Molecular Characterization and Experimental Utility of Monoclonal Antibodies with Specificity for Aliphatic Di- and Polyisocyanates.

Aliphatic di- and polyisocyanates are crucial chemical ingredients in many industrial processes and are a well-recognized cause of occupational asthma. Serologic detection of "chemical epitopes" in biological samples could serve as an exposure surveillance approach toward disease prevention, and thus we sought to generate aliphatic isocyanate-specific monoclonal antibodies (mAbs). Three hybridomas were generated from Balb/c mice immunized with a commercial product containing a combination of uretdione, homopolymer, and monomeric forms of hexamethylene diisocyanate (HDI). Three stable hybridomas were subcloned by limiting dilution, two secreting IgG1κ and one secreting IgMκ mAb that bind aliphatic di- and polyisocyanates (conjugated to albumin), but not aromatic toluene or methylene diphenyl diisocyanate (TDI or MDI). Each mAb demonstrates slight differences in epitope specificity, for example, recognition of hydrogenated MDI (HMDI) or different carrier proteins (transferrin, actin) reacted with vapor phase HDI, and is encoded by unique recombination of different germline antibody genes, with distinct complementary determining regions. By western blot, all three mAbs detect a molecule with characteristics of an albumin adduct uniquely in urine from mice skin exposed to a mixture of aliphatic di- and polyisocyanate. Together, the data define molecular determinants of humoral immune recognition of aliphatic di- and polyisocyanates through new mAbs, which will serve as useful research reagents and may be applicable to future exposure surveillance efforts.

Nasal protein profiles in work-related asthma caused by different exposures.

BACKGROUND: The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. METHODS: We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. RESULTS: We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. CONCLUSIONS: Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma.

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.

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.

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.

Toluene diisocyanate and methylene diphenyl diisocyanate: asthmatic response and cross-reactivity in a mouse model.

Both 2,4-toluene diisocyanate (TDI) and 4,4-methylene diphenyl diisocyanate (MDI) can cause occupational asthma. In this study, we optimized our mouse model of chemical-induced asthma in the C57Bl/6 mice strain using the model agent TDI. Furthermore, we validated MDI in this mouse model and investigated whether cross-reactivity between TDI and MDI is present. On days 1 and 8, C57Bl/6 mice were dermally treated (20 µl/ear) with 3 % MDI, 2 % TDI or the vehicle acetone olive oil (AOO) (3:2). On day 15, they received a single oropharyngeal challenge with 0.04 % MDI, 0.01 % TDI or the vehicle AOO (4:1). One day later, airway hyperreactivity (AHR) and pulmonary inflammation in the bronchoalveolar lavage (BAL) were assessed. Furthermore, total serum IgE levels, lymphocyte subpopulations in auricular lymph nodes and cytokine levels in supernatants of lymphocytes were measured. Both dermal sensitization with TDI or MDI resulted in increased total serum IgE levels along with T and B cell proliferation in the auricular lymph nodes. The auricular lymphocytes showed an increased release of both Th2 and Th1 cytokines. Mice sensitized and challenged with either TDI or MDI showed AHR, along with a predominant neutrophil lung inflammation. Mice sensitized with MDI and challenged with TDI or the other way around showed no AHR, nor BAL inflammation. Both TDI and MDI are able to induce an asthma-like response in this mouse model. However, cross-reactivity between both diisocyanates remained absent.

Immunochemical detection of the occupational allergen, methylene diphenyl diisocyanate (MDI), in situ.

