Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer.

We have shown previously that PCNA, a nuclear factor involved in DNA replication and repair in proliferating cells, is localized exclusively in the cytoplasm of neutrophils, where it regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have shown that only monomeric PCNA can expose its NES to be relocalized from nucleus to cytosol during granulocyte differentiation. This study tested the hypothesis that monomeric PCNA could have a biological role in neutrophils. With the use of a combination of cross-linking and gel-filtration experiments, trimeric and monomeric PCNAs were detected in neutrophil cytosol. The promyelocytic cell line PLB985 was next stably transfected to express the monomeric PCNAY114A mutant to examine its function compared with the WT trimeric PCNA. Monomeric PCNAY114A mutant potentiated DMF-induced differentiation, as evidenced by an increased percentage of CD11b- and gp91phox-positive PLB985PCNAY114A cells and by an increased, opsonized zymosan-triggered NADPH oxidase activity compared with PLB985PCNA or PLB985 cells overexpressing WT PCNA or the empty plasmid, respectively. Regarding antiapoptotic activity, DMF-differentiated PLB985 cells overexpressing WT or the monomeric PCNAY114A mutant displayed a similar antiapoptotic activity following treatment with gliotoxin or TRAIL compared with PLB985. The molecular basis through which cytoplasmic PCNA exerts its antiapoptotic activity in mature neutrophils may, at least in part, be independent of the trimeric conformation

Sotatercept, a novel transforming growth factor beta ligand trap, improves anemia in beta-thalassemia: a phase 2, open-label, dose-finding study

\u3b2-thalassemia, a hereditary blood disorder caused by defective synthesis of hemoglobin \u3b2 globin chains, leads to ineffective erythropoiesis and chronic anemia that may require blood transfusions. Sotatercept (ACE-011) acts as a ligand trap to inhibit negative regulators of late-stage erythropoiesis in the transforming growth factor beta superfamily, correcting ineffective erythropoiesis. In this phase II, open-label, dose-finding study, 16 patients with transfusion-dependent \u3b2-thalassemia and 30 patients with non-transfusion-dependent \u3b2 thalassemia were enrolled at 7 centers in 4 countries from November 2012 to November 2014. Patients were treated with sotatercept at 0.1, 0.3, 0.5, 0.75, or 1.0 mg/kg to determine a safe and effective dose. Doses were administered by subcutaneous injection every 3 weeks. Patients were treated for 6422 months. Response was assessed as a 6520% reduction in transfusion burden sustained for 24 weeks in transfusion-dependent \u3b2-thalassemia patients, and an increase in hemoglobin level of 651.0 g/dL sustained for 12 weeks in non-transfusion-dependent \u3b2-thalassemia patients. Sotatercept was well tolerated. After a median treatment duration of 14.4 months (range 0.6-35.9), no severe life-threatening adverse events were observed; 13% of patients reported serious but manageable adverse events. The active dose of sotatercept was 650.3 mg/kg for non-transfusion-dependent \u3b2-thalassemia and 650.5 mg/kg for transfusion-dependent \u3b2-thalassemia patients. Of 30 non-transfusion-dependent \u3b2-thalassemia patients treated with 650.1 mg/kg sotatercept, 18 (60%) achieved a mean hemoglobin increase 651.0 g/dL, and 11 (37%) an increase 651.5 g/dL, sustained for 6512 weeks. Four (100%) transfusion-dependent \u3b2-thalassemia patients treated with 1.0 mg/kg sotatercept achieved a transfusion-burden reduction of 6520%. Sotatercept was effective and well tolerated in patients with \u3b2-thalassemia. Most non-transfusion-dependent \u3b2-thalassemia patients treated with higher doses achieved sustained increases in hemoglobin level. Transfusion-dependent \u3b2-thalassemia patients treated with higher doses of sotatercept achieved notable reductions in transfusion requirement. The registration number at ClinicalTrials.gov was NCT01571635

Up-Regulation of Annexin-A1 and Lipoxin A4 in Individuals with Ulcerative Colitis May Promote Mucosal Homeostasis

PubMed ID: 22723974This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wildtype genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model

