In vitro performance of spacers for aerosol delivery during adult mechanical ventilation.

BACKGROUND: During mechanical ventilation, different aerosol generators are employed with various interfaces. The objective of this study was to evaluate the performance of a range of spacers, including a new device called Combihaler® designed for connection with both nebulizers and pressurized Metered-Dose Inhalers (pMDIs). METHODS: To assess the spacers, we used a ventilator and the Dual Adult Training and Test Lung (model 5600i, Michigan Instruments). Ventilation parameters were measured with and without spacers in volume-controlled and pressure-controlled mode. A filter was placed at the end of the endotracheal tube to measure aerosol delivery. Amikacin (1 g/8 mL) and salbutamol (5 mg/5 mL) were nebulized with an Aeroneb Solo® connected to its T-adapter or the Combihaler® spacer. Salbutamol (100 µg/actuation with 10 actuations) and beclomethasone (250 µg/actuation with 10 actuations) were delivered with a pMDI connected to a Minispacer®, an ACE® spacer, or a Combihaler® spacer. Drug delivery measurements were performed in volume-controlled mode in dry and humidified conditions. Drug deposits on the filter were assayed. RESULTS: The use of spacers and the T-adapter did not change the ventilation parameters (p>0.9). Aerosol delivery of salbutamol and Amikacin by nebulization increased up to three-fold with the Combihaler® compared with the T-adapter in humidified and nonhumidified conditions (p<0.05). Aerosol delivery of salbutamol and beclometasone by pMDI increased up to three-fold with the Combihaler® and the ACE® spacer compared with the Minispacer® in humidified and nonhumidified conditions (p<0.05). Aerosol delivery by pMDIs and vibrating mesh nebulizers using either a T-adapter or spacers was reduced by up to 62.5% in a humidified circuit compared with a nonhumidified circuit. CONCLUSION: Aerosol delivery via pMDIs and vibrating mesh nebulizers is greater with large spacers (Combihaler® and ACE®) than with smaller spacers (Minispacer®) or a T-adapter, in both humidified and nonhumidified conditions. In humidified conditions, the aerosol delivery decreased with all spacers.

Methotrexate modulates tight junctions through NF-κB, MEK, and JNK pathways.

OBJECTIVES: Chemotherapy often induces intestinal mucositis, which is associated with an increase in intestinal permeability; however, underlying mechanisms remain incompletely understood. Thus, we aimed to study the regulation of 3 tight junction (TJ) proteins, claudin-1, occludin, and zonula occludens-1, after anticancer treatment. METHODS: Methotrexate (MTX) was subcutaneously injected for 3 consecutive days in Sprague-Dawley rats to induce intestinal mucositis and was applied on Caco-2 cell monolayers. TJ protein expression and cellular distribution were studied by Western blot and microscopy, respectively. In Caco-2 cells, the paracellular permeability was evaluated by both transepithelial electrical resistance and flux of fluorescein isothiocyanate-dextran marker. Cytokine production and signaling pathways were also assessed. RESULTS: In MTX-treated rats, the cellular distribution of the 3 TJ proteins was altered and claudin-1 and occludin expression was reduced during the acute phase of mucositis compared with controls. During the recovery phase, these parameters were restored. In vitro, MTX treatment led to an increase in proinflammatory cytokine production at the apical side but did not affect Caco-2 cell apoptosis and necrosis. Increase in paracellular permeability was associated with altered occludin and zonula occludens-1 expression and cellular distribution. All of these alterations were prevented by MEK1 and 2, JNK, and NF-κB inhibitors. CONCLUSIONS: MTX treatment induced an increase in intestinal permeability partially related to alteration of TJs protein expression and cellular distribution that may be mediated by MAPK and NF-κB pathways. These are potential targets to limit the adverse effects of chemotherapy.

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways.

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6 h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4 mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-(2)H(3)]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Intestinal inflammation influences α-MSH reactive autoantibodies: relevance to food intake and body weight.

