Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (deltaF508), causes severe CF and resides on 70% of mutant chromosomes. Severe CF is also caused by premature stop mutations, which are found on 5% of CF chromosomes. Here we report that two common, disease-associated stop mutations can be suppressed by treating cells with low doses of the aminoglycoside antibiotic G-418. Aminoglycoside treatment resulted in the expression of full-length CFTR and restored its cyclic AMP-activated chloride channel activity. Another aminoglycoside, gentamicin, also promoted the expression of full-length CFTR. These results suggest that treatment with aminoglycosides may provide a means of restoring CFTR function in patients with this class of mutation.
IntroductionHigh mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear binding protein that has recently been shown to be an early trigger of sterile inflammation in animal models of trauma-hemorrhage via the activation of the Toll-like-receptor 4 (TLR4) and the receptor for the advanced glycation endproducts (RAGE). However, whether HMGB1 is released early after trauma hemorrhage in humans and is associated with the development of an inflammatory response and coagulopathy is not known and therefore constitutes the aim of the present study.MethodsOne hundred sixty eight patients were studied as part of a prospective cohort study of severe trauma patients admitted to a single Level 1 Trauma center. Blood was drawn within 10 minutes of arrival to the emergency room before the administration of any fluid resuscitation. HMGB1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, von Willebrand Factor (vWF), angiopoietin-2 (Ang-2), Prothrombin time (PT), prothrombin fragments 1+2 (PF1+2), soluble thrombomodulin (sTM), protein C (PC), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and D-Dimers were measured using standard techniques. Base deficit was used as a measure of tissue hypoperfusion. Measurements were compared to outcome measures obtained from the electronic medical record and trauma registry.ResultsPlasma levels of HMGB1 were increased within 30 minutes after severe trauma in humans and correlated with the severity of injury, tissue hypoperfusion, early posttraumatic coagulopathy and hyperfibrinolysis as well with a systemic inflammatory response and activation of complement. Non-survivors had significantly higher plasma levels of HMGB1 than survivors. Finally, patients who later developed organ injury, (acute lung injury and acute renal failure) had also significantly higher plasma levels of HMGB1 early after trauma.ConclusionsThe results of this study demonstrate for the first time that HMGB1 is released into the bloodstream early after severe trauma in humans. The release of HMGB1 requires severe injury and tissue hypoperfusion, and is associated with posttraumatic coagulation abnormalities, activation of complement and severe systemic inflammatory response.
Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-1 (TGF-1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-1 on 22 Na ؉ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-1 significantly reduced the amiloride-sensitive fraction of 22 Na ؉ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-1 also significantly decreased ␣ENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the ␣ENaC promoter in lung epithelial cells. The inhibitory effect of TGF-1 on sodium uptake and ␣ENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane ␣ENaC expression mediated through an ERK1/2-dependent inhibition of the ␣ENaC promoter activity. Acute lung injury (ALI)1 is a devastating syndrome characterized by flooding of alveolar spaces with a protein-rich exudate that impairs pulmonary gas exchange, leading to arterial hypoxemia and respiratory failure (1). Epithelial injury can contribute to alveolar flooding, because the epithelial barrier is much less permeable under normal conditions than the endothelial barrier. Injury to alveolar epithelial cells can also disrupt normal epithelial fluid transport, impairing the removal of edema fluid from the alveolar space. Clinical studies have demonstrated that impaired alveolar fluid clearance is a characteristic feature of clinical lung injury (2, 3), but the mechanisms for this decrease in epithelial fluid transport have not been well worked out. The removal of edema fluid from the airspaces occurs via an active transport-dependent sodium concentration gradient across the distal lung epithelium. The ratelimiting step in the transport of fluid across the lung epithelium is the movement of sodium and chloride across the apical plasma membrane, specifically the movement of sodium through amiloride-sensitive and -insensitive channels (4). Among the sodium channels at the apical membrane of lung epithelial cells, amiloride-sensitive channels represent 50 -60% ...
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