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Background Although serial transverse enteroplasty (STEP) improves function of dilated short bowel, a significant proportion of patients require repeat surgery. To address underlying reasons for unsuccessful STEP, we compared small intestinal mucosal characteristics between initial and repeat STEP procedures in children with short bowel syndrome (SBS). Methods Fifteen SBS children, who underwent 13 first and 7 repeat STEP procedures with full thickness small bowel samples at median age 1.5 years (IQR 0.7–3.7) were included. The specimens were analyzed histologically for mucosal morphology, inflammation and muscular thickness. Mucosal proliferation and apoptosis was analyzed with MIB1 and Tunel immunohistochemistry. Results Median small bowel length increased 42% by initial STEP and 13% by repeat STEP (p = 0.05), while enteral caloric intake increased from 6% to 36% (p = 0.07) during 14 (12-42) months between the procedures. Abnormal mucosal inflammation was frequently observed both at initial (69%) and additional STEP (86%, p = 0.52) surgery. Villus height, crypt depth, enterocyte proliferation and apoptosis as well as muscular thickness were comparable at first and repeat STEP (p > 0.05 for all). Patients, who required repeat STEP tended to be younger (p = 0.057) with less apoptotic crypt cells (p = 0.031) at first STEP. Absence of ileocecal valve associated with increased intraepithelial leukocyte count and reduced crypt cell proliferation index (p Conclusions No adaptive mucosal hyperplasia or muscular alterations occurred between first and repeat STEP. Persistent inflammation and lacking mucosal growth may contribute to continuing bowel dysfunction in SBS children, who require repeat STEP procedure, especially after removal of the ileocecal valve. Level of evidence Level IV, retrospective study.

Feeding strategies to care for patients who transition from enteral nutrient deprivation while on total parenteral nutrition (TPN) to enteral feedings generally proceed to full enteral nutrition once the gastrointestinal tract recovers; however, an increasing body of literature suggests that a subgroup of patients may actually develop an increased incidence of adverse events, including death. To examine this further, we studied the effects of acute refeeding in a mouse model of TPN. Interestingly, refeeding led to some beneficial effects, including prevention in the decline in intestinal epithelial cell (IEC) proliferation. However, refeeding led to a significant increase in mucosal expression of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), as well as an upregulation in Toll-like receptor 4 (TLR-4). Refeeding also failed to prevent TPN-associated increases in IEC apoptosis, loss of epithelial barrier function, and failure of the leucine-rich repeat-containing G protein-coupled receptor 5-positive stem cell expression. Transitioning from TPN to enteral feedings led to a partial restoration of the small bowel microbial population. In conclusion, while acute refeeding led to some restoration of normal gastrointestinal physiology, enteral refeeding led to a significant increase in mucosal inflammatory markers and may suggest alternative strategies to enteral refeeding should be considered.

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.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.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.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.

Small intestine luminal nutrient sensing may be crucial for modulating physiological functions. However, its mechanism of action is incompletely understood. We used a model of enteral nutrient deprivation, or total parenteral nutrition (TPN), resulting in intestinal mucosal atrophy and decreased epithelial barrier function (EBF). We examined how a single amino acid, glutamate (GLM), modulates intestinal epithelial cell (IEC) growth and EBF. Controls were chow-fed mice, T1 receptor-3 (T1R3)-knockout (KO) mice, and treatment with the metabotropic glutamate receptor (mGluR)-5 antagonist MTEP. TPN significantly changed the amount of T1Rs, GLM receptors, and transporters, and GLM prevented these changes. GLM significantly prevented TPN-associated intestinal atrophy (2.5-fold increase in IEC proliferation) and was dependent on up-regulation of the protein kinase pAkt, but independent of T1R3 and mGluR5 signaling. GLM led to a loss of EBF with TPN (60% increase in FITC-dextran permeability, 40% decline in transepithelial resistance); via T1R3, it protected EBF, whereas mGluR5 was associated with EBF loss. GLM led to a decline in circulating glucagon-like peptide 2 (GLP-2) during TPN. The decline was regulated by T1R3 and mGluR5, suggesting a novel negative regulator pathway for IEC proliferation not previously described. Loss of luminal nutrients with TPN administration may widely affect intestinal taste sensing. GLM has previously unrecognized actions on IEC growth and EBF. Restoring luminal sensing via GLM could be a strategy for patients on TPN.—Xiao, W., Feng, Y., Holst, J. J., Hartmann, B., Yang, H., Teitelbaum, D. H. Glutamate prevents intestinal atrophy via luminal nutrient sensing in a mouse model of total parenteral nutrition.

