Effect of low level direct current on in vivo tumor growth in hamsters.

A preliminary study has been carried out on the effect of low level direct current on tumor growth using an experimental tumor model developed from an amelanotic melanoma (T1-4) in the hamster. An inoculum of 2 X 10(6) viable cells was injected s.c. on day 0; on day 7 the tumor-bearing animals were randomly divided into treatment and control groups. On days 7 through 11 inclusive, the treatment group was subjected to electrical current (direct current) at levels from 0.1 to 2.4 mA, for 1 h/day under general anesthesia. Control groups were subjected to the same procedures, with the exception that the electrodes were not connected to the current source. On day 14, the animals were killed and autopsied; their tumors were removed, weighed, and sectioned. Treated tumors decreased in mass (as a percentage of controls) from 89% at 0.1 mA to 2% at 2.4 mA. Increased necrosis of the treated tumors was noted macroscopically and microscopically. On histological examination, it was observed that a thin rim of viable cells remained around the periphery after treatment even at the highest current levels. Similar results were obtained with both stainless steel and platinum-30% iridium electrodes. In separate experiments where the animals were allowed to survive after a treatment period (1 h/day for 5 days at 2.4 mA), the viable cells at the periphery developed into tumors whose mass at 28 days posttreatment averaged only 52% of that of the control tumors. The mechanism of growth reduction is unknown but hyperthermia was shown not to be a factor.

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins.

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.

Determination of the Surface tension of proteins. I. Surface tension of native serum proteins in aqueous media.

The desorption patterns of serum proteins in hydrophobic chromatography suggest that serum proteins that remain immersed in an aqueous medium and do not become in a protein-air interface are very hydrophilic. Contact angle measurements on fairly thick layers of hydrated serum proteins, formed on ultrafiltration membranes, yield surface tensions that correlate well with the degree of hydrophilicity derived from desorption data obtained by hydrophobic chromatography. For further confirmation the absorptivity of four human serum proteins was measured with respect to surfaces of different polymers of various surface tensions, for solution in aqueous solvents of different surface tensions. The surface tension of the solvent from which a dissolved protein adsorbs to precisely the same extent onto all solid substrates (regardless of their surface tensions) is equal to the surface tension of that protein. The surface tensions found by the contact angle (first value given) and by the protein adsorption methods (second value given) were. in erg/cm2; alpha 2-macroglobulin, 71.0, 71.0; serum albumin, 70.5, 70.2; immunoglobulin M, 69.5, 69.4; immunoglobulin G, 67.4, 67.7.

Determination of surface tensions of proteins. II. Surface tension of serum albumin, altered at the protein-air interface.

Serum albumin, which itself has a surface tension of congruent to 70.3 erg/cm2, when dissolved in water lowers the surface tension of water from 72.5 to congruent to 50 erg/cm2, as measured by a variety of means, including the pendant drop, the Wilhelmy plate and the platinum ring methods. Equally low and even lower surface tensions are found with the contact angle method, on a thin layer of albumin that had been adsorbed onto a low energy surface and subsequently exposed to air. Surface tensions of drops of albumin solutions varying in concentration from 0.01 to 5.5% (w/v) yielded, with a contact angle method, values that only varied between 67 and 61 erg/cm2. With the pendant drop, the Wilhelmy plate and the platinum ring methods, one essentially measures the surface tension at the air-liquid interface, at which proteins tend to adsorb, and where reversible or irreversible reorientation can be expected. The same holds for a thin layer of protein adsorbed onto a low energy surface, exposed to air. Thus, when through the very act of surface tension measurement, or after adsorbing protein onto a substrate, protein is exposed at the air-liquid interface, it apparently loses the pronounced hydrophilicity characteristic of its native hydrated state and manifests through reorientation a much more hydrophobic tertiary configuration.

Interaction of phagocytes with other blood cells and with pathogenic and nonpathogenic microbes.

