Evaluation of chemical-specific IgG antibodies in male workers from a urethane foam factory.

BACKGROUND: Plastic resins are complex chemicals that contain toluene diisocyanate (TDI) and/or trimellitic anhydride (TMA), which cause occupational allergies (OA), including respiratory allergies. Serum IgGs against TDI and TMA have been suggested as potential markers of the exposure status and as exploring cause of OA. Although TDI-specific IgG has been examined for suspected OA, TMA-specific IgG is not commonly evaluated in a urethane foam factory. This study therefore investigated both TDI- and TMA-specific IgGs in suspected OA patients and to evaluate the usefulness of the measurement of multiple chemical-specific IgG measurement for practical monitoring. METHODS: Blood samples were collected from two male workers who developed respiratory allergies supposedly caused by occupational exposure to TDI and/or TMA for the presence of TDI- and TMA-specific IgGs. In addition, blood samples from 75 male workers from a urethane foam factory, along with 87 male control subjects, were collected in 2014 and tested for the same IgGs in 2014. The presence and levels of TDI- and TMA-specific serum IgGs were measured using dot blot assays. RESULTS: We found that controls had mean concentrations of TDI- and TMA-specific IgGs of 0.98 and 2.10 µg/mL, respectively. In the two workers with respiratory allergies, the TDI-specific IgG concentrations were 15.6 and 9.51 µg/mL, and TMA-specific IgG concentrations were 4.56 and 14.4 µg/mL, which are clearly higher than those in controls. Mean concentrations of TDI- and TMA-specific IgGs in the factory workers were 1.89 and 2.41 µg/mL, respectively, and are significantly higher than those of the controls (P < 0.001 and P < 0.026 for TDI- and TMA-specific IgGs, respectively). CONCLUSION: The workers suspected of OA showed an evidently high level of TDI- and TMA-specific IgG, and these levels in workers at the urethane foam factory were also significantly higher than those in controls. In conclusion, the measurement of TDI- and TMA-specific IgG among workers using plastic resins is helpful to monitor their exposure status.

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.

Serum specific IgG response to toluene diisocyanate-tissue transglutaminase conjugate in toluene diisocyanate-induced occupational asthmatics.

BACKGROUND: Tissue transglutaminase (tTG) is a post-translational modifying enzyme located in airway epithelial cells. A potential contribution of serum specific IgG (sIgG) to tTG in airway inflammation of toluene diisocyanate (TDI)-induced occupational asthma (OA) has been suggested. OBJECTIVE: To prepare a TDI-tTG conjugate and detect serum specific antibodies in sera of patients with TDI-OA to understand this mechanism. METHODS: Ninety-nine patients with TDI-OA, 76 asymptomatic exposed controls, 208 patients with non-OA, and 74 unexposed controls were enrolled for this study. The TDI-tTG conjugate was prepared and confirmed by a native gel. Serum sIgG and/or sIgE antibodies to tTG, TDI-tTG, TDI conjugated to human serum albumin, cytokeratin 19, and serum cytokine levels, such as interleukin-8, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1, were measured by enzyme-linked immunosorbent assay. The level of interleukin-8 produced from airway epithelial cells (A549) treated with tTG was evaluated to investigate the inflammatory effect of tTG and TDI-tTG. RESULTS: In the TDI-OA group, the prevalence of serum sIgG to TDI-tTG (17.2%) was higher than that of sIgG to tTG (11.1%), which were significantly higher than those of the 3 control groups (P < .05 for all groups). TDI-exposed subjects with high levels of serum sIgG to TDI-tTG had a high prevalence of sIgG to cytokeratin 19 and higher serum levels of transforming growth factor-ß1 and tissue inhibitor of metalloproteinase-1. The tTG and TDI-tTG dose-dependently increased interleukin-8 production from A549 cells. CONCLUSION: These findings suggest that TDI exposure in the workplace binds to tTG to form a conjugate that can induce serum sIgG antibody production, airway inflammation, and airway remodeling in patients with TDI-OA.

Inception cohort study of workers exposed to toluene diisocyanate at a polyurethane foam factory: initial one-year follow-up.

