Serum vascular endothelial growth factor is a potential biomarker for acute mountain sickness.

Background: Acute mountain sickness (AMS) is the most common disease caused by hypobaric hypoxia (HH) in high-altitude (HA) associated with high mortality when progressing to high-altitude pulmonary edema (HAPE) and/or high-altitude cerebral edema (HACE). There is evidence for a role of pro- and anti-inflammatory cytokines in development of AMS, but biological pathways and molecular mechanisms underlying AMS remain elusive. We aimed to measure changes in blood cytokine levels and their possible association with the development of AMS. Method: 15 healthy mountaineers were included into this prospective clinical trial. All participants underwent baseline normoxic testing with venous EDTA blood sampling at the Bangor University in United Kingdom (69 m). The participants started from Beni at an altitude of 869 m and trekked same routes in four groups the Dhaulagiri circuit in the Nepali Himalaya. Trekking a 14-day route, the mountaineers reached the final HA of 5,050 m at the Hidden Valley Base Camp (HVBC). Venous EDTA blood sampling was performed after active ascent to HA the following morning after arrival at 5,050 m (HVBC). A panel of 21 cytokines, chemokines and growth factors were assessed using Luminex system (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-1ra, sIL-2Rα, IFN-γ, TNF-α, MCP-1, MIP-1α, MIP-1ß, IP-10, G-CSF, GM-CSF, EGF, FGF-2, VEGF, and TGF-ß1). Results: There was a significant main effect for the gradual ascent from sea-level (SL) to HA on nearly all cytokines. Serum levels for TNF-α, sIL-2Rα, G-CSF, VEGF, EGF, TGF-ß1, IL-8, MCP-1, MIP-1ß, and IP-10 were significantly increased at HA compared to SL, whereas levels for IFN-γ and MIP-1α were significantly decreased. Serum VEGF was higher in AMS susceptible versus AMS resistant subjects (p < 0.027, main effect of AMS) and increased after ascent to HA in both AMS groups (p < 0.011, main effect of HA). Serum VEGF increased more from SL values in the AMS susceptible group than in the AMS resistant group (p < 0.049, interaction effect). Conclusion: Cytokine concentrations are significantly altered in HA. Within short interval after ascent, cytokine concentrations in HH normalize to values at SL. VEGF is significantly increased in mountaineers suffering from AMS, indicating its potential role as a biomarker for AMS.

Vitamin A Supplementation by Endotracheal Application of a Nano-encapsulated Preparation Is Feasible in Ventilated Preterm Lambs.

BACKGROUND: Vitamin A (VA) is crucial for lung growth and development. In premature infants, inadequate VA levels are associated with an increased risk of bronchopulmonary dysplasia (BPD). Intramuscular VA supplementation has been shown to decrease the incidence of BPD, but is not widely used in the clinical setting due to concerns about feasibility and pain. We studied VA kinetics, distribution, and the induction of early genetic expression of retinoid homeostatic genes in the lung after endotracheal and intravenous application in a preterm lamb model. METHODS: Lambs were delivered prematurely after 85% of gestation, intubated, and ventilated for 3 hours. The animals were randomized to receive no VA ("control"), a bolus of VA intravenously ("i.v."), or VA endotracheally directly after administration of surfactant ("e.t."). RESULTS: Animals treated with VA endotracheally directly after administration of surfactant showed significant increases of VA in serum and lung compared to controls. Animals treated with a bolus of VA intravenously showed significant increases of VA in serum, lung, and liver; however, peak serum concentrations and mRNA levels of homeostatic genes raised concerns about toxicity in this group. CONCLUSIONS: Endotracheal VA supplementation in preterm lambs is feasible and might offer advantages in comparison to i.v. Further studies are warranted to explore biological effects in the context of BPD.

Influence of prenatal hypoxia and postnatal hyperoxia on morphologic lung maturation in mice.

