Minimally invasive repair of a late stricture in a reversed gastric tube.

Ingestion of caustic agents may result in severe scarring and stricture formation of the esophagus. Reversed gastric tube esophagoplasty is an option for definitive surgical management of severe esophageal stricture refractory to medical treatment. Delayed complications from this procedure have rarely been reported. We present a case involving a patient who developed a delayed gastric stricture three decades after receiving a reversed gastric tube esophagoplasty. A historical review of the reversed gastroplasty is presented as well.

Surgical management of type III and IV laryngotracheoesophageal clefts: the three-layered approach.

OBJECTIVE: The objective of this paper is to present our surgical experience with a cohort of four infants with laryngotracheoesophageal cleft (LTEC) in order to highlight our early failures and complications and to propose a comprehensive, three-layered approach in treating stages III and IV LTEC. METHOD: An IRB approved, retrospective chart review was carried out of a cohort of four cases occurring within a 2-year period of time. RESULTS: All patients had other significant anomalies, and the mortality rate was high: 75%. After our initial failures and difficulties with recurrent fistulas, tracheomalacia and tracheotomy dependence, we were able to achieve a successful outcome of a functional separation of the airway and the digestive tract without a tracheotomy in our last two patients. Unfortunately, both these patients died from factors not directly associated with the LTEC so long-term follow up was not possible. We now have one survivor, our second case, who is tracheotomy dependent. CONCLUSION: Laryngotracheoesophageal clefts are rare congenital anomalies with high morbidity and mortality despite various forms of surgical repair. Fistulas, tracheostomy dependence, tracheomalacia, and chronic lung disease secondary to aspiration are frequent problems following LTEC repairs. We advocate an anterior approach to the cleft repair, a three-layered closure of the cleft to include an interpositional muscular flap, and a physiologic repair of the posterior larynx with a standard cartilage graft technique used in laryngotracheal reconstruction.

The fibroblast growth factor pathway serves a regulatory role in proliferation and apoptosis in the pathogenesis of intestinal atresia.

BACKGROUND/PURPOSE: Intestinal atresia occurs in 1:5000 live births and is a neonatal challenge. Fibroblast growth factor receptor 2b (Fgfr2b) is a critical developmental regulator of proliferation and apoptosis in multiple organ systems including the gastrointestinal tract (GIT). Fgfr2b invalidation results in an autosomal recessive intestinal atresia phenotype. This study evaluates the role of Fgfr2b signaling in regulating proliferation and apoptosis in the pathogenesis of intestinal atresia. METHODS: Wild-type and Fgfr2b-/- embryos were harvested from timed pregnant mice. The GIT was harvested using standard techniques. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) was used to evaluate apoptosis and bromodeoxyuridine to assess proliferation by standard protocols. Photomicrographs were compared (Institutional Animal Care and Use Committee-approved protocol 32-02). RESULTS: Wild-type and mutant GIT demonstrate that deletion of the Fgfr2b gene results in inhibition of epithelial proliferation and increased apoptosis. Inhibited proliferation and increased apoptosis are specific to those tissues of normal Fgfr2b expression, corresponding to the site of intestinal atresia. CONCLUSIONS: The absence of embryonic GIT Fgfr2b expression results in decreased proliferation and increased apoptosis resulting in GIT atresia. The regulation of proliferation and apoptosis in intestinal cells as a genetically based cause of intestinal atresia represents a novel consideration in the pathogenesis of intestinal atresia.

Fibroblast growth factor-10 serves a regulatory role in duodenal development.

PURPOSE: Duodenal obstruction occurs in 1 of 6000 live births and requires urgent surgical intervention. Duodenal atresia previously has been ascribed to a developmental failure of luminal recanalization; however, the cause of duodenal atresia remains incompletely understood. Although familial intestinal atresias have been described and syndromic associations are known, no specific genetic link has been established. Fibroblast growth factor-10 (Fgf10) is a known regulatory molecule relevant to mesenchymal-epithelial interactions, and mice deficient in Fgf10 demonstrate congenital anomalies in several organ systems including the gastrointestinal tract. The authors hypothesized that Fgf10 could serve a regulatory role in establishing normal duodenal development. METHODS: Wild-type mice with beta-galactosidase under the control of the Fgf10 promoter were harvested from timed-pregnancy mothers. The expression of Fgf10 in the duodenum during development was evaluated by developing the embryos in X-Gal solution. Wild-type and mutant Fgf10(-/-) embryos were harvested from timed-pregnancy mothers at 18.5 days postconception (near term) and were analyzed for duodenal morphology (Institutional Animal Care and Use Committee-approved protocol 32-02). Photomicrographs were reviewed. RESULTS: Fibroblast growth factor-10 is active in the duodenum at a late stage of development. The Fgf10(-/-) mutants demonstrate duodenal atresia with a variable phenotype similar to clinical findings. The duodenum fails to develop luminal continuity and has proximal dilation. The phenotype occurs in an autosomal recessive pattern with incomplete penetrance (38%). CONCLUSIONS: Fibroblast growth factor-10 serves as a regulator in normal duodenal growth and development. Its deletion leads to duodenal atresia and challenges traditionally accepted theories of pathogenesis. This novel, genetically mediated duodenal malformation reflects an animal model that will allow further evaluation of the pathogenesis of this surgically correctable disease. By studying the mechanism of Fgf10 function in foregut development, the authors hope to better understand these anomalies and to explore possible therapeutic alternatives.

Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway.

BACKGROUND/PURPOSE: Colonic atresia occurs in 1:20,000 live births, offering a neonatal surgical challenge. Prenatal expression of fibroblast growth factor 10 (Fgf10), acting through fibroblast growth factor receptor 2b (Fgfr2b), is critical to the normal development of the colon. Invalidation of the Fgf10 pathway results in colonic atresia, inherited in an autosomal recessive pattern. Classically, disturbance of the mesenteric vasculature has been thought to cause many forms of intestinal atresia. The purpose of this study was to evaluate the role of vascular occlusion in the pathogenesis of colonic atresia. METHODS: Wild type (Wt), Fgf10(-/-), and Fgfr2b(-/-) mutant mouse embryos were harvested from timed pregnant mothers. Immediately following harvest, filtered India ink was infused via intracardiac microinjection. The gastrointestinal tract was dissected, and photomicrographs of the mesenteric arterial anatomy were taken at key developmental time points. RESULTS: Photomicrographs after India ink microinjections demonstrate normal, patent mesenteric cascades to the atretic colon at the time points corresponding to the failure of colonic development in the Fgf10(-/-) and Fgfr2b(-/-) mutants. The mesenteric arterial anatomy of the colon demonstrates no difference between the Wt and mutant colonic atresia. CONCLUSIONS: The absence of embryonic expression of Fgf10 or its receptor Fgfr2b results in colonic atresia in mice. India ink microinjection is a direct measure of mesenteric arterial patency. Colonic atresia in the Fgf10(-/-) and Fgfr2b(-/-) mutants occurs despite normal mesenteric vascular development. Thus the atresia is not the result of a mesenteric vascular occlusion. The patent colonic mesentery of the Fgf10(-/-) and Fgfr2b(-/-) mutants challenges an accepted pathogenesis of intestinal atresia. Although colonic atresia can occur as a result of vascular occlusion, new evidence exists to suggest that a genetic mechanism may play a role in the pathogenesis of this disease.

Fibroblast growth factor receptor 2 IIIb invalidation--a potential cause of familial duodenal atresia.

BACKGROUND/PURPOSE: Duodenal atresia (DA) occurs in 1 in every 6,000 live births and represents a significant surgically correctable cause of intestinal obstruction in the neonate. Familial or congenital DA has been reported, implying that at least some cases of DA are the result of genetic, heritable abnormalities. The genes controlling duodenal development are incompletely understood. Fibroblast growth factor receptor 2IIIb (Fgfr2b) is known to play a critical role in the development of multiple organ systems including other gastrointestinal tract (GIT) structures. This study shows the key role of Fgfr2b in normal duodenal development and the pathogenesis of DA. METHODS: Wild type (Wt) and Fgfr2b-/- embryos were harvested from timed pregnant mothers at stage E18.5 and were analyzed for duodenal phenotype. RESULTS: Inactivation of Fgfr2b results in DA. DA is present in the Fgf2b-/- mutants with a 35% penetrance. The duodenal phenotype of the Fgf2b-/- mutants ranges from normal to a mucosal web, type I, and type III atresia. CONCLUSIONS: Fgfr2b is a critical regulatory gene in the development of the duodenum. Fgfr2b invalidation (Fgfr2b-/- mutant) results in a reproducible, autosomal recessive duodenal atresia phenotype with incomplete penetrance and a variable phenotype.

Fibroblast growth factor 10 (Fgf10) invalidation results in anorectal malformation in mice.

BACKGROUND/PURPOSE: Anorectal malformations occur in 1 per 4,000 live births and represent a surgical challenge. Although critically important, the basic mechanisms of normal anorectal union are incompletely understood. Fgf10 signaling is known to serve a key role in mesenchymal/epithelial interactions in many organ systems including the gastrointestinal tract (GIT). The authors therefore hypothesized that Fgf10 signaling has a central role in normal anorectal development. METHODS: Fgf10 expression in wild-type (Wt) embryos was evaluated using whole-mount in situ hybridization. Wt and Fgf10-/- embryos were harvested from timed pregnant mothers at E12.5 through E17.5 and were analyzed for anorectal phenotype. RESULTS: Wt development of union between anorectal structures is completed between E12.5 and E13.5 with luminal communication between distal rectal epithelium and anus. Fgf10 is discretely expressed at E12.5 in the distal rectum. Fgf10-/- mutants show failure of union of the rectum and anus at an early stage (E13.5) and near term (E17.5). CONCLUSIONS: Fgf10 is expressed in the rectum at the time when anorectal continuity is established, indicating a role in normal anorectal development. Fgf10 invalidation (Fgf10-/- mutant) results in a genetically reproducible anorectal malformation phenotype. Fgf10 function is critical for normal anorectal development.

Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins.

Early embryonic lung branching morphogenesis is regulated by many growth factor-mediated pathways. Bone morphogenetic protein 4 (BMP4) is one of the morphogens that stimulate epithelial branching in mouse embryonic lung explant culture. To further understand the molecular mechanisms of BMP4-regulated lung development, we studied the biological role of Smad-ubiquitin regulatory factor 1 (Smurf1), an ubiquitin ligase specific for BMP receptor-regulated Smads, during mouse lung development. The temporo-spatial expression pattern of Smurf1 in mouse embryonic lung was first determined by quantitative real-time PCR and immunohistochemistry. Overexpression of Smurf1 in airway epithelial cells by intratracheal introduction of recombinant adenoviral vector dramatically inhibited embryonic day (E) 11.5 lung explant growth in vitro. This inhibition of lung epithelial branching was restored by coexpression of Smad1 or by addition of soluble BMP4 ligand into the culture medium. Studies at the cellular level show that overexpression of Smurf1 reduced epithelial cell proliferation and differentiation, as documented by reduced PCNA-positive cell index and by reduced mRNA levels for surfactant protein C and Clara cell protein 10 expression. Further studies found that overexpression of Smurf1 reduced BMP-specific Smad1 and Smad5, but not Smad8, protein levels. Thus overexpression of Smurf1 specifically promotes Smad1 and Smad5 ubiquitination and degradation in embryonic lung epithelium, thereby modulating the effects of BMP4 on embryonic lung growth.

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells.

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.

TACE is required for fetal murine cardiac development and modeling.

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.

Burkholderia cepacia-induced IL-8 gene expression in an alveolar epithelial cell line: signaling through CD14 and mitogen-activated protein kinase.

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.

Nongenomic estrogen action regulates tyrosine phosphatase activity and tuberin stability.

Estrogen action and tuberin function has been suggested to play a crucial role in the proliferation of lung smooth muscle-like cells and/or myofibroblasts in pulmonary lymphangioleiomyomatosis (LAM). Tuberin is a tumor suppressor phosphoprotein, which also regulates fluid phase endocytosis. Its activity, turnover and complex association with hamartin depends on its phosphorylation status. We have recently reported that nongenomic estrogen action regulates the phosphorylation status of several cytoplasmic proteins. Herein, we demonstrate that estrogen increases tyrosine phosphatase activity, which can be abrogated by antiestrogen ICI 182780 and tyrosine phosphatase inhibitor bpV(phen), but not by the protein synthesis inhibitor cyclohexamide. Furthermore, we show that estrogen transiently enhances the turnover of tuberin, which follows an inverse pattern to that observed for tyrosine phosphatase and endocytosis activity. We showed that tuberin phosphorylation protects it from degradation and induces its accumulation in female human lung fibroblasts and myofibroblasts. Our results suggest that nongenomic estrogen action induces tyrosine phosphatase activity that regulates stability of tyrosine phosphorylated proteins, including tuberin, which may play a crucial role in cellular specific functions such as endocytosis.

Differential expression of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in early and late gestational mouse skin and skin wounds.

Early gestation fetal mouse skin heals without scars. Plasminogen activator inhibitor-1 (PAI-1) has been associated with postnatal organ fibrosis. We hypothesized that the relative balance between urokinase-type plasminogen activator (uPA) and PAI-1 expression in favor of uPA prevents scarring in early fetal skin wounds, whereas a change in favor of PAI-1 in late gestation results in wound scarring. To evaluate uPA and PAI-1 expression, 1-mm skin wounds were made in E14.5 and E18 mice and harvested 24, 48, or 96 hours postwounding. Aprotinin (2 mg/ml)-coated beads were injected into selected E14.5 wounds. Normal skin and skin wounds were evaluated for uPA, PAI-1, and collagen expression. We showed that in normal skin uPA level is higher in E14.5 than in E18 mice, while PAI-1 is lower in E14.5 than in E18 mice. After wounding, E14.5 wounds show a moderate increase in uPA and a minimal increase in PAI-1. E18 wounds show a transient increase in uPA but a significant, sustained increase in PAI-1. Addition of aprotinin to E14.5 wounds causes an increase in collagen deposition. We conclude that the differential expression of uPA and PAI-1 in the skin of early vs. late gestation mice may contribute to the degree of scar formation seen after cutaneous injury.

