bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron Chelation, Cell Cycle-Dependent Kinases and HIV-1 Transcription.
Authors: Ammosova Tatyana , Debebe Zufan , Niu Xiaomei , Richardson Des R. , Jerebtsova Marina , Ray Patricio , Gordeuk Victor R. , Nekhai Sergei
Published: October 2019

Iron chelation leads to reduced cell cycle-dependent kinase 2 (CDK2) activity (reviewed in Biochim Biophys Acta2002;1603:31–46). Elongation of HIV-1 transcription is mediated by the interaction of HIV Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II, and our recent studies indicate that CDK2 is also required for Tat-dependent transcription. We hypothesized that iron chelation may inhibit HIV transcription via reduced activity of cell cycle-dependent kinases. We utilized 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311; previously shown to inhibit CDK2 expression) and 4-[3,5-bis-(hydroxyphenyl) -1,2,4-triazol-1-yl]-benzoic acid (ICL670) to chelate intracellular iron. We analyzed the effect of these chelators on HIV-1 transcription using HeLa MAGI and CEM-GFP T-cells containing an integrated HIV-1 promoter and infected with adenovirus expressing HIV-1 Tat protein. Both chelators inhibited Tat-induced HIV-1 transcription, most profoundly in CEM-GFP T-cells. The chelators also inhibited one round of HIV-1 replication in CEM-T cells infected with pseudotyped HIV-1 virus. Treatment of HeLa MAGI and CEM-GFP T-cells with iron chelators decreased CDK9 protein levels and, to a lesser extent, CDK2 protein levels. Our findings provide evidence that iron chelators may inhibit HIV-1 transcription by altering expression of CDK9 and CDK2.
Myoglobin Is a Nitrite Reductase That Generates NO and Regulates Mitochondrial Respiration.
Authors: Shiva Sruti , Huang Zhi , MacArthur Peter H. , Ringwood Lorna A. , Gladwin Mark T.
Published: October 2019

Previous studies have demonstrated that nitrite is an endocrine store of NO that may play an important role in hypoxic vasodilation. Nitrite can be converted to NO enzymatically by deoxyhemoglobin, in a reaction that is under allosteric control with a maximum reaction rate near the hemoglobin P50. In this study, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 50-times faster than deoxyhemoglobin due to its lower redox potential. Spectrophotometric measurements show the deoxymyoglobin-nitrite reaction to be second order and linearly dependent on deoxymyoglobin, nitrite and proton concentration with a bimolecular rate constant of 11.7 M−1s−1 at pH 7.4 at 37 degrees Celsius. Using this bimolecular rate constant, we calculate that at physiological concentrations of nitrite (20uM) and myoglobin (25uM), NO will be generated at a rate of 2.85 nM/sec. Since the IC50 of inhibition of mitochondrial respiration is approximately 100nM at physiological oxygen tensions (5–10 μM), we hypothesized that the myoglobin-dependent reduction of nitrite could regulate mitochondrial respiration. Indeed, the addition of deoxymyoglobin in conjunction with nitrite to isolated rat liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate, which contains both myoglobin and mitochondria, resulted in a measurable production of NO (with 1mM nitrite addition) and the inhibition of mitochondrial respiration (with 25μM nitrite addition) that was not significantly changed by the addition of the xanthine oxidase inhibitor allopurinol. These data corroborate previous studies demonstrating that NO generated from nitrite reduction can escape heme autocapture to mediate biological responses and confirm that the regulation of mitochondrial respiration by the nitrite-myoglobin reaction is relevant even in a physiological milieu. These data have implications for the modulation of hypoxic respiration, nitrite-dependent hypoxic signaling in tissue, and the regulation of oxygen gradients in the microcirculation.
Ascorbic Acid Catalyzes Nitric Oxide Production from Nitrite Ions.
Authors: Sibmooh Nathawut , Piknova Barbora , Schechter Alan N.
Published: October 2019

We have previously shown that nitrite ions can be reduced by hemoglobin to nitric oxide (NO), a ubiquitous signaling molecule and potent vasodilator. Nitrite serves as a stable tissue and vascular source for NO production; the reduction reaction is maximal at about 50% oxygen saturation values and is enhanced at low pH but little is known about other effectors of this reaction. In the current work, we studied the effect of ascorbic acid on nitrite reduction under physiological conditions using chemiluminescence to quantify NO production. In physiological buffer, this reaction has a rate constant of about 1×10−5 M−1.s−1. Thus, a significant production of NO would likely occur in plasma only at pharmacological levels of ascorbic acid (> 1 mM) although lowering pH below 7.0 markedly enhances this reaction. Loading human erythrocytes with 0.5 mM dehydroascorbic acid, which is in redox equilibrium with ascorbic acid and which can significantly raise intracellular ascorbic acid levels, increased basal levels of nitrite ions from 42±9.0 nM to 98±56 nM. Uptake of nitrite ions into erythrocytes by incubation in 10 μM nitrite was increased about 1.5 fold by dehydroascorbic acid and the half-time of nitrite loss was slowed to the same extent. Ascorbic acid also reduced free ferric heme in erythrocytes and plasma to ferrous heme which catalyzed the reduction of nitrite to NO with a rate constant of 2.3×103 M−1.s−1 under physiological conditions. However, free ferrous ions did not significantly produce NO in physiological buffer (rate constant = 1.8×10−2 M−1.s−1). The reaction of ferrous heme with nitrite was not affected by heme binding to proteins such as hemopexin and albumin, or erythrocyte membranes. These results suggest that physiological levels of ascorbic acid (20–80 μM in plasma and erythrocytes) may act to catalyze NO production in the blood by promoting the reduction of nitrite ions by free ferrous heme and by increasing intra-erythrocytic levels of nitrite ions which can be reduced to NO by deoxyhemoglobin.
Induction of Human γ Globin Gene Expression by Histone Deacetylase Inhibitors.
Authors: Cao Hua
Published: October 2019

In previous studies we have showed that HDAC inhibitors including hydroxamic acid derivatives of short chain fatty acids butyryl hydroxamate, propionyl hydroxamate, subericbis hydroxamic acid (SBHA), and suberoylanilide hydroxamic acid (SAHA), are potent inducers of γ globin gene expression in in vitro luciferase assays and in cultures of human adult erythroid progenitor cells. In this present study, we used μLCR Aγ transgenic mice to test whether these compounds can also induce γ gene expression in vivo. We found that in addition to γ gene induction these compounds have considerable erythropoiesis activity. Thus, Propionyl and butyryl hydroxamate increased reticulocytes of mice by 71% and 139%, the in vivo BFUe counts by 75% and 51% and the in vivo γ gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. We conclude that Hydroxamic acid derivatives can stimulate both the in vivo erythropoiesis and fetal globin production in a thalassemic murine model.

Cyclic depsipeptide FK228 is a highly potent histone deacetylase inhibitor, currently in clinical trials in cancer patients. We investigated whether FK228 also functions as inducer of human γ globin gene expression and compared Hb F induction by FK228 to that of four other HDAC inhibitors, including hydroxamic acids (TSA), synthetic benzamides (MS-275), and two cyclic tetrapeptides, Apicidin and HC-Toxin. Our results showed that FK228 is the most potent fetal hemoglobin inducer among all the HDAC inhibitors tested in our laboratory. In a concentration of 0.84 nanomolar, FK228 induces γ gene promoter activity in the dual luciferase assay by 7.81 fold. In the human erythroid progenitor cell cultures it increases the levels of γ mRNA by 8.48 fold in a concentration of 0.143 nM. In contrast, fetal hemoglobin induction by other HDAC inhibitors is achieved in concentrations that are 100 to 1000 fold higher. We conclude that FK228 is a promising compound for induction of Hb F in patients with sickle cell disease and thalassemia.
Evolution of Transfusion Requirement in Algerian Thalassemic Major (TM) and Intermediate (TI) Patients Treated with Hydroxyurea (HU).
Authors: Bradai Mohamed , Pissard Serge , Abad Mohand Tayeb , Dechartres Agnes , Ribeil Jean-Antoine , Landais Paul , De Montalembert Mariane
Published: October 2019

Context: Benefits of HU treatment in thalassemic patients are controversial. Most studies agree that HU decreases transfusion requirements in patients with TI, whereas results in patients with TM are inconsistent.

Objectives: To assess the proportion of good responders to HU and the factors associated with good response.

Methods: We initiated HU treatment in 54 thalassemic patients (9 TI and 45 TM). Median ages at first transfusion were 57 months (32–252) in TI and 13 months (4–60) in TM patients. Median ages at inclusion were 12.4 years (4.4–54.9) in TI and 9.9 years (2.0–21.0) in TM patients. Median numbers of erythrocyte concentrates (EC) transfused the year before HU were 3 (1–9) in TI and 12 (8–16) in TM patients (10–65). Good response was defined as more than 70% decrease of transfusional needs one year after beginning HU, partial response as a decrease between 40 and 70%, and non response as a decrease of less than 40%. Patients were transfused when their hemoglobin (Hb) level was < 6g/dL; not transfused when Hb was > 7 g/dL; decision to transfuse was based on clinical status for Hb level between 6 and 7 g/dL. Mean HU dose was 17 ± 2 mg/kg/d (13–21). Mean follow-up was 29.3 ± 14 months.

Results: Eight (90%) TI and 20 (44.5%) TM patients had a good response to HU. No TI and 9 (20%) TM patients had a partial response to HU. One (10%) TI and 16 (35,5%) TM patients had no response to HU. Clinical and hematological tolerance were good except for one 55 yr old TI patient who developed in the 3rd year of treatment an acute myeloblastic leukemia. Retrospectively, a malignant transformation of a pre-existent myeloproliferative disorder can not be excluded and the responsibility of HU is questionable. A better response to HU was associated with a later beginning of transfusion (p=0.02), a higher pre-HU Hb (p= 0.0004), codon 6(-A) mutation (p= 0.002), and with TI diagnosis (p=0.03). On the contrary, Gγ-158 C/C genotype (Xmn1−/−) (p= 0.0001) was associated with a worse response. After adjustment, decrease of transfusional needs was associated with the number of CE transfused the year before HU treatment (p= 0.001), Xmn1 polymorphism (p= 0.003) and the age at the beginning of HU (p=0.03) but not with the type of thalassemia. In conclusion, HU may represent an alternative to transfusions for both TI and TM patients in countries with limited blood supplies when beginning early.
Splenic Synthesis of Factor VIII: Basis for Transplant Therapy of Hemophilia A.
Authors: Suh Matthew Y. , Dada Shirine , Azpurua Humberto , Flynn Evelyn , Briscoe David M. , Kim Heung B.
Published: October 2019

