A new cryptic host defense peptide identified in human 11-hydroxysteroid dehydrogenase-1 ß-like: from in silico identification to experimental evidence.

BACKGROUND: Host defence peptides (HDPs) are evolutionarily conserved components of innate immunity. Human HDPs, produced by a variety of immune cells of hematopoietic and epithelial origin, are generally grouped into two families: beta structured defensins and variably-structured cathelicidins. We report the characterization of a very promising cryptic human HDP, here called GVF27, identified in 11-hydroxysteroid dehydrogenase-1 ß-like protein. METHODS: Conformational analysis of GVF27 and its propensity to bind endotoxins were performed by NMR, Circular Dichroism, Fluorescence and Dynamic Light Scattering experiments. Crystal violet and WST-1 assays, ATP leakage measurement and colony counting procedures were used to investigate antimicrobial, anti-biofilm, cytotoxicity and hemolytic activities. Anti-inflammatory properties were evaluated by ELISA. RESULTS: GVF27 possesses significant antibacterial properties on planktonic cells and sessile bacteria forming biofilm, as well as promising dose dependent abilities to inhibit attachment or eradicate existing mature biofilm. It is unstructured in aqueous buffer, whereas it tends to assume a helical conformation in mimic membrane environments as well as it is able to bind lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Notably it is not toxic towards human and murine cell lines and triggers a significant innate immune response by attenuating expression levels of pro-inflammatory interleukins and release of nitric oxide in LPS induced macrophages. CONCLUSION: Human GVF27 may offer significant advantages as leads for the design of human-specific therapeutics. GENERAL SIGNIFICANCE: Human cryptic host defence peptides are naturally no immunogenic and for this they are a real alternative for solving the lack of effective antibiotics to control bacterial infections.

Avian cathelicidins: paradigms for the development of anti-infectives.

The broad-spectrum defense system based on host defense peptides (HDPs) is evolutionary very old and many invertebrates rely on this system for protection from bacterial infections. However, in vertebrates the system remained important in spite of the superposition of a very sophisticated adaptive immune system. The cathelicidins comprise a major group of HDPs in mammals. About six years ago it was first described that cathelicidins are also present in birds. Here we review the properties and biological activities of the recently discovered avian cathelicidins and their potential to be used as a paradigm for the development of anti-infectives. Like the mammalian cathelicidins, avian cathelicidins exert direct antimicrobial activities but can also selectively boost host immune responses by regulation of cytokine production and recruitment of immune cells. In addition, it was found that chicken cathelicidins bind endotoxins and dampen the endotoxin-mediated inflammatory response. Molecular dissection has allowed identification of different structural elements involved in bacterial killing and immunomodulation. These studies have enabled the design of small HDP-based antibiotics with specific functions, i.e. having primarily immunomodulatory or antimicrobial activities. Since the immunomodulatory effects may, to a certain degree, be species-specific, we hypothesize that poultry-specific antibiotics can be developed based on avian cathelicidins.

Dose-dependent effects of leucine supplementation on preservation of muscle mass in cancer cachectic mice.

Cancer cachexia, which is characterized by muscle wasting, is associated with increased morbidity and mortality. Because muscle protein synthesis may be increased and protein breakdown reduced by leucine supplementation, we used the C26 tumor-bearing cachectic mouse model to assess the effects of dietary supplementation with leucine on muscle weight and the markers of muscle protein breakdown (mRNA of atrogin and murf). Male CD2F1 mice were subcutaneously inoculated with tumor cells (tumor-bearing mice; TB) or were sham injected (control; C). They were fed standard diets or diets supplemented with leucine [1 gr (TB1Leu) or 8 gr (TB8Leu) supplemented leucine per kg feed]; TB and C received 8.7% Leu/g protein, TB1Leu received 9.6% Leu/g protein and TB8Leu received 14.6 Leu/g protein. After 21 days, the following were determined: body weights, plasma amino-acid concentrations, tumor size and muscle mass of the gastrocnemius (mG), tibialis anterior (mTA), extensor digitorum longus (mEDL) and soleus (mS) muscles. In tumor-bearing (TB) mice, carcass and skeletal muscle masses decreased, and levels of atrogin and murf mRNA in the mEDL increased. Muscle-mass loss was counteracted dose-dependently by leucine supplementation: relative to TB, the mass of the mG was +23% in TB8Leu, and +22% in mTA (p<0.05). However, leucine supplementation did not change atrogin and murf mRNA levels. Total plasma amino acid concentrations increased in TB, especially for taurine, lysine, arginine and alanine (p<0.05). Leucine supplementation attenuated the increase in total plasma amino-acid concentrations (p<0.05). Irrespective of changes in muscle protein breakdown markers, leucine supplementation reduced muscle wasting in tumor-bearing cachectic mice and attenuated changes in plasma amino acids.

