Non-digestible oligosaccharides modulate intestinal immune activation and suppress cow's milk allergic symptoms.

BACKGROUND: Cow's milk allergy is a common food allergy in childhood and no effective preventive or curative treatment is available. This study aimed at comparing single short-chain galacto- (scGOS), long-chain fructo- (lcFOS) or pectin-derived acidic oligosaccharides (pAOS) and/or mixtures of scGOS/lcFOS (GF) or scGOS/lcFOS/pAOS (GFA) to prevent or treat food allergy. METHODS: In the preventive protocol, C3H/HeOuJ mice were fed diets containing single oligosaccharides or mixtures GF or GFA throughout the study protocol. In the treatment protocol, GF or GFA was provided for 4 wk starting after the last sensitization. The allergic skin response and anaphylaxis scores were determined, after oral challenge whey-specific immunoglobulins were measured, and qPCR for T-cell markers and Foxp3 counts using immunohistochemistry were performed on the small intestine and colon. RESULTS: Only in the preventive setting, the GF or GFA mixture, but not the single oligosaccharides, reduced the allergic skin response and whey-IgG(1) levels in whey-sensitized mice, compared to the control diet. Both GF and GFA increased the number of Foxp3+ cells in the proximal small intestine of whey - compared to sham-sensitized mice. Expression of Th2 and Th17 mRNA markers increased in the middle part of the small intestine of whey-sensitized mice, which was prevented by GF. By contrast, GFA enhanced Tbet (Th1), IL-10 and TGF-ß mRNA expression compared to GF which was maintained in the distal small intestine and/or colon. CONCLUSIONS: Dietary supplementation with scGOS/lcFOS or scGOS/lcFOS/pAOS during sensitization, both effectively reduce allergic symptoms but differentially affect mucosal immune activation in whey-sensitized mice.

Dietary long chain n-3 polyunsaturated fatty acids prevent allergic sensitization to cow's milk protein in mice.

BACKGROUND: Cow's milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease. OBJECTIVE: Assess whether dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy. METHODS: Mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed. RESULTS: The acute allergic skin response was reduced by more than 50% in sensitized mice fed the fish oil diet compared to the control diet. In addition, anti-whey-IgE and anti-whey-IgG1 levels were decreased in the fish oil group. Serum transfer confirmed that the Th2-type humoral response was suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic effector response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization in fish oil fed naïve recipient mice. In addition, the percentage of activated Th1 cells was reduced by fish oil in spleen and MLN of sham mice. The percentage of activated Th2 cells was reduced in both sham- and whey-sensitized mice. In contrast, whey-sensitized mice showed an increased percentage of CD11b+CD103+CD8α- DC in MLN in association with enhanced FoxP3+ regulatory T cells (Treg) in spleen and intestine of fish oil fed whey-sensitized mice compared to sham mice. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary n-3 LCPUFA largely prevented allergic sensitization in a murine model for cow's milk allergy by suppressing the humoral response, enhancing local intestinal and systemic Treg and reducing acute allergic symptoms, suggesting future applications for the primary prevention of food allergy.

CD25+ regulatory T cells transfer n-3 long chain polyunsaturated fatty acids-induced tolerance in mice allergic to cow's milk protein.

BACKGROUND: Recently, we have shown that dietary long-chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) largely prevent allergic sensitization in a murine model for cow's milk allergy. The aim of this study was to assess the contribution of regulatory T cells (Treg) in the prevention of food allergy by n-3 LCPUFA. METHODS: C3H/HeOuJ female donor mice were fed a control or fish oil diet before and during oral sensitization with cow's milk protein whey. Acute allergic skin response (ASR), anaphylaxis, body temperature, serum immunoglobulins, and mouse mast cell protease-1 (mmcp-1) were assessed. Splenocytes of sham- or whey-sensitized donor mice fed either control or fish oil diet were adoptively transferred to naïve recipient mice. Recipient mice received a whole splenocyte suspension, splenocytes ex vivo depleted of CD25+ cells, or MACS-isolated CD4+ CD25+ Treg. Recipient mice were sham- or whey-sensitized and fed control diet. RESULTS: The ASR as well as whey-specific IgE and whey-specific IgG1 levels were reduced in whey-sensitized donor mice fed the fish oil diet as compared to the control diet. Splenocytes of control-diet-fed whey-sensitized donors transferred immunologic memory. By contrast, splenocytes of fish-oil-fed whey-sensitized - but not sham-sensitized - donors transferred tolerance to recipients as shown by a reduction in ASR and serum mmcp-1, and depletion of CD25+ Treg abrogated this. Transfer of CD25+ Treg confirmed the involvement of Treg in the suppression of allergic sensitization. CONCLUSIONS: CD25+ Treg are crucial in whey allergy prevention by n-3 LCPUFA.

