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The measured surface tension of a binary liquid is found to depend strongly on the constituents of the adjacent vapor and on whether equilibrium has been achieved, giving insight into the complex interfacial configuration. This dependence is quantified by three techniques that offer complementary insights: surface tension measurements with a constrained sessile drop surrounded by different vapors, surface tension measurements by surface light scattering spectroscopy in a sealed cell at equilibrium, and molecular dynamics simulations of the equilibrium surface tension and excess surface concentration. Ensuring homogeneity of the binary liquid, which is essential for surface light scattering, was found to be nontrivial and was assured by high-sensitivity Schlieren imaging. Two pairs of liquids, n -pentane with 2-methylpentane and n -pentane with n -hexane, were investigated. The first pair was motivated by the observed improvement in the effectiveness of binary fluids versus a single constituent in wickless heat pipes studied in microgravity. The second pair was used for comparison. Experimental evaluation of different volume fractions of the two liquids showed strong dependence of surface tension on the relative concentration of different molecules near the interfacial region. For the above pairs of liquids, which appear to form ideal mixtures in bulk, we present sufficiently precise surface tension measurements to indicate unexpectedly complex behaviors at interfaces.

A self-assembled phospholipid monolayer at an air-water interface is a well-defined model system for studying surface thermodynamics, membrane biophysics, thin-film materials, and colloidal soft matter. Here we report a study of two-dimensional phase transitions in the dipalmitoylphosphatidylcholine (DPPC) monolayer at the air-water interface using a newly developed methodology called constrained drop surfactometry (CDS). CDS is superior to the classical Langmuir balance in its capacity for rigorous temperature control and leak-proof environments, thus making it an ideal alternative to the Langmuir balance for studying lipid polymorphism. In addition, we have developed a novel Langmuir-Blodgett (LB) transfer technique that allows the direct transfer of lipid monolayers from the droplet surface under well-controlled conditions. This LB transfer technique permits the direct visualization of phase coexistence in the DPPC monolayer. With these technological advances, we found that the two-dimensional phase behavior of the DPPC monolayer is analogous to the three-dimensional phase transition of a pure substance. This study has implications in the fundamental understanding of surface thermodynamics as well as applications such as self-assembled monolayers and pulmonary surfactant biophysics.

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis-Constrained Sessile Drop (ADSA-CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression-Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Drop shape techniques are widely used for surface tension measurement. As the shape becomes close to spherical, the performance of drop shape techniques deteriorates. A quantitative criterion called shape parameter was previously introduced to quantify the meaning of "well-deformed" drops and "close to spherical" drops. In this paper, a modified definition of the shape parameter that does not depend on the radius of curvature at the drop apex is proposed. Such definition is applicable to different constellations of pendant and sessile drops/bubbles. Dimensional analysis is used to describe similarity in constrained sessile drop shapes and to express the problem using appropriate dimensionless groups. The proposed shape parameter is found to depend only on two dimensionless groups: the dimensionless volume (drop volume normalized by the cube of the holder radius) and the Bond number (using the drop holder's radius as the length scale). A critical shape parameter (minimum value of the shape parameter that guarantees a specified accuracy) is shown to depend only on Bond number. A set of experiments were performed with pure liquids to illustrate the change of the critical shape parameter with the Bond number.

Chitosan, a cationic polysaccharide, has been found to improve the surface activity of lung surfactant extracts in the presence of various inhibitors. It has been proposed that chitosan binds to anionic lipids (e.g. phosphatidyl glycerols) in lung surfactants, producing stable lipid films at the air-water interface. This binding also reverses the net charge of the surfactant aggregates, from negative to positive. Unfortunately, positively charged aggregates may adsorb or interact with the negatively charged epithelial tissue, leading to poor surfactant performance. To address this issue an anionic polysaccharide, dextran sulfate (dexS), was used as a secondary coating to reverse the charge of chitosan-lung surfactant extracts without affecting the surface activity of the preparation. The dynamic surface tension and zeta potential of bovine lipid extract surfactant (BLES) containing chitosan chloride (chiCl) and dexS were evaluated as a function of dexS concentration. These studies were conducted in the absence and presence of sodium bicarbonate buffer, and in the absence and presence of bovine serum used as model inhibitor. It was determined that using an appropriate concentration of dexS, especially at physiological pH, it is possible to restore the negative charge of the surfactant aggregates, and retain their surface activity, even in the presence of bovine serum. High concentrations of dexS affect the binding of chiCl to BLES, and the surface activity of the preparation., (2010 Elsevier B.V. All rights reserved.)

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113kDa and 213kDa. The other two additives were cationic peptides, polylysine 50kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive-surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50 microl/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive-surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500 microl/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant)., (Copyright 2010 Elsevier B.V. All rights reserved.)

