Intestinal inflammation marker calprotectin regulates epithelial intestinal zinc metabolism and proliferation in mouse jejunal organoids.

Calprotectin (CP), a heterodimer of S100A8 and S100A9, is expressed by neutrophils and a number of innate immune cells and is used widely as a marker of inflammation, particularly intestinal inflammation. CP is a ligand for toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE). In addition, CP can act as a microbial modulatory agent via a mechanism termed nutritional immunity, depending on metal binding, most notably Zn2+. The effects on the intestinal epithelium are largely unknown. In this study we aimed to characterize the effect of calprotectin on mouse jejunal organoids as a model epithelium, focusing on Zn2+ metabolism and cell proliferation. CP addition upregulated the expression of the Zn2+ absorptive transporter Slc39a4 and of methallothionein Mt1 in a Zn2+-sensitive manner, while downregulating the expression of the Zn2+ exporter Slc30a2 and of methallothionein 2 (Mt2). These effects were greatly attenuated with a CP variant lacking the metal binding capacity. Globally, these observations indicate adaptation to low Zn2+ levels. CP had antiproliferative effects and reduced the expression of proliferative and stemness genes in jejunal organoids, effects that were largely independent of Zn2+ chelation. In addition, CP induced apoptosis modestly and modulated antimicrobial gene expression. CP had no effect on epithelial differentiation. Overall, CP exerts modulatory effects in murine jejunal organoids that are in part related to Zn2+ sequestration and partially reproduced in vivo, supporting the validity of mouse jejunal organoids as a model for mouse epithelium.

Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting.

It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.

FXR-dependent and -independent interaction of glucocorticoids with the regulatory pathways involved in the control of bile acid handling by the liver.

Treatment with glucocorticoids (GCs) may cause adverse effects, including cholestasis. The ability of dexamethasone, prednisolone and budesonide to affect the liver handling of bile acids (BAs) has been investigated. In rats treated with GCs for 4 days, altered serum and bile BA levels, changed conjugation pattern, and delayed and decreased ability to conjugate/secrete exogenously administered deoxycholate, were found using HPLC-MS/MS. RT-QPCR analyses revealed that GC treatment also induced a down-regulation of liver nuclear receptors (Fxr, Gr and Shp), transporters (Ntcp, Mrp4 and Bcrp) and enzymes (Cyp7a1 and Baat), whereas Bsep, Mrp2 and Cyp27a1 were up-regulated. Human HepG2 and Alexander cell lines were used as in vitro models of liver cells with and without constitutive FXR expression, respectively. In HepG2 cells, GCs induced a decreased expression of FXR and SHP, and inhibited the regulatory effect of GW4064 on FXR-target genes. In Alexander cells, only when they were transfected with FXR+RXR, GW4064 caused up-regulation of SHP and OSTß, and a down-regulation of CYP27A1. GCs had the opposite effect on these genes, both in the absence and in the presence of FXR expression. Co-transfection of Alexander cells with IR-1-Luc and FXR+RXR revealed that GCs did not inhibit but moderately enhanced FXR activity. Moreover, GCs have a synergistic effect on GW4064-induced FXR activation, whereas chenodeoxycholate and GW4064 have an additive effect. In conclusion, GCs are able to directly or indirectly activate FXR but they also antagonize, through FXR-independent mechanisms, the expression of FXR and FXR target genes involved in the hepatic handling of BAs.

Exogenous alkaline phosphatase treatment complements endogenous enzyme protection in colonic inflammation and reduces bacterial translocation in rats.