Depletion of CD4+CD25+ T cells switches the whey-allergic response from immunoglobulin E- to immunoglobulin free light chain-dependent.

BACKGROUND: Symptoms of allergy are largely attributed to an IgE-mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig-free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4+CD25+ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens. OBJECTIVE: To examine the contribution of CD4+CD25+ T cells and IgLC towards the whey-allergic response. METHODS: Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25+ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey-specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4+CD25+ T cells was confirmed in the spleen. RESULTS: Anti-CD25 treatment strongly reduced whey-specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3(+) regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25-depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti-CD25-treated mice, indicating that these responses were mainly IgLC dependent. CONCLUSIONS: Depletion of CD4+CD25+ T cells resulted in a switch from an IgE- to an IgLC-dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25+ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE-independent responses and both mechanisms might play a role in allergic responses to the same allergen.

Mouse genetic model for antigen-induced airway manifestations of asthma.

Allergic asthma is a genetically complex disease characterized by allergen-specific immunoglobulin (Ig)E, eosinophilic inflammation of the lungs and airway hyper-responsiveness to bronchospasmogenic stimuli. In this study, we compared 13 recombinant congenic (RC) mouse strains in an ovalbumin model of allergic asthma. Different intensities and types of responses are observed throughout the RC strains. Intensities range from resistance to asthma in CcS05, to a very severe bronchoconstrictive reaction upon methacholine challenge for the parental STS strain. All strains show a 'modified' Th2 response except CcS14, which shows a 'true' Th2 response. When data from all strains are pooled, airway reactivity shows significant correlations with the serum Ig levels and the levels of interleukin (IL)-4, IL-5 and IL-13 in the broncho-alveolar lavage (BAL), at low dosage of methacholine (below 25 mg/ml), whereas at high dosage airway reactivity only correlates with BAL neutrophil levels. This indicates that at least two different mechanisms are involved in the airway reactivity to methacholine. None of these correlations can be found in every individual strain, which demonstrates that the asthma traits in this mouse model are genetically dissociated and that the loci can be genetically mapped.

Macrophages induce an allergen-specific and long-term suppression in a mouse asthma model.

Increasing evidence suggests that macrophages (Mphi) play a crucial downregulatory role in the initiation and progression of allergic asthma. Recently, the current authors demonstrated that ovalbumin (OVA)-loaded Mphi (OVA-Mphi) suppress subsequent OVA-induced airway manifestations of asthma and that this effect could be potentiated upon selective activation. In the present study, the authors further delineated the underlying pathway by which Mphi exert this immunosuppressive effect. To examine the migration of OVA-Mphi, cells were labelled with 5'chloromethylfluorescein diacetate (CMFDA) and were administered (i.v.) into OVA-sensitised BALB/c mice. After 20 h, the relevant organs were dissected and analysed using fluorescent microscopy. Allergen-specificity was investigated by treating OVA-sensitised mice with keyhole limpet haemocyanin (KLH)-Mphi activated with immunostimulatory sequence oligodeoxynucleotide (ISS-ODN). By lengthening the period between treatment and challenge to 4 weeks it was examined whether OVA-Mphi exerted an immunosuppressive memory response. Strikingly, CMFDA-labelled Mphi were not trapped in the lungs, but migrated to the spleen. ISS-ODN-stimulated KLH-Mphi failed to suppress OVA-induced airway manifestations of asthma. Moreover, treatment with ISS-ODN-stimulated OVA-Mphi was still effective after lengthening the period between treatment and challenge. These data demonstrate that allergen-loaded macrophages can induce an indirect immunosuppressive response that is allergen-specific and long lasting, which are both hallmarks of a memory lymphocyte response.

Decarboxylation of L-dopa in the rat isolated vascularly perfused small intestine: contribution to systemic elimination and dose-dependent first pass effect.

The contribution of the rat small intestine to systemic and presystemic elimination of L-dopa was studied. When L-dopa was administered into the vascular perfusate, a systemic extraction ratio of 0.38 was found, the major part being decarboxylated to dopamine. The intestinal L-dopa clearance was estimated to be 17.1 mL min-1 kg-1. Thus, L-dopa intestinal clearance in rat represents up to at least 20% of the total body clearance. After luminal administration of L-dopa 83-88% of the administered dose was absorbed within 60 min. The total amount of L-dopa appearing in the vascular perfusate increased more than proportionally to the increase in the dose. In contrast, the amount of dopamine increased less than proportionally to the dose. As a result, the intestinal first pass appeared to be strongly dose-dependent. Since the total percentage absorbed from the lumen was independent of the administered dose and the total amount that appeared in the vascular perfusate increased linearly with the dose, the dose dependency was probably due to saturation of intestinal L-dopa decarboxylation.

