Alveolar Clearance Research Articles

Aerosol Delivery of siRNA to the Lungs. Part 1: Rationale for Gene Delivery Systems.

This article reviews the pulmonary route of administration, aerosol delivery devices, characterization of pulmonary drug delivery systems, and discusses the rationale for inhaled delivery of siRNA. Diseases with known protein malfunctions may be mitigated through the use of siRNA therapeutics. The inhalation route of administration provides local delivery of siRNA therapeutics for the treatment of various pulmonary diseases, however barriers to pulmonary delivery and intracellular delivery of siRNA exists. siRNA loaded nanocarriers can be used to overcome the barriers associated with the pulmonary route, such as anatomical barriers, mucociliary clearance, and alveolar macrophage clearance. Apart from naked siRNA aerosol delivery, previously studied siRNA carrier systems comprise of lipidic, polymeric, peptide, or inorganic origin. Such siRNA delivery systems formulated as aerosols can be successfully delivered via an inhaler or nebulizer to the pulmonary region. Preclinical animal investigations of inhaled siRNA therapeutics rely on intratracheal and intranasal siRNA and siRNA nanocarrier delivery. Aerosolized siRNA delivery systems may be characterized using in vitro techniques, such as dissolution test, inertial cascade impaction, delivered dose uniformity assay, laser diffraction, and laser Doppler velocimetry. The ex vivo techniques used to characterize pulmonary administered formulations include the isolated perfused lung model. In vivo techniques like gamma scintigraphy, 3D SPECT, PET, MRI, fluorescence imaging and pharmacokinetic/pharmacodynamics analysis may be used for evaluation of aerosolized siRNA delivery systems. The use of inhalable siRNA delivery systems encounters barriers to their delivery, however overcoming the barriers while formulating a safe and effective delivery system will offer unique advances to the field of inhaled medicine.

Glycosylation-dependent activation of epithelial sodium channel by solnatide

Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

Update in Mechanical Ventilation, Sedation, and Outcomes 2014.

Novel approaches to the management of acute respiratory distress syndrome include strategies to enhance alveolar liquid clearance, promote epithelial cell growth and recovery after acute lung injury, and individualize ventilator care on the basis of physiological responses. The use of extracorporeal membrane oxygenation (ECMO) is growing rapidly, and centers providing ECMO must strive to meet stringent quality standards such as those set out by the ECMONet working group. Prognostic tools such as the RESP score can assist clinicians in predicting outcomes for patients with severe acute respiratory failure but do not predict whether ECMO will enhance survival. Evidence continues to grow that novel modes of mechanical ventilation such as neurally adjusted ventilatory assist are feasible and improve patient physiology and patient-ventilator interaction; data on clinical outcomes are limited but supportive. Critical illness causes long-term psychological and function sequelae: the risk of a new psychiatric diagnosis and severe physical impairment is significantly increased in the months after discharge from the intensive care unit. These long-term effects might be amenable to changes in sedation practice and increased early mobilization. Daily sedation discontinuation enhances the validity of routine delirium assessment. Many critically ill patients merit assessment by palliative care clinicians; the demand for palliative care services among critically ill patients is expected to grow. Future trials to test therapies for critical illness must ensure that study designs are adequately powered to detect benefit using realistic event rates. Integrating "big data" approaches into treatment decisions and trial designs offers a potential means of individualizing care to enhance outcomes for critically ill patients.

IL-1α induces CD11b(low) alveolar macrophage proliferation and maturation during granuloma formation.

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL-1-deficient mice, we found that the absence of IL-1α, but not IL-1β, was associated with reduced CD11b(high) phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL-1α(-/-) mice with recombinant IL-1α restored lung clearance functions and the pulmonary accumulation of CD11b(high) phagocytic macrophages. Mechanistically, IL-1α induced the proliferation of CD11b(low) alveolar macrophages and differentiated these cells into CD11b(high) macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL-1α triggers lung responses requiring macrophage proliferation and maturation from tissue-resident macrophages.

Importance of bacterial replication and alveolar macrophage-independent clearance mechanisms during early lung infection with Streptococcus pneumoniae.

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Electroporation-mediated gene delivery of Na+,K+ -ATPase, and ENaC subunits to the lung attenuates acute respiratory distress syndrome in a two-hit porcine model.