Autoantibodies reacting with alpha-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are involved in regulation of feeding. In this work we studied if intestinal inflammation (mucositis) may influence α-MSH autoantibodies production relevant to food intake and body weight. Mucositis and anorexia were produced in Sprague-Dawley rats by methotrexate (MTX, 2.5mg/kg/day, for three days, subcutaneously). Plasma levels of total IgG and of α-MSH autoantibodies were measured during and after MTX-induced mucositis and were compared with pair-fed and ad libitum-fed controls. Effects of intraperitoneal injections of rabbit anti-α-MSH IgG (3 or 10 µg/day/rat) on MTX-induced anorexia and on plasma α-MSH peptide concentration were separately studied. Here we show that in MTX rats, intestinal mucositis and anorexia were accompanied by decreased plasma levels of both total IgG and of α-MSH autoantibodies while refeeding was characterized by their elevated levels. In spite of similar food intake in MTX and pair-fed rats, recovery of body weight was delayed by at least 1 week in the MTX group. During refeeding and body weight deficit in MTX rats, α-MSH IgG autoantibody levels correlated negatively with food to water intake ratios. Injections of anti-α-MSH IgG induced a dose-dependent attenuation of food intake and body weight regain in MTX-treated rats accompanied by increased concentrations of α-MSH peptide which correlated positively with plasma levels of α-MSH autoantibodies. These data show that intestinal inflammation, independently from food restriction, affects general humoral immune response which may influence food intake and body weight control via modulation of α-MSH plasma concentration by α-MSH reactive autoantibodies.

Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturated fatty acids in intestinal microvascular endothelium.

BACKGROUND & AIMS: The role of endothelial cells in inflammatory bowel disease has been recently emphasized. Endothelial activation and expression of adhesion molecules are critical for leukocytes recruitment into the inflammatory wall. Compelling evidence demonstrated anti-inflammatory effects of long chain n-3 PUFA in inflammatory models. We previously showed that long chain n-3 PUFA (EPA and DHA) inhibited inflammatory response in epithelial and dendritic cells. As long chain n-3 PUFA treatment led to a decreased expression of adhesion molecules in endothelial cells from other organs, we have now investigated their effect on intestinal endothelial cells in vitro and in colitic rats. METHODS: In vitro study: Primary culture of human intestinal microvascular endothelial cells (HIMEC) were pre-treated with DHA and then incubated with IL-1ß. In vivo study: Colitis was induced in 2 groups at day0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS). Rats received by gavage either fish oil, rich in EPA and DHA (TNBS+n-3) or an isocaloric isolipidic oil formula for 14 days. RESULTS: DHA led to a decreased VCAM-1, TLR4, cyclooxygenase-2 and VEGFR2 expression and a decreased production of IL-6, IL-8 and GM-CSF and a reduced production of PGE(2) and LTB(4) (p < 0.001) in IL-1ß-induced HIMEC. Similarly, dietary intervention with fish oil rich in EPA and DHA significantly decreased colon production of PGE(2) and LTB(4,) endothelial VCAM-1 and VEGFR2 in rats with colitis. CONCLUSIONS: Data obtained from in vitro and in vivo studies reveal a potential anti-angiogenic role of long chain n-3 PUFA in intestinal endothelial cells. This protective effect of long chain n-3 PUFA may partly explain the observed benefit of dietary intake of long chain n-3 PUFA in IBD development.

Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum1,3.

BACKGROUND: Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. OBJECTIVE: We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. DESIGN: Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 µmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. RESULTS: Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/µg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/ß, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. CONCLUSION: Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/ß-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.

Gastric electrical stimulation increases ghrelin production and inhibits catecholaminergic brainstem neurons in rats.

Gastric electrical stimulation (GES) is a new therapeutic option for functional dyspepsia and gastroparesis. In addition to ameliorating nausea and vomiting, GES results in improved appetite which is not always associated with accelerated gastric emptying. To explore the central and peripheral factors underlying GES-associated improvement of appetite we developed a GES model in anaesthetized Wistar rats. During laparotomy, two electrodes were implanted into the stomach and high-frequency low-energy GES (14 Hz, 5 mA) was applied. The effects of 1 h GES were compared with sham stimulation. After GES, c-Fos expression was increased in the mucosal and submucosal layers of the stimulated area (174%). In the stomach, GES increased ghrelin mRNA (178%) and doubled the number of ghrelin-positive cells, resulting in elevated plasma levels of ghrelin (2.3 ± 0.2 vs. 1.6 ± 0.2 ng/mL). In the arcuate nucleus of the hypothalamus, GES increased c-Fos (277%) and agouti-related protein (AgRP) mRNA expression (135%). GES reduced the number of c-Fos-positive cells throughout the nucleus of the solitary tract (between 93 and 75% from rostral to caudal levels) including catecholaminergic neurons (81% at caudal level). Gastric emptying, plasma glucose and heart rate variability were not affected by GES. This study shows that GES may improve appetite via stimulation of main orexigenic pathways, including ghrelin production in the stomach and AgRP in the hypothalamus, as well as by reducing the activity of catecholaminergic brainstem neurons.

