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1.
Sci Rep ; 10(1): 2265, 2020 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-32041992

RESUMEN

ROBO2 gene disruption causes vesicoureteric reflux (VUR) amongst other congenital anomalies. Several VUR patient cohorts have been screened for variants in the ubiquitously expressed transcript, ROBO2b, but, apart from low levels in a few adult tissues, ROBO2a expression is confined to the embryo, and might be more relevant to VUR, a developmental disorder. ROBO2a has an alternative promoter and two alternative exons which replace the first exon of ROBO2b. We screened probands from 251 Irish VUR families for DNA variants in these. The CpG island of ROBO2a, which includes the non-coding first exon, was found to contain a run of six variants abolishing/creating CpG dinucleotides, including a novel variant, present in the VUR cases in one family, that was not present in 592 healthy Irish controls. In three of these positions, the CpG was created by the non-reference allele, and the reference allele was not the nucleotide that would result from spontaneous deamination of methylcytosine to thymine, suggesting that there might have been selection for variability in number of CpGs in this island. This is in marked contrast to the CpG island at the start of ROBO2b, which only contained a single variant that abolishes a CpG.

2.
J Pediatr Surg ; 2020 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-32087933

RESUMEN

PURPOSE: Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. The pathophysiology of UPJ obstruction and the exact mechanism of pelviureteral peristalsis are poorly understood. Anoctamin-1 (ANO1), a Ca2+-activated chloride channel, has been shown to play a key role in muscle wall contractions in the gastrointestinal tract. We designed this study to investigate the hypothesis that ANO1 is expressed in smooth muscle cells (SMCs) of the human UPJ and that tyrosine phosphorylation is altered in UPJ obstruction. MATERIALS AND METHODS: Fresh frozen specimens of UPJ obstruction (n = 28) and control specimens from patients who underwent Wilms' tumor nephrectomy (n = 20) were prepared. Western blot (WB) was performed to evaluate levels of ANO1 protein expression and changes in tyrosine phosphorylation. In addition analysis of ANO1 and phalloidin using confocal-immunofluoresence-double staining and 3D reconstruction were carried out. RESULTS: Our WB results revealed increased tyrosine phosphorylation in UPJ obstruction samples compared to controls, and decreased ANO1 expression in UPJ obstruction. Confocal microscopy showed that ANO1 immunoreactivity was decreased in SMCs of UPJ obstruction compared to controls. CONCLUSIONS: We provide evidence, for the first time, of the presence of ANO1 expression in the human UPJ. We speculate that altered tyrosine phosphorylation, observed in UPJ obstruction, may lead to a failure of transmission of peristaltic waves in UPJ obstruction by inhibiting Ca2+-activated chloride channels in SMCs.

3.
Eur J Pediatr Surg ; 30(1): 59-63, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31707728

RESUMEN

INTRODUCTION: "Tuft" cells, also known as brush or caveolated cells, are characteristically fusiform shaped, with a distinct apical "tuft" of microvilli extending into the lumen. Double cortin-like kinase 1 (DCLK1) is a microtubule kinase and is a specific marker of intestinal tuft cells. DCLK1-positive tuft cells have been shown to play a key role in gastrointestinal chemosensation, inflammation, and neurotransmission. DCLK1 and Choline acetyltransferase (ChAT), the enzymes responsible for acetylcholine production, are reported to be coexpressed within the gastrointestinal tract. We designed this study to investigate the hypothesis that DCLK1 gene expression is altered in Hirschsprung's disease (HSCR). MATERIALS AND METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was undertaken to quantify DCLK1 gene expression, and immunolabeling of DCLK1-positive tuft cells was visualized using confocal microscopy. RESULTS: qRT-PCR analysis revealed significant downregulation of the DCLK1 gene in both aganglionic and ganglionic HSCR specimens compared with controls (p < 0.05). Confocal microscopy revealed DCLK1-positive tuft cell expression within the colonic mucosa, with a reduction in expression in both aganglionic and ganglionic HSCR colon compared with controls. CONCLUSION: DCLK1 is significantly downregulated in HSCR colon, suggesting a role for tuft cells in cholinergic neurotransmission of the distal colon. The marked decrease in DCLK1 expression within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients.

