The Antibacterial and Anti-inflammatory Activity of Chicken Cathelicidin-2 combined with Exogenous Surfactant for the Treatment of Cystic Fibrosis-Associated Pathogens.

Cystic fibrosis (CF) is characterized by recurrent airway infections with antibiotic-resistant bacteria and chronic inflammation. Chicken cathelicin-2 (CATH-2) has been shown to exhibit antimicrobial activity against antibiotic-resistant bacteria and to reduce inflammation. In addition, exogenous pulmonary surfactant has been suggested to enhance pulmonary drug delivery. It was hypothesized that CATH-2 when combined with an exogenous surfactant delivery vehicle, bovine lipid extract surfactant (BLES), would exhibit antimicrobial activity against CF-derived bacteria and downregulate inflammation. Twelve strains of CF-pathogens were exposed to BLES+CATH-2 in vitro and killing curves were obtained to determine bactericidal activity. Secondly, heat-killed bacteria were administered in vivo to elicit a pro-inflammatory response with either a co-administration or delayed administration of BLES+CATH-2 to assess the antimicrobial-independent, anti-inflammatory properties of BLES+CATH-2. CATH-2 alone exhibited potent antimicrobial activity against all clinical strains of antibiotic-resistant bacteria, while BLES+CATH-2 demonstrated a reduction, but significant antimicrobial activity against bacterial isolates. Furthermore, BLES+CATH-2 reduced inflammation in vivo when either co-administered with killed bacteria or after delayed administration. The use of a host-defense peptide combined with an exogenous surfactant compound, BLES+CATH-2, is shown to exhibit antimicrobial activity against antibiotic-resistant CF bacterial isolates and reduce inflammation.

Lung Lavage and Surfactant Replacement During Ex Vivo Lung Perfusion for Treatment of Gastric Acid Aspiration-Induced Donor Lung Injury.

BACKGROUND: Ex vivo lung perfusion (EVLP) provides opportunities to treat injured donor lungs before transplantation. We investigated whether lung lavage, to eliminate inflammatory inhibitory components, followed by exogenous surfactant replacement, could aid lung recovery and improve post-transplant lung function after gastric aspiration injury. METHODS: Gastric acid aspiration was induced in donor pigs, which were ventilated for 6 hours to develop lung injury. After retrieval and 10 hours of cold preservation, EVLP was performed for 6 hours. The lungs were randomly divided into 4 groups (n = 5, each): (1) no treatment (control), (2) lung lavage, (3) surfactant administration, and (4) lung lavage, followed by surfactant administration. After another 2-hour period of cold preservation, the left lung was transplanted and reperfused for 4 hours. RESULTS: Physiologic lung function significantly improved after surfactant administration during EVLP. The EVLP perfusate from the lavage + surfactant group showed significantly lower levels of interleukin (IL)-1ß, IL-6, IL-8, and secretory phospholipase A2. Total phosphatidylcholine was increased, and minimum surface tension was recovered to normal levels (≤5 mN/m) in the bronchioalveolar fluid after surfactant administration. Lysophosphatidylcholine in bronchioalveolar fluid was significantly lower in the lavage + surfactant group than in the surfactant group. Post-transplant lung function was significantly better in the lavage + surfactant group compared with all other groups. CONCLUSIONS: Lung lavage, followed by surfactant replacement during EVLP, reduced inflammatory mediators and prevented hydrolysis of phosphatidylcholine, which contributed to the superior post-transplant function in donor lungs with aspiration injury.

A respiratory-gated micro-CT comparison of respiratory patterns in free-breathing and mechanically ventilated rats.