Synthesis of factor VIII by liver sinusoidal endothelial cells has been documented, and liver transplantation for end stage liver disease has been shown to cure concomitant hemophilia A. Given the irreversible nature of liver transplantation, the spleen is an alternate organ of interest in transplant therapy to cure hemophilia A. Factor VIII gene expression in the spleen has been documented by RT-PCR (Hollestelle, M. J. et al, 2001, Thromb Haemost86:855–61), and a recent report of living related splenic transplantation for hemophilia A has shown symptom-free survival up to 4 years (Jiang, H. C. et al, 2006, Transplant Proc38:1483). However, it is unclear which cell populations in the spleen synthesize factor VIII. Immunohistochemistry of murine spleen showed factor VIII staining of the sinusoidal endothelial cells as expected. Surprisingly, clusters of large mononuclear cells in the red pulp also intensely stained for factor VIII. CD31 staining revealed same pattern of sinusoidal endothelial cells and large mononuclear cells in the red pulp. Single cell suspensions of murine spleen analyzed by flow cytometry corroborated significant factor VIII expression (47–70% of all splenic cells). At least 60% of splenic cells were CD31+ CD45+, and these CD31+ CD45+ cells showed significant factor VIII expression (38–48%). When CD31+ cells were depleted by magnetic cell sorting, there was still a subgroup of cells expressing factor VIII (approximately 50%). In summary, we have shown that a significant proportion of splenic cells synthesize factor VIII by flow cytometry and immunohistochemistry. In addition to the sinusoidal endothelial cells, CD31+ CD45+ cells (possibly the large mononuclear cells seen on immunohistochemistry) contribute to splenic synthesis of factor VIII. Based on CD31 depletion study, there is still an unidentified group of cells (CD31-) that contributes to factor VIII synthesis. Further characterization of these cells and an in vivo study in a murine hemophilia A model have been initiated. We conclude that the spleen is a viable source of factor VIII and may be useful in cell-based and/or organ transplant therapy of hemophilia A.
Neutrophils from Cystic Fibrosis Patients Are Defective in Killing of Phagocytosed Pseudomonas aeruginosa.
Authors: Wang Guoshun , Valentine Vincent G. , Lanson Nicholas A. , Leidal Kevin , Zhang Qiang , Lombard Gisele , Thompson Connie , Viswanathan Anand , Nauseef William M. , Wang Guangdi , Painter Richard G.
Published: October 2019

Cystic fibrosis (CF), the most common genetic disease in Caucasians, is caused by mutations of the gene encoding the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. CF has long been recognized as an epithelial disease whose most severe complications often occur in the lung. The clinical manifestations include persistent bacterial infection, prominent neutrophil infiltration and small airway obstruction. Even though dramatic advances have been made towards understanding of CF pathogenesis, the link between the CFTR chloride channel defect and a clinical defect in bacterial eradication has not been fully established. Our published data demonstrated that CFTR is expressed in human neutrophils and their phagolysosomes. CF neutrophils are defective in the chlorination of phagocytosed Pseudomonas aeruginosa (PAO1), indicating defective intraphagolysosomal hypochlorous acid (HOCl) production. In the current report, we assessed the bacterial killing abilities of neutrophils from CF and normal individuals. Percoll-purified peripheral blood neutrophils were incubated with opsonized PAO1 at a ratio of 1:1 or 1:50. To define the role of chloride in the killing process, two different Ringer’s buffers with either 0 mM chloride or 135 mM chloride were exploited. At various time points (0, 15, 30 and 60 minutes) after incubation of neutrophils with bacteria, samples were aliquoted to assay for viable bacteria. After correction for bacterial growth over the experimental period, the bacterial viability at each time point relative to that of the initial value was obtained. Two-way ANOVA tests indicated that CF neutrophils had a significantly lower initial rate of killing of PAO1 than that in normal controls. This defect is more pronounced under the condition of high bacterial load and low extracellular chloride. Surprisingly, the low extracellular chloride significantly affected neutrophil-mediated bacterial killing even for the normal neutrophils, suggesting the dependence of this ion in the killing process. Our data provide evidence to suggest the potential role of CFTR in supplying intraphagolysosomal chloride to produce hypochlorous acid (HOCl), an oxidant essential for the killing of HOCl-sensitive bacteria.
A Phase II Study of Nilotinib, a Novel Tyrosine Kinase Inhibitor Administered to Imatinib-Resistant and -Intolerant Patients with Chronic Myelogenous Leukemia (CML) in Chronic Phase (CP).
Authors: le Coutre Philipp , Bhalla Kapil , Giles Francis , Baccarani Michele , Ossenkoppele Gert J. , Hochhaus Andreas , Gattermann Norbert , Rafferty Teresa , Haque Ariful , Weitzman Aaron , Kantarjian Hagop
Published: October 2019

Nilotinib is a potent, highly selective, aminopyrimidine inhibitor which in vitro is 30-fold more potent than imatinib and active against 32/33 imatinib resistant Bcr-Abl mutations. This open-label study was designed to evaluate the safety and efficacy, as defined by hematologic/cytogenetic response rates (HR/CyR) of nilotinib administered at a daily dose of 400 mg bid to imatinib resistant or intolerant CML-CP patients. Daily doses of nilotinib could be escalated to 600 mg BID for patients who did not adequately respond to treatment, and in the absence of safety concerns. Data are available for 132 patients including 91 (69%) with imatinib resistance and 41 (31%) imatinib intolerant. Of the 132 patients, 45 (34%) had a CHR at baseline and 87 (66%) did not. Overall 77 (58%) patients had additional chromosomal abnormalities at baseline, and 16 (12%) had extramedullary involvement. Median age was 58 (range 26–85) years and the median time from first diagnosis to treatment was 57 (range 5–275) months. There were 72 males and 60 females. Nearly half (49%) of the patients had CML for ≥ 5 years and the median overall duration of prior imatinib was 978 days (range 9 to 3468). Treatment with nilotinib is ongoing for 82 of the 132 (62%) patients. A total of 50 (38%) patients have discontinued treatment (26 for adverse events, 15 for disease progression, 7 for other/administrative reasons, and there were two patient deaths (one myocardial infarction in a patient with previous infarctions noted on autopsy, and one with progressive disease). The median duration of nilotinib exposure was 226 (range 3–379) days. The median average dose intensity (mg/days) for all patients, with and without dose escalations, was 800 (range 134 to 1106). CHR was reported in 60 of 87 (69%) patients without a baseline CHR. The median time to CHR was 1.4 months (range 0.3 to 13). Major CyR was observed in 55 (42%), of which 33 (25%) were complete, 22 (17%) partial, 10 (8%) minor, 15 (11%) minimal, 21 (16%) had no cytogenetic response and 4 (3%) had disease progression. The median time to MCyR was 2.6 months (range 0.9 to 8.4). Overall the most frequent Grade 3 or 4 adverse events included thrombocytopenia 34 (26%), neutropenia in 24 (18%), elevated serum lipase in 10 (8%) and anemia in 9 (7%). In summary, nilotinib has demonstrated significant clinical activity as defined by 69% CHR and 42% MCR rates, and an acceptable safety and tolerability profile in patients with imatinib resistant or intolerant CML-CP. Updated information will be presented at the meeting.
Isolation of a Common Natural Type-I Interferon Producing Cell and Dendritic Cell Progenitor Population in Mouse Bone Marrow.
Authors: Onai Nobuyuki , Onai Aya , Manz Markus G.
Published: October 2019