This paper presents a continuation of the development of a drop shape method for film studies, ADSA-CSD (Axisymmetric Drop Shape Analysis-Constrained Sessile Drop). ADSA-CSD has certain advantages over conventional methods. The development presented here allows complete exchange of the subphase of a spread or adsorbed film. This feature allows certain studies relevant to lung surfactant research that cannot be readily performed by other means. The key feature of the design is a second capillary into the bulk of the drop to facilitate addition or removal of a secondary liquid. The development will be illustrated through studies concerning lung surfactant inhibition. After forming a sessile drop of a basic lung surfactant preparation, the bulk phase can be removed and exchanged for one containing different inhibitors. Such studies mimic the leakage of plasma and blood proteins into the alveolar spaces altering the surface activity of lung surfactant in a phenomenon called surfactant inhibition. The resistance of the lung surfactant to specific inhibitors can be readily evaluated using the method. The new method is also useful for surfactant reversal studies, i.e. the ability to restore the normal surface activity of an inhibited lung surfactant film by using special additives. Results show a distinctive difference between the inhibition when an inhibitor is mixed with and when it is injected under a preformed surfactant film. None of the inhibitors studied (serum, albumin, fibrinogen, and cholesterol) were able to penetrate a preexisting film formed by the basic preparation (BLES and protasan), while all of them can alter the surface activity of such preparation when mixed with the preparation. Preliminary results show that reversal of serum inhibition can be easily achieved and evaluated using the modified methodology.

A drop shape technique using a constrained sessile drop constellation (ADSA-CSD) has been introduced as a superior technique for studying spread films specially at high collapse pressures [Saad et al. Langmuir 2008, 24, 10843-10850]. It has been shown that ADSA-CSD has certain advantages including the need only for small quantities of liquid and insoluble surfactants, the ability to measure very low surface tension values, easier deposition procedure, and leak-proof design. Here, this technique was applied to investigate mixed DPPC/DPPG monolayers to characterize the role of such molecules in maintaining stable film properties and surface activity of lung surfactant preparations. Results of compression isotherms were obtained for different DPPC/DPPG mixture ratios: 90/10, 80/20, 70/30, 60/40, and 50/50 in addition to pure DPPC and pure DPPG at room temperature of 24 degrees C. The ultimate collapse pressure of DPPC/DPPG mixtures was found to be 70.5 mJ/m2 (similar to pure DPPC) for the cases of low DPPG content (up to 20%). Increasing the DPPG content in the mixture (up to 40%) caused a slight decrease in the ultimate collapse pressure. However, further increase of DPPG in the mixture (50% or more) caused a sharp decrease in the ultimate collapse pressure to a value of 59.9 mJ/m2 (similar to pure DPPG). The change in film elasticity was also tracked for the range of mixture ratios studied. The physical reasons for such changes and the interaction between DPPC and DPPG molecules are discussed. The results also show a change in the film hysteresis upon successive compression and expansion cycles for different mixture ratios.

Collapse pressure of insoluble monolayers is a property determined from surface pressure/area isotherms. Such isotherms are commonly measured by a Langmuir film balance or a drop shape technique using a pendant drop constellation (ADSA-PD). Here, a different embodiment of a drop shape analysis, called axisymmetric drop shape analysis-constrained sessile drop (ADSA-CSD) is used as a film balance. It is shown that ADSA-CSD has certain advantages over conventional methods. The ability to measure very low surface tension values (e.g., <2 mJ/m2), an easier deposition procedure than in a pendant drop setup, and leak-proof design make the constrained sessile drop constellation a better choice than the pendant drop constellation in many situations. Results of compression isotherms are obtained on three different monolayers: octadecanol, dipalmitoyl-phosphatidyl-choline (DPPC), and dipalmitoyl-phosphatidyl-glycerol (DPPG). The collapse pressures are found to be reproducible and in agreement with previous methods. For example, the collapse pressure of DPPC is found to be 70.2 mJ/m2. Such values are not achievable with a pendant drop. The collapse pressure of octadecanol is found to be 61.3 mJ/m2, while that of DPPG is 59.0 mJ/m2. The physical reasons for these differences are discussed. The results also show a distinctive difference between the onset of collapse and the ultimate collapse pressure (ultimate strength) of these films. ADSA-CSD allows detailed study of this collapse region.

The interaction between a cationic polyelectrolyte, chitosan, and an exogenous bovine lung extract surfactant (BLES) was studied using dynamic compression/expansion cycles of dilute BLES preparations in a Constrained Sessile Drop (CSD) device equipped with an environmental chamber conditioned at 37 degrees C and 100% R.H. air. Under these conditions, dilute BLES preparations tend to produce variable and relatively high minimum surface tensions. Upon addition of "low" chitosan to BLES ratios, the minimum surface tension of BLES-chitosan preparations were consistently low (i.e. <5 mJ/m2), and the resulting surfactant monolayers (adsorbed at the air-water interface) were highly elastic and stable. However, the use of "high" chitosan to BLES ratios induced the collapse of the surfactant monolayer at high minimum surface tensions (i.e. >15 mJ/m2). The zeta potential of the lung surfactant aggregates in the subphase suggests that chitosan binds to the anionic lipids (phosphatidyl glycerols) in BLES, and that this binding is ultimately responsible for the changes in the surface activity (elasticity and stability) of these surfactant-polyelectrolyte mixtures. Furthermore the transition from "low" to "high" chitosan to BLES ratios correlates with the flocculation and de-flocculation of surfactant aggregates in the subphase. It is proposed that the aggregation/segregation of "patches" of anionic lipids in the surfactant monolayer produced at different chitosan to BLES ratios explains the enhancing/inhibitory effects of chitosan. These observations highlight the importance of electrostatic interactions in lung surfactant systems.

