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Background: Balanced composition of a well-functioning pulmonary surfactant is crucial and essential for normal breathing. Here, we explored whether the composition of lipids recovered by broncho-alveolar lavage (BAL) in children with cystic fibrosis (CF) differ from children with protracted bacterial bronchitis (PBB) and controls. We wanted to differentiate, if alterations are primarily caused by the disease process or secondary due to an increased amount of cell-membrane lipids derived from inflammatory cells., Methods: Comprehensive lipidomics profiles of BAL fluid from children diagnosed with CF, PBB and controls were generated by electrospray ionization tandem mass spectrometry analysis. BAL cell differential and numbers were examined., Results: 55 children (37 patients with CF, 8 children with PBB and 10 controls) were included in this study. Results showed comparable total quantities of lipids in all groups. Phospholipids were the major lipid fraction and similar in all groups, whereas the fractions of cholesteryl esters were less and of free cholesterol were increased in children with CF. Among the phospholipids, patients with CF had higher proportion of the non-surfactant membrane-lipids in the classes phosphatidylethanolamine based plasmalogens (PE P), phosphatidylethanolmine (PE) and phosphatidylserine (PS), but a lower proportion of phosphatidylcholine (PC) compared to healthy controls. No such changes were identified in the BAL fluid of children diagnosed with PBB. No differences were observed for the surfactant lipids dipalmitoyl-phosphatidylcholin (PC 32:0) and phosphatidylglycerol (PG)., Conclusions: In CF patients with neutrophilic airway inflammation the lipid composition for surfactant phospholipid components were unchanged, whereas alteration in lipid profile were characteristic for those found in membranes of inflammatory cells. We suspect that the changes in CF were caused by the prolonged inflammation in contrast to a relatively short standing process in PBB., (Copyright © 2019 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: The state of oligomerization of surfactant associated protein-A (SP-A) monomers differs between individuals. This likely affects SP-A's functional properties and could thereby influence clinical status in patients with lung diseases. In this study we focus on SP-A structure in cystic fibrosis (CF) compared to both healthy subjects and disease controls., Methods: SP-A composition and function were assessed in both bronchoalveolar lavage (BAL) fluid and serum of 46 CF patients with mild disease, 25 patients with chronic bronchitis and 22 healthy subjects by gel chromatography and a functional agglutination assay. Relation of SP-A agglutination ability to disease severity of the subjects was explored., Results: SP-A was present in seven major oligomeric forms with the majority of SP-A being structurally organized as complex oligomeric forms. More complex oligomeric forms were associated with better SP-A function with regard to its agglutination ability. These forms were more frequently observed in BAL than in serum, but there were no differences between disease groups. In CF patients, more complex forms of SP-A were associated with better lung function., Conclusions: Organizational structure of SP-A affects its functional activity and is linked to disease severity in CF.

Cystic fibrosis (CF) lung disease is primarily a disease of the small airways. We hypothesized that even in patients with normal lung function, a reduced surfactant function would be present and favor small airway obstruction. Bronchoalveolar lavages from 76 patients with CF (5-31 years, median 11) with well-conserved lung function (FEV1 94% predicted, range 78-121) and from 10 healthy control subjects were investigated. The deviation of the biophysical surfactant performance from normal, assessed in a bubble surfactometer, was small; however, the ability of the surfactant to maintain the patency of a narrow airway (% open) was significantly reduced. Surfactant protein (SP)-C level was increased, SP-B and SP-D were unchanged, whereas SP-A was decreased. Among the patients with CF, neutrophilic inflammation was modestly related to a poorer surfactant activity, but not to lung function. SP-D was reduced in proportion to the degree of inflammation and in the presence of bacteria. These findings in a large cohort of patients with CF with normal lung function show that the endobronchial airway inflammation is linked to early perturbations of the biophysical properties and immunologic components of pulmonary surfactant and opens fields for novel therapeutic interventions.