Alkaline phosphatase (AP) inactivates bacterial lipopolysaccharide and may therefore be protective. The small intestine and colon express intestinal (IAP) and tissue nonspecific enzyme (TNAP), respectively. The aim of this study was to assess the therapeutic potential of exogenous AP and its complementarity with endogenous enzyme protection in the intestine, as evidenced recently. IAP was given to rats by the oral or intrarectal route (700U/kgday). Oral budesonide (1mg/kgday) was used as a reference treatment. Treatment with intrarectal AP resulted in a 54.5% and 38.0% lower colonic weight and damage score, respectively, and an almost complete normalization of the expression of S100A8, LCN2 and IL-1ß (p<0.05). Oral AP was less efficacious, while budesonide had a more pronounced effect on most parameters. Both oral and intrarectal AP counteracted bacterial translocation effectively (78 and 100%, respectively, p<0.05 for the latter), while budesonide failed to exert a positive effect. AP activity was increased in the feces of TNBS colitic animals, associated with augmented sensitivity to the inhibitor levamisole, suggesting enhanced luminal release of this enzyme. This was also observed in the mouse lymphocyte transfer model of chronic colitis. In a separate time course study, TNAP was shown to increase 2-3 days after colitis induction, while dextran sulfate sodium was a much weaker inducer of this isoform. We conclude that exogenous AP exerts beneficial effects on experimental colitis, which includes protection against bacterial translocation. AP of the tissue-nonspecific isoform is shed in higher amounts to the intestinal lumen in experimental colitis, possibly aiding in intestinal protection.

Sexual dimorphism in clock genes expression in human adipose tissue.

BACKGROUND: This study was carried out to investigate whether sex-related differences exist in the adipocyte expression of clock genes from subcutaneous abdominal and visceral fat depots in severely obese patients. METHODS: We investigated 16 morbidly obese patients, eight men and eight women (mean age 45 ± 20 years; mean BMI 46 ± 6 kg/m(2)), undergoing laparoscopic gastric bypass surgery. Biopsies were taken as paired samples [subcutaneous and visceral adipose tissue (AT)] at the beginning of the surgical process at 11:00 h in the morning. Metabolic syndrome features such as waist circumference, plasma glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also studied. The expression of clock genes (PER2, BMAL1, and CRY1) was measured by quantitative real-time PCR, Western blot, and immunohistochemical analysis. RESULTS: Gene expression was significantly higher in women than in men for the three genes studied in both ATs (P < 0.05). In visceral fat, these differences were more marked. (P < 0.001). Western blot analysis partially confirmed these results since statistical differences were observed for PER2 in both ATs and for CRY1 in subcutaneous adipose tissue. There were no differences in BMAL1 protein expression. Interestingly, clock gene expression level was correlated with LDL-C and HDL-C (P < 0.05). Moreover, we found significant associations with body fat mass in women and with age in men. CONCLUSIONS: Clock genes expression is sex dependent in human adipose tissue from morbidly obese subjects and correlates to a decreased in metabolic syndrome-related traits. These preliminary results make necessary to go deep into the knowledge of the molecular basis of the sexual dimorphism in chronobiology.

Effects of flavonoids and other polyphenols on inflammation.

Flavonoids are a family of polyphenolic compounds which are widespread in nature (vegetables) and are consumed as part of the human diet in significant amounts. There are other types of polyphenols, including, for example, tannins and resveratrol. Flavonoids and related polyphenolic compounds have significant antiinflammatory activity, among others. This short review summarizes the current knowledge on the effects of flavonoids and related polyphenolic compounds on inflammation, with a focus on structural requirements, the mechanisms involved, and pharmacokinetic considerations. Different molecular (cyclooxygenase, lipoxygenase) and cellular targets (macrophages, lymphocytes, epithelial cells, endothelium) have been identified. In addition, many flavonoids display significant antioxidant/radical scavenging properties. There is substantial structural variation in these compounds, which is bound to have an impact on their biological profile, and specifically on their effects on inflammatory conditions. However, in general terms there is substantial consistency in the effects of these compounds despite considerable structural variations. The mechanisms have been studied mainly in myeloid cells, where the predominant effect is an inhibition of NF-κB signaling and the downregulation of the expression of proinflammatory markers. At present there is a gap in knowledge of in vitro and in vivo effects, although the pharmacokinetics of flavonoids has advanced considerably in the last decade. Many flavonoids have been studied for their intestinal antiinflammatory activity which is only logical, since the gastrointestinal tract is naturally exposed to them. However, their potential therapeutic application in inflammation is not restricted to this organ and extends to other sites and conditions, including arthritis, asthma, encephalomyelitis, and atherosclerosis, among others.

Flavonoids exert distinct modulatory actions on cyclooxygenase 2 and NF-kappaB in an intestinal epithelial cell line (IEC18).

