Calf Lung Research Articles

Bovine respiratory syncytial virus lacking the virokinin or with a mutation in furin cleavage site RA(R/K)R109 induces less pulmonary inflammation without impeding the induction of protective immunity in calves

The BRSV fusion (F) protein is cleaved at two furin consensus sequence sites, resulting in the generation of disulphide-linked F1 and F2 subunits and the release of an intervening peptide of 27 amino acids (pep27), which is converted into a biologically active tachykinin (virokinin). The role of the virokinin and the importance of one of the furin cleavage sites, FCS-2 [RA(R/K)R109], in the pathogenesis of BRSV infection and in the subsequent development of immunity was studied in gnotobiotic calves infected with a recombinant BRSV (rBRSV) lacking pep27 (rBRSVdelta p27) or with rBRSV108/109, which contains two amino acid substitutions in FCS-2 (RANN109). Although replication of the mutant viruses and the parental wild-type (WT) rBRSV in the lungs was similar, the extent of gross and microscopic lesions induced by the mutant viruses was less than that induced by WT rBRSV. Furthermore, the numbers of eosinophils in the lungs of calves infected with the mutant viruses were significantly less than that in calves infected with WT virus. These observations suggest a role for the virokinin in the pathogenesis of BRSV infection. Following mucosal immunization with rBRSVdelta p27, the levels of BRSV-specific serum antibodies were similar to those induced by WT virus. In contrast, the level of neutralizing antibodies induced by rBRSV108/109 was 10-fold lower than that induced by WT virus. Nevertheless, resistance to BRSV challenge induced by the mutant and WT viruses was similar, suggesting that neither pep27 nor FCS-2 plays a major role in the induction of protective immunity.

Comparison of variable and conventional ventilation in a sheep saline lavage lung injury model*

There has recently been considerable interest in alternative lung-protective ventilation strategies such as variable ventilation (VV). We aimed at testing VV in a large animal lung injury model and exploring the mechanism of improvement in gas exchange seen with VV. Randomized, controlled comparative ventilation study. Research laboratory at a veterinary hospital. Female sheep weighing 59.8 +/- 10.57 kg and excised calf lungs. In a sheep saline lavage model of lung injury, we applied VV, whereby tidal volume (VT) and frequency (f) varied on each breath. Sheep were randomized into one of two groups (VV, n = 7; or control, n = 6) and ventilated for 4 hrs with all mean ventilation settings matched. Gas exchange, lung mechanics, and hemodynamic measures were recorded over the 4 hrs. VV sheep showed improvement in gas exchange (i.e., oxygenation and carbon dioxide elimination) and ventilation pressures (i.e., reduced mean and peak airway pressures) but control sheep did not. VV sheep also displayed lower-lung elastance and mechanical heterogeneity in comparison with control sheep from 2 to 4 hrs of ventilation. To study the mechanism behind improvements seen with VV, we examined the time course associated with the enhanced recruitment occurring during VV in eight saline-lavaged excised calf lungs. We found that the recruitment associated with a larger VT during VV lasted over 200 secs, nearly an order of magnitude greater than the average time interval between large VT deliveries during VV. The application of VV in a large animal model of lung injury results in improved gas exchange and superior lung mechanics in comparison with CV that can be explained at least partially by the long-lasting effects of the recruitments occurring during VV.

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO 2-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO 2-lipid and DEPN-8 reached maximum surface pressures of 72 mN/m (minimum surface tensions of <1 mN/m) in compressed films on the Wilhelmy balance (23 °C). Dispersions of DEPN-8 (0.5 mg/ml) and SO 2-lipid (2.5 mg/ml) also reached minimum surface tensions of <1 mN/m on a pulsating bubble surfactometer (37 °C, 20 cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO 2-lipid + 0.75% SP-B + 0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO 2-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10 wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO 2-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A 2 (PLA 2), while CLSE was severely inhibited by PLA 2. The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO 2-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.