BACKGROUND: Oxygen supply as a lifesaving intervention is frequently used to treat preterm infants suffering additionally from possible prenatal or perinatal pathogen features. The impact of oxygen and/or physical lung injury may influence the morphological lung development, leading to a chronic postnatal lung disease called bronchopulmonary dysplasia (BPD). At present different experimental BPD models are used. However, there are no systematic comparative studies regarding different influences of oxygen on morphological lung maturation. OBJECTIVE: We investigated the influence of prenatal hypoxia and/or postnatal hyperoxia on morphological lung maturation based on stereological parameters, to find out which model best reflects morphological changes in lung development comparable with alterations found in BPD. METHODS: Pregnant mice were exposed to normoxia, the offspring to normoxia (No/No) or to hyperoxia (No/Hyper). Furthermore, pregnant mice were exposed to hypoxia and the offspring to normoxia (Hypo/No) or to hyperoxia (Hypo/Hyper). Stereological investigations were performed on all pups at 14 days after birth. RESULTS: Compared to controls (No/No) 1) the lung volume was significantly reduced in the No/Hyper and Hypo/Hyper groups, 2) the volume weighted mean volume of the parenchymal airspaces was significantly higher in the Hypo/Hyper group, 3) the total air space volume was significantly lower in the No/Hyper and Hypo/Hyper groups, 4) the total septal surface showed significantly lower values in the No/Hyper and Hypo/Hyper groups, 5) the wall thickness of septa showed the highest values in the Hypo/Hyper group without reaching significance, 6) the volume density and the volume weighted mean volume of lamellar bodies in alveolar epithelial cells type II (AEII) were significantly lower in the Hypo/Hyper group. CONCLUSION: Prenatal hypoxia and postnatal hyperoxia differentially influence the maturation of lung parenchyma. In 14 day old mice a significant retardation of morphological lung development leading to BPD-like alterations indicated by different parameters was only seen after hypoxia and hyperoxia.

Hypoxia and the hypoxia-regulated transcription factor HIF-1α suppress the host defence of airway epithelial cells.

Chronic diseases of the respiratory tract, such as cystic fibrosis, are associated with mucosal and systemic hypoxia. Innate immune functions of airway epithelial cells are required to prevent and control infections of the lung parenchyma. The transcription factor hypoxia-inducible factor 1α (HIF-1α) regulates cellular adaptation to low oxygen conditions. Here, we show that hypoxia and HIF-1α regulate innate immune mechanisms of cultured human bronchial epithelial cells (HBECs). Exposure of primary HBECs to hypoxia or the prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG) resulted in a significantly decreased expression of inflammatory mediators (IL-6, IFN-γ-induced protein 10) in response to ligands for TLRs (flagellin, polyI:C) and Pseudomonas aeruginosa, whereas the expression of inflammatory mediators was not affected by hypoxia or DMOG in the absence of microbial factors. Small interfering RNA-mediated knockdown of HIF-1α in HBECs and in the bronchial epithelial cell line Calu-3 resulted in increased expression of inflammatory mediators. The inflammatory response was decreased in lungs of mice stimulated with inactivated P. aeruginosa under hypoxia. These data suggest that hypoxia suppresses the innate immune response of airway epithelial cells via HIF-1α.

Are Cytokines Useful Biomarkers to Determine Disease Severity in Neonates with Congenital Diaphragmatic Hernia?

Background This study investigates plasma cytokine levels in neonates with the more common left-sided congenital diaphragmatic hernia (CDH) and correlates them with severity of disease indicated by position of the liver. An intrathoracic part of liver is associated with higher need for extracorporeal membrane oxygenation (ECMO) and higher risk for chronic lung disease (CLD). Methods A total of 28 newborns with CDH were subdivided by their liver position in partially intrathoracic (n = 16) and only abdominal (n = 12) position. Only liver-up patients went on ECMO (n = 9) and developed severe/moderate CLD (n = 5). Controls consisted of 19 healthy matched-term neonates. Laboratory samples were extracted from umbilical cord blood and during the neonatal period. Results In umbilical cord blood, CDH patients showed decreased IL-8 values while MIP-1a (macrophage inflammatory protein-1) values were increased. Concerning the severity of CDH, we measured significantly higher levels of TGFb2 in CDH patients with liver-up than in liver-down cases and controls (p < 0.006). During the neonatal period, the concentration of IL-10 and vascular endothelial growth factor (VEGF) showed significant deviations in the liver-up group with need for ECMO (p < 0.009). Conclusion In neonates with CDH, plasma cytokine levels are already altered in utero. TGFb2 may work as an early predictor for severity of disease. VEGF and IL-10 could serve as potential biomarkers predicting the course of disease in CDH.

Down Syndrome: Gestational Age-Related Neonatal Anthropometrics for Germany.

