Fusion Expression and Fibrinolytic Activity of rPA/SP-B.

BACKGROUND: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). OBJECTIVE: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. METHODS: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. RESULTS: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. CONCLUSION: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Impact of synthetic surfactant CHF5633 with SP-B and SP-C analogues on lung function and inflammation in rabbit model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with diffuse inflammation, alveolar epithelial damage, and leakage of plasma proteins into the alveolar space, which together contribute to inactivation of pulmonary surfactant and respiratory failure. Exogenous surfactant delivery is therefore considered to hold potential for ARDS treatment, but clinical trials with natural derived surfactant or synthetic surfactant containing a surfactant protein C (SP-C) analogue have been negative. Synthetic surfactant CHF5633, containing analogues of SP-B and SP-C, may be effective against ARDS. The aim here was to compare treatment effects of CHF5633 and animal-derived surfactant poractant alfa in animal model of ARDS. ARDS was induced in adult New Zealand rabbits by mild lung lavages followed by injurious ventilation until respiratory failure (P/F ratio <26.7 kPa). The animals were then treated with intratracheal bolus of 200 mg/kg CHF5633 or poractant alfa (Curosurf® ), or air as control. The animals were subsequently ventilated for an additional 4 hr and respiratory parameters were recorded regularly. Postmortem, histological analysis, degree of lung edema, and levels of the cytokines TNFα, IL-6, and IL-8 in lung homogenates were evaluated. Both surfactant preparations improved lung function, reduced the levels of pro-inflammatory cytokines, and degree of lung edema to very similar degrees versus the controls. No significant differences in any of the analyzed parameters were observed between the CHF5633- and poractant alfa-treated groups. This study indicates that single dose of CHF5633 improves lung function and attenuates inflammation as effectively as poractant alfa in experimental ARDS caused by injurious ventilation.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

Anti-inflammatory effect of a curcumin-aspirin derivative on Ureaplasma-induced cytokine expressions in neonatal monocytes.

One of the major contributors to death in infants is infant respiratory distress syndrome (IRDS). The transition from early acute inflammation to fibrotic hyperplasia is important in the development of IRDS. However, there are no available and effective remedies for suppression of the early inflammation. In this study, the effect of a curcumin-aspirin (C-A) derivative on the expressions of interleukin (IL)-10, IL-8, tumor necrosis factor (TNF)-α, IL-1ß, toll-like receptor (TLR)4 and TLR2 was investigated in neonatal monocytes exposed to Ureaplasma parvum. The techniques used were qPCR and flow cytometry. The results revealed that C-A exerted anti-inflammatory effects on stimulated monocytes and significantly suppressed Ureaplasma-stimulated changes in IL-8, TNF-α and IL-1ß levels. It did not produce any inflammation or apoptosis in the neonatal cells. Moreover, C-A enhanced IL-10 expression and suppressed the expressions of TLR2 and TLR4, indicating that curcumin-aspirin derivative might hold therapeutic potential for early IRDS.

Metabolism of a synthetic compared with a natural therapeutic pulmonary surfactant in adult mice.

Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.

Paradoxical Bactericidal Effects of Hydrophobic Lung Surfactant Proteins and Their Peptide Mimics Using Liposome Molecular Trojan.

Lung surfactant, besides alveolar stability, also provides defence against pathogens by surfactant proteins (SP), SP-A and SP-D. The hydrophobic proteins SP-B and SP-C enhance surface activity. An unusual and paradoxical effect of bovine LS and synthetic model LS with SP-B/-C was bactericidal to Staphylococcus aureus and Escherichia coli. Bacterial proliferation were investigated with bovine lung surfactant extract (BLES), dipalmitoylphosphatdylcholine, palmitooleylglycerol, in combination with SP-B/-C using standard microbiological colony forming unit (CFU) counts and structural imaging. BLES and other surfactant-SP-B/-C mixtures inhibit bacterial growth in the concentration range of 0 -7.5 mg/mL, at > 10 mg/mL paradoxical growth of both the bacterial species suggest antibiotic resistance. The lipid only LS have no effect on bacterial proliferation. Smaller peptide mimics of SP-B or SP-B1-25, were less efficient than SP-Cff. Ultra structural studies of the bacterial CFU using electron and atomic force microscopy suggest some membrane damage of S. aereus at inhibitory concentration of BLES, and some structural alteration of E. coli at dividing zones, suggesting utilization and incorporation of surfactant lipid species by both bacteria. The results depicted from in vitro studies are also in agreement with protein-protein interactions obtained from PatchDock, FireDock and ClasPro algorithm. The MD-simulation decipher a small range fluctuation of gyration radius of the LS proteins and their peptide mimics. The studies have alarming implications in the use of high dosages (100 mg/mL/kg body weight) of exogenous surfactant for treatment of respiratory distress syndrome, genetic knock-out abnormalities associated with these proteins, and the novel roles played by SP-B/C as bactericidal agents.

