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Stern, Lawrence E., Richard A. Falcone, Jr., Christopher J. Kemp, Margaret C. Braun, Christopher R. Erwin, and Brad W. Warner. Salivary epidermal growth factor and intestinal adaptation in male and female mice. Am J Physiol Gastrointest Liver Physiol 278: G871-G877, 2000. --Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation. small bowel resection; kallikrein; salivary gland

Shin, Cathy E., Richard A. Falcone, Jr., Christopher J. Kemp, Christopher R. Erwin, David A. Litvak, B. Mark Evers, and Brad W. Warner. Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G717-G724, 1999.--Adaptation following small bowel resection (SBR) signals enterocyte proliferation and apoptosis. Because p53-induced [p21.sup.waf1/cip1] may be important for apoptosis in many cells, we hypothesized that these genes are required for increased enterocyte apoptosis during adaptation. Male C57BL/6 (wild-type) or p53-null mice underwent 50% proximal SBR or sham operation (bowel transection-reanastomosis). Adaptation (DNA-protein content, villus height-crypt depth, enterocyte proliferation), appearance of apoptotic bodies, and p53 and [p21.sup.waf1/cip1] protein expression were measured in the ileum after 5 days. Adaptation was equivalent after SBR in both wild-type and p53-null mice as monitored by significantly increased ileal DNA-protein content, villus height, and enterocyte proliferation. The number of crypt apoptotic bodies increased significantly after SBR evenly in both wild-type and p53-null mice. In the p53-null mice, SBR substantially induced the expression of [p21.sup.waf1/cip1] protein in villus enterocytes. The p53-independent induction of [p21.sup.waf1/cip1] may account for the similar intestinal response to SBR between wild-type and p53-null mice. Intestinal adaptation and increased enterocyte apoptosis following intestinal resection occur via a p53-independent mechanism. enterectomy; mice; intestinal resection; short-gut syndrome; programmed cell death

Erwin, Christopher R., Michael A. Helmrath, Cathy E. Shin, Richard A. Falcone, Jr., Lawrence E. Stern, and Brad W. Warner. Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G533-G540, 1999.--The effect of direct intestinal overexpression of epidermal growth factor (EGF) on postresection adaptation has been investigated by the production of transgenic mouse lines. A murine pro-EGF cDNA construct was produced, and expression of the EGF construct was targeted to the small intestine with the use of the rat intestinal fatty acid-binding protein promoter. An approximately twofold increase in intestinal EGF mRNA and protein was detected in heterozygous mice. No changes in serum EGF levels were noted. Except for a slightly shortened small intestine, no other abnormal phenotype was observed. Intestinal adaptation (increases in body weight, DNA, protein content, villus height, and crypt depth) was markedly enhanced after a 50% proximal small bowel resection in transgenic mice compared with nontransgenic littermates. This transgenic mouse model permits the study of intestinal adaptation and other effects of EGF in the small intestine in a more physiological and directed manner than has been previously possible. These results endorse a direct autocrine/paracrine mechanism for EGF on enterocytes as a means to enhance adaptation. intestinal resection; intestine development; short gut; RT-PCR; ribonuclease protection assay; epidermal growth factor

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]

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]

Background Congenital diaphragmatic hernia (CDH) is a prevalent major congenital anomaly with significant morbidity and mortality. Thirty to 40% mortality in CDH is largely attributed to pulmonary hypoplasia and pulmonary hypertension (PH). We hypothesized that the underlying genetic risk factors for hereditary PH are shared with CDH associated PH. Methods Participants were recruited as part of the Diaphragmatic Hernia Research & Exploration; Advancing Molecular Science (DHREAMS) study, a prospective cohort of neonates with a diaphragmatic defect enrolled from 2005 to 2012. PH affected patients with available DNA for sequencing had one of the following: moderate or severe PH on echocardiography at 3 months of age; moderate of severe PH at 1 month of age with death occurring prior to the 3 month echocardiogram; or on PH medications at 1 month of age. We sequenced the coding regions of the hereditary PH genes bone morphogenetic protein receptor type II ( BMPR2 ), caveolin 1 ( CAV1 ) and potassium channel subfamily K, member 3 ( KCNK3 ) to screen for mutations. Results There were 29 CDH patients with PH including 16 males and 13 females. Sequencing of BMPR2 , CAV1 , and KCNK3 coding regions did not identify any pathogenic variants in these genes. Type of study Prognosis study Level of evidence Level IV [ABSTRACT FROM AUTHOR]

Background Multicenter clinical research studies in pediatric surgery have been largely limited to relatively small case-series and retrospective reviews because of the rarity of many of the diseases we treat and difficulty coordinating and executing multi-institutional studies. Creation of a collaborative research network can provide the needed patient population and infrastructure to perform high quality multi-institutional studies. Methods In 2013, eleven academic pediatric surgery centers within the United States formed a research consortium to develop and conduct multicenter clinical research projects to advance the practice of pediatric surgery. Results We present our process for creating, developing, and maintaining this consortium including initial regional geographic limitation, charter development with by-laws and procedures for adopting studies, and research infrastructure including a central website for study monitoring and central reliance institutional review board process. Conclusion Our model could be reproduced or adapted by other institutions to develop or strengthen other research collaboratives. Level of evidence Type of study: retrospective, IV. [ABSTRACT FROM AUTHOR]

