Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes-Brocks syndrome 2.

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1-/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

Appendiceal Carcinoids in Children-Prevalence, Treatment and Outcome in a Large Nationwide Pediatric Cohort.

Background and Objectives: Appendiceal carcinoids are rare neuroendocrine tumors and mainly found incidentally during histopathological examination following appendectomy. This observational cohort study was performed to determine the prevalence, treatment modalities and outcomes in children diagnosed with an appendiceal carcinoid tumor. Materials and Methods: Data from the largest German statutory health insurance "Techniker Krankenkasse" were analyzed within an 8-year period: January 2010 to December 2012 and January 2016 to December 2020. Patient characteristics, surgical technique, type of surgical department, diagnostic management, and postoperative morbidity were analyzed. Results: Out of 40.499 patients following appendectomy, appendiceal carcinoids were found in 44 children, resulting in a prevalence of 0.11%. Mean age at appendectomy was 14.7 (±2.6) years. Laparoscopic approach was performed in 40 (91%) cases. Right-sided hemicolectomy was performed in 8 (18%) patients. Additional diagnostic work-up (CT and MRI) was recorded in 5 (11%) children. Conclusions: This large nationwide pediatric study shows that 1 in 1000 patients was found to have a neuroendocrine tumor of the appendix (prevalence 0.11%), emphasizing its low prevalence in the pediatric age group. The majority of patients were treated with appendectomy only. However, treatment modalities are still variable. Longer follow-up analyses are needed to evaluate published guidelines and recommendations to aim for a limited surgical approach.

Decreased Endoglin expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia rat model.

AIM OF THE STUDY: Pulmonary hypertension (PH) remains a therapeutical challenge in neonates born with congenital diaphragmatic hernia (CDH). Endoglin (Eng), an auxiliary receptor component of the transforming growth factor ß (TGFß) signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodelling. Genetic studies have linked TGFß and Eng mutations to human arterial PH and other cardiovascular syndromes. Eng interacts with the TGFß receptors 1 and 2 (Tgfßr1, Tgfßr2). We designed this study to investigate the hypothesis that Eng is altered in the pulmonary vasculature of rats with nitrofen-induced CDH subjected to its interdependency with Tgfßr1 and Tgfßr2. METHODS: After ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received either nitrofen or olive oil on gestational day (D9). The foetuses (n = 22) were sacrificed and divided into CDH and control group on D21. Gene and protein expressions of Eng, Tgfßr1 and Tgfßr2 were assessed via qRT-PCR and western blotting. Immunofluorescence staining for Eng was combined with CD34 to evaluate Eng expression in the pulmonary vasculature. MAIN RESULTS: Relative mRNA levels of Eng, Tgfßr1 and Tgfßr2 were significantly downregulated in CDH lungs compared to controls (Eng CDH 0.341 ± 0.022, Eng Ctrl 0.471 ± 0.031, p = 0.0015; Tgfßr1 CDH 0.161 ± 0.008, Tgfßr1 Ctrl 0.194 ± 0.01, p = 0.0114; Tgfßr2 CDH 0.896 ± 0.099, Tgfßr2 Ctrl 1.379 ± 0.081, p = 0.0006) Western blotting confirmed the reduced pulmonary protein expression of these three proteins in the CDH lungs. A markedly diminished endothelial expression of Eng in the pulmonary vasculature of nitrofen-exposed foetuses compared to controls was seen in laser scanning confocal-microscopy. CONCLUSION: This study demonstrates for the first time a reduced expression of Endoglin in the pulmonary vasculature of nitrofen-induced CDH. Abnormal Eng/Tgfßr1/Tgfßr2 signalling may contribute to impaired vascular remodelling and development of PH in this CDH animal model.