Chemotherapy-induced anorexia is accompanied by activation of brain pathways signaling dehydration.

BACKGROUND AND AIMS: Cancer chemotherapy is accompanied by anorexia and mucositis. To clarify the mechanisms of chemotherapy-induced anorexia, we studied the expression of c-fos and appetite-regulating neuropeptidergic and inflammatory mediators in the hypothalamus of rats treated with methotrexate (MTX). METHODS: Sprague-Dawley rats received MTX (2.5mg/kg, subcutaneously) on three consecutive days and were compared with ad libitum- and pair-fed control rats five days after the first injection. RESULTS: MTX administration inhibited food and water intake and induced lean and fat mass losses. MTX also induced mucositis and diarrhea without changes in plasma osmolality. Pair-fed rats lost a similar amount of body weight but had no mucositis or diarrhea. Increased number of c-fos positive hypothalamic vasopressin neurosecretory neurons as well as numerous c-fos positive cells in the subfornical organ and in the organum vasculosum of the lamina terminalis were found in MTX-treated as compared to control or pair-fed rats. In both MTX and pair-fed rats, a decrease of hypothalamic proopiomelanocortin mRNA expression and low plasma levels of interleukin-1ß (IL-1ß) were found reflecting probably the energy deficit. No significant changes of IL-1ß mRNA expression and intensity of microglial staining in the hypothalamus were found in MTX-treated rats. CONCLUSION: The pattern of c-fos expression in the hypothalamus during MTX treatment is similar to that seen with systemic dehydration, which is known to cause anorexia. No evidence of inflammatory origin of anorexia was found, suggesting that chemotherapy accompanied by mucositis and diarrhea may cause anorexia associated with systemic dehydration.

A diet containing whey protein, free glutamine, and transforming growth factor-beta ameliorates nutritional outcome and intestinal mucositis during repeated chemotherapeutic challenges in rats.

Anticancer chemotherapy often induces side effects such as mucositis. Recent data suggest that a diet, Clinutren Protect (CP), containing whey proteins, glutamine, and transforming growth factor-beta (TGFbeta)-rich casein limits intestinal mucositis and improves recovery after a single methotrexate (MTX) challenge in rats. Chemotherapy consists of alternating periods of treatment and rest. Thus, our study evaluated the effects of CP on nutritional outcome and intestinal mucositis in rats receiving repeated chemotherapeutic challenges. Thirty-six Sprague-Dawley rats received 3 cycles of MTX at 8-d intervals. Rats had free access to CP or control diet (Co) from 7 d before the first MTX injection until the end of the experiment at d 27. In Co, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein. L-Glutamine was replaced by L-alanine. Body composition was assessed by dual energy X-ray absorptiometry. Before MTX challenges, food intake and body weight were similar in both groups but became higher during MTX challenges in CP (P < 0.05). Fat mass decreased similarly in both groups. In contrast, the decrease of fat free mass between d -1 and d 27 was less pronounced in the CP group (-9.5 g) than in the Co group (-57.2 g) (P < 0.05). The intestinal damage score was lower in the CP group (0.6 +/- 0.3 vs. 2.1 +/- 0.6; P < 0.05). Fecal IgA increased over time in the CP group (P < 0.05) but not in the Co group. A diet containing whey proteins, glutamine, and TGFbeta improves nutritional outcome by limiting the reduction of fat free mass and reduces intestinal mucositis during repeated chemotherapeutic challenges in rats.

A diet containing whey protein, glutamine, and TGFbeta modulates gut protein metabolism during chemotherapy-induced mucositis in rats.