Tumour necrosis factor-α (TNF-α) has been reported to play a central role in intestinal barrier dysfunction in many diseases; however, the precise role of the TNF-α receptors (TNFRs) has not been well defined using in vivo models. Our previous data showed that enteral nutrient deprivation or total parenteral nutrition (TPN) led to a loss of intestinal epithelial barrier function (EBF), with an associated upregulation of TNF-α and TNFR1. In this study, we hypothesized that TNF-α plays an important role in TPN-associated EBF dysfunction. Using a mouse TPN model, we explored the relative roles of TNFR1 vs. TNFR2 in mediating this barrier loss. C57/BL6 mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Tumour necrosis factor-α receptor knockout (KO) mice, including TNFR1KO, TNFR2KO or TNFR1R2 double KO (DKO), were used. Outcomes included small intestine transepithelial resistance (TER) and tracer permeability, junctional protein zonula occludens-1, occludin, claudins and E-cadherin expression. In order to address the dependence of EBF on TNF-α further, exogenous TNF-α and pharmacological blockade of TNF-α (Etanercept) were also performed. Total parenteral nutrition led to a loss of EBF, and this was almost completely prevented in TNFR1R2DKO mice and partly prevented in TNFR1KO mice but not in TNFR2KO mice. The TPN-associated downregulation of junctional protein expression and junctional assembly was almost completely prevented in the TNFR1R2DKO group. Blockade of TNF-α also prevented dysfunction of the EBF and junctional protein losses in mice undergoing TPN. Administration of TPN upregulated the downstream nuclear factor-B and myosin light-chain kinase (MLCK) signalling, and these changes were almost completely prevented in TNFR1R2DKO mice, as well as with TNF-α blockade, but not in TNFR1KO or TNFR2KO TPN groups. Tumour necrosis factor-α is a critical factor for TPN-associated epithelial barrier dysfunction, and both TNFR1 and TNFR2 are involved in EBF loss. Nuclear factor-B and MLCK signalling appear to be important downstream mediators involved in this TNF-α signalling process.

BACKGROUND Injectable fat emulsions (FEs) are a clinically dependable source of essential fatty acids (FA). ω-6 FA is associated with an inflammatory response. Medium-chain triglycerides (MCT, ω-3 FA), fish oil, and olive oil are reported to decrease the inflammatory response. However, the effect of these lipids on the gastrointestinal tract has not been well studied. To address this, we used a mouse model of parenteral nutrition (PN) and hypothesized that a decrease in intestinal inflammation would be seen when either fish oil and MCT or olive oil were added. METHODS Three FEs were studied in adult C57BL/6 mice via intravenous cannulation: standard soybean-based FE (SBFE), 80% olive oil -supplemented FE (OOFE), or a combination of a soybean oil, MCT, olive oil, and fish oil emulsion (SMOF). PN was given for 7 days, small bowel mucosa-derived cytokines, animal survival rate, epithelial cell (EC) proliferation and apoptosis rates, intestinal barrier function and mucosal FA composition were analyzed. RESULTS Compared to the SBFE and SMOF groups, the best survival, highest EC proliferation and lowest EC apoptosis rates were observed in the OOFE group; and associated with the lowest levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β expression. Jejunal FA content showed higher levels of eicosapentaenoic and docosapentaenoic acid in the SMOF group and the highest arachidonic acid in the OOFE group. CONCLUSION The study showed that PN containing OOFE had beneficial effects to small bowel health and animal survival. Further investigation may help to enhance bowel integrity in patients restricted to PN.