Upregulation of S1P1 and Rac1 receptors in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Sphingolipids play a crucial role in pulmonary development. The sphingosine kinase 1 (SphK1) modulates the synthesis of sphingolipid sphingosine-1-phosphate (S1P). S1P regulates cell proliferation and angiogenesis via different receptors, S1P1, S1P2 and S1P3, which all influence the expression of Ras-related C3 botulinum toxin substrate 1 (Rac1). We designed this study to test the hypothesis that the S1P/Rac1 pathway is altered in the nitrofen-induced CDH model. METHODS: Pregnant rats received nitrofen or vehicle on D9. On D21, fetuses were killed and divided into nitrofen and control group (n = 12). QRT-PCR, western blotting and confocal-immunofluorescence microscopy were performed to reveal pulmonary gene and protein expression levels of SphK1, S1P1, S1P2, S1P3 and Rac1. RESULTS: Pulmonary gene expression of S1P1 and Rac1 was significantly increased in the CDH group compared to controls, whereas S1P2 and S1P3 expression was decreased. These results were confirmed by western blotting and confocal microscopy. SphK1 expression was not found to be altered. CONCLUSION: The increased expression of S1P1 and Rac1 in the pulmonary vasculature of nitrofen-induced CDH lungs suggests that S1P1 and Rac1 are important mediators of PH in this model.

Disruption of copper-dependent signaling pathway in the nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Normal development of the fetal diaphragm requires muscularization of the diaphragm as well as the structural integrity of its underlying connective tissue components. Developmental mutations that inhibit the formation of extracellular matrix (ECM) have been shown to result in congenital diaphragmatic hernia (CDH). Copper (Cu) is an important element during diaphragm morphogenesis by participating in cross-linking of collagen and elastin fibers. Cu transport is strictly regulated by two membrane proteins: Cu-uptake transporter 1 (CTR1) and the Cu-efflux pump ATP7A. Animals lacking Cu-dependent enzymes exhibit abnormal connective tissue with diaphragmatic defects. However, the molecular basis of disruptions in Cu-mediated ECM formation in CDH remains unclear. We designed this study to investigate the hypothesis that diaphragmatic expression of CTR1 and ATP7A is decreased in the nitrofen-induced CDH model. METHODS: Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D15 and D18. Microdissected fetal diaphragms (n = 48) were divided into control and nitrofen-induced CDH samples (n = 12 per experimental group and time-point). Diaphragmatic gene expression levels of CTR1 and ATP7A were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to evaluate CTR1 and ATP7A protein expression in fetal diaphragms, which was combined with specific rhodanine staining to determine diaphragmatic Cu content. RESULTS: Relative mRNA levels of CTR1 and ATP7A were significantly reduced in diaphragms of nitrofen-exposed fetuses on D15 (0.06 ± 0.02 vs. 0.18 ± 0.08; p < 0.05 and 0.04 ± 0.02 vs. 0.08 ± 0.02; p < 0.05) and D18 (0.10 ± 0.03 vs. 0.17 ± 0.02; p < 0.05 and 0.09 ± 0.03 vs. 0.16 ± 0.04; p < 0.05) compared to controls. Immunoreactivity of CTR1 and ATP7A was markedly decreased in the malformed diaphragmatic ECM of nitrofen-exposed fetuses on D15 and D18, which was associated with a significantly decreased diaphragmatic Cu content on D15 (7.22 ± 2.91 vs. 17.50 ± 3.09; p < 0.05) and D18 (17.60 ± 3.54 vs. 28.20 ± 4.63; p < 0.05) compared to controls. CONCLUSION: Reduced diaphragmatic expression of CTR1 and ATP7A during morphogenesis may impair the activity of Cu-dependent enzymes and thus contribute to defective ECM during diaphragmatic development.

Imbalance of caveolin-1 and eNOS expression in the pulmonary vasculature of experimental diaphragmatic hernia.

