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. OBJECTIVE: We sought to investigate the role that Lactobacillus casei Shirota (LcS) may play in modulating seasonal allergic rhinitis (SAR). METHODS: The study format was double-blinded, placebo-controlled with 10 SAR sufferers in each group. We have documented and compared changes in immune status arising through the daily ingestion of a milk drink with or without live LcS, over a period of 5 months. Pre-, peak- and post-grass pollen season blood samples were collected for determination of plasma total IgE and grass pollen-specific IgG and IgE levels by an enzyme immunoassay. At the same time, cytokine levels were determined by flow cytometric bead array technology following culture of peripheral blood mononuclear cells for 6 days in the presence or absence of specific grass pollen antigens. RESULTS: Volunteers treated with LcS showed a significant reduction in levels of antigen-induced IL-5, IL-6 and IFN-gamma production compared with volunteers supplemented with placebo. Meanwhile, levels of specific IgG increased and IgE decreased in the probiotic group. CONCLUSION: Changes in antigen-induced production of cytokines were observed in patients treated with probiotics. These data show that probiotic supplementation modulates immune responses in allergic rhinitis and may have the potential to alleviate the severity of symptoms.

Investigation of the interactions between beta-lactams and a metallo-beta-lactamase from bacillus cereus using a monoclonal antibody.

A monoclonal antibody recognizing the active site of a beta-lactamase from Bacillus cereus was identified and characterized. The binding of the monoclonal antibody to the active site was quantitatively inhibited by a broad spectrum of beta-lactam antibiotics. The levels of inhibition were found to be associated with particular structural features of the antibiotics and their ability to form stable enzyme/substrate complexes. A novel, broad specificity assay for beta-lactams was developed based on the inhibition of antibody binding of all the beta-lactams studied. The assay is applicable to detection of beta-lactams at or close to the MRL level and would be complementary to existing receptor-based assays. The approach described is relevant to the study of kinetic aspects of beta-lactamases and could prove a useful tool in future drug development.

Are whole extracts and purified glucosinolates from cruciferous vegetables antioxidants?

Fruits and vegetables contain several classes of compounds that can potentially contribute to antioxidant activity, including vitamins, simple and complex phenolics, sulphur-containing compounds and glucosinolates. The glucosinolates are found in high concentration in many cruciferous vegetables, and it is well established that their breakdown products induce endogenous antioxidant defences such as quinone reductase and glutathione S-transferase in cells and in vivo. Despite the anticarcinogenic effect of these compounds in animal models, the direct antioxidant properties of this class of compounds have not been systematically studied. We therefore examined the free radical-scavenging properties of representative extracts and of purified glucosinolates from cruciferous vegetables, by measuring their effect on ascorbate- or NADPH/iron-induced peroxidation of human liver microsomes, ascorbate/iron-induced peroxidation on phospholipid liposomes, iron chelation and hydroxyl radical scavenging using the deoxyribose assay, total antioxidant potential using ABTS (2,2'-azinobis(3-ethyl-benzothiazoline-6-sulphonate)) and the bleomycin assay. Most of the extracts from cruciferous vegetables exhibited some antioxidant properties, although extracts from cooked Brussels sprouts increased the rate of microsomal lipid peroxidation. The effects in these assays were dependent upon processing and species of crucifer, and the glucosinolate content appeared to play a minor role in these effects, since purified glucosinolates exhibited only weak antioxidant properties. The total antioxidant activities of extracts from cooked and autolysed Brussels sprouts were identical within experimental error. This is probably due to the content of phenolics which is unaltered by autolysis, despite the differences between these samples in other assays especially NADPH-iron-induced lipid peroxidation of human liver microsomes. The results demonstrate that glucosinolates are unlikely to account for the direct antioxidant effects of extracts from cruciferous vegetables.

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

We have examined the effect of human cytochrome P450's (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 P450's play a significant role in ascorbate/iron peroxidation.

Direct separation of hydroperoxy- and hydroxy-phosphatidylcholine derivatives: application to the assay of phospholipid hydroperoxide glutathione peroxidase.

We have developed a method for assaying the activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) which is both more sensitive and specific than the spectrophotometric assay. The assay is based on the direct detection of the enzymatic product 1-palmitoyl-2-(13-hydroxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine by HPLC. Under the conditions used, baseline separation is achieved for product and substrate. The utility of the method is demonstrated by the measurement of PHGPx activity in crude extracts from human lenses and from human Hep G2 hepatoma cells. This method is also suitable for measuring the specificity of PHGPx for cofactors apart from glutathione. The assay was used to demonstrate that cysteine alone at pH 7.4 mimics PHGPx activity.

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)

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.