Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells

BACKGROUND: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). METHODS: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. RESULTS: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. CONCLUSION: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells

Surfactantlipid biosynthesis: Regulation of transmembrane transport of palmitate

Titelblatt und Inhaltsverzeichnis Einführung in die Thematik Übersicht der eigenen Untersuchungen Zusammenfassung der Ergebnisse Zusammenfassung Literatur Abkürzungen Danksagung Eidesstattliche Versicherung Publizierte Forschungsergebnisse dieser ArbeitDie Senkung der Inzidenz des Atemnotsyndroms durch die pränatale Lungen-rei- feinduktion und die deutlich verringerte Letalität des Atemnotsyndroms seit Einführung der Surfactant-Therapie haben in erheblichem Umfang zur Abnahme der Säuglings-sterblich-keit beigetragen. Die Fortschritte innerhalb der letzten Jahre sind hinsichtlich der Co-Morbidität des Atemnotsyndroms allerdings deutlich geringer als in den Jahren vor 1990. Bei der Induktion der pränatalen Lungenreife wurde die Bedeutung der langketti-gen Fettsäuren (LCFA) als Substrat der Surfactantlipidsynthese nur am Rande unter-sucht. Mit dem Ziel, die körpereigene Surfactantbildung während der foetalen und/oder neonatalen Entwicklung gezielt zu steigern, haben wir Regulationsmechanismen der de novo Phosphatidylcholin-Synthese in Typ II Zellen untersucht. Der Schwerpunkt unserer Arbeit lag auf der Charakterisierung der Aufnahme von Palmitinsäure durch Typ II Zellen, da durch die ausreichende Bereitstellung dieses Substrates eine Steigerung der Surfac-tantlipidsynthese erzielbar ist. Zu Beginn unserer Untersuchungen wurde der Transport von LCFA durch die Zellmembran grundsätzlich kontrovers � passiv vs. proteinvermittelt - diskutiert. Wir untersuchten deshalb zunächst den transmembranären Transport von Palmitat hinsichtlich seiner Kinetik und Energieabhängigkeit und zeigten, daß FAT/CD36 für etwa 70 % der Palmitataufnahme isolierter Typ II Zellen verantwortlich ist. Die Palmitat-auf-nahme wird nicht über "coated pits", sondern über Caveolae-ähnliche Strukturen der Typ II Zellmembran, sogenannten DIGs, vermittelt. Durch Manipulation des zellulären Chole-sterolgehaltes gelang es uns, FAT/CD36 in DIGs anzureichern und dabei die Palmitatauf-nahme drastisch zu steigern. Die an FAT/CD36 gebundene Palmitinsäure braucht einen Lösungsvermittler um in das Cytosol zu gelangen. FABPs sind cytosolische Proteine, die die Lösung der LCFA vermitteln können. Folgerichtig ist die Palmitat-aufnahme und Phosphatidycholin-Synthese in Typ II Zellen von H-/E-FABP double knock out-Tieren verringert. Wird die Expression von FAT/CD36 und Caveolin-1 durch den PPARgamma-Aktivator Pioglitazon erhöht, steigen Palmitataufnahme und Phosphatidycholin-Synthese wieder auf das Niveau des Wildtypes. Die Kompensation des FABP-knock-out Effektes durch Pioglitazon beruht sehr wahrscheinlich auf einer Interaktion von Caveolin-1 und PPARgamma, die wir erstmals nachweisen konnten. Kürzlich wurde beschrieben, daß sich Vesikel, die Caveolin-1 enthalten, abschnüren und möglicherweise LCFA wie FABPs durch das Cytosol transportieren können. Diese und die von anderen Arbeitsgruppen gewonnenen Daten zeigen klar, daß membranständigen Fettsäuretransportern - hier FAT/CD36 - eine wichtige Rolle bei der Fettsäureaufnahme zukommt und daß die Zelle über verschiedene Mechanismen der Regulation der Palmitataufnahme verfügt. Die Beschleunigung der initialen Palmitatauf-nahme in Typ II Zellen geht mit einer Steigerung der Phosphatidylcholin-Synthese einher, die wahrscheinlich durch eine Aktivierung der Cytidylyltransferase vermittelt wird. Es ist aber auch denkbar, daß das vermehrte intrazelluläre Angebot an Fettsäure die Glycerin-3-phosphat Acyltransferase stimuliert, die ebenfalls geschwindigkeitsbestimmend bei der Phosphatidylcholin-Synthese sein kann. Wir konnten zeigen, daß Vitamin E-Depletion die Aktivität der Glycerin-3-phosphat Acyltransferase und die Synthese von Phosphatidyl-cholin in Typ II Zellen senkt. Die Oxidation von funktionellen SH-Gruppen des Enzyms bei Vitamin E-Mangel hemmt seine Aktivität. Es ist bekannt, daß die verringerte Phosphati-dylcholin-Synthese in Typ II Zellen unter Ozon mit einer isolierten Hemmung der Glycerin-3-phosphat Acyltransferase einhergeht. Die Kombination beider Faktoren, Vitamin E-Mangel und oxidative Belastung der Typ II Zelle, ist klinisch, vor allem bei unreifen Neugeborenen, relevant und beeinträchtigt sehr wahrscheinlich die Phosphatidylcholin-Synthese. Nach unserer Kenntnis ist der Effekt von Vitamin E auf die Inzidenz oder den Schweregrad des Atemnotsyndroms nicht untersucht. Ob unsere tierexperimentell gewonnenen Daten zur Phosphatidylcholin-Synthese bei Vitamin E-Mangel von klinischer Relevanz sind, läßt sich daher gegenwärtig nicht abschätzen.Considering the mechanisms by which antenatal maturation of lung can be induced, the role of long chain fatty acids as precursors of surfactant lipid synthesis has not been thoroughly investigated. To specifically increase surfactant synthesis during the fetal and/or neonatal period we studied the regulation of de novo phosphatidyl synthesis in type II pneumocytes. First, we characterised the transmembrane transport of palmitate, a long chain fatty acid prevalent in surfactant lipids, with regard to its kinetic and energy dependence and showed that FAT/CD36 facilitates around 70 % of palmitate uptake into type II pneumocytes. Palmitate uptake does not depend on coated pits but is mediated by membrane structures which are similar to caveolae, so called DIGs. Increase of cellular concentration of cholesterol caused a significant enrichment of FAT/CD36 in DIGs of type II cells which was accompanied by a drastically enhanced palmitate uptake. Once inside the cell, palmitate is bound to members of the family of fatty acid binding proteins (FABP). We identified two members, the epidermal (E-) and the heart (H-) type FABP in type II cells. Based on the assumption that E-FABP and H-FABP in alveolar type II cells mediate the synthesis of dipalmitoyl phosphatidylcholine, the main surfactant phospholipid, we analysed TII cells isolated from wild type (wt) and E/H-FABP double-knock out (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, β-oxidation, and incorporation into neutral lipids and total phosphatidylcholines of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs encoding fatty acid translocase (FAT), caveolin-1 and PPARγwas observed in the double-ko phenotype. As caveolin-1 interacted with PPARγ we assumed that FAT, caveolin-1, and PPARγ form a signalling chain for fatty acid or drug. Consequently, PPARγ-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARγ could �heal� the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells. These data and data obtained by other groups clearly indicate that membrane-bound fatty acid carriers, e.g. FAT/CD36, play an important role in long chain fatty acid uptake and that mammalian cells possess different mechanisms to regulate palmitate uptake. Increase of palmitate uptake is paralleled by an increased phosphatidylcholine synthesis, probably mediated by activation of cytidylyl transferase

