Comprehensive metabolomics identifies the alarmin uric acid as a critical signal for the induction of peanut allergy.

BACKGROUND: Food allergy, in particular peanut allergy, is a growing concern in Western countries. The prevalence of allergy to peanut, which currently stands at 1.4%, nearly tripled between 1997 and 2008. Allergic sensitization is a particularly difficult process to study as it is clinically silent. We sought to identify key pathways and mediators critically involved in the induction of allergic sensitization to peanut. METHODS: Comprehensive metabolomics analysis with liquid chromatography-mass spectrometry was used to detect metabolite changes in mice (C57BL/6) undergoing sensitization. Loss-of-function and gain-of-function studies were performed in mice subjected to two models of peanut sensitization and anaphylaxis that involved either oral or epicutaneous sensitization. Flow cytometric analyses on dendritic cells (DCs) in vitro and in vivo were used to investigate the mechanisms of immune activation. RESULTS: Elevated levels of uric acid (UA) were detected in mice undergoing sensitization as well as in peanut-allergic children who were not challenged with peanut. In mice, the depletion of UA during sensitization prevented the development of peanut-specific immunoglobulins IgE and IgG1 as well as anaphylaxis while exogenous delivery of UA crystals (monosodium urate, MSU) restored the allergic phenotype. Monosodium urate enhanced CD86 and OX40L expression on DCs, independent of Toll-like receptors 2 and 4, the NLRP3 inflammasome, and IL-1ß, via a PI3K signaling pathway. CONCLUSION: Overproduction of the UA alarmin in the local microenvironment plays a critical role in the induction of peanut-allergic sensitization, likely due to its ability to activate DCs. These finding suggest that cellular damage or tissue injury may be an essential requisite for the development of allergic sensitization to foods.

Sticky windows: chemical and biological characteristics of the organic film derived from particulate and gas-phase air contaminants found on an urban impervious surface.

A novel environmentally derived mixture that integrates exposure to atmospherically derived gas- and particle-phase compounds in urban areas-namely, the organic film that develops as a thin layer on urban impervious surfaces-was investigated for its ability to induce gene expression via the aryl hydrocarbon receptor (AhR). The organic film on window glass from 21 sites in downtown Toronto (Ontario, Canada) was found to contain a complex mixture of environmental contaminants typical of urban environments, notably PAHs, n-alkanes, PCBs, organochlorine (OC) pesticides, and polar constituents. Using a stably transfected reporter cell line, we found that the crude extract of organic film induces AhR-dependent gene expression in a dose-dependent fashion. Three subfractions of the crude extract induced significant luciferase expression: nonpolar aromatic > polar aromatic > nonpolar aliphatic. Recombination of the fractions did not lead to recovery of the full activity of the crude extract, which may indicate that some of the compounds lost during fractionation were significant contributors to the induction observed with the crude extract. The interactions between a tonic dose of B[ a]P (10(-7) M) and each of the aromatic fractions were determined to be antagonistic following analysis by the method of isoboles. Our results suggest that organic film makes up a diverse array of compounds active at the AhR and that these compounds may not interact in a strictly additive manner.

Atmospherically derived organic surface films along an urban-rural gradient.

Atmospherically derived organic films have been found on an impervious surface along an urban-rural gradient in the Toronto, Ontario, Canada area. Film thickness and concentrations, expressed on an aerial basis, of sigman-alkanes, sigmaPCB, sigmaPAH, and sigmaOC (organochlorine) pesticides decrease along this gradient, coincident with lower atmospheric emissions (PCB and PAH) and less accumulation in thinner rural films (OC pesticides). For PCBs and some OC pesticides, patterns of chemical abundance also shift, indicating a "fresh" pattern near emission sources (downtown) versus aged patterns at rural locations that are indicative of atmospheric transport. Plant-derived n-alkane concentrations were greater at urban than rural sites, and we hypothesize greater urban plant wax production and erosion due to air pollution. As expected, along the urban-rural gradient the concentration of particle-phase PAH decreased more rapidly than that of gas-phase compounds, but unexpectedly the contribution of alkylated PAH increased from urban to rural locations. Distances over which concentrations decline by 63% vary from 50 km for persistent gas-phase compounds (e.g., P1,2CB) to 10-20 km for reactive gas-phase compounds (e.g., naphthalene, fluoranthene) to <5-10 km for particle-phase compounds (e.g., P6-10CB, benzo[b]fluoranthene, benzo[e]pyrene).

Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts.

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides. which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted.

Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills.

Despite widespread industrial release of genotoxic contaminants, little is understood of their role in inducing germline mutations in natural populations. We used multilocus DNA fingerprinting to quantify germline minisatellite mutations in families of herring gulls (Larus argentatus) in three nesting categories: (a) near cities with large steel mills operating coking ovens; (b) near cities without steel mills; and (c) in rural locations removed from point sources of contamination. Gulls nesting near integrated steel mills showed significantly higher mutation rates than gulls from rural locations (Fisher's exact, P=0.0004); urban sites without steel mills fell midway between steel and rural sites (difference from rural; Fisher's exact, P=0.19). Distance of the nesting location of herring gulls from the steel industries' coking ovens was negatively correlated with minisatellite mutation rate demonstrating significant risk for induced germline mutations in cities with steel operations (Kendall Tau; tau=0.119; P<0.0001).

Characterization of ginseng saponins using electrospray mass spectrometry and collision-induced dissociation experiments of metal-attachment ions.

Electrospray mass spectrometry (ESMS) and collision-induced dissociation (CID) methodologies have been developed for the structural characterization of ginseng saponins (ginsenosides). Ginsenosides are terpene glycosides containing a triterpene core to which one to four sugars may be attached. They are neutral molecules which readily form molecular metal-attachment ions in positive ion ESMS experiments. In the presence of ammonium hydroxide intense deprotonated ions are generated. Both positive and negative ion ESMS experiments were found to be useful for molecular mass and structure determination of ten ginsenoside standards. Negative ion experiments made possible the determination of the molecular mass of each ginsenoside standard, the mass of the triterpene core and the masses and sequences of the sugar residues. Positive ion ESMS experiments with the alkali metal cations Li+ or Na+ and the transition metal cations Co2+, Ni2+ and Zn2+ were also useful in determining molecular masses. These alkali and transition metal cations form strongly bonded attachment ions with the ginsenosides. As a result, the CID mass spectra of the metal attachment ions show a variety of (structure characteristic) fragmentations. These experiments can be used to determine the identity of the triterpene core, the types and attachment points of sugars to the core and the nature of the O-glycosidic linkages in the appended disaccharides. Combining the results from the negative and positive ion experiments provides a promising approach to the structure analysis of this class of natural products.

Chemical and biological profiles of sediments as indicators of sources of genotoxic contamination in Hamilton Harbour. Part I: analysis of polycyclic aromatic hydrocarbons and thia-arene compounds.

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using polycyclic aromatic hydrocarbons (PAH) and thia-arenes as source apportionment tracers. Ratios of selected PAH and ratios of monomethyl and dimethyl/ethyl dibenzothiophenes to the parent dibenzothiophenes were calculated. Thia-arene and PAH profiles of Standard Reference Material SRM 1649 (urban dust/organics), SRM 1650 (diesel), SRM 1597 (coal tar), Hamilton coal tar and a composite Hamilton air particulate sample provided source sample data. The gas chromatography-mass spectrometry (GC-MS) chromatograms of all sample extracts were dominated by homocyclic PAH but interpretation of PAH profiles with respect to source was difficult. In contrast, thia-arene analyses revealed more distinct differences in profiles of samples collected in different areas of the harbour, including the tributary. These results indicated that areas of coal tar-contaminated sediment are potential contributors to the overall contaminant burden of sediments and suspended sediments in Hamilton Harbour. These data also indicated that contaminants related to mobile combustion sources were entering the harbour via a major tributary.

Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour. Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay.

Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach. Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9. Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses. The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH). Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH. Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo[a]pyrene, indeno[cd]pyrene and dibenz[a,h]anthracene. The YG1025+S9 mutagenic activity profiles were similar for all samples. Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different. The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary. Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.

Analysis of high-molecular-mass polycyclic aromatic hydrocarbons in environmental samples using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) with molecular masses higher than 300 u were analysed using LC-atmospheric pressure chemical ionization (APCI) MS in extracts of environmental samples from Hamilton, Canada including zebra mussels from Hamilton Harbour, air particulate and coal tar. The LC-APCI-MS profiles of three molecular mass classes of PAHs (326 u, 350 u and 374 u) were compared to identify potential sources of PAH contamination in Hamilton Harbour. The Hamilton air particulate profile was also compared with an urban air reference standard (NIST SRM 1649) from Washington, DC, USA. Profiles of all extracts were similar and suggested an environmental predominance of PAHs within the three isomeric molecular mass classes studied. However, PAHs of molecular mass 326 u and 350 u were detected in extracts of coal tar and zebra mussels from Hamilton Harbour but were not detected in Hamilton air. These results indicated that some high-molecular-mass PAHs may be characteristic of contamination by coal tar.