Direct effects of doxorubicin on skeletal muscle contribute to fatigue.

Chemotherapy-induced fatigue is a multidimensional symptom. Oxidative stress has been proposed as a working mechanism for anthracycline-induced cardiotoxicity. In this study, doxorubicin (DOX) was tested on skeletal muscle function. Doxorubicin induced impaired ex vivo skeletal muscle relaxation followed in time by contraction impediment, which could be explained by DOX-induced changes in Ca(2+) responses of myotubes in vitro. The Ca(2+) responses in skeletal muscle, however, could not be explained by oxidative stress.

Pulmonary surfactant from healthy Belgian White and Blue and Holstein Friesian calves: biochemical and biophysical comparison.

The biochemical composition and biophysical behaviour of pulmonary surfactant samples isolated from healthy Belgian White and Blue (BWB) and Holstein Friesian (HF) calves have been investigated and compared. Interesting differences in composition have been demonstrated. In particular, a higher level of total hydrophobic surfactant-associated proteins (SP) (due to higher levels of SP-B and SP-C) is reported in HF calves compared to BWB calves. Higher levels of phosphatidylcholine (PC) and especially the disaturated form of PC were also found in HF as compared to BWB calves. No immediate effect on the surface tension properties evaluated by the pulsating bubble surfactometer was found between the surfactant samples of the two breeds under physiological conditions. However, since a high content of disaturated PC and the presence of the SP-B and SP-C are thought to be essential for the surface activity, we propose that the reported modifications could contribute to the apparently lower resistance of the BWB calves to respiratory troubles in comparison with HF calves.

Localization and functions of SP-A and SP-D at mucosal surfaces.

Pulmonary surfactant protein A (SP-A) and D (SP-D), members of the collectin family, are implicated in innate host defense of the lung. Collectins consist of a collagen-like domain and a carbohydrate recognition domain. SP-A and SP-D recognize and interact with glycoconjugates on the surface of microorganisms. They protect the lung by interacting with a wide variety of potential pathogens, including viruses, bacteria, and fungi. This may result in enhanced killing and/or clearance by phagocytes. Although most extensively studied in the lung, both SP-A and SP-D, or proteins closely resembling SP-A and SP-D, are found in a number of other sites in the body and therefore may play a protective role at other sites than the lung. SP-A and SP-D protein and/or mRNA have been detected at various sites of the body: the respiratory tract, the gastrointestinal tract, the middle ear, and in the peritoneal cavity. The presence of SP-A and SP-D at these mucosal surfaces, in close contact with numerous potentially harmful microorganisms, supports a role for these "lung"-collectins in innate mucosal defense. SP-A and SP-D may be important molecules in a threefold innate defense, particularly in the neonatal period between maternally acquired immunity and a fully developed adaptive immune system; the time interval between first exposure to a pathogen and generation of specific antibodies; and states of impaired immune function.

The juxtamembrane lysine and arginine residues of surfactant protein C precursor influence palmitoylation via effects on trafficking.

Surfactant protein (SP)-C propeptide (proSP-C) becomes palmitoylated on cysteines 5 and 6 before mature SP-C is formed by several proteolytic steps. To study the structural requirements for the palmitoylation of proSP-C, his-tagged human proSP-C (his-proSP-C) and his-proSP-C mutants were expressed in Chinese hamster ovary cells and analyzed by metabolic labeling with [(3)H]palmitate and immunocytochemistry. Substitution of cysteines 5 and 6 by serines showed that these were the only two cysteine residues palmitoylated in his-proSP-C. Substitution of the juxtamembrane basic residues lysine and arginine by uncharged glutamines led to a large decrease in palmitoylation level of proSP-C. The addition of brefeldin A nearly abolished this decrease for the lysine and double mutant; the palmitoylation of the arginine mutant increased also, but not to wild-type (WT) levels. Fluorescence immunocytochemistry showed that WT proSP-C was localized in punctate vesicles throughout the cell, whereas the mutant lacking the juxtamembrane positive charges was found more perinuclear, probably in the endoplasmic reticulum (ER). This indicates that the two basic juxtamembrane residues influence palmitoylation of proSP-C by preventing the transport of proSP-C out of the ER, implying that proSP-C becomes palmitoylated normally in a compartment distal to the ER.

