Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D-amino acids were substituted for the L-amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L-peptide) rich in five arginine residues and consisting of an 11-amino acid peptide (residues 32-42) of human granulysin was synthesized. Subsequently, the L-amino acids of the 11-amino acid peptide were replaced partially (D-peptide) or wholly (AD-peptide) with D-amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D-amino acid substitutions were examined. Peptides with D-amino acid substitutions were found to lyse bacteria as efficiently as their all-L-amino acid parent, L-peptide. In addition, the peptide composed of L-amino acids was susceptible to trypsin, whereas peptides containing D-amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L-amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS-incubated peptide. Thus, D-amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D-amino acids are potential candidates for clinical therapeutic use.     

Influence of truncation of avian β-defensin-4 on biological activity and peptide-membrane interaction

Defensins are important components in host defense systems. The therapeutic use of β-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian β- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear β-defensins may be a promising candidate for future antibacterial agent.     

Lung surfactant proteins in the early human placenta

Pulmonary surfactant is a complex mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D). The biological functions of SP-A and SP-D are primarily twofold, namely surfactant homeostasis and host defense. The hydrophobic surfactant proteins, SP-B and SP-C, are required for achieving the optimal surface tension reducing properties of surfactant by promoting the rapid adsorption of surfactant phospholipids along the alveolar surface. Despite the promising findings, only little is known about the extrapulmonary distribution of these proteins. Therefore, in this study, the presence of SP-A, SP-B, SP-C and SP-D in early human placenta has been investigated. First-trimester placental tissues (22–56 days) were obtained from women undergoing curettage during normal pregnancies. In parallel tissue sections, vimentin, cytokeratin-7 and CD-68 immunostainings were used for the identification of mesenchymal cells, trophoblast cells and Hofbauer cells, respectively. According to immunohistochemistry (IHC) results, SP-A, SP-B, SP-C and SP-D immunoreactivities with different staining intensities were observed in trophoblastic layers of chorionic villous tree, trophoblastic cell columns, stromal cells, Hofbauer cells, angiogenic cell cords and vascular endothelium. Fetal hematopoietic cells showed a variable staining pattern for all four surfactant proteins ranging from none to strong intensity. Western blotting of tissue extracts confirmed our IHC results. The presence of surfactant glycoproteins in early human placenta may yield a very important feature of surfactants during first trimester and enables further studies of the role of surfactants in various pregnancy complications.     

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome.

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.     

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence.

Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.     

Critical structure-function determinants within the N-terminal region of pulmonary surfactant protein SP-B

Surfactant protein SP-B is absolutely required for the surface activity of pulmonary surfactant and postnatal lung function. The results of a previous study indicated that the N-terminal segment of SP-B, comprising residues 1–9, is specifically required for surface activity, and suggested that prolines 2, 4, and 6 as well as tryptophan 9, may constitute essential structural motifs for protein function. In this work, we assessed the role of these two motifs in promoting the formation and maintenance of surface-active films. Three synthetic peptides were synthesized including a peptide corresponding to the N-terminal 37 amino acids of native SP-B and two variants in which prolines 2, 4, 6, or tryptophan 9 were substituted by alanines. All three synthetic peptides were surface-active, as expected from their amphipathic structure. The peptides were also able to insert into dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol (7:3 w/w ratio) monolayers preformed at pressures >30 mN/m, indicating that they perturb and insert into membranes. Substitution of alanine for tryptophan at position 9 significantly decreased both the rate of adsorption/insertion of the peptide into the interface and reinsertion of surface-active material excluded from the film during successive compression-expansion cycles. Substitution of alanines for prolines at positions 2, 4, and 6 did not produce substantial changes in the rate of adsorption/insertion; however, reinsertion of surface-active material into the expanding interface film was not as effective as in the presence of the nativelike peptide. These results suggest that W9 is critical for optimal interface affinity, whereas prolines may promote a conformation that facilitates rapid insertion of the peptide into phospholipid monolayers compressed to the highest pressures during compression-expansion cycling.     

Location of structural transitions in an isotopically labeled lung surfactant SP-B peptide by IRRAS

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel beta-sheet formation in a synthetic peptide derived from human SP-B, residues 9-36 (SP-B(9-36)). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B(9-36), synthesized with (13)C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the beta-sheet marker band to approximately 1600 cm(-1) and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B(9-36) in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein.     