Most type-I interferon producing cells (IPCs) and dendritic cells (DCs) are non-dividing cells with a short in vivo half-live of several days, and thus need to be continuously replaced. A common differentiation pathway for IPCs and DCs, and accordingly, the existence of common IPC and DC progenitors remains controversial. Flt3-ligand (Flt3L) is a non-redundant cytokine for in vivo IPC and DC development: IPC and DC differentiation potential is confined to Flt3+-hematopoietic progenitors; Flt3L KO mice show massively reduced IPCs and DCs. In contrast to Flt3, the “myeloid” cytokines GM-CSF and M-CSF seem to be less relevant in steady-state IPC and DC differentiation, however, they might be critically important in inflammatory conditions.

To identify a candidate common IPC and DC progenitor population, we evaluated Flt3 and “myeloid” cytokine receptor expression in mouse bone marrow. We found that c-kitintlin− cells contained a Flt3+M-CSFR+ fraction that in Flt3L supplemented cultures gave rise to about 95% pure CD11c+MHC class II+ cells, consisting of both CD11c+B220+ IPCs and CD11c+B220− DCs, at a efficiency comparable to that of hematopoietic stem cells. In the presence of GM-CSF, Flt3+M-CSFR+c-kitintlin− cells gave rise to CD11c+CD11b+ DCs but not CD11c−CD11b+ macrophages/monocytes. Furthermore, Flt3+M-CSFR+c-kitintlin− cells possessed very poor, if any activity in myeloid colony forming assays, and lacked pre-B cell colony forming activity.

In both, lethally and sub-lethally irradiated mice, transferred Flt3+M-CSFR+c-kitintlin− cells differentiated into CD11c+B220+ IPCs, CD11c+CD8α+, and CD11c+CD8α− conventional DC subsets, while no other hematopoietic cells were detectable. In vivo reconstitution and CFSE-labeling experiments showed that Flt3+M-CSFR+c-kitintlin− cells extensively proliferate in the lethally irradiated mice, reaching peak progeny levels of IPC and DC at day 10 after transplantation, indicating high proliferative, but limited self-renewal capacity of these cells. Quantitative RT-PCR analysis revealed high expression of DC and IPC-development affiliated genes (such as PU.1, STAT3, GM-CSFR, and CX3CR1), but no lymphoid- and erythroid-development affiliated gene transcription. These data suggest the existence of common developmental intermediates for both IPCs and DCs in mouse bone marrow, and thus might provide new insights into the regulation of IPC and DC differentiation in steady-state and inflammation.
Highly Efficient Neural Differentiation of Human Peripheral Blood Haematopoietic Stem Cells.
Authors: Moebius Jeannette M. , Widera Darius , Kaltschmidt Barbara , Kaltschmidt Christian , Piechaczek Christoph
Published: October 2019

Neural stem cells are a potential source of cells for cell therapy of neurodegenerative diseases or drug screening. Ethical and practical considerations limit the application of neural stem cells derived from human embryonic stem cells or adult brain tissue. Therefore, alternative sources of adult human neural stem cells are of high interest for basic research as well as potential clinical use. These sources have to satisfy the demands of easy accession, rapid expansion in serum-free media and reliable induction to a neural fate.

CD133 positive hematopoietic stem cells (HSCs) isolated from mobilized leukapheresis were cultured and expanded in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2/bFGF) in serum-free medium. After two weeks of culture, neurosphere formation was observed. Subsequent culture in serum-free, EGF and FGF-2 containing medium resulted in large numbers of CD133−, Nestin+ and Sox-2+ neural stem cells. Neural stem cells derived from HSCs (hNSCs) are highly proliferative and are able to migrate in response to chemotactic stimuli described to induce migration of neural stem cells. We used immunocytochemical techniques and PCR to assess neural differentiation. Adherence to poly-D-lysine/laminin and growth factor deprivation resulted in cells of neuronal morphology and high expression of neuronal differentiation markers. We furthermore show that after retinoic acid treatment neuronal induction was greatly enhanced with more than 90% of cells expressing neuronal differentiation markers as beta-III-tubulin and neurofilaments. In addition, hNSCs were able to differentiate into cells of the glial lineage as shown by expression of glial fibrillary acidic protein (GFAP). Our novel method provides nearly limitless numbers of neural stem cells from an easily accessible autologous adult human source, which could be used as a starting point for further experimental studies and potential therapeutic use.
Phenotypic Comparison of Platelet- and Plasma-Derived Factor Va: Tyrosine Phosphorylation Differentiates the Two Cofactor Pools.
Authors: Wood Jeremy P. , Tracy Paula B.
Published: October 2019