Bronchoalveolar lavage (BAL) has been shown to be useful in the diagnosis of hypersensitivity pneumonitis (HP) in adults, the typical constellation being lymphocytosis with a decrease in the CD4/CD8 ratio. Only limited data exist for the diagnostic value of BAL cytology in paediatric patients with this disorder. Children aged 6-15 yrs (n=9) with acute HP were studied. BAL was performed before initiation of anti-inflammatory treatment via a flexible bronchoscope in the middle lobe with 3 mL x kg body weight(-1) normal saline warmed to body temperature; BAL cytology and lymphocyte surface markers were compared with age-matched controls. The percentage of lymphocytes was significantly increased in all patients with HP. No significant differences were observed in the CD4/CD8 ratio between children without lung disease and those with HP. Increased expression of human leukocyte antigen-DR was found in seven of eight children with HP, whereas natural killer cells were elevated in five of eight children. Every patient had at least one of these two alterations in BAL fluid in addition to lymphocytosis. It was concluded that while lymphocytosis is generally present in children with hypersensitivity pneumonitis, the CD4/CD8 ratio is not increased in these patients. Assessing natural killer cells and human leukocyte antigen-DR expression appears to be a helpful adjunct in the diagnosis of paediatric patients with this disorder.

Background: Matrix metalloproteinases (MMPs) are involved in the remodelling and degradation of extracellular matrix and may play a role in pulmonary tissue destruction in cystic fibrosis (CF)., Methods: Bronchoalveolar lavage (BAL) fluid levels of MMP-8, MMP-9, and their natural inhibitor TIMP-1 were measured on two occasions within 18 months in 23 children with mild CF, 13 of whom were treated with DNase., Results: MMP-8 (39.3 (6.8) v 0.12 (0.01) ng/ml), MMP-9 (58.0 (11.4) v 0.5 (0.02) ng/ml), and the molar ratio of MMP-9/TIMP-1 (0.36 (0.05) v 0.048 (0.01)) were significantly higher in patients with CF than in control children without lung disease. Gelatine zymography showed the typical banding pattern of neutrophil derived MMP-9, including 130 kDa NGAL-MMP-9 complex and 92 kDa latent MMP-9 bands; 85 kDa bands (corresponding to active MMP-9) were seen in all patients. There was a close correlation between BAL fluid concentrations of MMPs and alpha(2)-macroglobulin, a marker of alveolocapillary leakage. After 18 months MMP levels were increased in untreated patients and decreased in patients treated with DNase., Conclusions: Uninhibited MMPs may contribute to pulmonary tissue destruction even in CF patients with mild lung disease that may be positively affected by treatment with DNase.

Severe respiratory infections are a major cause of morbidity and mortality in children receiving immunosuppressive therapy for malignancies. The goal of this study was to assess the major changes in the protein patterns in these children. Bronchoalveolar lavage (BAL) fluids of seven control children and of ten children with malignancies and fever not responding to broad spectrum antibiotic treatment was separated by horizontal two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the isoelectric point range 3-9. We observed a large increase of alpha(1)-antitrypsin (p = 0.0004) and decreases of the immunoglobulin (Ig) binding factor, transthyretin and cystatin S. Significant changes occurred also in the small acidic proteins. The relative abundance of the IgG heavy and light chains may hinder the separation and identification of many minor protein spots located in the basic area of the gel, suggesting that their removal during sample preparation may be warranted. This study demonstrated significant alterations in BAL fluid proteome in immunosuppressed children with persistent fever and pulmonary infiltrates. Future target regions of interest were identified. Sample prefractionation and the selection of suitable narrow isoelectric point ranges will be necessary for optimized detection and separating conditions.

Bronchoalveolar lavage (BAL) is a well-established diagnostic tool for the assessment of pulmonary diseases in adults. How BAL contributes to the diagnostic process in childhood lung diseases is less clear. One of the problems in interpreting BAL findings in children is that there are few reference data for BAL fluid constituents in children. This report addresses some of the technical problems of bronchoalveolar lavage in children and summarizes current knowledge on cellular and noncellular bronchoalveolar lavage fluid components in children without lung disease.