Diisocyanate chemicals essential to polyurethane production are a well-recognized cause of occupational asthma. The pathogenesis of diisocyanate-induced asthma, including the pathways by which the chemical is taken up and its distribution in exposed tissue, especially the lung, remains unclear. We developed an antiserum with specificity for methylene diphenyl diisocyanate (MDI) the most abundantly produced and utilized diisocyanate world-wide, and established its ability to detect MDI in situ. Polyclonal MDI-specific IgG was induced by immunizing rabbits with MDI-conjugated to keyhole limpet hemocyanin (KLH) emulsified in complete Freund's adjuvant, followed by two booster injections with incomplete Freund's adjuvant. The antiserum contains IgG that recognize a variety of different MDI conjugated proteins, but not unconjugated or mock exposed proteins by dot blot analysis. The antiserum further demonstrates specificity for proteins conjugated with MDI, but not other commonly used diisocyanates. Immunochemical studies with cytospun airway cells and formalin-fixed paraffin embedded lung tissue sections from mice intranasally exposed to MDI (as reversibly reactive glutathione conjugates, e.g. GSH-MDI) demonstrated the antiserum's ability to detect MDI in tissue samples. The data demonstrate penetration of MDI into the lower airways, localized deposition in the epithelial region surrounding airways, and uptake by alveolar macrophages. The new immunochemical reagent should be useful for further studies delineating the uptake and tissue distribution of MDI, especially as it relates to adverse health effects from exposure.

Is the analysis of histamine and/or interleukin-4 release after isocyanate challenge useful in the identification of patients with IgE-mediated isocyanate asthma?

Isocyanates are a well-known and frequent cause of occupational asthma. The implementation of specific inhalation challenges (SICs) is the gold standard in asthma diagnosis supporting occupational case history, lung function testing, specific skin prick tests and the detection of specific IgE. However, the diagnosis is not always definitive. An interesting new approach, analyses of individual genetic susceptibilities, requires discrimination between a positive SIC reaction arising from IgE-mediated immune responses and one from other pathophysiological mechanisms. Hence, additional refinement tools would be helpful in defining sub-classes of occupational asthma and diagnosis. We used total IgE levels, specific IgE and SIC results for sub-classification of 27 symptomatic isocyanate workers studied. Some mutations in glutathione S-transferases (GSTs) are suspected either to enhance or to decrease the individual risk in the development of isocyanate asthma. Our patient groups were assessed for the point mutations GSTP1*I105V and GSTP1*A114V as well as deletions (null mutations) of GSTM1 and GSTT1. There seems to be a higher risk in developing IgE-mediated reactions when GSTM1 is deleted, while GSTT1 deletions were found more frequently in the SIC positive group. Blood samples taken before SIC, 30-60 min and 24h after SIC, were analyzed for histamine and IL-4, classical markers for the IgE-mediated antigen-specific activation of basophils or mast cells. We suggest that the utility of histamine measurements might provide an additional useful marker reflecting isocyanate-induced cellular reactions (although the sampling times require optimization). The promising measurement of IL-4 is not feasible at present due to the lack of a reliable, validated assay.

Effects of MBL2 polymorphisms in patients with diisocyanate-induced occupational asthma.

Diisocyanate (DI) is the most common cause of occupational asthma (OA) in Korea. Mannose-binding lectin (MBL) initiates the lectin complement activation pathway following oxidative stress and plays an important role in the regulation of inflammatory processes. To determine whether there is a genetic association between MBL2 polymorphisms and DI-OA, 99 patients with DI-OA, 99 asymptomatic exposed controls (AECs) and 144 unexposed normal controls were enrolled in this study. Three polymorphisms (-554 G>C, -431A>C and -225 G>C) in the MBL2 promoter were genotyped, and serum MBL levels were determined by enzyme-linked immunosorbent assay. Functional variabilities in the promoter polymorphisms were analyzed by a luciferase reporter assay and electrophoretic mobility shift assay (EMSA). A significantly higher frequency of haplotype (ht) 2 [CAG] was noted in the DI-OA group compared with the AEC group (P=0.044). The patients with DI-OA carrying ht2 [CAG] had significantly lower PC20 methacholine levels (P<0.001) than the non-carriers. The serum MBL levels were significantly higher in the DI-exposed subjects (both the DI-OA patients and AECs) carrying ht1 [GAG] (P=0.028). Luciferase activity was significantly enhanced in ht1 [GAG] compared with ht2 [CAG] in human hepatocarcinoma cells (Hep3B) (P=0.002). The EMSA showed that a -554G probe produced a specific shifted band compared with the -554C probe. These findings suggest that decreased serum MBL levels due to polymorphisms of the MBL2 gene may increase susceptibility to the development of DI-OA in DI-exposed individuals.

