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The unfolded protein response (UPR) is a complex adaptive signaling pathway activated by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). ER stress (ERS) triggers a cascade of responses that converge upon C/EBP homologous protein (CHOP) to drive inflammation and apoptosis. Herein, we sought to determine whether liver injury and fibrosis after small bowel resection (SBR) were mediated by a maladaptive hepatic ERS/UPR. C57BL/6 mice underwent 50% proximal SBR or sham operation. Markers of liver injury and UPR/ERS pathways were analyzed. These were compared with experimental groups including dietary fat manipulation, tauroursodeoxycholic acid (TUDCA) treatment, distal SBR, and global CHOP knockout (KO). At 10 wk, proximal SBR had elevated alanine aminotransferase/aspartate aminotransferase (ALT/AST) (P < 0.005) and greater hepatic tumor necrosis factor-α (TNFα) (P = 0.001) and collagen type 1 α1 (COL1A1) (P = 0.02) than shams. SBR livers had increased CHOP and p-eIF2α, but were absent in activating transcription factor 4 (ATF4) protein expression. Low-fat diet (LFD), TUDCA, and distal SBR groups had decreased liver enzymes, inflammation, and fibrosis (P < 0.05). Importantly, they demonstrated reversal of hepatic UPR with diminished CHOP and robust ATF4 signal. CHOP KO-SBR had decreased ALT but not AST compared with wild-type (WT)-SBR (P = 0.01, P = 0.12). There were no differences in TNFa and COL1A1 (P = 0.09, P = 0.50). SBR-induced liver injury, fibrosis is associated with a novel hepatic UPR/ERS response characterized by increased CHOP and decreased ATF4. LFD, TUDCA, and ileocecal resection rescued the hepatic phenotype and reversed the UPR pattern. Global CHOP KO only partially attenuated liver injury. This underscores the significance of disruptions to the gut/liver axis after SBR and potentiates targets to mitigate the progression of intestinal failure-associated liver disease. [ABSTRACT FROM AUTHOR]

Short bowel syndrome (SBS) is associated with diminished levels of serum fats caused by unknown mechanisms. We have shown that mesenteric lymphatics remodel to a more primitive state one week after small bowel resection (SBR); therefore, this study focuses on the effect of chronic lymphatic remodeling and magnitude of resection on intestinal lipid uptake and transport. C57BL6 and Prox1 creER-Rosa26LSLTdTomato (lymphatic reporter) mice underwent 50% or 75% proximal SBR or sham operations. Functional transport of lipids and fecal fat content was measured and lymphatic vasculature was compared via imaging. There was a significant reduction in functional transport of cholesterol and triglyceride after SBR with increasing loss of bowel, mirrored by a progressive increase in fecal fat content. We also describe significant morphological changes in the lymphatic vasculature in both the lamina propria and mesentery. Intestinal lymphatic drainage assay in vivo demonstrated a marked reduction of systemic absorption after resection. Intestinal lymphatic vessels significantly remodel in the setting of chronic SBS. This remodeling may account at least in part for impaired intestinal uptake and transport of fat via the compromised lymphatic architecture. We believe that these changes may contribute to the development of intestinal failure associated liver disease (IFALD), a major morbidity in patients with SBS. [ABSTRACT FROM AUTHOR]

Loss of functional small bowel surface area causes short bowel syndrome (SBS), intestinal failure, and parenteral nutrition (PN) dependence. The gut adaptive response following resection may be difficult to predict, and it may take up to 2 yr to determine which patients will wean from PN. Here, we examined features of gut microbiota and bile acid (BA) metabolism in determining adaptation and ability to wean from PN. Stool and sera were collected from healthy controls and from patients with SBS (n = 52) with ileostomy, jejunostomy, ileocolonic, and jejunocolonic anastomoses fed with PN plus enteral nutrition or who were exclusively enterally fed. We undertook 16S rRNA gene sequencing, BA profiling, and 7α-hydroxy-4-cholesten-3-one (C4) quantitation with LC-MS/MS and serum amino acid analyses. Patients with SBS exhibited altered gut microbiota with reduced gut microbial diversity compared with healthy controls. We observed differences in the microbiomes of patients with SBS with ileostomy versus jejunostomy, jejunocolonic versus ileocolonic anastomoses, and PN dependence compared with those who weaned from PN. Stool and serum BA composition and C4 concentrations were also altered in patients with SBS, reflecting adaptive changes in enterohepatic BA cycling. Stools from patients who were weaned from PN were enriched in secondary BAs including deoxycholic acid and lithocholic aicd. Shifts in gut microbiota and BA metabolites may generate a favorable luminal environment in select patients with SBS, promoting the ability to wean from PN. Proadaptive microbial species and select BA may provide novel targets for patient-specific therapies for SBS. NEW & NOTEWORTHY Loss of intestinal surface area causes short bowel syndrome, intestinal failure, and parenteral nutrition dependence. We analyzed the gut microbiota and bile acid metabolome of a large cohort of short bowel syndrome adult patients with different postsurgical anatomies. We report a novel analysis of the microbiome of patients with ileostomy and jejunostomy. Enrichment of specific microbial and bile acid species may be associated with the ability to wean from parenteral nutrition. [ABSTRACT FROM AUTHOR]