Surfactant-associated proteins: functions and structural variation.

Pulmonary surfactant is a barrier material of the lungs and has a dual role: firstly, as a true surfactant, lowering the surface tension; and secondly, participating in innate immune defence of the lung and possibly other mucosal surfaces. Surfactant is composed of approximately 90% lipids and 10% proteins. There are four surfactant-specific proteins, designated surfactant protein A (SP-A), SP-B, SP-C and SP-D. Although the sequences and post-translational modifications of SP-B and SP-C are quite conserved between mammalian species, variations exist. The hydrophilic surfactant proteins SP-A and SP-D are members of a family of collagenous carbohydrate binding proteins, known as collectins, consisting of oligomers of trimeric subunits. In view of the different roles of surfactant proteins, studies determining the structure-function relationships of surfactant proteins across the animal kingdom will be very interesting. Such studies may reveal structural elements of the proteins required for surface film dynamics as well as those required for innate immune defence. Since SP-A and SP-D are also present in extrapulmonary tissues, the hydrophobic surfactant proteins SP-B and SP-C may be the most appropriate indicators for the evolutionary origin of surfactant. SP-B is essential for air-breathing in mammals and is therefore largely conserved. Yet, because of its unique structure and its localization in the lung but not in extrapulmonary tissues, SP-C may be the most important indicator for the evolutionary origin of surfactant.

Functional tests for the characterization of surfactant protein B (SP-B) and a fluorescent SP-B analog.

Surfactant protein B (SP-B) enhances lipid insertion into the alveolar air/liquid interface upon inhalation. The aim of this study was (i) to apply a palette of tests for a detailed biochemical and biophysical characterization of SP-B and (ii) to use these tests to compare native SP-B with a fluorescent (Bodipy) SP-B analog. The method of labeling was fast and resulted in a covalent fluorophore-protein bond. The ability of both proteins to spread a surfactant film on top of a buffer surface was determined in a spreading tray using the Wilhelmy plate technique to allow detection of alterations in surface tension and calculation of spreading velocities. In a captive bubble surfactometer surface tensions of spread films were measured. Similar biophysical properties were found for both native and Bodipy-labeled SP-B. It is concluded that the combination of tests used allows detection of small differences in structure and activity between the two proteins.

Alveolar macrophages, surfactant lipids, and surfactant protein B regulate the induction of immune responses via the airways.

The influences of alveolar macrophages (AM) and pulmonary surfactant on the induction of immune responses via the airways were assessed. Mice were depleted of their AM by intratracheal instillation of multilamellar vesicles containing dichloromethylene-diphosphonate followed by intratracheal instillation of a T cell--dependent antigen, trinitrophenyl--keyhole limpet hemocyanin, in vesicles of various compositions. The primary immune response was determined in the spleen of these animals using an ELI-Spot assay. The secondary immune responses in the sera of the mice were assessed using enzyme-linked immunosorbent assays. An immune response was detected in animals depleted of their AM and intratracheally instilled with antigen in small unilamellar vesicles consisting of either phosphatidylcholine cholesterol or surfactant lipids. Incorporation of surfactant protein (SP)-B in the antigen vesicles enhanced the immune response, whereas SP-A or SP-C in the antigen vesicle did not have an effect. Strikingly, intratracheal instillation of SP-B containing antigen vesicles can induce an immunoglobulin M immune response in mice without depletion of AM. These results indicate that SP-B containing vesicles can enhance the induction of immune responses via the airways and further illustrate the important roles of both AM and pulmonary surfactant in the pulmonary immune system.

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer.

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.

The role of surfactant proteins in DPPC enrichment of surface films.