Bioassay-directed fractionation of PAH of molecular mass 302 in coal tar-contaminated sediment.

Bioassay-directed fractionation was used to characterise genotoxic polycyclic aromatic hydrocarbons (PAH) of molecular mass 302 amu in organic solvent extracts of coal tar-contaminated sediment from Sydney Harbour, Nova Scotia. A normal phase HPLC technique was employed to separate PAH-rich solvent extracts into fractions containing PAH of single molecular mass classes. The 302 amu molecular mass fraction was isolated and further separated using reversed phase HPLC; subfractions were collected every 30 s and subjected to bioassay analyses with Salmonella typhimurium strain YG1025 with the addition of oxidative metabolism (4% S9). Compounds eluting in the most active subfractions included naphtho[2,1-alpha]pyrene and naphtho[2,3-alpha]pyrene. The results of this study underscore the significant contribution that molecular mass 302 PAH make to the biological activity of complex environmental mixtures.

The Synthesis, NMR Spectroscopy, and X-ray Structure of a New Rhenium N(2)S(2) Chelate Complex.

A new chelate, mercaptoacetyl-L-histidinyl-S-benzyl-L-cysteine methyl ester was synthesized by standard peptide coupling techniques and reacted with ReOCl(3)(PPh(3))(2) to give two diastereomers, 7a and 7b. The two isomers were separated by reversed-phase HPLC and characterized by NMR spectroscopy and electrospray mass spectrometry. An X-ray structure of one isomer, 7a, confirmed that the chelated complex was analogous to other Re-N(2)S(2) compounds in that it formed a square pyramidal complex where the four donor atoms were the base of the pyramid and the oxygen attached to the rhenium was at the apex. The S-benzyl group, as expected, was cleaved during the formation of 7, and the resulting complex was a zwitterion where the rhenium was formally -1 and the counterion was the protonated imidazole ring.

Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene.

The recently described catecholamine-absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high-specific-activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high-specific-activity substituted tetrahydronaphthalene derivative (6-hydroxy-[125I]iodo-[N-(N-2',4'- dinitrophenyl)aminopropyl]-2-amino-1,2,3,4-tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.

Cyclic strabismus without binocular function.

Various forms of cyclic strabismus have been described. A cyclic variation in binocular function modifying a tendency to strabismus is said to be the underlying mechanism. Cyclic esotropia developed in two adult patients, one having no demonstrable binocular function and the other having suffered complete unilateral visual loss in the affected eye. In each case, on the "good days" there was still a manifest esotropia, in contrast to those patients with cyclic esotropia and good binocular function in which the eyes are straight on the "good days." The presence of binocular function is not required for the development of cyclic strabismus, although the level of binocular function is correlated with the ocular alignment on the "good days."

Chemico/biological investigation of contaminated sediment from the Hamilton Harbour area of western Lake Ontario.

Highly contaminated sediment from the Hamilton Harbour area of western Lake Ontario was examined using a bioassay-directed fractionation methodology. A sediment sample was extracted using a Soxhlet apparatus and the resulting extract was fractionated into compound classes using an alumina clean-up step and high performance liquid chromatographic techniques. The resulting fractions were subjected to bioassays using TA98- and TA100-like strains modified by the inclusion of genes for the activating enzymes nitroreductase and O-acetyl-transferase. The majority of the mutagenic activity displayed by the sample extract was found to be present in the fraction containing the polycyclic aromatic hydrocarbons (PAH). Extracts of the PAH-containing fraction displayed dramatically higher responses with the TA100 type strains with metabolic activation. Further separation of the PAH-containing fraction showed the majority of the biological activity coeluted with PAH having molecular masses of 276, 278, and 302 amu.

Identification of novel catecholamine absorbing proteins in the central nervous system.

