Atypical hemolytic uremic syndrome induced by SARS-CoV2 infection in infants with EXOSC3 mutation.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by systemic thrombotic microangiopathy mainly in the kidneys and mostly due to genetic disorders leading to uncontrolled activation of the complement system. Severe complications of SARS-CoV2 infection are linked to microvascular injury and complement activation is suspected to play a role in the pathogenesis of endothelial cell damage in severe COVID-19. METHODS: We present the first two cases of aHUS triggered by SARS-CoV-2 infection in two unrelated infants with the same mutation in the RNA exosome gene EXOSC3. This mutation is known to cause pontocerebellar hypoplasia type 1b, an autosomal-recessive neurodegenerative disease. So far, no kidney involvement in affected persons was reported. RESULTS: As eculizumab treatment was unsuccessful and complement-mediated disorders were ruled out, we suppose that the atypical HUS in our two patients is not due to complement-mediated thrombotic microangiopathy but rather due to a dysfunction of the RNA exosome. CONCLUSIONS: The RNA exosome is crucial for the precise processing and degradation of nuclear and cytoplasmatic RNA. We suspect that the SARS-CoV-2 infection led to changes in RNA that could not be offset by the defective RNA exosome in our two patients. The accumulation/wrong processing of the viral RNA must have led to the endothelial cell damage resulting in aHUS. This would be a new - "RNA-induced" - mechanism of aHUS.

A20 Is Increased in Fetal Lung in a Sheep LPS Model of Chorioamnionitis.

Chorioamnionitis is associated with an increased risk of preterm birth and aggravates adverse outcomes such as BPD. Development of BPD is associated with chronic inflammatory reactions and oxidative stress in the airways which may be antenatally initiated by chorioamnionitis. A20 is an immunomodulatory protein involved in the negative feedback regulation of inflammatory reactions and is a possible regulator protein in oxidative stress reactions. The influence of chorioamnionitis on A20 gene regulation in the fetal lung is unknown. We characterized the influence of LPS and proinflammatory cytokines on A20 expression in human lung endothelial (HPMEC-ST1.6R) and epithelial (A549) cells in vitro by real-time PCR and/or western blotting and used a sheep model of LPS-induced chorioamnionitis for in vivo studies. To study the functional role of A20, endogenous A20 was overexpressed in HPMEC-ST1.6R and A549 cells. LPS induced proinflammatory cytokines in HPMEC-ST1.6R and A549 cells. Both LPS and/or proinflammatory cytokines elevated A20 at transcriptional and translational levels. Intra-amniotic LPS transiently increased IL-1ß, IL-6, IL-8, and TNF-α mRNA levels in fetal lamb lungs, associated with an increase in A20 mRNA and protein levels. Overexpression of A20 reduced proinflammatory cytokines in vitro. Repeated LPS exposure induced LPS tolerance for proinflammatory cytokines and A20 in vitro and in vivo. Antenatal inflammation induced a transient increase in proinflammatory cytokines in the preterm fetal lung. The expression of proinflammatory cytokines increased expression of A20. Elevated A20 may have a protective role by downregulating chorioamnionitis-triggered fetal lung inflammation. A20 may be a novel target for pharmacological interventions to prevent chorioamnionitis-induced airway inflammation and lung damage, which can result in BPD later in life.

Acute Lung Functional and Airway Remodeling Effects of an Inhaled Highly Selective Phosphodiesterase 4 Inhibitor in Ventilated Preterm Lambs Exposed to Chorioamnionitis.

Phosphodiesterase (PDE) inhibition has been identified in animal studies as a new treatment option for neonatal lung injury, and as potentially beneficial for early lung development and function. However, our group could show that the inhaled PDE4 inhibitor GSK256066 could have dose-dependent detrimental effects and promote lung inflammation in the premature lung. In this study, the effects of a high and a low dose of GSK256066 on lung function, structure and alveolar development were investigated. In a triple hit lamb model of Ureaplasma-induced chorioamnionitis, prematurity, and mechanical ventilation, 21 animals were treated as unventilated (NOVENT) or 24 h ventilated controls (Control), or with combined 24 h ventilation and low dose (iPDE1) or high dose (iPDE10) treatment with inhaled GSK 256066. We found that high doses of an inhaled PDE4 inhibitor impaired oxygenation during mechanical ventilation. In this group, the budding of secondary septae appeared to be decreased in the preterm lung, suggesting altered alveologenesis. Ventilation-induced structural and functional changes were only modestly ameliorated by a low dose of PDE4 inhibitor. In conclusion, our findings indicate the narrow therapeutic window of PDE4 inhibitors in the developing lung.

