A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Acute Tubular Injury is Associated With Severe Traumatic Brain Injury: in Vitro Study on Human Tubular Epithelial Cells.

Acute kidney injury following traumatic brain injury is associated with poor outcome. We investigated in vitro the effects of plasma of brain injured patients with acute tubular kidney injury on kidney tubular epithelial cell function. we performed a prospective observational clinical study in ICU in a trauma centre of the University hospital in Italy including twenty-three ICU patients with traumatic brain injury consecutively enrolled. Demographic data were recorded on admission: age 39 ± 19, Glasgow Coma Score 5 (3-8). Neutrophil Gelatinase-Associated Lipocalin and inflammatory mediators were measured in plasma on admission and after 24, 48 and 72 hours; urine were collected for immunoelectrophoresis having healthy volunteers as controls. Human renal proximal tubular epithelial cells were stimulated with patients or controls plasma. Adhesion of freshly isolated human neutrophils and trans-epithelial electrical resistance were assessed; cell viability (XTT assay), apoptosis (TUNEL staining), Neutrophil Gelatinase-Associated Lipocalin and Megalin expression (quantitative real-time PCR) were measured. All patients with normal serum creatinine showed increased plasmatic Neutrophil Gelatinase-Associated Lipocalin and increased urinary Retinol Binding Protein and α1-microglobulin. Neutrophil Gelatinase-Associated Lipocalin was significantly correlated with both inflammatory mediators and markers of tubular damage. Patient' plasma incubated with tubular cells significantly increased adhesion of neutrophils, reduced trans-epithelial electrical resistance, exerted a cytotoxic effect and triggered apoptosis and down-regulated the endocytic receptor Megalin compared to control. Plasma of brain injured patients with increased markers of subclinical acute kidney induced a pro-inflammatory phenotype, cellular dysfunction and apoptotic death in tubular epithelial cells.

Detection of Angiotensin II type I-receptor antibodies in transplant glomerulopathy.

BACKGROUND: Transplant glomerulopathy (TG) is an important cause of late graft loss. The role of angiotensin type 1-receptor antibodies (AT1 R-Ab) in TG is not known. METHODS: All the TG cases (N = 137) between January 2007 and December 2014 (N = 1410) were analyzed. Donor-specific anti-HLA antibodies (DSA) at the time of biopsy and AT1 R-Ab IgG (positive, >17 UI/mL; "at risk," 10-17 UI/mL; negative, <10 UI/mL) in pre-transplant sera (PT-Ab) and at biopsy time (BT-Ab) were studied. RESULTS: AT1 R-PT-Ab+ and AT1 R-BT-Ab+ patients were 16.5% (51.5% "at risk") and 11.5% (27.4% "at risk"), respectively. Clinical correlations were found between AT1 R-Ab and HCV infection, number of transplants, and age. Considering Banff scores, ptc was higher in DSA+ patients vs AT1 R-PT-Ab+ (P = 0.002) or AT1 R-BT-Ab+ (P = 0.001) without differences in g and chronicity score (ci + ct); cg showed lower scores in DSA+ patients vs AT1 R-BT-Ab+ (P = 0.001). Graft survival was not influenced by the presence of AT1 R-Ab, AT1-R-Ab titer or MFI, but we observed a longer graft survival in patients with both AT1 R-BT-Ab+ or "at risk" and DSA+ vs patients positive only for DSA (P = 0.02), for AT1 R-BT-Ab (P = 0.019) or AT1 R-BT-Ab "at risk" (P = 0.039). CONCLUSION: AT1 R-Ab showed no independent prognostic role in TG in this pilot analysis.

Intratracheal Administration of Small Interfering RNA Targeting Fas Reduces Lung Ischemia-Reperfusion Injury.

OBJECTIVES: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice weighing 28-30 g. INTERVENTIONS: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. MEASUREMENTS AND MAIN RESULTS: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. CONCLUSIONS: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation.

Cerebrospinal fluid from patients with subarachnoid haemorrhage and vasospasm enhances endothelin contraction in rat cerebral arteries.

INTRODUCTION: Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ETA and ETB receptors. METHODS: Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ETA and ETB receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured. RESULTS: Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ETA receptors and new expression of ETB receptors was apparent; 5) reduction in the enhanced response to endothelin after ETB blockade in the low range and after ETA blockade in the high range of endothelin concentrations; 6) after combined ETA and ETB blockade a complete inhibition of endothelin contraction was observed. CONCLUSIONS: Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ETA and ETB receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.