Matthew W. Ralls

Loss of enteral nutrition in a mouse model results in intestinal epithelial barrier dysfunction

Total parenteral nutrition (TPN) administration in a mouse model leads to a local mucosal inflammatory response, resulting in a loss of epithelial barrier function (EBF). Although, the underlying mechanisms are unknown, a major contributing factor is a loss of growth factors and subsequent critical downstream signaling. An important component of these is the p‐Akt pathway. An additional contributing factor to the loss of EBF with TPN is an increase in proinflammatory cytokine abundance within the mucosal epithelium, including TNF‐α and IFN‐γ. Loss of critical nutrients, including glutamine and glutamate, may affect EBF, contributing to the loss of tight junction proteins. Finding protective modalities for the small intestine during TPN administration may have important clinical applications. Supplemental glutamine and glutamate may be examples of such agents.

Intestinal epithelial cell apoptosis and loss of barrier function in the setting of altered microbiota with enteral nutrient deprivation

Total parenteral nutrition (TPN), a commonly used treatment for patients who cannot receive enteral nutrition, is associated with significant septic complications due in part to a loss of epithelial barrier function (EBF). While the underlying mechanisms of TPN-related epithelial changes are poorly understood, a mouse model of TPN-dependence has helped identify several contributing factors. Enteral deprivation leads to a shift in intestinal microbiota to predominantly Gram-negative Proteobacteria. This is associated with an increase in expression of proinflammatory cytokines within the mucosa, including interferon-γ and tumor necrosis factor-α. A concomitant loss of epithelial growth factors leads to a decrease in epithelial cell proliferation and increased apoptosis. The resulting loss of epithelial tight junction proteins contributes to EBF dysfunction. These mechanisms identify potential strategies of protecting against TPN-related complications, such as modification of luminal bacteria, blockade of proinflammatory cytokines, or growth factor replacement.

Reoperative Surgery for Hirschsprung Disease

Despite most children undergoing a successful pull through for Hirschsprung disease, a small portion of children are left with persistent stooling issues. Most of these stooling issues can be addressed by nonoperative approaches. However, in a small group of remaining children, a reoperation may be necessary. Most children who may need a redo pull-through procedure may have a persistent area of aganglionosis, unremitting enterocolitis, or a torsion or stricture of the pull-through segment. Each of these influences the approach the surgeon must take to correct the presenting problem. The chapter details the diagnostic approach as well as the operative techniques, which best deal with each of these complications.

Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function

OBJECTIVE To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.

Intestinal microbial diversity and perioperative complications.

BACKGROUND AND AIMS Enteral nutrient deprivation via parenteral nutrition (PN) in a mouse model leads to a local mucosal inflammatory response. This proinflammatory response leads to a loss of epithelial barrier function and atrophy of the intestine. Although the underlying mechanisms are unknown, a potential contributing factor is the impact PN has on the intestinal microbiome. We recently identified a shift in the intestinal microbial community in mice given PN; however, it is unknown whether such changes occur in humans. We hypothesized that similar microbial changes occur in humans during periods of enteral nutrient deprivation. METHODS A series of small bowel specimens were obtained from pediatric and adult patients undergoing small intestinal resection. Mucosally associated bacteria were harvested and analyzed using 454 pyrosequencing techniques. Statistical analysis of microbial diversity and differences in microbial characteristics were assessed between enterally fed and enterally deprived portions of the intestine. Occurrence of postoperative infectious and anastomotic complications was also examined. RESULTS Pyrosequencing demonstrated a wide variability in microbial diversity within all groups. Principal coordinate analysis demonstrated only a partial stratification of microbial communities between fed and enterally deprived groups. Interestingly, a tight correlation was identified in patients who had a low level of enteric microbial diversity and those who developed postoperative enteric-derived infections or intestinal anastomotic disruption. CONCLUSIONS Loss of enteral nutrients and systemic antibiotic therapy in humans is associated with a significant loss of microbial biodiversity within the small bowel mucosa. These changes were associated with a number of enteric-derived intestinal infections and intestinal anastomotic disruptions.

Drug Shortage–Associated Increase in Catheter-Related Blood Stream Infection in Children

BACKGROUND: Ethanol lock therapy (ELT) has been shown to reduce the incidence of catheter-related blood stream infections (CRBSI) in intestinal failure (IF) patients. Dosing and frequency remains undefined. Scrutiny of pharmaceutical facilities by the Food and Drug Administration led to the voluntary shutdown of the sole supplier of ethanol, resulting in a nationwide shortage. To conserve supply, we reduced ELT frequency from a daily regimen. We examined the impact that reduction in ELT frequency had on CRBSI in pediatric IF patients. METHODS: We retrospectively reviewed our parenteral nutrition–dependent IF children. Primary outcome measure was CRBSI per 1000 catheter days after ELT frequency reduction. Data were compared (paired t test) to the same group over 1 year before ethanol shortage and to historical controls. RESULTS: During the shortage 13 outpatients received ELT. Eight met study criteria. Mean ± SD age was 9.1 ± 7.8 years. Mean CRBSI rate per 1000 catheter days was 0.7 ± 1.3 before ELT shortage. This increased to 6.2 ± 2.5 after frequency reduction (P < .001). This CRBSI rate was similar to historical IF children not on ELT (8.0 ± 5.4). Seven children developed CRBSI after frequency reduction, 6 requiring hospitalization, 2 to the ICU. Mean length of stay (15.5 days) averaged

Loss of ADAM17-Mediated Tumor Necrosis Factor Alpha Signaling in Intestinal Cells Attenuates Mucosal Atrophy in a Mouse Model of Parenteral Nutrition

104,783(± 111,034) in hospital charges. Organisms included Gram-negatives (6), methicillin-resistant Staphylococcus aureus (1), and Candida spp (1). CONCLUSIONS: ELT frequency reduction resulted in complete failure in CRBSI prophylaxis. The nationwide shortage of this drug has been costly both financially and in patient morbidity.

