Lung Gene Expression Suggests Roles for Interferon-Stimulated Genes and Adenosine Deaminase Acting against RNA-1 in Pathologic Responses to Diisocyanate.

Mechanisms underlying methylene diphenyl diisocyanate (MDI) and other low molecular weight chemical-induced asthma are unclear and appear distinct from those of high molecular weight (HMW) allergen-induced asthma. We sought to elucidate molecular pathways that differentiate asthma-like pathogenic vs nonpathogenic responses to respiratory tract MDI exposure in a murine model. Lung gene expression differences in MDI exposed immune-sensitized and nonsensitized mice vs unexposed controls were measured by microarrays, and associated molecular pathways were identified through bioinformatic analyses and further compared with published studies of a prototypic HMW asthmagen (ovalbumin). Respiratory tract MDI exposure significantly altered lung gene expression in both nonsensitized and immune-sensitized mice, vs controls. Fifty-three gene transcripts were altered in all MDI exposed lung tissue vs controls, with levels up to 10-fold higher in immune-sensitized vs nonsensitized mice. Gene transcripts selectively increased in MDI exposed immune-sensitized animals were dominated by chitinases and chemokines and showed substantial overlap with those increased in ovalbumin-induced asthma. In contrast, MDI exposure of nonsensitized mice increased type I interferon stimulated genes (ISGs) in a pattern reflecting deficiency in adenosine deaminase acting against RNA (ADAR-1), an important regulator of innate, as well as "sterile" or autoimmunity triggered by tissue damage. Thus, MDI-induced changes in lung gene expression were identified that differentiate nonpathogenic innate responses in nonsensitized hosts from pathologic adaptive responses in immune-sensitized hosts. The data suggest that MDI alters unique biological pathways involving ISGs and ADAR-1, potentially explaining its unique immunogenicity/allergenicity.

Occupational Risk Factors for SARS-CoV-2 Seropositivity in Healthcare Workers: Results of a Longitudinal Cohort.

OBJECTIVE: The aim of the study is to evaluate COVID-19 risk factors among healthcare workers (HCWs) before vaccine-induced immunity. METHODS: We conducted a longitudinal cohort study of HCWs ( N = 1233) with SARS-CoV-2 immunoglobulin G quantification by ELISA and repeated surveys over 9 months. Risk factors were assessed by multivariable-adjusted logistic regression and Cox proportional hazards models. RESULTS: SARS-CoV-2 immunoglobulin G was associated with work in internal medicine (odds ratio [OR], 2.77; 95% confidence interval [CI], 1.05-8.26) and role of physician-in-training (OR, 2.55; 95% CI, 1.08-6.43), including interns (OR, 4.22; 95% CI, 1.20-14.00) and resident physicians (OR, 3.14; 95% CI, 1.24-8.33). Odds were lower among staff confident in N95 use (OR, 0.55; 95% CI, 0.31-0.96) and decreased over the follow-up. CONCLUSIONS: Excess COVID-19 risk observed among physicians-in-training early in the COVID-19 pandemic was reduced with improved occupational health interventions before vaccinations.

Changes Over Time in COVID-19 Incidence, Vaccinations, Serum Spike IgG, and Viral Neutralizing Potential Among Individuals From a North American Gaming Venue: December 2020-August 2021.

OBJECTIVE: This study aims to evaluate COVID-19 cases and vaccine responses among workers in the gaming/entertainment industry. METHODS: Participants provided detailed information on occupational risk factors, demographics, COVID-19 history, and vaccination status through questionnaire. Enzyme-linked immunosorbent assays were used to measure serum antiviral antibodies and neutralizing capacity. RESULTS: Five hundred-fifty individuals participated with n = 228 (41.5%) returning for follow-up. At least 71% of participants were fully vaccinated within 8 months of vaccine availability and COVID-19 rates declined concomitantly. Serum anti-spike IgG levels and neutralizing capacity were significantly (P < 0.001) associated COVID-19 history and vaccine type, but not occupational risk factors, and declined (on average 36%) within 5 months. Few vaccine nonresponders (n = 12) and "breakthrough" infections (n = 1) were noted. CONCLUSIONS: COVID-19 vaccination was associated with a marked decrease in infections; however, individual humoral responses varied and declined significantly over time.

Glutathione reactivity with aliphatic polyisocyanates.