4.
Pediatr Surg Int ; 36(2): 235-239, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31602498

RESUMEN

INTRODUCTION: Bladder injury (BI) represents a rare complication of inguinal hernia surgery. Protrusions of the urinary bladder through the deep inguinal ring ("bladder ears") have been reported with an incidence of 9% in infants younger than 6 months of age and may be misinterpreted as the hernia sac. This literature review was designed to determine incidence and outcomes of bladder injuries during pediatric inguinal hernia repair. METHODS: A literature review of the literature (1967-2017) was performed using the keywords "bladder ears", "inguinal hernia", "iatrogenic bladder injury" and "bladder hernia". Publications were reviewed for epidemiology, presentation and extent of injury, treatment and outcome. RESULTS: Thirteen articles reporting on 30 cases of BI during inguinal hernia repair from 1967 to 2017 were included (19 boys, 2 girls, 9 unknown). Median age at herniotomy was 10.5 months (1 month-6 years). Out of 30 children, 14 (47%) experienced mild complications. Sixteen patients (53%) had severe complications after initial surgery and needed revisional surgery. Complications were noticed up to 4 years after the initial surgery. In 9 (56%) of the 16 severe cases, major damage to the bladder wall and impairment of bladder capacity occurred. In seven patients (44%), secondary closure was successful. In ten patients (63%), the bladder was partially resected, and in one child (6%), the entire bladder was removed. CONCLUSIONS: The degree of accidental BI during inguinal hernia repair was severe in in the majority of reported cases in the literature. Surgeons should be aware of the high prevalence of "bladder ears" in infants to prevent injury to the urinary tract.

5.
Pediatr Surg Int ; 36(1): 21-24, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31552492

RESUMEN

BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR) and anorectal malformation (ARM) are often associated with other congenital malformations, but the association of each other is rare. Some studies have reported the incidence of HSCR associated with ARM ranging from 2.0 to 3.4%. The purpose of this study was to update the current epidemiological and therapeutic features of this rare congenital association. METHODS: A systematic literature search for relevant articles was performed in four databases using a combination of the following terms "association of Hirschsprung's disease and anorectal malformation", "aganglionosis and anorectal malformation" and "congenital megacolon and anorectal malformation" for studies published between 1952 and 2019. Reference lists were screened for additional cases. RESULTS: Forty-three studies met the defined inclusion criteria, reporting a total of 126 patients who were diagnosed with HSCR with ARM. Thirty articles reported 42 single case reports of this association. Twelve articles reported 66 cases of HSCR in case series of 3309 ARM patients, resulting in an incidence of 2% of this association. Associated syndrome was found in 25 cases (20%): Currarino syndrome in 11, Down syndrome in 8, Cat eye syndrome in 4 and Pallister-Hall syndrome in 2 patients. Extent of aganglionosis was reported in 62 cases: short or rectosigmoid aganglionosis was reported in 44, long segment aganglionosis in 8, total colonic aganglionosis in 9 and total intestinal aganglionosis in 1 case. CONCLUSION: Although the association of ARM and HSCR is rare, the incidence of HSCR among ARM cases seems to be higher than in the general pediatric population. There was a high incidence of coexistence of ARM and HSCR with severe associated syndromes.

6.
J Pediatr Surg ; 2019 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-31859043

RESUMEN

PURPOSE: The pathophysiology of Hirschsprung's associated enterocolitis (HAEC) is not understood. Abnormal intestinal motility and altered intestinal epithelial barrier function have been suggested to play a key role in the causation of HAEC. Protease-activated receptors (PARs) 1 and 2, have been implicated in inflammatory reactions, intestinal permeability and modulation of motility in the gut. METHODS: We investigated PAR-1 and PAR-2 protein expression in aganglionic and ganglionic regions of patients with Hirschsprung's Disease (HSCR) (n = 10) versus normal control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression were quantified using quantitative real-time polymerase chain reaction (qPCR), western blot analysis, and densitometry. RESULTS: qPCR and Western blot analysis revealed that PAR-1 and PAR-2 expression was significantly increased in ganglionic and aganglionic bowel in HSCR compared to controls (p < 0.003). Confocal microscopy revealed strong PAR-1 and PAR-2 expression in smooth muscles, interstitial cells of Cajal (ICCs), platelet-derived growth factor-alpha receptor-positive (PDGFRα+) cells, enteric neurons and epithelium in the ganglionic and aganglionic bowel compared to controls. CONCLUSION: Increased PAR-1 and PAR-2 expression in the colon of patients with HSCR suggests that excessive local release of PAR activating proteases may trigger inflammatory responses leading to HAEC.