Case Report: Symptomatic Chronic Granulomatous Disease in the Newborn.

Chronic granulomatous disease (CGD) is a primary immunodeficiency, which is diagnosed in most patients between one and three years of age. Here we report on a boy who presented at birth with extensive skin lesions and lymphadenopathy which were caused by CGD. An analysis of the literature revealed 24 patients with CGD who became symptomatic during the first six weeks of life. Although pulmonary complications and skin lesions due to infection were the leading symptoms, clinical features were extremely heterogenous. As follow-up was not well specified in most patients, the long-term prognosis of children with very early onset of CGD remains unknown.

[A Newborn Suffering from Arhinia: Neonatologic Challenges During Primary Care of the Newborn With Bosma Arhinia Microphthalmia Syndrome (BAMS)]. / Neugeborenes mit Nasenagenesie: Neonatologische Herausforderungen bei der Versorgung eines Neugeborenen mit Bosma-Arhinie-Mikrophthalmie-Syndrom (BAMS).

The rare clinical picture of nasal agenesis is to be presented on the basis of a female newborn. Intrauterine growth restriction with polyhydramnios and midface hypoplasia were noted during pregnancy. Primary cesarean section at 38 + 4 weeks' gestation was done. Airway management was achieved by splinting through a Mayo tube which was subsequently replaced by a pharyngeal endotracheal tube without signs of respiratory failure. In addition to a complete nasal agenesis, hypertelorism, a Gothic palate, bilateral microphthalmus, and iris coloboma were found. Ultrasound scans of cerebral structures were normal. An orogastric tube was placed, and drinking training and a special pacifier improved coordination and drinking performance. We suspected a case of Bosma arhinia microphthalmia syndrome (BAMS). The structural maintenance of chromosomes flexible hinge domain (SMCHD) containing 1 gene plays a key role in the embryogenesis of the human nose and is known for mutations in BAMS. A heterozygous de novo mutation in the SMCHD1 gene (c.1043A > G; pHis348Arg) was confirmed by molecular genetic analysis. Initial stabilization after birth is often a challenge in patients with nasal agenesis. They are often intubated immediately postpartum and electively tracheotomized. In the absence of respiratory problems and appropriate growth, however, there is no urgent indication for early plastic surgical treatment, given the inherent risks of sepsis and growth disorders in the midface.

Diagnostic accuracy of interleukin-6 for early-onset sepsis in preterm neonates.

Background: Early-onset sepsis (EOS) is a leading cause of morbidity and mortality among neonates. Yet, accurate diagnosis remains a major challenge in clinical routine.Objective: The aim of this study was to evaluate the diagnostic accuracy of Interleukin-6 (IL-6) in combination with other objective perinatal data for early-onset sepsis (EOS) in preterm neonates.Methods: We conducted a retrospective nested case-control study with preterm neonates with a birth weight < 2000 g born in our NICU between January 2007 and June 2016. Differences of IL-6 levels and other perinatal clinical and laboratory data between neonates with and without EOS were statistically analyzed.Results: Sixty-seven preterm infants with and 115 neonates without EOS were included in this study. Specificity and sensitivity for IL-6 were 72.8% and 75.0%, respectively, with an area under the curve of 0.804 at a cut-off point of 40 ng/l. Depending on the statistical method applied, combining IL-6 with a second perinatal factor led either to an increase of specificity (82.4-100%) or sensitivity (75.0-92.2%).Conclusion: The combination of IL-6 with other perinatal factors can significantly increase specificity and sensitivity in the diagnosis of EOS. However, overall diagnostic accuracy cannot be notably improved as there is a tradeoff between sensitivity and specificity. Although these findings do not necessarily apply in clinical routine, they can be of substantial value in the assistance of individual decision making.

