Miquel Moretó

Dietary Plasma Protein Supplements Prevent the Release of Mucosal Proinflammatory Mediators in Intestinal Inflammation in Rats

Spray-dried plasma (SDP) is a complex mixture of active proteins that modulates the immune response of gut-associated lymphoid tissue. We examined whether SDP and Ig concentrate (IC) supplementation could modulate cytokine expression and inflammatory mediators in rats challenged with Staphylococcus aureus enterotoxin B (SEB). Wistar-Lewis rats were fed diets supplemented with SDP (8% wt:wt), IC (1.5% wt:wt), or milk proteins (control diet) from weaning (d 21) to d 34 after birth. On d 32 and 35, the rats were given SEB (0.5 mg/kg; intraperitoneal). Six hours after the second SEB dose, jejunal mucosa and Peyers patches (PP) from the small intestine were collected. The cytokines interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-6, IL-10, transforming growth factor-beta (TGFbeta), and leukotrienne B(4) (LTB(4)) were analyzed using commercial kits. SEB increased the release of proinflammatory mediators (IFNgamma, TNFalpha, IL-6, and LTB(4)) in PP (P < 0.05) and in the mucosa (P < 0.05). In both tissues, SDP prevented the increase in IFNgamma, IL-6, and LTB(4) induced by SEB (P < 0.05). IC reduced the expression of TNFalpha and LTB(4) in PP and mucosa (P < 0.05). SDP supplementation increased IL-10 and mature TGFbeta concentrations in intestinal mucosa from both inflamed and noninflamed rats. Both SDP and IC increased the mature:total TGFbeta ratio (all P < 0.05). Both supplements were effective at preventing the SEB-induced increase in proinflammatory:antiinflammatory cytokine ratios in PP and mucosa and in serum. The preventive effects of plasma supplements on intestinal inflammation involve modulation of intestinal cytokines, characterized by an increased expression of antiinflammatory cytokines.

Sequential Amino Acid Exchange across b 0,+ -like System in Chicken Brush Border Jejunum

Abstract. In the small intestine, cationic amino acids are transported by y+-like and b0,+-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b0,+-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b0,+-like system functions for l-arginine as an obligatory exchanger since its transport capacity increases 100–200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Argin efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Argout is higher than that of Argin, suggesting a possible order of binding (Argout first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed.

Lipid peroxidation induced by DHA enrichment modifies paracellular permeability in Caco-2 cells protective role of taurine

Dietary enrichment with docosahexaenoic acid (DHA) has numerous beneficial effects on health. However, the intake of high doses of polyunsaturated fatty acids can promote lipid peroxidation and the subsequent propagation of oxygen radicals. The purpose of this study was to evaluate the effect of DHA on lipid peroxidation and tight junction structure and permeability in Caco-2 cell cultures. Moreover, the effects of taurine, a functional ingredient with antioxidant properties, were also tested. Differentiated Caco-2 cell monolayers were maintained in DHA-supplemented conditions with or without added taurine. Incubation with 100 μM DHA increased lipid peroxidation and paracellular permeability, in parallel with a redistribution of the tight junction proteins occludin and ZO-1. Taurine partially prevented all of these effects. The participation of reactive oxygen and nitrogen species in increased paracellular permeability was also examined using various agents that modify the formation of superoxide radical, hydrogen peroxide, nitric oxide, and peroxynitrite. We conclude that hydrogen peroxide and peroxynitrite may be involved in the DHA-induced increase in paracellular permeability and that the protective role of taurine may be in part related to its capacity to counteract the effects of hydrogen peroxide.

Effect of a lysine-enriched diet onl-lysine transport by the brush-border membrane of the chicken jejunum

The influx ofl-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant ( K m) and features of system b0,+ and the other with relatively high K m forl-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g l-lysine/kg dietary protein, control animals 48 g/kg) onl-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediatedl-lysine influx was best fitted to the two-system model with y+and b0,+ activity. 3) In the presence of an Na+ gradient, totall-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K mb0,+6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity ( V max) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y+-like system are only observed on the V max and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+concentration and l-lysine influx. In conclusion, both b0,+- and y+-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y+-like carrier shows a V maxincrease without changes in K m, whereas the low-capacity b0,+-like system shows an increase in V max as well as in the K m.The influx of L-lysine into apical vesicles from the chicken jejunum occurs through two systems, one with low Michaelis constant (K(m)) and features of system b0,+ and the other with relatively high K(m) for L-lysine and with properties of system y+. In the present study the effect of a lysine-enriched diet (Lys, containing 68 g L-lysine/kg dietary protein, control animals 48 g/kg) on L-lysine uptake through both transport systems was investigated. Results show that 1) lysine enrichment had no effect on either body weight or the efficiency of food utilization. 2) In Lys-fed animals, the mediated L-lysine influx was best fitted to the two-system model with y+ and b0,+ activity. 3) In the presence of an Na+ gradient, total L-lysine uptake is significantly higher in Lys-fed animals than in control birds (about 40% increase). 4) Lys diet increases K(m)b0,+ 6-fold (KSCN gradient) and 12-fold (NaSCN gradient) and maximum velocity (Vmax) by 6- and 20-fold, respectively. The effects of Lys enrichment on the y(+)-like system are only observed on the Vmax and in the presence of a Na+ gradient (30% increase). 5) Na+ is involved in the activation of the transport process in the Lys-fed chickens, but there is no correlation between external Na+ concentration and L-lysine influx. In conclusion, both b(0,+)- and y(+)-like transport systems are upregulated by dietary lysine but with different kinetic profiles; the high-capacity y(+)-like carrier shows a Vmax increase without changes in K(m), whereas the low-capacity b(0,+)-like system shows an increase in Vmax as well as in the K(m).