Acute respiratory distress syndrome (ARDS) is a common cause of organ failure with an associated mortality rate of 40%. The initiating event is disruption of alveolar-capillary interface causing leakage of edema into alveoli. Electroporation-mediated gene delivery of epithelial sodium channel (ENaC) and Na+,K+ -ATPase into alveolar cells would improve alveolar clearance of edema and attenuate ARDS. Pigs were anesthetized and instrumented, and the superior mesenteric artery was clamped to cause gut ischemia/reperfusion injury and peritoneal sepsis by fecal clot implantation. Animals were ventilated according to ARDSnet protocol. Four hours after injury, animals were randomized into groups: (i) treatment: Na+,K+ -ATPase/ENaC plasmid (n = 5) and (ii) control: empty plasmid (n = 5). Plasmids were delivered to the lung using bronchoscope. Electroporation was delivered using eight-square-wave electric pulses across the chest. Following electroporation, pigs were monitored 48 h. The Pao2/Fio2 ratio and lung compliance were higher in the treatment group. Lung wet/dry ratio was lower in the treatment group. Relative expression of the Na+,K+ -ATPase transgene was higher throughout lungs receiving treatment plasmids. Quantitative histopathology revealed a reduction in intra-alveolar fibrin in the treatment group. Bronchoalveolar lavage showed increased surfactant protein B in the treatment group. Survival was improved in the treatment group. Electroporation-mediated transfer of Na+,K+ -ATPase/ENaC plasmids improved lung function, reduced fibrin deposits, decreased lung edema, and improved survival in a translational porcine model of ARDS. Gene therapy can attenuate ARDS pathophysiology in a high-fidelity animal model, suggesting a potential new therapy for patients.

0992. Effects of inhaled aerosolized insulin on acutely injured lungs under normoglycemia: insulin may contribute to enhance alveolar liquid clearance through epithelial sodium channel expression

We have previously shown that hyperglycemia enhances inflammatory responses and that aerosolized insulin ameliorates inflammatory responses compared to identical doses of intravenous insulin in an experimental model of acute lung injury [1].

A novel tumor necrosis factor-mediated mechanism of direct epithelial sodium channel activation.

Alveolar liquid clearance is regulated by Na(+) uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na(+)-K(+)-ATPase in type II alveolar epithelial cells. Dysfunction of these Na(+) transporters during pulmonary inflammation can contribute to pulmonary edema. In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na(+) uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na(+) uptake stimulatory activity. TIP peptide directly activates ENaC, but not the Na(+)-K(+)-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance

Pseudomonas aeruginosa infection is a hallmark of lung disease in cystic fibrosis. Acute infection with P. aeruginosa profoundly inhibits alveolar macrophage clearance of apoptotic cells (efferocytosis) via direct effect of virulence factors. During chronic infection, P. aeruginosa evades host defense by decreased virulence, which includes the production or, in the case of mucoidy, overproduction of alginate. The impact of alginate on innate immunity, in particular on macrophage clearance of apoptotic cells is not known.We hypothesized that P. aeruginosa strains that exhibit reduced virulence impair macrophage clearance of apoptotic cells and we investigated if the polysaccharide alginate produced by mucoid P. aeruginosa is sufficient to inhibit alveolar macrophage efferocytosis.Rat alveolar or human peripheral blood monocyte (THP-1)-derived macrophage cell lines were exposed in vitro to exogenous alginate or to wild type or alginate-overproducing mucoid P. aeruginosa prior to challenge with apoptotic human Jurkat T-lymphocytes. The importance of LPS contamination and that of structural integrity of alginate polymers was tested using alginate of different purities and alginate lyase, respectively.Alginate inhibited alveolar macrophage efferocytosis in a dose- and time-dependent manner. This effect was augmented but not exclusively attributed to lipopolysaccharide (LPS) present in alginates. Alginate-producing P. aeruginosa inhibited macrophage efferocytosis by more than 50%. A mannuronic-specific alginate lyase did not restore efferocytosis inhibited by exogenous guluronic-rich marine alginate, but had a marked beneficial effect on efferocytosis of alveolar macrophages exposed to mucoid P. aeruginosa.Despite decreased virulence, mucoid P. aeruginosa may contribute to chronic airway inflammation through significant inhibition of alveolar clearance of apoptotic cells and debris. The mechanism by which mucoid bacteria inhibit efferocytosis may involve alginate production and synergy with LPS, suggesting that alginate lyase may be an attractive therapeutic approach to airway inflammation in cystic fibrosis and other chronic obstructive pulmonary diseases characterized by P. aeruginosa colonization.

