Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-10 (of 10 Records) |
Query Trace: Bledsoe TA[original query] |
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The Influence of Diisocyanate Antigen Preparation Methodology on Monoclonal and Serum Antibody Recognition
Hagerman LM , Law BF , Bledsoe TA , Hettick JM , Kashon ML , Lemons AR , Wisnewski AV , Siegel PD . J Occup Environ Hyg 2016 13 (11) 829-39 Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences. |
Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin
Mhike M , Hettick JM , Chipinda I , Law BF , Bledsoe TA , Lemons AR , Nayak AP , Green BJ , Beezhold DH , Simoyi RH , Siegel PD . J Immunol Methods 2016 431 38-44 Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity. |
Respiratory morbidity in a coffee processing workplace with sentinel obliterative bronchiolitis cases
Bailey RL , Cox-Ganser JM , Duling MG , LeBouf RF , Martin SB Jr , Bledsoe TA , Green BJ , Kreiss K . Am J Ind Med 2015 58 (12) 1235-45 RATIONALE: Obliterative bronchiolitis in former coffee workers prompted a cross-sectional study of current workers. Diacetyl and 2,3-pentanedione levels were highest in areas for flavoring and grinding/packaging unflavored coffee. METHODS: We interviewed 75 (88%) workers, measured lung function, and created exposure groups based on work history. We calculated standardized morbidity ratios (SMRs) for symptoms and spirometric abnormalities. We examined health outcomes by exposure groups. RESULTS: SMRs were elevated 1.6-fold for dyspnea and 2.7-fold for obstruction. The exposure group working in both coffee flavoring and grinding/packaging of unflavored coffee areas had significantly lower mean ratio of forced expiratory volume in 1 s to forced vital capacity and percent predicted mid-expiratory flow than workers without such exposure. CONCLUSION: Current workers have occupational lung morbidity associated with high diacetyl and 2,3-pentanedione exposures, which were not limited to flavoring areas. |
Allergic sensitization in Canadian chronic rhinosinusitis patients
Green BJ , Beezhold DH , Gallinger Z , Barron CS , Melvin R , Bledsoe TA , Kashon ML , Sussman GL . Allergy Asthma Clin Immunol 2014 10 (1) 15 BACKGROUND: Chronic rhinosinusitis (CRS) is a societal burden and cause of morbidity in Canada; however, the prevalence of allergic sensitization in Canadian CRS patients has remained poorly characterized. OBJECTIVE: In this study, we used skin prick test (SPT) and specific immunoglobulin E (sIgE) and G (sIgG) titers to regionally relevant allergen sources in order to determine whether allergic sensitization is more prevalent in CRS patients compared to chronic idiopathic urticaria (CIU) control patients. METHODS: One hundred and fifty eight subjects (19-70 years of age) were recruited into the study. 101 subjects had a confirmed diagnostic history of CRS and 57 subjects with a clinical diagnosis of CIU were recruited as controls. Enrolled subjects underwent SPT to a panel of perennial and seasonal allergens and sIgE titers were quantified to selected environmental allergen mixes (grass, mold, and tree species) using Phadia ImmunoCAP. sIgG was additionally quantified to Alternaria alternata, Aspergillus versicolor, Cladosporium herbarum, and Stachybotrys atra. Differences between CRS and control CIU patient SPT and serological data were examined by chi-squared analysis and analysis of variance. RESULTS: Reactivity to at least one SPT extract occurred in 73% of CRS patients. Positive SPT reactivity to A. alternata (odds ratio (OR): 4.34, 95% confidence interval: 1.57, 12.02), cat (OR: 3.23, 95% CI: 1.16, 9.02), and ragweed (OR: 2.31, 95% CI: 1.02, 5.19) extracts were more prevalent in patients with CRS (p < 0.05). Although dust mite and timothy grass sensitization approached statistical significance in the chi-squared analysis of SPT data, other common perennial and seasonal allergens were not associated with CRS. No statistically significant differences were observed between mean sIgE and sIgG titers in CRS and control patients. CONCLUSIONS: This study supports previous data that suggests A. alternata sensitization is associated with CRS; however, these findings additionally highlight the contribution of other regionally important allergens including cat and ragweed. |
A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates
Lemons AR , Siegel PD , Mhike M , Law BF , Hettick JM , Bledsoe TA , Nayak AP , Beezhold DH , Green BJ . J Occup Environ Hyg 2014 11 (2) 101-10 Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 mug of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers. |
Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts
Lemons AR , Bledsoe TA , Siegel PD , Beezhold DH , Green BJ . J Immunol Methods 2013 397 66-70 Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures. |
Fungal and atopic sensitization are low among farmers in the Agricultural Health Study
Endres SM , Green BJ , Henneberger PK , Germolec DR , Bledsoe TA , Beezhold DH , London SJ , Alavanja MC , Beane Freeman LE , Hoppin JA . J Allergy Clin Immunol 2012 130 (1) 267-70 e1 Prevalence of fungal sensitization and atopy was lower among farmers than the US population. Fungal sensitization was related to growing specific agricultural commodities. |
