Temporal and long-term gut microbiota variation in allergic disease: A prospective study from infancy to school age.

BACKGROUND: Compositional changes in the early-life gut microbiota have been implicated in IgE-associated allergic diseases, but there is lack of longitudinal studies. We examined gut microbiota development from infancy to school age in relation to onset of IgE-associated allergic diseases. At 8 years of age, we also examined the relationship between gut microbiota and T-cell regulation, estimated as responses to polyclonal T-cell activation. METHODS: Stool samples were collected from 93 children at 4, 6, 13 months, and 8 years of age. The gut microbiota was profiled using 16S rRNA gene sequencing. Peripheral blood was drawn from all children, and mononuclear cells were polyclonally activated. Levels of IL-10 and FOXP3 mRNA copies were determined using real-time quantitative reverse transcriptase-PCR. RESULTS: At 8 years of age, 21 children were diagnosed with IgE-associated allergic disease and 90% displayed allergic comorbidity. Seventy-two children were nonallergic and nonsensitized. Statistical tests with multiple testing corrections demonstrated temporal underrepresentation of Ruminococcus and consistent underrepresentation of Bacteroides, Prevotella, and Coprococcus in allergic compared to nonallergic children from infancy to school age. The gut microbiota of the allergic 8-year-olds was enriched in Bifidobacterium and depleted of Lactobacillus, Enterococcus, and Lachnospira. In allergic 8-year-olds, Faecalibacterium correlated with IL-10 mRNA levels (rs  = 0.49, Padj  = 0.02) with the same trend for FOXP3 (rs  = 0.39, Padj  = 0.08). CONCLUSIONS: We identified both temporal and long-term variation in the differential abundance of specific bacterial genera in children developing IgE-associated allergic disease. Improved dietary interventions aiming at expanding immune-modulatory taxa could be studied for prevention of allergic disease.

Synthesis and enzymatic hydrolysis of esters, constituting simple models of soft drugs.

One way to minimise systemic side effects of drugs is to design molecules, soft drugs, in such a way that they are metabolically inactivated rapidly after having acted on their pharmacological target. Hydrolases (esterases, peptidases, lipases, glycosidases, etc.) are enzymes well suited to use for drug inactivation since they are ubiquitously distributed. Insertion of ester bonds susceptible to enzymatic cleavage may represent one approach to make the action of a drug more restricted to the site of application. The present study describes the chemical synthesis of fourteen model compounds comprising a bicyclic aromatic unit connected by an ester-containing bridge to another aromatic ring. Initial attempts to define a) the tissue selectivity of the hydrolytic metabolism and b) the molecular structural factors affecting the rate of enzymatic ester cleavage are presented. The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at hydrolysing the compounds. The rank order of the compounds was, however, very similar in the different biological systems. Commercially available pig liver carboxyl esterase and cholesterol esterase both reasonably well predict the rank order in the tissue fractions.

Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes.

Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.

Effects of N-acetylcysteine stereoisomers on oxygen-induced lung injury in rats.

The effects of the stereoisomers of N-acetylcysteine (L-NAC and D-NAC) on oxygen-induced lung oedema have been studied in rats. The NAC-isomers were given by an osmotic minipump in order to attain continuous administration, either intravenously or intragastrically. In some experiments, plasma concentrations of NAC, cysteine and glutathione (total concentrations, i.e., concentrations obtained after reduction of the samples with dithiothreitol) were recorded. Exposure to oxygen induced an almost two-fold increase of the lung wet weight. When L-NAC or D-NAC were given intravenously, in dose of 1.1 mmol/day/kg body weight, the increase of lung wet weight was prevented by 40-50%. The plasma concentrations were approximately 40 microM (L-NAC) and approximately 90 microM (D-NAC). Following intragastrical administration of the same doses, plasma concentrations of L-NAC and D-NAC reached approximately 3 and approximately 60 microM, respectively. Using this method of administration, only D-NAC significantly diminished the increase of the lung wet weight. The difference in plasma concentrations of the NAC isomers, particularly after intragastric administration, most likely reflects the fact that L-NAC is effectively hydrolysed in most tissues, while D-NAC is resistant to enzymatic hydrolysis, thus penetrating largely intact into the systemic circulation. The data presented shows that NAC, regardless of stereoconfiguration, will protect the lung against oxygen toxicity, provided sufficient systemic levels are obtained. Since D-NAC is not a precursor of L-cysteine, formation of glutathione cannot explain the protective effects of this isomer. L- and D-NAC may therefore act via direct antioxidant/radical scavenging mechanisms and not necessarily as precursors of glutathione in this model.

Glutathione in bronchoalveolar lavage fluid from smokers is related to humoral markers of inflammatory cell activity.

Cigarette smoking results in variable degrees of inflammation in the lower respiratory tract. Furthermore, smoking produces oxidant-mediated changes in the lung, important to the pathogenesis of emphysema. Since glutathione can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant and inflammatory injury. In the present study, broncholaveolar lavage (BAL) was performed in 27 smokers, and the concentrations of total glutathione as well as the cellular and humoral markers of inflammatory activity were studied. There were significant correlations between total glutathione and neutrophils; two neutrophil granule components, myeloperoxidase and elastase; and chemotactic activity for neutrophils. Moreover, the total glutathione correlated with the eosinophil cationic protein (ECP), a granule constituent of the eosinophil, with two locally produced antiproteases, secretory leukocyte protease inhibitor (SLPI) and antichymotrypsin (ACHY), but not with an alpha 1-protease inhibitor and albumin. These data suggest that the total glutathione levels in BAL fluid may reflect a degree of oxidative and inflammatory stress caused by cigarette smoke, and they are therefore likely to contribute to the protection against this stress.

Metabolism of N-acetyl-L-cysteine. Some structural requirements for the deacetylation and consequences for the oral bioavailability.

Rat liver, lung and intestine homogenates deacetylated N-acetyl-L-cysteine. Nearly stoichiometric amounts of L-cysteine were recovered. In rat liver, the enzyme activity was associated with the cytosolic fraction. Liver cytosol was much less active. N-Acetyl-D-cysteine or the disulphide of N-acetyl-L-cysteine were not deacetylated or in other ways consumed in vitro. Isolated, perfused rat liver did not retain or metabolize N-acetyl-L-cysteine to any measurable extent during single-pass experiments. N-Acetyl-L-cysteine or N-acetyl-D-cysteine were injected into a ligated segment of rat intestine in situ. After 1 hr 2% of the L-isomer and 35% of the D-isomer remained in the intestinal lumen. Systemic plasma levels were less than 3 microM of the L-form and congruent to 40 microM of the D-form. We conclude that deacetylation in the intestinal mucosa and possibly in the intestinal lumen is the major factor determining the low oral bioavailability of N-acetyl-L-cysteine. The deacetylation is discussed on the basis of the subcellular localization and the structural requirement of the reaction.

Streaming potential--a general affinity sensor.

Electrokinetic phenomena, such as streaming potential or streaming current, have so far been used for studies of unspecific adsorption of ionic compounds on various materials. This paper shows that streaming potential measurements can also be used for studies of biospecific interactions. Several different interactions were studied, e.g. lectin-carbohydrate, IgG-Protein A. Regardless of the type of interaction an increasing change in streaming potential was obtained with increasing concentration of the interacting molecule. The authors also observed a correlation between streaming potential and affinity constants for carbohydrate-induced desorption of Concanavalin A from partially hydrolyzed Sephadex G50. In conclusion, it is shown that streaming potential measurements can be used to study molecular interactions, and to determine concentrations and relative binding constants of interacting molecules.