In this study, we aim to quantify the differences in lung metrics measured in free-breathing and mechanically ventilated rodents using respiratory-gated micro-computed tomography. Healthy male Sprague-Dawley rats were anesthetized with ketamine/xylazine and scanned with a retrospective respiratory gating protocol on a GE Locus Ultra micro-CT scanner. Each animal was scanned while free-breathing, then intubated and mechanically ventilated (MV) and rescanned with a standard ventilation protocol (56 bpm, 8 mL/kg and PEEP of 5 cm H2O) and again with a ventilation protocol that approximates the free-breathing parameters (88 bpm, 2.14 mL/kg and PEEP of 2.5 cm H2O). Images were reconstructed representing inspiration and end expiration with 0.15 mm voxel spacing. Image-based measurements of the lung lengths, airway diameters, lung volume, and air content were compared and used to calculate the functional residual capacity (FRC) and tidal volume. Images acquired during MV appeared darker in the airspaces and the airways appeared larger. Image-based measurements showed an increase in lung volume and air content during standard MV, for both respiratory phases, compared with matched MV and free-breathing. Comparisons of the functional metrics showed an increase in FRC for mechanically ventilated rats, but only the standard MV exhibited a significantly higher tidal volume than free-breathing or matched MV Although standard mechanical ventilation protocols may be useful in promoting consistent respiratory patterns, the amount of air in the lungs is higher than in free-breathing animals. Matching the respiratory patterns with the free-breathing case allowed similar lung morphology and physiology measurements while reducing the variability in the measurements.

Differences in hyperpolarized (3) He ventilation imaging after 4 years in adults with cystic fibrosis.

PURPOSE: To evaluate cystic fibrosis (CF) subjects over 4 years using (3) He magnetic resonance imaging (MRI), pulmonary function tests, and track hospitalization and physician visits. MATERIALS AND METHODS: Five CF adults provided written informed consent to an approved protocol and underwent MRI, spirometry, and plethysmography at baseline, 7 days, and 4 ± 1 years later. (3) He MRI ventilation defect percent (VDP) was generated for all subjects and timepoints. RESULTS: After 4 years, mean forced expiratory volume in 1 second / forced vital capacity (FEV1 /FVC) was lower (P = 0.01) in all subjects and there were no other pulmonary function test changes. Two CF adults showed significantly elevated (worse) (3) He VDP at baseline and after 4 years they had significantly greater (worsened) VDP (P = 0.02), without a significant FEV1 decline (P = 0.06) but with a greater number of exacerbations (P < 0.05). Baseline VDP strongly correlated with FEV1 (r(2) = 0.98, P = 0.0007) at 4-year follow-up. CONCLUSION: For two CF subjects, VDP was significantly worse at baseline and worsened over 4 years, which was in agreement with a greater number of hospitalizations and clinic visits. These results are limited by the very small sample size, but the strong VDP correlation with longitudinal changes in FEV1 generates the hypothesis that abnormal VDP may temporally precede FEV1 decline in CF subjects; this must be tested in a larger CF study.

The effects of exogenous surfactant administration on ventilation-induced inflammation in mouse models of lung injury.

BACKGROUND: Mechanical ventilation (MV) is an essential supportive therapy for acute lung injury (ALI); however it can also contribute to systemic inflammation. Since pulmonary surfactant has anti-inflammatory properties, the aim of the study was to investigate the effect of exogenous surfactant administration on ventilation-induced systemic inflammation. METHODS: Mice were randomized to receive an intra-tracheal instillation of a natural exogenous surfactant preparation (bLES, 50 mg/kg) or no treatment as a control. MV was then performed using the isolated and perfused mouse lung (IPML) set up. This model allowed for lung perfusion during MV. In experiment 1, mice were exposed to mechanical ventilation only (tidal volume =20 mL/kg, 2 hours). In experiment 2, hydrochloric acid or air was instilled intra-tracheally four hours before applying exogenous surfactant and ventilation (tidal volume =5 mL/kg, 2 hours). RESULTS: For both experiments, exogenous surfactant administration led to increased total and functional surfactant in the treated groups compared to the controls. Exogenous surfactant administration in mice exposed to MV only did not affect peak inspiratory pressure (PIP), lung IL-6 levels and the development of perfusate inflammation compared to non-treated controls. Acid injured mice exposed to conventional MV showed elevated PIP, lung IL-6 and protein levels and greater perfusate inflammation compared to air instilled controls. Instillation of exogenous surfactant did not influence the development of lung injury. Moreover, exogenous surfactant was not effective in reducing the concentration of inflammatory cytokines in the perfusate. CONCLUSIONS: The data indicates that exogenous surfactant did not mitigate ventilation-induced systemic inflammation in our models. Future studies will focus on altering surfactant composition to improve its immuno-modulating activity.