Inhibition of surfactant activity byPneumocystis cariniiorganisms and components in vitro

This study examines the direct inhibitory effects of Pneumocystis carinii (Pc) organisms and chemical components on the surface activity and composition of whole calf lung surfactant (WLS) and calf lung surfactant extract (CLSE) in vitro. Incubation of WLS suspensions with intact Pc organisms (10(7) per milligram of surfactant phospholipid) did not significantly alter total phospholipid levels or surfactant protein A content. Incubation with intact Pc organisms also did not impair dynamic surface tension lowering in suspensions of WLS or centrifuged large surfactant aggregates on a bubble surfactometer (37 degrees C, 20 cycles/min, 0.5 and 2.5 mg phospholipid/ml). However, exposure of WLS or CLSE to disrupted (sonicated) Pc organisms led to severe detriments in activity, with minimum surface tensions of 17-19 mN/m vs. <1 mN/m for surfactants alone. Extracted hydrophobic chemical components from Pc (98.8% lipids, 0.1 mM) reduced the surface activity of WLS and CLSE similarly to sonicated Pc organisms, whereas extracted hydrophilic chemical components from Pc (primarily proteins) had only minor effects on surface tension lowering. These results indicate that in addition to surfactant dysfunction induced by inflammatory lung injury and edema-derived inhibitors in Pc pneumonia, disrupted Pc organisms in the alveolar lumen also have the potential to directly inhibit endogenous and exogenous lung surfactants in affected patients.

Mononuclear cell subsets in bronchoalveolar lavage fluid duringDictyocaulus viviparusinfection of calves: a potential role for γ/δ TCR‐expressing cells in airway immune responses?

Mononuclear cell populations in the lungs of calves infected with Dictyocaulus viviparus were studied during primary infection and reinfection in order to identify cells involved in development of protective immunity to parasitic bronchitis. Three groups of calves were either inoculated with 500 third-stage larvae at both weeks 0 and 10 (n = 6), inoculated only at week 10 (n = 6), or remained uninfected (n = 3). The animals were monitored weekly by collection of bronchoalveolar lavage fluid (BALF), blood and faeces. Among mononuclear BALF-cell populations, the gamma/delta TCR-expressing cells showed a pronounced transient increase in proportion as well as in relative cell size 2 weeks post primary infection, whereas CD4-, CD8-, Ig- and CD14-expressing cells showed no significant differences related to the infection. The increase in gamma/delta TCR-expressing cells coincided with significantly increased proportions of eosinophils and recovery of adult worms in BALF. After reinfection, gamma/delta TCR-expressing cells increased again, but not until week 3 post inoculation, whereas eosinophils were increased by week 2 and reached higher levels than after primary infection. After reinfection, establishment of D. viviparus was less successful than after primary infection. In conclusion, these results indicate a role for gamma/delta TCR-expressing lymphocytes in the pathogenesis of D. viviparus infection.

Lung Surfactant Gelation Induced by Epithelial Cells Exposed to Air Pollution or Oxidative Stress

Lung surfactant lowers surface tension and adjusts interfacial rheology to facilitate breathing. A novel instrument, the interfacial stress rheometer (ISR), uses an oscillating magnetic needle to measure the shear viscosity and elasticity of a surfactant monolayer at the air-water interface. The ISR reveals that calf lung surfactant, Infasurf, exhibits remarkable fluidity, even when exposed to air pollution residual oil fly ash (ROFA), hydrogen peroxide (H2O2), or conditioned media from resting A549 alveolar epithelial cells (AEC). However, when Infasurf is exposed to a subphase of the soluble fraction of ROFA- or H2O2-treated AEC conditioned media, there is a prominent increase in surfactant elasticity and viscosity, representing two-dimensional gelation. Surfactant gelation is decreased when ROFA-AEC are pretreated with inhibitors of cellular reactive oxygen species (ROS), or with a mitochondrial anion channel inhibitor, as well as when A549-rho0 cells that lack mitochondrial DNA and functional electron transport are investigated. These results implicate both mitochondrial and nonmitochondrial ROS generation in ROFA-AEC-induced surfactant gelation. A549 cells treated with H2O2 demonstrate a dose-dependent increase in lung surfactant gelation. The ISR is a unique and sensitive instrument to characterize surfactant gelation induced by oxidatively stressed AEC.

Outbreaks of pneumonia in beef calves associated with bovine viral diarrhea virus seroconversion and other respiratory pathogens

The present study describes the clinical, serological and bacteriolological findingsin calves from two beef herds experiencing outbreaks of pneumonia. The clinical signs were nasal discharge, cough, pyrexia and increased respiratory rates. The morbidity and mortality rates over a month period were 40.72% and 15.63% respectively. Laboratory investigations revealed that bovine viral diarrhea virus (BVDV) was involved in and probably initiated both outbreaks as indicated by a significant increase in antibody titers against BVDV in sera of convalescent calves (paired serum samples). No antibodies bovine herpesvirus-1 (BHV-1), bovine respiratory syncytial virus (BRSV) and parainfluenza-3 (BPIV-3) viruses were detected in both acute and convalescent sera. Mycoplasma bovis was concurrently demonstrated in lungs of affected calves as it was isolated from 13 (81.25%) of examined lungs suggesting that there may be a synergism between bovine viral diarrhea virus and Mycoplasma bovis in the pathogenesis of pneumonia. A total of 15 (68.18%) isolates of Mannheimia haemolytica, 5 (22.73%) Pasteurella multocida, 1 (4.54%) Pseudomonase aerugenosa, 3 (13.64%) Staphylococcus aureus, 3 (13.64%) Actinomycis pyogenes, 1 (4.54%) Klebsiella pneumonae, 1 (4.54%) Streptococcus pneumonae, 2 (9.09%) E. coli and 2 (9.09%) Aspergellus fumigatus were recovered from lungs of calves suffering from pneumonia.

