Comparison of Polymyxin E and Polymyxin B as an Additive to Pulmonary Surfactant in Escherichia coli Pneumonia of Ventilated Neonatal Rabbits.

BACKGROUND: Ascending maternofetal bacterial infections often result in premature birth and neonatal respiratory distress. These neonates are treated with exogenous pulmonary surfactant (SF) and systemic antibiotics. Polymyxins are antimicrobiotic peptides that may bind to SF phospholipids. OBJECTIVES: Does topical administration of SF/polymyxin reduce bacterial growth in neonatal rabbit pneumonia and improve pulmonary function? METHODS: Neonatal rabbits were tracheotomized and treated intratracheally with mixtures of porcine SF, SF/polymyxin E (PxE), or polymyxin B (PxB). Control animals received saline. Animals were then inoculated with Escherichia coli and ventilated for 4 h. During the experiment, peak insufflation pressures, dynamic lung compliance, and ECG were recorded. Pulmonary and renal bacterial load were determined. Lung histology was performed. Lung and kidney IL-8 were measured in subgroups. RESULTS: Eighty-five animals were included in 2 experimental series, of which 78% survived 4 h of ventilation. E. coli inoculation caused severe neonatal pneumonia with median IL-8 levels of 2.2 ng/g in the lungs compared to a median of 0.2 ng/g in the lungs of the saline controls (p < 0.01). Lung compliance after 4 h was significantly increased at a mean of 0.48 ml/(kg·cm H2O) in the SF group and 0.43 in the SF + PxE group compared to 0.35 in the E. coli group (p < 0.01). In direct comparison, bacterial growth found in the E. coli group was reduced 20-fold in the SF + PxB group compared to 75-fold in the SF + PxE group. CONCLUSION: Addition of polymyxin to SF effectively promotes antimicrobial treatment and improves lung function in neonatal pneumonia of rabbits.

Phospholipid Composition in Synthetic Surfactants Is Important for Tidal Volumes and Alveolar Stability in Surfactant-Treated Preterm Newborn Rabbits.

BACKGROUND: The development of synthetic surfactants for the treatment of lung pulmonary diseases has been going on for many years. OBJECTIVES: To investigate the effects of phospholipid mixtures combined with SP-B and SP-C analogues on lung functions in an animal model of respiratory distress syndrome. METHODS: Natural and synthetic phospholipid mixtures with/without SP-B and/or SP-C analogues were instilled in ventilated premature newborn rabbits. Lung functions were evaluated. RESULTS: Treatment with Curosurf or phospholipids from Curosurf combined with SP-B and SP-C analogues gave similar results. Treatment with phospholipids from adult rabbit lungs or liver combined with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG) gave tidal volumes (VT) well above physiological levels, but alveolar stability at end-expiration was only achieved when these phospholipids were combined with analogues of SP-B and SP-C. Treatment with egg yolk-PC mixed with DPPC with and without POPG gave small VT, but after addition of both analogues VT was only somewhat lower and lung gas volumes (LGV) similar to those obtained with Curosurf. Substitution of egg yolk-PC (≥99% PC) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and combining them with DPPC, POPG and 2% each of the SP-B and SP-C analogue gave a completely synthetic surfactant with similar effects on VT and LGV as Curosurf. CONCLUSIONS: Phospholipid composition is important for VT while the SP-B and SP-C analogues increase alveolar stability at end-expiration. Synthetic surfactant consisting of unsaturated and saturated phosphatidylcholines, POPG and the analogues of SP-B and SP-C has similar activity as Curosurf regarding VT and LGV in an animal model using preterm newborn rabbits ventilated without positive end-expiratory pressure.

Properties of modified natural surfactant after exposure to fibrinogen in vitro and in an animal model of respiratory distress syndrome.

BACKGROUND: Plasma proteins are known to interfere with pulmonary surfactant. Studies have proven the hypothesis that fibrinogen preserves exogenous surfactant subjected to long-term surface area cycling. METHODS: The exogenous surfactant Curosurf was subjected to long-term surface area cycling without or with fibrinogen (ratio 2:1 w/w) and was tested by captive bubble surfactometer and on newborn premature rabbits. RESULTS: Surface tension increased in Curosurf (80 mg/ml) samples without fibrinogen after 6-12 d of cycling. In samples with fibrinogen the cycling time had no effect on surface tension. Addition of fibrinogen to surfactant prevented lipid peroxidation. Lung gas volumes of animals with noncycled Curosurf or Curosurf cycled with fibrinogen for 6 d were comparable and higher than in rabbits with Curosurf cycled without fibrinogen. Alveolar volume density was higher in groups with noncycled Curosurf or Curosurf cycled with fibrinogen than in Curosurf cycled without fibrinogen (both P < 0.001). CONCLUSION: The effect of fibrinogen on pulmonary surfactant cycled at 37 °C depends both on surfactant concentration and cycling time. At high phospholipid concentration used in clinical practice fibrinogen has a protective effect on biophysical and physiological properties of natural modified surfactant subjected to surface area cycling. This effect is partially mediated by reduction in lipid peroxidation.

Alterations of the C-terminal end do not affect in vitro or in vivo activity of surfactant protein C analogs.

