Ureaplasma-Driven Neonatal Neuroinflammation: Novel Insights from an Ovine Model.

Ureaplasma species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including meningitis. Data on mechanisms involved in Ureaplasma-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to Ureaplasma parvum (UP) in utero. 7 days after intra-amniotic injection of UP (n = 10) or saline (n = 11), lambs were surgically delivered at gestational day 128-129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using ureB-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C-X-C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in Ureaplasma-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood-brain barrier function. According to these data and previous in vitro findings from our group, we speculate that Ureaplasma-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation.

Increase in CTGF mRNA expression by respiratory syncytial virus infection is abrogated by caffeine in lung epithelial cells.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infection in early childhood. Underlying pathomechanisms of elevated pulmonary morbidity in later infancy are largely unknown. We found that RSV-infected H441 cells showed increased mRNA expression of connective tissue growth factor (CTGF), a key factor in airway remodeling. Additional dexamethasone treatment led to further elevated mRNA levels, indicating additive effects. Caffeine treatment prevented RSV-mediated increase in CTGF mRNA. RSV may be involved in airway remodeling processes by increasing CTGF mRNA expression. Caffeine might abrogate these negative effects and thereby help to restore lung homeostasis.

Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells.

BACKGROUND: Expression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441. METHODS: Different quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D. RESULTS: SP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone. CONCLUSION: SP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research.

Caffeine modulates glucocorticoid-induced expression of CTGF in lung epithelial cells and fibroblasts.

BACKGROUND: Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells. METHODS: The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-ß1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition. RESULTS: Treatment with different glucocorticoids (1 µM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-ß1 mRNA in H441 cells, increased expression of TGF-ß2 and TGF-ß3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression. CONCLUSIONS: In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-ß1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes.

BACKGROUND: Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. AIM: The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes. METHODS: Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. RESULTS: Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4+ lymphocytes. CONCLUSION: For the first time, the immunomodulatory capacity of CHF5633 on CD4+ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4+ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.

Pitfalls in flow cytometric analyses of surfactant-exposed human leukocytes.

BACKGROUND: Surfactant replacement treatment is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome in preterm infants and may also improve oxygenation in acute respiratory distress syndrome in children, adolescents and adults. Beside surface tension- and mechanical shear-reducing functions, natural surfactants have been ascribed immunomodulatory capacities. Current in vitro studies on immunomodulatory effects of pulmonary surfactant preparations on human leukocytes rely on ELISA, Western blot and polymerase chain reaction. Data obtained by flow cytometry are missing, so far, most likely due to confounding phospholipid residues. Intracellular cytokine flow cytometry in surfactant-exposed immune cells would provide information on pro- and anti-inflammatory immunomodulation at the single-cell level and would allow for integrating detailed immunophenotyping, functional assays and assessment of viability. AIM: We implemented a flow cytometry protocol for reliable quantitative assessment of in vitro intracellular cytokine production in surfactant-exposed human lymphocytes (CD4(+)) and monocytes (CD14(+)). METHODS: Two different permeabilization techniques were tested for their ability to provide intracellular cytokine staining in surfactant-exposed CD14(+) monocytes and CD4(+) lymphocytes. Both a commercially available solution containing saponin and ice-cold methanol were used as permeabilization reagents. RESULTS: For both cell types, flow cytometry following saponin-based permeabilization revealed pronounced unspecific fluorescence signals in surfactant-exposed samples overlapping with the fluorescence spectra of the majority of conjugates. Autofluorescence of surfactant phospholipid particles interfered significantly with reliable quantification of fluorochrome-specific signals and conclusive analysis. Implementation of a methanol-based permeabilization protocol resulted in the elimination of confounding non-cell particle signals allowing for an accurate quantification of intracellular cytokine production. CONCLUSION: Reliable detection of intracellular cytokines by flow cytometry may be challenging in surfactant-exposed cell samples due to significant autofluorescence of aggregated phospholipid particles. This issue has been addressed for the first time and may be of high relevance for all types of surfactant research. We demonstrate that a methanol-based permeabilization approach completely removes interfering fluorescent surfactant micelles and allows for correct evaluation of data. The successful removal of confounding surfactant phospholipids opens up a wide variety of multiparameter flow cytometry; a method that has not been applied in the field of surfactant research, yet.

Poractant alfa (Curosurf®) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo.

BACKGROUND: Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages. METHODS: Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages. RESULTS: Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage. CONCLUSIONS: We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.

Airway angiopoietin-2 in ventilated very preterm infants: association with prenatal factors and neonatal outcome.