The surface activity of bovine lipid extracted surfactant (BLES) preparations used in surfactant replacement therapy is studied in dynamic film compression/expansion cycles as a function of relative humidity, surfactant concentration, compression rate, and compression periodicity. BLES droplets were formed in a constrained sessile droplet configuration (CSD). Images obtained during cycling were analyzed using axisymmetric drop shape analysis (ADSA) to yield surface tension, surface area, and drop volume data. The experiments were conducted in a chamber that allowed both humid (100% RH), and "dry" air (i.e. less than 20% RH) environments. It was observed that in humid environments BLES films are not stable and tend to have poor surface activity compared to BLES films exposed to dry air. Further analysis of the data reveal that if BLES films are compressed fast enough (i.e. at physiological conditions) to avoid film hydration, lower minimum surface tensions are achieved. A film hydration-relaxation mechanism is proposed to explain these observations.

The effect of humidity on the film stability of Bovine Lipid Extract Surfactant (BLES) is studied using the captive bubble method. It is found that adsorbed BLES films show distinctly different stability patterns at two extreme relative humidities (RHs), i.e., bubbles formed by ambient air and by air prehumidified to 100% RH at 37 degrees C. The differences are illustrated by the ability to maintain low surface tensions at various compression ratios, the behavior of bubble clicks, and film compressibility. These results suggest that 100% RH at 37 degrees C tends to destabilize the BLES films. In turn, the experimental results indicate that the rapidly adsorbed BLES film on a captive bubble presents a barrier to water transport that retards full humidification of the bubble when ambient air is used for bubble formation. These findings necessitate careful evaluation and maintenance of environmental humidity for all in vitro assessment of lung surfactants. It is also found that the stability of adsorbed bovine natural lung surfactant (NLS) films is not as sensitive as BLES films to high humidity. This may indicate a physiological function of SP-A and/or cholesterol, which are absent in BLES, in maintaining the extraordinary film stability in vivo.

Chitosan is a natural, cationic polysaccharide derived from fully or partially deacetylated chitin. Chitosan is capable of inducing large phospholipid aggregates, closely resembling the function of nonionic polymers tested previously as additives to therapeutic lung surfactants. The effects of chitosan on improving the surface activity of a dilute lung surfactant preparation, bovine lipid extract surfactant (BLES), and on resisting albumin-induced inactivation were studied using a constrained sessile drop (CSD) method. Also studied in parallel were the effects of polyethylene glycol (PEG, 10 kD) and hyaluronan (HA, 1240 kD). Both adsorption and dynamic cycling studies showed that chitosan is able to significantly enhance the surface activity of 0.5 mg/mL BLES and to resist albumin-induced inactivation at an extremely low concentration of 0.05 mg/mL, 1000 times smaller than the usual concentration of PEG and 20 times smaller than HA. Optical microscopy found that chitosan induced large surfactant aggregates even in the presence of albumin. Cytotoxicity tests confirmed that chitosan has no deleterious effect on the viability of lung epithelial cells. The experimental results suggest that chitosan may be a more effective polymeric additive to lung surfactant than the other polymers tested so far.

It has been reported in the literature that sugars such as dextrose and sucrose increase the surface tension of water. The effect was interpreted as a depletion of the solute molecules from the water-air interface. This paper presents accurate measurements of the surface tension of different concentrations of dextrose solution as well as its polymer (i.e., dextran). An automated drop shape technique called axisymmetric drop shape analysis (ADSA) was used for the surface tension determination. The surface tension measurement is presented as a function of a shape parameter, P(s), which has been used to quantify the range of the applicability of ADSA. The results of the above study show that dextrose solutions decrease the surface tension of water in contradiction to the results obtained from the weight drop method in the literature. The surface tension decreases continuously with increasing concentration. A similar effect was observed for the dextran solutions. To verify that the setup and the methodology are capable of accurately measuring increases in surface tension, a similar experiment was conducted with a sodium chloride solution with a concentration of 1 M. It is well-known that electrolyte solutions, e.g., sodium chloride, increase the surface tension of water. The results obtained from ADSA verify that the sodium chloride increases the surface tension of water by 1.6 mJ/m(2). It is concluded that dextrose and dextran decrease the surface tension of water. Thus, there is no evidence of depletion. To identify the sources of discrepancy between the results of ADSA and those reported in the literature, the experiments were repeated for different concentrations and the rate of drop formation using the drop weight method. It was found that the rate of drop formation is most likely the source of error in the results reported in the literature.