Intestinal failure-associated liver disease is a major morbidity associated with short bowel syndrome. We sought to determine if the obesity-resistant mouse strain (129S1/SvImJ) conferred protection from liver injury after small bowel resection (SBR). Using a parenteral nutrition-independent model of resection-associated liver injury, C57BL/6J and 129S1/SvImJ mice underwent a 50% proximal SBR or sham operation. At postoperative week 10, hepatic steatosis, fibrosis, and cholestasis were assessed. Hepatic and systemic inflammatory pathways were evaluated using oxidative markers and abundance of tissue macrophages. Potential mechanisms of endotoxin resistance were also explored. Serum lipid levels were elevated in all mouse lines. Hepatic triglyceride levels were no different between mouse strains, but there was an increased accumulation of free fatty acids in the C57BL/6J mice. Histological and serum markers of hepatic fibrosis, steatosis, and cholestasis were significantly elevated in resected C57BL/6J SBR mice as well as oxidative stress markers and macrophage recruitment in both the liver and visceral white fat in C57BL/6J mice compared with sham controls and the 129S1/SvImJ mouse line. Serum endotoxin levels were significantly elevated in C57BL/6J mice with significant elevation of hepatic TLR4 and reduction in PPARα expression levels. Despite high levels of serum lipids, 129S1/SvImJ mice did not develop liver inflammation, fibrosis, or cholestasis after SBR, unlike C57BL/6J mice. These data suggest that the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 contribute to the liver injury seen in C57BL/6J mice with short bowel syndrome. NEW & NOTEWORTHY Unlike C57BL/6 mice, the 129S1/SvImJ strain is resistant to liver inflammation and injury after small bowel resection. These disparate outcomes are likely due to the accumulation of hepatic free fatty acids as well as increased endotoxin-driven inflammatory pathways through PPARα and TLR4 in C57BL/6 mice with short bowel syndrome. [ABSTRACT FROM AUTHOR]

Background: Few studies have examined the metabolic consequences of short bowel syndrome (SBS) and its effects on body composition in adults. We hypothesized that body composition of SBS patients is altered compared to a normal age-, race-, and sex-matched population, regardless of parenteral nutrition (PN) dependence.Aim: To compare the body composition of adult patients with SBS to age-, sex-, and race-matched healthy controls.Methods: Twenty patients with SBS underwent body composition analysis using the GE Lunar iDXA scanner. Patients were age-, sex-, and race-matched to controls from the National Health and Nutrition Examination Survey (1999-2004). Mean differences in body mass index, fat-free mass, fat mass, percent body fat, visceral adipose tissue mass and volume, and bone mineral density were measured. Statistical analysis was performed using SAS 9.4 software.Results: Fifty-five percent of subjects had a history of PN use, and 30% were current PN users. Mean percent body fat for SBS patients was 35.1% compared to 30.9% for healthy controls (p = 0.043). Fat-free mass was reduced in SBS (p = 0.007). Patients with reduced bone mass had a trend toward significantly more years of PN exposure compared to those with normal bone mass (p = 0.094), and a trend toward older age (p = 0.075).Conclusions: SBS is associated with increased percent body fat and reduced fat-free mass, suggesting that improved dietary and therapeutic interventions are needed to restore normal metabolic indices and avoid risk of metabolic syndrome in SBS patients. [ABSTRACT FROM AUTHOR]

Background: Short bowel syndrome (SBS) results from significant loss of small intestinal length. In response to this loss, adaptation occurs, with Epidermal Growth Factor Receptor (EGFR) being a key driver. Besides enhanced enterocyte proliferation, we have revealed that adaptation is associated with angiogenesis. Further, we have found that small bowel resection (SBR) is associated with diminished oxygen delivery and elevated levels of hypoxia-inducible factor 1-alpha (HIF1α). Methods: We ablated EGFR in the epithelium and endothelium as well as HIF1α in the epithelium, ostensibly the most hypoxic element. Using these mice, we determined the effects of these genetic manipulations on intestinal blood flow after SBR using photoacoustic microscopy (PAM), intestinal adaptation and angiogenic responses. Then, given that endothelial cells require a stromal support cell for efficient vascularization, we ablated EGFR expression in intestinal subepithelial myofibroblasts (ISEMFs) to determine its effects on angiogenesis in a microfluidic model of human small intestine. Results: Despite immediate increased demand in oxygen extraction fraction measured by PAM in all mouse lines, were no differences in enterocyte and endothelial cell EGFR knockouts or enterocyte HIF1α knockouts by POD3. Submucosal capillary density was also unchanged by POD7 in all mouse lines. Additionally, EGFR silencing in ISEMFs did not impact vascular network development in a microfluidic device of human small intestine. Conclusions: Overall, despite the importance of EGFR in facilitating intestinal adaptation after SBR, it had no impact on angiogenesis in three cell types–enterocytes, endothelial cells, and ISEMFs. Epithelial ablation of HIF1α also had no impact on angiogenesis in the setting of SBS. [ABSTRACT FROM AUTHOR]

