bluuebunny/crossref_metadata_2025_split huggingface dataset:
AHSP (Alpha Hemoglobin Stabilizing Protein) Gene Expression during Normal and β-Thalassemic Erythroid Differentiation.
Authors: Ronzoni Luisa , Libani Ilaria V. , Giribaldi Giuliana , Turrini Franco , Rusconi Daniela , Refaldi Chiara , Cappellini Maria Domenica 
Published: October 2019

Abstract
β-thalassemia severity is stricktly related to the amount of free a-chains depending at least in part from different b-thalassemic genotype (β0 /β0, β0/β+, β+ /β+). Recently a new erythroid-specific protein, named AHSP (Alpha Hemoglobin Stabilizing Protein), able to form a stable complex with free α-hemoglobin, has been identified. This protein probably acts as a molecular chaperone to prevent the harmful aggregation and precipitation of α-globin that occurs during normal erythropoiesis and to a greater extent in different β-thalassemias. The aim of this study was to evaluate if the AHSP expression in erythroid cells depends on free α-chains amount. In order to verify this hypothesis, we studied the relative expression of AHSP and globin genes during differentiation of erythroid progenitors derived from peripheral blood of both normal subjects and β-thalassemic patients with different genotypes. Erythroid cultures were set up accordingly to the two phases liquid colture method described by Fibach. Total cytoplasmatic mRNA was extracted by the method of Chomczynsky-Sacchi and quantitative real-time PCR was performed using SYBRGreen to evaluate AHSP, α, β and γ globin genes expression. To quantify gene expression a mathematical model using calibration data (2-ΔΔct) was used and quantification of the house-keeping gene S14 was the internal control. In normal subjects AHSP expression was first detected at proerythroblast stage (on day 3 of culture), peaked at orthocromatic stage (on day 9) and declined to low levels by polychromatophilic-reticulocytes stage (day 13) (a decrease of 65% vs day 9 expression). Globin genes expression reached a steady state (a/non-a ratio value of 0.78 very near to a clinical equilibrium) on day 13. In this situation few free α-globin are detectable explaning the lower need of AHSP expression. In β-thalassemic subjects we observed a persistance of globin gene unbalance with an excess of free α-chains also in the advanced differentiation steps: interestingly the worst was the genotype, the highest was the globin ratio and higher was AHSP expression. In β+/β+ thalassemic patients AHSP expression declined on day 13 (a decrease of 15% vs day 9 expression) and globin gene ratio was 2.4, expression of a persistance of free α-chains excess. In β0 /β+ patients globin gene ratio on day 13 was 2.8 and AHSP expression increased of 2.5 times. Finally in β0/β0 patients globin gene ratio was very high with values up to 9–10; AHSP expression profiles instead were different between different subiects and related not only to globin unbalance but also to ineffective erythropoiesis rate and γ-globin chains persistance. All these findings underline an evident relation between AHSP and globin genes expression, suggesting a probable function of this protein during erythropoiesis.
Defective γδ T-Cell Function and Granzyme B Gene Polymorphism in Newly Diagnosed Breast Care Patients. Expansion with Zoledronic Acid Partially Compensates for This Deficiency.
Authors: Gaafar Ameera , Al-Sulaiman Abdulla , Iqniebi Alia , Al-Sayed Adher , Sahovic Entezam , Chaudhri Naeem A. , Al-Mohareb Fahad , Ajarim Dahish , Tbakhi Abdelghani , Al-Hussein Kkaled , Aljurf Mahmoud 
Published: October 2019