The in vitro adsorption kinetics of lung surfactant at air-water interfaces is affected by both the composition of the surfactant preparations and the conditions under which the assessment is conducted. Relevant experimental conditions are surfactant concentration, temperature, subphase pH, electrolyte concentration, humidity, and gas composition of the atmosphere exposed to the interface. The effect of humidity on the adsorption kinetics of a therapeutic lung surfactant preparation, bovine lipid extract surfactant (BLES), was studied by measuring the dynamic surface tension (DST). Axisymmetric drop shape analysis (ADSA) was used in conjunction with three different experimental methodologies, i.e., captive bubble (CB), pendant drop (PD), and constrained sessile drop (CSD), to measure the DST. The experimental results obtained from these three methodologies show that for 100% relative humidity (RH) at 37 degrees C the rate of adsorption of BLES at an air-water interface is substantially slower than for low humidity. It is also found that there is a difference in the rate of surface tension decrease measured from the PD and CB/CSD methods. These experimental results agree well with an adsorption model that considers the combined effects of entropic force, electrostatic interaction, and gravity. These findings have implications for the development and evaluation of new formulations for surfactant replacement therapy.

Shortage or malfunction of pulmonary surfactant in alveolar space leads to a critical condition termed respiratory distress syndrome (RDS). Surfactant replacement therapy, the major method to treat RDS, is an expensive treatment. In this paper, the effect of poly(ethylene glycol) (PEG) to improve dynamic surface activity of a bovine lipid extract surfactant (BLES) was studied by axisymmetric drop shape analysis (ADSA) and a captive bubble method. The activity of BLES+PEG mixtures was compared to that of a natural surfactant containing surfactant proteins A and D. When PEG was added into BLES mixtures, the surface tension hysteresis of BLES films was minimized when the films were compressed by more than 50%. PEG also helps to quickly restore surfactant films after film collapse. Thus, as far as surface tension effects go, the findings suggest that PEG might be used as a substitute for surfactant-associated protein SP-A in therapeutic surfactant products, and might also be used to reduce the amount of BLES required in clinical applications.

Existing methodology for surface tension measurements based on drop shapes suffers from the shortcoming that it is not capable to function at very low surface tension if the liquid dispersion is opaque, such as therapeutic lung surfactants at clinically relevant concentrations. The novel configuration proposed here removes the two big restrictions, i.e., the film leakage problem that is encountered with such methods as the pulsating bubble surfactometer as well as the pendant drop arrangement, and the problem of the opaqueness of the liquid, as in the original captive bubble arrangement. A sharp knife edge is the key design feature in the constrained sessile drop that avoids film leakage at low surface tension. The use of the constrained sessile drop configuration in conjunction with axisymmetric drop shape analysis to measure surface tension allows complete automation of the setup. Dynamic studies with lung surfactant can be performed readily by changing the volume of a sessile drop, and thus the surface area, by means of a motor-driven syringe. To illustrate the validity of using this configuration, experiments were performed using an exogenous lung surfactant preparation, bovine lipid extract surfactant (BLES) at 5.0 mg/ml. A comparison of results obtained for BLES at low concentration between the constrained sessile drop and captive bubble arrangement shows excellent agreement between the two approaches. When the surface area of the BLES film (0.5 mg/ml) was compressed by about the same amount in both systems, the minimum surface tensions attained were identical within the 95% confidence limits.

The primary role of lung surfactant is to reduce surface tension at the air-liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3,350; 8,000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m(2). PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8,000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy.

Pulmonary surfactant is crucial to maintain the proper functioning of the respiration system. Certain types of blood proteins (e.g. albumin) were found to inhibit the activity of pulmonary surfactant. Axisymmetric Drop Shape Analysis (ADSA) was used to study the effect of dextran to restore the activity of an albumin-inhibited pulmonary surfactant. It was found that dextran could effectively restore surface tension properties of the inhibited surfactant in vitro. Furthermore, dextran improved the performance of pulmonary surfactants when albumin was absent. It was found that when a surfactant film was under high compression (e.g. above 70% surface area reduction), the presence of dextran increased film stability, so that the film could sustain high surface pressures without being collapsing.

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 +/- 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 +/- 410 pg/ml, P < 0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2 degrees +/- 2.2 degrees and 41.4 degrees +/- 3.2 degrees for C57BL/6 and gld, respectively) compared to uninfected mice (43.2 degrees +/- 2.3 degrees and 39.5 degrees +/- 1.6 degrees, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.