Recombinant surfactant protein C-based surfactant for patients with severe direct lung injury.

RATIONALE: Patients with acute lung injury have impaired function of the lung surfactant system. Prior clinical trials have shown that treatment with exogenous recombinant surfactant protein C (rSP-C)-based surfactant results in improvement in blood oxygenation and have suggested that treatment of patients with severe direct lung injury may decrease mortality. OBJECTIVES: Determine the clinical benefit of administering an rSP-C-based synthetic surfactant to patients with severe direct lung injury due to pneumonia or aspiration. METHODS: A prospective randomized blinded study was performed at 161 centers in 22 countries. Patients were randomly allocated to receive usual care plus up to eight doses of rSP-C surfactant administered over 96 hours (n = 419) or only usual care (n = 424). MEASUREMENTS AND MAIN RESULTS: Mortality to 28 days after treatment, the requirement for mechanical ventilation, and the number of nonpulmonary organ failure-free days were not different between study groups. In contrast to prior studies, there was no improvement in oxygenation in patients receiving surfactant compared with the usual care group. Investigation of the possible reasons underlying the lack of efficacy suggested a partial inactivation of rSP-C surfactant caused by a step of the resuspension process that was introduced with this study. CONCLUSIONS: In this study, rSP-C-based surfactant was of no clinical benefit to patients with severe direct lung injury. The unexpected lack of improvement in oxygenation, coupled with the results of in vitro tests, suggest that the administered suspension may have had insufficient surface activity to achieve clinical benefit.

Surfactant protein-A reduces translocation of mediators from the lung into the circulation.

The objective of this study was to characterize a mouse model of lung inflammation and determine the effect of surfactant protein A (SP-A, or sftpa) on the transfer of inflammatory mediators from these injured lungs into the systemic circulation. Lung inflammation was induced in either sftpa-deficient (-/-) or wild-type (+/+) spontaneously breathing, adult mice via intranasal lipopolysaccharide (LPS). Four hours later, lungs were isolated, perfused, and mechanically ventilated for 2 hours. Perfusate was collected for analysis over the duration of ventilation and lung lavage was obtained in groups of animals immediately before and after mechanical ventilation (MV). Lavage analysis showed an increase in interleukin-6 (IL6) and tumor necrosis factor-alpha (TNFalpha) 4 hours after LPS, with a further increase in IL6 following MV. LPS and MV also caused an increase in total cell and neutrophil numbers as well as total protein in the lavage compared to controls. Perfusate analysis revealed a significant increase in IL6 and TNFalpha after LPS and MV, with significantly greater levels of these mediators in sftpa (-/-) versus (+/+) mice. The authors conclude that LPS followed by MV resulted in lung inflammation and injury, and that SP-A significantly influenced inflammatory mediator release from these inflamed lungs into the perfusate.

Elevated endogenous surfactant reduces inflammation in an acute lung injury model.

Acute lung injury (ALI) is associated with severe pulmonary inflammation and alterations to surfactant, and often results in overwhelming systemic inflammation, leading to multiple organ failure. The objective of this study was to determine the effect of increased endogenous surfactant pools on pulmonary and systemic inflammation in a model of lipopolysaccharide (LPS)-induced ALI. Mice received an instillation of liposome-encapsulated (i) dichloromethylene diphosphonic acid (DMDP) to increase surfactant pools via depletion of alveolar macrophages, or (ii) phosphate-buffered saline (PBS). Seven days after instillation, mice received an intranasal administration of LPS or saline. Following a 4-hour recovery period, mice were sacrificed and their lungs were isolated, mechanically ventilated, and perfused with 8 mL of recirculated perfusate through the pulmonary circulation for 2 hours. Perfusate and lavage fluid were collected for analysis of inflammatory mediators. Lavage analysis revealed a 5-fold increase in surfactant pools in DMDP-treated mice compared to PBS-treated controls. Lavage and perfusate analyses showed significant decreases in the concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1alpha, and IL-1beta cytokines in DMDP-LPS mice compared to PBS-LPS controls. Elevated endogenous surfactant pools are protective against both LPS- and mechanical ventilation-induced inflammation, in addition to inflammation associated with the combination of these two insults.