Introduction Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. NEC severity varies widely. Recent data have demonstrated a strong link between gut microbial dysbiosis and development of NEC. We tested the hypothesis that alterations in the gut microbiome at the time of diagnosis predict the severity of NEC. Methods We used prospectively collected fecal samples from very low birth weight infants who developed NEC, stratifying by NEC severity. Fecal bacterial DNA was sequenced using 16S rRNA pyrosequencing. A generalized Wald-type test based on the Dirichlet multinomial distribution was used to test for differences in microbial communities. Results Of 489 infants at risk, 30 NEC cases had 410 fecal samples collected in the 28 days prior to the onset of NEC available for analysis. There were no differences in the pre-NEC gut microbial community between infants treated medically vs. surgically, or those with NEC totalis. Furthermore, neither treatment of NEC significantly changed the gut microbiome post-NEC among the survivors. Conclusion We found no evidence that the gut microbiome, prior to the onset of disease, differentiates the clinical course of NEC. These data suggest that factors other than the gut microbiome may dictate disease severity. Level of evidence Level 4. [ABSTRACT FROM AUTHOR]

Purpose Previously, we demonstrated enhanced adaptation after small bowel resection (SBR) in intestinal-specific retinoblastoma (Rb)-deficient mice along with elevated levels of insulin-like growth factor 2 (IGF2) expression within the villi. The purpose of this study was to verify that the insulin-like growth factor 1 receptor (IGF1R) plays a role in this phenomenon. Methods Inducible and intestinal specific Rb and IGF1R double knockout mice (iRb/IGF1R-IKO) (n = 4) and Rb single knockout mice (iRb-IKO) (n = 5) underwent 50% mid SBR. On post-operative day 28, mice were harvested, and structural adaptation was measured as changes in crypt depth and villus height. Rates of enterocyte proliferation were recorded. IGF2 expression within the remnant villi was measured via RT-PCR. Results Both iRb-IKO and iRb/IGF1R-IKO mice demonstrated enhanced adaptation with at least a 45% increase in both crypt depth and villus height in the proximal and distal remnant bowel. Both groups showed elevation of IGF2 expression in the remnant villi, but there were no differences between the two groups. Conclusion Epithelial IGF1R is dispensable for IGF2-mediated enhanced intestinal adaptation in retinoblastoma-deficient mice. Our findings suggest that IGF2 signals for enhanced adaptation in cells outside of the epithelium. Further investigation is needed to study the IGF2/IGF1R signaling interaction within the mesenchyme. Level of Evidence Animal study - not clinical. [ABSTRACT FROM AUTHOR]

Background A significant number of children with short bowel syndrome experience intestinal failure-associated liver disease. We recently demonstrated accelerated hepatic steatosis after 50% small bowel resection (SBR) in mice. Since SBR is associated with alterations in the gut microbiome, the purpose of this study was to determine whether TLR4 signaling is critical to the development of resection-associated hepatic steatosis. Methods Male C57BL6 (control) and TLR4-knockout (KO) mice underwent 50% proximal SBR. Liver sections were analyzed to obtain the percent lipid content, and Ileal sections were assessed for morphological adaptation. Intestinal TLR4 mRNA expression was measured at 7 days and 10 weeks. Results Compared to controls, TLR4 KO mice demonstrated similar weight gain and morphological adaptation after SBR. Hepatic steatosis was decreased 32-fold in the absence of TLR4. Intestinal TLR4 mRNA expression was significantly elevated 7 days after SBR. We also found that TLR4 expression in the intestine was 20-fold higher in whole bowel sections compared with isolated enterocytes. Conclusions TLR4 signaling is critical for the development of resection-associated steatosis, but not involved in intestinal adaptation after massive SBR. Further studies are needed to delineate the mechanism for TLR4 signaling in the genesis of resection-associated liver injury. Level of evidence Animal study, not clinical. [ABSTRACT FROM AUTHOR]

Massive intestinal resection is a regrettably necessary but life-saving intervention for progressive or fulminant necrotizing enterocolitis (NEC). However, the resultant short bowel syndrome (SBS) poses its own array of challenges and complications. Within hours of such an abrupt loss of intestinal length, the intestine begins to adapt. Our ability to understand this process of intestinal adaptation has proven critical in our ability to clinically treat the challenging problem of short bowel syndrome. This review first highlights key data relating to intestinal adaptation including structural and functional changes, biochemical regulation, and other factors affecting the magnitude of intestinal adaptation responses. We then focus on intestinal rehabilitation as it relates to strategies to enhance intestinal adaptation while meeting nutritional needs and preventing complications of parenteral nutrition. [ABSTRACT FROM AUTHOR]