Exhaled nitric oxide in spray painters exposed to isocyanates: effect modification by atopy and smoking.

BACKGROUND: Isocyanate asthma is one of the most frequently identified forms of occupational asthma in industrialised countries. The underlying mechanisms have not been clarified. There is only limited information about the relationship between exhaled nitric oxide (eNO) and occupational exposure to isocyanates and asthma. OBJECTIVES: To investigate the association between isocyanate exposure and eNO levels in isocyanate-exposed workers and to elucidate whether eNO acts as a marker of airway inflammation controlling for smoking and atopy in an industry-wide survey. METHODS: Information on estimated personal isocyanate exposure, measured eNO levels, health effects and sensitisation were analysed in 229 workers from a cross-sectional study. Univariate and multiple regression analyses were used to explore the exposure-response relationships between isocyanate exposure and eNO, stratified by smoking and atopy. RESULTS: A marginally significant exposure-response relationship was found between isocyanate exposure and eNO in atopic, non-smokers (p=0.054). eNO was significantly associated with atopy and smoking, bronchial hyper-responsiveness (BHR), work-related conjunctivitis and rhinitis after adjustment for age, gender, atopy and smoking (p<0.05). A borderline significant association was found between eNO and asthma-like symptoms after adjustment for age, gender, atopy and current smoking (p=0.055). In a small group of isocyanate-exposed workers with positive serum-specific immunoglobulin E (IgE) antibodies to hexamethylene diisocyanate (HDI), elevated eNO levels were clearly exposure related. eNO was associated with the positive specific IgG antibodies to HDI in non-atopic, non-smokers (p=0.03). CONCLUSIONS: Increased eNO levels may indicate increased airway inflammation in atopic, non-smokers exposed to isocyanates especially at higher levels of isocyanate exposure.

A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates.

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 µg 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.

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 1.70 ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06 ng/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 26.92 ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93 ng/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.

Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure.

Methylene diphenyl diisocyanate (MDI) is among the leading chemical causes of occupational asthma world-wide, however, the mechanisms of disease pathogenesis remain unclear. This study tests the hypothesis that glutathione (GSH) reacts with MDI to form quasi-stable conjugates, capable of mediating the formation of MDI-conjugated "self" protein antigens, which may participate in pathogenic inflammatory responses. To test this hypothesis, an occupationally relevant dose of MDI (0.1%w/v) was reacted with varying concentrations of GSH (10µM-10mM), and the reaction products were characterized with regard to mass/structure, and ability to carbamoylate human albumin, a major carrier protein for MDI in vivo. LC-MS/MS analysis of GSH-MDI reaction products identified products possessing the exact mass of previously described S-linked bis(GSH)-MDI and its partial hydrolysis product, as well as novel cyclized GSH-MDI structures. Upon co-incubation of GSH-MDI reaction products with human albumin, MDI was rapidly transferred to specific lysines of albumin, and the protein's native conformation/charge was altered, based on electrophoretic mobility. Three types of modification were observed, intra-molecular MDI cross-linking, addition of partially hydrolyzed MDI, and addition of "MDI-GSH", where MDI's 2nd NCO had reacted with GSH's "N-terminus". Importantly, human albumin carbamoylated by GSH-MDI was specifically recognized by serum IgG from MDI exposed workers, with binding dependent upon the starting GSH concentration, pH, and NaCl levels. Together, the data define a non-enzymatic, thiol-mediated transcarbamoylating mechanism by which GSH may promote immune responses to MDI exposure, and identify specific factors that might further modulate this process.

Molecular determinants of humoral immune specificity for the occupational allergen, methylene diphenyl diisocyanate.

Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents.

Is specific IgE antibody analysis feasible for the diagnosis of methylenediphenyl diisocyanate-induced occupational asthma?