A Search for subgroups of patients with ARDS who may benefit from surfactant replacement therapy: a pooled analysis of five studies with recombinant surfactant protein-C surfactant (Venticute).

BACKGROUND: Studies to date have shown no survival benefit for the use of exogenous surfactant to treat patients with the ARDS. To identify specific patient subgroups for future study, we performed an exploratory post hoc analysis of clinical trials of recombinant surfactant protein-C (rSP-C) surfactant (Venticute; Nycomed GmbH; Konstanz, Germany). METHODS: We performed a pooled analysis of all five multicenter studies in which patients with ARDS due to various predisposing events were treated with rSP-C surfactant. Patients received either usual care (n = 266) or usual care plus up to four intratracheal doses (50 mg/kg) of rSP-C surfactant (n = 266). Factors influencing the study end points were analyzed using descriptive statistics, analysis of covariance, and logistic regression models. RESULTS: ARDS was most often associated with pneumonia or aspiration, sepsis, and trauma or surgery. For the overall patient population, treatment with rSP-C surfactant significantly improved oxygenation (p = 0.002) but had no effect on mortality (32.6%). Multivariate analysis showed age and acute physiology and chronic health evaluation (APACHE) II score to be the strongest predictors of mortality. In the subgroup of patients with severe ARDS due to pneumonia or aspiration, surfactant treatment was associated with markedly improved oxygenation (p = 0.0008) and improved survival (p = 0.018). CONCLUSIONS: rSP-C surfactant improved oxygenation in patients with ARDS irrespective of the predisposition. Post hoc evidence of reduced mortality associated with surfactant treatment was obtained in patients with severe respiratory insufficiency due to pneumonia or aspiration. Those patients are the focus of a current randomized, blinded, clinical trial with rSP-C surfactant.

Alterations to surfactant precede physiological deterioration during high tidal volume ventilation.

Lung injury due to mechanical ventilation is associated with an impairment of endogenous surfactant. It is unknown whether this impairment is a consequence of or an active contributor to the development and progression of lung injury. To investigate this issue, the present study addressed three questions: Do alterations to surfactant precede physiological lung dysfunction during mechanical ventilation? Which components are responsible for surfactant's biophysical dysfunction? Does exogenous surfactant supplementation offer a physiological benefit in ventilation-induced lung injury? Adult rats were exposed to either a low-stretch [tidal volume (Vt) = 8 ml/kg, positive end-expiratory pressure (PEEP) = 5 cmH2O, respiratory rate (RR) = 54-56 breaths/min (bpm), fractional inspired oxygen (Fi(O2)) = 1.0] or high-stretch (Vt = 30 ml/kg, PEEP = 0 cmH2O, RR = 14-16 bpm, Fi(O2) = 1.0) ventilation strategy and monitored for either 1 or 2 h. Subsequently, animals were lavaged and the composition and function of surfactant was analyzed. Separate groups of animals received exogenous surfactant after 1 h of high-stretch ventilation and were monitored for an additional 2 h. High stretch induced a significant decrease in blood oxygenation after 2 h of ventilation. Alterations in surfactant pool sizes and activity were observed at 1 h of high-stretch ventilation and progressed over time. The functional impairment of surfactant appeared to be caused by alterations to the hydrophobic components of surfactant. Exogenous surfactant treatment after a period of high-stretch ventilation mitigated subsequent physiological lung dysfunction. Together, these results suggest that alterations of surfactant are a consequence of the ventilation strategy that impair the biophysical activity of this material and thereby contribute directly to lung dysfunction over time.

Interleukin-6 has no effect on surfactant or lung function in different lung insults.

The current study determined if interleukin-6 (IL-6) had a causative role in the lung dysfunction and/or surfactant alterations associated with three different lung insults. IL-6 (or saline) was instilled into rats followed by mechanical ventilation in vivo for 4 hours. Also, IL-6 (-/-) and wild-type mice were subjected to 3 insults: ex vivo injurious mechanical ventilation; cecal ligation and perforation; and hyperoxia exposure. In all experiments, the presence or absence of IL-6 did not significantly influence gas exchange, lung compliance, or various surfactant measurements. These results suggest that IL-6 may have a limited role in the surfactant alterations observed in acute lung injury.

Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome.

BACKGROUND: Preclinical studies suggest that exogenous surfactant may be of value in the treatment of the acute respiratory distress syndrome (ARDS), and two phase 2 clinical trials have shown a trend toward benefit. We conducted two phase 3 studies of a protein-containing surfactant in adults with ARDS. METHODS: In two multicenter, randomized, double-blind trials involving 448 patients with ARDS from various causes, we compared standard therapy alone with standard therapy plus up to four intratracheal doses of a recombinant surfactant protein C-based surfactant given within a period of 24 hours. RESULTS: The overall survival rate was 66 percent 28 days after treatment, and the median number of ventilator-free days was 0 (68 percent range, 0 to 26); there was no significant difference between the groups in terms of mortality or the need for mechanical ventilation. Patients receiving surfactant had a significantly greater improvement in blood oxygenation during the initial 24 hours of treatment than patients receiving standard therapy, according to both univariate and multivariate analyses. CONCLUSIONS: The use of exogenous surfactant in a heterogeneous population of patients with ARDS did not improve survival. Patients who received surfactant had a greater improvement in gas exchange during the 24-hour treatment period than patients who received standard therapy alone, suggesting the potential benefit of a longer treatment course.

Physiological and inflammatory response to instillation of an oxidized surfactant in a rat model of surfactant deficiency.

Pulmonary surfactant is a mixture of phospholipids ( approximately 90%) and surfactant-associated proteins (SPs) ( approximately 10%) that stabilize the lung by reducing the surface tension. One proposed mechanism by which surfactant is altered during acute lung injury is via direct oxidative damage to surfactant. In vitro studies have revealed that the surface activity of oxidized surfactant was impaired and that this effect could be overcome by adding SP-A. On the basis of this information, we hypothesized that animals receiving oxidized surfactant preparations would exhibit an inferior physiological and inflammatory response and that the addition of SP-A to the oxidized preparations would ameliorate this response. To test this hypothesis, mechanically ventilated, surfactant-deficient rats were administered either bovine lipid extract surfactant (BLES) or in vitro oxidized BLES of three doses: 10 mg/kg, 50 mg/kg, or 10 mg/kg + SP-A. When instilled with 10 mg/kg normal surfactant, the rats had a significantly superior arterial Po2 responses compared with the rats receiving oxidized surfactant. Interestingly, increasing the dose five times mitigated this physiological effect, and the addition of SP-A to the surfactant preparation had little impact on improving oxygenation. There were no differences in alveolar surfactant pools and the indexes of pulmonary inflammation between the 10 mg/kg dose groups, nor was there any differences observed between either of the groups supplemented with SP-A. However, there was significantly more surfactant and more inflammatory cytokines in the 50 mg/kg oxidized BLES group compared with the 50 mg/kg BLES group. We conclude that instillation of an in vitro oxidized surfactant causes an inferior physiological response in a surfactant-deficient rat.

Treatment of acute respiratory distress syndrome with recombinant surfactant protein C surfactant.

We performed a phase I/II trial in North America of a recombinant surfactant protein C-based surfactant (Venticute) as treatment for the acute respiratory distress syndrome. Patients were prospectively randomized to receive either standard therapy or standard therapy plus one of two doses of exogenous surfactant given four times over 24 hours. Surfactant administration was well tolerated. No significant treatment benefit was associated with surfactant treatment. Bronchoalveolar lavage of treated patients at 48 hours reflected the presence of exogenous surfactant components, did not show evidence of improved surface tension lowering function, and had interleukin-6 concentrations that were significantly lower than control group values, consistent with an antiinflammatory treatment effect. The presence of exogenous surfactant was not detected in lavage fluid obtained at 120 hours. Future studies might rationally employ larger surfactant doses and a more prolonged dosing schedule.

Early surfactant administration protects against lung dysfunction in a mouse model of ARDS.