Factor Va, the essential cofactor for thrombin formation via the Prothrombinase complex, exists in two pools in whole blood: 75–80% is found in the plasma, while platelet alpha-granules contain the remaining 20–25%. Platelet-derived factor Va possesses several modifications not present in its plasma counterpart, such as an O-glycosylation on Thr402, and is the more procoagulant molecule, exhibiting resistance to inactivation by activated protein C (APC) and plasmin when bound to the activated platelet membrane surface. As the platelet-derived cofactor pool originates from endocytosis of plasma-derived factor V by megakaryocytes, it can be hypothesized that, subsequent to its endocytosis, factor V is post-translationally modified, proteolytically processed, and packaged into alpha-granules to form the unique platelet-derived molecule. Platelet- and plasma-derived factor Va were purified from three individual donors. Platelets were isolated from whole blood and lysed with Triton X-100 in the presence of the tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid to prevent dephosphorylation by any released tyrosine phosphatases. Factor V in both the platelet lysates and the platelet-poor plasma was activated with thrombin, adsorbed onto a resin-coupled monoclonal antibody against human factor V, washed extensively, and eluted using 10% SDS. Western blotting analyses, using a monoclonal antibody against phosphotyrosine residues, revealed the presence of tyrosine phosphorylation on the light chain of plasma-derived, but not platelet-derived, factor Va. No such modification was observed on the heavy chain of either cofactor. Monoclonal antibodies against the heavy and light chains of factor V were used to verify that similar levels of factor V were present in all samples. This differential modification was observed in all three donors. Subsequent MALDI-TOF analyses of trypsin-digested plasma- and platelet-derived factor Va light chains were performed to identify the site(s) of tyrosine phosphorylation. These data identified phosphorylation of the C-terminal tyrosine residue (Tyr2196) in plasma-derived, but not platelet-derived, factor Va. The phosphorylated fragment (residues 2188–2196, m/z = 1152.5) is consistently present in spectra of plasma-derived factor Va, while only the non-phosphorylated fragment (m/z = 1072.5) has been observed in the platelet-derived cofactor. To demonstrate that the phosphatase inhibitors used were effective, the platelets were lysed in the presence of added plasma-derived factor Va. Tyrosine phosphorylation, as determined by immunoblotting, was still present on the light chain, indicating that the dephosphorylation was occurring prior to platelet lysis. Tyrosine phosphorylation of other proteins has been implicated in regulation of endocytosis. Further experiments will need to be performed to determine if the modification has a similar role in megakaryocyte endocytosis of factor V.
Identification of a Plasmin-Interactive Site in the Factor VIII Light Chain Responsible for Proteolytic Cleavage at Lys36 in the A1 Domain.
Authors: Nogami Keiji , Shima Midori , Takeyama Masahiro , Tatsumi Kohei , Fujita Yuri , Saenko Evgueni L. , Tanaka Ichiro , Yoshioka Akira
Published: October 2019

Factor VIII is inactivated by limited proteolytic cleavages at Arg336 and Lys36 by plasmin (Abst #1017 and 1018, BLOOD106, 2005). We recently identified a plasmin-interactive site in the A2 domain responsible for cleavage at Arg336, and also reported that the cleavage at Lys36 was selectively regulated by the light chain that might interact with its protease. In the current study, several approaches were employed to examine the localization of plasmin-interactive site(s) responsible for cleavage at Lys36. Rate constant of plasmin-catalyzed factor VIIIa inactivation by the addition of the 1649A3-C1-C2 (intact light chain) or 1690A3-C1-C2 fragment was reduced by ~2-folds in a dose-dependent manner using one-stage clotting assay, but not by the 1722A3-C1-C2. SDS-PAGE analysis revealed that Lys36 cleavage in A1/1722A3-C1-C2 dimer by plasmin was markedly slower than that in the A1/1649A3-C1-C2 or A1/1690A3-C1-C2 dimer. Surface plasmon resonance-based assay using anhydro-plasmin (Ah-plasmin), catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that the 1722A3-C1-C2 bound to Ah-plasmin (Kd; 191 nM) with an ~3-fold lower affinity compared with 1649A3-C1-C2 or 1690A3-C1-C2 (68 or 83 nM, respectively). Recombinant A3 domain, expressed in E.Coli, also bound to Ah-plasmin with similar affinity (Kd; 44 nM), but the C2 failed to bind, suggesting the presence of a plasmin-binding site within N-terminus of the A3 domain. On the other hand, Glu-Gly-Arg-active-site modified factor IXa blocked the light chain binding to Ah-plasmin by ~60% (Ki: 10.5 nM), supporting that another plasmin-binding site juxtaposes or overlaps with a factor IXa-binding site (residues 1804–1818) in the A3 domain. The 6-aminohexanoic acid, a competitor of lysine-binding site of plasmin, blocked by >90% the light chain and Ah-plasmin interaction (Ki; 6.8 microM). Based on the major contribution of lysine-binding site(s) of plasmin for interaction with the light chain, evaluation of the A3 sequence revealed two regions involving clustered lysine residues of 1690–1705 and 1804–1818. Two synthetic peptides corresponding to these regions blocked the light chain binding to Ah-plasmin by ~50% (Ki: ~20 microM). We conclude that an extended surface centered the 1690–1705 and 1804–1818 regions within the A3 domain interacts with plasmin for the cleavage at Lys36 within the A1 domain.
Targeted Depletion and Inhibition of Activated T Lymphocytes with SGN-70, a Humanized Antibody Specific for the Activation Antigen CD70.
Authors: McEarchern Julie A. , Klussman Kerry , Boursalian Tamar E. , Grewal Iqbal S. , Law Che-Leung
Published: October 2019