BACKGROUND: Caveolin-1 (Cav-1) exerts major regulatory functions on intracellular signaling pathways originating at the plasma membrane. Cav-1 is a key regulator in adverse lung remodeling and the development of pulmonary hypertension (PH) regulating vasomotor tone through its ability to reduce nitric oxide (NO) production. This low-output endothelial NO synthase (eNOS) derived NO maintains normal pulmonary vascular homeostasis. Cav-1 deficiency leads to increased bioavailability of NO, which has been linked to increased nitrosative stress. Inhibition of eNOS reduced oxidant production and reversed PH, supporting the concept that Cav-1 regulation of eNOS activity is crucial to endothelial homeostasis in lungs. We designed this study to investigate the hypothesis that expression of Cav-1 is downregulated while eNOS expression is upregulated by the pulmonary endothelium in the nitrofen-induced congenital diaphragmatic hernia (CDH). METHODS: Pregnant rats were exposed to nitrofen or vehicle on day 9.5 (D9.5). Fetuses were sacrificed on D21 and divided into nitrofen and control groups. Quantitative real-time polymerase chain reaction, Western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of Cav-1 and eNOS. RESULTS: Pulmonary Cav-1 gene expression levels were significantly decreased, while eNOS gene expression was significantly increased in nitrofen-induced CDH(+). Western blotting and confocal microscopy revealed decreased pulmonary Cav-1 protein expression, while eNOS protein expression was increased in CDH(+) compared to controls. CONCLUSION: The striking evidence of markedly decreased gene and protein expression of Cav-1 with concurrently increased eNOS gene and protein expression in the pulmonary vasculature suggests that activation of eNOS secondary to Cav-1 deficiency may play an important role in the pathogenesis of PH in the nitrofen-induced CDH.

Prevalence of Hirschsprung's disease in premature infants: a systematic review.

PURPOSE: Intestinal dysmotility in preterm infants has often been attributed to immature enteric nervous system. It is frequently reported that Hirschsprung's disease (HD) is rare in premature infants. The exact prevalence of HD in premature infants is not well documented. The classical signs of HD may often not be identified due to the complexity of symptoms of prematurity itself. This systematic review was designed to determine the prevalence and presentation of HD in premature infants. METHODS: A systematic review of the literature using the keywords "Hirschsprung's disease", "aganglionosis", "congenital megacolon", "premature" and "preterm" was performed. Resulting publications were reviewed for epidemiology and morbidity. Only infants born <37 weeks of gestation or described as preterm birth by the authors were included. Reference lists were screened for additional relevant studies. RESULTS: Twenty-six publications from 1964 to 2013 reported data on premature infants with HD. Out of a total number of 4,147 infants, prematurity was recorded in 257 cases, giving a prevalence rate of 6 % of preterm infants diagnosed with HD. During 1964-1999, reported prevalence of HD in premature infants ranged from 1.7 to 9.2 % (overall prevalence 5 %) and during 2000-2013 prevalence ranged from 4 to 19.4 % (overall prevalence 14 %). The prevalence of total colonic aganglionosis in premature infants was 13 % (15 out of 118 infants). Mean gestational age of preterm infants was 34.5 (± 0.7) weeks and mean age at diagnosis ranged from 18.3 days to 3.9 months. Abdominal distension was observed in 80 % of preterm infants, delayed passage of meconium in 57 and 37 % of premature infants presented with bile-stained vomiting. CONCLUSION: In recent years, higher prevalence of HD has been reported in premature infants compared to previous years. Hirschsprung's disease should be considered in preterm infants presenting with features of intestinal obstruction.

Congenital anomalies of the kidney and urinary tract (CAKUT) associated with Hirschsprung's disease: a systematic review.

