Recombinant expression of the precursor of rat lung surfactant protein B in Escherichia coli and its antibacterial mechanism.

While the discovery of antibiotics has made a huge contribution to medicine, bacteria that are resistant to many antibiotics pose new challenges to medicine. Antimicrobial peptides (AMPs), a new kind of antibiotics, have attracted people's attention because they are not prone to drug resistance. In this study, glutathione transferase (GST) was used as a fusion partner to recombinantly expressed rat lung surfactant protein B precursor (proSP-B) in E. coli pLySs. Cck-8 evaluated the cytotoxicity of the fusion protein and calculated its 50% inhibitory concentration (IC50). The purified peptides showed broad-spectrum antibacterial activity using filter paper method and MIC, and propidium iodide (PI) was used to explore the antibacterial mechanism against Staphylococcus aureus. In addition, the pEGFP-N2-proSP-B vector was constructed to explore the localization of proSP-B in CCL-149 cells. We found that proSP-B has obvious antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has broad-spectrum antibacterial activity. Besides, proSP-B fusion protein has low toxicity and can change the permeability of Staphylococcus aureus cell membrane to realize its antibacterial. For these reasons, proSP-B can be used as a potential natural antibacterial drug.

Lung Surfactant Accelerates Skin Wound Healing: A Translational Study with a Randomized Clinical Phase I Study.

Lung surfactants are used for reducing alveolar surface tension in preterm infants to ease breathing. Phospholipid films with surfactant proteins regulate the activity of alveolar macrophages and reduce inflammation. Aberrant skin wound healing is characterized by persistent inflammation. The aim of the study was to investigate if lung surfactant can promote wound healing. Preclinical wound models, e.g. cell scratch assays and full-thickness excisional wounds in mice, and a randomized, phase I clinical trial in healthy human volunteers using a suction blister model were used to study the effect of the commercially available bovine lung surfactant on skin wound repair. Lung surfactant increased migration of keratinocytes in a concentration-dependent manner with no effect on fibroblasts. Significantly reduced expression levels were found for pro-inflammatory and pro-fibrotic genes in murine wounds. Because of these beneficial effects in preclinical experiments, a clinical phase I study was initiated to monitor safety and tolerability of surfactant when applied topically onto human wounds and normal skin. No adverse effects were observed. Subepidermal wounds healed significantly faster with surfactant compared to control. Our study provides lung surfactant as a strong candidate for innovative treatment of chronic skin wounds and as additive for treatment of burn wounds to reduce inflammation and prevent excessive scarring.

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.

The novel surfactant protein SP-H enhances the phagocytosis efficiency of macrophage-like cell lines U937 and MH-S.

BACKGROUND: Surfactant proteins (SP) secreted by alveolar type 2 cells, play an essential role in maintaining the air-liquid barrier of the lung and are also involved in the opsonisation and clearance of bacteria by phagocytes. We have recently described a novel surfactant protein, SP-H (SFTA3). Expression of SP-H was earlier demonstrated to be upregulated by LPS and negatively regulated by IL-1ß and IL-23 in vitro. The influence of SP-H on phagocytosis was measured using a murine and a human phagocytic cell line and fluorescent latex beads. FINDINGS: SP-H markedly increases phagocytosis in vitro in the murine-derived alveolar macrophage cell lines MH-S and in human-derived differentiated U937 cells. CONCLUSION: It can be assumed that SP-H is involved in regulating phagocytic activity of macrophages. SP-H is a new player in pulmonary host defence.

Effect of natural porcine surfactant in Staphylococcus aureus induced pro-inflammatory cytokines and reactive oxygen species generation in monocytes and neutrophils from human blood.

Surfacen® is a clinical surfactant preparation of porcine origin. In the present study, we have evaluated the effect of Surfacen® in the modulation of oxidative burst in monocytes and neutrophils in human blood and pro-inflammatory cytokine production in peripheral blood mononuclear cells (PBMC). Reactive oxygen species (ROS) level was measured in monocytes and neutrophils by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA) as substrate, while, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels were estimated in PBMC supernatant by enzyme-linked immunosorbent assays (ELISA). Our results show that Staphylococcus aureus-induced ROS level was slightly affected by Surfacen® added to whole blood monocytes and neutrophils. The time course experiments of pre-incubation with Surfacen® showed no significant increase of ROS level at 2h; however, the ROS level decreased when pre incubated for 4h and 6h with Surfacen®. Pre-incubation of PBMC cells with Surfacen® at 0.125 and 0.5mg/mL showed a dose-dependent suppression of TNF-α levels measured after 4h of S. aureus stimulation, an effect less impressive when cells were stimulated for 24h. A similar behavior was observed in IL-6 release. In summary, the present study provides experimental evidence supporting an anti-inflammatory role of Surfacen® in human monocytes and neutrophils in vitro.