Effect of Cecropin B and a Synthetic Analogue on Propagation of Fish Viruses In Vitro

Abstract Cecropins and other natural antimicrobial peptides are widely distributed in animals from insects to mammals. These proteins have been shown to be major constituents of the innate immune systems of animals for nonspecific defense of the host against various bacteria and parasites. Therefore, exploitation of this natural innate defense system may lead to the development of effective methods for protecting fish from invasion by microbial pathogens. Recently, we have demonstrated that the introduction of cecropin transgenes into Japanese medaka (Oryzias latipes) conferred resistance to infection by fish bacterial pathogens. Aside from a few reports documenting the antiviral effect of antimicrobial peptides including cecropins against mammalian viruses, there is no evidence for the effect of these peptides against fish viruses. In this article we present results of in vitro characterization of native cecropin B and a synthetic analogue, CF17, against several important fish viral pathogens—namely, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), snakehead rhabdovirus (SHRV), and infectious pancreatic necrosis virus (IPNV). Upon coincubation of these peptides and viruses, the viral titers yielded in fish cells were reduced from several fold to 104-fold. Direct disruption of the viral envelope and disintegration of the viral capsids may be involved in the inhibition of viral replication by the peptides. Results of our studies demonstrate the potential of manipulating antimicrobial peptide genes by transgenesis to combat viral infection in fish.     

Liver carboxylesterase cleaves surfactant protein (SP-) B and promotes surfactant subtype conversion

Conversion of the biophysically active large surfactant aggregate subtype of alveolar surfactant into the less surface active small surfactant aggregates occurs in vitro and in vivo, possibly in dependency of a carboxylesterase, entitled surfactant convertase. The substrate has yet not been safely identified. Utilizing the in vitro cycling assay we investigated conversion of an organic rabbit lavage extract reconstituted with SP-A. Porcine liver carboxylesterase, which is closely related to surfactant convertase, induced subtype conversion to a similar degree as compared with native lavage fluid containing endogenous convertase. In addition, we asked for cleavage products of SP-B and identified a approximately 12 kDa band upon cycling with liver carboxylesterase, having the same N-terminus as mature SP-B. A band of same molecular weight was found in native lavage fluid after in vitro conversion mediated by the endogenous convertase. We conclude that SP-B plays a pivotal role during subtype conversion and represents the substrate for surfactant convertase.     

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.     

Mapping and analysis of the lytic and fusogenic domains of surfactant protein B

Surfactant protein B (SP-B) is a hydrophobic, 79 amino acid peptide that regulates the structure and function of surfactant phospholipid membranes in the airspaces of the lung. Addition of SP-B to liposomes composed of DPPC/PG (7:3) leads to membrane binding, destabilization, and fusion, ultimately resulting in rearrangement of membrane structure. The goal of this study was to map the fusogenic and lytic domains of SP-B and assess the effects of altered fusion and lysis on surface activity. Synthetic peptides were generated to predicted helices and/or interhelical loops of SP-B and tested for fusion, lytic, and surface activities. The N-terminal half of SP-B (residues 1-37), which includes the nonhelical N-terminal amino acids in addition to helices 1 and 2, promoted rapid liposome fusion whereas shorter peptides were significantly less effective. The requirements for optimal surface tension reduction were similar to those for fusion; in contrast, helix 1 (residues 7-22) alone was sufficient for liposome lysis. The C-terminal half of SP-B (residues 43-79), which includes helices 3, 4, and 5, exhibited significantly lower levels of fusogenic, lytic, and surface tension reducing activities compared to the N-terminal region. These results indicate that SP-B fusion, lytic and surface activities map predominantly to the N-terminal half of SP-B. Amino acid substitutions in synthetic peptides corresponding to the N-terminal half of SP-B indicated that, in general, decreased fusion or lytic activities were associated with altered surface tension reducing properties of the peptide. However, the presence of fusion and lytic activities alone could not account for the surface tension reducing property of SP-B. We propose a model in which association of helix 1 with lipids leads to membrane permeabilization but not aggregation; helix 2 mediates membrane cross-linking (aggregation), which, in turn, facilitates lipid mixing, membrane fusion, and interfacial adsorption/surface tension reduction.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Membrane activity of (Cys48Ser) lung surfactant protein B increases with dimerisation