PURPOSE: Congenital anomalies of the kidney and urinary tract (CAKUT), a term introduced in the late 1990 s accounts for 30-50 % of cases of end-stage renal disease in children. The association of urogenital anomalies and Hirschsprung's disease (HSCR) based on the common genetic background of enteric nervous system and human urinary tract development has been well described in the literature. However, the reported prevalence of HSCR associated with CAKUT seems to be underestimated. The aim of this systematic review was to determine the prevalence of this association and show its relationship to other syndromes. METHODS: A systematic literature search was conducted for relevant articles published between 1955 and 2014. Two online databases were searched for the terms "Hirschsprung's disease", "congenital anomalies of the kidney and urinary tract", "urogenital anomalies" and "urological anomalies". All published studies containing adequate clinical data were included. Resulting publications were reviewed for epidemiology, genetic testing, operative treatment and morbidity. Reference lists were screened for additional cases. RESULTS: A total of 32 articles reported 222 cases of HSCR associated with either CAKUT, "urological" or "urogenital" anomalies from 1955 to 2014. Gender was reported in a total of 68 cases, with 54 (79 %) males and 14 (21 %) females. Extent of aganglionosis was reported in 67 cases and included classical rectosigmoid disease in 38, long-segment aganglionosis in 12, total colonic aganglionosis in 12 and total intestinal aganglionosis in 5 patients. 18 articles reported 204 cases of either CAKUT, "urological" or "urogenital" anomalies in a case series of 5.693 HSCR patients, resulting in an overall prevalence of 3.6 % of this association. Within this collective of 18 studies only seven were, regardless of the date of publication compatible with CAKUT criteria introduced and published in the late 1990 s. These seven studies reported a total of 72 patients with associated CAKUT among 757 HSCR patients resulting in a prevalence of 9.5 %. After introduction of the CAKUT acronym, only three studies specifically investigated the association of HSCR and CAKUT stating a prevalence of 14.3 % resulting in an almost fivefold increase compared to the reported prevalence of HSCR and associated urological and urogenital anomalies. The remaining 14 publications reported 18 single cases of HSCR patients with associated CAKUT phenotypes. Of these 18 cases, 11 (61 %) cases were associated with other syndromes or syndromatic features or reported chromosomal anomalies. CONCLUSION: This review confirms that the recognition of CAKUT in HSCR patients has been underestimated in the past. The results suggest that when confronted with HSCR in a patient, a thorough urological investigation may be indicated. The high prevalence of associated syndromes in HSCR with CAKUT may further suggest a syndromic association.

Myogenin gene expression is not altered in the developing diaphragm of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Pleuroperitoneal folds (PPFs) represent the only source of muscle precursors cells (MPCs) in the primordial diaphragm. However, the exact pathogenesis of malformed PPFs and congenital diaphragmatic hernia (CDH) remains unclear. The muscle-specific transcription factor myogenin plays a key role during development and muscularization of the fetal diaphragm. Although myogenin knockout mice lack skeletal muscle fibers, the diaphragmatic musculature is intact without any defects. It has further been demonstrated that proliferation and differentiation of MPCs in PPFs and developing diaphragms are normal in rodent CDH models. We hypothesized that myogenin gene expression is not altered in malformed PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model. METHODS: Pregnant rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested during PPF formation (D13), diaphragmatic development (D14-15) and muscularization (D18-21). Fetal PPFs, developing diaphragms and diaphragmatic musculature were dissected and divided into nitrofen and control groups. Myogenin mRNA levels were analyzed by quantitative real-time polymerase chain reaction, while immunohistochemistry was performed to investigate myogenin protein expression and distribution. RESULTS: Relative mRNA expression of myogenin was not significant different in PPFs (0.30 ± 0.09 vs. 0.48 ± 0.09; P = 0.37), developing diaphragms (1.25 ± 0.29 vs. 1.60 ± 0.32; P=0.53) and diaphragmatic musculature (1.08 ± 0.24 vs. 1.59 ± 0.20; P = 0.15) of nitrofen-exposed fetuses compared to controls. Myogenin immunoreactivity was not altered in the muscular components of malformed PPFs, developing diaphragms and diaphragmatic musculature of nitrofen-exposed fetuses compared to controls. CONCLUSION: Myogenin gene expression is not altered in PPFs, developing diaphragms and diaphragmatic musculature in the nitrofen-induced CDH model, thus suggesting that diaphragmatic defects in this model develop independent of myogenic processes.

Downregulation of p300 gene expression in airway mesenchyme of nitrofen-induced hypoplastic lungs.