7.
Pediatr Surg Int ; 35(12): 1327, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31542826
8.
Pediatr Surg Int ; 35(12): 1431-1435, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31542828

RESUMEN

AIM OF THE STUDY: Potassium (K+) channels with a two-pore domain (K2P) are a large family of hyperpolarising ion channels which play a key role in cell excitability. This family comprises three members: TREK-1, TREK-2 and TRAAK. TRAAK channels have previously been reported to be expressed in murine enteric ganglia. To date, no data exist regarding TRAAK channel expression in the human colon. Thus, we designed this study to investigate TRAAK gene expression in the normal human colon and in Hirschsprung's disease (HSCR). METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify TRAAK gene expression, and immunolabelling of TRAAK proteins was visualized using confocal microscopy. MAIN RESULTS: Confocal microscopy revealed TRAAK protein expression within both neurons and interstitial cells of Cajal in the myenteric plexus, with a reduction in both ganglionic HSCR colon and aganglionic HSCR colon, compared to controls. qRT-PCR analysis revealed a significant downregulation of the TRAAK gene in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). CONCLUSIONS: TRAAK gene expression is significantly downregulated in HSCR colon, suggesting a role for these ion channels in colonic neurotransmission. TRAAK downregulation within ganglionic specimens highlights the dysfunctional nature of ganglia in this region.


Asunto(s)
Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/cirugía , Canales de Potasio/genética , Animales , Ano Imperforado/cirugía , Colon/diagnóstico por imagen , Colon/cirugía , Regulación hacia Abajo/genética , Femenino , Expresión Génica/genética , Humanos , Lactante , Masculino , Ratones , Microscopía Confocal/métodos
9.
Pediatr Surg Int ; 35(9): 929-934, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31256294

RESUMEN

BACKGROUND/PURPOSE: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is unclear. Caveolin-1 (Cav-1) regulates the functions of different nitric oxide synthase (NOS) isoforms, which play critical roles in inflammation and intestinal epithelial barrier function. We designed this study to investigate the hypothesis that Cav-1 expression is altered in the bowel of patients with Hirschsprung's disease (HSCR). METHODS: HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). qRT-PCR analysis was undertaken to quantify Cav-1 gene expression, and Western blot analysis was undertaken to determine Cav-1 protein quantification. Immunolabelling of Cav-1 proteins was visualized using confocal microscopy. RESULTS: qRT-PCR and Western blot analysis revealed that Cav-1 was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls (p < 0.01). Confocal microscopy revealed a markedly decreased expression of Cav-1 in colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls. CONCLUSION: To our knowledge, this is the first report of significantly decreased Cav-1 expression in patients with HSCR. Decreased expression of Cav-1 in the bowel of HSCR may increase susceptibility to HAEC in HSCR.


Asunto(s)
Caveolina 1/genética , Enfermedad de Hirschsprung/genética , Western Blotting , Colon/cirugía , Regulación hacia Abajo/genética , Femenino , Técnica del Anticuerpo Fluorescente , Enfermedad de Hirschsprung/cirugía , Humanos , Masculino
10.
Pediatr Surg Int ; 35(9): 923-927, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31263958

RESUMEN

AIM OF THE STUDY: Ryanodine receptors are the largest of all ion channels, named after their exogenous ligand, ryanodine. The ryanodine receptor calcium release channel is central to cytoplasmic Ca2+ signalling in skeletal muscle, the heart, and many other tissues, playing a vital role in muscular contraction. Three ryanodine receptors exist, Ryr1, Ryr2 and Ryr3. The ryanodine receptor, Ryr3, is encoded by the Ryr3 gene, which has been reported to be highly specific to colonic smooth muscle cells in mice. We designed this study to investigate Ryr1, Ryr2 and Ryr3 gene expression in the normal human colon and in Hirschsprung's disease (HSCR). METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Ryr1, Ryr2 and Ryr3 gene expression, and immunolabelling of Ryr1, Ryr2 and Ryr3 proteins was visualised using confocal microscopy. MAIN RESULTS: qRT-PCR analysis revealed a significant downregulation of the Ryr1 and Ryr3 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed Ryr1, Ryr2 and Ryr3 protein expressions within the smooth muscle, with a reduction in aganglionic and ganglionic HSCR colon compared to controls. CONCLUSIONS: Ryr1 and Ryr3 gene expression is significantly downregulated in HSCR colon, suggesting a role for these genes in colonic smooth-muscle motility. Ryr1 and Ryr3 downregulations within ganglionic specimens highlight the physiologically abnormal nature of this segment which may explain the occurrence of persistent bowel symptoms in some HSCR patients following a properly performed pull-through operation.