Cystic fibrosis (CF) is associated with a neutrophil dominated airway inflammation. So far bronchoalveolar lavage (BAL) studies in CF have used pooled BAL samples which may be more representative of the alveolar compartment rather than the airways. To assess whether the first sample of a BAL is more sensitive in the evaluation of airway inflammation, the authors have studied 105 stable CF patients aged 5-37 yrs with a mean forced expiratory volume in one second (FEV1) of 96+/-15% (mean+/-SD). BAL cytology of the first and pooled samples were compared to reference values obtained in children without respiratory disease. Absolute cell counts and the percentage of neutrophils were significantly increased in CF patients. If the 95% confidence interval was used as a cut-off point, 17/105 CF patients had a normal percentage of neutrophils in pooled BAL samples, but only three also had a normal percentage of neutrophils in the first BAL aliquot. Therefore, neutrophil dominated airway inflammation is more pronounced in the first, mainly bronchial, bronchoalveolar lavage sample suggesting that sequential analysis of bronchoalveolar lavage fluid may have a higher sensitivity to detect early inflammatory changes in CF patients.

This review summarizes current knowledge about the role of nitric oxide (NO) in cystic fibrosis (CF) lung disease. NO is endogenously produced by a group of enzymes, the NO synthases (NOSs). There are three isoforms of NOS, each encoded by different genes: neuronal (nNOS), immune or inducible (iNOS), and endothelial (eNOS) nitric oxide synthase.(1) They all form NO and L-citrulline by enzymatic oxidation of L-arginine. This reaction requires a number of cosubstrates, including molecular oxygen and tetrahydrobiopterin. It is now known whether all three isoenzymes are constitutively expressed in cells of the respiratory tract and that their gene expression is inducible.(2,3) NO production by iNOS, the "high-output" NOS, is stimulated by bacterial lipopolysaccharide (LPS) as well as proinflammatory cytokines such as interleukin (IL)-1gamma, IL-2, interferon (IFN)-gamma, and tumor necrosis factor (TNF). In contrast to nNOS and eNOS, activation of iNOS does not require an increase in intracellular Ca(2+) concentration., (Copyright 1999 Wiley-Liss, Inc.)

An increased lipid content in alveolar macrophages of bronchoalveolar lavage (BAL) fluid is thought to be a useful indicator for recurrent pulmonary aspiration. To assess whether pulmonary diseases unrelated to aspiration can raise the lipid content in alveolar macrophages, we evaluated Oil-Red-O-stained smears of BAL fluid in 18 children aged 3-15 years undergoing elective surgery for nonpulmonary illnesses under general anesthesia and in 18 children aged 1-16 years who had pulmonary diseases without clinical evidence of aspiration (pneumonia, exogenous allergic alveolitis, or cystic fibrosis). A semiquantitative lipid-laden macrophage (LLM) index was determined for each patient. LLM indices in children without pulmonary disease were higher than those published for healthy adults. In children with pulmonary diseases but without evidence of aspiration, a significantly higher LLM index was observed compared to controls. The LLM indices of children with pulmonary diseases were similar to those published by other authors for children with pulmonary aspiration. We conclude that an elevated LLM index in alveolar macrophages of BAL can be found in a variety of pulmonary diseases in which there is no clinical evidence of aspiration and is therefore unlikely to be a specific parameter for silent pulmonary aspiration.

Background: Pulmonary infections are associated with an increase in capillary permeability but information regarding age related differences in the local inflammatory response is lacking. To quantify the degree of capillary leakage during inflammation, the concentrations of the plasma proteins albumin, alpha1-antitrypsin, alpha2-macroglobulin and the locally produced proteins elastase, myeloperoxidase, lactoferrin and fibronectin were studied in the bronchoalveolar lavage (BAL) fluid of immunosuppressed children and adults with pneumonia., Methods: Sixteen children aged 2-16 years and 15 adults who developed pneumonia while receiving immunosuppressive therapy for haematological malignancies were included in the study. Bronchoalveolar lavage was performed via a flexible bronchoscope with three aliquots of 1 ml/kg body weight in children and 200 ml in adults. Protein concentrations in BAL fluid were determined using highly sensitive immunoluminometric assays., Results: Despite considerable variability, the median concentrations of all proteins in BAL fluid were significantly higher in both patient populations than in previously collected age adjusted reference values. The concentrations of serum derived proteins were significantly higher in children with pneumonia than in adult patients. In contrast, no differences were observed between the two groups for locally produced proteins., Conclusions: These data suggest that the degree of protein exudation is more pronounced in immunosuppressed children with pneumonia than in adults in a similar clinical situation. This is in agreement with our studies in healthy individuals and may reflect a greater permeability of the alveolar-capillary membrane in children, regardless of disease status.