Murine nitrofen-induced pulmonary hypoplasia does not involve induction of TGF-beta signaling.

BACKGROUND/PURPOSE: In the murine nitrofen-induced model of congenital diaphragmatic hernia (CDH), the lungs are primarily hypoplastic and immature even before diaphragmatic closure. Because excess transforming growth factor-beta (TGF-beta) signaling induces pulmonary hypoplasia, the authors hypothesized that primary hypoplasia after nitrofen exposure may be caused by aberrant signaling by the TGF-beta pathway. Therefore, abrogation of TGF-beta signaling might rescue the hypoplasia. METHODS: The authors performed intratracheal microinjections of a recombinant adenoviral vector encoding a dominant-negative TGF-beta type II receptor (AdIIR-DN) in nitrofen-exposed and control E12 mouse lungs, which then were cultured for 4 days in serumless chemically defined media. The mRNA expression of Smad2, 3, 4, and 7 in nitrofen-exposed and control E12 lungs after 4 days in culture were compared. RESULTS: ADIIR-DN increased terminal branching in control lungs by 28% compared with lungs injected with control virus (61.8 +/- 4.6 v. 48.4 +/- 4.7, P =.004). However, there was no difference between nitrofen-exposed lungs injected with ADIIR-DN and those injected with control virus. Compared with control lungs, Smad mRNA expression was decreased markedly in nitrofen-exposed lungs: Smad2 (40%, P =.16), Smad3 (29%, P =.02), Smad4 (25%, P =.07), and Smad7 (36%, P =.04). CONCLUSIONS: Because abrogation of TGF-beta signaling does not rescue the hypoplasia seen in the nitrofen model, and Smad expression is decreased in nitrofen-exposed lungs, the TGF-beta pathway does not appear to play a role in nitrofen-induced pulmonary hypoplasia.

Nitrofen inhibition of pulmonary growth and development occurs in the early embryonic mouse.

BACKGROUND/PURPOSE: It was believed previously that pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) was a consequence of the herniation of abdominal viscera into the chest. Using the murine nitrofen-induced model of CDH, the authors evaluated lung growth and development before diaphragm closure or herniation. METHODS: The authors examined nitrofen-exposed early embryonic lungs on embryonic day 12 (E12). Branching morphogenesis was quantified before and after 4 days in culture in serumless chemically defined media and compared with age-matched control lungs. The mRNA expression of proliferative and developmental markers in cultured lungs was then determined. RESULTS: Nitrofen-exposed lungs had 30% fewer total terminal branches than age-matched controls (9.3 +/- 1.9 nitrofen v 13.7 +/- 2.6 control; P <.001). Hypoplasia also was more profound in the left than the right lung. These effects persisted after culturing the lungs for 4 days in serumless chemically-defined media (31.7 +/- 6.8 nitrofen v 42.9 +/- 8.4 control, P <.001). Furthermore, the mRNA expression of proliferative and developmental markers was decreased in nitrofen-exposed E12 lungs cultured for 4 days (as a percentage of age-matched controls): cyclin A (69.28%; P =.04), Nkx2.1 (44.4%, 0.04), SP-A (24.1%; P =.008), SP-B (23.4%; P =.05), SP-C (20%; P =.06), and CC-10 (13.8%; P =.04). CONCLUSION: Nitrofen induces primary pulmonary hypoplasia and immaturity in the early embryonic mouse, and this effect persists in culture.

Application of the principles of myotomy and strictureplasty for treatment of esophageal strictures.

BACKGROUND/PURPOSE: Strictures of the esophagus in the pediatric population may be congenital or acquired. Regardless of the etiology, the treatment goal is to relieve the symptoms and allow patients to eat normally. The cornerstone of nonoperative management is repeated esophageal dilatations. However, when nonoperative management fails, operative intervention becomes necessary. In this report the authors present 4 cases of severe upper esophageal strictures managed by applying the principles of myotomy and strictureplasty. METHODS: Retrospective review of 4 patients undergoing esophageal stricturotomy from January 1, 1993 to January 1, 2000 was conducted at Childrens Hospital Los Angeles, with a mean follow-up period of 5 years. RESULTS: Three of the 4 patients reported in this report are doing well and have not required any further surgical intervention. One patient in whom additional strictures developed has had a microvascularized free jejunal graft and also is doing well. CONCLUSIONS: Currently accepted surgical management of strictures includes resection of the affected segment with end-to-end anastomosis or esophageal replacement. The authors propose that before embarking on a long and technically hazardous operation, consideration should be given to stricturotomy. In cases of isolated strictures, this may be the preferred approach.