Intestinal resident macrophages (Mϕs) regulate gastrointestinal homeostasis via production of an anti-inflammatory cytokine interleukin (IL)-10. Although a constant replenishment by circulating monocytes is required to maintain the pool of resident Mϕs in the colonic mucosa, the homeostatic regulation of Mϕ in the small intestine (SI) remains unclear. Here, we demonstrate that direct stimulation by dietary amino acids regulates the homeostasis of intestinal Mϕs in the SI. Mice that received total parenteral nutrition (TPN), which deprives the animals of enteral nutrients, displayed a significant decrease of IL-10-producing Mϕs in the SI, whereas the IL-10-producing CD4+ T cells remained intact. Likewise, enteral nutrient deprivation selectively affected the monocyte-derived F4/80+ Mϕ population, but not non-monocytic precursor-derived CD103+ dendritic cells. Notably, in contrast to colonic Mϕs, the replenishment of SI Mϕs and their IL-10 production were not regulated by the gut microbiota. Rather, SI Mϕs were directly regulated by dietary amino acids. Collectively, our study highlights the diet-dependent, microbiota-independent regulation of IL-10-producing resident Mϕs in the SI.

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.

Contains fulltext : 110369.pdf (author's version ) (Open Access) Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires Bcl10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with Bcl10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, Bcl10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that Bcl10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for Bcl10 in NF-kB signaling.

We report on a novel non-enzymatic sensor for hydrogen peroxide (HP) that is based on a biocomposite made up from chitosan (CS), hemoglobin (Hb), and silver nanoparticles (AgNPs). The AgNPs were prepared in the presence of CS and glucose in an ultrasonic bath, and CS is found to act as a stabilizing agent. They were then combined with Hb and CS to construct a carbon paste biosensor. The resulting electrode gave a well-defined redox couple for Hb, with a formal potential of about −0.17 V (vs. SCE) at pH 6.86 and exhibited a remarkable electrocatalytic activity for the reduction of HP. The sensor was used to detect HP by flow injection analysis, and a linear response is obtained in the 0.08 to 250 μM concentration range. The detection limit is 0.05 μM (at S/N = 3). These characteristics, along with its long-term stability make the sensor highly promising for the amperometric determination of HP.

A study of the response behavior of anion to surface plasmon resonance (SPR) based sensor, in which a Poly(pyrrole) (Ppy) modified thin gold film was used as a sensor chip is described. In situ surface plasmon resonance and electrochemical methods were used to investigate the electropolymerization and doping/dedoping processes of thin Ppy film. The electropolymerization of pyrrole was carried out under cyclic voltammetric conditions, and simultaneously monitored by in situ SPR. It has revealed that the transition between the reduced state and oxidized state of the Ppy, corresponding to the doping/dedoping of anions, can lead to very distinct changes in SPR signal at a fixed angle of incident laser beam. Furthermore, it has demonstrated that the concentration, the charge and size of anions, as well as the film thickness play important roles in the ingress/egress process of anions. Based on this, this combination of experimental approaches can be used to detect Cl−. SPR signal exhibited a good linear relationship with the concentration of Cl−.

Total parenteral nutrition (TPN) results in a number of derangements to the intestinal epithelium, including a loss of epithelial barrier function (EBF). As TPN supplemented with glutamine has been thought to prevent this loss, this article further defined the impact of glutamine on EBF, and investigated potential mechanisms that contributed to the preservation of EBF. C57BL/6J male mice were randomized to enteral nutrition (control), TPN, or TPN supplemented with glutamine (TPN+GLN). Changes in intraepithelial lymphocyte (IEL)-derived cytokine expression were measured, and EBF was assessed with electrophysiologic methods and assessment of junctional protein expression. TPN resulted in a significant decline in EBF, and this loss of EBF was significantly prevented in the TPN+GLN group. Coincident with these changes was a loss of intraepithelial lymphocyte (IEL, mucosal lymphocyte)-derived IL-10 and increase in interferon-gamma (IFN-gamma) expression, and a decline in IEL numbers in the TPN group. A prevention in the increase in IFN-gamma and decline in IL-10 expression was seen in the TPN+GLN group. To determine the mechanism responsible for these glutamine-associated cytokine changes, we tested whether blockade of the IL-7 signaling pathway between epithelial cells (EC) and IEL would prevent these changes; however, blockade failed to influence IEL-derived cytokine changes. Glutamine-supplemented TPN leads to a specific IEL-derived cytokine profile, which may account for the preservation of EBF; and such action may be due to a direct action of glutamine on the IEL.

