Richard Keijzer

Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.

Pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains a major therapeutic problem. Moreover, the pathogenesis of pulmonary hypoplasia in case of CDH is controversial. In particular, little is known about early lung development in this anomaly. To investigate lung development separate from diaphragm development we used an in vitro modification of the 2, 4-dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH. This enabled us to investigate the direct effects of Nitrofen on early lung development and branching morphogenesis in an organotypic explant system without the influence of impaired diaphragm development. Epithelial cell differentiation of the lung explants was assessed using surfactant protein-C and Clara cell secretory protein-10 mRNA expression as markers. Furthermore, cell proliferation and apoptosis were investigated. Our results indicate that Nitrofen negatively influences branching morphogenesis of the lung. Initial lung anlage formation is not affected. In addition, epithelial cell differentiation and cell proliferation are attenuated in lungs exposed to Nitrofen. These data indicate that Nitrofen interferes with early lung development before and separate from (aberrant) diaphragm development. Therefore, we postulate the dual-hit hypothesis, which explains pulmonary hypoplasia in CDH by two insults, one affecting both lungs before diaphragm development and one affecting the ipsilateral lung after defective diaphragm development.

Congenital diaphragmatic hernia: Comparison of animal models and relevance to the human situation

Congenital diaphragmatic hernia (CDH) occurs in 1 in 3,000 newborns. Mortality and morbidity are due to the amount of pulmonary hypoplasia (PH), the response on artificial ventilation and the presence of therapy-resistant pulmonary hypertension. The pathogenesis and etiology of CDH and its associated anomalies are still largely unknown despite all research efforts over the past years. Several animal models have been proposed to study CDH. In this review we compare surgical, pharmacological and transgenic models, and discuss their strengths and limitations to study PH.

Thoracoscopic repair in congenital diaphragmatic hernia: patching is safe and reduces the recurrence rate

PURPOSE Congenital diaphragmatic hernia (CDH) has traditionally been repaired via a laparotomy. More and more reports on thoracoscopic repair are being published. The aim of this study was to evaluate our series of thoracoscopic CDH repair and compare this group to an open repair group treated during the same period in the same institute. PATIENTS AND METHODS Between June 2006 and December 2008, 49 children with posterolateral CDH were admitted, of whom 23 (47%) were operated thoracoscopically and 23 (47%) using an open repair, depending on the discretion of the attending surgeon and the clinical condition of the patient. Three patients (6%) with CDH were not treated because of associated anomalies (twice Cornelia de Lange syndrome and once hypoplastic left heart syndrome). Six thoracoscopic operations (26%) were converted to open surgery. Nine defects (39%) were closed thoracoscopically without a patch. In 8 (35%) patients, a patch was used. We used a patch in 20 open procedures (87%). RESULTS Three (33%) of the 9 thoracoscopic repairs without patch and 1 (12%) of the 8 with a patch developed a recurrence. All these recurrences were repaired thoracoscopically. The 3 recurrences from the thoracoscopic primary repair were repaired using a patch. In the open group, 3 patients (13%) developed a recurrence, of whom 2 were repaired thoracoscopically. Mean operative time was significantly longer in the thoracoscopic patch repair group (158 minutes), when compared to the open repair group (125 minutes). CONCLUSION As in open repair, it seems wise to use large patches liberally, not only to reconstruct the dome of the diaphragm but also to avoid undue tension on the repair and prevent recurrences. The thoracoscopic approach is also considered feasible in case of a recurrence from either a thoracoscopic or open repair.

Pulmonary Surfactant Protein A, B, and C mRNA and Protein Expression in the Nitrofen-Induced Congenital Diaphragmatic Hernia Rat Model

Neonates with congenital diaphragmatic hernia (CDH) suffer from a diaphragmatic defect, lung hypoplasia, and pulmonary hypertension, with poor lung function forming the major clinical challenge. Despite prenatal diagnosis and advanced postnatal treatment strategies, the mortality rate of CDH is still high. CDH has been subject of extensive research over the past decades, but its etiology remains unknown. A major problem with CDH is the failure to predict the individual response to treatment modalities like high-frequency ventilation, inhaled nitric oxide, and extracorporeal membrane oxygenation. In this study, we tested the possibility that CDH lungs are surfactant protein deficient, which could explain the respiratory failure and difficulties in treating CDH infants. We investigated this hypothesis in the nitrofen-induced CDH rat model and assessed the cellular concentrations of surfactant protein (SP)-A, -B, and -C mRNA with a quantitative radioactive in situ hybridization technique. No differences were observed between control and CDH lungs for SP mRNA expression patterns. The cellular concentration (mean OD) of SP-A and SP-B mRNA was similar at all stages whereas the mean OD of SP-C mRNA and the volume fraction of cells (% Area) expressing SP mRNA was higher in CDH lungs at term. Immunohistochemical analysis revealed no differences between control and CDH lungs for SP protein expression. No differences in the mean OD or % Area for the SP mRNAs were found between the ipsi- and contralateral side of CDH lungs. We conclude that there is no primary deficiency of surfactant proteins in the nitrofen-induced CDH rat model.