Abstract
It has been well established that γδ T-cells play a role in innate anti-tumor immunity. However, the exact mechanism has not yet been fully elucidated. Most of these responses have been ascribed to Vγ9Vδ2 cells, which represent a major subset of the circulating γδ T-cells in humans (1–10%). IFN-γ and granzyme B are important molecules in the anti-tumor immune responses. Upon stimulation, γδ T-cells rapidly produce IFN-γ and cytotoxic molecules. In the present study we analyzed the immune responses by γδ T-cells in 30 newly diagnosed breast cancer patients and 30 normal controls before and after expansion with zoledronic acid. We also scanned the granzyme B gene polymorphism in breast cancer patients and controls. Our result revealed that γδ-T cells in PBMC were reduced in both frequency and function in breast cancer patients compared with the normal controls. Ex-vivo stimulation of γδ T-cells with zoledronic acid and IL-2 partially compensated for this deficiency, as it stimulates production of IFN- γ and release of cytotoxic molecules by these cells. However, the IFN- γ and granzyme B and cytotoxicity of the expanded γδ T-cells from breast cancer patients remained significantly below normal control. Genotypic analysis of granzyme B gene revealed significantly higher frequency of the RAH haplotype in breast cancer patients compared with normal controls. The prevalence of the wild genotype QPY/QPY was significantly higher in normal controls compared with the breast cancer patients. Cytotoxicity by γδ T-cells against various targets was reduced in breast cancer patients compared to normal controls. In conclusion, our analysis shows a defective immune function of γδ T-cells and granzyme B gene polymorphism in breast cancer patients. The γδ T-cell function defect in these patients can be partially corrected by zoledronic acid. Further studies of γδ T-cell function and granzyme B gene polymorphism in other cancers, as well as the therapeutic use of zoldedronic acid is warranted.
Micro-RNA Profiling in CD4+ T Cell Differentiation.
Authors: Banerjee Arnob , Schambach Felix , Hammond Scott , Reiner Steven 
Published: October 2019

Abstract
Micro-RNAs comprise a class of small noncoding RNAs which have been found to be important regulators of cellular differentiation in multiple species. Previous analysis of micro-RNA expression in the murine hematopoietic system has suggested a role in cell differentiation and the maintenance of cell identity. Naïve progenitor CD4+ T cells respond to a combination of appropriate antigen and other specific signals by undergoing proliferation and further differentiation into one of at least two subsets. T helper 1 (TH1) cells produce high levels of the cytokine IFN-γ and T helper 2 (TH2) cells produce high levels of IL-4, optimizing them for control of intracellular and extracellular pathogens, respectively. It is currently not known whether micro-RNA molecules influence CD4+ T cell differentiation. We have used oligonucleotide arrays to analyze micro-RNA expression profiles of freshly isolated murine CD4+ T cells compared to cells differentiating into TH1 and TH2 subsets. Expression profiles were found to differ significantly between naïve and stimulated CD4+ cells, with fewer differences between TH1 and TH2 subsets. Promising candidate micro-RNAs are being further evaluated by northern blot and genetic studies. Micro-RNA-155 is upregulated on stimulation of CD4+ T cells in multiple oligonucleotide array assays. Micro-RNA-155 is encoded by the BIC oncogene and has been implicated in lymphomagenesis as well as in other malignancies. We have verified the induction of micro-RNA-155 in stimulated helper T cells by northern blot and are studying the effects of this micro-RNA on CD4+ T cell differentiation. Our observations support a role for micro-RNAs in helper T cell differentiation during the immune response.
Serotonergic Mechanisms in Haemostasis: A Connection between Major Depression and Thrombotic Risk.
Authors: Lopez-Vilchez Irene , Serra Montserrat , Alonso Antonio , Navalon Fulgencio , Hernandez Rosa , Gasto Cristobal , Escolar Gines , Galan Ana M. 
Published: October 2019