Effective in vivo treatment of acute lung injury with helical, amphipathic peptoid mimics of pulmonary surfactant proteins.

Acute lung injury (ALI) leads to progressive loss of breathing capacity and hypoxemia, as well as pulmonary surfactant dysfunction. ALI's pathogenesis and management are complex, and it is a significant cause of morbidity and mortality worldwide. Exogenous surfactant therapy, even for research purposes, is impractical for adults because of the high cost of current surfactant preparations. Prior in vitro work has shown that poly-N-substituted glycines (peptoids), in a biomimetic lipid mixture, emulate key biophysical activities of lung surfactant proteins B and C at the air-water interface. Here we report good in vivo efficacy of a peptoid-based surfactant, compared with extracted animal surfactant and a synthetic lipid formulation, in a rat model of lavage-induced ALI. Adult rats were subjected to whole-lung lavage followed by administration of surfactant formulations and monitoring of outcomes. Treatment with a surfactant protein C mimic formulation improved blood oxygenation, blood pH, shunt fraction, and peak inspiratory pressure to a greater degree than surfactant protein B mimic or combined formulations. All peptoid-enhanced treatment groups showed improved outcomes compared to synthetic lipids alone, and some formulations improved outcomes to a similar extent as animal-derived surfactant. Robust biophysical mimics of natural surfactant proteins may enable new medical research in ALI treatment.

A Combination of Short and Simple Surfactant Protein B and C Analogues as a New Synthetic Surfactant: In Vitro and Animal Experiments.

PURPOSE: Pulmonary surfactants for preterm infants contain mostly animal-derived surfactant proteins (SPs), which are essential for lowering surface tension. We prepared artificial pulmonary surfactants using synthetic human SP analogs and performed in vitro and in vivo experiments. MATERIALS AND METHODS: We synthesized peptide analogues that resemble human SP-B (RMLPQLVCRLVLRCSMD) and SP-C (CPVHLKRLLLLLLLLLLLLLLLL). Dipalmitoylphosphatidylcholine (DPPC), phosphatidylglycerol (PG), and palmitic acid (PA) were added and mixed in lyophilized to render powdered surfactant. Synsurf-1 was composed of DPPC:PG:PA:SP-B (75:25:10:3, w/w); Synsurf-2 was composed of DPPC:PG:PA:SP-C (75:25:10:3, w/w); and Synsurf-3 was composed of DPPC:PG:PA:SP-B:SP-C (75:25:10:3:3, w/w). We performed in vitro study to compare the physical characteristics using pulsating bubble surfactometer and modified Wilhelmy balance test. Surface spreading and adsorption test of the surfactant preparations were measured. In vivo test was performed using term and preterm rabbit pups. Pressure-volume curves were generated during the deflation phase. Histologic findings were examined. RESULTS: Pulsating bubble surfactometer readings revealed following minimum and maximum surface tension (mN/m) at 5 minutes: Surfacten® (5.5±0.4, 32.8±1.6), Synsurf-1 (16.7±0.6, 28.7±1.5), Synsurf-2 (7.9±1.0, 33.1±1.6), and Synsurf-3 (7.1±0.8, 34.5±1.0). Surface spreading rates were as follows: Surfacten® (27 mN/m), Synsurf-1 (43 mN/m), Synsurf-2 (27 mN/m), and Synsurf-3 (27 mN/m). Surface adsorption rate results were as follows: Surfacten® (28 mN/m), Synsurf-1 (35 mN/m), Synsurf-2 (29 mN/m), and Synsurf-3 (27 mN/m). The deflation curves were best for Synsurf-3; those for Synsurf-2 were better than those for Surfacten®. Synsurf-1 was the worst surfactant preparation. Microscopic examination showed the largest aerated area of the alveoli in the Synsurf-3 group, followed by Synsurf-1 and Surfacten®; Synsurf-2 was the smallest. CONCLUSION: Synsurf-3 containing both SP-B and SP-C synthetic analogs showed comparable and better efficacy than commercially used Surfacten® in lowering surface tension, pressure-volume curves, and tissue aerated area of the alveoli.

Anti-inflammatory effects of the new generation synthetic surfactant CHF5633 on Ureaplasma-induced cytokine responses in human monocytes.