Ventilator-Associated Pneumonia During Weaning From Mechanical Ventilation: Role of Fluid Management

Pulmonary edema may alter alveolar bacterial clearance and infectivity. Manipulation of fluid balance aimed at reducing fluid overload may, therefore, influence ventilator-associated pneumonia (VAP) occurrence in intubated patients. The objective of the present study was to assess the impact of a depletive fluid-management strategy on ventilator-associated complication (VAC) and VAP occurrence during weaning from mechanical ventilation.We used data from the B-type Natriuretic Peptide for the Fluid Management of Weaning (BMW) randomized controlled trial performed in nine ICUs across Europe and America. We compared the cumulative incidence of VAC and VAP between the biomarker-driven, depletive fluid-management group and the usual-care group during the 14 days following randomization, using specific competing-risk methods (the Fine and Gray model).Among the 304 patients analyzed, 41 experienced VAP, including 27 (17.8%) in the usual-care group vs 14 (9.2%) in the interventional group (P = .03). From the Fine and Gray model, the probabilities of VAC and VAP occurrence were both significantly reduced with the interventional strategy while adjusting for weaning outcome as a competing event (subhazard ratios [25th-75th percentiles], 0.44 [0.22-0.87], P = .02 and 0.50 [0.25-0.96], P = .03, respectively).Using proper competing risk analyses, we found that a depletive fluid-management strategy, when initiating the weaning process, has the potential for lowering VAP risk in patients who are mechanically ventilated.ClinicalTrials.gov; No.: NCT00473148; URL: www.clinicaltrials.gov.

Clearance of carbon nanotubes in the human respiratory tract-a theoretical approach.

Theoretical knowledge of carbon nanotube clearance in the human respiratory tract represents an essential contribution to the risk assessment of artificial airborne nanomaterials. Thus, single phases of nanotube clearance were simulated with the help of a theoretical model. In this study, clearance of single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT) was simulated by using a validated mathematical approach that includes all clearance mechanisms known hitherto. Fast mucociliary clearance is approximated by a steady-state steady-flow mucus model, whereas slow clearance mechanisms are modeled by definition of related clearance half-times. Clearance may be subdivided into three phases, including fast bronchial clearance (mucociliary escalator), slow bronchial clearance (particle uptake by airway macrophages, transcytosis), and alveolar clearance (phagocytosis by alveolar macrophages, endocytosis by alveolar epithelium). According to the clearance model used in this study, mucociliary clearance is completed within the first 24 h after exposure, whereas slow bronchial clearance is characterized by a half-time of 5 d. Alveolar clearance is marked by half-times >100 d. As a result of their different deposition patterns, SWCNT and MWCNT show some discrepancies with regard to their clearance insofar as long SWCNT reside significantly longer in the lungs than MWCNT. This circumstance is among other expressed by higher 24-h, 10-d, and 100-d retentions computed for SWCNT compared to MWCNT. Due to their partly high residence times in distal lung regions, carbon nanotubes may bear the potential to act as triggers of inflammatory reactions or fibrotic modifications of the lung structure. Further they may also induce malignant transformations of lung cells, resulting in the development of lung tumours.