Post-hire asthma among insect-rearing workers
Suarthana E , Shen A , Henneberger PK , Kreiss K , Leppla NC , Bueller D , Lewis DM , Bledsoe TA , Janotka E , Petsonk EL . J Occup Environ Med 2012 54 (3) 310-317 OBJECTIVE: To evaluate the incidence of post-hire asthma (PHA) among insect-rearing workers, defined as asthma, the symptoms of which appeared after hire at the current workplace. METHODS: We surveyed the health of workers at three insect-rearing facilities and an associated office facility. We calculated the incidence and estimated hazard ratios for PHA. RESULTS: Post-hire asthma incidence in 157 insect-rearing workers was 16.2 per 1000 person-years compared with 9.2 per 1,000 person-years in 70 office workers. Workers with predominant exposure to Lepidoptera had an incidence of 26.9 per 1000 person-years and a hazard ratio of 5.5 (95% confidence interval: 1.6 to 23.9) adjusted for sex, race, and parental asthma. In contrast, the presence of specific immunoglobulin E to Lepidoptera antigens was not associated with PHA. CONCLUSION: Insect-rearing workers had a high incidence of PHA, primarily accounted for by workplace exposure to Lepidoptera. |
Occupational sensitization to soy allergens in workers at a processing facility
Green BJ , Cummings KJ , Rittenour WR , Hettick JM , Bledsoe TA , Blachere FM , Siegel PD , Gaughan DM , Kullman GJ , Kreiss K , Cox-Ganser J , Beezhold DH . Clin Exp Allergy 2011 41 (7) 1022-30 BACKGROUND: Exposure to soy antigens has been associated with asthma in community outbreaks and in some workplaces. Recently, 135 soy flake processing workers (SPWs) in a Tennessee facility were evaluated for immune reactivity to soy. Allergic sensitization to soy was common and was five times more prevalent than in health care worker controls (HCWs) with no known soy exposure. OBJECTIVE: To characterize sensitization to soy allergens in SPWs. Methods Sera that were positive to soy ImmunoCAP (n=27) were tested in IgE immunoblots. Wild-type (WT) and transgenic (TG) antigens were sequenced using nanoscale Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (nanoUPLC MS/MS). IgE reactivity towards 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSP), a protein found in TG soy, was additionally investigated. De-identified sera from 50 HCWs were used as a control. Results Immunoblotting of WT and TG soy flake extracts revealed IgE against multiple soy antigens with reactivity towards 48, 54, and 62 kDa bands being the most common. The prominent proteins that bound SPW IgE were identified by nanoUPLC MS/MS analysis to be the high molecular weight soybean storage proteins, beta-conglycinin (Gly m 5), and Glycinin (Gly m 6). No specific IgE reactivity could be detected to lower molecular weight soy allergens, Gly m 1 and Gly m 2, in soybean hull (SH) extracts. IgE reactivity was comparable between WT and TG extracts; however, IgE antibodies to CP4-EPSP could not be detected. CONCLUSIONS AND CLINICAL RELEVANCE: SPWs with specific IgE to soy reacted most commonly with higher molecular weight soybean storage proteins compared with the lower molecular weight SH allergens identified in community asthma studies. IgE reactivity was comparable between WT and TG soy extracts, while no IgE reactivity to CP4-EPSP was observed. High molecular weight soybean storage allergens, Gly m 5 and Gly m 6, may be respiratory sensitizers in occupational exposed SPWs. |
Allergen content of patient problem and nonproblem gloves: relationship to allergen-specific patch-test findings
Siegel PD , Fowler Jr JF , Storrs FJ , Sasseville D , Pratt M , Bledsoe TA , Law BF , Beezhold D , Zug K , Fowler LM . Dermatitis 2010 21 (2) 77-83 BACKGROUND: Identification of putative contact allergen and source material is often done by a combination of patch testing and manufacturer-supplied product information. The accuracy of the identification of allergen-source material and level of allergen in that allergen-source material is not known. OBJECTIVE: The objectives of the study were to survey the chemical allergen content of glove allergic contact dermatitis (ACD) patient-identified problem and nonproblem gloves and to evaluate the ability of the patient to discriminate between problem and nonproblem gloves. METHODS: Gloves from patch-tested rubber allergen-positive ACD patients were analyzed for species and amount of rubber allergen. RESULTS: Approximately half the subjects were able to correctly identify their problem and nonproblem gloves. Correct association of a glove with ACD was directly related to patch-test reaction severity and inversely related to the number of glove brands being used by the patient. Of note, thiurams were not detected in any of the gloves examined. CONCLUSIONS: Although patch testing is invaluable in identifying individual allergen sensitivities, the identification of the ACD-causative specific chemical allergen and source material remains problematic. All glove brands used within days prior to and during an ACD episode should be considered potential sources of the contact allergen. |
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