Owing to the high surface tension of blood cells and to the equally high surface tension of their liquid habitat, the Hamaker coefficients A131 of blood cells (subscript 1) in blood (subscript 3), are unusually small; they are of the order of 0.25 to 2.5 X 10(-16) ergs. The very small van der Waals attractions such low Hamaker coefficients give rise to, coupled to the medium low but still sizable negative xi-potentials (-11 to -18 mV) of the cells, which cause an appreciable mutual electrostatic repulsion between blood cells, have been used to elaborate potential energy vs. distance diagrams, which closely reflect the unusual stability of blood cells in blood. When bacteria find their way into the bloodstream, they initially form an almost equally stable suspension. However, relatively hydrophobic nonpathogenic bacteria quickly aspecifically adsorb immunoglobulin G (IgG) molecules from blood serum, whilst hydrophilic pathogenic bacteria sooner or later also become coated with specific antibody molecules of the IgG-class. Through receptor sites on the surface of phagocytic blood cells, which can specifically bind to the Fc tails of IgG molecules, bacteria are first bound and then removed from the blood circulation and surrounding tissues. These Fc-receptor bonds presumably also are of a combined van der Waals and electrostatic nature. Thus in the normal course of events and by purely physicochemical mechanisms, phagocytic leukocytes will neither interfere with other leukocytes nor with any other blood cells, whilst they specifically interact with microorganisms and other unwanted foreign particles via IgG-IgG-receptor interactions. Also discussed, in the light of the principles elaborated above, are: some of the antiphagocytic mechanisms developed by certain pathogenic bacteria; the phagocytic disposal of aged, weak, or abnormal blood cells; and the role played by immunoglobulins other than IgG, and by complement, in the removal of bacteria and viruses.

Nebulized vs. instilled exogenous surfactant in an adult lung injury model.

Three days after subcutaneous injection of N-nitroso-N-methylurethane (NNNMU) to induce lung injury, adult rabbits were mechanically ventilated and lung function was evaluated. Each animal then received either nebulized Survanta (Neb Surv), nebulized saline (Neb Saline), nebulized gas alone (Neb Gas), or tracheally instilled Survanta (Inst Surv). The ventilation efficiency index (VEI) value increased significantly compared with pretreatment values (P less than 0.01) over a 3-h treatment period for the Neb Surv animals, whereas VEI values for the other three groups decreased after treatment (P less than 0.05). Arterial PO2-to-fraction of inspired O2 ratios and dynamic compliance values significantly decreased after treatment for the Inst Surv group (P less than 0.05). Pressure-volume curves demonstrated a significantly greater volume at maximal pressure for the Neb Surv group compared with each of the other groups studied (P less than 0.01). The calculated quantity of surfactant recovered in lung tissue for the Neb Surv group was only 4.9 +/- 1.0 mg lipid/kg compared with 100 mg lipid/kg delivered to the Inst Surv group. Surfactant administered as an aerosol resulted in modest physiological improvements in this model of lung injury and was superior to the tracheal instillation technique.

Lung function and surfactant distribution in saline-lavaged sheep given instilled vs. nebulized surfactant.

Adult sheep (35 +/- 3 kg) underwent saline lung lavage and 1.5 h of mechanical ventilation to induce acute lung injury. Animals received 100 mg lipid/kg body wt of tracheally instilled surfactant (Inst Surf) or either nebulized surfactant (Neb Surf) or nebulized saline (Neb Saline) and were killed 3 h later. Inst Surf and Neb Surf groups had significant improvements in oxygenation (P < 0.01) and peak inspiratory pressures (PIP) (P < 0.05) compared with pretreatment values. Improvements in oxygenation and PIP for Inst Surf animals were significantly greater than for Neb Surf animals (P < 0.05). Volumes of maximal pressure of quasi-static pressure-volume curves measured at the time the animals were killed were significantly greater for Inst Surf and Neb Surf animals than for animals given Neb Saline (P < 0.05). Alveolar recovery of exogenous surfactant was 100 times greater for Inst Surf animals (1,732 +/- 70 mg) than for Neb Surf animals (15.3 +/- 2.9 mg) at the time they were killed. Although there were no differences in exogenous surfactant distribution patterns at the lobar level between the two surfactant-treated groups, distribution histograms calculated for 10-g lung pieces revealed the Neb Surf animals had significantly more pieces within 25% of the mean value of 1.0 (42.7 +/- 6.9%) than did Inst Surf animals (20.8 +/- 5.5%) (P < 0.01). Exogenous surfactant therapy improved lung function with significantly different quantities of surfactant deposited in lung tissue for the two delivery methods evaluated.