Several pharmacologically active catecholamines have been shown to react covalently with CNS proteins, namely species of 47, 40, 22, and 20 kDa. Of these, the 47-kDa protein showed the greatest incorporation of tritium following treatment with [3H]dopamine, [3H]ADTN, or [3H]N-propyl-norapomorphine. Labeling was accomplished by incubating the tritiated ligands with crude membrane preparations in the absence of reducing agents. These proteins displayed several unique characteristics: 1. The proteins are distributed throughout the CNS, but no evidence was found for their presence in other tissues; 2. The proteins have a unique pharmacological profile, interacting with dopamine, ADTN, N-propyl-norapomorphine, and apomorphine, but not with ligands specific for other proteins known to interact with these compounds; 3. The labeling of these proteins is not inhibited by several similar catecholamines and other catechols, suggesting specific structural requirements; and 4. These proteins exhibited stereoselectivity with respect to this labeling. These results demonstrate the existence of novel CNS proteins capable of covalently absorbing several physiologically important catecholamines in vitro.

The surgical treatment of traumatic Brown syndrome.

We present the results of surgical treatment in 13 patients who attended Moorfields Eye Hospital with traumatic Brown syndrome between 1975 and 1990. The average age was 34 years (range, 18 to 46 years), and in 12 cases the cause was a road traffic accident. Diplopia was the indication for surgery in all cases. Most patients had two surgical procedures; but in two patients, three were required. The outcome was assessed both subjectively and objectively. Objective assessment was by analysis of the preoperative and postoperative fields of binocular single vision and Hess charts which were available for 11 patients. The Hess charts were analyzed using a new scoring system. At final follow up, subjective improvement had occurred in 7 patients, 3 patients reported no improvement, and 3 patients thought their symptoms were worse. The field of binocular single vision improved in 8 patients and was worse in 3 patients. The Hess chart improved in 8 patients and was worse in 3 patients. The management of this difficult condition is discussed.

A scoring method for Hess charts.

A new scoring system for Hess charts is described. Scores for both horizontal and vertical deviations are calculated from the displacement of individual points on the Hess chart, using weighting factors for the centre, inner and outer zones. The scoring system was validated in a group of 24 patients undergoing surgery for paralytic or restrictive strabismus. Subjective scores were obtained from patients both before and after corrective surgery, and corresponding Hess charts were scored using the new system. There was a good correlation between the changes in subjective and objective score. Our scoring system has applications in both research and clinical practice, allowing objective analysis of changes in muscle balance in a variety of motility disorders.

Management of dysthyroid eye disease.

Once the diagnosis of thyroid eye disease has been made the doctor's problems begin because the management is long term with a condition that has many ramifications over a period of years. Short-term heroics to save vision by use of high-dose systemic steroids, orbital decompression or radiotherapy may have to be followed by control of diplopia by muscle surgery and prisms. Lid retraction, both upper and lower, may merit surgery, and even those patients who have come to terms with their facial appearance may still complain of lacrimation, irritation and grittiness. The advantages of a thyroid clinic, run jointly by a physician and an ophthalmologist, are presented and the patient is helped to live with this distressing condition.

Metabolic activation of 3-nitroperylene in the Salmonella/S9 assay.

The mutagenicity of 3-nitroperylene (nitroPer), a nitrated polycyclic aromatic hydrocarbon (PAH) has been extensively studied using the Ames Salmonella test. In accord with previous work, nitroPer proved to be a potent frameshift mutagen which requires activation by mixed function oxidases (MFOs) in the microsomes of rat liver S9 preparations. The concentration of S9 required for optimal activation of this mutagen is several times lower than that recommended for routine screening by the Ames test. Studies with the MFO inducers Aroclor 1254, phenobarbital, 3-methylcholanthrene and beta-naphthoflavone, as well as with some MFO inhibitors indicate that both P450b and P450c appear to be involved in the activation of nitroPer. Two non-mutagenic PAHs (perylene and benzo[e]pyrene) inhibited the mutagenicity of nitroPer in a competitive fashion. The mutagenic activity of nitroPer was greatly decreased in a strain of bacteria (TA98/1,8-DNP6) that lacks an acetyltransferase needed for the activation of many nitroarenes. Incubation of nitroPer with microsomes from Aroclor-treated rats plus appropriate cofactors led to the formation of several metabolites which could be separated from one another and from nitroPer by h.p.l.c. Three of these were direct-acting mutagens with Salmonella typhimurium strain TA98, while another required microsomal activation. We postulate that the metabolic activation of nitroPer requires three steps: (i) metabolism by MOF enzymes to yield a ring-oxidized compound which is absorbed by the bacteria; (ii) reduction of this compound to the hydroxylamine by a bacterial nitroreductase; and (iii) O-acetylation of the hydroxylamine to yield a reactive ultimate mutagen.