Flow-dependent extraction of 1-naphthol by the rat isolated perfused kidney.

The influence of variation of perfusion flow rate on the renal clearance of p-aminohippuric acid and 1-naphthol was studied with an isolated perfused rat kidney preparation. Kidney functions were well maintained at low perfusion flow rates by the use of a fluorocarbon emulsion to increase the oxygen capacity of the perfusion buffer. Renal extraction of p-aminohippuric acid decreased with increasing perfusion flow. Our data show that at high perfusion flow rates maximal extractable perfusion flow forms only a small part of the total perfusion flow. 1-Naphthol is rapidly metabolized to its glucuronide and sulfate conjugate in the isolated perfused rat kidney. Using PAH as a marker for the maximal extractable perfusion flow, 1-naphthol could be regarded as a high-extraction compound even at high perfusion flow rates. Our results suggest that p-aminohippuric acid clearance, rather than total perfusion flow rate, should be used as the measure of maximal extractable blood flow for the estimation of extraction ratio in the isolated perfused kidney of compounds excreted or metabolized by the proximal tubules.

Nephrotoxicity of the glutathione conjugate of menadione (2-methyl-1, 4-naphthoquinone) in the isolated perfused rat kidney. Role of metabolism by gamma-glutamyltranspeptidase and probenecid-sensitive transport.

The renal processing of the glutathione conjugate of menadione, 2-methyl-3-S-glutathionyl-1,4-naphthoquinone (thiodione) was studied in the isolated perfused rat kidney. Thiodione at an initial concentration of 600 microM was eliminated rapidly from the perfusate (clearance = 6.0 ml/min). Renal disposition could be ascribed to metabolism and transport of the glutathione conjugate. Renal metabolism by gamma-glutamyltranspeptidase was inhibited by AT-125 [L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] (0.5 mM) resulting in a reduction of the thiodione clearance to 0.86 ml/min. Further reduction of the renal clearance of thiodione was achieved by a combination of AT-125 (0.5 mM) and probenecid (0.5 mM), resulting in a renal clearance of 0.58 ml/min which equalled glomerular filtration rate. Addition of thiodione to the perfusate caused loss of renal function and cellular damage, as reflected by a decreased glucose reabsorption and an increased urinary secretion of lactate dehydrogenase, respectively. Thiodione-induced nephrotoxicity was ameliorated by AT-125 and prevented completely by a combination of AT-125 and probenecid. Aminooxyacetic acid (0.5 mM), an inhibitor of beta-lyase, did not afford protection against the nephrotoxic action of thiodione. From our results it can be concluded that the thiodione-mediated toxicity in the isolated perfused rat kidney can be linked to cellular uptake by anionic transport systems and metabolism by gamma-glutamyltranspeptidase.

Absorption and presystemic glucuronidation of 1-naphthol in the vasculary fluorocarbon emulsion perfused rat small intestine: the influence of the luminal flow rate and intraluminal binding.

Using an isolated vasculary perfused rat small intestine we studied the role of luminal flow rate and intraluminal binding on the absorption of 1-naphthol (1-N) and the intestinal metabolism of 1-N to 1-naphthol-beta-D-glucuronide (1-NG). Raising the luminal perfusion rate resulted in a decrease in the luminal 1-N extraction ratio and an increase in the luminal 1-N clearance Cllum. The dependency of Cllum on flow rate appeared to conform to a convective diffusion model. A differential susceptibility of 1-N absorption and the total 1-NG appearance to the luminal flow rate resulted in a flow-dependent first-pass effect of 1-N. Next, the effect of intraluminal binding on 1-N disposition was studied in experiments in which albumin was added to the luminal perfusion fluid. The unbound concentration, as the driving force for the uptake of 1-N, seems not to be rate-limiting for the appearance of 1-NG. The total appearance of 1-NG in the presence of albumin was greater than would be anticipated from the free concentration of 1-N. As a result the extent of presystemic extraction increased with increasing albumin concentration. The precise mechanisms responsible for the phenomenona are not entirely clear. Consideration of the heterogeneity in the glucuronidation capacity along the rat small intestine and along the crypt-villus axis can help to explain the obtained results.

Systemic intestinal metabolism of 1-naphthol. A study in the isolated vascularly perfused rat small intestine.

