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% ...

Abstract-Interleukin (IL)-1␤ has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1␤-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1␤ production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the ␣v␤6 integrin and/or transforming growth factor-␤ attenuated the IL-1␤-induced ALI. The results of in vitro studies indicated that IL-1␤ caused the activation of transforming growth factor-␤ via RhoA/␣v␤6 integrin-dependent mechanisms and the inhibition of the ␣v␤6 integrin and/or transforming growth factor-␤ signaling completely blocked the IL-1␤-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1␤ increased protein permeability across lung endothelial cell monolayers via RhoA-and ␣v␤5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the ␣v␤5 and ␣v␤6 integrins had an additive protective effect against IL-1␤-induced ALI. In summary, these results demonstrate a critical role for the ␣v␤5/␤6 integrins in mediating the IL-1␤-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI. Key Words: lung Ⅲ cytokines Ⅲ inflammation Ⅲ endothelial cells Ⅲ epithelial cells Ⅲ rodents A cute lung injury (ALI) is a devastating clinical syndrome in critically ill patients with an overall mortality rate of 30% to 40%. 1 The syndrome is characterized by alveolar epithelial and lung endothelial injury leading to increased permeability across the alveolar-capillary barrier, pulmonary edema, and acute respiratory failure. 2 Despite an improved understanding of the pathogenesis of ALI in recent years, the molecular steps regulating the development of increased lung endothelial and epithelial permeability remain poorly understood, and no specific pharmacological therapies are currently available.During the early phase of ALI, a variety of inflammatory mediators are released into the distal air spaces. 2 Among those, interleukin (IL)-1␤ has been shown to be among the most biologically active cytokines in the lungs early after the onset of ALI. 3-5 Furthermore, IL-1␤ stimulates the production of a variety of chemokines (eg, IL-8, monocyte chemotactic protein [MCP]-1, and macrophage inflammatory protein [MIP]-1␣) 6 involved in epithelial wound repair 7,8 and is a potent inducer of lung fibrosis. 9,10 It has been previously shown in rats that lung vascular permeability increases after short-term exposure of IL-1␣ and IL-1␤ protein when ...

Hsp 72 can be detected in the serum of severely traumatized patients within 30 minutes after injury. Elevated initial serum levels of Hsp 72 (serum levels > 15 ng/mL) are associated with survival after severe trauma, but are not related to the incidence or severity of the postinjury inflammatory response or organ dysfunction.

We examined the relation between Cl current (Icl) stimulation and cell membrane capacitance (Cm) when cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in Xenopus oocytes. ICl and Cm increased in parallel when oocytes expressing CFTR were stimulated by forskolin (10 microM) and 3-isobutyl-1-methylxanthine (1 mM). The adenosine 3',5'-cyclic monophosphate (cAMP)-induced increase in surface area detected by Cm was confirmed by morphometry in the same oocytes used for the electrical recordings. These increases in ICl and Cm were reversible and were absent from control oocytes not injected with CFTR cRNA. The time to reach peak ICl lagged slightly behind the peak in Cm. ICl was varied by altering CFTR expression level or agonist dose or by expressing different CFTR mutants. In all cases, there was a close correlation between ICl and Cm, and the kinetics of ICl and Cm stimulation were more rapid the larger the magnitude of the stimulated current. The Cm-ICl relation for wild-type CFTR saturated, consistent with a limited capacity of cells to increase their surface area. These results indicate that stimulation of the CFTR ICl is linked closely to increases in membrane area. This suggests that CFTR is present in the membrane vesicles whose insertion is stimulated by cAMP. The contents of these vesicles may provide a link between activation of CFTR and its cAMP-dependent regulation of other channels.

SUMMARYSynthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed ⌬F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of ⌬F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the ⌬F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that ⌬F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact ⌬F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted ⌬F508 CFTR maturation and function. S-Nitrosoglutathione-corrected ⌬F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting ⌬F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize ⌬F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why ⌬F508 homozygous patients do not present with kidney dysfunction.