PURPOSE: Congenital diaphragmatic hernia (CDH) is a relatively common developmental abnormality causing life-threatening respiratory distress at birth. The nitrofen model has been widely used to investigate the pathogenesis of hypoplastic lungs associated with CDH. Embryos lacking p300 and CBP genes are significantly smaller in lung formation. We hypothesized that pulmonary gene expression of p300 and CBP is downregulated during late gestation in the nitrofen-induced CDH model. METHODS: Time-pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 (n = 8 at each time point). Pulmonary gene expression of p300 and CBP was analyzed by quantitative real-time PCR. Immunohistochemistry was performed to investigate expression and localization of pulmonary p300 and CBP proteins. RESULTS: Relative mRNA expression levels of p300 were significantly decreased in nitrofen-induced hypoplastic lungs on D18 compared to controls (3.00 ± 0.20 vs. 3.76 ± 0.14; p = 0.0039), while CBP levels were not altered. p300 immunoreactivity was markedly diminished in surrounding mesenchymal compartments and nuclei of proximal and distal airway cells, while CBP expression was not altered. CONCLUSION: Downregulation of p300 gene expression during the early canalicular stage may disrupt epithelial-mesenchymal signaling interactions, contributing to the development of hypoplastic lungs in the nitrofen-induced CDH model.

Increased pulmonary vascular expression of Krüppel-like factor 5 and activated survivin in experimental congenital diaphragmatic hernia.

AIM OF THE STUDY: The high morbidity and mortality in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by increased pulmonary artery smooth muscle cell (SMC) proliferation, suppressed apoptosis as well as endothelial dysfunction. Krüppel-like factor 5 (KLF5) belongs to a family of transcription factors that has diverse functions during cell differentiation and embryonic development. KLF5 is preferentially expressed in proliferating SMCs but reduced in differentiated cells. KLF5 induces the expression of Survivin, a 16.5 kDa protein overexpressed in almost all malignancies but hardly detected in normal differentiated tissues. Survivin has been shown to inhibit apoptosis, promote cell proliferation, and enhance angiogenesis. Recent studies have implicated activation of KLF5 and Survivin in the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that KLF5 and Survivin expression are increased in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen (n = 16) and control group (n = 16). Quantitative real-time PCR, western blotting, and confocal immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of KLF5, Survivin, and phosphorylated Survivin (p-Survivin). MAIN RESULTS: Confocal microscopy revealed markedly increased pulmonary vascular KLF5 and p-Survivin expression in lungs of nitrofen-exposed fetuses compared to controls. These results were confirmed by western blotting, showing increased pulmonary expression of KLF5 and p-Survivin. Furthermore, the relative pulmonary gene expressions of KLF5 and Survivin were significantly increased in the CDH group compared to controls (p < 0.005 rsp. p < 0.01). CONCLUSION: This study provides striking evidence of increased gene and protein expression of KLF5 and activated Survivin in the pulmonary vasculature of nitrofen-induced CDH, suggesting that increased expression of KLF5 may activate p-Survivin expression and play an important role in the pathogenesis of PH in nitrofen-induced CDH.

Prenatal administration of all-trans retinoic acid upregulates leptin signaling in hypoplastic rat lungs with experimental congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypoplasia (PH), characterized by alveolar immaturity, is one of the leading causes of respiratory insufficiency in newborns with congenital diaphragmatic hernia (CDH). Leptin (Lep) and its receptor (Lep-R) play an important role in fetal lung growth by stimulating alveolar differentiation and maturation. Lep and Lep-R are strongly expressed by alveolar cells during the saccular stage of fetal lung development. Lep-deficient mice exhibit decreased alveolarization with reduced pulmonary surfactant phospholipid synthesis, similar to human and nitrofen-induced PH. Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced PH. Recent studies have demonstrated that Lep and Lep-R expression in developing lungs is regulated by ATRA. We hypothesized that prenatal treatment with ATRA increases pulmonary Lep and Lep-R expression in the nitrofen model of CDH-associated PH. METHODS: Time-mated rats received either 100 mg nitrofen or vehicle via oral-gastric lavage on embryonic day 9.5 (E9.5). Control and nitrofen-exposed dams were randomly assigned to either intraperitoneal ATRA (5 mg/kg/d) or placebo administration on E18.5, E19.5 and E20.5. Fetal lungs were harvested on E21.5, and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo and Nitrofen+ATRA. Alveolarization was assessed using stereo- and morphometric analysis techniques. Surfactant phospholipid synthesis was analyzed by labeling for surfactant protein B (SP-B). Pulmonary gene expression levels of Lep and Lep-R were determined using quantitative real-time polymerase chain reaction. Immunohistochemical staining for Lep and Lep-R was performed to evaluate alveolar protein expression and localization. RESULTS: In vivo administration of ATRA resulted in significantly increased lung-to-body weight ratio with enhanced radial alveolar count and decreased mean linear intercept compared to placebo treatment. Immunofluorescence analysis demonstrated markedly increased pulmonary SP-B expression in Nitrofen+ATRA compared to Nitrofen+Placebo. Relative mRNA expression of Lep and Lep-R was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo. Lep and Lep-R immunoreactivity was markedly increased in interstitial and alveolar epithelial cells of Nitrofen+ATRA compared to Nitrofen+Placebo. CONCLUSION: Increased Lep and Lep-R expression after prenatal administration of ATRA in nitrofen-induced PH suggests that ATRA may have therapeutic potential in attenuating CDH-associated PH by stimulating alveolarization and de novo surfactant production.