BACKGROUND: Neonates with Down syndrome (DS) weigh less, are smaller and have increased first-year mortality, especially if born small for gestational age (GA). DS-specific GA-related neonatal anthropometrics for Germany are lacking. OBJECTIVES: To construct reference tables and centile curves for birth weight (g), crown-heel length (cm) and head circumference (cm) by sex and GA for German DS neonates. METHODS: Retrospective anthropometric data from live-born singleton DS neonates born in Germany from January 1966 to June 2010 were collected using standardized questionnaires and patient records. Reference tables were created based on means and standard deviations. The 3rd, 10th, 25th, 50th, 75th, 90th and 97th centile curves were constructed and smoothed using running medians and Cole's LMS method. RESULTS: Anthropometric measurements were obtained for 1,304 DS neonates [males/females: 713/591 (54.7%/45.3%)]. Reference tables and centile charts were constructed from 3,542 (males/females: 1,932/1,610) observations for GA 32-41 weeks. Compared with general-population newborns, prematurity was increased (21.1 vs. 6.3%) at GA 32-36 weeks. Term-born (GA 40 weeks) male and female DS neonates were 352.5 and 223.5 g lighter and 1.5 and 1.4 cm smaller than general-population neonates, and head circumference was also 1.4 and 1.5 cm smaller, respectively. CONCLUSION: This is the first study to report GA-related, sex-specific reference tables and centile charts of birth weight, length and head circumference for DS neonates born in Germany. Compared with the general German population, DS newborns are more frequently born prematurely, weigh less, are smaller and have a smaller head circumference at birth.

Human umbilical cord blood mononuclear cells in a double-hit model of bronchopulmonary dysplasia in neonatal mice.

BACKGROUND: Bronchopulmonary dysplasia (BPD) presents a major threat of very preterm birth and treatment options are still limited. Stem cells from different sources have been used successfully in experimental BPD, induced by postnatal hyperoxia. OBJECTIVES: We investigated the effect of umbilical cord blood mononuclear cells (MNCs) in a new double-hit mouse model of BPD. METHODS: For the double-hit, date mated mice were subjected to hypoxia and thereafter the offspring was exposed to hyperoxia. Human umbilical cord blood MNCs were given intraperitoneally by day P7. As outcome variables were defined: physical development (auxology), lung structure (histomorphometry), expression of markers for lung maturation and inflammation on mRNA and protein level. Pre- and postnatal normoxic pups and sham treated double-hit pups served as control groups. RESULTS: Compared to normoxic controls, sham treated double-hit animals showed impaired physical and lung development with reduced alveolarization and increased thickness of septa. Electron microscopy revealed reduced volume density of lamellar bodies. Pulmonary expression of mRNA for surfactant proteins B and C, Mtor and Crabp1 was reduced. Expression of Igf1 was increased. Treatment with umbilical cord blood MNCs normalized thickness of septa and mRNA expression of Mtor to levels of normoxic controls. Tgfb3 mRNA expression and pro-inflammatory IL-1ß protein concentration were decreased. CONCLUSION: The results of our study demonstrate the therapeutic potential of umbilical cord blood MNCs in a new double-hit model of BPD in newborn mice. We found improved lung structure and effects on molecular level. Further studies are needed to address the role of systemic administration of MNCs in experimental BPD.

Bronchopulmonary dysplasia in a double-hit mouse model induced by intrauterine hypoxia and postnatal hyperoxia: closer to clinical features?

Despite increased survival of very preterm newborns, bronchopulmonary dysplasia (BPD) remains a major threat, as it affects long-term pulmonary function and neurodevelopmental outcome. Recent research focused on mechanisms of lung repair. Animal models of BPD in term rodents use postnatal hyperoxia in order to mimic features observed in very preterm human neonates: reduced alveolarization and impaired septal architecture without profound inflammatory changes. In contrast, BPD in very preterm human neonates involves prenatal hits e.g. infections and growth restriction plus postnatal ventilation. BPD induced in rodents by postnatal hyperoxia also exhibits reduced alveolarization however without septal pathology but with marked inflammation. We therefore aimed to establish an animal model combining prenatal growth restriction (FiO2 0.1 for 4 days) with postnatal hyperoxia (FiO2 0.7 for 2 weeks). In double-hit mice the development was retarded: body weight and length, lung and brain weight were significantly reduced by day P14 compared with normoxic controls. Histomorphometric analysis revealed reduced alveolarization and increased septal thickness without pronounced inflammatory lesions. A down-regulation of SftpB and SftpC genes was observed in double-hit animals compared with controls. Thus, we established a new model of BPD using pre- and postnatal hits.

Levels of cytokines in umbilical cord blood in relation to spontaneous term labor.