Asunto(s)
Expresión Génica/genética , Enfermedad de Hirschsprung/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Colon/cirugía , Regulación hacia Abajo/genética , Enfermedad de Hirschsprung/cirugía , Humanos , Lactante
11.
Curr Urol Rep ; 20(9): 49, 2019 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-31289951

RESUMEN

PURPOSE OF REVIEW: Endoscopic injection of bulking agents for the treatment of vesicoureteral reflux (VUR) has become a therapeutic alternative to antibiotic prophylaxis and ureteral reimplantation. Although considered as a safe and efficient procedure, several studies have reported cases of ureteral obstruction (UO) after endoscopic correction of VUR. This review article evaluates the present VUR literature to estimate the incidence of UO following endoscopic injection of different substances, while also discussing the impact of injection technique and implant volume. RECENT FINDINGS: Twenty-five publications were identified that provided detailed information on 64 females and 32 males (age range, 7 months-48 years) that developed UO after endoscopic treatment of VUR using dextranomer/hyaluronic acid (Dx/HA), polyacrylate polyalcohol (PP), polydimethylsiloxane (PDMS), calcium hydroxyapatite (CaHA), polytetrafluoroethylene (PTFE), or collagen. There was some variation in the reported incidence of UO among these materials: Dx/HA (0.5-6.1%), PP (1.1-1.6%), PDMS (2.5-10.0%), CaHA (1.0%), and PTFE (0.3%). Postoperative UO was described following subureteric transurethral injection (STING), intraureteric hydrodistension implantation technique (HIT), combined HIT/STING and double HIT. The injected volume ranged widely, also depending on the type of bulking agent: Dx/HA (0.3-3.0 mL), PP (0.3-1.2 mL), PDMS (1.0-2.2 mL), CaHA (0.4-0.6 mL), and PTFE (1.5-2.0 mL). The timing of UO varied from immediately after the procedure to 63 months. Over half of patients showed asymptomatic hydroureteronephrosis on follow-up imaging, whereas the remaining presented with symptoms of acute UO or fever. UO remains a rare complication after endoscopic correction of VUR, generally reported in less than 1% of treated cases, which appears to be independent of the injected substance, volume, and technique. However, long-term follow-up is recommended as asymptomatic or delayed UO can occur, potentially leading to deterioration of renal function.


Asunto(s)
Materiales Biocompatibles/efectos adversos , Endoscopía/efectos adversos , Obstrucción Ureteral/etiología , Reflujo Vesicoureteral/cirugía , Resinas Acrílicas/efectos adversos , Adolescente , Adulto , Niño , Preescolar , Colágeno/efectos adversos , Dextranos/efectos adversos , Dimetilpolisiloxanos/efectos adversos , Durapatita/efectos adversos , Femenino , Humanos , Ácido Hialurónico/efectos adversos , Lactante , Inyecciones , Masculino , Persona de Mediana Edad , Politetrafluoroetileno/efectos adversos , Resultado del Tratamiento , Adulto Joven
12.
J Pediatr Surg ; 54(11): 2318-2324, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31079866

RESUMEN

BACKGROUND: Retained transition zone is a leading cause of obstructive symptoms after pull-through operation in Hirschsprung's disease. OBJECTIVE: We aimed to evaluate the extent of the histological transition zone in patients with Hirschsprung's disease. DESIGN: We performed an observational study. DAB+ immunohistochemistry for Protein Gene Product 9.5 was used to evaluate the neuronal networks in serial sections of pull-through specimens obtained from children with Hirschsprung's disease (n = 12). Reference ranges for ganglion size/density and nerve trunk diameter were statistically determined using healthy controls obtained from colostomy specimens from children with anorectal malformations (n = 8). The transition zone was defined as ganglionic bowel exhibiting ganglion hypoplasia, hypertrophic nerve trunks, or partial circumference aganglionosis. RESULTS: The mean submucosal nerve trunk diameter in controls was 19.56 µm +/- 3.87 µm. The median age at pull-through for Hirschsprung's disease was 5 months (3-14 months). The median length of the transition zone across the population was 8 cm (4-22 cm). Median transition zone extent was significantly longer in patients with long-segment aganglionosis (n = 6) compared to rectosigmoid aganglionosis (n = 6, 13 cm vs 6 cm, p = 0.041). Due to the age of the patients enrolled, long-term follow-up of bowel function is not yet available. CONCLUSION: Our data suggest that, in children with rectosigmoid Hirschsprung's disease, the transition zone can extend for up to 13 cm. In children with long-segment disease, a longer transition zone is possible. Extended resection at a minimum 5 cm beyond the most distal ganglionic intra-operative biopsy and intra-operative histological examination of the proximal resection margin are required to minimize transition zone pull-through. LEVEL OF EVIDENCE: 2.