The role ofTc‐99mDTPAaerosol scintigraphy in the differential diagnosis ofCOPDand asthma

Chronic obstructive lung disease (COPD) and asthma are characterized as similar to each other in causing airway obstruction and being an inflammatory process. The purpose of this study was to investigate whether technetium-99m diethylenetriaminepentaacetic acid ((99m) Tc-DTPA) aerosol scintigraphy could be used in the differential diagnosis of asthma and COPD. Eighty-four patients (male/female: 32/52; mean age 50.2 ± 12.7 years) with obstructive lung disease and 30 healthy volunteers as the control group were enrolled in the study. The patients were divided into two groups as COPD and asthma and also smoking subgroups. Alveolar clearance study was performed using a radiolabeled aerosol of (99m) Tc-DTPA. Mucociliary clearance was evaluated with T½ , cap value and penetration index parameters. All patient underwent pulmonary function tests and Forced expiratory volume (FEV1 ), forced vital capacity (FVC) and FEV1 /FVC parameters were obtained. The mean of T½ values of (99m) Tc-DTPA aerosol and FEV1 /FVC value among spirometric tests of the nonsmoking COPD patients were significantly lower than nonsmoking asthma patients (46.1 ± 14.3, 62.3 ± 18.7, P = 0.02; 65.2 ± 10.8, 81.4 ± 16.5, P = 0.04, respectively). The cap value was significantly higher in nonsmoking COPD patients (1.21 ± 0.49, 0.76 ± 0.22, P = 0.03). While there were no statistically and significantly different between control and asthmatic groups at the scintigraphic parameters and spirometric parameters, the mean of T½ values, cap value and spirometric parameters were statistically different between control and COPD groups (P < 0.05). We showed that assessment of mucociliary permeability with (99m) Tc-DTPA aerosol scintigraphy was a useful, easy to apply and a noninvasive technique to use in the differential diagnosis of nonsmoker COPD and asthma.

Assessment of ventilation inhomogeneity during mechanical ventilation using a rapid-response oxygen sensor-based oxygen washout method.

PurposeVentilatory inhomogeneity indexes in critically ill mechanically ventilated patients could be of importance to optimize ventilator settings in order to reduce additional lung injury. The present study compared six inhomogeneity indexes calculated from the oxygen washout curves provided by the rapid oxygen sensor of the LUFU end-expiratory lung volume measurement system.MethodsInhomogeneity was tested in a porcine model before and after induction of acute lung injury (ALI) at four different levels of positive end-expiratory pressure (PEEP; 15, 10, 5 and 0 cm H2O). The following indexes were assessed: lung clearance index (LCI), mixing ratio, Becklake index, multiple breath alveolar mixing inefficiency, moment ratio and pulmonary clearance delay.ResultsLCI, mixing ratio, Becklake index and moment ratio were comparable with previous reported values and showed acceptable variation coefficients at baseline with and without ALI. Moment ratio had the highest precision, as calculated by the variation coefficients. LCI, Becklake index and moment ratio showed comparable increases in inhomogeneity during decremental PEEP steps before and after ALI.ConclusionsThe advantage of the method we introduce is the combined measurement of end-expiratory lung volume (EELV) and inhomogeneity of lung ventilation with the LUFU fast-response medical-grade oxygen sensor, without the need for external tracer gases. This can be combined with conventional breathing systems. The moment ratio and LCI index appeared to be the most favourable for integration with oxygen washout curves as judged by high precision and agreement with previous reported findings. Studies are under way to evaluate the indexes in critically ill patients.

Reflection of stem cell therapy: An epilogue to the ‘Stem cells and the lung’ review series

Reflection of stem cell therapy: An epilogue to the ‘Stem cells and the lung’ review series

Disruption of airway lymphatics as a novel cause of impairment of airway clearance in severe asthma

Background We previously reported the phenotypic distribution patterns of airway smooth muscles in severe asthmatics; with smooth muscle bundle thickening only in large airways and in whole airways [1] , by different mechanisms, hypertrophy and/or hyperplasia [2], which was reexamined recently to fit in 55 fatal asthmatics by James AL, et al. [3]. We also revealed that pulmonary lymphatics distributed in interlobular septa and subpleural lesions were destroyed by increased fibrosis in patients with idiopathic pulmonary fibrosis which would impair alveolar clearance [4] . In this context, we were interested in the alteration of airway lymphatics in severe asthmatics and hypothesized that increased smooth muscle bundles and fibrosis in the airway walls would disrupt airway lymphatics and impair airway clearance in these patients.

AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride-sensitive Na+ current in primary dog, pig and rat alveolar type II cells

Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung conditions, such as ALI and ARDS. Apart from ventilation strategies, no specific therapy yet exists for treatment of these potentially fatal illnesses. The oedema-reducing capacity of the lectin-like domain of TNF (TIP) and of synthetic peptides, mTIP and hTIP, which mimic the TIP domain of mouse and human TNF, have been demonstrated in various studies in rodents. Cell-based electrophysiological studies have revealed that the alveolar fluid clearing capacity of TNF and the TIP peptides is due to activation of the amiloride-sensitive Na+ current in alveolar epithelial cells and that the primary site of action is on the apical side of these cells.AP301, a synthetic cyclic peptide mimicking the TIP domain of human TNF is currently undergoing clinical trials as a therapy for pulmonary permeability oedema. AP301 has been shown to improve alveolar liquid clearance and lung function in a porcine model of ALI.For non-clinical regulatory assessment, dog, pig and rat are standard animal models; accordingly, pre-clinical toxicological and pharmacological safety studies have been conducted with AP301 in dogs and rats. Hitherto, no studies have assessed the pharmacodynamic effect of AP301 on primary canine or porcine type II AEC. The current study describes the effect of AP301 on the amiloride-sensitive Na+ current in type II AEC isolated from dog, pig and rat lungs. In whole cell patch clamp experiments with dog type II AEC, an increase in the amiloride-sensitive Na+ current from 3.7 pA to 49.4 pA was observed in the presence of AP301; in pig type II AEC, an increase from 10.0 pA to 159.6 pA was observed, and in rat AEC, from 6.9 pA to 62.4 pA. In whole cell patch clamp experiments in A549 cells, AP301-induced enhancement of the amiloride-sensitive current was eliminated when Na+ in the bath solution was replaced with N-methyl-d-glucamine (NMDG), and when the cells were pre-incubated with 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn2+ or l-cis-diltiazem prior to AP301.These results provide strong evidence that AP301 activates the amiloride-sensitive Na+ current through ENaC in type II AEC from dog, pig and rat. To our knowledge, this is the first cell-based analysis of the oedema-clearing effect of AP301 observed in the porcine model of pulmonary oedema. Furthermore, the results validate the dog and pig models in non-clinical assessment of AP301.

A Computational Study of the Oligosaccharide Binding Sites in the Lectin-Like Domain of Tumor Necrosis Factor and the TNF-derived TIP Peptide

The lectin-like domain of Tumor Necrosis Factor (TNF), mimicked by the TIP peptide, activates amiloride-sensitive sodium uptake in type II alveolar epithelial cells and as such increases alveolar liquid clearance in dysfunctional lungs. This protective effect is blunted upon mutation of residues T105, E107 and E110 in human TNF into alanine or upon pre-incubation of the cytokine with the disaccharide N,N'-diacetylchitobiose. In this study, we used molecular docking and molecular dynamics simulation to predict the binding sites for N,N'-diacetylchitobiose and trimannose-O-ethyl in the lectin-like domain of TNF and in the TIP peptide. Specific sites (K98, S99, P100, Q102 and E116) in the three loops of the lectin-like domain provide specific binding for both oligosaccharides, but none of the residues crucial for anti-edema activity are involved in hydrogen bonding with oligosaccharides or are subjected to steric hindrance by them. These results thus suggest that neither chitobiose nor trimannose affect crucial amino acids, while they occupy the cavity in the lectin-like domain. Consequently, both crucial amino acids and the emptiness of the cavity in the lectin-like domain may be critical for TNF's lectin-like activity. Analogously, the R4, E5, P7, Y16 amino acids of the TIP peptide are involved in forming hydrogen bonds with both oligosaccharides, whereas residues T6, E8 and E11 (corresponding to T105, E107 and E110 in hTNF) play an important role in stabilizing the peptide-oligosaccharide complex, supporting the hypothesis that amino acids in the polar region (TPEGAE) of the TIP peptide represent only a partial binding motif for sugars.

Influenza-virus infection impacts on alveolar edema clearance: role of virus- and host factors in regulation of alveolar epithelial cell ENaC and Na,K-ATPase expression

Influenza A viruses (IV) can cause primary viral pneumonia in humans resulting in acute lung injury (ALI/ARDS) with fatal outcome. One of the hallmarks of IV-induced ALI is the accumulation of edema fluid in the alveolar compartment, which under normal conditions is cleared along a sodium gradient established by basolateral Na,K-ATPase and the apical epithelial sodium channel (ENaC). IV-infection was found to be associated with a decrease in alveolar fluid clearance (AFC). Therefore we aim to investigate the regulation of ENaC and Na,K-ATPase in IV-infection by viral and host factors to characterize the molecular mechanisms affecting AFC.

