Receptor-independent, direct membrane binding leads to cell-surface lipid sorting and Syk kinase activation in dendritic cells.

Binding of particulate antigens by antigen-presenting cells is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. By using atomic force microscopy as a tool for real-time single-cell activation analysis, we report that uric acid crystals could directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein-based cellular contacts. Binding of particulate substances activated Syk kinase-dependent signaling in dendritic cells. These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell-surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation, and adjuvanticity.

Methyl-beta-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol.

Pulmonary surfactant, a defined mixture of lipids and proteins, imparts very low surface tension to the lung-air interface by forming an incompressible film. In acute respiratory distress syndrome and other respiratory conditions, this function is impaired by a number of factors, among which is an increase of cholesterol in surfactant. The current study shows in vitro that cholesterol can be extracted from surfactant and function subsequently restored to dysfunctional surfactant films in a dose-dependent manner by methyl-beta-cyclodextrin (MbetaCD). Bovine lipid extract surfactant was supplemented with cholesterol to serve as a model of dysfunctional surfactant. Likewise, when cholesterol in a complex with MbetaCD ("water-soluble cholesterol") was added in aqueous solution, surfactant films were rendered dysfunctional. Atomic force microscopy showed recovery of function by MbetaCD is accompanied by the re-establishment of the native film structure of a lipid monolayer with scattered areas of lipid bilayer stacks, whereas dysfunctional films lacked bilayers. The current study expands upon a recent perspective of surfactant inactivation in disease and suggests a potential treatment.

A comparative study of mechanisms of surfactant inhibition.

Pulmonary surfactant spreads to the hydrated air-lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air-lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air-water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.

Pulmonary surfactant function is abolished by an elevated proportion of cholesterol.

A molecular film of pulmonary surfactant strongly reduces the surface tension of the lung epithelium-air interface. Human pulmonary surfactant contains 5-10% cholesterol by mass, among other lipids and surfactant specific proteins. An elevated proportion of cholesterol is found in surfactant, recovered from acutely injured lungs (ALI). The functional role of cholesterol in pulmonary surfactant has remained controversial. Cholesterol is excluded from most pulmonary surfactant replacement formulations, used clinically to treat conditions of surfactant deficiency. This is because cholesterol has been shown in vitro to impair the surface activity of surfactant even at a physiological level. In the current study, the functional role of cholesterol has been re-evaluated using an improved method of evaluating surface activity in vitro, the captive bubble surfactometer (CBS). Cholesterol was added to one of the clinically used therapeutic surfactants, BLES, a bovine lipid extract surfactant, and the surface activity evaluated, including the adsorption rate of the substance to the air-water interface, its ability to produce a surface tension close to zero and the area compression needed to obtain that low surface tension. No differences in the surface activity were found for BLES samples containing either none, 5 or 10% cholesterol by mass with respect to the minimal surface tension. Our findings therefore suggest that the earlier-described deleterious effects of physiological amounts of cholesterol are related to the experimental methodology. However, at 20%, cholesterol effectively abolished surfactant function and a surface tension below 15 mN/m was not obtained. Inhibition of surface activity by cholesterol may therefore partially or fully explain the impaired lung function in the case of ALI. We discuss a molecular mechanism that could explain why cholesterol does not prevent low surface tension of surfactant films at physiological levels but abolishes surfactant function at higher levels.

The surface activity of pulmonary surfactant from diving mammals.