After 50% proximal small bowel resection (SBR) in mice, we have demonstrated hepatic steatosis, impaired glucose metabolism without insulin resistance, and increased pancreatic islet area. We sought to determine the consequences of SBR on pancreatic β-cell morphology, proliferation, and expression of a key regulatory hormone, glucagon-like peptide-1 (GLP-1). C57BL/6 mice underwent 50% SBR or sham operation. At 10 wk, pancreatic insulin content and secretion was measured by ELISA. Immunohistochemistry was performed to determine structural alterations in pancreatic α-and β-cells. Western blot analysis was used to measure GLP-1R expression, and immunoassay was used to measure plasma insulin and GLP-1. Experiments were repeated by administering a GLP-1 agonist (exendin-4) to a cohort of mice following SBR. After SBR, there was pancreatic islet hypertrophy and impaired glucose tolerance. The proportion of α and β cells was not grossly altered. Whole pancreas and pancreatic islet insulin content was not significantly different; however, SBR mice demonstrated decreased insulin secretion in both static incubation and islet perfusion experiments. The expression of pancreatic GLP-1R was decreased approximately twofold after SBR, compared with sham and serum GLP-1, was decreased. These metabolic derangements were mitigated after administration of the GLP-1 agonist. Following massive SBR, there is significant hypertrophy of pancreatic islet cells with morphologically intact α- and β-cells. Significantly reduced pancreatic insulin release in both static and dynamic conditions demonstrate a perturbed second phase of insulin secretion. GLP-1 is a key mediator of this amplification pathway. Decreased expression of serum GLP-1 and pancreatic GLP-1R in face of no change in insulin content presents a novel pathway for enteropancreatic glucose regulation following SBR. NEW & NOTEWORTHY Metabolic changes occur following intestinal resection; however, the effects on pancreatic function are unknown. Prior studies have demonstrated that glucagon-like protein-1 (GLP-1) signaling is a crucial player in the improved insulin sensitivity after bariatric surgery. In this study, we explore the effect of massive small bowel resection on gut hormone physiology and provide novel insights into the enteropancreatic axis. [ABSTRACT FROM AUTHOR]

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10-8), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH. [ABSTRACT FROM AUTHOR]

Enteric duplications have been described throughout the entire gastrointestinal tract. The usual perinatal presentation is an abdominalmass. Duplications associated with the foregut have associated respiratory symptoms, whereas duplications in the midgut and hindgut can present with obstructive symptoms, perforation, nausea, emesis, hemorrhage, or be asymptomatic, and identified as an incidental finding. These are differentiated from other cystic lesions by the presence of a normal gastrointestinal mucosal epithelium. Enteric duplications are located on the mesenteric side of the native structures and are often singular with tubular or cystic characteristics. Management of enteric duplications often requires operative intervention with preservation of the native blood supply and intestine. These procedures are usually very well tolerated with low morbidity. [ABSTRACT FROM AUTHOR]

Ventilation practices have changed significantly since the initial reports in the mid 1980 of successful use of permissive hypercapnia and spontaneous ventilation [often called gentle ventilation (GV)] in infants with congenital diaphragmatic hernia (CDH). However, there has been little standardization of these practices or of the physiologic limits that define GV. We sought to ascertain among Diaphragmatic Hernia Research and Exploration; Advancing Molecular Science (DHREAMS) centers' GV practices in the neonatal management of CDH. Pediatric surgeons and neonatologists from DHREAMS centers completed an online survey on GV practices in infants with CDH. The survey gathered data on how individuals defined GV including ventilator settings, blood gas parameters and other factors of respiratory management. A total of 87 respondents, from 12 DHREAMS centers completed the survey for an individual response rate of 53% and a 92% center response rate. Approximately 99% of the respondents defined GV as accepting higher carbon dioxide (PCO2) and 60% of the respondents also defined GV as accepting a lower pH. There was less consensus about the use of sedation and neuromuscular blocking agents in GV, both within and across the centers. Acceptable pH and PCO2 levels are broader than the goal ranges. Despite a lack of formal standardization, the results suggest that GV practice is consistently defined as the use of permissive hypercapnia with mild respiratory acidosis and less consistently with the use of sedation and neuromuscular blocking agents. GV is the reported practice of surveyed neonatologists and pediatric surgeons in the respiratory management of infants with CDH. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N’ terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. [ABSTRACT FROM AUTHOR]

Background/Aims: Expression of the transcriptional co-regulator tis7 is markedly increased in the adaptive small intestine in a mouse model of short bowel syndrome. Transgenic mice with enterocytic overexpression of tis7 (tis7tg) have accelerated triglyceride absorption, with increased adiposity yet reduced skeletal muscle mass. To further explore this phenotype, we examined whether tis7 also regulates amino acid and carbohydrate absorption. Methods: Small intestinal glucose and amino acid uptake were quantified in wild type (WT) and tis7tg mice. Amino acid transporter expression was assessed by qRT-PCR and immunoblot. Apical cell surface transporter expression was quantified by cell surface biotinylation. Results: Active glucose uptake rates were unchanged. Uptake of proline but not leucine was significantly reduced in tis7tg vs. WT jejunum. Expression of serum and glucocorticoid-induced kinase 1 (SGK1), a solute carrier activator, was inhibited in tis7tg jejunum. Apical membrane expression of the proline transporter SLC6A20 was reduced in tis7tg jejunum. Conclusions: Tis7 overexpression in enterocytes inhibits proline uptake, associated with decreased expression of activated SGK1 and reduced cell surface expression of SLC6A20. Consistent with the observed tis7tg phenotype, tis7 overexpression increases triglyceride absorption but has adverse effects on the uptake of selected amino acids. Tis7 has pleiotropic effects on nutrient absorption. [ABSTRACT FROM AUTHOR]

Introduction: Short bowel syndrome (SBS) is a morbid clinical condition that results from massive small-bowel resection (SBR). After SBR, there is a dramatic weight loss in the acute postoperative period. Our aim was to determine the impact of a high-protein diet (HPD) on weight gain and body composition in mice after SBR. Methods: C57BL/6 mice underwent 50 % proximal SBR. Postoperatively, mice were randomly selected to receive standard rodent liquid diet (LD) ( n = 6) or an isocaloric HPD ( n = 9) for 28 days. Mice weights were recorded daily. Body composition analyses were obtained weekly. Student's t test was used for statistical comparisons with p < 0.05 considered significant. Results: Mice that were fed HPD after SBR returned to baseline weight on average at postoperative day (POD) 8 versus mice that were fed LD that returned to baseline weight on average at POD 22. Total fat mass and lean mass were significantly greater by POD 14 within the HPD group. Both groups of mice demonstrated normal structural adaptation. Conclusion: HPD results in greater weight gain and improved body composition in mice after SBR. This finding may be clinically important for patients with SBS since improved weight gain may reduce the time needed for parenteral nutrition. [ABSTRACT FROM AUTHOR]