Cross Adaptation - Heat and Cold Adaptation to Improve Physiological and Cellular Responses to Hypoxia

To prepare for extremes of heat, cold or low partial pressures of O2, humans can undertake a period of acclimation or acclimatization to induce environment specific adaptations e.g. heat acclimation (HA), cold acclimation (CA), or altitude training. Whilst these strategies are effective, they are not always feasible, due to logistical impracticalities. Cross adaptation is a term used to describe the phenomenon whereby alternative environmental interventions e.g. HA, or CA, may be a beneficial alternative to altitude interventions, providing physiological stress and inducing adaptations observable at altitude. HA can attenuate physiological strain at rest and during moderate intensity exercise at altitude via adaptations allied to improved oxygen delivery to metabolically active tissue, likely following increases in plasma volume and reductions in body temperature. CA appears to improve physiological responses to altitude by attenuating the autonomic response to altitude. While no cross acclimation-derived exercise performance/capacity data have been measured following CA, post-HA improvements in performance underpinned by aerobic metabolism, and therefore dependent on oxygen delivery at altitude, are likely. At a cellular level, heat shock protein responses to altitude are attenuated by prior HA suggesting that an attenuation of the cellular stress response and therefore a reduced disruption to homeostasis at altitude has occurred. This process is known as cross tolerance. The effects of CA on markers of cross tolerance is an area requiring further investigation. Because much of the evidence relating to cross adaptation to altitude has examined the benefits at moderate to high altitudes, future research examining responses at lower altitudes should be conducted given that these environments are more frequently visited by athletes and workers. Mechanistic work to identify the specific physiological and cellular pathways responsible for cross adaptation between heat and altitude, and between cold and altitude, is warranted, as is exploration of benefits across different populations and physical activity profiles

L'érythropoïèse : un paradigme pour l'étude du rôle des caspases dans la mort et la différenciation cellulaire

La différenciation érythroïde est sous la dépendance du facteur de transcription GATA-1 qui régule l'expression des gènes érythroïdes (hémoglobine, glycophorine, récepteur à l'érythropoïétine) et de l'érythropoïétine. La différenciation érythroïde terminale est caractérisée par des modifications morphologiques comprenant une réduction progressive du volume cellulaire et du noyau associée à une condensation marquée de la chromatine. Les changements morphologiques sont en partie comparables à ceux qui sont observés au cours de l'apoptose. La production de globules rouges dépend du taux d'apoptose des progéniteurs et des précurseurs érythroïdes. La privation en érythropoïétine ou l'induction de la voie Fas aboutissent à l'activation de la caspase-3, ce qui a pour conséquence la protéolyse de GATA-1, l'arrêt de maturation et l'apoptose des érythroblastes immatures. Récemment nous avons mis en évidence qu'en présence d'érythropoïétine, l'activation de la caspase- 3 est également indispensable aux modifications morphologiques caractéristiques observées au cours de la différenciation érythroïde terminale chez l'Homme. Les protéines clivées par les caspases, lors de l'érythropoïèse, comprennent la Lamine B et Acinus impliquées dans la condensation de la chromatine. Par contre, alors que la caspase-3 est activée, le clivage de GATA-1 et l'apoptose ne sont pas observées. Ainsi, le devenir des précurseurs érythroïdes est déterminé en aval de l'activation des caspases en fonction des substrats qu'elles clivent. Il semble donc qu'existent lors de l'érythropoïèse, des mécanismes de protection sélective des substrats vis à vis des caspases activées. Cette nouvelle fonction des caspases que nous décrirons dans cette revue pourrait jouer un rôle dans d'autres systèmes hématopoïétiques et non hématopoïétiques

Fanconi syndrome due to deferasirox

Deferasirox is an innovative iron-chelating treatment. However, preliminary data have suggested that kidney toxicity may be a major issue in the management of patients receiving this drug. We report a case of Fanconi syndrome associated with acute renal insufficiency in a patient receiving deferasirox. The latter has to be added to the expanding list of drugs that may induce Fanconi syndrome. Careful monitoring of kidney function and markers of proximal tubular injury are mandatory in patients undergoing treatment with deferasirox

Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation