Neonatal maternal deprivation (NMD) increases gut paracellular permeability (GPP) through mast cells and nerve growth factor (NGF), and modifies corticotrophin-releasing factor (CRF) and corticosterone levels. CRF, corticosterone and mast cells are involved in stress-induced mucosal barrier impairment. Consequently, this study aimed to specify whether corticosteronaemia and colonic expression of both preproCRF and CRF are modified by NMD, and to determine if altered expression may participate in the elevated GPP in connection with NGF and mast cells. Male Wistar rat pups were either separated from postnatal days 2-14, or left undisturbed with their dam. At 12 weeks of age, adult rats were treated with mifepristone (an antagonist of corticoid receptors), α-helical CRF (9-41) (a non-specific CRF receptor antagonist), or SSR-125543 (CRF-R 1 receptor antagonist). We also determined corticosteronaemia and both colonic preproCRF and CRF expression. Then, control rats were treated by CRF, doxantrazole (mast cell stabilizer), BRX-537A (a mast cell activator) and anti-NGF antibody. NMD did not modify colonic CRF level but increased colonic preproCRF expression and corticosteronaemia. Peripheral CRF, via CRF-R 1 receptor, but not corticosterone, was involved in the elevated GPP observed in these rats, through a mast-cell-mediated mechanism, since the increase of GPP induced by exogenous CRF was abolished by doxantrazole. Anti-NGF antibody treatment also reduced the elevated GPP induced by CRF or BRX-537A. CRF acts through CRF-R 1 receptors to stimulate NGF release from mast cells, which participates in the elevated GPP observed in NMD adult rats. This suggests that early traumatic experience induced neuro-endocrine dysfunction, involved in alterations of gut mucosal barrier.

Sepsis is associated with bacterial translocation (BT)and changes in colonic paracellular permeability (CPP), but the link between these effects is unknown. The present study aimed to identify whether changes in CPP after lipopolysaccharide (LPS) administration triggers BT, colonic inflammation, visceral pain, and sickness behavior and to evaluate the role of myosin light chain kinase (MLCK) in colonocyte cytoskeleton contraction. Rats received the MLCK inhibitor ML-7 alone or combined with LPS. CPP was measured for 6 hours after administration. Visceral pain, food intake, BT, electron microscopy of tight junctions of colonocytes, cytokine levels, and Western blotting of phosphorylated MLC from colonic mucosa were assessed in a time range of 0 to 3 hours after treatment. Sepsis increased CPP at 0 to 6 hours after LPS and associated with tight junction morphological changes, increased MLC phosphorylation, and mucosal release of proinflammatory cytokines. Massive BT, visceral hyperalgesia, and reduced food intake were also observed. Addition of ML-7 prevented all LPSinduced effects, except for changes in food intake. In conclusion, LPS-mediated effects on CPP include gut inflammation, BT, and visceral hyperalgesia. Inhibition of MLCK-dependent colonocyte cytoskeleton contraction by ML-7 prevents the LPS-induced alterations of CPP and its subsequent effects. The gastrointestinal mucosal barrier plays a pivotal role in the body's protection against luminal pathogens and antigenic molecules. The impairment of the gastrointestinal barrier function with massive bacterial translocation (BT) occurs during sepsis, both in human and in animal models. [1][2][3][4][5] In these circumstances, bacteria and their lysis products, such as lipopolysaccharide (LPS), gain access to portal and systemic circulations, producing critical deleterious effects. Sepsis is the most common cause of death for hospitalized patients. The gastrointestinal barrier includes secreted mucus and the epithelial cell layer itself, which prevent BT of intestinal flora through both transcellular and paracellular pathways.7 Tight junction (TJ) opening is the major limiting factor of the paracellular pathway. Examination of isolated human and rodent small intestine cells and cell lines have shown that TJ opening is driven by myosin light chain (MLC) phosphorylation, which depends on myosin light chain kinase (MLCK) activation. 8,9 MLC phosphorylation occurs within the perijunctional actomyosin ring and co-localizes with the TJ.9 Studies using an inducible active MLCK have shown that this activity is sufficient to activate the downstream events necessary for TJ regulation. 10Moreover MLCK-mediated regulation of TJ opening appears to be a common intermediate in a variety of physiological and pathophysiological pathways related to altered paracellular permeability both in vitro and in vivo. 10 -12 For example, enteropathogenic Escherichia coli infection causes diarrhea in children. An in vitro model has demonstrated that enteropathogenic E. coli dramatically ...