OBJECTIVE: Pulmonary angiogenesis is a prerequisite for lung development. Angiopoietin-2 (Ang2) destabilizes endothelial cells through its endothelial receptor TIE-2, enabling vascular sprouting. Ang1 stabilizes new blood vessels. Soluble TIE-2 (sTIE-2) modulates these effects. We hypothesized that histological funisitis is associated with alterations of Ang2 in airways and of the systemic angiopoietin-TIE-2 homeostasis in very low birth weight (VLBW) infants, contributing to pulmonary morbidity and mortality. METHODS: We measured Ang2 in tracheobronchial aspirate fluid (TAF) of 42 VLBW <30 weeks of gestation from day 1 through 15 and Ang1, Ang2, and sTIE-2 in umbilical cord serum of 28 infants by enzyme-linked immunosorbent assay. Histological examination distinguished three groups: funisitis, chorioamnionitis, and controls. RESULTS: Funisitis was associated with lower Ang2 values in TAF but not with changes of Ang1, Ang2, and sTIE-2 in umbilical cord serum. Infants who developed bronchopulmonary dysplasia (BPD) or died had a persistently decreased ratio of previously measured Ang1 to Ang2 in TAF on days 1-5 and increased cord serum concentrations of sTIE-2. Moderate BPD/death was associated with an increase of Ang2 in TAF on day 10 and decreased Ang1/Ang2 ratio from day 3-15. Small for gestational age (SGA) infants had increased Ang2 in TAF on day 1-7 and a lower Ang1/Ang2 ratio on days 5-7. CONCLUSIONS: The predominance of Ang2 in airway fluid of infants with BPD/death and SGA infants suggests a link between disrupted placental and fetal pulmonary angiogenesis. Histological funisitis with reduced Ang2 in TAF was of minor relevance for outcome in our cohort.

Funisitis is associated with increased interleukin-10 gene expression in cord blood mononuclear cells in preterm infants ≤32 weeks of gestation.

OBJECTIVES: A systemic inflammatory response after intrauterine funisitis is assumed to be an important priming factor for acute and chronic pulmonary morbidity and neurological impairment in premature infants. Fetal lymphocytes and monocytes modulate the primary immune response. Genetic regulation of the cytokine-mediated process is partially known. The objective of our study was to examine the pro-inflammatory and anti-inflammatory responses in umbilical cord blood mononuclear cells (CBMC) of preterm infants on the transcriptional level. STUDY DESIGN: Fifteen preterm infants with a gestational age ≤32 weeks were enrolled in this prospective study. Funisitis was diagnosed in five of the 15 by histological examination. Gene expression of pro-inflammatory cytokines (TNF-α, IL-8, IL-1ß and IL-17) and anti-inflammatory cytokines (IL-10 and TGF-ß1) was examined in CBMC by real time reverse transcription polymerase chain reaction. RESULTS: Gene expression of IL-10 was significantly higher in the funisitis group compared to unexposed controls (p<0.008). Expression of TGF-ß1, TNF-α, IL-8 and IL-1ß did not differ significantly between the funisitis and control group. IL-17 was detectable in only two samples. CONCLUSIONS: Funisitis is associated with increased IL-10 gene expression in CBMC of preterm infants with a gestational age ≤32 weeks. This might contribute to modulation of postnatal immunoreactions and immunoregulation in these individuals.

Soluble receptor for advanced glycation end products (sRAGE) in tracheobronchial aspirate fluid and cord blood of very low birth weight infants with chorioamnionitis and funisitis.

BACKGROUND: A systemic fetal inflammatory response, reflected by histological funisitis is associated with pulmonary morbidity and increased mortality after premature birth. The receptor for advanced glycation end products (RAGE) is a membrane-bound multiligand receptor with a key role in inflammation. Soluble RAGE (sRAGE) is created by alternative mRNA splicing or shedding of the receptor's extracellular domain and can inhibit RAGE-activation. AIMS: To assess the association of funisitis with airway and systemic concentrations of sRAGE in very premature infants. METHODS: Forty-two ventilated infants (gestational age: 27.4 +/- 1.8weeks, birth weight: 1017 +/- 229 g [mean +/- SD]) were studied. sRAGE concentrations were measured in tracheobronchial aspirate fluid (TAF) on days of life 1, 3, 5, 7 and 10 and in umbilical cord serum of 28 infants by ELISA. The secretory component for IgA (SC) served as reference protein in TAF. Placental tissue, membranes and umbilical cords were examined microscopically to distinguish three groups: chorioamnionitis (n=9), funisitis (n=17) and controls (n=16). RESULTS: The funisitis group had lower sRAGE concentrations than both other groups in cord blood serum (median: 0.52 ng/ml [25th-75th centile: 0.32-0.91]; control, 1.72 [1.02-2.69]; chorioamnionitis, 1.44 [0.92-1.63], p<0.01) and TAF on day 1 (290 ng/ngSC [140-400]; control, 2750 [1470-28920]; chorioamnionitis, 2150 [1220-7140], p<0.01). sRAGE in TAF remained lower in the funisitis than in the chorioamnionitis group on days 3 and 10, p<0.01 respectively. CONCLUSIONS: Decreased sRAGE in airways and circulation after funisitis may contribute to an imbalance between pro- and anti-inflammatory factors priming very premature infants for pulmonary injury and increasing the risk of adverse outcome.