Using a vascularly and luminally perfused rat small intestine, we studied the systemic intestinal metabolism of the model substrate 1-naphthol (1-N) to 1-naphthol-beta-D-glucuronide (1-NG). An intestinal extraction ratio of 0.30 +/- 0.02 was found for 1-N. This implies that intestinal metabolism represents up to 14% of the total plasma clearance of 1-N in the rat in vivo. The formed 1-NG was preferentially released into the vascular perfusate, suggesting specialized transport carriers for 1-NG in brushborder and basolateral membrane. When the vascular flow rate was decreased from 10 to 0.5 ml/min, the clearance of 1-N appeared to be completely flow dependent. The apparent conflict between a low extraction ratio (0.30 +/- 0.02 at all flows investigated) and a flow rate-limited 1-N clearance can be explained by the presence of an intestinal vascular bed with a high extraction ratio. We suggest that 1-N is completely extracted from the mucosal blood flow. This view was confirmed by the results of experiments in which the capillary flow of the intestinal mucosa was decreased by infusion of noradrenaline. As a result a temporary decrease in the 1-N extraction ratio was observed. The contribution of the intestine to the total clearance can be masked by the hepatic clearance, because the blood supply to the intestine and liver is coupled in series. An equation was derived to describe the relative contribution of the intestine to the mesenteric clearance of the intestine-liver system. It appears that the effective contribution of the intestine to the mesenteric clearance is of little interest for high extraction drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption and presystemic glucuronidation of 1-naphthol in the vascularly fluorocarbon emulsion perfused rat small intestine. The influence of 1-naphthol concentration, perfusate flow and noradrenaline.

Using the isolated vascularly fluorocarbon emulsion perfused rat small intestine some factors which determine the extent of the intestinal glucuronidation of 1-naphthol to 1-naphthol-beta-D-glucuronide were studied. Increasing the luminal 1-naphthol concentration resulted in a concomitant increase in the 1-naphthol appearance in the vascular perfusate. In contrast, the total appearance of 1-naphthol-beta-D-glucuronide increased less than proportional to the increase in the luminal 1-naphthol concentration. About 88% of the total amount of 1-naphthol-beta-D-glucuronide excreted was released into the vascular perfusate. The capacity-limited intestinal glucuronide efflux is most likely due to saturation of the excretory mechanism for 1-naphthol-beta-D-glucuronide. Decreasing the vascular flow rate influenced both the appearance of 1-naphthol and 1-naphthol-beta-D-glucuronide in the vascular perfusate, whereas the appearance of 1-naphthol-beta-D-glucuronide in the luminal perfusate was essentially flow-independent. A noradrenaline-induced change in the haemodynamic state of the vascular bed (with the total flow kept constant) resulted in a marked decrease in the naphthol vascular concentration. The vascular 1-naphthol-beta-D-glucuronide concentration was only slightly affected. These results indicate that changes in blood flow and blood flow distribution within the intestinal wall can affect the extent of presystemic intestinal metabolism by interfering with the absorption of the parent compound and the efflux of formed conjugates. These parameters can be of paramount importance for causing variable intestinal first-pass effects of drugs in vivo.

Bio-analysis and preliminary pharmacokinetics of the experimental antitumour drug LL-D49194 alpha 1.

LL-D49194 alpha 1 is a recently discovered compound, produced by Streptomyces vinaceus-drappus. This micro-organism produces a number of antibiotics, all showing antibacterial and antitumour activity, of which LL-D49194 alpha 1 is one of the main compounds. The compounds' antitumour effectiveness has been proven in vitro and the drug is undergoing further tests. For the assay of the drug in plasma a high-performance liquid chromatographic (HPLC) system has been developed, preceded by a clean-up step. The drug is extracted from the biological matrix with ethyl acetate followed by direct HPLC analysis of the organic layer via an analytical RP8 column preceded by a guard column to retain endogenous plasma compounds. Detection of drug and metabolites was carried out by fluorescence with reference to a non-fluorescent internal standard detected by UV absorption. The detection limit was 1 ng ml-1 plasma (using 1 ml sample; signal-to-noise ratio, 3), i.e. 1 ng on column. The method has been utilized in a preliminary pharmacokinetic study in rat.

Conjugative clearance of 1-naphthol and disposition of its glucuronide and sulfate conjugates in the isolated perfused rat kidney.