Fetal Tracheal Occlusion for Severe Pulmonary Hypoplasia in Isolated Congenital Diaphragmatic Hernia: A Systematic Review and Meta-analysis of Survival

Objective: To evaluate fetal survival after tracheal occlusion in fetuses with severe pulmonary hypoplasia and isolated congenital diaphragmatic hernia (CDH). Background: Despite recent advances in neonatal intensive care, CDH still has a high mortality and morbidity. Fetoscopic endoluminal tracheal occlusion (FETO) stimulates lung growth and improves gas exchange in animal models of CDH, but the effects in humans are still under investigation. Methods: We searched Pubmed, Cochrane, EMBASE, and Scopus databases for clinical studies on tracheal occlusion and CDH. All studies comparing FETO and a contemporary control group were included. The primary outcome was survival, with the need for oxygen on discharge the secondary outcome. Meta-analysis of outcome measures was performed and odds ratios, relative risk ratios, and 95% confidence intervals were estimated with a fixed-effects model and were reported in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidance. Results: Between 1997 and 2015, five eligible studies describing 211 patients were included (101 control and 110 FETO). All studies selected isolated severe CDH fetuses with a lung-to-head ratio 1.0 or less and liver herniation into the thoracic cavity. FETO favored survival outcome (odds ratio 13.32; 95% confidence interval, 5.40–32.87). Meta-analysis of the secondary outcome oxygen need at discharge could not be calculated, because it was not reported in all included studies. Conclusions: FETO improves survival in isolated CDH with severe pulmonary hypoplasia compared with the standard perinatal management.

MicroRNAs and lung development

MicroRNAs (miRNAs) constitute a large group of small (∼22 nucleotides), non‐coding RNA sequences that are highly conserved among animals, plants and microorganisms, suggesting that microRNAs represent a highly conserved and important regulatory mechanism. They have been demonstrated to play an important role in gene regulation. Recently, miRNAs have become a major focus of interest for research in organ development. Research focusing on the potential role of microRNAs during lung development is slowly starting to emerge. A number of miRNAs have been demonstrated to play important roles during early and late lung development. Several studies have begun to profile miRNA expression at various stages of lung development and this article provides an overview of the various miRNAs that have been implicated in lung organogenesis. Pediatr Pulmonol. 2013; 48:317–323.

Spatial and temporal expression of glucocorticoid, retinoid, and thyroid hormone receptors is not altered in lungs of congenital diaphragmatic hernia.

The degree of associated pulmonary hypoplasia and persistent pulmonary hypertension are major determination factors for survival in congenital diaphragmatic hernia (CDH) patients. Glucocorticoids, thyroid hormone, and vitamin A have been shown to be involved in human lung development. To determine their therapeutic potential in hypoplastic lungs of CDH patients, the temporal and spatial expression of glucocorticoid receptor, thyroid hormone receptors, retinoic acid receptors, and retinoid X receptors were evaluated in lungs of CDH patients, hypoplastic lungs from other causes, and normal lungs. As a series of supportive experiments, the expressions of these receptors were analyzed in lungs of nitrofen-induced CDH rats. Immunohistochemistry (human and rat) and in situ hybridization (rat) demonstrated no overt difference between CDH, hypoplastic, and control lungs, either in the localization nor the timing of the first expression of all analyzed receptors. The mRNA expression of each receptor was detected in all human CDH lungs by quantitative PCR. Our results suggest that, as far as receptors are concerned, hypoplastic lungs of fetuses and newborns with CDH are potentially as responsive to glucocorticoids, thyroid hormone, and retinoic acid as the lungs of normal children.