Sepsis can predispose the lung to insults such as mechanical ventilation (MV). It was hypothesized that treating the lung with exogenous surfactant early in the development of sepsis will reduce the lung dysfunction associated with MV 18 h later. Mice underwent sham or cecal ligation and perforation (CLP) surgery. Immediately after surgery, mice were either untreated or given 100 mg/kg of bovine lipid extract surfactant intratracheally. Eighteen hours later, the lungs were removed and analyzed either immediately or following ventilation ex vivo for 2 h by an "injurious" mode of ventilation (20 ml/kg, 0 cm positive end-expiratory pressure). In nonventilated lungs, exogenous surfactant had no impact on compliance or IL-6 concentrations in the lungs. In the ventilated groups, the administered surfactant had a significant protective effect on the lung dysfunction induced by MV, but only in the CLP lungs. We conclude that administration of exogenous surfactant at the time of a systemic insult can protect the lung from the damaging effects of MV 18 h later.

Glutathione replacement preserves the functional surfactant phospholipid pool size and decreases sepsis-mediated lung dysfunction in ethanol-fed rats.

BACKGROUND: Alcohol abuse increases the incidence of acute respiratory distress syndrome (ARDS). Previous evidence from our laboratory links ethanol-mediated glutathione depletion to impaired surfactant production by alveolar epithelial cells in vitro and to endotoxin-mediated edematous injury in isolated lungs ex vivo. ARDS patients have an imbalance between the inactive small aggregate (SA) and the bioactive large aggregate (LA) forms of surfactant phospholipid (as reflected by increased SA/LA ratios). Therefore, we hypothesized that ethanol ingestion, via glutathione depletion, could alter surfactant phospholipid distribution between LA and SA forms and thereby exacerbate sepsis-mediated lung dysfunction in vivo. METHODS: Rats fed an isocaloric diet with or without ethanol (36% total calories) for 6 weeks were made septic via cecal ligation and perforation. Some ethanol-fed rats had their diets supplemented with the glutathione precursor procysteine (>L-2-oxothiaxolidine-4-carboxylate). Sepsis physiology was assessed by determining respiratory rates, arterial blood pressures, and plasma lactate levels, and lung dysfunction was assessed by determining lung lavage fluid protein levels (index of alveolar endothelial/epithelial barrier disruption), arterial hypoxemia (index of impaired gas exchange) and surfactant phospholipid SA and LA fractions (index of the alveolar epithelium's ability to maintain a functional surfactant pool during sepsis). RESULTS: Ethanol ingestion decreased (p< 0.05) lung lavage fluid glutathione levels, and this defect was prevented by procysteine. Although ethanol ingestion had no effect (p< 0.05) on any of the indices of sepsis, it increased (p< 0.05) lung lavage fluid protein levels, worsened hypoxemia, and decreased the functional (LA) surfactant phospholipid pool after sepsis, all of which was prevented by procysteine. CONCLUSIONS: Ethanol ingestion, via glutathione depletion, increased sepsis-mediated lung dysfunction, and these effects could contribute to the increased risk of ARDS seen in alcoholic patients.

Pulmonary surfactant and inflammation in septic adult mice: role of surfactant protein A.

Surfactant alterations, alveolar cytokine changes, and the role of surfactant protein (SP)-A in septic mice were investigated. Sepsis was induced via cecal ligation and perforation (CLP). Septic and sham mice were euthanized at 0, 3, 6, 9, 12, 15, and 18 h after surgery. Mice deficient in SP-A and mice that overexpressed SP-A were euthanized 18 h after surgery. In wild-type, sham-operated mice, surfactant pool sizes were similar at all time points, whereas in the CLP groups there was a significant decrease in small-aggregate surfactant pool sizes beginning 6 h after CLP. Interleukin-6 concentrations in bronchoalveolar lavage fluid from septic animals increased from 6 to 18 h after surgery. Identical surfactant alterations and concentrations of cytokines were observed in septic mice that were SP-A deficient or that overexpressed SP-A. In conclusion, alterations of pulmonary surfactant and alveolar cytokines occur simultaneously, 6 h after a systemic insult. In addition, we did not detect a role for SP-A in regulating surfactant phospholipid pool sizes or pulmonary inflammation in septic mice.