Clara cell secretory protein in tracheobronchial aspirates and umbilical cord serum of extremely premature infants with systemic inflammation.

BACKGROUND: A systemic fetal inflammatory response, reflected by chorioamnionitis with funisitis, is a risk factor for bronchopulmonary dysplasia. Clara cell secretory protein (CC10), a product of pulmonary Clara cells, has anti-inflammatory properties. Local down-regulation of CC10 has been associated with inflammatory lung disease. Increased serum levels of CC10 can indicate injury to alveolar-capillary integrity. OBJECTIVE: We hypothesized that extremely premature infants with a systemic fetal inflammatory response would have decreased concentrations of CC10 in tracheobronchial aspirates and that CC10 concentrations in umbilical cord serum of these infants would be increased, reflecting alveolar epithelial damage. METHODS: We measured CC10 concentrations in tracheobronchial aspirates of 42 ventilated extremely premature infants during their first week of life and in umbilical cord serum of 24 of them by ELISA. Standardized histological examination of the placenta, membranes and umbilical cord was used to identify infants with funisitis. RESULTS: Seventeen infants with funisitis had lower CC10 concentrations in tracheobronchial aspirates on days 1 (p < 0.01) and 3 (p < 0.05) than the remaining 25. Exogenous surfactant treatment was associated with higher CC10 concentrations on day 1 (p < 0.05). Initial leukocyte count correlated inversely with CC10 in tracheobronchial aspirates on days 1-5. Umbilical cord serum concentrations of CC10 did not differ between the infants with funisitis and the controls. CONCLUSIONS: Reduced anti-inflammatory CC10 concentrations in airways of extremely premature infants with a fetal inflammatory response might make their lungs susceptible for further postnatal injuries. Umbilical cord serum CC10 is not an indicator for a fetal systemic inflammatory reaction.

Airway concentrations of angiopoietin-1 and endostatin in ventilated extremely premature infants are decreased after funisitis and unbalanced with bronchopulmonary dysplasia/death.

A systemic inflammatory response of the fetus, reflected by histologic funisitis, is a risk factor for bronchopulmonary dysplasia (BPD). Impaired pulmonary angiogenesis accompanied by simplification and rarification of alveoli is a histologic hallmark of BPD. Angiopoietin-1 mediates vascular development, maturation, and stabilization. Endostatin mainly acts as an angiostatic factor. We hypothesized that funisitis was associated with changes of endostatin and angiopoietin-1 concentrations in the airways and that an imbalance between the factors might be associated with BPD or death. We measured concentrations of angiopoietin-1 and endostatin by enzyme-linked immunosorbent assay in tracheobronchial aspirate fluid samples of 42 ventilated preterm infants during postnatal days 1 through 15. The secretory component for IgA served as reference protein. A standardized histologic examination was used to distinguish three groups: chorioamnionitis, funisitis, and controls without inflammation. Concentrations of the mediators steadily decreased. Funisitis was associated with lower concentrations of both proteins, which might impair their physiologic activities in pulmonary angiogenesis. An increase of the ratio angiopoietin-1/endostatin until day 7 of life indicated a shift of the mediators potentially favoring angiogenesis. However, infants, who developed BPD or died, had a decreased ratio on days 1, 3, and 15, suggesting an imbalance toward inhibition of pulmonary angiogenesis.

Systemic fetal inflammation and reduced concentrations of macrophage migration inhibitory factor in tracheobronchial aspirate fluid of extremely premature infants.

OBJECTIVE: Macrophage migration inhibitory factor is a proinflammatory mediator of innate immunity, enhances cell growth, and plays a role in preterm delivery. We speculated that funisitis, reflecting fetal systemic inflammation, would be associated with higher concentrations of macrophage migration inhibitory factor in airways of extremely premature infants. STUDY DESIGN: We measured macrophage migration inhibitory factor by enzyme linked immunosorbent assay in tracheobronchial aspirate fluid of 35 ventilated infants less than 30 weeks' gestational age, throughout the first week of life. Three groups were distinguished histologically: chorioamnionitis, funisitis, and control. RESULTS: Unexpectedly, funisitis was associated with significantly decreased macrophage migration inhibitory factor in tracheobronchial aspirate fluid on day 1 (P < .01) and levels remained lower than in the chorioamnionitis group thereafter. For the 35 patients in total, macrophage migration inhibitory factor steadily declined. CONCLUSION: Decreased macrophage migration inhibitory factor concentrations in airways of extremely premature infants with systemic fetal inflammation early in life might predispose them to pulmonary infection and interfere with maturation of the lung, contributing to adverse pulmonary outcome.