Short courses of dual-strain probiotics appear to be effective in reducing necrotising enterocolitis

AIM: Prophylactic probiotics to reduce necrotising enterocolitis (NEC) are mostly given for at least 28 days or until discharge. We describe the effects of a shorter duration dosing strategy. METHODS: Retrospective cohort study of neonates (birth weight 400-1,500 g) in three neonatal intensive care units in Switzerland and Germany that embarked on probiotic prophylaxis given for 10 or 14 days, employing a fixed combination (Lactobacillus acidophilus plus Bifidobacterium infantis, each 10(9) CFU/d) licensed as a drug in Switzerland. Probiotics were initiated upon discontinuation of antibiotics, or on day 1-3 in infants without antibiotics. Repeat probiotic courses were given whenever antibiotics had been instituted and were discontinued. RESULTS: Birth weight and gestational age were similar in the two 24-month pre- and post-implementation cohorts. NEC rates fell from 33/633 (5.2%) to 8/591 infants alive at 3 days (1.4%; risk ratio (RR) 0.26, 95% confidence interval (CI) 0.12-0.55). The drop in NEC was significant both for infants of 400-999 g (6.4% to 2.5%) and 1,000-1,500 g birth weight (4.4% to 0.6%). Mortality was 5.1% (32/633) without, as opposed to 3.5% (21/591) with probiotics, respectively (RR 0.69, 95% CI 0.41-1.19). CONCLUSION: Short courses of a dual-strain probiotics appear to be effective in reducing NEC. This article is protected by copyright. All rights reserved

Anatomic accuracy, physiologic characteristics, and fidelity of very low birth weight infant airway simulators