Total parenteral nutrition (TPN) leads a loss of epithelial barrier function, decline in epithelial cell (EC) proliferation, and decreased expression of E-cadherin. As a large portion of intracellular β-catenin is tightly associated with E-cadherin, we hypothesized that the loss of E-cadherin would result in a redistribution of intracellular β-catenin, and could be a contributing mechanism for this TPN-associated loss of EC proliferation. An assessment of small bowel epithelium was performed in mice given either enteral nutrition (Control) or intravenous nutrition (TPN). TPN significantly down-regulated E-cadherin and β-catenin expression, and resulted in a loss of a colocalization of these factors. TPN also up-regulated phosphorylated (p)-β-catenin (Ser31/33,Thr41) and down-regulated (p)-β-catenin(Ser552) expression. To further address mechanisms driving this, we observed a significant decrease in the abundance of p-AKT and p-GSK3β expression, and an associated decline in tcf-4 transcription factors (cyclin D1, c-myc and Axin2), as well as a loss of EC proliferation by 7 days. To address whether the mechanism driving these changes was the absence of nutritional factors, glutamine was added to the TPN solution. This resulted in a partial restoration of β-catenin expression and EC proliferation, suggesting that an alteration in nutrient delivery may affect many of these changes. Based on these findings, the loss of EC proliferation with TPN may well be due to a loss of total β-catenin, as well as a concomintant change in the differential expression of β-catenin phosphorylation sites, and a reduction in β-catenin mediated tcf-4 transcription. This potential pathway may well explain many of the findings of mucosal atrophy associated with TPN.

Loss of intestinal epithelial barrier function (EBF) is a major problem associated with total parenteral nutrition (TPN) administration. We have previously identified intestinal intraepithelial lymphocyte (IEL)-derived interferon-γ (IFN-γ) as a contributing factor to this barrier loss. The objective was to determine whether other IEL-derived cytokines may also contribute to intestinal epithelial barrier breakdown. C57BL6J male mice received TPN or enteral nutrition (control) for 7 days. IEL-derived interleukin-10 (IL-10) was then measured. A significant decline in IEL-derived IL-10 expression was seen with TPN administration, a cytokine that has been shown in vitro to maintain tight junction integrity. We hypothesized that this change in IEL-derived IL-10 expression could contribute to TPN-associated barrier loss. An additional group of mice was given exogenous recombinant IL-10. Ussing chamber experiments showed that EBF markedly declined in the TPN group. TPN resulted in a significant decrease of IEL-derived IL-10 expression. The expression of several tight junction molecules also decreased with TPN administration. Exogenous IL-10 administration in TPN mice significantly attenuated the TPN-associated decline in zonula occludens (ZO)-1, E-cadherin, and occludin expression, as well as a loss of intestinal barrier function. TPN administration led to a marked decline in IEL-derived IL-10 expression. This decline was coincident with a loss of intestinal EBF. As the decline was partially attenuated with the administration of exogenous IL-10, our findings suggest that loss of IL-10 may be a contributing mechanism to TPN-associated epithelial barrier loss.

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.

Total parenteral nutrition (TPN), a necessary treatment for patients who cannot receive enteral nutrition, is associated with infectious complications due in part to a loss of intestinal epithelial barrier function (EBF). Using a mouse model of TPN, with enteral nutrient deprivation, we previously demonstrated an increase in mucosal interferon-γ and tumor necrosis factor-α; these cytokine changes are a major mediator driving a reduction in epithelial tight junction (TJ) protein expression. However, the exact ultrastructural changes to the intestinal epithelial barrier have not been previously described. We hypothesized that TPN dependence results in ultrastructural changes in the intestinal epithelial TJ meshwork. C57BL/6 mice underwent internal jugular venous cannulation and were given enteral nutrition or TPN with enteral nutrient deprivation for 7 days. Freeze-fracture electron microscopy was performed on ileal tissue to characterize changes in TJ ultrastructure. EBF was measured using transepithelial resistance and tracer permeability, while TJ expression was measured via Western immunoblotting and immunofluorescence staining. While strand density, linearity, and appearance were unchanged, TPN dependence led to a mean reduction in one horizontal strand out of the TJ compact meshwork to a more basal region, resulting in a reduction in meshwork depth. These findings were correlated with the loss of TJ localization of claudin-4 and tricellulin, reduced expression of claudin-5 and claudin-8, and reduced ex vivo EBF. Tight junction ultrastructural changes may contribute to reduced EBF in the setting of TPN dependence.