BACKGROUND AND PURPOSE: Cyclooxygenase 2 (COX-2) is involved in inflammatory bowel disease, but the effect of flavonoids at the intestinal epithelial level is unknown. We aimed to characterize the effect and structure-activity relationship of nine selected flavonoids on COX-2 expression in intestinal epithelial cell (IEC)18 cells. We also investigated the signal transduction pathway(s) responsible for the effects observed. EXPERIMENTAL APPROACH: Intestinal epithelial cell 18, a non-tumour cell line with intestinal epithelial phenotype, was used. COX-2 was measured by Western blot and the involvement of the NF-kappaB pathway assessed by Western blot, pharmacological inhibition, luciferase reporter assays and nuclear translocation experiments. KEY RESULTS: The effect of flavonoids on COX-2 expression depended on the experimental conditions tested [non-stimulated and lipopolysaccharide (LPS)-stimulated]. Flavonoids caused an increase in COX-2 expression and NF-kappaB-dependent gene transcription under basal conditions. Conversely, under LPS stimulation flavonoids increased, decreased or did not affect COX-2 levels depending on the specific type. Variable effects were observed on extracellular signal regulated kinase/p38/c-Jun N-terminal kinase phosphorylation and p50/65 nuclear translocation. CONCLUSION AND IMPLICATIONS: The effect of flavonoids on COX-2 expression depended on the balance of the interference with IkappaB-alpha phosphorylation and other signalling targets, and therefore depends on the experimental conditions and on the type of flavonoids. This is expected to result in different effects in inflammatory conditions. In general, flavonoids may limit epithelial COX-2 expression in inflammatory conditions while favouring it when inflammation is not present.

Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17.

BACKGROUND AND PURPOSE: Bovine glycomacropeptide (BGMP) is an inexpensive, non-toxic milk peptide with anti-inflammatory effects in rat experimental colitis but its mechanism of action is unclear. It is also unknown whether BGMP can ameliorate inflammation in proximal regions of the intestine. Our aim was therefore two-fold: first, to determine the anti-inflammatory activity of BGMP in the ileum; second, to characterise its mechanism of action. EXPERIMENTAL APPROACH: We used a model of ileitis induced by trinitrobenzenesulphonic acid in rats. Rats were treated orally with BGMP and its efficacy compared with that of oral 5-aminosalicylic acid or vehicle, starting 2 days before ileitis induction. KEY RESULTS: BGMP pretreatment (500 mg kg(-1) day(-1)) resulted in marked reduction of inflammatory injury, as assessed by lower extension of necrosis and damage score, myeloperoxidase, alkaline phosphatase, inducible nitric oxide synthase, interleukin 1beta, tumour necrosis factor and interleukin 17. These effects were generally comparable to those of 5-aminosalicylic acid (200 mg kg(-1) day(-1)). Neither compound affected the production of interferon gamma, tumour necrosis factor and interleukin 2 by mesenteric lymph node cells isolated from animals with ileitis. The expression of Foxp3 was increased in ileitis and not reduced significantly by BGMP or aminosalicylate treatment. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that BGMP has anti-inflammatory activity in the ileum with similar efficacy to 5-aminosalicylic acid. The mechanism of action may involve Th17 and regulatory T cells and perhaps macrophages but probably not Th1 lymphocytes. Patients with Crohn's ileitis may benefit from treatment with BGMP.

The bisphosphonate alendronate improves the damage associated with trinitrobenzenesulfonic acid-induced colitis in rats.

BACKGROUND AND PURPOSE: The nitrogen-containing bisphosphonates are drugs used successfully in the treatment of osteoporosis. They act inhibiting farnesyl diphosphate synthase. This mechanism may also produce anti-inflammatory effects. The therapeutic activity of alendronate was tested in vivo using a model of inflammatory bowel disease. EXPERIMENTAL APPROACH: The trinitrobenzenesulfonic acid model of colitis in the rat was used. Rats were treated orally with alendronate and its efficacy compared with that of oral sulphasalazine or vehicle, starting 2 h after colitis induction. The status of the animals was assessed 5 days later. KEY RESULTS: Alendronate treatment (25 or 75 mg kg(-1) day(-1)) resulted in a decrease in the colonic damage score and loss of body weight (at 25 mg kg(-1) day(-1) only). This was associated to a dramatic reduction in the mRNA levels of interleukin 1 beta (IL-1 beta), monocyte chemoattractant protein 1 (MCP-1) and interleukin 1 receptor antagonist (IL-1 ra). The magnitude of the beneficial effect was comparable to that of sulphasalazine (at a 6-20 fold higher dose). Thus sulphasalazine post-treatment reduced the mRNA levels of IL-1 beta/IL-1 ra and MCP-1 to the same extent as alendronate and additionally lowered colonic alkaline phosphatase activity, but failed to affect body weight loss or colonic damage score. Alendronate failed to exert beneficial effects when administered intraperitoneally. CONCLUSIONS AND IMPLICATIONS: Oral but not intraperitoneal alendronate significantly protected the colon in experimental rat colitis. Inflammatory bowel disease patients might benefit from exposure to oral alendronate.