GLO:4CR/01aug21:jpen2241-fig-0001.jpg PHOTO (COLOR): 1 Dr Josef E. Fischer. gl Dr Josef Fischer, one of the giants in surgery, as well as in the creation and propagation of nutrition support in surgical patients, died June 14, 2021, shortly after his 84th birthday. During his 23 years at the helm of the department, Dr Fischer firmly established the University of Cincinnati as an outstanding surgical training program and one of the foremost nutrition research centers in the world. The synergy between these great leaders (Fischer, Hasselgren, Bower, and Alexander) culminated in a unique and innovative program for advancing the field of nutrition support. [Extracted from the article]

Previously, we have demonstrated that genetically disrupting retinoblastoma protein (Rb) expression in enterocytes results in taller villi, mimicking resection-induced adaption responses. Rb deficiency also results in elevated insulin-like growth factor-2 (IGF-2) expression in villus enterocytes. We propose that postoperative disruption of Rb results in enhanced adaptation which is driven by IGF-2. Inducible, intestine-specific Rb-null mice (iRbIKO) and wild-type (WT) littermates underwent a 50 % proximal small-bowel resection (SBR) at 7-9 weeks of age. They were then given tamoxifen on postoperative days (PODs) 4-6 and harvested on POD 28. The experiment was then repeated on double knockouts of both IGF-2 and Rb (IGF-2 null/iRbIKO). iRbIKO mice demonstrated enhanced resection-induced adaptive villus growth after SBR and increased IGF-2 messenger RNA (mRNA) in ileal villus enterocytes compared to their WT littermates. In the IGF-2 null/iRbIKO double-knockout mice, there was no additional villus growth beyond what was expected of normal resection-induced adaptation. Adult mice in which Rb is inducibly deleted from the intestinal epithelium following SBR have augmented adaptive growth. IGF-2 expression is necessary for enhanced adaptation associated with acute intestinal Rb deficiency. [ABSTRACT FROM AUTHOR]

Effective therapies are limited for patients with parenteral nutritiondependent short bowel syndrome. We previously showed that intestinal expression of the transcriptional coregulator tetradecanoyl phorbol acetate-induced sequence 7 (tis7) is markedly increased during the adaptive response following massive small bowel resection and tis7 plays a role in normal gut lipid metabolism. Here, we further explore the functional implications of tis7 deletion in intestinal lipid metabolism and the adaptive response following small bowel resection. Intestinal tis7 transgenic (tis7tg), tis7-/-, and wild-type (WT) littermates were subjected to 50% small bowel resection. Mice were fed a control or a high-saturated-fat (42% energy) diet for 21 days. Survival, body weight recovery, lipid absorption, mucosal lipid analysis, and the morphometric adaptive response were analyzed. Quantitative real-time PCR was performed to identify tis7 downstream gene targets. Postresection survival was markedly reduced in high-fat, but not control, diet-fed tis7-/- mice. Decreased survival was associated with anastomotic inflammation and intestinal obstruction postresection. High-fat, but not control, diet-fed tis7-/- mice had increased intestinal IL-6 expression. Intestinal lipid trafficking was altered in tis7-/- compared with WT mice postresection. In contrast, high-fat diet-fed tis7tg mice had improved survival postresection compared with WT littermates. High-fat diet feeding in the setting of tis7 deletion resulted in postresection anastomotic inflammation and small bowel obstruction. Tolerance of a calorie-rich, high-fat diet postresection may require tis7 and its target genes. The presence of luminal fat in the setting of tis7 deletion promotes an intestinal inflammatory response postresection. [ABSTRACT FROM AUTHOR]

We previously demonstrated increased villus height following genetic deletion, or knockout, of retinoblastoma protein (Rb) in the intestinal epithelium (Rb-IKO). Here we determined the functional consequences of augmented mucosal growth on intestinal fat absorption and following a 50% small bowel resection (SBR). Mice with constitutively disrupted Rb expression in the intestinal epithelium (Rb-IKO) along with their floxed (wild-type, WT) littermates were placed on a high-fat diet (HFD, 42% kcal fat) for 54 wk. Mice were weighed weekly, and fat absorption, indirect calorimetry, and MRI body composition were measured. Rb-IKO mice were also subjected to a 50% SBR, followed by HFD feeding for 33 wk. In separate experiments, we examined intestinal fat absorption in mice with conditional (tamoxifen-inducible) intestinal Rb (inducible Rb-IKO) deletion. Microarray revealed that the transcriptional expression of lipid absorption/transport genes was significantly reduced in constitutive Rb-IKO mice. These mice demonstrated greater mucosal surface area yet manifested paradoxically impaired intestinal long-chain triglyceride absorption and decreased cholesterol absorption. Despite attenuated lipid absorption, there were no differences in metabolic rate, body composition, and weight gain in Rb-IKO and WT mice at baseline and following SBR. We also confirmed fat malabsorption in inducible Rb-IKO mice. We concluded that, despite an expanded mucosal surface area, Rb-IKO mice demonstrate impaired lipid absorption without compensatory alterations in energy homeostasis or body composition. These findings underscore the importance of delineating structural/functional relationships in the gut and suggest a previously unknown role for Rb in the regulation of intestinal lipid absorption. [ABSTRACT FROM AUTHOR]

