Stephen J. Chambers

Oral delivery of Lactobacillus casei Shirota modifies allergen‐induced immune responses in allergic rhinitis

Background Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements.

Design and development of immunoassays for detection of proteins

Abstract Immunoassay methodology is currently the method of choice for the quantitative and semi-quantitative detection of many types of proteins in complex mixtures. A combination of sensitivity, specificity and cost-effectiveness in terms of analytical performance are allied to a diverse array of assay formats suitable for laboratory and field use. In the present article, the problems of setting up immunoassays for novel food proteins, and the questions that need to be answered if a successful outcome is to be achieved, are discussed. Speculation on future developments in immunochemistry leads to the conclusion that antibody technology will play an important role in detection of novel proteins from genetically modified organisms.

Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy

Specific molecular‐receptor interactions with gut epithelium cells are important in understanding bioactivity of food components and drugs, binding of commensal microflora, attachment and initiation of defense mechanisms against pathogenic bacteria and for development of targeted delivery systems to the gut. However, methods for probing such interactions are lacking. Methodology has been developed and validated to measure specific molecular‐receptor interactions on living human colorectal cancer cells as in vitro models for the gut epithelium. Atomic force microscopy (AFM) was used to measure ligand‐receptor interactions and to map receptor locations on cell surfaces. Measurements were made using silica beads attached to the AFM tip‐cantilever assembly, which were functionalized by coupling of ligands to the bead surface. Wheat germ agglutinin (WGA) binds to the glycosylated extracellular domain III of the epidermal growth factor receptor. Methodology was tested by measuring binding of WGA to the surface of confluent monolayers of living Caco‐2 human intestinal epithelial cells. The measured modal detachment force of 125 pN is typical of values expected for single molecule interactions. Adhesive events were used to map the location of binding sites on the cell surface revealing heterogeneity in their distribution within and between cells within the monolayer.—Gunning, A. P., Chambers, S., Pin, C., Man, A. L., Morris, V. J., Nicoletti, C. Mapping specific adhesive interactions on living human intestinal epithelial cells with atomic force microscopy. FASEB J. 22, 2331–2339 (2008)

Use of the indirect competitive ELISA for the detection of Brazil nut in food products

Food related allergic reactions following inadvertent ingestion are increasingly common, with nuts, including Brazil nut, placed firmly in the top 10 food groups whose presence within a product should be declared. The presence of hidden allergens as a result of adulteration or contamination of ingredients presents a problem for both the food industry and the consumer. A sensitive and specific immunoassay for Brazil nut is described with a limit of detection of 1 ppm. Based upon the detection of the abundant 2S protein the assay is suitable for detection of raw and roasted Brazil nut in a range of food matrices.

Adoptive transfer of dendritic cells from allergic mice induces specific immunoglobulin E antibody in naïve recipients in absence of antigen challenge without altering the T helper 1/T helper 2 balance.

Dendritic cells (DCs) are important in the regulation of immune responses and it has been proposed that these cells play an important role in asthma; however, their role in food allergy is still largely unknown. Our aim was to study specific immunoglobulin E (IgE) and immunoglobulin G (IgG) responses in naïve recipients following adoptive transfer of myeloid DCs from allergic and control mice. The phenotypic features and lymphokine production of DCs were also investigated. CD11c+/hi B220− DCs isolated from spleen and Peyers patches (PP) of cows milk (CM) allergic and control mice were transferred intravenously (i.v.) into naïve syngeneic recipients, and IgE‐ and IgG‐specific responses were evaluated. Experiments were also carried out to determine the levels of interferon‐γ (IFN‐γ) and interleukin (IL)‐4 produced by splenocytes from naïve recipients following the adoptive transfer, and CD40 ligand (CD40L)‐mediated IL‐10 production by DCs from allergic and control mice. DCs isolated from spleen and PP of allergic mice, but not control groups, induced CM‐specific IgG and IgE antibody production in naïve recipients in the absence of previous immunization, but did not modify the T helper 1 (Th1) and T helper 2 (Th2) balance. Furthermore, although no difference was observed in the expression of canonical DC surface markers, PP DCs from allergic mice produced less IL‐10 than DCs from controls. We interpret these data as showing that DCs play a pivotal role in allergen‐specific IgE responses and that a Th2‐skewed response may not be involved in the early phase of allergic responses. The identification of the mechanisms underlying these events may help to design novel strategies of therapeutic intervention in food allergy.