Unlabelled: Nitric oxide (NO) is produced in the upper and lower respiratory tract and can be detected in exhaled air of both healthy individuals and subjects with pulmonary diseases. Recent studies have shown that exhaled NO is mainly derived from the upper airways. There is, however, evidence that in aqueous solutions NO is rapidly converted to distinct oxides of nitrogen. We therefore studied the stable NO metabolites nitrate and nitrite in broncho-alveolar lavage (BAL) fluid and serum as indicators of NO formation in the lower respiratory tract. The study population consisted of 31 healthy children undergoing elective surgery for non-pulmonary illnesses and 13 immunosuppressed children with pneumonia. Nitrate and nitrite were determined photometrically. Nitrate was found in BAL fluid of all children. In children with pneumonia, nitrate concentrations in BAL fluid were significantly higher than in healthy children. A significant correlation was observed between nitrate in BAL fluid and serum of immunosuppressed children with pneumonia. Nitrite was not detected in any of the BAL fluid or serum samples., Conclusions: Our results suggest that in the lower airways significant amounts of NO are metabolised to nitrate. Studies on NO in pulmonary diseases should therefore include determination of nitrate in lower airway fluids.

(BAL) has been increasingly used in recent years also in children. In paediatric pulmonary diseases, however, interpretation has proved difficult in the absence of standard reference values for BAL in childhood. It is the purpose of the present publication to discuss the methodological aspects of BAL in childhood and to proviced criteria by means of standard reference values for the assessment of BAL in paediatric patients. In a group of 50 children aged 3 to 15 years with healthy lungs, who were subjected to BAL during an elective surgical intervention, we examined both cellular and non-cellular components of the BAL. Whereas the percentage of macrophages and lymphocytes in the total population did not differ significantly from the standard values in adults, the absolute and relative granulocyte count was found to be elevated in children under 8 years of age. The CD4/CD8 quotient of BAL was 0.7 on the average, and hence clearly below the standard reference values defined for adults. In the non-cellular components we found an increase in the level of all proteins from plasma, whereas locally produced proteins did not differ from the findings in healthy adults. The total phospholipid content of BAL was markedly enhanced in children below 8 years of age and decreased with increasing age. These differences between children and adults should be taken into account when interpreting pathological findings. They are the basis for better defining the ranking of BAL in paediatric pulmonary diseases.

Unlabelled: Quantification of proteins in bronchoalveolar lavage fluid (BALF) is being performed increasingly, because of their potency in elucidating pathophysiological processes and their possible clinical relevance., Study Population: 37 normal children (12 female, 25 male, mean age +/- SD 7.9 +/- 3.5 years, range 3-16 years) undergoing elective surgery for nonpulmonary illnesses and 16 healthy adults (all male, mean age +/- SD 24.7 +/- 2.6 years, range 20-28 years, 8 smokers, 8 nonsmokers) have been studied after approval by the ethics committees of both institutions., Methods: BAL was performed with normal saline in the right middle lobe or one of its segments. Lavage volume was 15 x 20 ml in adults and 3 x 1 ml/kg in children. In adults the first aliquot was processed separately, 2 aliquots of the following were pooled (resulting in 8 different fractions) so that different lavage protocols could be calculated. The concentrations of the plasma-proteins alpha 1-proteinase inhibitor (alpha-1PI), alpha 2-macroglobulin (alpha 2PI), albumin (Alb) and of the locally released proteins myeloperoxidase (MPO), lactoferrin (Lf), elastase (Ela) and fibronectin (Fib) were determined in BALF using chemiluminescence immunoassays., Results: The BALF concentrations of the plasma proteins were higher in children compared to adults. In contrast, concentrations of the locally released proteins were similar in both groups. Protein concentrations in the first recovered aliquot were different from the following in healthy adults with higher values of MPO, Lf and Ela and lower concentrations of alpha 1PI, alpha 2PI and Alb. Subsequent aliquots did not show a progressive dilution of these proteins with increasing lavage volumes. In smokers, BALF concentrations of albumin, alpha 1PI and MPO were higher compared to nonsmokers., Conclusion: BALF protein concentrations are different in normal children and adults. These results underscore the necessity to establish independent reference values for children, as could be demonstrated earlier with regard to BAL cell composition.