Safety of the production process of SURFACEN(®) to inactivate and remove virus.

SURFACEN(®) is a biological product produced from pig lungs. Since these animals can be potential sources of microbial pathogens such as viruses, the manufacturing process of this product should guarantee safety from health hazards. The SURFACEN(®) production procedure is capable of effective viral clearance (inactivation/removal) by involving two stages of organic solvent extraction followed by acetone precipitation and heat treatment. In this study, we evaluated the clearance capacity of these four stages for a wide range of viruses by performing spiking experiments. Residual contamination was assessed using a Tissue Culture Infectious Dose assay (log10 TCID50). The validation study demonstrated that, for all viruses tested, the TCID50 titers were reduced by more than 2 log10 in each stage. Total log reduction values achieved were between ≥17.82 log10 and ≥27.93 log10, depending on the virus physical properties, titer, and the number of processing stages applied. Results indicated that the production procedure of SURFACEN(®) can inactivate or remove contaminant viruses from the raw material.

Effects of keratin and lung surfactant proteins on the surface activity of meibomian lipids.

PURPOSE: In vitro studies indicate that surface tension and surface viscosity of the tear film lipid layer (TFLL) are governed by interactions between meibomian lipids and proteins from the aqueous layer. The role of minor tear proteins with strong lipophilic properties or those correlated with pathological states is still unknown. The discovery of lung surfactant proteins (SPs) in tears and keratin in normal and abnormal meibomian gland excretions warrants investigation into their effects on the surface activity of meibomian lipid films. METHODS: Commercial keratin and bovine lung SPs were used in vitro to assess the surface pressure of meibomian lipid films using a Langmuir trough. RESULTS: The pressure-area profiles of meibomian lipid films seeded with SPs (2.5 µL; 0.1 µg) demonstrated hybrid characteristics between meibomian lipid films alone and SPs alone but reached much higher maximum surface pressures (approximately 30 vs. 24 mN/m). Microscopically, the appearance of meibomian lipid films was not altered by SPs. Maximum surface pressure of meibomian films premixed with keratin was much higher than meibum alone. The pressure-area isocycles appeared more like those of meibomian lipids with a low concentration of protein and more like pure keratin films at a high concentration. CONCLUSIONS: The data strongly indicate that SPs and keratin likely interact with the TFLL. SPs are likely to act as strong surfactants and to reduce the surface tension of the lipid layer. Excess concentrations of keratin as identified in patients with meibomian gland dysfunction could disrupt the normal structure of the meibomian lipid film.

In vitro interaction between SURFACEN® and surfactant protein A against Leishmania amazonensis.

Leishmaniasis is caused by a parasite of the Leishmania genus, affecting more than 12 million people in 98 countries. The control of leishmaniasis remains a serious problem. There are currently no vaccines for leishmaniasis. The drugs available are toxic, expensive and frequently ineffective. The in vitro activity of SURFACEN® and SP-A against Leishmania amazonensis was evaluated. The combination of both products resulted in a synergic pharmacology effect, demonstrated by a fractional inhibitory concentration index <0.5. A more effective combination was a SURFACEN/SP-A ratio of 4:1, using a method of fixed ratio. The therapeutic effect of SURFACEN and SP-A as antileishmanial compounds was demonstrated, with a potentiation of activity when they were incubated in conjunction. Our results propose an exploration of these products in order to design new formulations against the Leishmania parasite.

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.

In vitro activity of the clinical pulmonary surfactant Surfacen® against Leishmania amazonensis.

Surfacen® is an exogenous natural lung surfactant, composed by phospholipids and hydrophobic proteins, which is applied successfully in Newborn Respiratory Distress Syndrome. In this paper, in vitro activity of Surfacen® against Leishmania amazonensis is described. The product showed activity against the amastigote form found in peritoneal macrophages from BALB/c mice, with an IC50 value of 17.9 ± 3.0 µg/mL; while no toxic effect on host cell was observed up to 200 µg/mL. This is the first report about the antileishmanial activity of Surfacen®.