BACKGROUND: Mucositis, a common side effect of chemotherapy, is characterized by compromised digestive function, barrier integrity and immune competence. AIMS: Our aim was to evaluate the impact of a specifically designed diet Clinutren Protect (CP), which contains whey proteins, TGFbeta-rich casein, and free glutamine, on mucositis in rats. METHODS: Mucositis was induced by three consecutive injections (day 0, day 1, day 2) of methotrexate (2.5 mg/kg). Rats had free access to CP or placebo diets from days -7 to 9. In the placebo diet, whey proteins and TGFbeta-rich casein were replaced by TGFbeta-free casein and glutamine by alanine. Intestinal parameters were assessed at day 3 and 9. Values, expressed as mean +/- SEM, were compared using two-way ANOVA. RESULTS: At day 3, villus height was markedly decreased in the placebo (296 +/- 11 microm) and CP groups (360 +/- 10 microm) compared with controls (464 +/- 27 microm), but more markedly in the placebo as compared to CP group. The intestinal damage score was also reduced in the CP compared with the placebo group. Glutathione content increased in the CP compared with the placebo group (2.2 +/- 0.2 vs. 1.7 +/- 0.2 micromol/g tissue). Gut protein metabolism was more affected in the placebo than in the CP group. The fractional synthesis rate was decreased in the placebo group (93.8 +/- 4.9%/day) compared with controls (121.5 +/- 12.1, P < 0.05), but not in the CP group (106.0 +/- 13.1). In addition, at day 9, rats exhibited improved body weight and food intake recovery in the CP compared to the placebo group. CONCLUSIONS: Clinutren Protect feeding reduces intestinal injury in the acute phase of methotrexate-induced mucositis in rats and improves recovery.

Increased proteasome-mediated degradation of occludin in irritable bowel syndrome.

OBJECTIVES: Proteasome-mediated protein degradation may contribute to the regulation of intestinal inflammation. At the same time, low-grade inflammation and increased intestinal permeability seem to be involved in the pathophysiology of irritable bowel syndrome (IBS). Thus, we aimed to evaluate proteasome composition and activities in colonic mucosa of IBS patients and its putative pathogenic role. METHODS: Proteasome activities and proteasome subunit expression were measured in colonic mucosa of IBS, Crohn's disease (CD), and control patients by fluorometric assays and western blot, respectively. Expression of inhibitor of kappa B factor (IkappaB alpha) and occludin, a tight junction protein, was also evaluated in colonic biopsies. The degradation of recombinant occludin incubated with protein extracts from colonic mucosa was evaluated in the presence or absence of proteasome inhibitor, MG132. RESULTS: Proteasome trypsin-like activity was increased in IBS patients compared with CD and controls, whereas chymotrypsin-like activity was upregulated in CD patients only. Caspase-like activity was reduced both in IBS and CD patients. IkappaB alpha expression was similar between IBS and controls. In contrast, occludin expression was lower in IBS than in controls, but occludin mRNA level was similar. Protein extracts from IBS patients but not from controls degraded recombinant occludin (20% over 160 min), which was blocked by MG132. Although mast cell number was increased in IBS patients, no correlation was found between this number and proteasome alterations. CONCLUSIONS: Our study shows that proteasome alterations are present in the colonic mucosa of IBS patients and may contribute to the pathophysiology of IBS by increasing occludin degradation.

Methotrexate induces intestinal mucositis and alters gut protein metabolism independently of reduced food intake.

One of the main secondary toxic side effects of antimitotic agents used to treat cancer patients is intestinal mucositis. This one is characterized by compromised digestive and absorptive functions, barrier integrity, and immune competence. At the same time, food intake is decreased, which may induce intestinal damages per se. The aim of the study was to characterize which alterations are specific to methotrexate, independently of the anorexic effect of the drug. Male Sprague-Dawley rats received subcutaneously saline solution as control group or 2.5 mg/kg of methotrexate during 3 days (D0-D2). Methotrexate-treated rats were compared with ad libitum and pair-fed controls. Histological examinations and specific markers of the immune and nonimmune gut barrier function were assessed at D4 or D7. Compared with ad libitum and pair-fed controls, methotrexate induced at D4 villus atrophy associated with epithelial necrosis. Mucosal protein synthesis rate and mucin contents of methotrexate treated rats were reduced. At the same time, cathepsin D proteolytic activity was increased compared with ad libitum and pair-fed controls, whereas calpain activity was increased when compared with the only pair-fed controls. These intestinal lesions were associated with various metabolic disturbances such as increased TNF-alpha level and inflammation score in the jejunum but also disturbances of amino acid concentrations in the duodenum and plasma. At D7, these alterations were partially or completely normalized. In addition to the consequences of a low food intake, methotrexate further impairs different biological processes leading to a dramatic loss of gut homeostasis. Targeted nutritional management of chemotherapy receiving patients should be set up to prevent or limit such alterations.