PURPOSE: Early recognition improves the prognosis of isocyanate asthma. A major unanswered question is whether IgE-dependent mechanisms are of diagnostic value? Our objective was to appraise serological methods using various methylenediphenyl diisocyanate (MDI)-albumin conjugates and weigh up the data versus the outcome of standardized comprehensive clinical diagnostics to evaluate the viability of immunological analysis in supportive MDI-asthma diagnosis (OAI). METHODS: Specific IgE (sIgE) and IgG (sIgG) binding was measured with fluorescence enzyme immunoassay in 43 study subjects (using conjugates prepared in-vapor, in-solution and commercial preparations). The differential clinical diagnosis included standardized measurement of pulmonary function, non-specific bronchial hyper-responsiveness, specific MDI-prick test (MDI-SPT) and specific inhalation challenge (MDI-SIC). RESULTS: Detailed diagnostic scheme allows the differential OAI and MDI-induced hypersensitivity pneumonitis (PI). The presumed OAI diagnoses were confirmed in 84 % (45 % cases having demonstrable sIgE antibodies) with RR 5.7, P > 0.001, when OAI diagnosis is correlated with MDI-SIC/MDI-SPT (RR 1.28 for MDI-SIC alone); sIgG antibodies were clinically relevant for PI and not for the OA diagnosis. MDI-specific IgE data generated with commercial ImmunoCAP preparations show high correlation with our in-vapor generated MDI conjugates. CONCLUSIONS: Isocyanate-specific IgE antibodies are not always detectable but their presence is strongly predictive of OAI and supportive for the diagnosis. MDI-SPT can be a valuable parameter differentiating OAI and PI. We have confirmed and extended published data showing that isocyanate-albumin conjugates perform better in specific antibody assays when prepared with volatile phase formulations and would like to stress additionally the necessity for further refinements and standardization in clinical diagnostics procedures.

Pro/Con debate: Is occupational asthma induced by isocyanates an immunoglobulin E-mediated disease?

Isocyanates, low-molecular weight chemicals essential to polyurethane production, are one of the most common causes of occupational asthma, yet the mechanisms by which exposure leads to disease remain unclear. While isocyanate asthma closely mirrors other Type I Immune Hypersensitivity (Allergic) disorders, one important characteristic of hypersensitivity ('allergen'-specific IgE) is reportedly absent in a large portion of affected individuals. This variation from common environmental asthma (which typically is induced by high-molecular weight allergens) is important for two reasons. (1) Allergen-specific IgE is an important mediator of many of the symptoms of bronchial hyper-reactivity in 'allergic asthma'. Lack of allergen-specific IgE in isocyanate hypersensitive individuals suggests differences in pathogenic mechanisms, with potentially unique targets for prevention and therapy. (2) Allergen-specific IgE forms the basis of the most commonly used diagnostic tests for hypersensitivity (skin prick and RAST). Without allergen-specific IgE, isocyanates may go unrecognized as the cause of asthma. In hypersensitive individuals, chronic exposure can lead to bronchial hyperreactivity that persists years after exposure ceases. Thus, the question of whether or not isocyanate asthma is an IgE-mediated disease, has important implications for disease screening/surveillance, diagnosis, treatment and prevention. The present Pro/Con Debate, addresses contemporary, controversial issues regarding IgE in isocyanate asthma.

Monoclonal antibodies against toluene diisocyanate haptenated proteins from vapor-exposed mice.

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.

Immunochemical detection of food-derived isothiocyanate as a lysine conjugate.

In a previous study we prepared monoclonal antibody against allyl isothiocyanate (AITC)-modified lysine (Lys), and found that AITC reacted with Lys under physiological conditions in vitro (T. Nakamura et al., Chem. Res. Toxicol., 22, 536-542 (2009)). In the present study, antibodies against benzyl isothiocyanate (ITC), 6-methylsulfinylhexyl ITC and phenethyl ITC modified protein were prepared, and the respective monoclonal antibodies, B6C9, 6MS3D10, and PE3A10 were obtained. These antibodies were applied to ITC detection in food using shredded Wasabia japonica (wasabi) and ground Carica papaya (papaya) seed by trapping ITC with biotin-labeled bovine serum albumin. ITC formation from the wasabi and papaya seed samples was confirmed using the antibodies in a dose-dependent manner. These antibodies might be applicable in identifying food-derived ITC.