BACKGROUND: Isocyanates are one of the most commonly reported causes of occupational asthma; however, the risks of developing isocyanate asthma in modern production facilities remain poorly defined. We evaluated TDI exposure and respiratory health among an inception cohort of workers during their first year of employment at a new polyurethane foam production factory. METHODS: Forty-nine newly hired workers were evaluated pre-employment, 6-months, and 12-months post-employment through questionnaire, spirometry, and TDI-specific serology. Airborne TDI levels were monitored by fixed-point air sampling and limited personal sampling. Qualitative surface SWYPE™ tests were performed to evaluate potential sources of skin exposure. RESULTS: Airborne TDI levels overall were low; over 90% of fixed-point air measurements were below the limit of detection (0.1 ppb). Over the first year of employment, 12 of the 49 original workers (24.5%) were lost to follow-up, no additional workers were enrolled, and seven of the 49 original workers (14.2%) developed either new asthma symptoms (N = 3), TDI-specific IgG (N = 1), new airflow obstruction (N = 1) and/or a decline in FEV1 ≥ 15% (N = 3), findings that could indicate TDI-related health effects. The prevalence of current asthma symptoms was significantly higher in the workers lost to follow-up compared to those who completed the 12-month follow-up (25% vs. 2.7%; P = 0.04). CONCLUSIONS: The findings suggest possible early TDI-related health effects in a modern polyurethane production plant. These findings also highlight the need for further longitudinal evaluation of these workers and the challenges of studying workers at risk for isocyanate asthma.

Mild improvement in symptoms and pulmonary function in a long-term follow-up of patients with toluene diisocyanate-induced asthma.

BACKGROUND: Long-term follow-up of diisocyanate-induced occupational asthma has been occasionally reported. METHODS: We studied the outcome of toluene diisocyanate (TDI)-induced asthma in 46 patients at diagnosis and after a follow-up of 11 ± 3.6 years. Symptoms, anti-asthma therapy, forced expiratory volume in 1 s (FEV1) and bronchial hyperresponsiveness to methacholine were assessed. RESULTS: A significant improvement in FEV1 (% predicted) and PD20FEV1 methacholine was observed at follow-up in comparison with diagnosis. Anti-asthma treatment was performed by 42% of patients at diagnosis and by 70% at follow-up. At the time of follow-up, 32 subjects had been removed from exposure for 6.0 ± 6.9 years, whereas 14 subjects continued to work with reduced exposure to TDI. There was a significant reduction in the prevalence of attacks of shortness of breath and dyspnoea at follow-up, but only in unexposed patients. PD20FEV1 was significantly improved only in patients with a lower FEV1 at diagnosis and in those who have ceased work. Logistic regression analysis, using different models with some independent variables, showed that there were no significant determinants of improvement in FEV1 at follow-up, while a shorter duration of symptoms before diagnosis was a significant predictor of improvement in PD20FEV1 at follow-up. CONCLUSIONS: Asthma-like symptoms, bronchial hyperresponsiveness and airway obstruction improved, but did not normalize, after a long-term follow-up with cessation or reduction in TDI exposure, mainly in subjects with an early diagnosis of occupational asthma and in patients with a lower baseline FEV1 no longer exposed to TDI.

Leukocyte nicotinamide adenine dinucleotide phosphate-reduced oxidase is required for isocyanate-induced lung inflammation.

BACKGROUND: Isocyanates are low-molecular-weight compounds noted for inducing occupational and environmental asthma. Isocyanate-induced lung disease, an oxidant stress-dependent pulmonary inflammation, is the leading cause of occupational asthma. OBJECTIVES: To address the role of leukocyte-produced oxidants in airway inflammation induced by toluene diisocyanate (TDI), and to elucidate the role of leukocyte nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase in pathogenesis by TDI. METHODS: Wild-type mice and NADPH oxidase-deficient mice (neutrophil cytosolic factor 1 mutant, Ncf1(-/-)) were intranasally injected, challenged with inhalatory TDI, and then investigated for lung inflammation. RESULTS: Cell infiltration in lung tissue and leukocytes in bronchoalveolar lavage, airway reactivity to a methacholine challenge, and TDI-induced inflammatory cytokine expression and nuclear factor activation in the lung tissue were all markedly lower in Ncf1(-/-) mice. Wild-type mice treated with blocking antibodies against CD4 and IL-17 showed markedly lower TDI-induced airway hyperresponsiveness. CONCLUSION: Leukocyte NADPH oxidase is an essential regulator in TDI-induced airway inflammation through redox modification of immune responses.