CD70 (CD27L) is a member of the tumor necrosis factor superfamily that is predominantly expressed on activated lymphocytes and is important for generation of B and T memory and effector responses. High levels of CD70 are also found on chronically activated T cells and lymphocytes in patients with autoimmune disorders. Blockade of CD70 interaction with its receptor has been shown to inhibit the onset of experimental autoimmune encephalomyelitis and cardiac allograft rejection in mice. We have engineered a humanized anti-CD70, SGN-70, that mediates Fc-dependent antibody effector functions and blocks binding of CD70 to its receptor. In this report we show that SGN-70 inhibits co-stimulation of T lymphocytes, and selectively depletes CD70+ activated T cells. Depletion of antigen-specific T cells by SGN-70 was demonstrated using CD8+ T cells specific for a peptide (M58-66) derived from the influenza A matrix protein M1. Stimulation of peripheral blood mononuclear cells with the M1 peptide markedly induced CD70 expression within a discrete expanding of M1 peptide-specific CD8+Vβ17+ T cell subset. Treatment of peptide-stimulated cultures with SGN-70 (0.01–1 μg/mL) decreased the number of CD70+CD8+Vβ17+ cells by >80%. This depletion was dependent on the activity of CD16+ cells within the culture since blocking CD16 completely eliminated the depleting effect of SGN-70. Non-activated Vβ17 negative cells were not affected by SGN-70 and were functionally equivalent to untreated control cultures in subsequent response to mitogenic re-stimulation. Together, these data demonstrate the capacity of SGN-70 to selectively target activated cells and its potential to limit expansion of antigen-specific T lymphocytes. Thus, SGN-70 may have therapeutic potential to target T cell-mediated autoimmune and inflammatory diseases.
CD4+CD25+ Marrow Infiltrating Lymphocytes in Myeloma Patients Display an Activated Phenotype and Lack Suppressive Function.
Authors: Noonan Kimberly A. , Mgebroff Stephanie , Serafini Paolo , Meckel Kristen , Bonyhadi Mark , Borrello Ivan
Published: October 2019

We have previously shown that marrow infiltrating lymphocytes (MILs) from myeloma patients proliferate upon stimulation with anti-CD3/CD28 beads more efficiently and exhibit greater specificity to autologous tumor than their peripheral blood lymphocyte (PBL) counterparts. In attempting to dissect the mechanisms responsible for greater tumor specificity of MILs, we examined the frequency and function of the putative CD4+/CD25+ regulatory T cells (Tregs) in both compartments. Phenotypic and functional differences in the CD4+/CD25+ populations were examined in MILs and PBLs from 12 multiple myeloma patients and 3 normal donors. CD4+/CD25+ cells were stained for the suppressive marker: FoxP3; or the activation markers: CD40L, CD71 (transferrin receptor). CD4+/CD25+ MILs of myeloma patients are CD25lo, FoxP3lo and predominantly express activation markers. They expand more readily upon anti-CD3/CD28 stimulation, produce high amounts of the Th1 cytokines: IFNγ and IL-2, and low amounts of IL-10. Importantly, they fail to suppress the tumor specific T cell proliferation. These findings are all consistent with an activated T cell phenotype. In contrast, CD4+/CD25+ cells from PBLs of myeloma patients are CD25hi, FoxP3+, primarily produce IL-10 and IL-4 and markedly suppress T cell expansion in a tumor specific assay, suggestive of a regulatory T cell phenotype. Although MILs possess a lower number of FoxP3+ cells as compared to PBLs, the higher MFI may indicate the presence of a small yet highly regulatory T cell population. Interestingly, MILs from normal donors possess a more regulatory phenotype then their myeloma counterparts as determined by high FoxP3 and low CD40L and CD71 expression as well as reduced expansion upon activation. Taken together, these data underscore major differences in the immuno-inhibitory pathways present in blood as compared to marrow. Notably, MILs of myeloma patients demonstrate a paucity of Tregs. These differences may explain the disparities seen in the tumor-specificity of T cells from these two compartments. The ability to expand a T cell population with fewer endogenous Tregs and heightened tumor-specificity may have significant implications for the implementation of adoptive immunotherapy.

%CD25 (MFI) %FoxP3 (MFI) %CD40L %CD71 Fold Expansion Ability to Suppress Myeloma CD4/CD25 MILs 4.7 (29.6) 2.2 (2932.1) 88.8 90.1 12.2 − CD4/CD25 PBLs 19.1 (115.2) 52.3 (163.7) 15.7 19.6 2.5 +++++ Normal CD4/CD25 MILs 14.0 (56.8) 70.1 (82.7) 37.3 6.4 3.3 +++ CD4/CD25 PBLs 24.0 (19.4) 30.9 (261.8) 12.1 4.2 4.0 ++
Reaction of Factor VIIa (VIIa) with Plasma Inhibitors Ex Vivo and In Vivo.
Authors: Petersen Lars C. , Christoffersen Carsten L. , Krogh Thomas N. , Elm Torben
Published: October 2019

