation of Blood Cells from Human Ips Cells in Vitro through the Hematopoietic Progenitors Concentrated within the Unique Structures, Ips-Sac.
Authors: Takayama Naoya , Eto Koji , Nakauchi Hiromitsu , Yamanaka Shinya 
Published: April 2020

Abstract
Human embryonic stem cells (hESCs) are proposed as an alternative source for transfusion therapy or studies of hematopoiesis. We have recently established an in vitro culture system whereby hESCs can be differentiated into hematopoietic progenitors within the ‘unique sac-like structures’ (ES-sacs), that are able to produce megakaryocytes and platelets (Takayama et al., Blood, 111, 5298–306, 2008). However there is a little concern that repetitive transfusion with same human ESC-derived platelets may induce immunological rejection against transfused platelets expressing allogenic HLA. Meanwhile, induced pluripotent stem (iPS) cells established from donor with identical HLA are well known as a potential and given source on platelet transfusion devoid of rejection. To examine if human iPS cells could generate platelets as well as from hESCs, we utilized 3 different human iPS cell lines; two were induced by transduction of 4 genes (Oct3/4, Klf4, Sox2, and c-Myc) in adult dermal fibroblasts, and one was by 3 genes without c-Myc. Sac-like structures (iPS-sac), inducible from 3 iPS cell lines, concentrated hematopoietic progenitors that expressed early hemato-endothelial markers, such as CD34, CD31, CD41a (integrin αIIb) and CD45. These progenitors were able to form hematopoietic colonies in semi-solid culture and differentiate into several blood cells including leukocytes, erythrocytes or platelets. Of these, obtained platelets responded to agonist stimulation, in which the function was as much as human ESC-derived platelets, as evidenced by PAC-1 binding with activated αIIbβ3 integrin or full spreading onto fibrinogen. These results collectively indicated that human dermal fibroblasts could generate functional and mature hematopoietic cells through the reprogramming process and this method may be useful for basic studies of hematopoietic disorders and clinical therapy in the future.