Background Congenital diaphragmatic hernia (CDH) is a common birth defect affecting 1 in 3000 births. It is characterised by herniation of abdominal viscera through an incompletely formed diaphragm. Although chromosomal anomalies and mutations in several genes have been implicated, the cause for most patients is unknown. Methods We used whole exome sequencing in two families with CDH and congenital heart disease, and identified mutations in GATA6 in both. Results In the first family, we identified a de novo missense mutation (c.1366C>T, p.R456C) in a sporadic CDH patient with tetralogy of Fallot. In the second, a nonsense mutation (c.712G>T, p.G238") was identified in two siblings with CDH and a large ventricular septal defect. The G238" mutation was inherited from their mother, who was clinically affected with congenital absence of the pericardium, patent ductus arteriosus and intestinal malrotation. Deep sequencing of blood and saliva-derived DNA from the mother suggested somatic mosaicism as an explanation for her milder phenotype, with only approximately 15% mutant alleles. To determine the frequency of GATA6 mutations in CDH, we sequenced the gene in 378 patients with CDH. We identified one additional de novo mutation (c.1071delG, p.V358Cfs34"). Conclusions Mutations in GATA6 have been previously associated with pancreatic agenesis and congenital heart disease. We conclude that, in addition to the heart and the pancreas, GATA6 is involved in development of two additional organs, the diaphragm and the pericardium. In addition, we have shown that de novo mutations can contribute to the development of CDH, a common birth defect. [ABSTRACT FROM AUTHOR]

Previous studies have shown that high-fat diet (HFD) enhances adaptation if provided immediately following small bowel resection (SBR). The purpose of this study was to determine if HFD could further enhance villus growth after resection-induced adaptation had already taken place. C57/Bl6 mice underwent a 50 % proximal SBR or sham operation and were then provided a standard rodent liquid diet (LD) ad lib. After a typical period of adaptation (7 days), SBR and sham-operated mice were randomized to receive either LD or HFD (42 % kcal fat) for an additional 7 days. Mice were then harvested, and small intestine was collected for analysis. Adaptation occurred in both SBR groups; however, the SBR/HFD had significantly increased villus height compared to SBR/LD. Reverse transcription-polymerase chain reaction of villus enterocytes showed a marked increase in CD36 expression in the SBR/HFD group compared with SBR/LD mice. While exposure to increased enteral fat alone did not affect villus morphology in sham-operated mice, HFD significantly increased villus growth in the setting of resection-induced adaptation, supporting the clinical utility of enteral fat in augmenting adaptation. Increased expression of CD36 suggests a possible mechanistic role in dietary fat metabolism and villus growth in the setting of short gut syndrome. [ABSTRACT FROM AUTHOR]

Congenital diaphragmatic hernia (CDH) is characterized by incomplete formation of the diaphragm occurring as either an isolated defect or in association with other anomalies. Genetic factors including aneuploidies and copy number variants are important in the pathogenesis of many cases of CDH, but few single genes have been definitively implicated in human CDH. In this study, we used whole exome sequencing (WES) to identify a paternally inherited novel missense GATA4 variant (c.754C>T; p.R252W) in a familial case of CDH with incomplete penetrance. Phenotypic characterization of the family included magnetic resonance imaging of the chest and abdomen demonstrating asymptomatic defects in the diaphragm in the two 'unaffected' missense variant carriers. Screening 96 additional CDH patients identified a de novo heterozygous GATA4 variant (c.848G>A; p.R283H) in a non-isolated CDH patient. In summary, GATA4 is implicated in both familial and sporadic CDH, and our data suggests that WES may be a powerful tool to discover rare variants for CDH. [ABSTRACT FROM AUTHOR]

Background Congenital diaphragmatic hernia (CDH) is a common birth defect with significant morbidity and mortality. Although the aetiology of CDH remains poorly understood, studies from animal models and patients with CDH suggest that genetic factors play an important role in the development of CDH. Chromosomal anomalies have been reported in CDH. Methods In this study, the authors investigated the frequency of chromosomal anomalies and copy number variants (CNVs) in 256 parent-child trios of CDH using clinical conventional cytogenetic and microarray analysis. The authors also selected a set of CDH related training genes to prioritise the genes in those segmental aneuploidies and identified the genes and gene sets that may contribute to the aetiology of CDH. Results The authors identified chromosomal anomalies in 16 patients (6.3%) of the series including three aneuploidies, two unbalanced translocation, and 11 patients with de novo CNVs ranging in size from 95 kb to 104.6 Mb. The authors prioritised the genes in the CNV segments and identified KCNA2, LMNA, CACNA1S, MYOG, HLX, LBR, AGT, GATA4, SOX7, HYLS1, FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, HOMER2, BNC1, BID, and TBX1 as genes that may be involved in diaphragm development. Gene enrichment analysis identified the most relevant gene ontology categories as those involved in tissue development (p=4.4×10-11) or regulation of multicellular organismal processes (p=2.8×10-10) and 'receptor binding' (p=8.7×10-14) and 'DNA binding transcription factor activity' (p=4.4×10-10). Conclusions The present findings support the role of chromosomal anomalies in CDH and provide a set of candidate genes including FOXC1, FOXF2, PDGFA, FGF6, COL4A1, COL4A2, SOX7, BNC1, BID, and TBX1 for further analysis in CDH. [ABSTRACT FROM AUTHOR]