Modifying Calf Lung Surfactant by Hexadecanol

Monolayer properties such as phase behavior, collapse pressure, and surface viscosity are determined by monolayer composition. Learning how to control these properties through simple additives is important to understanding lung surfactant monolayers and to designing optimal replacement surfactants for treatment of disease. The properties of Infasurf, a replacement lung surfactant derived from calf lung lavage organic extract, can be modified in a controlled fashion by adding hexadecanol (HD). Grazing incidence X-ray diffraction shows that the HD preferentially interacts with dipalmitoylphosphatidylcholine (DPPC), the main phospholipid component of Infasurf, in the same way as in binary mixtures of DPPC and HD. HD intercalates between the DPPC chains, which leads to a tighter packing of the two-dimensional lattice and greater stability of the solid phase. This molecular reorganization triggers changes at the macroscopic scale, leading to a greatly increased solid-phase fraction at a given surface pressure and order of magnitude increases in the surface shear viscosity. However, the collapse pressure decreases, and, hence, the minimum surface tension of the film increases.

COMPARISON OF THREE LIPID FORMULATIONS FOR SYNTHETIC SURFACTANT WITH A SURFACTANT PROTEIN B ANALOG

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic dimeric SP-B1–25 (SP-B1–25), a peptide based on the N-terminal domain of human SP-B, efficiently mimics the functional properties of SP-B. The authors investigated the optimum lipid composition for SP-B1–25 by comparing the effects of natural lung lavage lipids (NLL), a synthetic equivalent of NLL (synthetic lavage lipids SLL), and a standard lipid mixture (TL) on the activities of SP-B1–25. Surfactant preparations were formulated by mixing 2 mol% SP-B1–25 in NNL, SLL, and TL. Calfactant, a calf lung lavage extract with SP-B and SP-C, was a positive control and lipids without peptide were negative controls. Minimum surface tension measured on a captive bubble surfactometer was similar for the three SP-B1–25 surfactant preparations and calfactant. The effects on lung function were compared in ventilated, lavaged, surfactant-deficient rats. Oxygenation and lung volumes were consistently higher in rats treated with calfactant and SP-B1–25 in NLL or SLL than in rats treated with SP-B1–25 in TL. Fourier transform infrared spectra observed abnormal secondary conformations for SP-B1–25 in TL as a possible cause for the reduced lung function. Lipid composition plays a crucial role in the in vitro and in vivo functions of SP-B1–25 in surfactant preparations.

SP-B and SP-C Alter Diffusion in Bilayers of Pulmonary Surfactant

The hydrophobic proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface by an unknown mechanism. We tested the hypothesis that these proteins accelerate adsorption by disrupting the structure of the lipid bilayer, either by a generalized increase in fluidity or by a focal induction of interfacial boundaries within the bilayer. We used fluorescence recovery after photobleaching to measure diffusion of nitrobenzoxadiazolyl-dimyristoyl-phosphatidylethanolamine between 11 and 54°C in multilayers containing the complete set of lipids and proteins in calf lung surfactant extract (CLSE), or the complete set of neutral and phospholipids without the proteins. Above 35°C, Arrhenius plots of diffusion were parallel for CLSE and neutral and phospholipids, but shifted to lower values for CLSE, suggesting that the proteins rigidify the lipid bilayer rather than producing the proposed increase in membrane fluidity. The slopes of the Arrhenius plots for CLSE were steeper below 35°C, suggesting that the proteins induce phase separation at that temperature. The mobile fraction fell below 27°C, consistent with a percolation threshold of coexisting gel and liquid-crystal phases. The induction of lateral phase separation in CLSE, however, does not correlate with apparent changes in adsorption kinetics at this temperature. Our results suggest that SP-B and SP-C accelerate adsorption through a mechanism other than the disruption of surfactant bilayers, possibly by stabilizing a high-energy, highly curved adsorption intermediate.