Detrimental Effects of an Inhaled Phosphodiesterase-4 Inhibitor on Lung Inflammation in Ventilated Preterm Lambs Exposed to Chorioamnionitis Are Dose Dependent.

Background: Treatment of bronchopulmonary dysplasia in preterm infants is challenging due to its multifactorial origin. In rodent models of neonatal lung injury, selective inhibition of phosphodiesterase 4 (PDE4) has been shown to exert anti-inflammatory properties in the lung. We hypothesized that GSK256066, a highly selective, inhalable PDE4 inhibitor, would have beneficial effects on lung injury and inflammation in a triple hit lamb model of Ureaplasma parvum (UP)-induced chorioamnionitis, prematurity, and mechanical ventilation. Methods: Twenty-one preterm lambs were surgically delivered preterm at 129 days after 7 days intrauterine exposure to UP. Sixteen animals were subsequently ventilated for 24 hours and received endotracheal surfactant and intravenous caffeine citrate. Ten animals were randomized to receive twice a high (10 µg/kg) or low dose (1 µg/kg) of nebulized PDE4 inhibitor. Results: Nebulization of high, but not low, doses of PDE4 inhibitor led to a significant decrease in pulmonary PDE activity, and was associated with lung injury and vasculitis, influx of neutrophils, and increased proinflammatory cytokine messenger RNA levels. Conclusion: Contrary to our hypothesis, we found in our model a dose-dependent proinflammatory effect of an inhaled highly selective PDE4 inhibitor in the lung. Our findings indicate the narrow therapeutic range of inhaled PDE4 inhibitors in the preterm population.

Progesterone Antagonizes Dexamethasone-Regulated Surfactant Proteins In Vitro.

Pregnant women at risk of preterm labor routinely receive glucocorticoids (GCs) and frequently also progesterone. Administration of GCs accelerates intrauterine surfactant synthesis and lung maturation, thereby reducing the incidence of neonatal respiratory distress syndrome; progesterone has the potential to prevent preterm birth. Little is known about possible interactions of GCs and progesterone. Our aim was to clarify whether progesterone can affect dexamethasone (DXM)-regulated expression of surfactant protein A (SP-A), SP-B, and SP-D in lung epithelial cells. H441 cells were exposed to DXM and progesterone and expression of SPs was analyzed by quantitative real-time polymerase chain reaction and immunoblotting. Although progesterone had no direct effect on the expression of SP-B, DXM-mediated induction was inhibited dose dependently on the transcriptional (64 µM [P < .0001], 32 µM [P = .0005], 16 µM [P = .0019]) and the translational level. Furthermore, progesterone inhibited stimulatory effects of other GCs as well. While exogenous tissue growth factor ß1 (TGF-ß1) inhibited DXM-induced SP-B expression (messenger RNA [mRNA]: P = .0014), progesterone itself did not influence TGF-ß1 mRNA expression and/or TGF-ß1/Smad signaling, demonstrating that TGF-ß1 and/or Smad activation is not involved. The inhibitory effect of progesterone could be imitated by the GC and progesterone receptor (PR) antagonist RU-486, but not by the specific PR antagonist PF-02413873, indicating that progesterone acts as a competitive antagonist of DXM. The effect of progesterone on DXM-regulated genes was not specific for SP-B, as expression of SP-A and SP-D mRNAs was also antagonized. The present study highlights a new action of progesterone as a potential physiological inhibitor of GC-dependent SP expression in lung epithelial cells. The clinical relevance of this in vitro finding is currently unknown.

Effect of progesterone on Smad signaling and TGF-ß/Smad-regulated genes in lung epithelial cells.