Intestinal adaptation is an important compensatory response to massive small bowel resection (SBR) and occurs because of a proliferative stimulus to crypt enterocytes by poorly understood mechanisms. Recent studies suggest the enteric nervous system (ENS) influences enterocyte proliferation. We, therefore, sought to determine whether ENS dysfunction alters resection-induced adaptation responses. Ret+/- mice with abnormal ENS function and wild-type (WT) littermates underwent sham surgery or 50% SBR. After 7 days, ileal morphology, enterocyte proliferation, apoptosis, and selected signaling proteins were characterized. Crypt depth and villus height were equivalent at baseline in WT and Ret+/- mice. In contrast after SBR, Ret+/- mice had longer villi (Ret+/- 426.7 μ± 46.0 m vs. WT 306.5 ± 7.7 μm, P < 0.001) and deeper crypts (Ret+/- 119 ± 3.4 μm vs. WT 82.4 ± 3.1 μm, P < 0.001) than WT. Crypt enterocyte proliferation was higher in Ret+/- (48.8 ± 1.3%) than WT (39.9 ± 2.1%; P < 0.001) after resection, but apoptosis rates were similar. Remnant bowel of Ret+/- mice also had higher levels of glucagon-like peptide 2 (6.2-fold, P < 0.005) and amphiregulin (4.6-fold, P < 0.001) mRNA after SBR, but serum glucagon- like peptide 2 protein levels were equal in WT and Ret+/- mice, and there was no evidence of increased c-Fos nuclear localization in submucosal neurons. Western blot confirmed higher crypt epidermal growth factor receptor (EGFR) protein levels (1.44-fold; P < 0.001) and more phosphorylated EGFR (2-fold; P < 0.003) in Ret+/- than WT mice after SBR. These data suggest that Ret heterozygosity enhances intestinal adaptation after massive SBR, likely via enhanced EGFR signaling. Reducing Ret activity or altering ENS function may provide a novel strategy to enhance adaptation attenuating morbidity in patients with short bowel syndrome. [ABSTRACT FROM AUTHOR]

Increased apoptosis in crypt enterocytes is a key feature of intestinal adaptation following massive small bowel resection (SBR). Expression of the proapoptotic factor Bax has been shown to be required for resection-induced apoptosis. It has also been demonstrated that p38-α MAPK (p38) is necessary for Bax activation and apoptosis in vitro. The present studies were designed to test the hypothesis that p38 is a key regulator of Bax activation during adaptation after SBR in vivo. Enterocyte expression of p38 was deleted by tamoxifen administration to activate villin-Cre in adult mice with a floxed Mapk14 (p38-α) gene. Proximal 50% SBR or sham operations were performed on wild-type (WT) and p38 intestinal knockout (p38-IKO) mice under isoflurane anesthesia. Mice were killed 3 or 7 days after operation, and adaptation was analyzed by measuring intestinal morphology, proliferation, and apoptosis. Bax activity was quantified by immunoprecipitation, followed by Western blotting. After SBR, p38-IKO mice had deeper crypts, longer villi, and accelerated proliferation compared with WT controls. Rates of crypt apoptosis were significantly lower in p38-IKO mice, both at baseline and after SBR. Levels of activated Bax were twofold higher in WT mice after SBR relative to sham. In contrast, activated Bax levels were reduced by 67% in mice after p38 MAPK deletion. Deleted p38 expression within the intestinal epithelium leads to enhanced adaptation and reduced levels of enterocyte apoptosis after massive intestinal resection. p38-regulated Bax activation appears to be an important mechanism underlying resection-induced apoptosis. [ABSTRACT FROM AUTHOR]

Background: Adaptation following massive intestinal loss is characterized by increased villus height and crypt depth. Previously, we demonstrated that p21-null mice do not adapt after small bowel resection (SBR). As retinoblastoma protein (Rb) levels are elevated in p21-null crypt cells, we first sought to determine whether Rb is required for normal adaptation. Next, we tested whether Rb expression is responsible for blocked adaptation in p21-nulls. Methods: Genetically manipulated mice and wild-type (WT) littermates underwent either 50% SBR or sham operation. The intestine was harvested at 3, 7, or 28 days later and intestinal adaptation was evaluated. Enterocytes were isolated and protein levels evaluated by Western blot and quantified by optical density. Results: Rb-null mice demonstrated increased villus height, crypt depth, and proliferative rate at baseline, but there was no further increase following SBR. Deletion of one Rb allele lowered Rb expression and restored resection-induced adaptation responses in p21-null mice. Conclusion: Rb is specifically required for resection-induced adaptation. Restoration of adaptation in p21-null mice by lowering Rb expression suggests a crucial mechanistic role for Rb in the regulation of intestinal adaptation by p21. [ABSTRACT FROM AUTHOR]