[Three cases of isocyanate-induced hypersensitivity pneumonitis with different HRCT findings].

We reported 3 cases of hypersensitivity pneumonitis who had been exposed to polyurethane material containing diphenylmethane diisocyanate (MDI) for 1-12 weeks. They had cough, fever and dyspnea before admission. All 3 cases were diagnosed immunologically using anti-MDI antibodies. Chest HRCT findings of the 3 cases were different. Case 1 showed an organizing pneumonia (OP) pattern : patchy peripheral consolidation with air bronchogram and ground-glass opacities (GGO), and case 2 showed a nonspecific interstitial pneumonia (NSIP) pattern : consolidation and reticular shadows in both lower lobes. In addition, case 3 showed a hypersensitivity pneumonitis (HP) pattern : centrilobular GGO and diffuse granular shadows. All cases were successfully treated by corticosteroids alone. This suggests that different amounts of inhaled antigen can cause different HRCT findings.

Rats repeatedly exposed to toluene diisocyanate exhibit immune reactivity against methyl isocyanate-protein conjugates.

BACKGROUND: Volatile monoisocyanates are formed through thermal degradation when products containing polyurethane are heated. Repeated exposure to diisocyanates, such as toluene diisocyanate (TDI) are a well-known cause of occupational asthma. However, although monoisocyanates are abundant in occupational settings, there are few data concerning their ability to provoke immune reactions and asthma. We compared immune reactivity and respiratory disease following single or repeated inhalation exposures to the monoisocyanates methyl isocyanate (MIC) and isocyanic acid (ICA) with the effects of TDI. METHODS: Isocyanates were administrated either as single vapor exposures or as repeated intranasal instillations in rats. Adverse health effects were monitored by analyzing airway inflammation, respiratory function and weight gain. Immune reactivity caused by repeated exposures was studied by analysis of isocyanate-specific antibodies and airway infiltration of immune competent cells. RESULTS: Repeated exposures to TDI induced airway infiltration of neutrophils and lymphocytes, while neither MIC nor ICA provoked a detectable inflammatory response. Antibodies against isocyanate-albumin conjugates were detected in serum after both exposures to TDI and MIC, but not to ICA. TDI-exposed rats also displayed IgG antibodies against MIC-albumin conjugates. Even though MIC did not induce airway inflammation, single exposure provoked an increase in airway resistance and repeated exposures caused weight loss similar to that of TDI. CONCLUSIONS: Airway exposure to TDI produces an antibody response not only against TDI but also against MIC-protein conjugates. This indicates that immune reactivity against abundant monoisocyanates in occupational environments can occur in individuals pre-sensitized with low abundance but highly sensitizing diisocyanates.

Lung/skin connections in occupational lung disease.

PURPOSE OF REVIEW: Exposure to occupational and environmental agents can cause a spectrum of lung diseases that are predominantly immune-mediated. Research and prevention have focused primarily on the respiratory tract. Recent studies, however, suggest that the skin may also be an important route of exposure and site of sensitization. This article highlights key findings, focusing on isocyanate asthma and chronic beryllium disease. RECENT FINDINGS: Occupational lung diseases such as isocyanate asthma and chronic beryllium disease continue to occur despite reduced airborne exposures. Although challenging to quantify, recent studies have documented isocyanate and beryllium skin exposure, even with the use of personal protective clothing. Factors that impair skin barrier function, such as trauma, may promote sensitization to such agents. Animal studies demonstrate that skin exposure to isocyanates and protein allergens is highly effective at inducing sensitization, with subsequent inhalation challenge eliciting asthmatic responses. Limited clinical studies suggest a similar role for human skin exposure to certain sensitizing agents. SUMMARY: Recent findings support a greater focus on the role of skin exposure in the development of certain occupational and environmental lung diseases. Although further research is needed, it is prudent to reduce both skin and inhalation exposures.