Analysis of cytokine production by peanut-reactive T cells identifies residual Th2 effectors in highly allergic children who received peanut oral immunotherapy.

BACKGROUND: Only limited evidence is available regarding the cytokine repertoire of effector T cells associated with peanut allergy, and how these responses relate to IgE antibodies to peanut components. OBJECTIVE: To interrogate T cell effector cytokine populations induced by Ara h 1 and Ara h 2 among peanut allergic (PA) children in the context of IgE and to evaluate their modulation during oral immunotherapy (OIT). METHODS: Peanut-reactive effector T cells were analysed in conjunction with specific IgE profiles in PA children using intracellular staining and multiplex assay. Cytokine-expressing T cell subpopulations were visualized using SPICE. RESULTS: Ara h 2 dominated the antibody response to peanut as judged by prevalence and quantity among a cohort of children with IgE to peanut. High IgE (> 15 kU(A)/L) was almost exclusively associated with dual sensitization to Ara h 1 and Ara h 2 and was age independent. Among PA children, IL-4-biased responses to both major allergens were induced, regardless of whether IgE antibodies to Ara h 1 were present. Among subjects receiving OIT in whom high IgE was maintained, Th2 reactivity to peanut components persisted despite clinical desensitization and modulation of allergen-specific immune parameters including augmented specific IgG4 antibodies, Th1 skewing and enhanced IL-10. The complexity of cytokine-positive subpopulations within peanut-reactive IL-4(+) and IFN-γ(+) T cells was similar to that observed in those who received no OIT, but was modified with extended therapy. Nonetheless, high Foxp3 expression was a distinguishing feature of peanut-reactive IL-4(+) T cells irrespective of OIT, and a correlate of their ability to secrete type 2 cytokines. CONCLUSION: Although total numbers of peanut-reactive IL-4(+) and IFN-γ(+) T cells are modulated by OIT in highly allergic children, complex T cell populations with pathogenic potential persist in the presence of recognized immune markers of successful immunotherapy.

Sensitization to food and inhalant allergens in relation to age and wheeze among children with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is common in children; however, persistence of AD with or without asthma is less common. Longitudinal studies remain limited in their ability to characterize how IgE antibody responses evolve in AD, and their relationship with asthma. OBJECTIVE: To use a cross-sectional study design of children with active AD to analyse age-related differences in IgE antibodies and relation to wheeze. METHODS: IgE antibodies to food and inhalant allergens were measured in children with active AD (5 months to 15 years of age, n = 66), with and without history of wheeze. RESULTS: Whereas IgE antibodies to foods persisted at a similar prevalence and titre throughout childhood, IgE antibodies to all aeroallergens rose sharply into adolescence. From birth, the chance of sensitization for any aeroallergen increased for each 12-month increment in age (OR ≥ 1.21, P < 0.01), with the largest effect observed for dust mite (OR = 1.56, P < 0.001). A steeper age-related rise in IgE antibody titre to dust mite, but no other allergen was associated with more severe disease. Despite this, sensitization to cat was more strongly associated with wheeze (OR = 4.5, P < 0.01), and linked to Fel d 1 and Fel d 4, but not Fel d 2. Comparison of cat allergic children with AD to those without, revealed higher IgE levels to Fel d 2 and Fel d 4 (P < 0.05), but not Fel d 1. CONCLUSIONS AND CLINICAL RELEVANCE: Differences in sensitization to cat and dust mite among young children with AD may aid in identifying those at increased risk for disease progression and development of asthma. Early sensitization to cat and risk for wheeze among children with AD may be linked to an increased risk for sensitization to a broader spectrum of allergen components from early life. Collectively, our findings argue for early intervention strategies designed to mitigate skin inflammation in children with AD.

Application of isolated hepatocytes to studies of drug metabolism in large food animals.

A definitive hazard assessment of xenobiotics translocated through food animals into edible products such as meat or milk requires a complete analysis of metabolism in food animals. However, large animal metabolism studies present many experimental difficulties. None of several in vitro alternatives such as subcellular fractions has been established as an acceptable predictor of in vivo metabolism. The feasibility of using isolated hepatocytes to predict the metabolism of xenobiotics, both quantitatively and qualitatively, in large ruminant animals (e.g. cattle) is being studied in our laboratory. A procedure was developed for isolating hepatocytes aseptically from the caudate process of the liver which was obtained surgically from 100-125 kg calves. A modified two-step vascular perfusion procedure provides hepatocyte suspensions that are typically greater than or equal to 85% viable and greater than or equal to 1 X 10(7) viable hepatocytes/g of liver (wet wt). Xenobiotic metabolism has been evaluated in suspensions and primary cultures using aldrin epoxidation, ethoxycoumarin O-deethylation, and 7-hydroxycoumarin glucuronidation and sulfation. Metabolic activities are relatively short-lived in suspensions less than or equal to 4 h, but quite stable up to 10 h when cultured on collagen-coated plates in chemically defined medium. Bovine hepatocytes behave similarly in culture to rodent hepatocytes. Although primary culturing of hepatocytes is more difficult than suspensions, primarily due to the asepsis requirements, it is the method of choice for xenobiotic metabolism determinations in isolated hepatocytes of cattle.