Abstract
Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p<0.05 vs. controls). Treatment of patients with the SSRI normalized the expression of these antigens vs. control samples. Studies performed under flow conditions only showed a significant increase of the fibrin formation on vascular damaged vessels of patients with major depression vs. controls (71.1±9.5 % vs. 45.8±5.3 %, p<0.05) with corresponding elevations in F1+2 levels. In vitro incubation of blood samples with a SSRI was followed by a 30% of reduction in fibrin formation. Similar tendencies were confirmed in studies performed along clinical treatment of patients with SSRI. A decrease of 20% and 80% of covered surface by fibrin was observed after 8 and 24 weeks of treatment respectively. Levels of whole blood TF of patients suffering depression were found elevated vs. control samples (1.3±0.4 ng/ml vs. 1.0± 0.4 ng/ml respectively) and tended to decrease during the treatment (p<0.05). Our results reveal a more thrombogenic state in patients diagnosed of major depression associated to serotonergic mechanisms. Strategies aimed to control serotonergic mechanisms in platelets may offer a potential therapy for the regulation of the prothrombotic state of patients with major depression, but also in patients with cardiovascular risk.
Contribution of Lipopolysaccharide to Prolylcarboxypeptidase Activation in Endothelial Cells.
Authors: Perkins Ryan , Ngo My-Linh D. , Mahdi Fakhri , Shariat-Madar Zia 
Published: October 2019

Abstract
Bradykinin (BK) participates in the etiology of vascular inflammation and the regulation of local blood flow and blood pressure by causing a vascular hyperemia. The bradykinin generating pathway of human plasma consists of prolylcarboxypeptidase (PRCP), heat shock protein 90 (HSP90), coagulation factor XII (FXII, Hageman factor), and the heteromer complex of prekallikrein (PK) and high molecular weight kininogen (HK). In the presence of HK, PRCP activates PK to generate kallikrein on endothelial cells. Formed kallikrein then cleaves HK to liberate BK. PRCP-induced PK activation is independent of the activated FXII. The activity of PRCP leads to vasodilation. In inflamed tissues, tissue injury or hypotensive bacteremia, BK has been shown to accumulate in the blood or plasma of these patients. In this investigation, we have assessed the possible contribution of lipopolysaccharide (LPS) to PRCP activation in primary cultures of human umbilical vein endothelial cells (HUVECs) using multiple approaches. The role of LPS in HK binding to HUVECs was assessed. The effect of LPS on the endothelial permeability injury was evaluated by histological changes and analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The release of von Willebrand factor (VWF), endothelin-1 (ET-1) and intercellular adhesion molecule-1 (ICAM-1) induced by LPS in HUVEC was determined. PK activation on LPS-pretreated endothelial cells was higher than untreated cell. PRCP inhibitor, z-pro-prolinal, inhibited the production of kallikrein from PK compared to the control. These results suggest that LPS contributes to PRCP overactivation which results in the acceleration of the inflammatory process, exerting its effects via PK activation. Inhibiting the activity of PRCP may exert some beneficial therapeutic action in systemic inflammation, at least in part by inhibiting the production of BK and thus preventing vascular dysfunction.
The Use of Recombinant Activated Factor VII in Australia and New Zealand.
Authors: Dunkley Scott , Cameron Peter A. , Phillips Louise E. , Isbister James 
Published: October 2019

Abstract
Introduction:
The role of Recombinant activated factor VII (rFVIIa, NovoSeven) for off-label indications, including trauma, cardiac surgery and other critical bleeding episodes, remains controversial. Through the Haemostasis Registry, we report on the Australian and New Zealand experience of using rFVIIa.
Methods:
Monash University has established the Haemostasis Registry (with an educational grant from NovoNordisk Pharmaceuticals) to monitor the use of rFVIIa throughout Australia and New Zealand. More than 50 hospitals are contributing data to the Registry.
Results:
Over 600 cases have been reported to the Register. In those where complete data has been received, major areas of use are cardiac surgery (~42%), other surgery (~19%) and trauma (~13%). The reported efficacy of rFVIIa was high (where reported, in >70% cases bleeding decreased or stopped after first dose of rFVIIa) while the reported Adverse Events “possibly” or “probably” linked to rFVIIa use were relatively low (~6%). There were no directly linked adverse events. Despite suffering from conditions where mortality is high, the survival rate in rFVIIa recipients was 69% at 28 days post treatment. These results will be compared with international experience and published trials.
Conclusions:
Although randomized controlled trials are important in establishing the safety and efficacy of new treatments, they do not replace the need for registries, especially for treatments where clinicians believe that withholding treatment may be unethical because of potential life threatening consequences. This problem is made more difficult where there are a wide range of applications. As more data becomes available, the Haemostasis Registry data will help to elucidate the safety and efficacy of rFVIIa and provide important feedback to doctors and hospitals.
Results of a One-Year Registry of Bleeding Complications of Vitamin K Antagonist Therapy outside of a Controlled Population.
Authors: Halbritter Kai M. , Beyer Jan , Oettler Wolfram , Kleinstück Babett , Schellong Sebastian M. 
Published: October 2019