Evidence for decreased lipofibroblast expression in hypoplastic rat lungs with congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypoplasia (PH) is a serious condition in newborns with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) play an essential role in fetal lung maturation by stimulating alveolarization and lipid homeostasis. In rodents, LIFs are first evident during the canalicular phase of lung development with a significant increase over the last 4 days of gestation. Adipocyte differentiation-related protein (ADRP), a functional lipogenic molecular marker characterizing LIFs, is highly expressed in fetal lungs during this critical time period. We hypothesized that LIF expression in hypoplastic rat lungs is decreased in the nitrofen-induced CDH model, which is accompanied by reduced alveolar ADRP expression and lipid content. METHODS: On embryonic day 9.5 (E9.5), time-mated rats received either nitrofen or vehicle. Fetuses were sacrificed on selected time points E18.5 and E21.5, and dissected lungs were divided into controls and CDH-associated PH. Pulmonary gene expression levels of ADRP were determined by quantitative real-time polymerase chain reaction. ADRP immunohistochemistry and oil red O staining were used to assess pulmonary protein expression and lipid content. Immunofluorescence double staining for alpha smooth muscle actin, which is known to be absent in LIFs, and lipid droplets was performed to evaluate the pulmonary expression of this specific subset of fibroblasts. RESULTS: Relative mRNA expression of ADRP was significantly reduced in lungs of CDH-associated PH on E18.5 and E21.5 compared to controls. ADRP immunoreactivity and lipid staining were markedly diminished in alveolar mesenchymal cells of CDH-associated PH on E18.5 and E21.5 compared to controls. Confocal laser scanning microscopy demonstrated markedly decreased LIF expression in alveolar interstitium of CDH-associated PH on E18.5 and E21.5 compared to controls. CONCLUSION: Decreased pulmonary LIF expression during late gestation suggests impaired LIF functioning in the nitrofen-induced CDH model, which may cause disruption in fetal alveolarization and lipid homeostasis, and thus contribute to the development of PH.

Decreased apelin and apelin-receptor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: The high morbidity and mortality in congenital diaphragmatic hernia (CDH) are attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by structural changes in pulmonary arteries, resulting in adventitial and medial thickness. These effects are triggered by abnormal apoptosis and proliferation of pulmonary vascular endothelial and smooth muscle cells (SMCs). Apelin (APLN), a target gene of bone morphogenic protein receptor 2 (BMPR2), is known to play an important and manifold role in regulating pulmonary homeostasis promoting endothelial cell (EC) survival, proliferation and migration. In addition to these autocrine effects of apelin, it displays a paracrine function attenuating the response of pulmonary SMCs to growth factors and promoting apoptosis. Apelin exerts its effect via its G-protein-coupled receptor (APLNR) and is solely expressed by pulmonary vascular EC, whereas APLNR is co-localized in pulmonary ECs and SMCs. Dysfunction of BMPR2 and downstream signalling have been shown to disturb the crucial balance of proliferation of SMCs contributing to the pathogenesis of human and experimentally induced PH. We designed this study to investigate the hypothesis that apelin and APLNR signalling are disrupted in the pulmonary vasculature of rats in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9 of gestation. Foetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA levels of APLN and APLNR were determined by quantitative real-time PCR. Protein expression of apelin and APLNR was investigated by western blotting. Confocal immunofluorescence double staining for apelin, APLNR and SMCs were performed. RESULTS: Relative mRNA level of APLN and APLNR were significantly decreased in the CDH group compared to control lungs. Western blotting and confocal microscopy confirmed the qRT-PCR results showing decreased pulmonary protein expression of apelin and APLNR in lungs of nitrofen-exposed foetuses compared to controls. CONCLUSION: This study provides striking evidence of markedly decreased gene and protein expression of apelin and its receptor APLNR in the pulmonary vasculature of nitrofen-induced CDH. The disruption of the apelin-APLNR signalling axis in the pulmonary vasculature may lead to extensive vascular remodelling and contribute to PPH in the nitrofen-induced CDH model.