BACKGROUND: Synthetic surfactants represent a promising alternative to animal-derived preparations in the treatment of neonatal respiratory distress syndrome. The synthetic surfactant CHF5633 has proven biophysical effectiveness and, moreover, demonstrated anti-inflammatory effects in LPS-stimulated monocytes. With ureaplasmas being relevant pathogens in preterm lung inflammation, the present study addressed immunomodulatory features on Ureaplasma-induced monocyte cytokine responses. METHODS: Ureaplasma parvum-stimulated monocytes were exposed to CHF5633. TNF-α, IL-1ß, IL-8, IL-10, TLR2 and TLR4 expression were analyzed using qPCR and flow cytometry. RESULTS: CHF5633 did not induce pro-inflammation, and did not aggravate Ureaplasma-induced pro-inflammatory cytokine responses. It suppressed U. parvum-induced intracellular TNF-α (p < 0.05) and IL-1ß (p < 0.05) in neonatal monocytes and inhibited Ureaplasma-induced TNF-α mRNA (p < 0.05), TNF-α protein (p < 0.001), and IL-1ß (p = 0.05) in adult monocytes. Ureaplasma-modulated IL-8, IL-10, TLR2 and TLR4 were unaffected. CONCLUSION: CHF5633 does neither act pro-apoptotic nor pro-inflammatory in native and Ureaplasma-infected monocytes. Suppression of Ureaplasma-induced TNF-α and IL-1ß underlines anti-inflammatory features of CHF5633.

Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes.

BACKGROUND: New generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes. METHODS: Purified neonatal CD14(+) cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1ß, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry. RESULTS: CHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1ß. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected. CONCLUSION: Our data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1ß cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.

A small key unlocks a heavy door: The essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes.

BACKGROUND: Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. AIM: The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. METHODS: Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. RESULTS: Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4+ lymphocytes. CONCLUSION: For the first time, the immunomodulatory capacity of CHF5633 on CD4+ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4+ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.

Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes.

BACKGROUND: Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes. METHODS: Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1ß, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry. RESULTS: Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1ß mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes. CONCLUSION: The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.

Natural Anti-Infective Pulmonary Proteins: In Vivo Cooperative Action of Surfactant Protein SP-A and the Lung Antimicrobial Peptide SP-BN.

The anionic antimicrobial peptide SP-B(N), derived from the N-terminal saposin-like domain of the surfactant protein (SP)-B proprotein, and SP-A are lung anti-infective proteins. SP-A-deficient mice are more susceptible than wild-type mice to lung infections, and bacterial killing is enhanced in transgenic mice overexpressing SP-B(N). Despite their potential anti-infective action, in vitro studies indicate that several microorganisms are resistant to SP-A and SP-B(N). In this study, we test the hypothesis that these proteins act synergistically or cooperatively to strengthen each other's microbicidal activity. The results indicate that the proteins acted synergistically in vitro against SP-A- and SP-B(N)-resistant capsulated Klebsiella pneumoniae (serotype K2) at neutral pH. SP-A and SP-B(N) were able to interact in solution (Kd = 0.4 µM), which enabled their binding to bacteria with which SP-A or SP-B(N) alone could not interact. In vivo, we found that treatment of K. pneumoniae-infected mice with SP-A and SP-B(N) conferred more protection against K. pneumoniae infection than each protein individually. SP-A/SP-B(N)-treated infected mice showed significant reduction of bacterial burden, enhanced neutrophil recruitment, and ameliorated lung histopathology with respect to untreated infected mice. In addition, the concentrations of inflammatory mediators in lung homogenates increased early in infection in contrast with the weak inflammatory response of untreated K. pneumoniae-infected mice. Finally, we found that therapeutic treatment with SP-A and SP-B(N) 6 or 24 h after bacterial challenge conferred significant protection against K. pneumoniae infection. These studies show novel anti-infective pathways that could drive development of new strategies against pulmonary infections.

New surfactant with SP-B and C analogs gives survival benefit after inactivation in preterm lambs.

BACKGROUND: Respiratory distress syndrome in preterm babies is caused by a pulmonary surfactant deficiency, but also by its inactivation due to various conditions, including plasma protein leakage. Surfactant replacement therapy is well established, but clinical observations and in vitro experiments suggested that its efficacy may be impaired by inactivation. A new synthetic surfactant (CHF 5633), containing synthetic surfactant protein B and C analogs, has shown comparable effects on oxygenation in ventilated preterm rabbits versus Poractant alfa, but superior resistance against inactivation in vitro. We hypothesized that CHF 5633 is also resistant to inactivation by serum albumin in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Nineteen preterm lambs of 127 days gestational age (term = 150 days) received CHF 5633 or Poractant alfa and were ventilated for 48 hours. Ninety minutes after birth, the animals received albumin with CHF 5633 or Poractant alfa. Animals received additional surfactant if P(a)O(2) dropped below 100 mmHg. A pressure volume curve was done post mortem and markers of pulmonary inflammation, surfactant content and biophysiology, and lung histology were assessed. CHF 5633 treatment resulted in improved arterial pH, oxygenation and ventilation efficiency index. The survival rate was significantly higher after CHF 5633 treatment (5/7) than after Poractant alfa (1/8) after 48 hours of ventilation. Biophysical examination of the surfactant recovered from bronchoalveolar lavages revealed that films formed by CHF 5633-treated animals reached low surface tensions in a wider range of compression rates than films from Poractant alfa-treated animals. CONCLUSIONS: For the first time a synthetic surfactant containing both surfactant protein B and C analogs showed significant benefit over animal derived surfactant in an in vivo model of surfactant inactivation in premature lambs.