Asunto(s)
Enfermedad de Hirschsprung , Procedimientos Quirúrgicos del Sistema Digestivo , Ganglios/patología , Ganglios/fisiopatología , Enfermedad de Hirschsprung/patología , Enfermedad de Hirschsprung/fisiopatología , Enfermedad de Hirschsprung/cirugía , Humanos , Lactante , Complicaciones Posoperatorias
13.
J Pediatr Surg ; 54(8): 1573-1577, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-30262203

RESUMEN

PURPOSE: Hirschsprung's associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's Disease (HSCR). The pathogenesis of HAEC remains unsatisfactorily understood. Mounting evidence of an altered microbiome in patients with HSCR adds a new angle to the pathogenesis of HAEC. NLRP6 is a member of the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) innate immune receptor family, a multiprotein complex that functions as a sensor of damage-associated molecular patterns. Known as inflammasomes they have been implicated in the regulation of colonic microbial ecology and by alteration, regulators of inflammation. We designed this study to test the hypothesis that NLRP6 expression is altered in the colon of patients with HSCR. METHODS: We investigated NLPR6 protein expression in both the aganglionic and ganglionic regions of HSCR patients (n = 10) versus healthy control colon (n = 10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expressions were quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. MAIN RESULTS: qPCR and Western blot analysis revealed that NLRP6 is expressed in the colon of patients with HSCR. NLRP6 expression was significantly decreased (p < 0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that NLRP6 expression in colonic epithelium was markedly decreased in HSCR specimens compared to controls. CONCLUSION: We demonstrate for the first time the expression and distribution of NLRP6 in patients with HSCR. The observed decreased expression of NLRP6 may contribute to an altered colonic microbiome in patients with HSCR and increases the susceptibility to develop HAEC.


Asunto(s)
Colon , Enfermedad de Hirschsprung , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Colon/química , Colon/metabolismo , Colon/patología , Enterocolitis , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Humanos
14.
Eur J Pediatr Surg ; 29(1): 113-119, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30469162

RESUMEN

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization. RESULTS: Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium. CONCLUSION: Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial-mesenchymal interactions, thus causing diaphragmatic defects and PH.


Asunto(s)
Diafragma/embriología , Efrina-B1/genética , Hernias Diafragmáticas Congénitas/embriología , Hernias Diafragmáticas Congénitas/genética , Pulmón/embriología , Receptor EphB2/genética , Receptor EphB4/genética , Animales , Diafragma/metabolismo , Efrina-B1/metabolismo , Femenino , Expresión Génica , Hernias Diafragmáticas Congénitas/inducido químicamente , Hernias Diafragmáticas Congénitas/metabolismo , Pulmón/metabolismo , Éteres Fenílicos , Ratas Sprague-Dawley , Receptor EphB2/metabolismo , Receptor EphB4/metabolismo
15.
Eur J Pediatr Surg ; 29(1): 102-107, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30536263