[Recent development in animal testing to predict the skin and respiratory sensitizing potential of chemicals].

The identification of chemicals with skin and/or respiratory sensitizing potential is important for the prevention of allergic diseases in both living and work environments. Although a number of animal models for respiratory allergic diseases have been reported, none of these models meets the goals of broad assessments of chemical sensitizing potential. We are attempting to develop a test for predicting the respiratory sensitization of chemicals. In the evaluation of skin sensitization of chemicals, the mostly used predictive tests are the guinea pig maximization test, Buehler test, and mouse local lymph node assay (LLNA). However, only LLNA has been validated formally and independently. Recent studies have revealed that EC3 estimated by LLNA correlates well with human skin sensitizing potency and the threshold for the induction of skin sensitization in the human repeat patch test. Thus, LLNA can predict the potency of skin sensitizing potential of a chemical and its risk in humans.

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.

The HLA DRB1*1501-DQB1*0602-DPB1*0501 haplotype is a risk factor for toluene diisocyanate-induced occupational asthma.

BACKGROUND: Although the pathogenesis of toluene diisocyanate (TDI)-induced occupational asthma (TDI-OA) is incompletely understood, several studies have suggested immunologic mechanisms, including specific IgE responses. A few studies have suggested human leukocyte antigen (HLA) associations with TDI-induced asthma in Western countries, but this is the first investigation of associations between HLA class I and II alleles and TDI-induced asthma patients in Asia, using high-resolution analysis. METHODS: Patients with TDI-OA (n = 84), asymptomatic exposed controls (AECs, n = 47) and unexposed normal controls (NCs, n = 127) were enrolled. HLA class I and II genotyping was performed by the direct DNA sequencing analysis. Specific serum IgE antibodies to the vapor type TDI-albumin conjugate were measured by ELISA. RESULTS: There was no significant association between the allele frequencies and the phenotype of TDI-OA. However, the frequency of the HLA DRB1*1501-DQB1*0602-DPB1*0501 haplotype was significantly higher in TDI-OA patients (19%) than in AEC (2.1%, p = 0.007, OR 4.429, CI 1.497-13.103) or NC (3.1%, p < 0.001, OR 7.235, CI 2.236-22.510) subjects, with statistical significance persisting after correction for multiple comparisons. DQB1*0402 was significantly associated with the presence of specific IgE to TDI-albumin conjugates in serum (p = 0.006, OR 4.552, CI 1.540-13.449). This p value remained significant after correction for multiple comparison. CONCLUSION: The HLA DRB1*1501-DQB1*0602-DPB1*0501 haplotype may be a genetic marker for the development of TDI-induced asthma in Koreans. Several HLA alleles that enhance specific IgE sensitization in exposed subjects are indicated.

Oropharyngeal aspiration: an alternative route for challenging in a mouse model of chemical-induced asthma.

BACKGROUND: To assess the importance of the route of challenge in an existing mouse model of chemical-induced asthma, we replaced intranasal instillation by oropharyngeal aspiration. To our knowledge, oropharyngeal aspiration as a challenge route has not yet been investigated in a mouse model of chemical-induced asthma. METHODS: On days 1 and 8, mice were dermally sensitized with toluene diisocyanate (TDI) (0.3%) [or vehicle (acetone/olive oil)] and on day 15 they received a single challenge, via oropharyngeal aspiration, with TDI (0.01%) or vehicle. One day after challenge, airway reactivity to methacholine was measured by a forced oscillation technique (FlexiVent) and total and differential cell counts, as well as levels of KC, IL-5, IL-17 and TNF-alpha, were assessed in the bronchoalveolar lavage (BAL) fluid. Lymphocytes from the auricular and mediastinal lymph nodes were cultured to determine the concanavaline A-induced secretion of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN-gamma. Total serum IgE was measured. RESULTS: In TDI-sensitized mice, a significant increase in airway reactivity was found after a single oropharyngeal challenge with TDI. BAL neutrophils and eosinophils were increased 7- and 5-fold, respectively. An upregulation of Th1 (IFN-gamma), Th2 (IL-4, IL-10, IL-13) and Th17 (IL-17) cytokines was found in the auricular lymph nodes, in the mediastinal lymph nodes only IL-4 was upregulated. The total serum IgE level in TDI-sensitized mice was significantly increased when compared to control mice. CONCLUSION: We conclude that challenging mice via oropharyngeal aspiration mimics the characteristics of human asthma well, without the possible drawbacks of other techniques.