Activated proteases are typically short-lived in the circulation where they react with plasma inhibitors and are removed as complexes by scavenger receptors. In contrast VIIa is characterized by a relatively long in vivo clearance half-life of 2–3 hr. Free VIIa exists in a latent state that is fully activated only upon binding to tissue factor (TF). Ex vivo VIIa is described to react predominantly with anti-thrombin (AT) in a slow reaction enhanced by heparin and by TF. Free VIIa is assumed to be rather inert towards plasma inhibitors also in vivo, and this may account for its slow clearance. Still it is not known whether the reaction with AT or other plasma inhibitors is an obligatory step in the in vivo clearance of VIIa. To clarify this question we studied the interaction between VIIa and plasma inhibitors in human and murine serum and plasma, and also studied this interaction during in vivo clearance of VIIa in mice. The VIIa-inhibitor complexes formed after spiking of serum with VIIa for 24 h were purified by affinity chromatography and subjected to SDS-PAGE and characterized by MALDI analysis. In the absence of heparin we identified VIIa complexes with alpha 2-macroglobulin (alpha 2M), AT, alpha 2-plasmin inhibitor and inter-alpha-trypsin inhibitor with the complex with alpha 2M as the most prominent component, whereas the AT complex was pre-dominant in the presence of heparin. The interaction between VIIa and plasma inhibitors might be differently affected by contact with the vasculature in the circulation. We therefore next studied VIIa complex formation in vivo in mice after i.v. injection of 125I-VIIa. Complex formation was followed by SDS PAGE and autoradiography, and clearance was followed by measurements of the VII antigen level. The data suggest that alpha 2M and AT complexes are formed in vivo, however, without affecting VIIa antigen clearance. This was confirmed by studies of in vivo clearance of VIIa or a preformed VIIa-AT complex. Measurements of VIIa clot activity, and of VII- and VIIa-AT antigen concentrations suggestedthat in vivo clearance of VIIa from circulation does not include complex formation with a plasma inhibitor as an obligatory intermediate,that the VIIa-AT complex is cleared with the same rate as VIIa andthat a transient VIIa-AT complex is formed subsequent to VIIa bolus injection which may account for a faster clearance of VIIa when measured by clot activity relative to VII clearance when this is measured by antigen concentration.
Heat Shock Prevents Tissue Factor Induction after Rewarming from Lethal Hypothermia in a c-Jun-Dependent Manner.
Authors: Egorina Elena M. , Sovershaev Mikhail A. , Kondratiev Timofei V. , Olsen Jan O. , Tveita Torkjel , Østerud Bjarne
Published: October 2019

Decrease in body core temperature is associated with life threatening coagulation disturbances in humans. Although rewarming from hypothermia is a prerequisite for survival, it can lead to mobilization of large amounts of tissue factor (TF) from damaged tissues, which in turn can induce disseminated intravascular coagulation. Using immunoblotting, In-Cell Western assay, fluorescence activated cell sorting (FACS) and TF activity assay, we studied post-rewarming TF levels, activity and surface redistribution, along with the regulation of TF gene transcription in mononuclear cells (MNCs) obtained from an in vivo rat model. Twenty-four male Wistar rats were randomly assigned into four groups: normothermic control group (n=6); hypothermic followed by rewarming group (n=6); heat shock pretreated hypothermic followed by rewarming group (n=6); and heat shock pretreated normothermic group (n=6). Rewarming after a 5-hour episode of 15oC hypothermia caused a 5-fold rise of TF protein levels and 2-fold increase in surface TF signal intensity and serface redistribution of TF antigen, judged by 2-fold increase in surface-to-total TF ratio. TF activity was enhanced from 5.6±0.7 mU 106/cells in normothermic group to 13.9±3.6 mU 106/cells after hypothermia/rewarming. This was accompanied by a dramatic elevation of c-Jun and c-Jun-N-terminal kinase (JNK) phosphorylation, and the absence of early growth response-1 and NF-kB activation. Reflecting certain limitations of the method when dealing with detection of low levels of antigens, FACS analysis of TF-positive population of MNCs failed to detect any differences between the groups. To search for a stimulus to counteract c-Jun-mediated induction of TF activity in MNCs from rewarmed rats, we applied heat shock pretreatment one day before the hypothermia/rewarming experiment. Heat shock pretreatment restored post-rewarming TF activity, protein levels and surface-to-total TF ratio in rat MNCs to normothermic levels. Furthermore, in heat shock-pretreated animals, rewarming from hypothermia failed to increase phosphorylated c-Jun and JNK levels. We attribute this to the profound overexpression of heat shock protein 70 and inhibition of JNK. Taken together, in our present study, we report an induction of TF activity, protein levels and redistribution of TF antigen to the surface of MNCs, isolated from rats rewarmed from hypothermia. This increase was counteracted by a full body heat shock 24 hours prior to hypothermia via suppression of hypothermia-induced activation of c-Jun.
O-Linked Glycosylation with Sialylated T-Antigen: A Novel Carbohydrate Determinant of von Willebrand Factor Antigen Levels.
Authors: Lenting Peter J. , van Schooten Carina J.M. , Lisman Ton , van den Berg Marijke , Eikenboom Jeroen C.J. , Leebeek Frank W.G. , Goudemand Jenny , Fressinaud Edith , de Groot Philip G. , Denis Cécile V.
Published: October 2019

The glycosylation-profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of the sialylated T-antigen (NeuAc( α 2–3)Gal( β 1–3)[NeuAc( α 2–6)]GalNAc). It is unknown yet if O-linked carbohydrates affect VWF levels. We therefore developed an immunosorbent-based assay using the lectin Peanut Agglutinin (PNA) to determine O-linked T-antigen on VWF. Control experiments showed that PNA-binding was: (i) specific to T-antigen that was de-sialylated via neuramidase-treatment; (ii) proportional to the number of O-linked glycosylation sites and (iii) independent of the extent of multimerisation. Using this assay, we found a strong correlation between PNA binding and VWF-antigen levels in a series of randomly selected plasma samples (252±125% for VWF<0.5 U/ml (n=15); 131±36% for VWF between 0.5 and 1.5 U/ml (n=32); 92±40% for VWF>1.5 U/ml (n=19); p<0.003). Reduced or increased PNA binding was also observed for patients characterized by increased (liver cirrhosis; n=58) or reduced (von Willebrand disease (VWD)-type 1; n=32) VWF-antigen levels, respectively. VWD-type 1 patients further displayed increased ratios of propeptide over mature VWF-antigen levels (0.38±0.18 versus 0.17±0.03 for patients (n=32) and controls (n=20), respectively; p<0.0001), which is indicative for reduced VWF survival in these patients. Interestingly, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank= 0.50; p=0.0039), suggesting a potential association between T-antigen glycosylation and VWF survival. Finally, we observed a marked decrease in PNA binding in post-DDAVP samples from various patients (131±45% versus 85±33% in pre- and post DDAVP, respectively; n=18; p=0.0013). Since these patients had distinct underlying pathologies (primary platelet function disorder, hemophilia A or VWD type 1, 2A or 2N), this seems to represent a general phenomenon that is not restricted to VWD. Moreover, it indicates that the O-linked glycosylation profile of VWF stored in endothelial storage-organelles may differ from that of constitutively secreted VWF.
Formation of Platelet Strings and Microthrombi in the Presence of ADAMTS13 Inhibitor Does Not Require P-Selectin or β3 Integrin.
Authors: Chauhan Anil K. , Wagner Denisa D.
Published: October 2019