Immunohistopathological findings in the lungs of calves naturally infected with Mycoplasma bovis.

Histology and immunohistochemistry were used to analyse the lesions and distribution of Mycoplasma bovis antigen in the lungs of 18 naturally infected calves. Microscopic examination of pneumonic lungs revealed two distinct patterns of necrosis and inflammation. The first pattern was observed in six of 18 (33.3%) calves in which microscopic lesions were characterized by large irregular areas of coagulative necrosis surrounded by a dense zone of degenerated neutrophils. Moderate amounts of mycoplasmal antigen were in the centre and periphery of these necrotic foci and, to a lesser extent, in mononuclear cells of the peribronchial lymphoid tissue. The second pattern was observed in 18 of 18 (100%) calves and consisted of rounded foci of caseous necrosis composed by granular eosinophilic material surrounded by a rim of granulation tissue. Large amounts of M. bovis antigen were detected in the centre and periphery of these necrotic foci and, to a lesser extent, in the peribronchial lymphoid tissue, and alveolar and interstitial macrophages. It was concluded that both caseous and coagulative necrosis of the lung parenchyma was primarily caused by M. bovis. Infection with M. bovis should be suspected in bovine necrotic bronchopneumonia, particularly in cases in which the pulmonary necrosis is part of a pyogranulomatous inflammation centred around airways. The pattern of caseous necrosis with pyogranulomatous inflammation is characteristic of M. bovis infection while the pattern of coagulative necrosis is similar to and must be differentiated from Mannheimia haemolytica and Haemophilus somnus infection.

Surfactant abnormalities after single lung transplantation in dogs: impact of bronchoscopic surfactant administration

Objective Disturbances of the alveolar surfactant system have been implicated in the pathogenesis of reperfusion injury. The aim of this study was to evaluate the influence of exogenous surfactant administration on surfactant properties in a model of single lung transplantation. Methods We performed heterologous, left lung transplantation (+4°C ischemia; 24 hours, Euro-Collins solution) in 6 foxhounds (untreated) and in 6 animals that received calf lung surfactant extract (Alveofact) prior to explantation (only donor lung; 50 mg/kg body weight) and immediately after onset of reperfusion (both lungs, 200 mg/kg body weight). Separate but synchronized ventilation of each lung was performed, in a volume-controlled, pressure-limited mode, with animals in prone position. Bronchoalveolar lavage fluids were collected in pretransplantation lungs (control), after 24 hours of ischemia prior to transplantation (0 hours) and 6 and 12 hours after reperfusion in both the grafts and the recipient native lungs. Results Ischemic storage per se did not provoke any changes of the surfactant system; however, severe alterations occurred within 6 hours of reperfusion, resulting in a severe loss of surface activity, including a decrease in the percentage of the large surfactant aggregate fraction, reduction of the surfactant apoproteins SP-B and SP-C, the dipalmitoyl molecular species of phosphatidylcholine and phosphatidylglycerol within the large surfactant aggregate fraction. These abnormalities were restricted to the graft, with virtually normal surfactant function and composition being found in the recipient native lung. Surfactant administration fully normalized the biochemical and largely improved the biophysical surfactant properties, alongside maintenance of lung gas exchange properties. Conclusions Severe surfactant abnormalities occur exclusively in the graft when performing separate, synchronized ventilation of each lung to attenuate ventilator-induced lung injury. Bronchoscopic surfactant administration provides protection against these abnormalities and may be a therapeutic strategy in lung transplantation.

Non-cooperative effects of lung surfactant proteins on early adsorption to an air/water interface

Two small hydrophobic proteins, SP-B and SP-C, are responsible for rapid adsorption of pulmonary surfactant to the air/water interface. Despite their physiological importance, the number of protein molecules required to trigger an absorption event remains unknown. To investigate this issue, we varied the protein content of calf lung surfactant extract (CLSE) by dilution with protein-depleted surfactant lipids (neutral and phospholipids, N&PL). Vesicles of a constant size and of composition ranging between 100% N&PL and 100% CLSE were generated by probe sonication. Their adsorption kinetics to an air/water interface were monitored at different temperatures using a Wilhelmy plate to measure surface tension. When plotted versus protein concentration, the adsorption rates during the initial change in surface tension exhibit a diphasic behavior, first increasing rapidly and linearly between 0% and 25% CLSE, and then more slowly at higher concentrations. Direct linearity at low protein content (0–5% CLSE ratio) was confirmed at 37 °C. These observations argue against cooperative behavior, for which the adsorption rate would first rise slowly with the protein content, and then increase suddenly once the critical number of proteins on each vesicle is reached. The apparent activation energy Ea and the free energy of activation ΔG0*, calculated from the temperature dependence of adsorption, further support the view that at least the early stages of protein-induced surfactant adsorption proceeds through a sequence of events involving not several, but a single surfactant protein.

Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine.

Surface activity and sensitivity to inhibition from phospholipase A2 (PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions <1 mN/m under dynamic compression on the Wilhelmy balance and on a pulsating bubble surfactometer (37 degrees C, 20 cycles/min, 50% area compression). DEPN-8 + 1.5% SP-B/C exhibited even greater adsorption and had overall dynamic surface tension lowering equal to CLSE on the bubble. In addition, films of DEPN-8 + 1.5% SP-B/C on the Wilhelmy balance had better respreading than CLSE after seven (but not two) cycles of compression-expansion at 23 degrees C. DEPN-8 is structurally resistant to degradation by PLA2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2 led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.

Liquid-Crystalline Collapse of Pulmonary Surfactant Monolayers

During exhalation, the surfactant film of lipids and proteins that coats the alveoli in the lung is compressed to high surface pressures, and can remain metastable for prolonged periods at pressures approaching 70 mN/m. Monolayers of calf lung surfactant extract (CLSE), however, collapse in vitro, during an initial compression at ∼45 mN/m. To gain information on the source of this discrepancy, we investigated how monolayers of CLSE collapse from the interface. Observations with fluorescence, Brewster angle, and light scattering microscopies show that monolayers containing CLSE, CLSE-cholesterol (20%), or binary mixtures of dipalmitoyl phosphatidylcholine(DPPC)-dihydrocholesterol all form bilayer disks that reside above the monolayer. Upon compression and expansion, lipids flow continuously from the monolayer into the disks, and vice versa. In several respects, the mode of collapse resembles the behavior of other amphiphiles that form smectic liquid-crystal phases. These findings suggest that components of surfactent films must collapse collectively rather than being squeezed out individually.

Coordinated expression of tracheal antimicrobial peptide and inflammatory-response elements in the lungs of neonatal calves with acute bacterial pneumonia.

Lung tissue removed from neonatal calves with acute Mannheimia haemolytica pneumonia showed that rapid up-regulation of the basal mRNA expression of tracheal antimicrobial peptide (TAP), NF-kappa B, and intercellular adhesion molecule 1 occurred after infection; TAP and interleukin 8 expression were highly correlated. This work suggests that the coordinated expression of beta-defensin and inflammatory elements occurs during bacterial pneumonia.

Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on Binary Phospholipid Monolayers: II. Infrared External Reflectance-Absorption Spectroscopy

In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d 62:DPPG, and 7:1 DPPC-d 62:DOPG, alone and in the presence of 0.5–10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH 2 antisymmetric and symmetric stretching bands (∼2920 and 2852 cm −1) along with the analogous CD 2 stretching bands (∼2194 and 2089 cm −1) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1–2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH 2 and CD 2 stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d 62:DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d 62:DOPG monolayers, but in this case an increase in CH 2 band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD 2 band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d 62 chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d 62:DPPG. SP-B/C had no significant effect on DPPC-d 62 structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.

A selectin inhibitor decreases neutrophil infiltration during acute Mannheimia haemolytica pneumonia.

The degree to which the selectin inhibitor TBC1269 reduces neutrophil infiltration in specific microscopic locations of the lung during acute pneumonia of neonates was determined. Neonatal calves were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 2 and 6 hours postinoculation (PI). One 6-hour group inoculated with M. haemolytica received TBC1269 intravenously before and after inoculation with M. haemolytica. Infiltrates of neutrophils were significantly higher in the alveolar lumen and septae but lower in the bronchial lumen and epithelium at 6 hours PI than at 2 hours PI. Significantly fewer neutrophils (P < 0.05) were present in the alveolar lumen and septae, and the bronchiolar lumen and lamina propria in the lungs of TBC1269-treated calves compared with untreated calves at 6 hours PI. TBC1269 did not alter the infiltration into bronchi and blood vessels or the expression of the selectin-independent adhesion molecule, ICAM-1. This work suggests that during acute pneumonia of neonates 1) neutrophil infiltrates progressively increase in the alveolar lumens and septae but decrease in the bronchial lumen and epithelium with time, 2) TBC1269 reduces neutrophil infiltration into specific regions of alveoli and bronchioles rather than uniformly throughout the lung, and 3) selectin inhibition does not affect the location and intensity of ICAM-1 expression.

Content-dependent activity of lung surfactant protein B in mixtures with lipids.

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.

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

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