Disruption of THY-1 signaling in alveolar lipofibroblasts in experimentally induced congenital diaphragmatic hernia.

PURPOSE: Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 (-/-) mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs. METHODS: Timed-pregnant Sprague-Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs. RESULTS: Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls. CONCLUSION: Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.

Association of Hirschsprung's disease and anorectal malformation: a systematic review.

BACKGROUND: The association of Hirschsprung's disease (HD) and anorectal malformation (ARM) is rare. The exact incidence of this association is not known but HD coexisting with ARM has been reported in 2.3 to 3.4% of ARM cases. Most of the reported cases in the literature have been single case reports. The aim of this systematic review was to determine the incidence of HD associated with ARM and its relationship to other syndromes. METHODS: A systematic review of the literature was performed for the keywords "association of Hirschsprung's disease and anorectal malformation", "aganglionosis and anorectal malformation" as well as "congenital megacolon and anorectal malformation". Resulting publications were reviewed for epidemiology, operative treatment and morbidity. Reference lists were screened for additional cases. RESULTS: A total of 38 articles reported 90 cases of HD coexisting with ARM from 1952 to 2013. Twenty eight articles reported 40 single case reports of this association. Ten articles reported 50 cases of HD in case series of 2,465 ARM patients, resulting in an incidence of 2% of this association. Gender was reported in 63 cases, with 30 males (48%) and 33 females (52%). Associated syndromes were reported in 23 patients: Currarino syndrome in 11, Down syndrome in 8, Cat eye syndrome in 3 and Pallister-Hall syndrome in one case. Extent of aganglionosis was reported in 49 cases and included classical rectosigmoid disease in 36, long segment aganglionosis in 5, total colonic aganglionosis in 7 and total intestinal aganglionosis in one patient. In 35% of the patients stoma was created in the aganglionotic region and failed to work. There was a median delay of 8 months for the diagnosis of HD from initial diagnosis of ARM. Various surgical techniques were employed for the pull-through operation for HD. CONCLUSION: The review confirms that the recognition of HD is often delayed because of the initial diagnosis of ARM and the fact that the dysfunctional colostomy is usually proximal to the affected aganglionotic bowel. There is a high incidence of associated syndromes when HD coexists with ARM.

Pulmonary FGF-18 gene expression is downregulated during the canalicular-saccular stages in nitrofen-induced hypoplastic lungs.

PURPOSE: Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. However, the molecular pathogenesis of PH is still poorly understood. In developing fetal lungs, fibroblast growth factor 18 (FGF-18) plays a crucial role in distal airway maturation. FGF-18 knockouts show smaller lung sizes with reduced alveolar spaces and thicker interstitial mesenchymal compartments, highlighting its important function for fetal lung growth and differentiation. We hypothesized that pulmonary FGF-18 gene expression is downregulated during late gestation in nitrofen-induced hypoplastic lungs. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18 and D21, and lungs were divided into three groups: controls, hypoplastic lungs without CDH [CDH(-)], and hypoplastic lungs with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary FGF-18 gene expression levels were analyzed by qRT-PCR. Immunohistochemistry was performed to investigate FGF-18 protein expression/distribution. RESULTS: Relative mRNA levels of pulmonary FGF-18 gene expression were significantly decreased in CDH(-) and CDH(+) on D18 and D21 compared to controls (p < 0.05 and p < 0.01, respectively). Immunoreactivity of FGF-18 was markedly diminished in mesenchymal cells surrounding the airway epithelium on D18 and D21 compared to controls. CONCLUSION: Downregulation of FGF-18 gene expression in nitrofen-induced hypoplastic lungs suggests that decreased FGF-18 expression during the canalicular-saccular stages may interfere with saccular-alveolar differentiation and distal airway maturation resulting in PH.

