© 2002 American Thoracic Society
Surfactant lavage demonstrates protein fibrils in a neonate with congenital surfactant protein b deficiencyTo the Editor:Genetic deficiency of surfactant protein B (SP-B) is a rare condition (1), resulting in severe respiratory failure in combination with accumulation of proteinaceous material in the terminal airspaces in term neonates. Short-term improvement has been reported after surfactant instillation (2). We describe a term male neonate (weight: 3,900 g) with congenital SP-B deficiency who was treated with a novel technique for bronchoalveolar lavage (BAL) with diluted surfactant. The baby was transferred on Day 7 of life with the presumed diagnosis of group B streptococcal sepsis and pulmonary hypertension. Chest x-rays demonstrated bilateral opacification. Surfactant bolus instillation had already been given on Day 3, resulting in short-term improvement. From the third week of life on, progressive respiratory failure developed that did not respond to high-frequency oscillatory ventilation. In BAL fluid, high protein content but no mature SP-B (ELISA; Professor Seeger and Dr. Günther, Giessen, Germany) was found. However, before the results of the DNA analysis for hereditary SP-B deficiency became available, the child developed hypoxemia on Day 21. A Portex 4.0 endotracheal tube was inserted, and separate surfactant lavage of the right and the left lung lobe was performed under continued mechanical ventilation using a 8 F cardiac balloon-tipped catheter placed in the right or the left main bronchus over a bronchoscopy adapter by positioning of the child (Figure 1) . One hundred-twenty milliliters of warmed saline containing 5 mg/ml of a modified porcine natural surfactant (Curosurf; Nycomed Pharma, Munich, Germany) were instilled in 15-ml aliquots and aspirated. The entire procedure was completed within 50 minutes without significant drop in PaO2 or increase in PaCO2. After the lavage procedure, 60 mg/kg bw of Curosurf were given as bolus. PaO2/fraction of inspired oxygen (FIO2) improved from 64.5 to 99.5 (at 1 hour) and 173.9 mm Hg (at 12 hours after surfactant treatment), but then deteriorated gradually to pretreatment values during the following 72 hours. Unfortunately, a 121ins2 mutation of the SP-B gene was confirmed (Professor L. M. Nogee, Johns Hopkins Hospital, Baltimore, MD), but lung transplantation was not considered as a therapeutic option by the parents. The infant died on Day 26. A healthy girl was born to the heterozygous parents 13 months later, after prenatal exclusion of homozygosity for the 121ins2 mutation.
Abnormal surfactant function is characteristic of hereditary SP-B deficiency. Surfactant instillation results in short-term improvement in gas exchange in SP-B–deficient babies, and more than 80 doses of surfactant have been given to individual infants (2). The abundant proteins present in the airways inactivate endogenous surfactant. We found up to 10 mg/ml of total protein on analysis of the lavage fluid. Minimum surface tension (  min), as measured in a pulsating bubble surfactometer, was high in the initial samples, indicating abnormal surfactant function, but was reduced to near-zero values after repeated lavage with diluted surfactant. However, min rapidly increased again in the following days. Under conditions of surfactant inactivation (e.g. meconium aspiration syndrome, pneumonia, or acute respiratory distress syndrome [ARDS]), lavage with diluted surfactant may be more effective than bolus instillation. The technique described here may be considered as an alternative in small infants with ARDS due to abundance of surfactant inhibitors in the bronchoalveolar space. It is relatively minimally invasive, inexpensive, and allows continued ventilation of one lung during lavage of the other lung.
Apart from the absence of SP-B, abnormal processing of proSP-C and reduced levels of mature SP-C peptide have been described in SP-B knock-out mice and in SP-B–deficient humans (3). We therefore analyzed the lavage material for its protein composition and electron microscopic appearance. The lavage fluid was solubilised in 1% (weight/volume) sodium dodecyl sulphate (SDS) and centrifuged 100,000 x g for 20 minutes at 20°C. The supernatant containing soluble proteins was then removed and the pellet resolubilised in 1% SDS. This procedure, which has been used for isolation of amyloid fibrils from an adult patient with pulmonary alveolar proteinosis (4), was repeated four times. Electron microscopy of the final pellet revealed abundant amyloid fibrils (Figure 2) . Aberrant processing of proSP-C and SP-B deficiency results in accumulation of a 10–12-kD processing intermediate that contains the SP-C sequence (3). SP-C avidly forms amyloid fibrils depending on its poly-valine sequence (5). SDS–polyacrylamide gel electrophoresis under reducing conditions of the pellet material demonstrated one weak band at a molecular mass of about 5 kD, and one stronger band at about 12 kD. Amino acid sequence analysis of these bands identified neutrophil defensin 1 and a fragment of
To our knowledge the presence of amyloid fibrils has not been described before in BAL from neonates. Fibril formation is associated with severe disease, as these fibrils are difficult or impossible to degrade once they are formed. We speculate that surfactant lavage, which removes fibrils, can be more effective than simple bolus instillation.
a Georg August University Göttingen, Germany Shahparak Zaltash REFERENCES
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-1-fetoprotein, respectively, as the major constituents. The ability of proteins to form amyloid appears to be more widespread than initially thought (