In a recent paper,(1) we demonstrated that molecular interactions between biopolymers and other smaller molecules can be detected by means of dynamic surface tension measurements. In the present paper, we demonstrate that the same methodology can be employed for investigating dose effects and specificity of molecular interactions. Three similar lipids were chosen for this study: 12(S)-hydroxy-5(Z), 8(Z),10(E),14(Z)-eicosatetraenoic acid (12(S)-HETE-free acid), methyl 12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoate (12(S)-HETE-methyl ester), and 5(Z),8(Z),11(Z), 14(Z)-eicosatetraenoic acid (arachidonic acid-free acid). These substances were added to a fatty acid free bovine serum albumin (BSA) aqueous solution at different lipid concentrations. The characteristic tension response indicates that molecular interactions between 12(S)-HETE-free acid and BSA exist. The detected interactions are concentration dependent: at a molecular ratio of lipid to protein of 1:1, the binding of 12(S)-HETE-free acid to BSA is hydrophobic in nature; at the molecular ratio of lipid to protein of 10:1, a secondary binding occurs and is hydrophilic in nature. Similar molecular interactions were not detected between 12(S)-HETE-methyl ester or arachidonic acid-free acid and BSA, indicating that the interactions between 12(S)-HETE-free acid and BSA are specific. As an independent means, surface elasticity is used to probe the molecular interactions at the interface. In the case of 12(S)-HETE-free acid but not its methyl ester or arachidonic acid, distinct higher surface elasticities were observed at lipid concentrations in excess of a molecular ratio of lipid to protein of 1:1. This finding reinforces the above stipulations.

Patients with cystic fibrosis (CF) have abnormal concentrations and composition of electrolytes and macromolecules in gastrointestinal secretions. Such alterations could change intestinal surface properties, such as surface hydrophobicity, and may influence the adhesion of macromolecules, bacteria, or microbial toxins to the intestinal surface. The objective of this study was to compare the surface hydrophobicity of the gastrointestinal tract in wild type and CF mice. We used axisymmetric drop shape analysis-contact diameter to determine surface hydrophobicity by measuring contact angles of sessile water droplets placed onto epithelial surfaces. In wild type mice, there were no differences in contact angles between the duodenum, upper jejunum, lower jejunum, and ileum. The contact angle of the gastric mucosa was lower than the rest of the gastrointestinal tract. Contact angles of the proximal colon and distal colon were both higher than that of the gastric mucosa and those of the small intestinal sections. In CF mice, contact angles along the gastrointestinal tract followed the same pattern as in wild type mice. However, contact angles in the ileum and proximal colon of CF mice were greater than those from wild type mice. This study of the murine intestine showed regional differences in surface hydrophobicity comparable to those observed in other mammalian species. In addition, we showed that the ileum and proximal colon of CF mice were more hydrophobic than the corresponding segments in wild type mice. These observations are of potential clinical relevance because patients with CF exhibit clinical manifestations of gastrointestinal disease primarily in the ileum and proximal colon.

Helicobacter mustelae is a gastric pathogen in ferrets that adheres to epithelial cells both in vitro and in vivo. In this study, the authors examine the role of surface hydrophobic properties in the adhesion of these organisms to eukaryotic cell surfaces. The surface properties of six H. mustelae strains were characterized by hydrophobic interaction chromatography (HIC), salt aggregation testing (SAT) and contact-angle measurement by axisymmetric drop-shape analysis (ADSA). Contact angles in multiple regions of the gastrointestinal tract, obtained from infected and uninfected ferrets, were also measured. The cell surface of H. mustelae was found to be hydrophilic by SAT but relatively hydrophobic by HIC. Contact-angle measurements for H.mustelae (mean 22.5 degrees, 95% confidence interval [CI] 9.3 degrees to 35.7 degrees) were higher than values previously reported for Helicobacter pylori (mean 12.1 degrees, 95% CI 2.0 degrees to 22.2 degrees, p < 0.05). The body of the stomach was more hydrophilic in infected ferrets (mean contact angle 59.9 degrees, 95% CI 52.5 degrees to 67.3 degrees) than in uninfected animals (mean contact angle 94.2 degrees, 95% CI 84.4 degrees to 104.0 degrees, p < 0.05). Reductions in the surface hydrophobicity of the ferrets' stomachs were correlated with the degree of mucosal inflammation (p < 0.01). These findings demonstrate that H. mustelae has surface properties comparable to those of H. pylori strains. Like the human stomach infected by H. pylori, there is a reduction in surface hydrophobicity of the ferret antrum associated with H. mustelae infection and the resulting mucosal inflammatory cell response.