Expression of Sproutys and SPREDs is decreased during lung branching morphogenesis in nitrofen-induced pulmonary hypoplasia.

PURPOSE: Pulmonary hypoplasia (PH) is a life-threatening condition associated with congenital diaphragmatic hernia (CDH), characterized by defective lung development. Sproutys and Sprouty-related proteins (SPREDs) play a key role in lung branching morphogenesis through modification of epithelial-mesenchymal interactions. During the pseudoglandular stage, Sproutys are highly expressed in distal airway epithelium, while SPREDs within the surrounding mesenchyme. Sprouty2/4 knockouts show severe defects in branching morphogenesis with reduced number of distal airways. SPRED-1 and SPRED-2 are strongly expressed in regions of new airway formation, highlighting their important function in branching pattern. We hypothesized that expression of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 is decreased during lung branching morphogenesis in nitrofen-induced PH. METHODS: Timed-pregnant rats received either nitrofen or vehicle on E9.5. On E15.5 (n = 16), fetal lungs were micro-dissected and divided into controls and PH, while on E18.5 (n = 24) groups were: control, PH without CDH [CDH(-)], and PH with CDH [CDH(+)]. Pulmonary gene expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were analyzed by qRT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution. RESULTS: On E18.5, relative mRNA expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were significantly decreased in CDH(-) and CDH(+) groups compared to controls (P < 0.05). Immunoreactivity of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 was markedly diminished on E18.5 in nitrofen-induced PH. CONCLUSION: Decreased expression of Sproutys and SPREDs during the terminal pseudoglandular stage may disrupt lung branching morphogenesis by interfering with epithelial-mesenchymal interactions contributing to PH.

Laparoscopic versus Open Inguinal Hernia Repair Is Feasible in Infants with Caudal Anesthesia and Spontaneous Respiration.

INTRODUCTION: Minimally invasive surgery (i.e., laparoscopy) and minimally invasive anesthesia (i.e., caudal anesthesia with spontaneous respiration) have separately shown benefits for inguinal hernia repair in infants, yet to what degree these techniques can be combined remains unknown. This study investigated whether laparoscopy impacts the feasibility of performing caudal anesthesia with spontaneous respiration in infants. METHODS: Prospectively collected data of all infants less than 12 months old and over 3 kg weight who underwent laparoscopic indirect hernia repair (LAP) at our department from 2019 to 2021 were compared with a historical control-matched group of infants who underwent open repair (OPEN) from 2017 to 2021. We assessed the patients' characteristics, anesthesia, and surgical data as well as intra- and postoperative complications. RESULTS: A total of 87 infants were included (LAP n = 29, OPEN n = 58). Caudal anesthesia with spontaneous respiration was feasible in 62.1% of cases (LAP n = 55.2%, OPEN n = 65.5%; nonsignificant). Neither group registered anesthetic intra- or postoperative complications. Sedatives were utilized in 97% of LAP patients versus 56.9% of OPEN patients (p < 0.00001). The airway was secured with a laryngeal mask in 89.7% of patients during LAP versus 41.4% during OPEN (p < 0.00001). No significant differences were found regarding the use frequency of opioids (48.3% LAP vs. 34.5% OPEN; nonsignificant) or neuromuscular blockers (6.9% LAP vs. 5.2% OPEN; nonsignificant). CONCLUSION: This is the first comparative study on caudal anesthesia and spontaneous respiration in infants undergoing laparoscopic versus open inguinal hernia surgery. Laparoscopy increased the need for ventilatory support and sedatives but did not significantly impair the feasibility of caudal anesthesia and spontaneous respiration.