Abstract
Background: The registry was designed to collect data on bleeding complications in patients under Vitamin-K-antagonist therapy (VKA) during a one year period in a predefined area outside a controlled population and to describe clinical management of these complications.
Patients and methods: Between 1.1.05 and 31.12.05, bleeding complications requiring hospital stay in 23 hospitals in eastern saxonia, Germany, were documented. Indication for VKA therapy, type of bleeding, INR at hospital entry, application of packed erythrocytes and fresh-frozen plasma, clinical management and outcome at the end of hospital stay and after 3 months were recorded.
Results: 311 episodes of VKA-associated bleeding were documented. Patients were male in 49%, mean age was 73±10 years. Indication for VKA therapy was atrial fibrillation in 64%, mechanical heart valves in 10%, impaired LV-function in 1%, secondary prophylaxis of VTE in 18% and others in 6%. Bleeding affected the gastrointestinum in 23%, 22% were superficial hematomas, 14% were intracranial, 10% epistaxis, 10% hematuria, 7% intraabdominal/retroperitoneal, 5% were overdoses without clinical signs, 10% involved other sites.
INR on admission was 3.76±2.45, 40% of INR values were above, and 30% were in the targeted INR range. Vitamin K was administered orally in 26% of episodes, FFP in 9%, factor concentrate in 6%. Per episode, 3±2 units of packed erythrocytes were necessary. 8% of patients died during hospital stay. Surgical therapy was chosen in 25% of episodes, 20% were controlled by endoscopy. 3% of episodes were followed by ongoing detoriation of health status. Mean hospital stay was 13±9 days. Mortality was highest in patients with intracranial bleeding (24%). Retroperitoneal/intraabdominal bleedings required the largest amount of blood products and stabilization of coagulation. Three months after the index episode, mortality was 14%.
Conclusions: Complications of VKA-therapy outside of a controlled population occur most often at a supratherapeutic INR level. Gastrointestinal bleeding constitutes the major part of the complications, intra-hospital mortality of 8% is mainly caused by intracranial bleeding.
PT and INR Interference by the Antibiotic Daptomycin in Commercial Thromboplastin Reagents: Dependence on Phospholipid Composition.
Authors: Cawthern Kevin , Bottenus Ralph , Rawal Dharini , Triscott Mark 
Published: October 2019

