Targeting cytosolic proliferating cell nuclear antigen in neutrophil-dominated inflammation.

New therapeutic approaches that can accelerate neutrophil apoptosis under inflammatory conditions to enhance the resolution of inflammation are now under study. Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells is a key regulator of neutrophil survival. The nuclear-to-cytoplasmic relocalization occurred during granulocytic differentiation and is dependent on a nuclear export sequence thus strongly suggesting that PCNA has physiologic cytoplasmic functions. In this review, we will try to put into perspective the physiologic relevance of PCNA in neutrophils. We will discuss key issues such as molecular structure, post-translational modifications, based on our knowledge of nuclear PCNA, assuming that similar principles governing its function are conserved between nuclear and cytosolic PCNA. The example of cystic fibrosis that features one of the most intense neutrophil-dominated pulmonary inflammation will be discussed. We believe that through an intimate comprehension of the cytosolic PCNA scaffold based on nuclear PCNA knowledge, novel pathways regulating neutrophil survival can be unraveled and innovative agents can be developed to dampen inflammation where it proves detrimental

Heat and Hypoxic Acclimation Increase Monocyte Heat Shock Protein 72 but Do Not Attenuate Inflammation following Hypoxic Exercise

Acclimation to heat or hypoxic stress activates the heat shock response and accumulation of cytoprotective heat shock proteins (HSPs). By inhibiting the NF-κB pathway HSP72 can preserve epithelial function and reduce systemic inflammation. The aim of this study was to determine the time course of mHSP72 accumulation during acclimation, and to assess intestinal barrier damage and systemic inflammation following hypoxic exercise. Three groups completed 10 × 60-min acclimation sessions (50% normoxic VO2peak) in control (n = 7; 18°C, 35% RH), hypoxic (n = 7; FiO2 = 0.14, 18°C, 35% RH), or hot (n = 7; 40°C, 25% RH) conditions. Tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 10 (IL-10), and intestinal fatty acid binding protein (I-FABP) were determined at rest and following a cycling normoxic stress test (NST; ~2 weeks before acclimation), pre-acclimation hypoxic stress test (HST1; FiO2 = 0.14, both at 50% normoxic VO2peak; ~1 week before acclimation) and post-acclimation HST (48 h; HST2). Monocyte HSP72 (mHSP72) was determined before and after exercise on day 1, 3, 5, 6, and 10 of acclimation. Accumulation of basal mHSP72 was evident from day 5 (p < 0.05) of heat acclimation and increased further on day 6 (p < 0.01), and day 10 (p < 0.01). In contrast, basal mHSP72 was elevated on the final day of hypoxic acclimation (p < 0.05). Following the NST, plasma TNF-α (–0.11 ± 0.27 ng.mL−1), IL-6 (+0.62 ± 0.67 ng.mL−1) IL-10 (+1.09 ± 9.06 ng.mL−1) and I-FABP (+37.6 ± 112.8 pg.mL−1) exhibited minimal change. After HST1, IL-6 (+3.87 ± 2.56 ng.mL−1), IL-10 (+26.15 ± 26.06 ng.mL−1) and I-FABP (+183.7 ± 182.1 pg.mL−1) were elevated (p < 0.01), whereas TNF-α was unaltered (+0.08 ± 1.27; p > 0.05). A similar trend was observed after HST2, with IL-6 (+3.09 ± 1.30 ng.mL−1), IL-10 (+23.22 ± 21.67 ng.mL−1) and I-FABP (+145.9 ±123.2 pg.mL−1) increased from rest. Heat acclimation induces mHSP72 accumulation earlier and at a greater magnitude compared to matched work hypoxic acclimation, however neither acclimation regime attenuated the systemic cytokine response or intestinal damage following acute exercise in hypoxia

Ineffective Erythropoiesis in β

In humans, β-thalassemia dyserythropoiesis is characterized by expansion of early erythroid precursors and erythroid progenitors and then ineffective erythropoiesis. This ineffective erythropoiesis is defined as a suboptimal production of mature erythrocytes originating from a proliferating pool of immature erythroblasts. It is characterized by (1) accelerated erythroid differentiation, (2) maturation blockade at the polychromatophilic stage, and (3) death of erythroid precursors. Despite extensive knowledge of molecular defects causing β-thalassemia, less is known about the mechanisms responsible for ineffective erythropoiesis. In this paper, we will focus on the underlying mechanisms leading to premature death of thalassemic erythroid precursors in the bone marrow

Carboxy-terminal fragment of fibroblast growth factor 23 induces heart hypertrophy in sickle cell disease

International audienc

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

Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival

Cytosolic proliferating cell nuclear antigen (PCNA) binds to procaspases and protects human neutrophils from apoptosis

Preclinical Development of a Lentiviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the interleukin-2 receptor \u3b3 chain gene (IL2RG), and it is characterized by profound defects in T, B, and natural killer (NK) cell functions. Transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with early murine leukemia retrovirus (MLV)-derived gammaretroviral vectors showed restoration of T cell immunity in patients, but it resulted in vector-induced insertional oncogenesis. We developed a self-inactivating (SIN) lentiviral vector carrying a codon-optimized human IL2RG cDNA driven by the EF1\u3b1 short promoter (EFS-IL2RG), and we tested its efficacy and safety in vivo by transplanting transduced Il2rg-deficient Lin 12 HSPCs in an Il2rg 12/ 12/Rag2 12/ 12 mouse model. The study showed restoration of T, B, and NK cell counts in bone marrow and peripheral blood and normalization of thymus and spleen cellularity and architecture. High-definition insertion site analysis defined the EFS-IL2RG genomic integration profile, and it showed no sign of vector-induced clonal selection or skewing in primarily and secondarily transplanted animals. The study enables a phase I/II clinical trial aimed at restoring both T and B cell immunity in SCID-X1 children upon non-myeloablative conditioning

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