Objective: Bronchoalveolar lavage (BAL) is a well established technique for the detection of pathogens in immunosuppressed children, but its diagnostic yield is variable. The aim of this study was to investigate whether BAL differential cell counts are helpful in the evaluation of pulmonary infiltrates in immunocompromised children., Study Design: BAL was performed 28 times in 27 febrile immunocompromised children with pulmonary infiltrates. All patients were pretreated with broad spectrum antibiotics; 11 children also received amphotericin B. BAL was conducted with a flexible bronchoscope wedged in the area of maximal pathology as suggested by the chest radiograph or in the middle lobe in patients with diffuse interstitial radiographic changes. Differential cell counts were performed from cell smears obtained after centrifugation of BAL fluid., Results: Bacterial or fungal organisms were detected in BAL fluid of 12 patients. Patients with bacterial or fungal infections (group 1) had a significantly higher percentage of granulocytes in BAL fluid both compared with patients with sterile BAL cultures (group 2) and with a control group of children without pulmonary disease (p < 0.001, Wilcoxon test). The proportion of lymphocytes was not different from the control group in group 1 but significantly increased in group 2 (p < 0.001, Wilcoxon test). Blood differential cell counts were not different in the two patient groups. Lymphocyte subsets of BAL fluid obtained in a subgroup of patients were not significantly different from controls., Conclusion: These data suggest that BAL differential cell counts may be a useful adjunct in the differential diagnosis of pulmonary infection in immunocompromised children.

Whilst alterations in surfactant components of bronchoalveolar lavage fluid (BALF) have been described in a variety of pulmonary diseases in adults, no information is available on the surfactant composition in children beyond the neonatal period. In order to obtain reference values for the paediatric age group, we have studied phospholipid profiles of BALF in children without pulmonary disease. The study population consisted of 38 children aged 3-15 yrs (mean +/- SD 8 +/- 4 yrs) undergoing elective surgery for nonpulmonary illnesses. Surfactant composition was analysed by high pressure liquid chromatography using both an ultraviolet and a light-scattering detector. Total phospholipid concentration was higher in children under 8 yrs of age and decreased with age. In contrast, the total protein concentration remained constant throughout the age range. No age-related changes in the relative composition of phospholipid fractions could be observed. Surfactant protein A concentrations were highly variable in all age groups. This study provides the first data on surfactant components in bronchoalveolar lavage fluid of healthy children and will be the basis for future studies of children with pulmonary diseases.

Immunoglobulins play an important role in the pulmonary host defense, but little information is available about immunoglobulin and beta 2-microglobulin concentrations in the lung of normal children. Using bronchoalveolar lavage (BAL) we have studied immunoglobulin and beta 2-microglobulin levels in 30 children 3-15 years old undergoing elective surgery for nonpulmonary illnesses and in 15 healthy adult volunteers. BAL was performed with 3 x 1 ml/kg of body weight normal saline through an endotracheal tube after induction of anesthesia in children and under local anesthesia in adults. Similar concentrations of IgA and IgG were found in BAL fluid of children and adults even though serum levels were lower in children. As comparable results were obtained for albumin, a serum-derived protein, these data suggest that the permeability of the alveolar membrane is higher in children. IgE and IgM were detected in BAL fluid in only a fraction of children. beta 2-microglobulin levels were higher in both blood and BAL fluid of children. These data provide the first reference data for immunoglobulin and beta 2-microglobulin in children and can serve as a basis for future studies of children with pulmonary diseases.

