Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Ferroportin Q248H mutation has an allele frequency of 2.2–13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. Some investigators have reported that ferroportin Q248H is degraded after exposure to hepcidin in exactly the same manner as wild-type ferroportin, but supraphysiological concentrations of hepcidin were used. The aim of our study was to determine whether ferroportin Q248H may have reduced sensitivity to physiological concentrations of hepcidin. The sensitivity of ferroportin Q248H to hepcidin was determined in 293T cells transiently expressing ferroportin using immunoblotting and fluorescence analysis. Ferritin concentrations were measured in these cells and also in human primary monocytes derived from humans with different ferroportin genotypes. The effect of Q248H on serum iron measures was examined in patients with sickle cell anemia. Immunoblotting and fluorescence analysis showed decreased sensitivity of ferroportin Q248H to physiological concentrations of hepcidin. Lower ferritin concentrations were observed after incubation with iron and hepcidin in 293T cells expressing ferroportin Q248H and in primary monocytes from ferroportin Q248H subjects. In sickle cell anemia, ferroportin Q248H heterozygotes had lower serum ferritin concentrations than wild-type subjects, consistent with enhanced iron release by macrophage ferroportin Q248H. A clinical benefit of ferroportin Q248H was suggested by lower echocardiographic estimates of pulmonary artery pressure in patients carrying mutant alleles. In conclusion, our results suggest that ferroportin Q248H protein is resistant to physiological concentrations of hepcidin and that this mutation has discernible effects on iron metabolism-related clinical complications of sickle cell anemia. They provide a mechanistic explanation for the effect of ferroportin Q248H on iron status in individuals of African descent and suggest that these changes in iron metabolism may be beneficial under certain disease-specific circumstances.(ClinicalTrials.gov Identifier:{"type":"clinical-trial","attrs":{"text":"NCT00011648","term_id":"NCT00011648"}}NCT00011648).     

Immediate anti‐tumor necrosis factor‐α (etanercept) therapy enhances axonal regeneration after sciatic nerve crush

Peripheral nerve regeneration begins immediately after injury. Understanding the mechanisms by which early modulators of axonal degeneration regulate neurite outgrowth may affect the development of new strategies to promote nerve repair. Tumor necrosis factor‐α (TNF‐α) plays a crucial role in the initiation of degenerative cascades after peripheral nerve injury. Here we demonstrate using real‐time Taqman quantitative RT‐PCR that, during the time course (days 1–60) of sciatic nerve crush, TNF‐α mRNA expression is induced at 1 day and returned to baseline at 5 days after injury in nerve and the corresponding dorsal root ganglia (DRG). Immediate therapy with the TNF‐α antagonist etanercept (fusion protein of TNFRII and human IgG), administered systemically (i.p.) and locally (epineurially) after nerve crush injury, enhanced the rate of axonal regeneration, as determined by nerve pinch test and increased number of characteristic clusters of regenerating nerve fibers distal to nerve crush segments. These fibers were immunoreactive for growth associated protein‐43 (GAP‐43) and etanercept, detected by anti‐human IgG immunofluorescence. Increased GAP‐43 expression was found in the injured nerve and in the corresponding DRG and ventral spinal cord after systemic etanercept compared with vehicle treatments. This study established that immediate therapy with TNF‐α antagonist supports axonal regeneration after peripheral nerve injury. © 2009 Wiley‐Liss, Inc.     

Expression of metalloproteinases (MMP-2, MMP-9, and TACE) and TNF-alpha in the nerves of leprosy patients

Matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) play important and related roles in the pathogenesis of nerve injury. MMP-dependent and TNF-alpha-dependent processes of neurodegeneration, such as blood-nerve breakdown and immune cell recruitment, are characteristic of leprosy nerve damage. Our work has contributed to the understanding of the role of cytokines in the process, but the role of MMPs in the pathogenesis of neuritic leprosy has not been investigated. This study analyzed the changes in mRNA expression and immunodistribution of MMP-2, MMP-9, TNF-alpha-converting enzyme (TACE), TNF-alpha in nerves of 27 pure neuritic leprosy (PNL) patients, both acid-fast bacilli positive (AFB(+)) and acid-fast bacilli negative (AFB(-)), and 8 non-leprosy patients with control peripheral neuropathic conditions. MMP-2, MMP-9, and TNF-alpha mRNA expression was significantly induced in the AFB(-) relative to the AFB(+) neuritic leprosy group and nonlepritic controls; TACE levels were also elevated in the AFB(-) group, but this change was not statistically significant. Immunoreactive profiles for TNF-alpha and MMPs demonstrated strong reactivity of myelinated axons, infiltrating macrophages, Schwann cells, endothelial cells, and perineurial cells in neuritic leprosy biopsies. This study provides the evidence of the involvement of MMPs in the pathogenesis of PNL neuropathy.     

