Margaret C. Bruce

Elevation of fibronectin levels in lung secretions of infants with respiratory distress syndrome and development of bronchopulmonary dysplasia

To determine whether the level of fibronectin in lung secretions correlates with the severity of lung injury or with the development of bronchopulmonary dysplasia, or both, serial tracheal aspirate samples were collected from 32 preterm infants with severe respiratory distress syndrome. Levels of total fibronectin, cellular fibronectin, plasma fibronectin, albumin, and secretory component of IgA (SC) were determined for the first 1 to 2 weeks of life in the 14 infants who recovered without pulmonary sequelae, and for weeks 1 to 4 in the 18 infants in whom bronchopulmonary dysplasia developed. Secretory component was chosen as the reference protein because its concentration in lung secretions is minimally influenced by capillary leak and does not vary with gestational or postnatal age. Albumin/SC and plasma fibronectin/SC ratios in tracheal aspirates were significantly higher (p less than 0.05) during the first 2 weeks of life in infants in whom bronchopulmonary dysplasia developed, suggesting greater capillary permeability in these infants. Cellular fibronectin/SC ratios in aspirates from infants with bronchopulmonary dysplasia were also significantly higher in the first 2 weeks, 9.0 +/- 1.7 and 7.4 +/- 2.0 micrograms/microgram SC in weeks 1 and 2, respectively, in comparison with values from infants without bronchopulmonary dysplasia, 1.6 +/- 0.4 and 1.1 +/- 0.8 micrograms/microgram SC (p less than 0.01), suggesting increased synthesis of fibronectin in the lungs of infants with subsequent bronchopulmonary dysplasia. Elevated levels of both plasma and cellular fibronectin in tracheal aspirate samples may provide an early index of the severity of lung injury in infants with severe respiratory distress syndrome.

Impaired Surface Membrane Expression of C3bi but Not C3b Receptors on Neonatal Neutrophils

ABSTRACT: Because increased complement receptor expression is necessary for optimal function of adult neutrophils, we tested the hypothesis that the increased susceptibility of neonates to infection might be due to an impaired ability of neonatal neutrophils to increase expression of complement receptors in response to chemotactic stimuli. We used monoclonal antibodies and flow cytometry to compare surface expression of the receptors for the complement components C3b (CR1) and C3bi (CR3) on adult and neonatal cord blood neutrophils (PMNs). We also compared receptor expression on PMNs from infants delivered by cesarean section without labor versus infants delivered vaginally. Expression of both CR1 and CR3 was minimal on resting adult and neonatal PMNs maintained at 0° C. There was a modest increase in expression of both receptors when PMNs were warmed to 37° C. This increase was similar on adult and neonatal cells, both unfractionated in whole blood and after isolation with Percoll density centrifugation, with one exception. Expression of CR1 was greater on isolated PMNs from vaginally delivered infants versus adults when the cells were warmed to 37° C. This difference was not observed with cells from infants delivered by cesarean section without labor, suggesting this modest increase in receptor expression may be due to factors associated with labor. When isolated cells were stimulated with either N-formyl-methionyl-Ieucyl-phenylalanine or zymosan-activated serum, expression of CR1 increased to the same extent in both neonatal and adult PMNs. In contrast, maximal CR3 expression on cord PMNs stimulated with N-formyl-methionyl-leucyl-phenylalanine or zymosan-activated serum was only 75% of the adult values. This was significantly less than CR3 expression in adult PMNs across a broad range of N-formylmethionyl- Ieucyl-phenylalanine concentrations. Maximal stimulated expression of both receptors was the same in PMNs from infants delivered by cesarean section without labor and from infants delivered vaginally. The diminished CR3 expression on cord PMNs was not due to decreased responsiveness to N-formyl-methionyl-leucyl-phenylalanine per se since deficient CR3 expression was observed at all concentrations tested and also when zymosan-activated serum was the stimulus. Our observations of impaired expression of CR3, which is necessary for adherence and directed migration, may explain some of the impairment of these functions in neonatal neutrophils.

Comparison of secretory component for immunoglobulin A with albumin as reference proteins in tracheal aspirate from preterm infants

OBJECTIVES To determine whether the concentration of secretory component (SC) in tracheal aspirate samples is less altered by changes in alveolar-capillary permeability and thus is a more reliable reference standard than albumin for the measurement of other components obtained by saline lavage in preterm infants. METHODS A total of 1229 tracheal aspirate and 1530 blood samples were collected from 195 neonates to evaluate the effects of advancing postnatal and gestational age, resolution of acute respiratory distress syndrome (RDS), steroid therapy for chronic lung disease, and acute sepsis on tracheal aspirate SC and albumin levels. The tracheal aspirate and blood samples were analyzed by enzyme-linked immunosorbent assay techniques for SC and albumin concentrations. RESULTS The mean values for the concentrations of aspirate and plasma SC did not vary significantly during an 8-week study period (n = 100) and did not vary with either gestational age (23 to 36 weeks) or postnatal age. Albumin concentration significantly decreased in aspirate samples from 1.67 +/- 0.77 mg/dl at week 1 to 0.41 +/- 0.21 mg/dl at week 8 (p < 0.001), whereas serum levels increased from 2.65 +/- 0.36 to 2.99 +/- 0.54 gm/dl (p < 0.001), suggesting a decrease in alveolar-capillary leakage with advancing postnatal age. The concentration of SC in aspirate samples from 51 infants who received dexamethasone remained constant during the first week of therapy, whereas the concentration of albumin decreased from 1.33 +/- 0.91 mg/dl at the initiation of therapy to 0.51 +/- 0.34 mg/dl on treatment day 7 (p < 0.001). The onset of sepsis (n = 40) was not accompanied by a significant change in either aspirate SC or albumin levels. However, in infants who had a deterioration in respiratory status concomitant with the onset of sepsis (n = 10), the levels of aspirate albumin increased whereas serum levels decreased (p < 0.001), suggesting an increase in alveolar-capillary leakage; the levels of aspirate SC remained unaltered. CONCLUSIONS Secretory component may serve as a more valid reference protein for the standardization of tracheal aspirate collection in preterm infants during evaluation of changes in inflammatory mediators in disease states and therapeutic interventions that alter alveolar-capillary integrity.