Efficacy of repeated pretreatment with olopatadine hydrochloride on rhinitis induced by intranasal instillation of toluene-2,4-diisocyanate in rats.

It is well known that starting treatment for cedar pollinosis therapy with second-generation antihistamines before the initial day of pollen scattering can relieve nasal symptom severity during the pollen season. Olopatadine hydrochloride (olopatadine) is an antiallergic agent with histamine H(1) receptor antagonistic action. We have evaluated the effects of repeated preadministration of olopatadine on the toluene-2,4-diisocyanate-induced rhinitis in rats. A single administration of olopatadine suppressed sneezing and the increases in histamine, nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) production in nasal lavage fluid. When olopatadine was administered repeatedly once a day for 7 days before provocation, its inhibitory effects were enhanced compared to the effect of a single administration. Although the repeated administration of fexofenadine enhanced the inhibitory effects on sneezing, it did not inhibit the increases in NGF and VEGF production. These results show that the suppression of the increase in NGF and VEGF might partially be involved in the improvement of nasal allergy signs by the treatment with olopatadine. It is expected that the early treatment with olopatadine may achieve stable therapeutic effects.

How long do the systemic and ventilatory responses to toluene diisocyanate persist in dermally sensitized mice?

BACKGROUND: Years after removal from exposure, workers with occupational asthma still show respiratory symptoms and airway hyperresponsiveness on re-exposure to the offending agent. OBJECTIVE: We investigated the persistence of the respiratory responsiveness to toluene diisocyanate (TDI) in a mouse model. METHODS: BALB/C mice received dermal applications of TDI on days 1 and 8, and a single intranasal instillation of TDI on day 10, 15, 20, 25, 30, 40, 50, 60, or 90. After instillation, early (1 hour) changes in ventilatory function and methacholine responsiveness (22 hours) were assessed. Cell counts and macrophage inflammatory protein 2 were measured in bronchoalveolar lavage. Total serum IgE, IgG(1), and IgG(2a) were quantified. Lymphocyte subpopulations were assessed in auricular and cervical lymph nodes, and release of IL-4 and IFN-gamma by these lymph node cells was measured. RESULTS: Toluene diisocyanate-treated mice showed immediate ventilatory changes, increased methacholine reactivity, and an influx of neutrophils and macrophage inflammatory protein 2 in bronchoalveolar lavage as long as 50 days after initial treatment. These mice also showed a relative increase in CD19(+) cells and a decrease in CD4(+) and CD8(+) cells in auricular lymph nodes. Increased release of IL-4 and IFN-gamma in auricular lymph node cells was observed only until 20 days after sensitization. Total serum IgE, IgG(1), and IgG(2a) remained significantly elevated in TDI-sensitized mice until 90 days after dermal sensitization. CONCLUSION: Ventilatory and lung inflammatory responses decrease with increasing delay between sensitization and challenge, despite persistent humoral signs of sensitization.

Effect of olopatadine hydrochloride, an anti-histamine drug, on rhinitis induced by intranasal instillation of toluene-2,4-diisocyanate in rats.