Intravenous and Intratracheal Mesenchymal Stromal Cell Injection in a Mouse Model of Pulmonary Emphysema

Abstract The aim of this study was to characterize the evolution of lung function and -structure in elastase-induced emphysema in adult mice and the effect of mesenchymal stromal cell (MSC) administration on these parameters. Adult mice were treated with intratracheal (4.8 units/100 g bodyweight) elastase to induce emphysema. MSCs were administered intratracheally or intravenously, before or after elastase injection. Lung function measurements, histological and morphometric analysis of lung tissue were performed at 3 weeks, 5 and 10 months after elastase and at 19, 20 and 21 days following MSC administration. Elastase-treated mice showed increased dynamic compliance and total lung capacity, and reduced tissue-specific elastance and forced expiratory flows at 3 weeks after elastase, which persisted during 10 months follow-up. Histology showed heterogeneous alveolar destruction which also persisted during long-term follow-up. Jugular vein injection of MSCs before elastase inhibited deterioration of lung function but had no effects on histology. Intratracheal MSC treatment did not modify lung function or histology. In conclusion, elastase-treated mice displayed persistent characteristics of pulmonary emphysema. Jugular vein injection of MSCs prior to elastase reduced deterioration of lung function. Intratracheal MSC treatment had no effect on lung function or histology.

Unique Tracheal Fluid MicroRNA Signature Predicts Response to FETO in Patients With Congenital Diaphragmatic Hernia

Objective and Background: Our objective was to determine the fetal in vivo microRNA signature in hypoplastic lungs of human fetuses with severe isolated congenital diaphragmatic hernia (CDH) and changes in tracheal and amniotic fluid of fetuses undergoing fetoscopic endoluminal tracheal occlusion (FETO) to reverse severe lung hypoplasia due to CDH. Methods: We profiled microRNA expression in prenatal human lungs by microarray analysis. We then validated this signature with real-time quantitative polymerase chain reaction in tracheal and amniotic fluid of CDH patients undergoing FETO. We further explored the role of miR-200b using semiquantitative in situ hybridization and immunohistochemistry for TGF-&bgr;2 in postnatal lung sections. We investigated miR-200b effects on TGF-&bgr; signaling using a SMAD-luciferase reporter assay and Western blotting for phospho-SMAD2/3 and ZEB-2 in cultures of human bronchial epithelial cells. Results: CDH lungs display an increased expression of 2 microRNAs: miR-200b and miR-10a as compared to control lungs. Fetuses undergoing FETO display increased miR-200 expression in their tracheal fluid at the time of balloon removal. Future survivors of FETO display significantly higher miR-200 expression than those with a limited response. miR-200b was expressed in bronchial epithelial cells and vascular endothelial cells. TGF-&bgr;2 expression was lower in CDH lungs. miR-200b inhibited TGF-&bgr;-induced SMAD signaling in cultures of human bronchial epithelial cells. Conclusions: Human fetal hypoplastic CDH lungs have a specific miR-200/miR-10a signature. Survival after FETO is associated with increased miR-200 family expression. miR-200b overexpression in CDH lungs results in decreased TGF-&bgr;/SMAD signaling.

Analysis of a Parent-Initiated Social Media Campaign for Hirschsprung’s Disease

Background Social media can be particularly useful for patients or families affected by rare conditions by allowing individuals to form online communities across the world. Objective Our aim in this study was to conduct a descriptive and quantitative analysis of the use of a social media community for Hirschsprung’s Disease (HD). Methods In July 2011, a mother of a child with HD launched the “Shit Happens” campaign. The campaign uses social media (blogs, Twitter, and Facebook) to engage other families affected by HD. Internet analytics including Google Analytics and Facebook Insights were used to evaluate the reach and responsiveness of this campaign. Results On the day the HD campaign was launched, 387 people viewed the blog “Roo’s Journey”. Blog views have now exceeded 5400 views from 37 countries. The Facebook page extends to 46 countries, has an average post reach of 298 users, 1414 “likes”, and an overall reach of 131,032 users. The campaign has 135 Twitter followers and 344 tweets at the time of writing. The most common question posted on the Facebook page is related to treatment for extreme diaper rash. Responsiveness assessment demonstrated that within 2 hours of posting, a question could receive 143 views and 20 responses, increasing to 30 responses after 5 hours. Conclusions Social media networks are well suited to discussion, support, and advocacy for health-related conditions and can be especially important in connecting families affected by rare conditions. The HD campaign demonstrates the reach and responsiveness of a community that primarily relies on social media to connect families affected by HD. Although responsive, this community is currently lacking consistent access to evidence-based guidance for their common concerns. We will explore innovative consumer-researcher partnerships to offer a solution in future research.