Extensive extrahepatic metabolism of 1-naphthol (1-N) was shown to occur in the rat in vivo (clearance = 377 ml/min/kg). The renal contribution to the metabolism of 1-N was investigated in the isolated perfused rat kidney (IPK). 1-N was metabolized to its sulfate (1-NS) and glucuronide (1-NG) conjugate by the IPK. The renal clearance of 1-N ranged from 5.36 to 6.24 ml/min/kidney and was 80 to 95% of the p-aminohippuric acid clearance by the IPK. This indicated that the renal extraction ratio of 1-N was 0.80 to 0.95, showing an important contribution of renal metabolic clearance to the total clearance. Nonlinear kinetics, due to saturation of the metabolic pathways, was shown at 1-N concentrations higher than 10 microM. 1-NS and 1-NG are both secreted in the urine and perfusate, although the urinary excretion of 1-NS is very small whereas the excretion of 1-NG into the urine is extensive, due to differences in the active secretion and protein binding of both conjugates.

Presystemic and systemic intestinal metabolism of fenoterol in the conscious rat.

The intestinal and liver first pass metabolism of fenoterol.HBr (Berotec, Partusisten) was investigated in the conscious rat. Fenoterol plasma concentrations (2-1000 ng/ml) were measured with a new HPLC determination using electrochemical detection. After intraduodenal administration, fenoterol was incompletely absorbed (47-66% not absorbed). Presystemic intestinal (EGpre) and liver (EH) extraction ratios, EGpre = 0.93 +/- 0.01, EH = 0.67 +/- 0.04, were calculated from AUC values after intraduodenal, intraportal, and iv administration. Saturation of intestinal and/or liver metabolism was checked by using three dose levels at different administration routes. Total systemic availability after intraduodenal administration ranged from 0.8 (10 mg/kg) to 1.2% (40 mg/kg). The contribution of the splanchnic region to the systemic clearance of fenoterol was assessed by measuring fenoterol and fenoterol-glucuronide concentrations in arterial and portal venous blood under steady state conditions. During iv infusion (30 micrograms fenoterol/min X kg), an intestinal extraction ratio of EG = 0.26 was observed. After iv administration of fenoterol (1 and 2 mg/kg), dose-dependent pharmacokinetics were observed. Doubling of the dose resulted in an increase of systemic clearance (Cl = 53.8 +/- 2.7 and 74.4 +/- 1.8 ml/min X kg) and distribution volume (Vss = 0.95 +/- 0.13 and 1.21 +/- 0.11 liters/kg); the mean residence time (17.9 +/- 2.4 and 16.3 +/- 1.4 min) and terminal half-life (45.8 +/- 5.5 and 46.8 +/- 2.8 min) were not changed.

First-pass metabolism of pentamethylmelamine in the rat liver.

The disposition of pentamethylmelamine (PMM) was studied in the male Wistar rat. PMM (5 mg/kg) was administered intraarterially, i.v. (5 and 10 mg/kg), via the portal vein, and into the duodenum to cannulated and unanesthetized rats (n greater than or equal to 4) via infusion. Parent compound and metabolites were quantified by gas chromatography. The areas under the plasma concentration-time curves of PMM after intraarterial and i.v. administration were equal and twice as large as the areas after portal vein and intraduodenal administration. This indicated insignificant lung metabolism for PMM; the low bioavailability of PMM when given via the portal vein or intraduodenally (in both cases, some 50% of an i.v. dose) was the result of presystemic metabolism in the liver. PMM was completely absorbed after intraduodenal administration, and no intestinal metabolism was observed. Linear kinetic behavior of i.v. PMM was observed in the 5- to 10-mg/kg dose range. The area under the plasma concentration-time curve of the first metabolite N2,N2,N4,N6-tetramethylmelamine was significantly greater when PMM was given via the portal vein or intraduodenally than when given intraarterially or i.v. This indicated either extrahepatic elimination/renal excretion of PMM or the existence of an additional metabolic pathway. However, experiments with adrenalectomized rats and rats with ligated blood flow to the kidneys did not alter the area for the first metabolite. These findings may be explained by the formation of unknown metabolites and/or reactive intermediates of PMM.

A rational superfusion medium for the bioassay of E-type prostaglandins on the rat stomach strip.

The optimum composition of a mixture of antagonist to be used in the bioassay of E-type prostaglandins was determined for the rat stomach strip (RSS). In the presence of atropine (10(-7)M), indomethacin (10(-6)M), propranolol (10(-4)M), and tolazoline (10(-4)M) the sensitivity of the RSS to muscarinic, alpha and beta adrenergic and serotonergic agonists was greatly reduced whereas its responsiveness to PGE1 and PGE2 was unaltered. Using the oil-immersion-superfusion technique with this drug mixture, the bioassay of prostaglandins from samples also containing other agonists gave accurate estimates of the PG concentration of the samples using small amounts of 10(-6)M PGE1 or PGE2.