To quantitate surface hydrophobicity of the stomach, we measured contact angles formed with water droplets in different regions of rabbit stomach at varying ages (suckling, weanling, and adult). Contact angles were measured using novel methods: axisymmetric drop-shape analysis-contact diameter for contact angles < 90 degrees and axisymmetric drop-shape analysis-maximum diameter for contact angles > 90 degrees. To determine whether gastric mucus was responsible for the physical properties of the surface mucosa, the surface tension of mucus derived from the body of stomach was measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult antrum 82.9 degrees +/- 5.5 degrees (mean +/- SEM) were greater than in the body of stomach (36.1 degrees +/- 2.6 degrees, p = 0.0001). Contact angles on mucosa obtained from the body of the stomach of both suckling rabbits (76.4 degrees +/- 2.7 degrees) and weanling rabbits (84.2 degrees +/- 2.9 degrees) were greater than in adult animals (ANOVA, p < 0.05). Pendant drop analysis of mucus derived from the body of stomach showed a high surface tension (57.72 +/- 0.06 mJ/m2, mean +/- SD). We conclude that there are maturational changes and regional differences in the surface hydrophobicity of the lapine stomach. These changes are likely caused by changes in the overlying mucus layer.

To quantitate surface hydrophobicity of the intestine, we measured contact angles formed with water droplets in multiple regions of rabbit intestine at varying ages (suckling, weanling, and adult) and after dinitrochlorobenzene-induced colitis. Contact angles were measured using novel methods: axisymmetric drop-shape analysis-contact diameter for contact angles less than 90 degrees and axisymmetric dropshape analysis-maximum diameter for contact angles greater than 90 degrees. To determine whether mucus was present on the surface of intestine used, indirect immunofluorescence was performed using antibody specific to goblet cell mucin. To confirm that intestinal mucus could be responsible for the physical properties of surface mucosa, surface tensions of mucus prepared from distal ileum, distal colon, and inflamed distal colon of adult rabbits were measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult small intestine [duodenum, 38.0 +/- 11.2 degrees (SD); jejunum, 44.0 +/- 22.9 degrees; ileum, 56.4 +/- 23.3 degrees] were less than proximal colon (93.2 +/- 6.7 degrees; P less than 0.05) and distal colon (86.4 +/- 24.2 degrees; P less than 0.05). Contact angles on proximal colon from suckling rabbits (53.2 +/- 8.4 degrees) were less than both weanling (93.2 +/- 23.3 degrees; P less than 0.05) and adult rabbits (93.2 +/- 6.7 degrees; P less than 0.05). Contact angles on inflamed adult distal colon (54.7 +/- 20.6 degrees) were decreased from values on normal distal colons (86.4 +/- 24.2 degrees). Indirect immunofluorescence demonstrated that mucin was present in both vacuoles of goblet cells and on the colonic surface.(ABSTRACT TRUNCATED AT 250 WORDS)

There appears to be a particular association between Helicobacter pylori and the gastric antrum, but the mechanisms by which the organism adheres to and colonizes the gastric mucosa are unclear. Surface hydrophobicity and surface charge mediate the adherence of other bacterial pathogens to mucosal epithelial cell surfaces. Therefore, in this study we characterized both the surface hydrophobicity and the surface charge of 10 H. pylori strains grown in broth culture. Four complementary methods were used to determine hydrophobicity: hydrophobic interaction chromatography, the salt aggregation test, comparison of bacterial adherence to polystyrene with adherence to sulfonated polystyrene, and measurement of contact angle with droplets of water. Three of the methods (salt aggregation test, adherence to polystyrene, and contact angles) indicated that each of the 10 strains expressed a relatively hydrophilic cell surface. In contrast, hydrophobic interaction chromatography determinations with both phenyl- and octyl-Sepharose suggested that the H. pylori strains were relatively hydrophobic. However, tetramethyl urea (0.4 M) did not reduce the binding of H. pylori to phenyl-Sepharose columns. DEAE-cellulose ion-exchange chromatography showed that each of the 10 strains of H. pylori had a surface which, overall, was highly negatively charged. We conclude that H. pylori expresses an overall relatively hydrophilic and negatively charged surface in vitro.

The aim was to clarify whether or not sudden spike concentrations of plasma growth hormone (GH) can affect the endocrine pancreas in vivo. The peaking of GH was reproduced by an injection (10 mg/kg iv) of bovine GH to anesthetized normal, pancreatectomized, and alloxan-diabetic dogs. In portal but not in peripheral blood, immunoreactive plasma glucagon (IRG), glucagon-like activity (GLI), and immunoreactive insulin (IRI), were significantly elevated within 10 min in normal and alloxan-diabetic dogs. In pancreatectomized dogs, GH did not affect either IRG or GLI. When a physiological dose of GH (6 microgram/kg) calculated to produce ambient peak plasma concentrations of 40 ng/ml was given to four conscious, normal dogs with indwelling portal catheters, a rise of IRG from 108 +/- 19 to 170 +/- 17 pg/ml and of IRI from 20 +/- 12 to 67 +/- 19 muU/ml (mean +/- SE) occurred within 2 min. GLI was not affected. Thus a sudden rise in GH concentration can stimulate the release of a) GLI in the presence but not in the absence of the pancreas, and b) pancreatic IRG and IRI but not extrapancreatic IRG.