Activation of umbilical cord endothelial cells and fetal inflammatory response in preterm infants with chorioamnionitis and funisitis.

Chorioamnionitis and funisitis are associated with preterm labor and postnatal morbidity. Activation of endothelium resulting in up-regulation of adhesion molecules seems to be a key mechanism in development of organ damage. We investigated whether chorioamnionitis with or without funisitis in preterm infants induced expression and shedding of adhesion molecules in the umbilical cord and resulted in increased concentrations of E-selectin, intercellular adhesion molecule (ICAM)-1, IL-1beta, IL-6, and IL-8 in the cord blood. Data were obtained by using immunohistochemistry and ELISA. Thirty-two preterm infants were divided into three groups according to histology: chorioamnionitis with funisitis, chorioamnionitis without funisitis, and controls without signs of inflammation. ICAM-1 expression on arterial endothelium was higher with funisitis compared with chorioamnionitis alone or with the control group. Similar results for ICAM-1 expression were found in venous endothelium, vascular walls, Wharton's jelly, and amnion epithelium. Endothelial E-selectin and vascular cell adhesion molecule (VCAM)-1 expression was only induced significantly with funisitis. Serum-concentrations of soluble ICAM-1 were higher with funisitis compared with chorioamnionitis alone or control group. Similarly, concentrations of soluble E-selectin, IL-1beta, IL-6, and IL-8 were increased exclusively with funisitis. In conclusion, only chorioamnionitis with funisitis was associated with systemic inflammation and endothelial activation with up-regulation and shedding of umbilical cord adhesion molecules. We speculate that this activation of endothelium may not be limited to the umbilical cord but may also involve other organs resulting in neonatal morbidity. This underlines the importance of funisitis as a risk factor for adverse outcome.

Intratracheal albumin reduces interleukin-8 in tracheobronchial aspirates in piglets after meconium aspiration.

Meconium aspiration induces pulmonary inflammation and reduces surfactant function. We hypothesized that albumin mixed with meconium attenuates pulmonary inflammation and improves surfactant function after meconium aspiration. We measured the concentration of free fatty acids (FFA) in the meconium (110 mg dry weight/mL) and added albumin to provide a molar FFA:albumin ratio of 1:1. Newborn piglets, 0-2 day of age, artificially ventilated and exposed to hypoxemia by ventilation with 8% O2, were randomized to group A receiving meconium (n = 12), or group B receiving meconium + albumin (n = 12), 3 ml/kg intratracheally. The animals were reoxygenated for 8 h. Reoxygenation was started when mean arterial blood pressure was < 20 mm Hg or base excess was < -20 mmol/L. During 8 h of reoxygenation the interleukin-8 concentrations in tracheobronchial aspirates increased 5-fold more in the meconium vs. the meconium + albumin groups (93 +/- 56 vs. 18 +/- 4 pg/mL, p < 0.005). There were no differences between the groups for tumor necrosis factor alpha in tracheobronchial aspirates, recruitment of inflammatory cells in the airspaces or surfactant function in bronchoalveolar lavage fluid. In conclusion, albumin significantly decreased interleukin-8 concentrations in tracheobronchial aspirates after meconium aspiration.

Comparison of pulmonary and inflammatory effects of lipid- and water-soluble components in meconium in newborn piglets.

To understand the pathogenesis of meconium aspiration syndrome, we compared the pulmonary and inflammatory effects of the water and lipid extracts of human meconium instilled into the lungs of newborn piglets. The piglets were artificially ventilated, made hypoxemic, and randomized into three groups. At start of reoxygenation, 3 ml/kg of one of the following mixtures was instilled intratracheally: (1) meconium (n = 12); (2) water extract of meconium (n = 12), and (3) lipid extract of meconium (n = 12). During 8 h of reoxygenation, hemodynamics, pulmonary gas exchange, lung mechanics, and interleukin-8 concentrations in tracheobronchial aspirates were monitored. Oxygenation index (p = 0.04) and mean airway pressure (p = 0.04) increased more in the lipid extract group than in the water extract group. Dynamic compliance and mean arterial blood pressure decreased (p < 0.05) in the meconium and lipid extract groups, but not in the water extract group. At 8 h of reoxygenation, the interleukin-8 concentration in the tracheobronchial aspirates was three times higher in the lipid extract group as compared with the water extract group (110 +/- 102 vs. 37 +/- 27 pg/ml; p = 0.02). In conclusion, pulmonary dysfunction in meconium aspiration syndrome is caused by both the water- and lipid-soluble fractions of meconium, with stronger inflammatory and more detrimental effects promoted by the lipid extract than the water extract.