The effect of endogenous progesterone and/or exogenous pre- or postnatal progesterone application on lung function of preterm infants is poorly defined. While prenatal progesterone substitution may prevent preterm birth, in vitro and in vivo data suggest a benefit of postnatal progesterone replacement on the incidence and severity of bronchopulmonary dysplasia (BPD). However, the molecular mechanisms responsible for progesterone's effects are undefined. Numerous factors are involved in lung development, airway inflammation, and airway remodeling: the transforming growth factor beta (TGF-ß)/mothers against decapentaplegic homolog (Smad) signaling pathway and TGF-ß-regulated genes, such as connective tissue growth factor (CTGF), transgelin (TAGLN), and plasminogen activator inhibitor-1 (PAI-1). These processes contribute to the development of BPD. The aim of the present study was to clarify whether progesterone could affect TGF-ß1-activated Smad signaling and CTGF/transgelin/PAI-1 expression in lung epithelial cells. The pharmacological effect of progesterone on Smad signaling was investigated using a TGF-ß1-inducible luciferase reporter and western blotting analysis of phosphorylated Smad2/3 in A549 lung epithelial cells. The regulation of CTGF, transgelin, and PAI-1 expression by progesterone was studied using a promoter-based luciferase reporter, quantitative real-time PCR, and western blotting in the same cell line. While progesterone alone had no direct effect on Smad signaling in lung epithelial cells, it dose-dependently inhibited TGF-ß1-induced Smad3 phosphorylation, as shown by luciferase assays and western blotting analysis. Progesterone also antagonized the TGF-ß1/Smad-induced upregulation of CTGF, transgelin, and PAI-1 at the promoter, mRNA, and/or protein levels. The present study highlights possible new molecular mechanisms involving progesterone, including inhibition of TGF-ß1-activated Smad signaling and TGF-ß1-regulated genes involved in BPD pathogenesis, which are likely to attenuate the development of BPD by inhibiting TGF-ß1-mediated airway remodeling. Understanding these mechanisms might help to explain the effects of pre- or postnatal application of progesterone on lung diseases of preterm infants.

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.

Is sweat testing for cystic fibrosis feasible in patients with down syndrome?

BACKGROUND: Recurrent airway infections are common in patients with Down's syndrome (DS). Hence, ruling out Cystic Fibrosis (CF) in these patients is often required. In the past, the value of sweat testing - the gold standard to diagnose CF - has been questioned in DS as false positive results have been reported. However, these reports are based on measurements of sweat osmolality or sodium concentrations, not chloride concentrations. This study analyses sweat secretion rate and chloride concentration in sweat samples of patients with DS in comparison to healthy controls. METHODS: We assessed sweat samples in 16 patients with DS and 16 healthy controls regarding sweat secretion rate (SSR) and sweat chloride concentration. RESULTS: All measured chloride concentrations were within the normal range. The chloride concentrations were slightly, but not significantly lower in patients with DS (15,54 mmol/l (±4,47)) compared to healthy controls (18,31 mmol/l (±10,12)). While no gender gap in chloride concentration could be found, chloride concentration increased with age in both groups. Insufficient sweat was collected in 2 females with DS (12.5% of the study group) but not in an individual of the control group. A significant lower sweat secretion rate was found in the DS group (27,6 µl/30 min (± 12,18)) compared to the control group (42,7 µl/30 min (± 21,22)). In a sub-analysis, female patients produced significantly less sweat (20,8 ± 10,6 µl/30 min) than male patients with DS (36,4 ± 7,8 µl/30 min), which accounts for the difference between patients and controls. Furthermore, while the sweating secretion rate increased with age in the control group, it did not do so in the DS group. Once again this was due to female patients with DS, who did not show a significant increase of sweat secretion rate with age. CONCLUSIONS: Sweat chloride concentrations were within the normal range in patients with DS and therefore seem to be a reliable tool for testing for CF in these patients. Interestingly, we found a reduced sweat secretion rate in the DS group. Whether the last one has a functional and clinical counterpart, possibly due to a disturbed thermoregulation in DS patients, requires further investigation.

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.

Double Peripheral Venous and Arterial Cannulation for Extracorporeal Membrane Oxygenation in Combined Septic and Cardiogenic Shock.