Proteins and peptides in enteral nutrition.

A protein is an essential macronutrient for the growth and maintenance of corporal structures. An important concept in proteic nutrition is the protein's quality, mainly determined by the profile and proportion of the amino acids making up the protein, although other factors such as solubility and degree of glycosylation may be involved. There are different ways to evaluate protein quality that can be classified as chemical, biological and microbiological. Currently Protein Digestibility-Corrected Amino Acid Score (PDCAAS) is routinely used. Protein quality can be altered by the technological and culinary processes to which food is subjected and also by the presence in food of anti-nutritional factors affecting the bioavailability of amino acids. Protein complementation through the formulation of low-quality protein mixtures lets us improve bioavailability, and therefore the quality of this protein mix. In the past few years, nutrition and food technology are undergoing a profound transformation due to the development of the concept of functional and nutraceutic foods. Functional proteins and bioactive peptides are gaining in importance since, in addition to their nutritional role as a source of amino acids, they are capable of exerting different biological effects on the immune system, the cardiovascular system or the gastrointestinal tract. In addition, these peptides and proteins have been described as having anticancer, antibacterial or antiviral effects. This paper reviews the most relevant functional proteins and bioactive peptides from a functional standpoint, with special emphasis on those coming from milk, eggs and soy.

Proteínas y péptidos en nutrición enteral / Proteins and peptides in enteral nutrition

La proteína es un macronutriente esencial para el crecimiento y el mantenimiento de las estructuras corporales. Un concepto importante en nutrición proteica es la calidad de la proteína que viene, principalmente, determinada por el perfil y proporción de los aminoácidos que la componen, aunque pueden intervenir otros factores como la solubilidad y el grado glicosilación. Para evaluar la calidad de la proteína existen diversos métodos que se pueden clasificar en químicos, biológicos y microbiológicos. Actualmente se utiliza, de rutina el cómputo de aminoácidos corregido con la digestibilidad de la proteína (PDCAAS). La calidad de una proteína puede modificarse por los tratamientos tecnológicos y culinarios a los que son sometidos los alimentos que la contiene y también por la presencia en ellos de factores antinutricionales que afectan a la biodisponibilidad de los aminoácidos. La complementación proteica permite, mediante la formulación de mezclas de proteínas de baja calidad, mejorar la biodisponibilidad, y por tanto la calidad de esa mezcla proteica. En los últimos años la nutrición y la tecnología de los alimentos están experimentando una profunda transformación debido al desarrollo del concepto de alimentos funcionales y de nutracéuticos. Tanto las proteínas funcionales como los péptidos bioactivos están cobrando gran importancia ya que, además de su papel nutricional por ser fuente de aminoácidos, son capaces de ejercer diferentes efectos biológicos específicos sobre el sistema inmune, el sistema cardiovascular o el tracto gastrointestinal. Además, se ha descrito que estos péptidos y proteínas pueden tener efectos anticancerígenos, antibacterianos o antivirales.En este trabajo se revisan las proteínas funcionales y los péptidos bioactivos más relevantes desde el punto de vista de su funcionalidad, haciendo especial hincapié en aquellos procedentes de la leche, el huevo y la soja (AU)