Objective To determine the prenatal factors associated with the need for extracorporeal membrane oxygenation (ECMO) and neonatal survival in congenital diaphragmatic hernia (CDH). Study design A retrospective cohort study of all cases of CDH seen in our center between 1998 and 2008. Prenatal ultrasound and neonatal records were reviewed. Both univariable and logistic regression analyses were performed to determine the significant factors associated with the use of ECMO and survival. Results Among 107 cases of CDH seen during the study period, 62 were evaluated prenatally in our center and 49 had information on all variables evaluated. The overall rate of ECMO use was 27/107 (25%) and survival rate was 53/107 (49.5%). The lung area to head circumference ratio (LHR) and gestational age (GA) at delivery were the only significant factors associated with ECMO use, and the LHR and absence of liver herniation were significantly associated with survival. LHR values under 1.0 were associated with 57% need for ECMO and 100% neonatal death. Although, overall, the observed:expected LHR (O:E LHR) was not significantly associated with ECMO use or survival, levels below 65% were associated with 58% need for ECMO ( p = 0.004) and 100% neonatal death ( p = 0.002). Conclusion The study confirms the LHR, GA at delivery and liver herniation as significant prenatal predictors of the need for ECMO or survival in cases with CDH. This information is helpful for counseling women with fetuses complicated by CDH. Copyright © 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Introduction: Liver mass is regulated in precise proportion to body mass in health and is restored by regeneration following acute injury. Despite extensive experimental analyses, the mechanisms involved in this regulation have not been fully elucidated. Previous investigations suggest that signals from the bowel may play an important role. The purpose of the studies reported here was to determine the effect of proximal partial small bowel resection on liver mass in a murine model.Methods: Mice were subjected to a 50% proximal small bowel resection or sham surgery followed by primary anastomosis, then sacrificed at serial times for determination of liver:body mass ratio and analyses of liver tissue.Results: Liver:body weight ratio was significantly decreased 72 h after small bowel resection, and this decrease correlated with reduced functional liver mass as assessed by determination of total hepatic tissue protein and alanine transaminase (ALT) activity. Liver from bowel-resected animals demonstrated increased expression of LC3-II, a marker of autophagy, and also of pro-apoptotic Bax compared to anti-apoptotic Bcl-2.Conclusion: These data support a role for signals from the intestine in liver mass regulation, and they have potential implications regarding the pathogenesis of liver injury following small bowel resection. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that the proapoptotic protein Bax plays an important role in the elevated enterocyte apoptosis that occurs during the intestinal adaptation response to massive small bowel resection (SBR). Additionally, epidermal growth factor receptor (EGFR) activation prevents SBR-induced enterocyte apoptosis. The present study aims to delineate the relationship between EGFR activity and intestinal epithelial cell apoptosis. Treatment of model intestinal epithelial cells (RIEC-18) with both a selective EGFR inhibitor (ZD1839) and EGFR small interfering RNA knockdown resulted in a dramatic increase in apoptosis, accompanied by rapid phosphorylation of p38α. Concurrently, Bax underwent conformational changes consistent with activation and translocated to mitochondria. In contrast, EGF stimulation enhanced cell survival by attenuating p38α phosphorylation, Bax conformational change, mitochondrial trafficking, and apoptosis. These results demonstrate that that diminished EGFR activity initiates the intrinsic pathway of apoptosis through p38α-dependent Bax activation in intestinal epithelial cells. These finding provide mechanistic insight into the role that EGFR signaling plays in the regulation of enterocyte apoptosis following massive intestinal loss. [ABSTRACT FROM AUTHOR]

Previous work in our group has demonstrated that mouse salivary gland has the highest concentration of salivary-derived VEGF protein compared with other organs and is essential for normal palatal mucosal wound healing. We hypothesize that salivary VEGF plays an important role in maintaining the integrity of the gastrointestinal mucosa following small bowel resection (SBR). Thirty-five 8- to 10-wk-old C57BL/6 female mice were divided into seven treatment groups: 1) sham (transaction and anastomosis, n = 5); 2) SBR (n = 8); 3) sialoadenectomy and small bowel resection (SAL+SBR, n = 8); 4) sialoadenectomy and small bowel resection with EGF supplementation (SAL+SBR+EGF, n = 9); 5) sialoadenectomy and small bowel resection with VEGF supplementation (SAL+SBR+VEGF, n = 9); 6) sialoadenectomy and small bowel resection supplemented with EGF and VEGF (SAL+ SBR+VEGF+EGF, n = 6); 7) selective inhibition of VEGF in the submandibular gland by Ad-VEGF-Trap following small bowel resection (Ad-VEGF-Trap+SBR, n = 7). Adaptation was after 3 days by ileal villus height and crypt depth. The microvascular response was evaluated by CD31 immunostaining and for villus-vessel area ratio by FITC-labeled von Willebrand factor immunostaining. The adaptive response after SBR was significantly attenuated in the SAL group in terms of villus height (250.4 ± 8.816 vs. 310 ± 19.35, P = 0.01) and crypt depth (100.021 ± 4.025 vs. 120.541 ± 2.82, P = 0.01). This response was partially corrected by orogastric VEGF or EGF alone. The adaptive response was completely restored when both were administered together, suggesting that salivary VEGF and EGF both contribute to intestinal adaptation. VEGF increases the vascular density (6.4 ± 0.29 vs. 6.1 ± 0.29 vs. 5.96 ± 0.20) and villus-vessel area ratio (0.713 ± 0.01 vs. 0.73 ± 0.01) in the adapting bowel. Supplementation of both EGF and VEGF fully rescues adaptation, suggesting that the adaptive response may be dependent on VEGF-driven angiogenesis. These results support a previously unrecognized role for VEGF in the small bowel adaptive response. [ABSTRACT FROM AUTHOR]