Redo pullthrough for Hirschsprung disease: A single surgical group's experience

ABSTRACT Total parenteral nutrition (TPN) is commonly used clinically to sustain patients; however, TPN is associated with profound mucosal atrophy, which may adversely affect clinical outcomes. Using a mouse TPN model, removing enteral nutrition leads to decreased crypt proliferation, increased intestinal epithelial cell (IEC) apoptosis and increased mucosal tumor necrosis factor alpha (TNF-α) expression that ultimately produces mucosal atrophy. Upregulation of TNF-α signaling plays a central role in mediating TPN-induced mucosal atrophy without intact epidermal growth factor receptor (EGFR) signaling. Currently, the mechanism and the tissue-specific contributions of TNF-α signaling to TPN-induced mucosal atrophy remain unclear. ADAM17 is an ectodomain sheddase that can modulate the signaling activity of several cytokine/growth factor receptor families, including the TNF-α/TNF receptor and ErbB ligand/EGFR pathways. Using TPN-treated IEC-specific ADAM17-deficient mice, the present study demonstrates that a loss of soluble TNF-α signaling from IECs attenuates TPN-induced mucosal atrophy. Importantly, this response remains dependent on the maintenance of functional EGFR signaling in IECs. TNF-α blockade in wild-type mice receiving TPN confirmed that soluble TNF-α signaling is responsible for downregulation of EGFR signaling in IECs. These results demonstrate that ADAM17-mediated TNF-α signaling from IECs has a significant role in the development of the proinflammatory state and mucosal atrophy observed in TPN-treated mice.

Bacterial nutrient foraging in a mouse model of enteral nutrient deprivation: insight into the gut origin of sepsis

INTRODUCTION This study presents our surgical experience for redo-pullthrough (RedoPT) for Hirschsprung disease (HD). It reviews the patients clinical outcomes and assesses stooling patterns after RedoPT. METHODS A retrospective review of our institutions RedoPTs as well as one authors overseas cases was performed. Stooling scores were tabulated using an established survey tool and compared to primary PT matched patients. RESULTS Between 1974 and 2012, 46 individuals (52% males) underwent RedoPT, representing 3 percent of all HD pullthroughs. Median age at primary PT and RedoPT was 1year (range 1week-18years) and 3.5years (range 8weeks-41years), respectively. Indications for RedoPT were predominately for aganglionosis/transition zone pathology (71%); followed by stricture or an obstructing Duhamel pouch (19%), tight cuff (8%) and a twisted PT (4%). None were performed for an isolated clinical diagnosis of repeated bouts of enterocolitis. RedoPT surgical approach depended upon the initial pullthrough technique and any previous complications. Stooling scores were significantly (P<0.05) worse in the RedoPT patients compared to the historically-matched group of children undergoing a primary PT for HD (5.5±1.2 vs. 12.2±1.4, primary PT versus RedoPT, respectively). When breaking down this total score into individual parameters, stooling pattern scores (1.0±0.2 vs. 4.1±0.4, P=0.001) and enterocolitis scores (2.0±0.4 vs. 4.2±0.4, P=0.001) were statistically worse in the RedoPT group. Patients in both groups had similar overall continence rates. CONCLUSION Appropriately selected children undergoing a RedoPT can achieve good results, with comparable continence rates to those undergoing a primary PT.

Redo pullthrough for Hirschsprung disease

Total parenteral nutrition (TPN) leads to a shift in small intestinal microbiota with a characteristic dominance of Proteobacteria This study examined how metabolomic changes within the small bowel support an altered microbial community in enterally deprived mice. C57BL/6 mice were given TPN or enteral chow. Metabolomic analysis of jejunal contents was performed by liquid chromatography/mass spectrometry (LC/MS). In some experiments, leucine in TPN was partly substituted with [13C]leucine. Additionally, jejunal contents from TPN-dependent and enterally fed mice were gavaged into germ-free mice to reveal whether the TPN phenotype was transferrable. Small bowel contents of TPN mice maintained an amino acid composition similar to that of the TPN solution. Mass spectrometry analysis of small bowel contents of TPN-dependent mice showed increased concentration of 13C compared with fed mice receiving saline enriched with [13C]leucine. [13C]leucine added to the serosal side of Ussing chambers showed rapid permeation across TPN-dependent jejunum, suggesting increased transmucosal passage. Single-cell analysis by fluorescence in situ hybridization (FISH)-NanoSIMS demonstrated uptake of [13C]leucine by TPN-associated bacteria, with preferential uptake by Enterobacteriaceae Gavage of small bowel effluent from TPN mice into germ-free, fed mice resulted in a trend toward the proinflammatory TPN phenotype with loss of epithelial barrier function. TPN dependence leads to increased permeation of TPN-derived nutrients into the small intestinal lumen, where they are predominately utilized by Enterobacteriaceae The altered metabolomic composition of the intestinal lumen during TPN promotes dysbiosis.