OBJECTIVE: Inflammatory mechanisms are thought to play an important role in the process of labor, both in preterm and spontaneous term delivery. We aimed to determine whether normal spontaneous vaginal delivery (SVD) at term was associated with an inflammatory activation compared with elective cesarean section (ECS) without previous onset of labor. METHODS: Cytokine concentrations were measured in venous cord blood obtained from 60 term newborns (ECS, n=35; SVD, n=25) born to mothers with clinically uneventful pregnancy and without signs of infection. RESULTS: Both study groups showed no differences in birth weight, umbilical artery pH, Apgar at 5 min, and gender distribution. Infants delivered by ECS had lower gestational age: mean, 38.5 weeks (range, 37.0-39.6 weeks) vs. mean, 39.8 weeks (range, 37.9-42.4 weeks) (P<0.001). Concentrations of interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-17, IL-1 receptor antagonist, soluble IL-2 receptor α, granulocyte-macrophage colony-stimulating factor, interferon (IFN)-α2, IFN-γ, tumor necrosis factor (TNF)-α, TNF-ß, and interferon inducible protein 10 were not different between ECS and SVD. Newborns after SVD displayed increased levels of transforming growth factor ß1 (TGF-ß1): mean, 8580 pg/mL (95% CI, 5554-11,606) vs. mean, 4864 pg/mL (95% CI, 2471-7257) (P<0.0012). CONCLUSIONS: Our findings suggest that, in healthy, term neonates, the exposition to normal spontaneous delivery and labor is not associated with systemic activation of different inflammatory mediators compared with ECS except for TGF-ß1. Further studies are needed to evaluate the immunomodulatory role of labor-associated TGF-ß1 increase in fetal cord blood.

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages.

BACKGROUND: Investigations on pulmonary macrophages (MΦ) mostly focus on alveolar MΦ (AM) as a well-defined cell population. Characteristics of MΦ in the interstitium, referred to as lung interstitial MΦ (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. METHODS: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. RESULTS: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both MΦ populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6. CONCLUSION: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung MΦ in the inflammatory response.

Transient tachypnea of the newborn (TTN): a role for polymorphisms in the beta-adrenergic receptor (ADRB) encoding genes?

AIM: Transient tachypnea of the newborn (TTN) is a common cause of early respiratory distress in the neonatal period of term infants. Delayed resorption of foetal lung fluid after birth is considered as the main pathophysiological factor. As resorption of foetal lung fluid is a catecholamine dependent process, we aimed at investigating, whether beta1- and beta2-adrenoreceptor (ADRB1, ADRB2) polymorphisms, known to alter catecholamine activity, are operative in TTN. METHODS: DNA was collected for genotyping from 73 term newborns suffering from TTN and 55 healthy controls from a Caucasian cohort. RESULTS: TTN infants were more likely to be male (70% vs. 49%; p < 0.05), had a lower mean birthweight (3120 +/- 450 vs. 3396 +/- 504 g; p < 0.001) and gestational age (GA) (38.4 +/- 1.2 vs. 39.4 +/- 1.3 weeks; p < 0.001) and were more often delivered by caesarean section (CS) (71% vs. 26%; p < 0.001). The beta1Ser49Gly polymorphism differed significantly between cases and controls. Multivariate analysis provided beta1Gly49 homozygotes with higher risk for TTN (OR 18.5; 95%CI 1.5-229; p = 0.023) than beta1Ser49 allele carrier. Further analysis showed significant association of T-47C, A46G, C79G and C491T (TACC) haplotype in ADRB2 gene with TTN (p = 0.048). CONCLUSION: We conclude that beta1Gly49 homozygosity and TACC haplotype of ADRB2 gene, both loss-of-function genetic variations, may predispose to TTN.

Complex humoral immune response against a benign tumor: frequent antibody response against specific antigens as diagnostic targets.

There are numerous studies on the immune response against malignant human tumors. This study was aimed to address the complexity and specificity of humoral immune response against a benign human tumor. We assembled a panel of 62 meningioma-expressed antigens that show reactivity with serum antibodies of meningioma patients, including 41 previously uncharacterized antigens by screening of a fetal brain expression library. We tested the panel for reactivity with 48 sera, including sera of patients with common-type, atypical, and anaplastic meningioma, respectively. Meningioma sera detected an average of 14.6 antigens per serum and normal sera an average of 7.8 antigens per serum (P = 0.0001). We found a decline of seroreactivity with malignancy with a statistical significant difference between common-type and anaplastic meningioma (P < 0.05). We detected 17 antigens exclusively with patient sera, including 12 sera that were reactive against KIAA1344, 9 against natural killer tumor recognition (NKTR), and 7 against SRY (sex determining region Y)-box2 (SOX2). More than 80% of meningioma patients had antibodies against at least one of the antigens KIAA1344, SC65, SOX2, and C6orf153. Our results show a highly complex but specific humoral immune response against a benign tumor with a distinct serum reactivity pattern and a decline of complexity with malignancy. The frequent antibody response against specific antigens offers new diagnostic and therapeutic targets for meningioma. We developed a statistical learning method to differentiate sera of meningioma patients from sera of healthy donors.