Influence of fruit and vegetable extracts on lipid peroxidation in microsomes containing specific cytochrome P450s

Abstract We have examined the effect of 12 food extracts on iron/ascorbate-induced lipid peroxidation using microsomes enriched with specific cytochrome P450 isoenzymes, namely 1A1 and 3A4, prepared from human lymphoblastic cells. As a comparison, we also studied control microsomes, which contained negligible amounts of cytochrome P450. We observed antioxidant effects with both control and P450-containing microsomes in the case of grapefruit, green tea, coffee, tarragon and rosemary extracts. Pro-oxidant effects were observed for the brassica extracts (cabbage, cauliflower and Brussels sprouts) for all three microsome groups. Differences in the degree of lipid peroxidation between microsomes containing P450s 1 A 1 3 A 4 and control microsomes were seen for apple, tomato and parsnip peel extracts. The antioxidant properties of some of the food extracts were therefore modified by the presence of specific P450s. The results demonstrate the potential of P450-enriched microsomes in determining the antioxidant properties of food extracts and components.

Human cytochrome P450's are pro-oxidants in iron/ascorbate-initiated microsomal lipid peroxidation.

We have examined the effect of human cytochrome P450s (1A1,1A2,3A4,2A6,2B6,2D6,2E1) on ascorbate/iron-induced lipid peroxidation. Using microsomes prepared from human lymphoblastic cells enriched in recombinant cytochrome P450 isoenzymes, we have shown that the degree of peroxidation is a function of the amount of P450 present rather than the presence of any specific isoenzyme. Incorporated P450 increased the amount of peroxidation products by up to 2.1-fold compared to the control microsomes with no P450. It is therefore concluded that cytochrome P450s play a significant role in ascorbate/iron peroxidation.

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation of microsomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 micrograms was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37 degrees C, pH 7.6) on (beta-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-gamma-palmitoyl)-L-alpha-phosphatidylcho line was 91 mol mol-1 s-1. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK +/- CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4-5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the antioxidant properties of a methanolic extract from 'Juice Plus fruit' and 'Juice Plus vegetable' (dietary supplements)

Abstract The antioxidant properties of methanolic extracts from ‘Juice Plus fruit’ and ‘Juice Plus vegetable’, which are sold in capsule form as dietary supplements, were evaluated using a range of established in vitro antioxidant assays. The values are compared to representative extracts from equivalent amounts (by dry weight) of selected fruits and vegetables prepared by the same method. Although there are some differences between the results of each assay, in general Juice Plus performed about equally to the fresh fruit and vegetables on a dry weight basis. This implies that, based on in vitro assays to measure antioxidant potential, one capsule of ‘Juice Plus fruit’ or ‘Juice Plus vegetable’ (weighing 1 g) is equivalent to about 10 g (fresh weight) of fruit or vegetable.

An investigation of the dissociation and denaturation of legumin by salts using laser light scattering and circular dichroism spectroscopy

The dissociation of legumin, a 12 S seed storage globulin from Pisum sativum, has been studied by laser light scattering and circular dichroism spectroscopy. Salts from the Hofmeister series, in particular sodium perchlorate, were used as dissociating agents. The Mr 360,000 hexameric protein was found to dissociate first to trimers and further to monomers and the number of amino acids involved in the trimer-trimer interaction estimated to be 23(+/-4). Native legumin appears to be more strongly bound together than some analogous seed storage globulins from other plant species such as Arachis hypogaea or Sesamum indicum and the dissociation process was accompanied by some changes in conformation.