RESUMEN

INTRODUCTION: Persistent pulmonary hypertension (PPH) is a major cause of morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). PPH is characterized by increased vascular resistance and smooth muscle cell (SMC) proliferation, leading to obstructive changes in the pulmonary vasculature. Nitric oxide (NO), generated by endothelial NO synthase (eNOS), is an important regulator of vascular tone and plays a key role in pulmonary vasodilatation. NO synthase interacting protein (NOSIP), which is strongly expressed by pulmonary SMCs, has recently been identified to reduce the endogenous NO production by interacting with eNOS. We designed this study to investigate the pulmonary vascular expression of NOSIP in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and lung specimens divided into CDH and control (n = 6 for each group). Quantitative real-time polymerase chain reaction and Western blotting were performed to analyze pulmonary gene and protein expression of NOSIP. Immunofluorescence double staining for NOSIP was combined with a specific SMC marker to evaluate protein expression in the pulmonary vasculature. RESULTS: Relative messenger ribonucleic acid and protein expression of NOSIP was significantly decreased in nitrofen-exposed CDH lungs compared with controls. Confocal laser scanning microscopy revealed markedly diminished NOSIP immunofluorescence in nitrofen-exposed CDH lungs compared with controls, mainly in the muscular and endothelial components of the pulmonary vasculature. CONCLUSION: This study demonstrates for the first time decreased NOSIP expression in the pulmonary vasculature of the nitrofen-induced CDH. These findings suggest that NOSIP underexpression may interfere with NO production, contributing to abnormal vascular remodeling and PPH.


Asunto(s)
Hernias Diafragmáticas Congénitas/enzimología , Músculo Liso Vascular/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/enzimología , Animales , Animales Recién Nacidos , Proteínas Portadoras , Femenino , Expresión Génica , Hernias Diafragmáticas Congénitas/inducido químicamente , Hernias Diafragmáticas Congénitas/complicaciones , Pulmón/enzimología , Masculino , Síndrome de Circulación Fetal Persistente/etiología , Éteres Fenílicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Ubiquitina-Proteína Ligasas
16.
Pediatr Surg Int ; 35(2): 175-180, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30386895

RESUMEN

BACKGROUND/PURPOSE: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare congenital and generally fatal cause of functional intestinal obstruction in the newborn. The cause of this syndrome is unknown. Familial occurrence and reports of consanguinity in MMIHS implies that genetic factors may have an important role in the pathogenesis of this syndrome. The aim of the study was to determine the consequence of consanguinity for the incidence of MMIHS. METHODS: A literature search was performed using the keywords "megacystis microcolon intestinal hypoperistalsis" for studies published between 1976 and 2018. Retrieved articles, including additional studies from reference lists, were reviewed for consanguinity between parents and recurrence of MMIHS between siblings. Data were extracted for cases where familial MMIHS was present. RESULTS: A total of 450 patients with the diagnosis of MMIHS have been reported in the literature. There were 56 (12%) cases in which familial MMIHS was confirmed, 25 families with multiple siblings and 3 families with single affected infant. Of the 25 families with multiple siblings, 22 families had 2 siblings with confirmed MMIHS and 3 families had 3 children each with MMIHS. Consanguinity between parents was confirmed in 30 cases (18 siblings and 12 individual cases). Female-to-male ratio in the 30 patients was 4.4:1. CONCLUSION: The occurrence of MMIHS in the offspring of consanguineous parents and recurrence in siblings of healthy parents suggest that MMIHS is an autosomal recessive disorder. Pre-marital and pre-conception counselling of consanguineous populations is recommended to prevent harmful consequences.


Asunto(s)
Anomalías Múltiples/genética , Colon/anomalías , Consanguinidad , Seudoobstrucción Intestinal/genética , Vejiga Urinaria/anomalías , Humanos , Incidencia , Hermanos
18.
Pediatr Surg Int ; 35(1): 9-14, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30386899

RESUMEN

PURPOSE: Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, to regulate smooth muscle contraction. Recent studies have reported that the voltage-gated sodium channel type 1ß (Scn1b), and the chloride channel subunit, Fxyd1, are highly expressed by both ICC and PDGFRα+ cells in the mouse colon. We designed this study to investigate the expression of the Scn1b and Fxyd1 genes in the normal human colon and in HSCR. METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Scn1b and Fxyd1 gene expression, and immunolabelling of Scn1b and Fxyd1 proteins were visualized using confocal microscopy. RESULTS: qRT-PCR analysis revealed significant downregulation of Scn1b and Fxyd1 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed a reduction in Scn1b and Fxyd1 protein expression in both aganglionic and ganglionic HSCR colon compared to controls. CONCLUSION: Scn1b and Fxyd1 expression was significantly downregulated in HSCR colon. These results add to mounting evidence suggesting that the pulled-through ganglionic segment of bowel in these patients is abnormal, despite the presence of ganglion cells.