Normal dogs were injected i.v. with a single dose of 0.25 mg/kg sodium salt of HB 419 (Glibenclamide). Plasma insulin and glucose concentrations were measured at stated intervals over a period of two hours. The rise in insulin, but not the hypoglycemic response was abolished in peripheral blood when the animals were pretreated with a single i.v. injection of either 0.1 mg/kg or 0.3 mg/kg dl-propranolol, 30 min prior to the administration of HB 419. The d-isomer of propranolol was ineffective in this respect. These results indicate that a) the mechanism by which propranolol inhibits sulphonylurea-stimulated insulin secretion involves beta adrenergic receptors; and b) the hypoglycemia produced by HB 419 in the presence of propranolol could be the result of extrapancreatic effects. Since the possibility of an early rise in the insulin concentration of portal blood was not excluded in our present series of experiments, final proof will have to be provided by studies in totally pancreatectomized dogs.

Sampling portal blood every 15 min by means of indwelling cannulae, we have found evidence that basal insulin secretion in nonanesthetized dogs takes place in six or seven major secretory episodes over a 24-h period. Reproducible patterns were obtained in four experiments conducted on three normal animals. When frequency of peaks was plotted against the log of insulin concentration, a normal distribution was obtained (lognormal distribution). The means and variances were nearly the same in the three animals. Low levels of insulin (10--25 microU/ml) alternated with peaks which were occasionally higher than 200 microU/ml. The average duration of a peak was 30 min and seemed to be independent of ambient glucose levels. It is suggested that these peaks are the result of surges in plasma growth hormone concentrations and/or spontaneous repetitive discharges along neuronal pathways functionally related to insulin secretion.

For a given temperature the extent of platelet adhesion increased with increasing substrate surface tension, in agreement with earlier findings. For a given substrate platelet adhesion decreases linearly with decreasing temperature. Morphometric evaluation of the adhering platelets indicated that the extent of platelet spreading as a function of temperature follows the same pattern as the extent of platelet adhesion. Smaller quantities of ADP were required to induce aggregation at the reduced temperatures. Substrate roughness did not appear to influence the extent of platelet adhesion to any of the surfaces for any of the temperatures examined.

The lipolytic responses to dihydroergotamine (5 mug/mi) of isolated rat adipocytes andto epinephrine (50 ng/m1 and 1 mug/m1) of both rat and dog adipocytes were measured over a period of two hours. Glycerol release was calculated as a function of either glycerol ester content or of cell number per incubation vial. The conventional calculation based on glyceride content gave the following results: a) epididymal fat cells from rats weighing less than 160 gm were more sensitive to dihydroergotamine and epinephrine than were fat cells from heavier animals weighing close to 200 gm; b) dog adipocytes from abdominal subcutaneous tissue were less sensitive to eipnephrine than were rat adipocytes. No such differences were noted when the same data were calculated on the basis of cell numbers per incubation vial: the lipolytic response was uniform with rat adipocytes obtained either from the lighter or the heavier weight groups, and so was the response of dog and rat cells. These results indicate that in the type of studies where receptor-dependent hormone and drug effects are measured, the number of cells per incubation vial is an important parameter to be considered in evaluating a lipolytic response.

The insulinogenic response to a standard i.v. dose of a sulfonylurea can be markedly augmented in normal, conscious dogs if they are given 30 min earlier a single i.v. dose of dihydroergotamine (DHE). Since the parent substance ergotamine posssed no such amplifying properties, further experiments were conducted to clarify the essential structural requirements that have to be fulfilled for an ergot alkaloid to act as an amplifier of sulfonylurea-stimulated insulin secretion. Amine alkaloids ergonovine, dihydroergonovone and dihydromethylergonovine had no amplifying potency, but the hydrogenated amino acid alkaloids dihydroergocornine, dihydroergocristine and dihydroergokryptine (Hydergine) were almost as potent amplifiers as was DHE. The data indicate that (a) DHE, Hydergine and by inference all hydrogenated amino acid alkaloids are potent amplifiers of sulfonylurea-stimulated insulin secretion; (b) saturation of the double bond at C9 and C10 of the lysergic acid moiety and the pressence of an amino acid side chain are essential structual requirements for an ergot alkaloid to function as an amplifier of the action of sulfonylureas; and (c) it appears that these compounds are acting chiefly by mechanisms other than alpha-adrenergic and serotonergic receptor blockade, perhaps as regulatory molecules inducing positive cooperative changes in integral proteins of the plasma membrane of beta cells.

Normal dogs were injected i.v. with 0.25 mg/kg sodium salt of HB 419 (glibenclamide) and plasma insulin concentrations were measured over a period of 2 hrs. When the animals were given a single i.v. injection of 0.2 mg/kg dihydroergotamine tartrate (DHE) 30 min prior to the administration of HB 419, the insulinogenic effect of the sulfonylurea was considerably amplified (192 muU/ml vs 34 muU/ml at 45 min). No augmentation of the insulinogenic effect of HB 419 was observed when the same experiments were conducted with 0.05, 0.025 or 0.01 mg/kg ergotamine tartrate. At the dose level of 0.1 mg/kg the insulinogenic effect of HB 419 was suppressed. Since the structural difference between these two ergot alkaloids consists of the presence or absence of the double bond at C9 and C10 of the lysergic acid moiety, it appears that saturation of this double bond is an essential structural requirement for DHE to function as an amplifier of sulfonylurea-stimulated insulin release.