A protein is an essential macronutrient for the growth and maintenance of corporal structures. An important concept in proteic nutrition is the protein's quality, mainly determined by the profile and proportion of the amino acids making up the protein, although other factors such as solubility and degree of glycosylation may be involved. There are different ways to evaluate protein quality that can be classified as chemical, biological and microbiological. Currently Protein Digestibility-Corrected Amino Acid Score (PDCAAS) is routinely used. Protein quality can be altered by the technological and culinary processes to which food is subjected and also by the presence in food of anti-nutritional factors affecting the bioavailability of amino acids. Protein complementation through the formulation of low-quality protein mixtures lets us improve bioavailability, and therefore the quality of this protein mix. In the past few years, nutrition and food technology are undergoing a profound transformation due to the development of the concept of functional and nutraceutic foods. Functional proteins and bioactive peptides are gaining in importance since, in addition to their nutritional role as a source of amino acids, they are capable of exerting different biological effects on the immune system, the cardiovascular system or the gastrointestinal tract. In addition, these peptides and proteins have been described as having anticancer, antibacterial or antiviral effects. This paper reviews the most relevant functional proteins and bioactive peptides from a functional standpoint, with special emphasis on those coming from milk, eggs and soy (AU)

Experimental inflammation of the rat distal colon inhibits ion secretion in the proximal colon by affecting the enteric nervous system.

Intestinal inflammation causes hyporesponsiveness of the inflamed tissue to secretagogues but little is known about the behaviour of the areas proximal to the site of inflammation. We studied the responses of the proximal segment of the colon to carbachol, histamine, isobutylmethylxanthine (IBMX) and vasoactive intestinal peptide (VIP) in rats with trinitrobenzenesulphonic acid (TNBS)-induced, chronic inflammation of the distal colon. Macroscopic and biochemical analysis ruled out the presence of inflammation in the proximal colon. When mounted in Ussing chambers under voltage-clamp conditions, basal transport and conductance were not affected. However, the maximum response in the concentration/response curves (short-circuit current) for carbachol and histamine was reduced in TNBS-treated rats, without changes in the EC(50). This effect corresponded to reduced chloride secretion, as demonstrated by ion substitution experiments. The responses to IBMX and VIP were virtually unaffected. The inhibitory effect was abolished by pretreatment with the neural blockers tetrodotoxin and lidocaine but not indomethacin, suggesting that the enteric nervous system is responsible for the inhibition. In conclusion, chronic distal inflammation of the distal colon results in inhibition of calcium-dependent secretion in the proximal colon via a reduction of the contribution of the enteric nervous system.

Arginina, óxido nítrico y función endotelial / Arginine, nitric oxide and endothelial function

La arginina es un aminoácido semiesencial con importantes funciones fisiológicas. Entre ellas destaca su papel como precursora del óxido nítrico, una molécula producida a partir de la arginina por la enzima óxido nítrico sintasa en muchos tejidos y que en el endotelio vascular se comporta como vasodilatadora, antiaterogénica y antiagregante plaquetaria. El estudio detallado de esta reacción enzimática indica que la óxido nítrico sintasa tiene una gran afinidad por su sustrato, la arginina, que se encuentra en concentraciones altas en el endotelio. Por tanto, resultaba sorprendente que el funcionamiento de esta enzima estuviera condicionado por las variaciones en las concentraciones de arginina debidas al aporte nutricional. A esto se le llamó «paradoja de la arginina». Sin embargo, se ha demostrado recientemente la existencia de un inhibidor endógeno de la óxido nítrico sintasa denominado dimetilarginina asimétrica. Este compuesto disminuiría la formación del óxido nítrico por inhibición competitiva con el sustrato natural, la arginina. De ahí la importancia de la suplementación con arginina para contrarrestar este efecto. Además de la arginina, existen otros componentes de la dieta que pueden influir también en la síntesis de óxido nítrico por el endotelio vascular. el endotelio vascular (AU)