Asunto(s)
Colon/patología , Ganglios/metabolismo , Regulación de la Expresión Génica , Enfermedad de Hirschsprung/genética , Proteínas de la Membrana/genética , Fosfoproteínas/genética , ARN/genética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética , Western Blotting , Colon/metabolismo , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente , Ganglios/patología , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/cirugía , Humanos , Lactante , Proteínas de la Membrana/biosíntesis , Microscopía Confocal , Fosfoproteínas/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/biosíntesis
19.
Pediatr Surg Int ; 35(2): 193-197, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30386900

RESUMEN

PURPOSE: Voltage-gated potassium ion channels have long been implicated in gastrointestinal motility. Recent studies have highlighted the role of voltage-gated channel subfamily G member 3 (KCNG3) and 4 (KCNG4) genes in the electrical functioning of interstitial cells of Cajal and PDGFRα+ cells of the mouse colon. We designed this study to investigate KCNG3 and KCNG4 expression in the normal human colon and in Hirschsprung's disease (HSCR). METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify KCNG3 and KCNG4 gene expression, and immunolabelling of KCNG3 and KCNG4 proteins was visualized using confocal microscopy. RESULTS: qRT-PCR analysis revealed significant downregulation of the KCNG3 and KCNG4 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed KCNG3 and KCNG4 expression within neurons, ICC and PDGFRα+ cells of the myenteric plexus and smooth muscle layers, with a reduction in both proteins in aganglionic and ganglionic HSCR colon compared to controls. CONCLUSION: KCNG3 and KCNG4 gene expression is significantly downregulated in HSCR colon, suggesting a role for these genes in colonic motility. KCNG3 and KCNG4 downregulation within ganglionic specimens highlights the physiologically abnormal nature of this segment in HSCR patients.


Asunto(s)
Colon/metabolismo , Enfermedad de Hirschsprung/genética , Enfermedad de Hirschsprung/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Estudios de Casos y Controles , Regulación hacia Abajo , Enfermedad de Hirschsprung/cirugía , Humanos , Lactante , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa
20.
Pediatr Surg Int ; 35(1): 15-20, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30386901

RESUMEN

AIM OF THE STUDY: The pathogenesis of Hirschsprung's disease-associated enterocolitis (HAEC) is poorly understood. Inflammasomes are a large family of multiprotein complexes that act to mediate host immune responses to microbial infection and have a regulatory or conditioning influence on the composition of the microbiota. Inflammasomes and the apoptosis-associated speck-like protein (ASC) lead to caspase-1 activation. The activated caspase-1 promotes secretion of pro-inflammatory cytokines (IL-1ß and IL-18) from their precursors (pro-IL-1ß and pro-IL-18). Inflammasomes have been implicated in a host of inflammatory disorders. Among the inflammasomes, NLRP3, NLRP12 and NLRC4 are the most widely investigated. Knock-out mice models of inflammasomes NLRP3, NLRP12, NLRC4, caspase-1 and ASC are reported to have higher susceptibility to experimental colitis. The purpose of this study was to investigate the expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 in the bowel specimens from patients with HSCR and controls. METHODS: Pulled-through colonic specimens were collected from HSCR patients (n = 6) and healthy controls from the proximal colostomy of children with anorectal malformations (n = 6). The gene expression of NLRP3, NLRP12, NLRC4, caspase-1, ASC, pro-IL-1ß and pro-IL-18 was assessed using qPCR. The protein distribution was assessed using immunofluorescence and confocal microscopy. MAIN RESULTS: qRT-PCR analysis revealed that NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions was significantly downregulated in the aganglionic and ganglionic colon of patients with HSCR compared to controls. Confocal microscopy revealed a markedly decreased expression of NLRP3, NLRP12, NLRC4 and ASC protein in the colonic epithelium of aganglionic and ganglionic bowel of patients with HSCR compared to controls. CONCLUSIONS: To our knowledge, this is the first study analyzing NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß gene expressions in patients with HSCR. Decreased expression of NLRP3, NLRP12, NLRC4, ASC and pro-IL-1ß in the aganglionic and ganglionic bowel may increase susceptibility of HSCR patients to develop HAEC.


Asunto(s)
Regulación de la Expresión Génica , Enfermedad de Hirschsprung/genética , Inflamasomas/genética , ARN/genética , Niño , Ganglios/metabolismo , Ganglios/patología , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Humanos , Inflamasomas/biosíntesis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Microscopía Confocal , Reacción en Cadena de la Polimerasa
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