A pressure-driven captive bubble surfactometer was used to determine the role of surfactant proteins in refinement of the surface film. The advantage of this apparatus is that surface films can be spread at the interface of an air bubble with a different lipid/protein composition than the subphase vesicles. Using different combinations of subphase vesicles and spread surface films a clear correlation between dipalmitoylphosphatidylcholine (DPPC) content and minimum surface tension was observed. Spread phospholipid films containing 50% DPPC over a subphase containing 50% DPPC vesicles did not form stable surface films with a low minimum surface tension. Addition of surfactant protein B (SP-B) to the surface film led to a progressive decrease in minimum surface tension toward 1 mN/m upon cycling, indicating an enrichment in DPPC. Surfactant protein C (SP-C) had no such detectable refining effect on the film. Surfactant protein A (SP-A) had a positive effect on refinement when it was present in the subphase. However, this effect was only observed when SP-A was combined with SP-B and incubated with subphase vesicles before addition to the air bubble containing sample chamber. Comparison of spread films with adsorbed films indicated that refinement induced by SP-B occurs by selective removal of non-DPPC lipids upon cycling. SP-A, combined with SP-B, induces a selective adsorption of DPPC from subphase vesicles into the surface film. This is achieved by formation of large lipid structures which might resemble tubular myelin.

Role of pulmonary surfactant components in surface film formation and dynamics.

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.

Very low surfactant protein C contents in newborn Belgian White and Blue calves with respiratory distress syndrome.

We have studied a respiratory distress syndrome (RDS) occurring in newborn calves of the Belgian White and Blue (BWB) breed that represents the large majority of beef cattle in Belgium. Pulmonary surfactant isolated from 14 BWB newborn calves that died from RDS and from 7 healthy controls was analysed for composition and surface activity. An extremely low content or, in some instances, an absence of surfactant protein C (SP-C) was detected in the RDS samples by Western blotting and differential amino acid analysis [0.03+/-0.01% (w/w) relative to total phospholipids, compared with 0.39+/-0.06% for healthy controls (means+/-S.E.M., P < 0.001)]. The contents of surfactant protein B (SP-B) were similar in RDS and control samples. The crude surfactant samples isolated from RDS calves had higher ratios of total protein to total phospholipid, altered phospholipid profiles and lower SP-A contents. Both crude and organic extracts of RDS surfactant samples showed increased dynamic surface tension compared with healthy controls when evaluated with a pulsating-bubble surfactometer. The addition of purified SP-C to organic extracts of RDS surfactant samples lowered surface tension. Strongly decreased levels of mature SP-C associated with fatal RDS and altered surface activity in vitro have, to the best of our knowledge, not been previously reported. The mechanisms underlying RDS and the decrease in SP-C in BWB calves remain to be established.

Role of pulmonary surfactant protein D in innate defense against Candida albicans.

Pulmonary surfactant protein D (SP-D), which is a member of the collectin family, is implicated in pulmonary defense against pathogens. To determine whether SP-D is involved in first-line immunity against Candida albicans, an important respiratory fungus, the interaction of SP-D with C. albicans was studied. SP-D was found to bind C. albicans, resulting in agglutination of the microorganisms. Binding was calcium dependent and was inhibited by competing sugars maltose or mannose. Incubation of C. albicans with SP-D resulted in profound fungal growth inhibition and decreased hyphal outgrowth. Furthermore, it was found that SP-D inhibited phagocytosis of C. albicans by alveolar macrophages. These data suggest that the lung collectin SP-D has an important role in first-line defense against C. albicans in the lung, by agglutinating C. albicans and limiting their growth, without the need for macrophage activation.

Pulmonary surfactant is altered during mechanical ventilation of isolated rat lung.