VWF is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies respectively. VWF released from the storage granules upon cellular activation or stimulation with secretagogues are extremely large and known as Ultra-Large von Willebrand factor (UL-VWF). Such filaments were first shown by Dong and colleagues (Dong et. al., 2002) and platelets were seen to align as string of beads on these UL-VWF multimers. These platelet strings are then rapidly cleaved to smaller size by the metalloprotease ADAMTS-13 (A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats-13). Recently, we have similarly shown that in Adamts13−/− mice platelet strings form and remain on stimulated endothelium in veins, where they wave in the blood stream for many minutes (Motto et. al., 2005). We have also shown that antibody to ADAMTS13 induces platelet strings in activated venules and transient thrombi formation in activated microvenules of wild type similar to Adamts13−/− mice (Chauhan et. al., 2006). The obvious question is what anchors the UL-VWF mulimers to the endothelial surface? In a flow chamber model, using polyclonal antibodies to P-selectin or soluble P-selectin, it was proposed that P-selectin anchors the UL-VWF multimers to the endothelial surface. The present study was designed to address the role of P-selectin and that of the platelet and endothelial integrin β3 in platelet string formation in vivo using intravital microscopy and genetically engineered mice. We demonstrate that in histamine activated venules, causing Weibel-Palade bodies secretion, P-selectin is not required for formation or retention of VWF platelet strings on endothelium in vivo. The mean time the platelet strings (~30–100 μm long) anchored onto the endothelium was 45 s. This duration was significantly longer than in P-selectin−/− mice infused with control Ig (P<0.001) and similar to WT mice treated with the inhibitory antibody. Similar to wild type, 45 s after topical superfusion of calcium ionophore A23187, platelet strings forming transient thrombi formation was seen in microvenules (25–30 μm in diameter) of P-selectin−/− mice when the ADAMTS13 activity was inhibited. Platelet strings were either not seen or were very short lived in the β3−/− mice infused with control Ig, whereas in the mice infused with anti- ADAMTS13 antibody platelet strings could be seen that anchored to the endothelium with a mean time of 50 s (P<0.0001). These observations show that platelet strings are not held by β3 integrin and platelets lacking β3 integrin can adhere firmly to the VWF strings in the histamine activated venules. VWF containing platelet strings could also entangle and form emboli in the absence of β3 integrin.
CD34+AC133+VEGFR-2+ Cells Are Primitive Hematopoietic Progenitors, Not Functional Endothelial Progenitor Cells.
Authors: Case Jamie , Mead Laura E. , White Hilary A. , Saadatzadeh Mohammad R. , Yoder Mervin C. , Haneline Laura S. , Ingram David A.
Published: October 2019

Endothelial progenitor cells (EPCs) are currently used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk and progression. However, there is no uniform definition of an EPC, which complicates interpretation of prior EPC studies. EPCs are primarily defined by expression of cell surface antigens. The most widely cited definition of an EPC is a cell which co-expresses CD34, AC133 and VEGFR-2. Importantly, these antigens are also expressed on the most primitive population of hematopoietic progenitor cells (HPCs), including high proliferative potential- (HPP-) and low proliferative potential-colony forming cells (LPP-CFCs). Remarkably, CD34+AC133+VEGFR-2+ cells have never been isolated and plated in endothelial cell (EC) or hematopoietic cell clonogenic assays to determine what cell progeny can be derived from a CD34+AC133+VEGFR-2+ cell. Utilizing human umbilical cord blood (CB), an enriched source of both EPCs and HPCs, we isolated and purified CD34+AC133+VEGFR-2+ cells by FACS and assayed for the presence of clonogenic endothelial CFCs (ECFCs) plus HPP- and LPP-CFCs. Surprisingly, CD34+AC133+VEGFR-2+ cells do not form ECFCs under any culture conditions previously described for outgrowth of EPCs. However, consistent with a HPC phenotype, CD34+AC133+VEGFR-2+ cells formed both HPP- and LPP-CFCs in multiple independent assays. In addition, all CD34+AC133+VEGFR-2+ cells were shown to co-express the specific hematopoietic cell surface antigen, CD45, which is not present on ECs. Based on this information, we plated CD34+CD45+ or CD34+CD45− cells to determine if EPCs could be separated from HPCs on the basis of CD45 expression. In multiple independent assays, CD34+CD45+ cells consistently formed both HPP- and LPP-CFCs but not EC colonies. In contrast, CD34+CD45− cells form EC colonies but not hematopoietic cell colonies. Taken together, these data demonstrate that circulating CD34+AC133+VEGFR-2+ cells are HPCs and the biologic mechanism for their correlation with cardiovascular disease needs to be re-examined. Furthermore, studies focused on determining the angiogenic potential of CD34+CD45− cells are needed given that this cell population harbors ECFCs.