The main symptoms of allergic rhinitis (AR) are sneezing, rhinorrhea and nasal obstruction. In patients with AR, levels of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) increase. Olopatadine hydrochloride (olopatadine) is an anti-allergic agent with histamine H1 receptor antagonistic action. To investigate whether olopatadine has an effect on inflammatory reactions, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as an animal model of nasal allergy. Nasal allergy signs (sneezing, rhinorrhea and inflammation) were induced after TDI challenge. Amounts of NGF and VEGF in the nasal lavage fluid increased. Olopatadine reduced nasal allergy signs and inhibited increases in NGF and VEGF. These findings suggest that the increases in NGF and VEGF production are involved in the mechanism responsible for nasal allergy signs in TDI-challenged rats. Other histamine H1 receptor antagonists did not inhibit and instillation of histamine did not increase TDI-induced NGF and VEGF production. Therefore, olopatadine appears to exert additional biological effects other than its blockade of the histamine H(1) receptor. These results suggest that suppression of neurogenic inflammatory reactions might be partially involved in the improvement of allergy signs after treatment with olopatadine.

Use of long term dermal sensitization followed by intratracheal challenge method to identify low-dose chemical-induced respiratory allergic responses in mice.

The inhalation of many types of chemicals, including pesticides, perfumes, and other low-molecular weight chemicals, is a leading cause of allergic respiratory diseases. We attempted to develop a new test protocol to detect environmental chemical-related respiratory hypersensitivity at low and weakly immunogenic doses. We used long-term dermal sensitization followed by a low-dose intratracheal challenge to evaluate sensitization by the well-known respiratory sensitizers trimellitic anhydride (TMA) and toluene diisocyanate (TDI) and the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them intratracheally with TMA, TDI, or DNCB, we assayed differential cell counts and chemokine levels in bronchoalveolar lavage fluid (BALF); lymphocyte counts, surface antigen expression of B cells, and local cytokine production in lung-associated lymph nodes (LNs); and antigen-specific IgE levels in serum and BALF. TMA induced marked increases in antigen-specific IgE levels in both serum and BALF, proliferation of eosinophils and chemokines (MCP-1, eotaxin, and MIP-1beta) in BALF, and proliferation of Th2 cytokines (interleukin (IL)-4, IL-10, and IL-13) in restimulated LN cells. TDI induced marked increases in levels of cytokines (IL-4, IL-10, IL-13, and IFN-gamma) produced by restimulated LN cells. In contrast, DNCB treatment yielded, at most, small, nonsignificant increases in all parameters. Our protocol thus detected respiratory allergic responses to low-molecular weight chemicals and may be useful for detecting environmental chemical-related respiratory allergy.

Detection of low-level environmental chemical allergy by a long-term sensitization method.

Multiple chemical sensitivity (MCS) is characterized by various signs, including neurological disorders and allergy. Exposure may occur through a major event, such as a chemical spill, or from long-term contact with chemicals at low levels. We are interested in the allergenicity of MCS and the detection of low-level chemical-related hypersensitivity. We used long-term sensitization followed by low-dose challenge to evaluate sensitization by well-known Th2 type sensitizers (trimellitic anhydride (TMA) and toluene diisocyanate (TDI)) and a Th1 type sensitizer (2,4-dinitrochlorobenzene (DNCB)). After topically sensitizing BALB/c mice (9 times in 3 weeks) and challenging them with TMA, TDI or DNCB, we assayed their auricular lymph nodes (LNs) for number of lymphocytes, surface antigen expression of B cells, and local cytokine production, and measured antigen-specific serum IgE levels. TMA and TDI induced marked increases in levels of antigen-specific serum IgE and of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) produced by ex vivo restimulated lymph node cells. DNCB induced a marked increase in Th1 cytokine (IL-2, IFN-gamma, and TNF-alpha) levels, but antigen-specific serum IgE levels were not elevated. All chemicals induced significant increases in number of lymphocytes and surface antigen expression of B cells. Our mouse model enabled the identification and characterization of chemical-related allergic reactions at low levels. This long-term sensitization method would be useful for detecting environmental chemical-related hypersensitivity.

Oral Administration of Bisphenol A Directly Exacerbates Allergic Airway Inflammation but Not Allergic Skin Inflammation in Mice.