OBJECTIVE: To test the hypothesis that the lung injury induced by certain mechanical ventilation strategies is associated with changes in the pulmonary surfactant system. DESIGN: Analysis of the pulmonary surfactant system from isolated rat lungs after one of four different ventilatory strategies. SETTING: A research laboratory at a university. SUBJECTS: A total of 45 Sprague-Dawley rats. INTERVENTIONS: Isolated lungs were randomized to either no ventilation (0-TIME) or to ventilation at 40 breaths/min in a humidified 37 degrees C chamber for either 30 mins or 120 mins with one of the following four strategies: a) control (CON, 7 mL/kg, 3 cm H2O positive end-expiratory pressure); b) medium volume, zero end-expiratory pressure (MVZP, 15 mL/kg, 0 cm H2O end-expiratory pressure); c) medium volume, high positive end-expiratory pressure (MVHP, 15 mL/kg, 9 cm H2O positive end-expiratory pressure); and d) high volume, zero end-expiratory pressure (HVZP, 40 mL/kg, 0 cm H2O end-expiratory pressure). MEASUREMENTS: Pressure-volume curves were determined before and after the ventilation period, after which the lungs were lavaged for surfactant analysis. MAIN RESULTS: Compared with 0-TIME, 30 mins of ventilation with the HVZP strategy or 120 mins of ventilation with CON and MVZP strategies caused a significant decrease in compliance. Groups showing a decreased compliance had significant increases in the amount of surfactant, surfactant large aggregates, and total lavage protein compared with 0-TIME. CONCLUSIONS: A short period of injurious mechanical ventilation can cause a decrease in lung compliance that is associated with a large influx of proteins into the alveolar space and with alterations of the pulmonary surfactant system. The changes of surfactant in these experiments are different from those seen in acute lung injury, indicating that they may represent an initial response to mechanical ventilation.

Cell-specific expression of CCAAT/enhancer-binding protein delta (C/EBP delta) in epithelial lung cells.

CCAAT/enhancer-binding proteins (C/EBP) constitute a family of transcription factors that are involved in regulation of proliferation and differentiation in several cell types. In epithelial lung cells the C/EBP alpha isoform seems to play a role in the regulation of surfactant proteins (SP) and Clara cell specific protein (CCSP), whereas the roles of C/EBP beta and C/EBP delta are unclear. We have examined the protein levels of C/EBP delta in bronchiolar Clara cells and alveolar type 2 cells, and its relation to the expression of lung specific proteins and cell proliferation. The protein expression of C/EBP delta was high in freshly isolated Clara cells compared to type 2 cells. In both cell types C/EBP delta levels increased during culture. Alterations of the levels of C/EBP delta did not correspond with the proliferation levels of Clara cells, but seemed to correspond in type 2 cells. Clara cell secretory protein (CCSP) was highly expressed in freshly isolated Clara cells, in contrast to type 2 cells. SP-D and CYP2B1 were expressed at somewhat higher levels in Clara cells than in type 2 cells, whereas SP-A exhibited highest expression in type 2 cells. During culture the levels of all these lung proteins were strongly reduced. However, compared to with serum we found an increase in CCSP in Clara cell cultures without serum, and this correlated with an increase in C/EBP delta. Overall our in vitro data suggest that C/EBP delta alone is not related to the maintenance of proteins involved in differentiation.

Dimeric N-terminal segment of human surfactant protein B (dSP-B(1-25)) has enhanced surface properties compared to monomeric SP-B(1-25).

Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.

Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution.

Porcine organs and lung surfactant have medically important applications in both xenotransplantation and therapy. We have started to characterize porcine lung surfactant by cloning the cDNA of porcine surfactant protein D (SP-D). SP-D and SP-A are important mediators in innate immune defense for the lung and possibly other mucosal surfaces. Porcine SP-D will also be an important reagent for use in existing porcine animal models for human lung infections. The complete cDNA sequence of porcine SP-D, including the 5' and 3' untranslated regions, was determined from two overlapping bacteriophage clones and by PCR cloning. Three unique features were revealed from the porcine sequence in comparison to SP-D from other previously characterized species, making porcine SP-D an intriguing species addition to the SP-D/collectin family. The collagen region contains an extra cysteine residue, which may have important structural consequences. The other two differences, a potential glycosylation site and an insertion of three amino acids, lie in the loop regions of the carbohydrate recognition domain, close to the carbohydrate binding region and thus may have functional implications. These variations were ruled out as polymorphisms or mutations by confirming the sequence at the genomic level in four different pig breeds. Porcine SP-D was shown to localize primarily to the lung and with less abundance to the duodenum, jejunum, and ileum. The genes for SP-D and SP-A were also shown to colocalize to a region of porcine chromosome 14 that is syntenic with the human and murine collectin loci.