Non-technical summary The enteric nervous system (ENS) is an autonomous nervous system integrated along the gut that controls major gastrointestinal (GI) functions such as motility. Increasing data have demonstrated that nutritional factors can modulate the ENS phenotype and consequently impact upon GI functions. Western diet is central in the development of obesity but surprisingly no study has characterized its impact upon ENS phenotype and functions. We show that Western diet-induced obesity (DIO) prevented age-associated loss in a specific population of enteric neurons leading to an acceleration of gastric emptying. In addition, we showed that neuroprotective effects of DIO likely involved leptin and glial cell line-derived neurotrophic factor (GDNF). This is the first study demonstrating an impact of DIO upon the ENS. These DIO-induced neuroplastic changes in the ENS could be involved in the physiopathology of obesity.Abstract Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca 2+ response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

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Key points• We investigated altered colonic functions in high fat diet-induced obesity in pre-diabetic mice.• After feeding an adipogenic diet for 12 weeks, accelerated colonic transit was associated with upregulation and enhanced signalling of acetylcholine and serotonin, two key mediators in the enteric nervous system (ENS). Importantly, these changes occurred without signs of impaired mucosal integrity or immune cell infiltration in the gut wall. Neuronal sensitization was not observed in obese mice fed for 4 weeks.• Weight gain correlated positively with the level of adipocyte markers and the degree of neuronal sensitization.• We conclude that enhanced neural excitation in the colon by acetylcholine and serotonin is a key feature of a later phase of obesity and is involved in altered ENS functions and abnormal colonic transit.• Furthermore, the results suggest that the occurrence of altered gut functions in obesity is independent of inflammation in the gut wall.Abstract Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.

BackgroundSuboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1].Methodology/Principal FindingsIn this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-γ and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-γ and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-γ and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-γ; and iv) anti-melanoma specific cytotoxic cells.Conclusions/SignificanceThese data indicate that pharmacological treatment of melanoma cell lines with IFN-γ and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

LPS-induced endotoxemia is associated with gut immune stimulation, mucosal inflammation, colonic paracellular permeability (CPP) alteration, and it promotes bacterial translocation (BT). Gut permeability increase linked to LPS promotes mucosal barrier dysfunction resulting to BT. However, the mechanisms involved in these alterations remain unknown. We aimed to evaluate the role of colonic mucosal mast cells and luminal serine protease activity (PA) in the alterations of CPP and BT induced by LPS. Rats receiving doxantrazole, a mast cell stabilizer, combined or not with LPS from Escherichia coli and CPP as well as BT were evaluated after each treatment. Mucosal mast cell activation was assessed by histological methods and by rat mast cell protease 2 level measurement in colonic content. Colonic luminal PA and mucosal inflammation (myeloperoxidase activity) were biochemically determined. In addition, the ability of luminal contents to act on CPP was evaluated in vitro in Ussing chambers. Peripheral administration of LPS promoted mast cell degranulation and increased CPP, BT, mucosal myeloperoxidase activity as well as rat mast cell protease 2 levels, and PA in colonic content. LPS-induced CPP increase and BT were prevented by doxantrazole. In vitro, exposure of the apical side of colonic tissues with supernatants from colonic contents of LPS-treated rats increased CPP. This effect was blocked by the serine protease inhibitor soybean trypsin inhibitor. Our data bring evidence of a key role of mucosal mast cells in LPS-induced increase of CPP and BT through the release of serine proteases into the colonic lumen.