Bronchoalveolar lavage (BAL) is increasingly used in the pediatric age group. However, normal values for BAL fluid (BALF) constituents are lacking. As part of an investigation to define reference values in children, we studied lymphocyte surface markers of BALF in 28 children 3 to 16 yr of age without bronchopulmonary disease. All of them were undergoing elective surgery for nonpulmonary illnesses. BAL was performed under general anaesthesia with tracheal intubation. A flexible bronchoscope (Pentax 3.5 or 4.9 mm) was wedged into the middle lobe or into one of its segments, and 3 x 1 ml/kg body weight normal saline warmed to body temperature were instilled and immediately withdrawn. The first sample was studied separately; subsequent samples were pooled for analysis. Studies on lymphocyte surface markers were performed on the pooled sample only. The distribution of B-cells, pan T-cells, and CD57 positive cells was within the range reported for adult subjects. However, CD4/CD8 ratios were lower than in adults (0.7 +/- 0.4, mean +/- SD). This decrease in the CD4/CD8 ratio was caused by an increase in CD8 cells. Comparative analysis of blood and BALF lymphocytes in a subgroup of children showed that CD4/CD8 ratios in blood were within the normal range reported for this age group and significantly higher when compared with BALF. The lower CD4/CD8 ratios in normal children have to be considered in the interpretation of BALF in children with pulmonary diseases.

Bronchoalveolar lavage (BAL) is increasingly used in the assessment of pulmonary diseases in children. However, reference values for cellular and non-cellular constituents of BAL fluid in children are lacking. We have studied the differential cytology of BAL fluid in 48 children aged 3-16 years (mean age +/- SD 7.9 +/- 3.5 yrs) undergoing elective surgery for nonpulmonary illnesses. A flexible bronchoscope (Pentax 3.5 or 4.9 mm) was wedged in the middle lobe or one of its segments. BAL was performed with 3x1 ml-kg-1 body weight of normal saline warmed to body temperature. The first sample was studied separately; subsequent samples were pooled for analysis. The mean recovery was 58 +/- 15%. Significantly more granulocytes and less lymphocytes were found in the first, as compared to the pooled, sample. Total cell counts were highly variable and ranged from 0.5-57.1 x 10(4) ml-1. Macrophages were the predominant cell type, with a mean percentage of 81.2 +/- 12.7%. The relative proportion of lymphocytes was higher than that reported in most studies of adult volunteers (16.1 +/- 2.4%). No age dependency was observed for either cell type. The mean percentage of granulocytes was 2.5 +/- 3.3%. Absolute granulocyte counts were significantly higher in children under 8 yrs of age. This study provides the first reference data on BAL differential cytology in children without pulmonary disease and will be the basis for future investigations of BAL in paediatric lung diseases.

Abstract: Reduced glutathione (GSH), a major antioxidant and modulator of cell proliferation, is decreased in the bronchoalveolar lavage fluid (BALF) of cystic fibrosis (CF) patients. We previously have shown that GSH inhalation in CF patients significantly increased GSH levels in BALF and improved lung function (M. Griese et al., 2004, Am. J. Respir. Crit. Care Med. 169, 822–828). GSH depletion in vitro enhances susceptibility to oxidative stress, increases inflammatory cytokine release, and impairs T cell responses. We therefore hypothesized that an increase in GSH in BALF reduces oxidative stress, decreases inflammation, and modulates T cell responses in lungs of CF patients. BALF from 17 CF patients (median FEV1 67% (43–105%) of predicted) was assessed before and after GSH inhalation for total protein, markers of oxidative stress (8-isoprostane, myeloperoxidase, and ascorbic and uric acid), pattern of protein oxidation, prostaglandin E2 (PGE2), and proinflammatory cytokines. BALF cells were differentiated using cytospin slides, and lymphocytes were further analyzed by flow cytometry. Inhalation of GSH decreased BALF levels of PGE2 and increased CD4+ and CD8+ lymphocytes in BALF significantly but had no effect on markers of oxidative stress. BALF lymphocytes correlated positively with lung function, whereas levels of PGE2 showed an inverse correlation. The patients with the greatest improvement in lung function after GSH treatment also had the largest decline in PGE2 levels. We conclude that GSH inhalation in CF patients increases lymphocytes and suppresses PGE2 in the bronchoalveolar space. Thus, GSH primarily affected the pulmonary immune response rather than the oxidative status in CF patients. The effect of GSH inhalation on PGE2 levels and lymphocytes in CF warrants further investigation. [Copyright &y& Elsevier]