Palmitoylation of pulmonary surfactant protein SP-C is critical for its functional cooperation with SP-B to sustain compression/expansion dynamics in cholesterol-containing surfactant films.

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.

Different effects of surfactant proteins B and C - implications for development of synthetic surfactants.

Treatment of premature newborn rabbits with synthetic surfactants containing a surfactant protein C analogue in a simple phospholipid mixture gives similar tidal volumes as treatment with poractant alfa (Curosurf(R)) but ventilation with a positive end-expiratory pressure (PEEP) is needed for this synthetic surfactant to stabilize the alveoli at end-expiration. The effect on lung gas volumes seems to depend on the structure of the peptide since treatment with a synthetic surfactant containing the 21-residue peptide (LysLeu(4))(4)Lys (KL(4)) gives low lung gas volumes in experiments also performed with PEEP. Surfactant preparations containing both surfactant proteins B and C or their analogues prevent alveolar collapse at end-expiration even if ventilated without PEEP. Treatment of premature newborn rabbits with different natural surfactants indicates that both the lipid composition and the proteins are important in order to stabilize the alveoli at end-expiration. Synthetic surfactants containing two peptides may be able to replace natural surfactants within the near future but more trials need to be performed before any conclusion can be drawn about the ideal composition of this new generation of synthetic surfactants.

Hydrophobic surfactant proteins induce a phosphatidylethanolamine to form cubic phases.

The hydrophobic surfactant proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface. Previous evidence suggests that they achieve this effect by facilitating the formation of a rate-limiting negatively curved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipid leaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by 1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transition at 71 degrees C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57 degrees C, the proteins induce formation of bicontinuous inverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidence of other structures above 0.1% and 62 degrees C, but no change in the lattice-constants of the lamellar or cubic phases. The induction of the bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypothetical stalk-intermediate, supports the proposed model of how the surfactant proteins promote adsorption.

Protein- and lipid modification of natural bovine surfactant. Effects in experimental lung failure with special consideration of the response in neonates.

UNLABELLED: Surfactant therapy does not lead to a uniform, optimal and sustained effect on gas exchange in neonatal respiratory distress syndrome (RDS) and acute respiratory distress syndrome (ARDS). The aim of the present study therefore was to compare the effects of a lipid-enriched and protein-modified natural surfactant preparations with a licensed, clinically used bovine surfactant preparation - SF-Ril (Alveofact). METHODS: SF-Ril was enriched with phosphatidylglycerol, sphingomyeline, phosphatidylethanolamine plasmalogen, phosphatidylethanolamine. Furthermore, SF-Ril was modified with increased surfactant protein B (SP-B)/surfactant protein C (SP-C) content and finally a mercaptoethanol (ME) treated preparation for breaking the sulfhydril bondage of SP-B/SP-C by chemical reduction in methylene chloride using ME was developed. Finally ME was removed by vacuum extraction. These modified surfactants were tested at a dosage of 100 mg/ kg each in a model of respiratory failure induced by lung lavage in male adult rats and compared with SF-Ril at an identical dosage. Mechanical ventilation was standardised with fraction of inspiratory oxygen (FiO2) 1.0 and time-cycled pressure limited ventilation 16/0.8 cmH2O before and 26/6 cmH2O (peak inspiratory pressure/positive endexpiratory pressure) after lung lavage (target arterial oxygen pressure [pa02] < 100 mmHg), respiratory rate 36/min, inspiration/expiration time ratio 1:2. RESULTS: During the observation period of 120 min, the sphingomyeline substituted and protein-modified (ME reduced) surfactant preparations exerted improved and sustained oxygenation compared with SF-Ril. Similar effects were observed for tidal volumes. All other preparations were equal or inferior to SF-Ril in our model. CONCLUSION: For the development of surfactant preparations less prone for inactivation the above mentioned data may provide useful information, provided they can be confirmed in further investigations employing other alternative models.