Recent studies suggest a close interaction between epidermal growth factor (EGF) and TLR signaling in the modulation of intestinal epithelial cell (IEC) proliferation; however, how these signaling pathways adjust IEC proliferation is poorly understood. We utilized a model of total parenteral nutrition (TPN), or enteral nutrient deprivation, to study this interaction as TPN results in mucosal atrophy due to decreased IEC proliferation and increased apoptosis. We identified the novel finding of decreased mucosal atrophy in TLR4 knockout (TLR4KO) mice receiving TPN. We hypothesized that EGF signaling is preserved in TLR4KO-TPN mice and prevents mucosal atrophy. C57Bl/6 and strain-matched TLR4KO mice were provided either enteral feeding or TPN. IEC proliferation and apoptosis were measured. Cytokine and growth factor abundances were detected in both groups. To examine interdependence of these pathways, ErbB1 pharmacologic blockade was used. The marked decline in IEC proliferation with TPN was nearly prevented in TLR4KO mice, and intestinal length was partially preserved. EGF was significantly increased, and TNF-α decreased in TLR4KO-TPN versus wild-type (WT)-TPN mice. Apoptotic positive crypt cells were 15-fold higher in WT-TPN versus TLR4KO-TPN mice. Bcl-2 was significantly increased in TLR4KO-TPN mice, while Bax decreased 10-fold. ErbB1 blockade prevented this otherwise protective effect in TLR4KO-sTPN mice. TLR4 blockade significantly prevented TPN-associated atrophy by preserving proliferation and preventing apoptosis. This is driven by a reduction in TNF-α abundance and increased EGF. Potential manipulation of this regulatory pathway may have significant clinical potential to prevent TPN-associated atrophy.—Freeman, J. J., Feng, Y., Demehri, F. R., Dempsey, P. J., Teitelbaum, D. H. TPN-associated intestinal epithelial cell atrophy is modulated by TLR4/EGF signaling pathways.

Tunneling planer Hall (TPH) effect in Ni81 Fe19/Al2O3/NixFe1−x trilayer junction is different from general planer Hall effect in single-layer film or two-layer junction. This effect concerns the spin-polarized transport, so that the TPH voltage depends on the angle between magnetic vectors of two ferromagnetic layers. The TPH effect is reported to be influenced by composition and magnetic properties of FM layers and the thickness of the insulating layer.

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.

Tumour necrosis factor-α (TNF-α) has been reported to play a central role in intestinal barrier dysfunction in many diseases; however, the precise role of the TNF-α receptors (TNFRs) has not been well defined using in vivo models. Our previous data showed that enteral nutrient deprivation or total parenteral nutrition (TPN) led to a loss of intestinal epithelial barrier function (EBF), with an associated upregulation of TNF-α and TNFR1. In this study, we hypothesized that TNF-α plays an important role in TPN-associated EBF dysfunction. Using a mouse TPN model, we explored the relative roles of TNFR1 vs. TNFR2 in mediating this barrier loss. C57/BL6 mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Tumour necrosis factor-α receptor knockout (KO) mice, including TNFR1KO, TNFR2KO or TNFR1R2 double KO (DKO), were used. Outcomes included small intestine transepithelial resistance (TER) and tracer permeability, junctional protein zonula occludens-1, occludin, claudins and E-cadherin expression. In order to address the dependence of EBF on TNF-α further, exogenous TNF-α and pharmacological blockade of TNF-α (Etanercept) were also performed. Total parenteral nutrition led to a loss of EBF, and this was almost completely prevented in TNFR1R2DKO mice and partly prevented in TNFR1KO mice but not in TNFR2KO mice. The TPN-associated downregulation of junctional protein expression and junctional assembly was almost completely prevented in the TNFR1R2DKO group. Blockade of TNF-α also prevented dysfunction of the EBF and junctional protein losses in mice undergoing TPN. Administration of TPN upregulated the downstream nuclear factor-B and myosin light-chain kinase (MLCK) signalling, and these changes were almost completely prevented in TNFR1R2DKO mice, as well as with TNF-α blockade, but not in TNFR1KO or TNFR2KO TPN groups. Tumour necrosis factor-α is a critical factor for TPN-associated epithelial barrier dysfunction, and both TNFR1 and TNFR2 are involved in EBF loss. Nuclear factor-B and MLCK signalling appear to be important downstream mediators involved in this TNF-α signalling process.

Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88−/− mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88−/− mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80+CD11b+CD11cdull/− macrophage–derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88−/− TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.

Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of β-catenin and glycogen synthase kinase-3β (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis.

Glial cell line-derived trophic factor (GDNF) is a peptide with pleiotropic survival and growth-promoting effects on neurons. We found that intraspinal injection of a non-replicating herpes simplex virus-based vector coding for GDNF 2 h after blunt trauma to the thoraco-lumbar spinal cord produced sustained improvement in motor behavioral outcomes up to 5 weeks following injury. The improvement in behavior correlated with an increase in synaptophysin and glutamic acid decarboxylase (GAD) in the spinal cord at the level of injury. Addition of recombinant GDNF protein to primary spinal cord neurons in-vitro resulted in enhanced neurite growth and a marked increase in protein levels of GAD65 and GAD67, synapsin I and synaptophysin. GDNF-mediated increases in GAD and the synaptic markers were blocked by the MEK inhibitor UO126, but not by the phosphoinositide 3-kinase inhibitor LY294002. These results suggest that GDNF, acting through the MEK-ERK pathway enhances axonal sprouting, synaptic connectivity, and GABAergic neurotransmission in the spinal cord, that result in improved behavioral outcomes after spinal cord contusion injury.

Total parenteral nutrition (TPN), or the complete absence of enteral nutrients, is commonly used in a clinical setting. However, a major consequence of TPN administration is the development of mucosal atrophy and a loss of epithelial barrier function (EBF); and this loss may lead to an increase in clinical infections and septicemia. Our laboratory has investigated the mechanism of this TPN-associated loss of EBF using a mouse model. We have demonstrated that the mucosal lymphoid population significantly changes with TPN, and leads to a rise in interferon gamma (IFN-gamma) and decline in interleukin-10 (IL-10) expression-both of which contribute to the loss of EBF. Associated with these cytokine changes is a dramatic decline in the expression of tight junction and adherens junction proteins. This article discusses the potential mechanisms responsible for these changes, and potential strategies to alleviate this loss in EBF.

Total parenteral nutrition (TPN) results in intestinal mucosal atrophic changes due to an absence of enteral nutrition; however, the mechanisms responsible for this are not fully understood. It has been shown that bone morphogenetic protein (BMP) activation inhibits intestinal epithelial cell (EC) proliferation. Therefore, we hypothesized that the BMP pathway could be upregulated by TPN. To address this, we randomly assigned mice to receive TPN or to be enterally fed (control) for 7 d. Mucosal EC isolates were harvested from the entire length of small intestine for RNA and protein measurements. Full-thickness, mid-small bowel was processed for histological examination. TPN increased the abundance of BMP2, BMP4, and BMP type II receptor at the RNA and protein levels. Phosphorylation of Smad1, Smad5, and Smad8 also was greater in the TPN group than in the control, which helped to confirm activation of this pathway. Interestingly, the TPN and control groups did not differ in the mRNA expression of the extracellular soluble bmp antagonists, noggin, gremlin, chordin, or follistatin. Compared to the control group, the expression of c-Myc (cellular myelocytomatosis) mRNA was lower, whereas the level of p21(WAF1/CIP1) was greater, in the TPN group. Because the BMP family may function through suppression of Wnt-beta-catenin signaling, this pathway was also examined. mRNA expression of Wnt 3, Wnt5a, and the Wnt receptor Lrp5 were lower in the TPN group compared to controls. The results suggest that the BMP signaling pathway may be involved in the development of intestinal mucosal atrophy due to TPN administration.