One of the possible functions of lung surfactant protein B (SP-B), an hydrophobic membrane-associated saposin-like protein, is to reduce the alveolar surface tension by promoting insertion of phospholipids into the air/liquid interface of the lung. SP-B is a covalent homodimer; Cys48 of two polypeptides form an intermolecular disulphide bond. In order to test whether dimerisation of SP-B is important for surfactant function, transgenic mice which express (Cys48Ser) human SP-B in a mouse SP-B null background were generated. In previous studies (Cys48Ser)SP-B showed a concentration-dependent in vitro activity, suggesting that it may form non-covalent dimers. Here (Cys48Ser)SP-B isolated from bronchoalveolar lavage of transgenic mice was studied at different concentrations by circular dichroism (CD) spectroscopy, pulsating bubble surfactometry, mass spectrometry and reversed-phase HPLC. The results indicate that (Cys48Ser)SP-B, both in a phospholipid environment and in organic solvents, is largely monomeric and exhibits low activity at concentrations lower than 1 -2 microM, while at higher concentrations it forms non-covalent dimers, which are nearly functionally equivalent to native SP-B in vitro. Furthermore, electrospray mass spectrometry showed that more dimers were found relative to the monomer when the polarity of the solvent was decreased, and when the concentration of SP-B increased. (Cys48Ser)SP-B also eluted earlier than native SP-B in reversed-phase HPLC. Taken together, these results indicate that a polar surface is buried upon dimerisation, thereby promoting formation of interchain ion pairs between Glu51-Arg52' and Glu51'-Arg52.     

Surfactant in respiratory distress syndrome and lung injury

A deficiency in alveolar surfactant due to immaturity of alveolar type II epithelial cells causes respiratory distress syndrome (RDS). In contrast to animals, the fetal maturation of surfactant in human lungs takes place before term, exceptionally large quantities of surfactant accumulating in the amniotic fluid. The antenatal development of surfactant secretion is very variable but corresponds closely to the risk of RDS. The variation in SP-A and SP-B genes, race, sex and perinatal complications influence susceptibility to RDS. Surfactant therapy has improved the prognosis of RDS remarkably. Abnormalities in alveolar or airway surfactant characterize many lung and airway diseases. In the acute respiratory distress syndrome, deficiencies in surfactant components (phospholipids, SP-B, SP-A) are evident, and may be caused by pro-inflammatory cytokines (IL-1, TNF) that decrease surfactant components. The resultant atelectasis localizes the disease, possibly allowing healing (regeneration, increase in surfactant). In the immature fetus, cytokines accelerate the differentiation of surfactant, preventing RDS. After birth, however, persistent inflammation is associated with low SP-A and chronic lung disease. A future challenge is to understand how to inhibit or redirect the inflammatory response from tissue destruction and poor growth towards normal lung development and regeneration.     

Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella

Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung’s host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (μM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50 = 50 nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection.     

Fibrinolysis-inhibitory capacity of clot-embedded surfactant is enhanced by SP-B and SP-C

Incorporation of pulmonary surfactant into fibrin inhibits its plasmic degradation. In the present study we investigated the influence of surfactant proteins (SP)-A, SP-B, and SP-C on the fibrinolysis-inhibitory capacity of surfactant phospholipids. Plasmin-induced fibrinolysis was quantified by means of a (125)I-fibrin plate assay, and surfactant incorporation into polymerizing fibrin was analyzed by measuring the incorporation of (3)H-labeled L-alpha-dipalmitoylphosphatidylcholine into the insoluble clot material. Incorporation of a calf lung surfactant extract (Alveofact) and an organic extract of natural rabbit large surfactant aggregates (LSA) into a fibrin clot revealed a stronger inhibitory effect on plasmic cleavage of this clot than a synthetic phospholipid mixture (PLX) and unprocessed LSA. Reconstitution of PLX with SP-B and SP-C increased, whereas reconstitution with SP-A decreased, the fibrinolysis-inhibitory capacity of the phospholipids. The SP-B effect was paralleled by an increased incorporation of phospholipids into fibrin. We conclude that the inhibitory effect of surfactant incorporation into polymerizing fibrin on its susceptibility to plasmic cleavage is enhanced by SP-B and SP-C but reduced by SP-A. In the case of SP-B, increased phospholipid incorporation may underlie this finding.