Bisphenol A (BPA) is used in various areas of daily life as a major component of plastic products. However, it is also known as a strong endocrine disruptor that affects the human immune system. Studies have indicated that BPA possibly exacerbates allergic diseases such as atopic dermatitis and asthma. The main aim of this study was to elucidate whether BPA is directly involved in the exacerbation of allergic inflammation. Initially, in vivo experiments with mouse models of allergic inflammation induced by Th2 type hapten toluene-2, 4-diisocyanate (TDI) was performed. Mice were subjected to oral administration of BPA 48, 24, and 4 h before challenge with TDI. Dermal challenge of TDI onto the ear auricle was performed for the allergic dermatitis model, and intratracheal challenge of TDI was performed for the allergic airway inflammation model. In the allergic dermatitis model, ear-swelling response was significantly downregulated by high doses of BPA. The opposite reaction was observed in the allergic airway inflammation model, including significant exacerbation of red coloration in the lung, local cytokine levels, and total IgE levels in serum by BPA administration. To confirm the in vivo results, in vitro experiments with human epidermal keratinocytes (HEKs) and bronchial epithelial (BEAS-2B) cells were carried out. Significant enhancement of cytokine release from BEAS-2B cells but not HEKs in the BPA-treated group supported the in vivo observations. Our results imply that exposure to BPA directly exacerbates allergic airway inflammation but not allergic dermatitis.

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.

Genetic variants in TNFα, TGFB1, PTGS1 and PTGS2 genes are associated with diisocyanate-induced asthma.

Diisocyanates are the most common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants in inflammatory response genes (TNFα, IL1α, IL1ß, IL1RN, IL10, TGFB1, ADAM33, ALOX-5, PTGS1, PTGS2 and NAG-1/GDF15) are associated with increased susceptibility to diisocyanate asthma (DA). These genes were selected based on their role in asthmatic inflammatory processes and previously reported associations with asthma phenotypes. The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate-exposed workers in two groups: workers with specific inhalation challenge (SIC) confirmed DA (DA(+), n = 95) and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5' nuclease PCR assay. After adjusting for potentially confounding variables of age, smoking status and duration of exposure, the PTGS1 rs5788 and TGFB1 rs1800469 single nucleotide polymorphisms (SNP) showed a protective effect under a dominant model (OR = 0.38; 95% CI = 0.17, 0.89 and OR = 0.38; 95% CI = 0.18, 0.74, respectively) while the TNFα rs1800629 SNP was associated with an increased risk of DA (OR = 2.08; 95% CI = 1.03, 4.17). Additionally, the PTGS2 rs20417 variant showed an association with increased risk of DA in a recessive genetic model (OR = 6.40; 95% CI = 1.06, 38.75). These results suggest that genetic variations in TNFα, TGFB1, PTGS1 and PTGS2 genes contribute to DA susceptibility.

A novel benzofuran, 4-methoxybenzofuran-5-carboxamide, from Tephrosia purpurea suppressed histamine H1 receptor gene expression through a protein kinase C-δ-dependent signaling pathway.

Histamine H1 receptor (H1R) gene is upregulated in patients with allergic rhinitis (AR), and its expression level is strongly correlated with the severity of allergic symptoms. We previously reported isolation of the putative anti-allergic compound, 4-methoxybenzofuran-5-carboxamide (MBCA) from Tephrosia purpurea and its chemical synthesis (Shill et al., Bioorg Med Chem 2015;23:6869-6874). However, the mechanism underlying its anti-allergic activity remains to be elucidated. Here, we report the mechanism of MBCA on phorbol 12-myristate-13-acetate (PMA)- or histamine-induced upregulation of H1R gene expression in HeLa cells, and in vivo effects of MBCA were also determined in toluene-2,4-diisocyanate (TDI)-sensitized rats. MBCA suppressed PMA- and histamine-induced upregulation of H1R expression at both mRNA and protein levels and inhibited PMA-induced phosphorylation of PKCδ at Tyr(311) and subsequent translocation to the Golgi. Furthermore, MBCA ameliorated allergic symptoms and suppressed the elevation of H1R and helper T cell type 2 (Th2) cytokine mRNAs in TDI-sensitized rats. Data suggest that MBCA alleviates nasal symptoms in TDI-sensitized rats through the inhibition of H1R and Th2 cytokine gene expression. The mechanism of its H1R gene suppression underlies the inhibition of PKCδ activation.