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.

Structure and functions of a dimeric form of surfactant protein SP-C: a Fourier transform infrared and surfactometry study.

Surfactant proteins SP-B (M(r) = 8700, reduced) and SP-C (M(r) = 3000-6000, major form, non-reduced) interact with surfactant phospholipids to enhance their surface active properties. In the present study, we describe the structural and functional characteristics of a novel dimeric form of bovine SP-C (M(r) = 9000, non-reduced), which is identified as [SP-C]2. Dimeric SP-C exhibits surface tension-lowering properties differing from those of monomeric SP-C and enhances the surface properties of bovine SP-B/phospholipid mixtures. Chemical analysis indicated that [SP-C]2 was not acylated at the cysteinyl residues. Fourier transform-infrared spectroscopy (FT-IR) was utilized to determine the secondary structures of [SP-C]2 in DPPC films. Relative percentages of alpha-helical, beta-sheet, beta-turn and random coil structures were calculated by peak fit analysis of the amide I band of the FT-IR spectra indicating that, in contrast to the helical structure of monomeric SP-C, [SP-C]2 exhibits almost exclusively beta-sheet structure. In addition, only 10% of the amide (backbone) hydrogens exchanged with deuterium of D2O, indicating that the remaining 90% of amide hydrogens were not accessible to D2O due to strong hydrogen bonding or their location in a hydrophobic environment. Dimerization of SP-C effects a major change in secondary structure, a factor which may play a role in the interaction of SP-C with phospholipids in pulmonary surfactant.

The role of bacterial hydrophobicity in infection: bacterial adhesion and phagocytic ingestion.

The role that bacterial surface hydrophobicity (surface tension) plays in determining the extent of adhesion of polymer substrates and phagocytic ingestion is reviewed. The early attachment phase in bacterial adhesion is shown to depend critically on the relative surface tensions of the three interacting phases; i.e., bacteria, substrate, and suspending liquid surface tension. When suspended in a liquid with a high surface tension such as Hanks balanced salt solution, the most hydrophobic bacteria adhere to all surfaces to the greatest extent. When the liquid surface tension (gamma LV) is larger than the bacterial surface tension (gamma BV), then for any single bacterial species the extent of adhesion decreases with increasing substrate surface tension (gamma SV). When gamma LV less than gamma BV then adhesion increases with increasing gamma SV. Bacterial surface tension also determines in part the extent of phagocytic ingestion and the degree to which antibodies specifically adsorb onto the bacterium resulting in opsonization. The nonspecific adsorption of antibodies results in a considerable modification in the surface properties of the bacteria. Bacterial surface hydrophobicity can be altered significantly through exposure to subinhibitory concentrations of antibiotics, surfactants, lectins, etc. The effect of these changes on subsequent phagocytic ingestion is discussed.

The nature of the antigen-antibody bond and the factors affecting its association and dissociation.

Considering that generally both Coulombic and van der Waals' bonds occur in AG-AB interactions, for AG-AB dissociation both interactions have to be made repulsive simultaneously. The theory and practice of repulsive van der Waals' interactions are outlined. Also treated are the hysteresis of AG-AB dissociation contrasted with the inhibition of association; dissociation of hydrogen bonds; dissociation by haptens; dissociation by alteration of tertiary and secondary configurations; and physical methods of dissociation.