Quantitation of radiolabeled mucous glycoproteins secreted by tracheal explants

Abstract An agarose gel column filtration method has been developed to quantitate labeled mucous glycoprotein secreted into culture medium by explants of tracheal epithelium. A multichannel pump is employed to simultaneously analyze 15 samples. Compared to other methods, such as dialysis and trichloroacetic acid/phosphotungstic acid precipitation methods, the column method not only quantitates high-molecular-weight mucous glycoprotein specifically but also offers the advantages of rapid processing of samples, low-background radioactivity, linearity and reproducibility even with small amounts of labeled mucous glycoproteins, and recovery of samples for chemical analysis. This method can be used to quantitate any substances that can be isolated by a one-step column chromatographic technique.

Elevated laminin concentrations in lung secretions of preterm infants supported by mechanical ventilation are correlated with radiographic abnormalities

OBJECTIVE The objective of this study was to test the hypothesis that the presence of laminin in neonatal tracheal aspirates would be indicative of damage to the structural integrity of the basal laminae of the lung caused by barotrauma and hyperoxia. We predicted that disruption of the basal laminae would be a critical determinant of lung injury and fibrotic repair in the preterm infant whose lungs were ventilated with supplemental oxygen. STUDY DESIGN The study group consisted of 23 premature infants in the neonatal intensive care unit whose lungs were ventilated by supplemental oxygen. We quantitated concentrations of laminin and fibronectin from sequential tracheal aspirates by enzyme-linked immunosorbent assays. A two-way analysis of variance was used to compare laminin and fibronectin concentrations in infants with and without radiographic evidence of coarse pulmonary markings indicative of fibrotic repair of lung injury. RESULTS The concentrations of laminin, but not fibronectin, were significantly higher throughout the first 5 weeks of life in infants with abnormal chest radiographs at 36 weeks after conception. The concentrations of laminin in infant serum were approximately 1/30 that of tracheal aspirate laminin concentrations, suggesting that little if any of the laminin detected in the tracheal aspirates was derived from the serum. CONCLUSIONS Increased concentrations of laminin in tracheal aspirates may be an indication of lung injury and fibrotic repair in the preterm infant.

Immunofluorescence Studies of Lung Tissue in Cystic Fibrosis

Previous studies have suggested that immune mechanisms contribute to lung injury in cystic fibrosis (CF); however, there have been no comprehensive studies of immunofluorescent staining patterns in CF lung tissue. We performed immunofluorescence (IF) studies for immunoglobulins, C3, and fibrinogen on autopsy frozen lung tissue from 21 CF patients. Results were compared with lung findings in patients without CF. In CF-derived lung tissue fibrinogen was ubiquitous along the alveolar wall, alveolar space, and interstitium. Free immunoglobulin G (IgG) and IgA coated the alveolar surface segmentally in 14 and 6 cases, respectively. Unequivocal interstitial deposits were infrequent and IgM was present in blood vessels in one patient only. Intra-alveolar and interstitial inflammatory cells demonstrated cytoplasmic IgG, IgA, and IgM, respectively, in 18, 14, and 6 patients. C3 was seen only segmentally along the alveolar wall in two patients and in blood vessels in one. Antinuclear antibody (ANA) staining of interstitial cells for C3 and immunoglobulins was seen in five patients, four of whom had interstitial pneumonitis. Insignificant amounts of alveolar or interstitial fibrinogen and immunoglobulins in inflammatory cells were seen in controls in the absence of lung inflammation. The IF patterns were similar in the inflammatory lesions of CF and control specimens. The IF patterns observed in CF lung tissue are consistent with nonspecific vascular leakage and chronic inflammation with little evidence of immune complex deposition in the interstitium or blood vessels. This study confirms previous reports of ANA activity in CF patients, although the significance of this finding is unknown.

1061 DISTRIBUTION OF TRANSFUSED LEUKOCYTES IN NEWBORN VS ADULT

Transfused polymorphonuclear leukocytes(PMNs) have been proposed as therapy for septic neonates, yet their distribution to lung(pneumonia) and brain(meningitis) is unknown. To determine this, we injected 104-105cfu/gm type III group B Streptococcus (strain GBS 130) into 52 newborn(NB) and 20 adult(A) rats ip. and phosphate buffered saline(PBS) into 19 NB and 10 A rats as controls. 51Cr-labeled human PMNs were given to all rats (A,0.2×106/gm; NB,1.1×106/gm) at 7 hrs. Lung, spleen, kidney, liver and brain γ counts were measured after sacrifice at 13 hrs. Organ indices were calculated as actual cpm/expected cpm, based upon each organs % wt. All GBS-NB rats had 102-106cfu/gm GBS in both lung and brain, whereas only 32% of adults had 102-104cfu/gm in either lung or brain. The white cell count × 106 cells/ml (WBC) after transfusion(Tx) did not change in A and increased (p<.01) in NB PBS-rats, but increased (p<.05) in A and decreased (p<.001) in NB GBS-rats. In both A and NB rats, the distribution of PMNs to lung and brain did not change with either PBS or GBS injections. However, significantly more PMNs were present in both lung and brain of NB compared to A rats, p<.001. We conclude that significantly more transfused PMNs are distributed to lung & brain of infected or uninfected NB rats compared to A.