Arginine is a semi-essential amino acid with major physiological functions. One of the most outstanding of such is its role as an amino acid precursor of nitric oxide, a molecule produced, in many tissues, from arginine by the nitric oxide synthase enzyme. Within the vascular endothelium, nitric oxide behaves as a vasodilator, antiatherogenic, and anti-plaque aggregation agent. The detailed study of this enzymatic reaction indicates that nitric oxide synthase presents high affinity, aggregation agent. The detailed study of this enzymatic reaction indicates that nitric oxide synthase presents high affinity, for its substrate, arginine, which is found in high concentrations in the endothelium. Consequently, it is surprising that for its substrate, arginine, which is found in high concentrations in the endothelium. Consequently, it is surprising that the functionality of this enzyme is conditioned by variations in concentrations of arginine, produced by nutritional intake. the functionality of this enzyme is conditioned by variations in concentrations of arginine, produced by nutritional intake. This is known as «the arginine paradox». However, the existence of an endogenous nitric oxide synthase inhibitor, This is known as «the arginine paradox». However, the existence of an endogenous nitric oxide synthase inhibitor, known as asymmetric dimethylarginine, has been recently demonstrated. This compound would decrease the formation of nitric oxide through competitive inhibition with the natural substrate, arginine. It is for this reason that dietary supplementation with arginine would be important as a means to counteracting such an effect. In addition to arginine, supplementation with (..) (AU)

Intestinal anti-inflammatory activity of dietary fiber (Plantago ovata seeds) in HLA-B27 transgenic rats.

BACKGROUND & AIMS: Dietary fiber has been proven to be beneficial in maintaining remission in human ulcerative colitis, an effect related with an increased luminal production of short-chain fatty acids (SCFA). The aim of the present study was to further investigate the mechanisms involved in the intestinal anti-inflammatory effects of dietary fiber in an experimental model of rat colitis. METHODS: HLA-B27 transgenic rats (8-10 weeks old) were fed a fiber-supplemented diet (5% Plantago ovata seeds) for 13 weeks before evaluation of the colonic inflammatory status, both histologically and biochemically. The luminal colonic production of SCFA was quantified. In vitro studies were also performed to test the interaction between two SCFA (butyrate and propionate) as inhibitors of cytokine production in THP-1 cells. RESULTS: Dietary fiber supplementation ameliorated the development of colonic inflammation in transgenic rats as evidenced by an improvement of intestinal cytoarchitecture. This effect was associated with a decrease in some of the pro-inflammatory mediators involved in the inflammatory process: nitric oxide, leukotriene B(4), tumor necrosis factor alpha (TNFalpha). The intestinal contents from fiber-treated colitic rats showed a significant higher production of SCFA, butyrate and propionate, than non-treated colitic animals. In vitro studies revealed a synergistic inhibitory effect of butyrate and propionate on TNFalpha production. CONCLUSIONS: Dietary fiber supplementation ameliorated colonic damage in HLA-B27 transgenic rats. This effects was associated with an increased production of SCFA, which can act synergistically in inhibiting the production of pro-inflammatory mediators.

Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP.

We used the trinitrobenzenesulphonic acid (TNBS) rat model of experimental colitis to study the alterations in electrogenic ion transport in the inflamed distal colon. The distal colon exhibited decreased basal transport and reduced short-circuit current responses to carbachol and isobutylmethylxanthine (IBMX). The concentration/response curve for IBMX was also shifted to the right. Ion substitution experiments indicated that electrogenic transport was attributable chiefly to Cl(-) secretion. The mucosal layer of the inflamed distal colon (devoid of the submucosa) exhibited normal maximal responses to carbachol and IBMX, although the concentration/response curve for the latter was again shifted to the right. Tetrodotoxin markedly increased the response of the normal distal colon to both secretagogues and nullified the inhibition of the response to carbachol, but not that to IBMX, in the inflamed colon. The response of the mucosal preparation to 8-bromoadenosine 3',5'-cyclic monophosphate was similar in the normal and inflamed intestine, while the G protein activator NaF had a greater effect in the latter. The expression of the cystic fibrosis transmembrane conductance regulator (CFTR), as assessed by Northern blotting, was unchanged. cAMP levels in isolated colonocytes were markedly reduced by inflammation. We conclude that colonic inflammation produces disturbances of the enteric nervous system resulting in defective mucosal cAMP production and inhibition of ionic secretion.

Effect of psychogenic stress on gastrointestinal function.

This review summarizes the studies published over the last twenty years on the effects of psychogenic stress on gastrointestinal function, using animal models. The effects of stress on gastric ulceration have received wide attention and the central and local mechanisms of mucosal damage have been, for the most part, clearly delineated. In comparison, relatively few studies have focused on the impact of stress on intestinal and colonic physiology, even though its influence on intestinal motility, mucosal permeability and inflammation has been established. More work is necessary in this field, especially considering the importance of irritable bowel syndrome in modern society.