The secondary structure, orientation and hydrogen/deuterium exchange of SP-C33, a surfactant protein C analog, in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/egg phosphatidylglycerol (8:2, wt./wt.) bilayers, was studied by attenuated total reflection Fourier transform infrared spectroscopy. This showed a transmembrane α-helix, in which about 55% of the amide hydrogens do not exchange for up to 20 h. Moreover, C-terminally modified SP-C33, either truncated after position 30, or having the methionine at position 31 exchanged for either lysine or isoleucine, showed the same secondary structure and orientation. The different peptides, suspended in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 68:31 (wt./wt.), were tested for surfactant activity in vitro in a captive bubble surfactometer and in vivo in an animal model of respiratory distress syndrome using premature rabbit fetuses. All preparations showed similar surface activity in the captive bubble surfactometer. Also, in the rabbit model, all preparations performed equally well and significantly better than non-treated controls, both regarding tidal volumes and lung gas volumes. Thus, truncation or residue replacements in the C-terminal part of SP-C33 do not seem to affect membrane association or surfactant activity.

Prophylactic intratracheal polymyxin B/surfactant prevents bacterial growth in neonatal Escherichia coli pneumonia of rabbits.

In neonatal pneumonia, the surface activity of pulmonary surfactant is impaired and microorganisms may invade by passing the air-liquid interface. Previously, we have shown that addition of the antimicrobial peptide polymyxin B (PxB) to modified porcine surfactant (pSF) improves resistance to surfactant inactivation in vitro while antimicrobial activity of PxB is maintained. In this study, we investigated pSF/PxB in vivo. Neonatal near-term rabbits were treated with intratracheal pSF and/or PxB. Rabbits treated with only saline served as controls. Animals were ventilated with standardized tidal volumes and received ∼10(7) Escherichia coli intratracheally. Plethysmographic pressure-volume curves were recorded every 30 min. After 240 min, animals were killed, the right lung and left kidney were excised, and bacterial growth was determined. The left lung was used for histologic analysis. Intratracheal administration of PxB ± pSF significantly reduced the growth of E. coli compared with control animals or animals receiving only pSF. This was accompanied by reduction of severe inflammatory tissue destruction and significantly reduced bacterial translocation to the left kidney. Animals receiving pSF + PxB had no difference in lung compliance compared with the pSF- or PxB-treated group. Mixtures of PxB and pulmonary surfactant show antimicrobial effects in neonatal rabbits and prevent systemic spreading of E. coli.

Inactivation of pulmonary surfactant by silicone oil in vitro and in ventilated immature rabbits.

OBJECTIVE: Surface activity of pulmonary surfactant is impaired by exposure to syringes lubricated with silicone oil (SO). These syringes are used daily in clinical practice. DESIGN: In vitro experiments were used for detection of SO, determination of surface activity, and semiquantitative measurement of surfactant protein (SP)-B and -C in SO/surfactant mixtures. Randomized, controlled animal studies were applied for determination of in vivo activity. SETTING: University research laboratory. INTERVENTIONS: Mass spectrometry of SO originating from syringes with and without surfactant was performed. The surface activity of SO plus surfactant phospholipids (PLs) or modified natural surfactant (Curosurf) was measured. SO/Curosurf preparations were further analyzed for changes in the content of SP-B and SP-C using immunoblotting. Neonatal rabbits received mixtures of SO/Curosurf (ratio 0-1.3 mg/mg PL) intratracheally and were then ventilated with a standardized sequence of peak insufflation pressures. Tidal volume curves were recorded, gas volumes of excised lungs were measured, and histologic analysis was performed. MEASUREMENTS AND MAIN RESULTS: Dissolved SO was found after rinsing syringes with organic solvents or Curosurf. Surface activity of Curosurf was significantly reduced after addition of 0.13-1.3 mg SO/mg PL. Immunoblotting revealed interference of SO with SP-B, but not with SP-C. With increasing SO/Curosurf ratios, patchy alveolar air expansion was observed, lung gas volumes were reduced, and time to inflate the lungs was increased, whereas compliance and tidal volumes remained unimpaired. CONCLUSIONS: In vitro SO impairs surface activity of Curosurf and leads to interference with SP-B. SO contamination of exogenous surfactant impairs lung function in animal studies and should be avoided.

Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome.

Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of approximately 5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.

Influence of partial liquid ventilation on bacterial growth and alveolar expansion in newborn rabbits with group B-streptococcal pneumonia.

Partial liquid ventilation (PLV) with perfluorocarbons has been considered as an alternative therapy for severe inflammatory lung disease. The present study was performed to test whether PLV influences bacterial growth and lung histology in a rabbit model of congenital pneumonia caused by group B streptococci. Near-term newborn rabbits were tracheotomized, inoculated via the airways with group B streptococci, and subsequently ventilated for 5 h with either PLV or conventional ventilation. At 30 min after group B streptococci administration, animals in the PLV group (n = 16) received 30 mL/kg body weight of perfluorocarbon (PF 5080) via the tracheal tube. Evaporative losses were substituted with 20 mL/kg perfluorocarbon at hourly intervals. Identical volumes of air were injected in control animals at the same times (n = 15). The number of colony-forming units in left lung homogenate, evaluated at the end of the experiments, tended to be lower in PLV-treated animals than in controls (6.8 x 109 versus 6.4 x 1010 colony-forming units/g body weight; p = 0.06). Comparison of these numbers with the colony-forming units injected at the beginning of the experiments revealed a reduction in bacterial number in the PLV group and proliferation in the controls (-2.2 x 108 versus +5.6 x 1010 colony-forming units/g body weight; p < 0.05). Histologic examination demonstrated less inflammation and more homogeneous lung expansion in PLV-treated animals. Two animals in the PLV group had focal interstitial emphysema. Our results suggest that PLV with PF 5080 reduces bacterial proliferation in experimental group B streptococcal pneumonia.