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

Abstract.The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (KmA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (KmB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na+ (KiB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b0,+ and y+, respectively.

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na+-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (α-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.

Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted to high and low NaCl intakes

1 The effect of a low‐NaCl diet (LS diet) on the properties of hexose transport across the brush‐border and basolateral membranes of enterocytes from jejunum, ileum and rectum of the chicken was investigated. 2 In the brush‐border membrane, LS adaptation had no effect on Km for α‐methyl‐d‐glucoside while Vmax values were significantly reduced in the ileum and in the rectum. All Scatchard plots of specific [3H]phlorizin binding give a straight line, consistent with a single population of binding sites. Phlorizin binding vs.α‐methyl‐d‐glucoside maximal transport rates showed a linear correlation. 3 In the basolateral membrane, the LS diet did not modify the Km for d‐glucose but reduced the Vmax in the ileum and in the rectum. Scatchard plots of [3H]cytochalasin B binding support the view that there is a single transport system in this membrane. There was a linear correlation between cytochalasin B binding and d‐glucose Vmax values. 4 The response of the chicken intestine to LS intake consists of a dramatic reduction in the number of glucose transporters in both apical and basolateral membranes of the rectum, an intermediate response in the ileum and no significant effects in the jejunum.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564-575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402-420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum

1 The intestinal transport of L‐methionine has been investigated in brush‐border membrane vesicles isolated from the jejunum of 6‐week‐old chickens. L‐Methionine influx is mediated by passive diffusion and by Na+‐dependent and Na+‐independent carrier‐mediated mechanisms. 2 In the absence of Na+, cis‐inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L‐methionine influx: one sensitive to L‐lysine and the other sensitive to 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid (BCH). The L‐lysine‐sensitive flux is strongly inhibited by L‐phenylalanine and can be broken down into two pathways, one sensitive to N‐ethylmaleimide (NEM) and the other to L‐glutamine and L‐cystine. 3 The kinetics of L‐methionine influx in Na+‐free conditions is described by a model involving three transport systems, here called a,b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km= 2.2 ± 0.3 μM and Vmax= 0.13 ± 0.005 pmol (mg protein)−1 (2 s)−1; system b: Km= 3.0 ± 0.3 mM and Vmax= 465 ± 4.3 pmol (mg protein)−1 (2 s)−1); system c is specific for neutral amino acids, has a Km of 1.29 ± 0.08 mM and a Vmax of 229 ± 5.0 pmol (mg protein)−1 (2 s)−1 and is sensitive to BCH inhibition. 4 The Na+‐dependent component can be inhibited by BCH and L‐phenylalanine but cannot interact either with cationic amino acids or with α‐(methylamino)isobutyrate (MeAIB). 5 The kinetic analysis of L‐methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3‐fold decrease in the Michaelis constant and a 1.4‐fold increase in Vmax. In the presence of Na+, the BCH‐sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 ± 0.013 mM and a Vmax of 391 ± 2.3 pmol (mg protein)−1 (2 s)−1. 6 It is concluded that L‐methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L‐methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.

Dietary plasma proteins attenuate the innate immunity response in a mouse model of acute lung injury

We examined whether oral plasma protein supplements affect the innate immune response in a model of acute lung inflammation. Mice were fed diets supplemented with 8 % spray-dried plasma (SDP) or 2 % plasma Ig concentrate (IC) from day 19 (weaning) until day 34. The mice were challenged with intranasal lipopolysaccharide (LPS) at day 33 (and killed 24 h later for cytokine and leucocyte analyses) or at day 34 (and killed 6 h later for cytokine determinations). In bronchoalveolar lavage fluid (BALF), LPS increased the number of leucocytes by twenty-sevenfold, an effect that was partly prevented by both SDP and IC, and by twentyfold the percentage of activated monocytes, which was partly prevented by SDP. In the lung tissue, LPS increased the infiltrated leucocytes, and this effect was prevented in part by SDP. In unchallenged mice, both SDP and IC diets reduced the percentage of resident neutrophils and monocytes (P < 0·05). In the blood, both SDP and IC completely prevented LPS-dependent monocyte activation (CD14⁺; P < 0·05). LPS dramatically increased the concentration of cytokines (TNF-α, IL-1α, IL-6, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor) and chemokines (CXCL1, CCL2, CCL3 and CCL4) in BALF. The acute response of cytokine production was reduced by 20-80 % by both SDP and IC. For chemokines, plasma supplements had no effect on LPS-induced CXCL1 expression but significantly reduced CCL2, CCL3 and CCL4 production (P < 0·05). The results support the view that dietary plasma proteins can be used to attenuate endotoxin-associated lung inflammation.