We investigated whether substrates with different surface tensions would induce a different degree of conformational change in adsorbed protein molecules, which would be reflected by differences in the surface tension of the adsorbed layers. The solidification front technique allowed this study without requiring to expose the protein coated-particle to an air interface which would induce conformational changes in the adsorbed protein layer. With a low bulk albumin concentration (0.1%) decreasing surface tension of the adsorbed protein layer with increasing hydrophobicity of the substrate, suggested more extensive conformational changes on the more hydrophobic surfaces. At high bulk concentrations (0.5% and above) the surface tension of the adsorbed albumin layer was independent of the substrate material and increased to a value of approximately 70.2 ergs/cm2. This was consistent with the surface tension of albumin derived from other independent techniques such as contact angle measurements on thick layers of the protein material or from adsorption data. Freezing front measurements with albumin (BSA or HSA), immunoglobulin G (IgG), and fibrinogen adsorbed onto one and the same substrate material, octyl-sepharose beads, indicate that the hydrophobicity of the protein coated sepharose increased in the following order: BSA less than HSA less than IgG less than Fibrinogen. This was in good agreement with the relative hydrophobicity of these proteins determined by other independent methods.

The role of hydrophobicity in the attachment of enteropathogens to gastrointestinal mucosa is controversial. In vitro binding of Escherichia coli RDEC-1 to rabbit intestine is dependent on the expression of pili. We examined in vitro adherence of piliated RDEC-1 after altering either the hydrophobicity of the organisms, the hydrophobicity of the substrate for attachment, or the surface tension of the suspending liquid. Hydrophobicity of RDEC-1 was determined using four complementary methods. In each assay piliated RDEC-1 demonstrated relatively more hydrophobic properties compared with both organisms grown to suppress pilus expression and a mutant that cannot express mannose-resistant pili. When piliated RDEC-1 were pretreated with tetramethyl urea to disrupt hydrophobic bonds surface hydrophobicity decreased. Concurrently, bacterial adherence to rabbit ileal microvillus membranes, mucus and mucin was reduced. Binding of piliated organisms to hydrophobic surfaces was significantly higher compared to both nonpiliated bacteria and the adherence of piliated RDEC-1 to relatively hydrophilic surfaces. Addition of propanol reduced the surface tension of the suspending liquid, and decreased adhesion of piliated RDEC-1 to polystyrene by 80%. Conversely, adherence of piliated organisms to a hydrophilic surface increased 12-fold after lowering the surface tension of the suspending liquid. We conclude that hydrophobic properties have a role in mediating in vitro adherence of this E. coli enteric pathogen.

The adhesion of five strains of bacteria, i.e., Staphylococcus aureus (strain 049), Staphylococcus epidermidis (strain 047), Escherichia coli (strains 055 and 2627), and Listeria monocytogenes, to various polymeric surfaces was studied. The design of the experimental protocol was dictated by thermodynamic considerations. From the thermodynamic model for the adhesion of small particles from a suspension onto a solid substratum, it follows that the extent of adhesion is determined by the surface properties of all three phases involved, i.e., the surface tensions of the adhering particles, of the substrate, and of the suspending liquid medium. In essence, adhesion is more extensive to hydrophilic substrata (i.e., substrata of relatively high surface tension) than to hydrophobic substrata, when the surface tension of the bacteria is larger than that of the suspending medium. When the surface tension of the suspending liquid is larger than that of the bacteria, the opposite pattern of behavior prevails. Suspensions of bacteria at a concentration of 10(8) microorganisms per ml were brought into contact with several polymeric surfaces (Teflon, polyethylene, polystyrene, and acetal and sulfonated polystyrene) for 30 min at 20 degrees C. After rinsing, the number of bacteria adhering per unit surface area was determined by image analysis. The surface tension of the suspending medium. Hanks balanced salt solution, was modified through the addition of various amounts of dimethyl sulfoxide. It was found that the number of bacteria adhering per unit surface area correlates well with the thermodynamic predictions and that these data may be used to determine the surface tension of the different bacterial species. The surface tensions of the bacteria obtained in this fashion are in excellent agreement with those obtained by other methods.

The solidification front technique is employed to determine the surface tension of fixed erythrocytes of dog, horse, human, chicken, and turkey. The results range from 65.5 erg/cm2 for dog erythrocytes to 67.6 erg/cm2 for turkey erythrocytes. A detailed error analysis shows that the differences obtained are statistically significant. Since cellular interactions are governed to a considerable extent by surface tension effects, it is concluded that caution needs to be exercised when results obtained for one species are used to predict the behavior of cells of another species.