Background!#!Medical simulation training requires realistic simulators with high fidelity. This prospective multi-center study investigated anatomic precision, physiologic characteristics, and fidelity of four commercially available very low birth weight infant simulators.!##!Methods!#!We measured airway angles and distances in the simulators Premature AirwayPaul (SIMCharacters), Premature Anne (Laerdal Medical), Premie HAL S2209 (Gaumard), and Preterm Baby (Lifecast Body Simulation) using computer tomography and compared these to human cadavers of premature stillbirths. The simulators' physiologic characteristics were tested, and highly experienced experts rated their physical and functional fidelity.!##!Results!#!The airway angles corresponded to those of the reference cadavers in three simulators. The nasal inlet to glottis distance and the mouth aperture to glottis distance were only accurate in one simulator. All simulators had airway resistances up to 20 times higher and compliances up to 19 times lower than published reference values. Fifty-six highly experienced experts gave three simulators (Premature AirwayPaul: 5.1 ± 1.0, Premature Anne 4.9 ± 1.1, Preterm Baby 5.0 ± 1.0) good overall ratings and one simulator (Premie HAL S2209: 2.8 ± 1.0) an unfavorable rating.!##!Conclusion!#!The simulator physiology deviated significantly from preterm infants' reference values concerning resistance and compliance, potentially promoting a wrong ventilation technique.!##!Impact!#!Very low birth weight infant simulators showed physiological properties far deviating from corresponding patient reference values. Only ventilation with very high peak pressure achieved tidal volumes in the simulators, as aimed at in very low birth weight infants, potentially promoting a wrong ventilation technique. Compared to very low birth weight infant cadavers, most tested simulators accurately reproduced the anatomic angular relationships, but their airway dimensions were relatively too large for the represented body. The more professional experience the experts had, the lower they rated the very low birth weight infant simulators

Anatomic accuracy, physiologic characteristics, and fidelity of very low birth weight infant airway simulators

Background Medical simulation training requires realistic simulators with high fidelity. This prospective multi-center study investigated anatomic precision, physiologic characteristics, and fidelity of four commercially available very low birth weight infant simulators. Methods We measured airway angles and distances in the simulators Premature AirwayPaul (SIMCharacters), Premature Anne (Laerdal Medical), Premie HAL S2209 (Gaumard), and Preterm Baby (Lifecast Body Simulation) using computer tomography and compared these to human cadavers of premature stillbirths. The simulators' physiologic characteristics were tested, and highly experienced experts rated their physical and functional fidelity. Results The airway angles corresponded to those of the reference cadavers in three simulators. The nasal inlet to glottis distance and the mouth aperture to glottis distance were only accurate in one simulator. All simulators had airway resistances up to 20 times higher and compliances up to 19 times lower than published reference values. Fifty-six highly experienced experts gave three simulators (Premature AirwayPaul: 5.1 +/- 1.0, Premature Anne 4.9 +/- 1.1, Preterm Baby 5.0 +/- 1.0) good overall ratings and one simulator (Premie HAL S2209: 2.8 +/- 1.0) an unfavorable rating. Conclusion The simulator physiology deviated significantly from preterm infants' reference values concerning resistance and compliance, potentially promoting a wrong ventilation technique. Impact Very low birth weight infant simulators showed physiological properties far deviating from corresponding patient reference values. Only ventilation with very high peak pressure achieved tidal volumes in the simulators, as aimed at in very low birth weight infants, potentially promoting a wrong ventilation technique. Compared to very low birth weight infant cadavers, most tested simulators accurately reproduced the anatomic angular relationships, but their airway dimensions were relatively too large for the represented body. The more professional experience the experts had, the lower they rated the very low birth weight infant simulators

Symptom Burden and Factors Associated with Acute Respiratory Infections in the First Two Years of Life-Results from the LoewenKIDS Cohort.

Acute respiratory infections (ARIs) are the most common childhood illnesses worldwide whereby the reported frequency varies widely, often depending on type of assessment. Symptom diaries are a powerful tool to counteract possible under-reporting, particularly of milder infections, and thus offer the possibility to assess the full burden of ARIs. The following analyses are based on symptom diaries from participants of the German birth cohort study LoewenKIDS. Primary analyses included frequencies of ARIs and specific symptoms. Factors, which might be associated with an increased number of ARIs, were identified using the Poisson regression. A subsample of two hundred eighty-eight participants were included. On average, 13.7 ARIs (SD: 5.2 median: 14.0 IQR: 10-17) were reported in the first two years of life with an average duration of 11 days per episode (SD: 5.8, median: 9.7, IQR: 7-14). The median age for the first ARI episode was 91 days (IQR: 57-128, mean: 107, SD: 84.5). Childcare attendance and having siblings were associated with an increased frequency of ARIs, while exclusive breastfeeding for the first three months was associated with less ARIs, compared to exclusive breastfeeding for a longer period. This study provides detailed insight into the symptom burden of ARIs in German infants