Background: Spontaneous pneumothorax (SPTX) is a relatively common condition. In patients with SPTX, CT has been advocated to identify blebs and bullae (BB) to help in management planning.Purpose: The study was designed to assess our experience with CT evaluation for underlying BB in children with SPTX as compared to normal controls.Materials and Methods: Forty-three children (mean age 16 years, range 13-19 years) with 50 SPTX events with both chest radiographs and CT scans were reviewed. CT findings were compared with those seen in 29 age- and gender-matched controls without SPTX. The parameters evaluated included size, number, location, and ipsi-/contralateral BB; apical lines; and surgical correlation.Results: In the study group, BB were identified in 14 imaged events (28%) (size 2.5-45 mm, one to six BB) with contralateral BB in 11 of the 14 (78.6%). All BB were confined to the apices. BB were sometimes difficult to differentiate from "apical lines"--a suspected normal variant seen in 28 imaged events (56%). Of blebs seen at surgery, 59% were identified on CT, and there were no false-positive CT findings. In the control group, no BB were identified but "apical lines" were seen in eight children (28%).Conclusion: BB were seen by CT in 28% of imaged events in children with SPTX and were always confined to the apices. When present, BB were commonly bilateral (78.6%). BB should not be confused with "apical lines," which were not only seen in 56% of imaged events in the SPTX group but also in 28% of the normal controls. [ABSTRACT FROM AUTHOR]

A toddler with a persistent congenital umbilical papule is described. The papule was found to contain both omphalomesenteric and urachal remnants. This patient's findings illustrate the importance of further evaluation of umbilical lesions when conventional therapy with silver nitrate fails. [ABSTRACT FROM AUTHOR]

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21waf1/cip1 is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27kip1, another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21waf1/cip1-null, p27kip1-null, and p21waf1/cip1/p27kip1 double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21waf1/cip1 and decreased p27kip1 within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27kip1-null mice; however, mice deficient in both p21waf1/cip1 and p27kip1 failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21waf1/cip1 protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27kip1 does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation. [ABSTRACT FROM AUTHOR]

Prior studies of intestinal adaptation after massive small bowel resection (SBR) have focused on growth factors and their effects on amplification of the gut mucosa. Because adaptive changes have also been described in intestinal smooth muscle, we sought to determine the effect of targeted smooth muscle growth factor overexpression on resectioninduced intestinal adaptation. Male transgenic mice with smooth muscle cell overexpression of insulin-like growth factor I (IGF-I) by virtue of an α-smooth muscle actin promoter were obtained. SMP8 IGF-I transgenic (IGF-I TG) and nontransgenic (NT) littermates underwent 50% proximal SBR or sham operation and were then killed after 3 or 28 days. NT mice showed the expected alterations in mucosal adaptive parameters after SBR, such as increased wet weight and villus height. The IGF-I TG mice had inherently taller villi, which did not increase significantly after SBR. In addition, IGF-I TG mice had a 50% postresection persistent increase in remnant intestinal length, which was associated with an early decline and later increase in relative mucosal surface area. These results indicate that growth factor overexpression within the muscularis layer of the bowel wall induces significant postresection adaptive intestinal lengthening and a unique mucosal response. IGF-I signaling within the muscle wall may play an important role in the pathogenesis of resection-induced adaptation. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of the study was to evaluate the impact of a clinical pathway on the volume of imaging studies performed in children with suspected clinical diagnosis of hypertrophic pyloric stenosis. The pathway suggested referral to surgeons for clinical evaluation for palpation of the olive prior to ordering imaging studies. Only those children in whom the olive could not be palpated would be referred for imaging, and it was anticipated that imaging volume would be reduced following guideline implementation.Materials and Methods: The database of the Health Policy and Clinical Effectiveness Department was used to evaluate all patients who had surgery for hypertrophic pyloric stenosis. The presence of a palpable olive and the type of imaging were evaluated both prior to and after the implementation of the clinical guideline.Results: Prior to the guideline, 85 infants had surgery for pyloric stenosis, with 83 of the 85 (97%) having imaging. After the implementation of the guideline, 90 infants had surgery for pyloric stenosis with 84 of 90 patients imaged (92%). A chi-square analysis demonstrated no significant difference in the percentage of children imaged in the two groups (P = 0.104). Approximately one in five children referred for vomiting were diagnosed with hypertrophic pyloric stenosis.Conclusion: No significant change in imaging volume occurred following initiation of a guideline which recommended clinical evaluation for palpation of the olive prior to ordering imaging studies. Multiple factors probably contributed to the lack of demonstrated changes. [ABSTRACT FROM AUTHOR]

Intestinal adaptation following small bowel resection (SBR) is associated with greater rates of enterocyte apoptosis by unknown mechanism(s). Because postresection adaptation is associated with increased translocation of luminal bacteria, we sought to characterize the role for the extrinsic, death receptor pathway for the activation of enterocyte apoptosis after massive SBR. We first performed SBR or sham operations in mice, and the temporal expression of caspases 8, 9, and 3, death receptors tumor necrosis factor receptor-1 (TNFR[sub 1]) and Fas and corresponding ligands (TNF and Fas ligand) was determined in the remnant intestine at various postoperative time points. Ileal TNFR[sub 1] and Fas expression were then measured after SBR in the setting of increased (waved-2 mice) or decreased (exogenous EGF administration) apoptosis. Finally, intestinal adaptation and apoptosis were recorded in the remnant ileum after SBR in TNFR[sub 1]-null and Fas-null mice. The expression of death receptor family proteins and caspases demonstrated only modest changes after SBR and did not correlate with the histological appearance of apoptosis. In the setting of accelerated apoptosis, TNFR[sub 1] and Fas expression were paradoxically decreased. Apoptotic and adaptive responses were preserved in both TNFR[sub 1]-null and Fas-null mice. These results suggest that the mechanism for increased enterocyte apoptosis following massive SBR does not appear to involve the extrinsic, death receptor-mediated pathway. [ABSTRACT FROM AUTHOR]

Examines the role of epidermal growth factor and its receptor in chemotherapy-induced intestinal injury. Weight recovery; Evidence showing that apoptosis was not decreased; Occurrence of mucositis.