BACKGROUND The use of venoarterial extracorporeal membrane oxygenation (va-ECMO) via peripheral cannulation for septic shock is limited by blood flow and increased afterload for the left ventricle. CASE REPORT A 15-year-old girl with acute myelogenous leukemia, suffering from severe septic and cardiogenic shock, was treated by venoarterial extracorporeal membrane oxygenation (va-ECMO). Sufficient extracorporeal blood flow matching the required oxygen demand could only be achieved by peripheral cannulation of both femoral arteries. Venous drainage was performed with a bicaval cannula inserted via the left V. femoralis. To accomplish left ventricular unloading, an additional drainage cannula was placed in the left atrium via percutaneous atrioseptostomy (va-va-ECMO). Cardiac function recovered and the girl was weaned from the ECMO on day 6. Successful allogenic stem cell transplantation took place 2 months later. CONCLUSIONS In patients with vasoplegic septic shock and impaired cardiac contractility, double peripheral venoarterial extracorporeal membrane oxygenation (va-va-ECMO) with transseptal left atrial venting can by a lifesaving option.

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.

The new generation synthetic reconstituted surfactant CHF5633 suppresses LPS-induced cytokine responses in human neonatal monocytes.

BACKGROUND: New generation synthetic surfactants represent a promising alternative in the treatment of respiratory distress syndrome in preterm infants. CHF5633, a new generation reconstituted agent, has demonstrated biophysical effectiveness in vitro and in vivo. In accordance to several well-known surfactant preparations, we recently demonstrated anti-inflammatory effects on LPS-induced cytokine responses in human adult monocytes. The present study addressed pro- and anti-inflammatory effects of CHF5633 in human cord blood monocytes. METHODS: Purified neonatal CD14(+) cells, either native or simultaneously stimulated with E. coli LPS, were exposed to CHF5633. TNF-α, IL-1ß, IL-8 and IL-10 as well as TLR2 and TLR4 expression were analyzed by means of real-time quantitative PCR and flow cytometry. RESULTS: CHF5633 did not induce pro-inflammation in native human neonatal monocytes and did not aggravate LPS-induced cytokine responses. Exposure to CHF5633 led to a significant decrease in LPS-induced intracellular TNF-α protein expression, and significantly suppressed LPS-induced mRNA and intracellular protein expression of IL-1ß. CHF5633 incubation did not affect cell viability, indicating that the suppressive activity was not due to toxic effects on neonatal monocytes. LPS-induced IL-8, IL-10, TLR2 and TLR4 expression were unaffected. CONCLUSION: Our data confirm that CHF5633 does not exert unintended pro-apoptotic and pro-inflammatory effects in human neonatal monocytes. CHF5633 rather suppressed LPS-induced TNF-α and IL-1ß cytokine responses. Our data add to previous work and may indicate anti-inflammatory features of CHF5633 on LPS-induced monocyte cytokine responses.

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.

Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes.

BACKGROUND: Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes. METHODS: Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1ß, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry. RESULTS: Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1ß mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes. CONCLUSION: The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.

Multiple Antenatal Dexamethasone Treatment Alters Brain Vessel Differentiation in Newborn Mouse Pups.

Antenatal steroid treatment decreases morbidity and mortality in premature infants through the maturation of lung tissue, which enables sufficient breathing performance. However, clinical and animal studies have shown that repeated doses of glucocorticoids such as dexamethasone and betamethasone lead to long-term adverse effects on brain development. Therefore, we established a mouse model for antenatal dexamethasone treatment to investigate the effects of dexamethasone on brain vessel differentiation towards the blood-brain barrier (BBB) phenotype, focusing on molecular marker analysis. The major findings were that in total brains on postnatal day (PN) 4 triple antenatal dexamethasone treatment significantly downregulated the tight junction protein claudin-5, the endothelial marker Pecam-1/CD31, the glucocorticoid receptor, the NR1 subunit of the N-methyl-D-aspartate receptor, and Abc transporters (Abcb1a, Abcg2 Abcc4). Less pronounced effects were found after single antenatal dexamethasone treatment and in PN10 samples. Comparisons of total brain samples with isolated brain endothelial cells together with the stainings for Pecam-1/CD31 and claudin-5 led to the assumption that the morphology of brain vessels is affected by antenatal dexamethasone treatment at PN4. On the mRNA level markers for angiogenesis, the sonic hedgehog and the Wnt pathway were downregulated in PN4 samples, suggesting fundamental changes in brain vascularization and/or differentiation. In conclusion, we provided a first comprehensive molecular basis for the adverse effects of multiple antenatal dexamethasone treatment on brain vessel differentiation.

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.