Interlobular distribution of hepatic xenobiotic-metabolizing enzyme activities in cattle, goats and sheep.

Microsomal and cytosolic enzymes that metabolize xenobiotics were measured in composite samples representing entire livers and in samples from three lobes, using livers of cattle, goats and sheep. Within individual species, concentrations of cytochrome P-450 and b5 and activities of NADPH cytochrome c reductase, aldrin epoxidase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase, microsomal and cytosolic stilbene oxide (epoxide) hydrolase and glutathione S-transferase were not different (P greater than .05) among the various hepatic lobes. Among species, several activities differed (P less than .05), with cattle livers generally having lower values than sheep or goats.

Xenobiotic metabolism in suspensions and primary cultures of isolated hepatocytes prepared from the caudate process of bovine liver.

Isolated hepatocytes were prepared from 100- to 125-kg Holstein male calves (n = 10) by perfusion of the caudate process of the caudate lobe of the liver. The 11th or 12th rib on the right side was resected to provide exposure of the caudate process. Complete postsurgical recovery of the donor from partial lobectomy was confirmed by growth data and serum chemical and hematologic criteria. Hepatocytes were isolated under aseptic conditions, using a 2-step collagenase vascular perfusion procedure. Hepatocyte preparations averaged 85% viability, and the yield averaged 1.2 X 10(7) viable hepatocytes/g of (wet weight) liver. Morphologic characteristics of hepatocytes examined under light and scanning electron microscopy were considered normal, except for occasional surface blebs. Freshly isolated hepatocytes in suspension rapidly decreased in viability and xenobiotic metabolizing capacity (aldrin epoxidation and ethoxycoumarin 0-deethylation and 7-hydroxycoumarin glucuronidation and sulfation), and hepatocytes surviving the initial 2 to 3 hours appeared to undergo repair. As an alternative, primary monolayer cultures on collagen-coated plates were evaluated. Hepatocytes attached to the collagen surface within 4 hours and appeared flattened by 12 hours. Although metabolic activity decreased about 30% over 8 hours in culture, the pattern of ethoxycoumarin metabolites was relatively constant. It was not determined to what extent the apparent loss of metabolic capacity was caused by hepatocyte detachment from the collagen surface. Although complicated by the requirement for asepsis, primary cultures were superior to suspensions for xenobiotic metabolism studies in cattle.

Biological and induction effects of phenobarbital and 3-methylcholanthrene in mink (Mustela vison).

Mink were injected (ip) daily with 20 mg/kg of 3-methylcholanthrene (MC) or 40 mg/kg of phenobarbital (PB) for 3 days and killed 48 hr after the last injection. The duration of anesthetic action of PB increased after each injection. MC-treated mink became anorexic and lost substantial body weight. PB caused enlargement of liver and lungs, whereas MC caused liver atrophy. No major treatment-related morphologic changes including amount of endoplasmic reticulum (ER) in liver were revealed by electron microscopic examination. Microsomal protein content was not increased and NADPH cytochrome P-450 reductase was not induced in liver by either PB or MC. Cytochrome P-450 (448) was increased 3.2-fold by PB and 2.5-fold by MC. Cytochrome b5 was increased 2.3-fold by MC but was not affected by PB. Aminopyrine N-demethylase was enhanced 5.1-fold in activity by PB whereas hexobarbital hydroxylase was not induced. MC-treatment moderately increased the activities of benzo(a)pyrene hydroxylase (1.7-fold) and ethoxyresorufin O-deethylase (2.1-fold) but had no effect on ethoxycoumarin O-deethylase. The most distinctive features of the mink revealed by this study are a) lack of PB induction of the ER, microsomal protein content, NADPH-cytochrome P-450 reductase, and hexobarbital hydroxylase, and b) lack of MC induction of cytochrome P-448-associated mixed function oxidases that are known to be highly responsive to MC in other species.