In vitro activity of the clinical pulmonary surfactant Surfacen® against Leishmania amazonensis / Actividad in vitro de surfactante pulmonar clínico Surfacen® frente a Leishmania amazonensis

Surfacen® is an exogenous natural lung surfactant, composed by phospholipids and hydrophobic proteins, which is applied successfully in Newborn Respiratory Distress Syndrome. In this paper, in vitro activity of Surfacen® against Leishmania amazonensis is described. The product showed activity against the amastigote form found in peritoneal macrophages from BALB/c mice, with an IC50 value of 17.9 ± 3.0 µg/mL; while no toxic effect on host cell was observed up to 200 µg/mL. This is the first report about the antileishmanial activity of Surfacen®.

Surfacen® es un surfactante natural exógeno extraído del pulmón, formado por fosfolípidos y proteínas hidrofóbicas, el cual es aplicado con éxito en el Síndrome de Distrés Respiratorio en Niños Recién Nacidos. En este trabajo, se describe la actividad in vitro del Surfacen® contra Leishmania amazonensis. El producto mostró actividad frente a amastigotes que se encuentran en macrófagos peritoneales de ratón BALB/c, con una CI50 de 17.9 ± 3.0 µg/mL, mientras no se observaron efectos tóxicos sobre la célula hospedera hasta 200 µg/mL. Este estudio constituye el primer reporte sobre la actividad antileishmania del Surfacen®.

Antimicrobial properties of MX-2401, an expanded-spectrum lipopeptide active in the presence of lung surfactant.

MX-2401 is an expanded-spectrum lipopeptide antibiotic selective for Gram-positive bacteria that is a semisynthetic analog of the naturally occurring lipopeptide amphomycin. It was active against Enterococcus spp., including vancomycin-sensitive Enterococcus (VSE), vanA-, vanB-, and vanC-positive vancomycin-resistant Enterococcus (VRE), linezolid- and quinupristin-dalfopristin-resistant isolates (MIC(90) of 4 µg/ml), methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) (MIC(90) of 2 µg/ml), coagulase-negative staphylococci, including methicillin-sensitive Staphylococcus epidermidis (MSSE) and methicillin-resistant S. epidermidis (MRSE) (MIC(90) of 2 µg/ml), and Streptococcus spp. including viridans group streptococci, and penicillin-resistant, penicillin-sensitive, penicillin-intermediate and macrolide-resistant isolates of Streptococcus pneumoniae (MIC(90) of 2 µg/ml). MX-2401 demonstrated a dose-dependent postantibiotic effect varying from 1.5 to 2.4 h. Furthermore, MX-2401 was rapidly bactericidal at 4 times the MIC against S. aureus and Enterococcus faecalis, with more than 99.9% reduction in viable bacterial attained at 4 and 24 h, respectively. The MICs of MX-2401 against MRSA, MSSA, VSE, and VRE strains serially exposed for 15 passages to sub- to supra-MICs of MX-2401 remained within three dilutions of the original MIC. In contrast to that of the lipopeptide daptomycin, the antibacterial activity of MX-2401 was not affected in vitro by the presence of lung surfactant, and MX-2401 was active in vivo in the bronchial-alveolar pneumonia mouse model, in which daptomycin failed to show any activity. Moreover, the activity of MX-2401 was not as strongly dependent on the Ca(2+) concentration as is the activity of daptomycin. In conclusion, MX-2401 is a promising new-generation lipopeptide for the treatment of serious infections with Gram-positive bacteria, including hospital-acquired pneumonia.

Recombinant human Clara cell secretory protein treatment increases lung mRNA expression of surfactant proteins and vascular endothelial growth factor in a premature lamb model of respiratory distress syndrome.