Erythropoietin reduces Schwann cell TNF-alpha, Wallerian degeneration and pain-related behaviors after peripheral nerve injury

Chronic sciatic nerve constriction injury (CCI) induces Wallerian degeneration and exaggerated pain-like behaviors. These effects are mediated in large part by pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha). In this study, we demonstrate that systemically administered recombinant human erythropoietin (rhEpo) facilitates recovery from chronic neuropathic pain associated with CCI in rats. Because TNF-alpha has been implicated in the development of pain-related behaviors, we measured TNF-alpha mRNA at the nerve injury site. Systemically or locally administered rhEpo decreased TNF-alpha mRNA, compared with that observed in untreated animals. RhEpo also significantly (P < 0.05) decreased axonal degeneration. Immunohistochemistry of CCI nerve showed abundant TNF-alpha in Schwann cells, axoplasm and macrophages. In rhEpo-treated animals, TNF-alpha immunopositivity was decreased selectively in Schwann cells. These results suggest a model in which rhEpo counteracts the effects of TNF-alpha in CCI by blocking expression of TNF-alpha in Schwann cells. To further test this model, we studied primary Schwann cell cultures. RhEpo inhibited TNF-alpha expression in response to lipopolysaccharide, supporting the conclusions of our in vivo CCI experiments. In addition, rhEpo directly counteracted Schwann cell death induced by exogenously added TNF-alphain vitro. These results indicated that rhEpo regulates TNF-alpha by multiple mechanisms; rhEpo regulates TNF-alpha mRNA expression by Schwann cells but also may directly counteract TNF-alpha signaling pathways that lead to injury, chronic pain and/or death.     

Tissue inhibitors of metalloproteases strike a nerve

正Signals of touch,pressure,pain,temperature,position,and vibration are transmitted from the peripheral nervous system(PNS)to the dorsal horn of the segmental spinal cord via pseudounipolar dorsal root ganglia(DRG)neurons.Sensory information gathering relies on functional integrity of DRG neurons and can be interrupted by PNS injury due to trauma,     

Pleiotropic effects of intravascular haemolysis on vascular homeostasis

The breakdown of senescent or defective red blood cells releases red cell contents, especially haemoglobin, which scavenges nitric oxide (NO) and decomposes to haem and free iron. These are potent oxidants, all of which have promoted the evolution of inducible and vasculoprotective compensatory pathways to rapidly clear and detoxify haemoglobin, haem and iron. Chronic haemolytic red cell disorders as diverse as sickle cell disease, thalassaemia, unstable haemoglobinopathy, cytoskeletal defects and enzymopathies have been linked to a clinical constellation of pulmonary hypertension, priapism, leg ulceration and possibly cerebrovascular disease and thrombosis. Besides free haemoglobin, haemolysis has been associated with extracellular arginase that limits substrate availability to NO synthase, endogenous inhibitors of NO synthase activity, and inappropriate activation of haemostatic pathways. This article reviews the haemolytic disorders that have been reported to manifest vascular complications, and explores the speculative possibility that haemolysis mediates some of the vascular complications of inflammation and diabetes.     

Characterization of novel Mycobacterium tuberculosis pncA gene mutations in clinical isolates from the Ukraine

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis cases in the Ukraine are increasing. Pyrazinamide (PZA) is critically important for first- and second-line tuberculosis (TB) treatment regimes. However, PZA drug susceptibility testing is time consuming and technically challenging. The present study utilized Next-generation sequencing (NGS) to identify mutations in the pncA gene from clinical isolates and to assess the prevalence of pncA gene mutations in MDR/XDR-TB patients. Clinical isolates were inactivated in molecular transport media and shipped from Kharkiv, Ukraine, to San Antonio, TX. Whole-genome and targeted pncA gene sequencing was carried out using Illumina MiSeq instrumentation. Mutations were noted in 67 of 91 (74%) clinical isolates comprising substitutions, insertions, and deletions in the pncA coding and upstream promoter region. Of 45 mutation types, there were 11 novel, i.e., to date unknown, pncA mutations identified of which 3 were confirmed PZA resistant. Seven isolates contained mixed base mutations, whereas 4 harbored doubled mutations. Data reported here further support use of NGS for pncA gene characterization and may contribute in significant fashion to PZA therapy, especially in MDR- and XDR-TB patients.