Ubiquitination-driven down-regulation of the cell adhesion molecule Na,K-ATPase β-subunit by hypercapnia in alveolar epithelial cells

Acute lung injury (ALI) is associated with alveolo-capillary barrier dysfunction leading to impaired gas exchange and thus elevated CO2 levels (hypercapnia). In addition to the well-studied role of the α-subunit of the Na,K-ATPase in alveolar edema fluid clearance during ALI, the Na,K-ATPase β-subunit located at the sites of cell-cell junction acts as a cell adhesion molecule and therefore might be an important player in the alveolar-capillary barrier repair. Here, we studied the effects of hypercapnia on the stability of Na,K-ATPase β-subunit and on cell junction formation. Exposing human alveolar epithelial A549 cells to elevated CO2 concentrations led to rapid ubiquitination of the Na,K-ATPase β-subunit in the plasma membrane and protein internalization, as assessed by cell-surface biotinylation. Both ubiquitination and endocytosis of the Na,K-ATPase β-subunit were prevented when Lys 5 and 7 were mutated to Arg, highlighting the critical role of these residues in targeting the Na,K-ATPase β-subunit by ubiquitin. After endocytosis, the Na,K-ATPase β-subunit underwent degradation, as evaluated by pulse-chase experiments and by blocking protein synthesis with cycloheximide and evaluating protein levels. The degradation of Na,K-ATPase β-subunit was prevented by inhibition of the proteasome system by MG-132, but not by inhibition of the lysosome by E-64 or chloroquine, suggesting that hypercapnia induces proteasome-mediated degradation of the Na,K-ATPase β-subunit. Similarly, the formation of cell-cell junctions, assessed by the hanging-drop technique, was markedly inhibited by elevated CO2 levels, which was completely prevented when the proteasome system was inhibited. Accordingly, we provide evidence that hypercapnia promotes ubiquitination, endocytosis and proteasomal degradation of the Na,K-ATPase β-subunit in alveolar epithelial cells. Additionally, we show that elevated CO2 concentrations inhibit alveolar epithelial cell junction formation, which may be mediated by surface instability of Na,K-ATPase β-subunit and may lead to persistent alveolar epithelial barrier dysfunction during ALI.

Lymphatic fluctuation in the parenchymal remodeling stage of acute interstitial pneumonia, organizing pneumonia, nonspecific interstitial pneumonia and idiopathic pulmonary fibrosis

Because the superficial lymphatics in the lungs are distributed in the subpleural, interlobular and peribroncovascular interstitium, lymphatic impairment may occur in the lungs of patients with idiopathic interstitial pneumonias (IIPs) and increase their severity. We investigated the distribution of lymphatics in different remodeling stages of IIPs by immunohistochemistry using the D2-40 antibody. Pulmonary tissue was obtained from 69 patients with acute interstitial pneumonia/diffuse alveolar damage (AIP/DAD, N = 24), cryptogenic organizing pneumonia/organizing pneumonia (COP/OP, N = 6), nonspecific interstitial pneumonia (NSIP/NSIP, N = 20), and idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP, N = 19). D2-40+ lymphatic in the lesions was quantitatively determined and associated with remodeling stage score. We observed an increase in the D2-40+ percent from DAD (6.66 ± 1.11) to UIP (23.45 ± 5.24, P = 0.008) with the advanced process of remodeling stage of the lesions. Kaplan-Meier survival curves showed a better survival for patients with higher lymphatic D2-40+ expression than 9.3%. Lymphatic impairment occurs in the lungs of IIPs and its severity increases according to remodeling stage. The results suggest that disruption of the superficial lymphatics may impair alveolar clearance, delay organ repair and cause severe disease progress mainly in patients with AIP/DAD. Therefore, lymphatic distribution may serve as a surrogate marker for the identification of patients at greatest risk for death due to IIPs.