Infant respiratory distress syndrome (IRDS) can lead to impaired alveolarization and dysmorphic vascularization of bronchopulmonary dysplasia. Clara cell secretory protein (CC10) has anti-inflammatory properties but is deficient in the premature infant. Because surfactant and vascular endothelial growth factor (VEGF) profiles are impaired by inflammation and CC10 inhibits lung inflammation, we hypothesized that CC10 may up-regulate surfactant protein (SP) and VEGF expression. Preterm lambs ( N = 24; 126 +/- 3 days [standard error] gestation) with IRDS were randomized to receive 100 mg/kg surfactant, 100 mg/kg surfactant followed by intratracheal 0.5, 1.5, or 5 mg/kg rhCC10 and studied for 4 hours. Gas exchange and lung mechanics were monitored; surfactant protein and VEGF mRNA profiles in lung were assessed. There was a significant rhCC10 dose-dependent increase in respiratory compliance and ventilation efficiency index; both parameters were significantly greater in animals treated with 5 mg/kg rhCC10 than those treated with surfactant alone. Similarly, there was a significant rhCC10 dose and protein-dependent increase in surfactant protein (SP-B > SP-C > SP-A) and dose- and isoform-dependent increase in VEGF (VEGF189 > VEGF165 > VEGF121). These data demonstrate that early intervention with rhCC10 up-regulates surfactant protein and VEGF expression, supporting the role of CC10 to protect against hyperoxia and mechanical ventilation in the immature lung.

Surfactant protein expression in human skin: evidence and implications.

The presence of surfactant proteins (SPs), critical to local barrier and defense functions and usually associated with the lung, was revealed in adult and fetal human skin complementary deoxyribonucleic acid, in skin samples from three adult female donors and also in cultured fibroblasts, keratinocytes, and melanocytes. Using reverse transcription-PCR, SP-A, SP-B, SP-C, and SP-D messenger ribonucleic acid expression was detected to varying extents in the different skin sources. The stronger expression of SP-C in fetal skin, compared to adult skin, suggested that the role of this protein alters with age. Immunohistochemical studies showed variable distribution of SPs in human epidermis and dermis, confirming that these proteins are indeed translated and expressed in skin tissue. In vitro studies showed that the surface tension of SP-deficient artificial sebum is (a) lowered by skin-extracted SP-B and (b) further reduced to a level comparable to normal sebum by the additional presence of skin-extracted SP-A and SP-D, consistent with their surface tension-lowering capabilities in lung. The possible roles of SPs in skin, based on their known functions in the lung are discussed. However, their potential as therapeutic targets or diagnostic markers of skin disease remains to be elucidated.

Lung surfactants.

PURPOSE: The pharmacology, clinical efficacy, and cost considerations of exogenous lung surfactants are reviewed. SUMMARY: Exogenous pulmonary surfactants, along with advancements in ventilatory care, have significantly reduced the incidence of respiratory distress syndrome (RDS) and its related complications in infants. The following exogenous surfactants are approved for the prophylaxis and treatment (rescue) of neonatal RDS: beractant, a modified natural surfactant; calfactant and poractant, both natural surfactants; and colfosceril, a synthetic surfactant that is not currently available in the United States. Lucinactant, a synthetic surfactant, is under investigation and received approvable status from the Food and Drug Administration in February 2005. The surfactants are delivered directly to their site of action, and only small amounts reach the systemic circulation. Bioavailability to the distal airways and alveoli depends on the method of delivery, the stage and severity of pulmonary disease, and the properties of the particular surfactant. According to data from clinical trials, the use of exogenous surfactant therapy for rescue within the first two hours of life appears to be as efficacious as prophylaxis in most premature infants. CONCLUSION: Comparative trials of surfactants have proven the efficacy of both synthetic and natural surfactants in the prevention and treatment of RDS. However, these trials have universally demonstrated greater reduction in the immediate need for ventilator support in infants who receive natural surfactants. Natural preparations cause less pneumothorax, bronchopulmonary dysplasia, and mortality compared with synthetic preparations. Synthetic agents offer the potential advantages of an unlimited supply with consistent pharmaceutical quality and no risk of transmitting infectious disease or causing immunologic sensitization.

Surfactant releases internal calcium stores in neutrophils by G protein-activated pathway.

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.

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.

The role of surfactant in asthma.

Pulmonary surfactant is a unique mixture of lipids and surfactant-specific proteins that covers the entire alveolar surface of the lungs. Surfactant is not restricted to the alveolar compartment; it also reaches terminal conducting airways and is present in upper airway secretions. While the role of surfactant in the alveolar compartment has been intensively elucidated both in health and disease states, the possible role of surfactant in the airways requires further research. This review summarizes the current knowledge on surfactant functions regarding the airway compartment and highlights the impact of various surfactant components on allergic inflammation in asthma.