Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-5 (of 5 Records) |
Query Trace: Ruwona TB[original query] |
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Production, characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins
Ruwona TB , Johnson VJ , Hettick JM , Schmechel D , Beezhold D , Wang W , Simoyi RH , Siegel PD . J Immunol Methods 2011 373 127-35 Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring. |
Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers
Chipinda I , Ruwona TB , Templeton SP , Siegel PD . Toxicology 2010 280 (3) 135-43 Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA),carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10muM for 24 and 48h. The direct-binding haptens dinitrochlorobenzene (DNCB) benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (+/-S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens. |
Monoclonal antibodies against toluene diisocyanate haptenated proteins from vapor-exposed mice
Ruwona TB , Johnson VJ , Schmechel D , Simoyi RH , Beezhold D , Siegel PD . Hybridoma (Larchmt) 2010 29 (3) 221-9 Toluene diisocyanate (TDI) is an industrially important polymer cross-linker used in the production of polyurethane. Workplace exposure to TDI and other diisocyanates is reported to be a leading cause of low molecular weight-induced occupational asthma (OA). Currently we have a limited understanding of the pathogenesis of OA. Monoclonal antibodies (MAbs) that recognize TDI bound proteins would be valuable tools/reagents, both in exposure monitoring and in TDI-induced asthma research. We sought to develop toluene diisocyanate (TDI)-specific MAbs for potential use in the development of standardized immunoassays for exposure and biomarker assessments. Mice were exposed 4 h/day for 12 consecutive weekdays to 50 ppb, 2,4;2,6 TDI vapor (80/20 mixture). Splenocytes were isolated 24 h after the last exposure for hybridoma production. Hybridomas were screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against a 2,4 TDI-human serum albumin (2,4 TDI-HSA) protein conjugate. Three hybridomas producing 2,4 TDI-HSA reactive IgM MAbs were obtained. The properties of these MAbs (isotype and reactivity to various protein-isocyanate conjugate epitopes) were characterized using ELISA, dot blot, and Western blot analyses. Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links, resulting in multimers and a change in the electrophoretic mobility of the conjugate. These antibodies may be useful tools for the isolation of endogenous diisocyanate-modified proteins after natural or experimental exposures and for characterization of the toxicity of specific dNCOs. |
Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol
Chipinda I , Ajibola RO , Morakinyo MK , Ruwona TB , Simoyi RH , Siegel PD . Chem Res Toxicol 2010 23 (5) 918-25 The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens. |
Structural elucidation of isocyanate-peptide adducts using tandem mass spectrometry
Hettick JM , Ruwona TB , Siegel PD . J Am Soc Mass Spectrom 2009 20 (8) 1567-75 Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when isocyanates are reacted with model peptides, tandem mass spectrometry was employed to unambiguously identify the binding site of four commercially-relevant isocyanates on model peptides. In each case, the isocyanates react preferentially with the N-terminus of the peptide. No evidence of side-chain/isocyanate adduct formation exclusive of the N-terminus was observed. However, significant intra-molecular diisocyanate crosslinking was observed between the N-terminal amine and a side-chain amine of arginine, when Arg was located within two residues of the N-terminus. Addition of multiple isocyanates to the peptide occurs via polymerization of the isocyanate at the N-terminus, rather than via addition of multiple isocyanate molecules to varied residues within the peptide. The direct observation of isocyanate binding to the N-terminus of peptides under these experimental conditions is in good agreement with previous studies on the relative reaction rate of isocyanate with amino acid functional groups. |
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