Notch signaling is required for the maintenance of intestinal epithelial proliferation. Dysfunction of this signaling pathway is associated with the loss of proliferated crypt epithelial cells.The aim of this study was to investigate the role of Notch signaling in small bowel resection (SBR)-associated crypt epithelial cell proliferation.Male Sprague-Dawley rats were subjected to sham operation (bowel transection and reanastomosis) or 70% mid-SBR. Intestinal tissue samples were collected at 0.5, 1, 6, 12, 24, 72, and 168 h after operation. The expression of Notch pathway mRNAs and proteins was analyzed using RT-PCR and Western blot. The expression of the Notch pathway proteins Jagged-1, NICD and Hes-1 was also determined through immunohistochemical staining using day 3 postoperative intestinal tissues. The degree of crypt epithelial cell proliferation was evaluated using the immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Furthermore, IEC-6 cells were used to examine the function of the Jagged-1 signaling system.SBR led to increased crypt epithelial cell proliferation and increased expression of Jagged-1 and Hes-1 mRNA and protein along with cleaved Notch-1. Immunohistochemical staining showed that Jagged-1, cleaved Notch-1 and Hes-1 colocalized in the same proliferated crypt epithelial cell population. Recombinant Jagged-1 significantly stimulated the proliferation of IEC-6 cells. Transient upregulation of Jagged-2 expression was found 1 h after SBR, and it was accompanied by cleaved Notch-1 and Hes-1 upregulation.The Jagged-1/Notch-1/Hes-1 signaling pathway is involved in intestinal adaptation through increasing crypt epithelial cell proliferation.

Introduction: Both GLP-2 and EGF can induce intestinal epithelial cell (IEC) growth. We previously showed that both growth factors (GFs) decrease with total parenteral nutrition (TPN) administration, and both exogenous GFs can prevent TPN-associated atrophy. However, the precise mechanisms driving this protective effect remain incompletely understood. This study investigated the role of PI3K/AKT and Wnt/β-catenin signaling mediating EC responses with TPN. Methods: C57BL/6 mice received enteral nutrition (Control) or TPN for 7 days. Wildtype, IEC-ErbB1KO and IEC-pik3r1KO (class IA subunit of PI3K) mice were treated with exogenous EGF or GLP-2. IEC proliferation was measured by PCNA staining. Nuclear and cytoplasm fractions were isolated from jejunal crypt cells and target proteins were measured by western blot. Results: TPN resulted in loss of both GFs expression and decreased EC proliferation. Exogenous EGF or GLP-2 restored IEC proliferation. We then explored differential mechanisms for EGF/GLP-2 responses. ErbB1: Both GFs actions weremarkedly decreased in IEC-ErbB1KOTPNmice, as well as in gefitinib (ErbB1 antagonist) treated TPN mice, demonstrating ErbB1-dependency for these GFs responses. P-AKT: Both GFs restored cytoplasmic p-Akt expression, but only EGF induced sustained nuclear pAKT expression. p-AKT expression was blocked in IEC-pi3kr1KO mice. However, in IECpi3kr1KO mice, GLP-2 induced EC proliferation but EGF. This indicated that GLP-2 acted by a PI3K-pAKT independent mechanism.Wnt-β-catenin: Increased cytoplasmic p-β-catenin (ser33/37) expression was found in TPNmice and both GFs decreased β-catenin ubiquination by preventing this increase. Nuclear p-β-catenin (ser552) expression was down-regulated in TPNmice and both GFs prevented this decline. Cytoplasmic p-GSK3βwas down-regulated in TPN mice and GLP-2 treatment prevented this decline but EGF did not. However, in IEC-pi3kr1KO TPN mice, nuclear p-β-catenin (ser552) and total cytoplasm β-catenin expression were maintained by GLP-2, whereas both β-catenin levels were still diminished in the presence of EGF. Stem cell: A marked loss of LGR5+ crypt cells (1% vs 20% , TPN vs Control) were observed in Lgr5-EGFP knock-in mice with TPN administration. EGF failed to prevent this loss, but LGR5+ was partially protected with GLP-2 (10%). Interestingly, although EC proliferation decreased in GLP-2 treated mice given gefinitib, LGR5+ crypts (10%) were still observed. Conclusions: Reduced EGF and GLP-2 signaling contributed to TPN-associated loss of EC proliferation. Both GFs required an intact ErbB1 signaling for their responses. However, EGF was dependent on PI3K/P-AKT signaling to promote EC proliferation, whereas GLP-2 was partially independent of the PI3K/p-AKT pathway to modulate EC proliferation and appeared to mediate proliferation viaWnt-β-catenin signaling.