Utilization of the indirect lysosome targeting pathway by lysosome-associated membrane proteins (LAMPs) is influenced largely by the C-terminal residue of their GYXXphi targeting signals.

A systematic study was conducted on the requirements at the C-terminal position for the targeting of LAMPs to lysosomes, examining the hypothesis that a bulky hydrophobic residue is required. Mutations deleting or replacing the C-terminal valine with G, A, C, L, I, M, K, F, Y, or W were constructed in a reporter protein consisting of the lumenal/extracellular domain of avian LAMP-1 fused to the transmembrane and cytoplasmic domains of LAMP-2b. The steady-state distribution of each mutant form in mouse L-cells was assessed by quantitative antibody binding assays and immunofluorescence microscopy; efficiency of internalization from the plasma membrane and delivery to the lysosome were also estimated. It is found that (a) only C-terminal V, L, I, M, and F mediated efficient targeting to lysosomes, demonstrating the importance hydrophobicity and an optimal size of the C-terminal residue in targeting; (b) efficiency of lysosomal targeting generally correlated with efficiency of internalization; and (c) mutant forms that did not target well to lysosomes showed unique distributions in cells rather than simply default accumulation in the plasma membrane. Interactions of the targeting signals with adaptor subunits were measured using a yeast two-hybrid assay. The results are consistent with the hypothesis that trafficking of LAMP forms in cells through the indirect pathway is determined by the affinities of their targeting signals, predominantly for the mu2 and mu3 adaptors involved at plasma membrane and endosomal cellular sorting sites, respectively.

Dietary nucleotides might influence the humoral immune response against cow's milk proteins in preterm neonates.

The objective of this study was to evaluate the influence of dietary nucleotide supplementation in preterm infants during the first month of life on the intestinal permeability to lactulose, mannitol and to beta-lactoglobulin and on the development of circulating antibodies to beta-lactoglobulin and alpha-casein. Twenty-seven preterm infants were enrolled in the study; 11 of them were fed a standard low-birth weight milk formula and 16 infants were fed the same formula supplemented with nucleotides at similar levels to those found in human milk. Blood and urine samples were obtained at 1, 7 and 30 days of age. Serum beta-lactoglobulin, serum IgG antibody to alpha-casein and serum IgG antibody to beta-lactoglobulin were measured by ELISA. The lactulose/mannitol urinary excretion rate was measured by gas liquid chromatography. Neither the intestinal permeability to saccharides nor the intestinal absorption of beta-lactoglobulin were affected by the nucleotide supplementation. However, serum concentrations of IgG antibody to beta-lactoglobulin were higher in preterm neonates fed the supplemented formula than in those fed the standard formula. According to these results, dietary nucleotides might influence the maturation of the humoral immune response in preterm newborn infants.

Exogenous nucleotides alter the proliferation, differentiation and apoptosis of human small intestinal epithelium.

The turnover of intestinal epithelial cells is a finely regulated process extending from undifferentiated crypt stem cells to terminally differentiated villus cells. The final phase of this maturation process is apoptosis and extrusion. Recent studies have shown that programmed cell death (PCD) occurs not only in senescent cells and in rapidly developing tissues but also in response to cellular stress preventing damaged cells from entering uncontrolled proliferation without repair. This study examined the role of exogenous nucleotides on cell proliferation, differentiation and apoptosis in organ cultures of human fetal small intestine. The addition of adenosine monophosphate (AMP), a putative stress reactant, to the culture media resulted in the suppression of crypt cell proliferation followed by the restoration of differentiation and the induction of apoptosis across a broad range of villus epithelium when compared with controls without added nucleotide. In contrast, the addition to cytidine monophosphate (CMP) to the culture media did not increase apoptosis, despite the nucleotide being taken up by the fetal small intestine in a similar dose- and time-dependent manner to AMP. Furthermore, Bax mRNA, an apoptosis-inducer gene, was increased with addition of AMP, suggesting that the induction of apoptosis may be channeled through the p53 pathway. These results suggest that a specific exogenous nucleotide, AMP, may have an important role in controlling the dynamic balance of cellular turnover in the developing human small intestine.