Abstract
The antibiotic, daptomycin (brand name Cubicin, Cubist Pharmaceuticals) is approved in the US for treatment of complicated skin and skin structure infections caused by gram-positive pathogens (e.g., MRSA, methicillin-resistant Staphylococcus aureus). Recently, this antibiotic has been shown to interfere with prothrombin time (PT) assays, resulting in a prolongation of clotting times and artificial increases in the INR (International Normalized Ratio). The level of interference varies with Daptomycin concentration in the plasma, the brand of prothrombin time reagent as well as the type (normal or abnormal) of patient sample being analyzed. The bactericidal action of the antibiotic is known and is through a depolarization of bacterial membranes in the presence of physiologic calcium (Silverman, et al, Antimicrobial Agents and Chemotherapy, Aug 2003); therefore, daptomycin interference with the PT clotting assay is likely mediated through the phospholipid component of the thromboplastin formulations. To examine the dependence of daptomycin interference for a number of commercial PT formulations, testing was performed with daptomycin spiked samples of normal pooled plasma as well as abnormal samples from oral anticoagulant therapy (OAT) patients. For spiked normal samples, a small degree of prolongation in clotting times was observed with most PT reagents, with one reagent showing a significant prolongation. In OAT samples spiked with Daptomycin, the PT prolongation was greater for all formulations, with the one reagent again showing greater prolongation as for the normals. Ultimately, this variation in response was attributed to differences in phospholipid composition among the reagents, supported by previous studies of daptomycin with neutral and acidic phospholipids in the presence of calcium (D. Jung, et al., Chemistry and Biology, Jul 2004). A four-dimensional experimental design matrix was constructed using a mixture of four synthetic phospholipids (DOPC, DOPS, DOPE and DOPG), and the relationship of daptomycin-PT interference to percent composition of the phospholipids was examined. In both normal and OAT plasmas spiked with daptomycin, the major contributor to PT prolongation was the concentration of DOPG (phosphatidylglycerol) in the lipid mixture, with additional contributions correlated with DOPC (phosphatidylcholine). Because of the ubiquitous use of these phospholipids in commercial PT formulations, this work suggests that daptomycin interference should be assessed for each combination of PT reagent and INR range used.
Peroxisome Proliferator-Activated Receptor gamma (PPARγ) Regulates CD36 Gene Expression in Megakaryocytic Cell Lines.
Authors: Thornton Michael A. , Poncz Mortimer , Badejogbin Victor 
Published: October 2019

Abstract
CD36 is a receptor for thrombospondin and is present on megakaryocytes and platelets. It may be critical for normal megakaryocyte-formation and platelet function. The human CD36 gene locus extends 38.3 kb along chromosome 7 at position 7q11.2. Six distinct promoters (1A-1F) have been described that flank corresponding alternatively spliced first exons. To date the activity of these six promoters has not been tested in the megakaryocytic lineage and understanding the regulated expression of the CD36 gene may provide additional insights into the biology of this receptor. The 1B promoter, which is active in monocytes and adipocytes possesses a PPAR γ Direct Repeat (DR-1) binding site at position -260bp that is inducible in those same lineages with PPAR γ ligands. Recent studies have shown that primary megakaryocytes, platelets and the megakaryocyte line MEG-01 cells express PPARγ. To elucidate the molecular basis for CD36 gene expression in megakaryocytes and to query whether PPARγ plays a role in CD36 expression in this lineage we treated two megakaryocytic cell lines HEL and CHRF with increasing doses of two PPARγ ligands, 15DPGJ2, an arachadonic acid (AA) metabolite and Troglitazone, a thiazolidinedione drug administered to diabetics. RT-PCR studies using an exon 1A/exon 3 primer set or an exon 1B/exon 3 primer set established that CD36 mRNA from both promoters was expressed in these two lineages. Further these studies showed that HEL cells exhibited ligand dependent transactivation of the 1B but not the 1A promoter to both 15DPGJ2 and Troglitazone. CHRF cells did not demonstrate a drug dependent induction of either promoter. However, flow cytometry studies using a FITC conjugated anti-human CD36 antibody revealed that both HEL and CHRF cells had significant increased surface expression of the CD36 receptor. CHRF cells in particular had a 23 fold increase in surface CD36 over vehicle treated controls. Western Blot Analysis demonstrated that this increase in surface receptor quantity was secondary to drug-induced increases in the steady state protein levels. The relative increase in protein level was consistent with the mRNA levels determined by our RT-PCR results. We conclude that the 1B promoter is PPARγ responsive in HEL cells and that an alternate promoter perhaps 1C, 1D, 1E, 1F or a novel promoter is PPAR gamma responsive in CHRF cells. These data may have clinical implications for diabetics currently undergoing thiazolidinedione drug treatment with troglitazone as CD36 increases on the platelet surface lead to increased AA uptake and circulating platelet hyperactivation. These represent the first studies showing the nuclear receptor PPARγ’s involvement in regulating gene expression in megakaryocytes.
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