Pinnipeds (seals and sea lions) have developed a specialised respiratory system to cope with living in a marine environment. They have a highly reinforced lung that can completely collapse and reinflate during diving without any apparent side effects. These animals may also have a specialised surfactant system to augment the morphological adaptations. The surface activity of surfactant from four species of pinniped (California sea lion, Northern elephant seal, Northern fur seal and Ringed seal) was measured using a captive bubble surfactometer (CBS), and compared to two terrestrial species (sheep and cow). The surfactant of Northern elephant seal, Northern fur seal and Ringed seal was unable to reduce surface tension (gamma) to normal levels after 5 min adsorption (61.2, 36.7, and 46.2 +/- 1.7 mN/m, respectively), but California sea lion was able to reach the levels of the cow and sheep (23.4 mN/m for California sea lion, 21.6 +/- 0.3 and 23.0 +/- 1.5 mN/m for cow and sheep, respectively). All pinnipeds were also unable to obtain the very low gamma(min) achieved by cow (1.4 +/- 0.1 mN/m) and sheep (1.5 +/- 0.4 mN/m). These results suggest that reducing surface tension to very low values is not the primary function of surfactant in pinnipeds as it is in terrestrial mammals, but that an anti-adhesive surfactant is more important to enable the lungs to reopen following collapse during deep diving.

A statistical analysis of the social behavior of the male stumptail macaque (Macaca arctoides).

The social behavior of male stumptail macaques was analyzed in terms of behavioral sequences recorded during paired encounters in a large test cage. Recurrent patterns of behavioral sequences were sought and used to hypothesize the structure of motivational systems of social behavior as has been done previously for other species. In addition to traditional statistical analyses to determine which dyadic behavioral sequences were nonrandom, there were several methodological innovations. Instead of analyzing behavior as a single channel of communications, we analyzed three independent channels and considered their inter-correlations: 1) acts and postures; 2) vocalizations; and 3) facial expressions. Also, we analyzed not only within-animal behavioral sequences but between-animal sequences as well. Results were derived from 40 tests, most of which included vigorous agonistic and sexual interactions and a behavioral repertoire similar to that of adult male stumptail macaques observed by previous investigators. There were 30 acts and postures, eight facial expressions, and seven vocalizations that occurred more than five times. Many acts and postures occurred in nonrandom sequences, 43 such sequences within-animal and 40 between-animal. From these sequences and their correlations with specific vocalizations and facial expressions, it was possible to differentiate six categories of social behavior that may correspond to six different motivational systems: offense, defense, submission, groom and contact, male sexual behavior, and display. Both the frequency of behaviors in each category and the nature of the behavioral sequences were affected by the relative dominance of the two animals.

Nebulizing poractant alfa versus conventional instillation: Ultrastructural appearance and preservation of surface activity.

BACKGROUND: Nebulized surfactant therapy has been proposed as an alternative method of surfactant administration. The use of a perforated vibrating membrane nebulizer provides a variety of advantages over conventional nebulizers. We investigated the molecular structure and integrity of poractant alfa pre- and post-nebulization. METHOD: Curosurf® was nebulized using an Investigational eFlow® Nebulizer System. Non-nebulized surfactant ("NN"), recollected surfactant droplets from nebulization through an endotracheal tube ("NT") and nebulization of surfactant directly onto a surface ("ND") were investigated by transmission electron microscopy. Biophysical characteristics were assessed by the Langmuir-Wilhelmy balance and the Captive Bubble Surfactometer. RESULTS: Volume densities of lamellar body-like forms (LBL) and multi-lamellar forms (ML) were high for "NN" and "NT" samples (38.8% vs. 47.7% for LBL and 58.2% vs. 47.8% for ML). In the "ND" sample, we found virtually no LBL's, ML's (72.6%) as well as uni-lamellar forms (16.4%) and a new structure, the "garland-like" forms (9.4%). Surface tension for "NN" and "NT" was 23.33 ± 0.29 and 25.77 ± 1.12 mN/m, respectively. Dynamic compression-expansion cycling minimum surface tensions were between 0.91 and 1.77 mN/m. CONCLUSION: The similarity of surfactant characteristics of nebulized surfactant via a tube and the non-nebulized surfactant suggests that vibrating membrane nebulizers are suitable for surfactant nebulization. Alterations in surfactant morphology and characteristics after nebulization were transient. A new structural subtype of surfactant was identified.