Isocyanate chemicals known to cause adverse health effects when inhaled are essential to making important products and are used in multiple industries. Glutathione (GSH), a major antioxidant of the lower airways with a well described role in xenobiotic metabolism, is a primary reaction target for di-isocyantes. However, GSHs reactivity with poly-isocyanates which have largely replaced diisocyanates (particularly aliphatic) in most end-user settings remains uncertain. We hypothesized aliphatic polyisocyanates would readily react with glutathione under physiologic conditions and the products could be identified using liquid chromatography (LC) coupled-mass spectrometry (MS) and tandem MS/MS. The data identified (tris)GSH-isocyanate adducts as the major reaction product of GSH with the most commonly used contemporary polymeric (tri-isocyanate) formulations of hexamethylene diisocyanate (HDI), the isocyanurate and biuret, as [M+H]+ ions of 1426.53 and 1400.55 m/z respectively in reverse phase LC-MS using electrospray in positive ion mode. The uretdione form of HDI, a stabilized dimer, formed two reaction products with GSH, a tris(GSH)-isocyanate reaction product recognized as a 1258.44 m/z [M+H]+ ion, and a bis(GSH)-isocyanate product identified as a 951.36 m/z [M+H]+ ion. Predicted structures for the newly described GSH-polyisocyanate reaction products, modeled based on collision induced dissociation (CID) fragmentation patterns in tandem MS/MS, support S-linkage of the GSH to N = C = O groups. In summary, industrially-used aliphatic polyisocyanates readily react with GSH to form primarily S-linked tris(GSH)-conjugates, a process that may play an important role in response to respiratory tract exposure.

Development and utilization of a surrogate SARS-CoV-2 viral neutralization assay to assess mRNA vaccine responses.

BACKGROUND: Tests for SARS-CoV-2 immunity are needed to help assess responses to vaccination, which can be heterogeneous and may wane over time. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring serum neutralizing antibodies but requires high level biosafety, live viral cultures and days to complete. We hypothesized that competitive enzyme linked immunoassays (ELISAs) based on SARS-CoV-2 spike protein's receptor binding domain (RBD) attachment to its host receptor, the angiotensin converting enzyme 2 receptor (ACE2r), would correlate with PRNT, given the central role of RBD-ACE2r interactions in infection and published studies to date, and enable evaluation of vaccine responses. METHODS AND RESULTS: Configuration and development of a competitive ELISA with plate-bound RBD and soluble biotinylated ACE2r was accomplished using pairs of pre/post vaccine serum. When the competitive ELISA was used to evaluate N = 32 samples from COVID-19 patients previously tested by PRNT, excellent correlation in IC50 results were observed (rs = .83, p < 0.0001). When the competitive ELISA was used to evaluate N = 42 vaccinated individuals and an additional N = 13 unvaccinated recovered COVID-19 patients, significant differences in RBD-ACE2r inhibitory activity were associated with prior history of COVID-19 and type of vaccine received. In longitudinal analyses pre and up to 200 days post vaccine, surrogate neutralizing activity increased markedly after primary and booster vaccine doses, but fell substantially, up to <12% maximal levels within 6 months. CONCLUSIONS: A competitive ELISA based on inhibition of RBD-ACE2r attachment correlates well with PRNT, quantifies significantly higher activity among vaccine recipients with prior COVID (vs. those without), and highlights marked declines in surrogate neutralizing activity over a 6 month period post vaccination. The findings raise concern about the duration of vaccine responses and potential need for booster shots.

Severe asthma and death in a worker using methylene diphenyl diisocyanate MDI asthma death.

Diisocyanates are well-recognized to cause occupational asthma, yet diisocyanate asthma can be challenging to diagnose and differentiate from asthma induced by other allergens. The present study assesses the potential contribution of methylene diphenyl diisocyanate (MDI) to a workplace fatality. Examination of medical records, tissue, and blood from the deceased worker were undertaken. Formalin-fixed paraffin-embedded lung tissue sections were assessed through histologic and immunochemical stains. Serum MDI-specific IgE and IgG, and total IgE, were measured by enzyme-linked immunosorbent assays and/or Western blot. Information about potential chemical exposures and industrial processes in the workplace were provided by the employer and through interviews with co-workers. Review of the worker's medical records, occupational history, and autopsy findings were consistent with severe asthma as the cause of death, and ruled out cardiac disease, pulmonary embolism, or stroke. Lung pathology revealed hallmarks of asthma including smooth muscle hypertrophy, eosinophilia, basement membrane thickening, and mucus plugging of bronchioles. Immunochemical staining for MDI was positive in the thickened basement membrane of inflamed airways. MDI-specific serum IgE and IgG were significantly elevated and demonstrated specificity for MDI versus other diisocyanates, however, total serum IgE was normal (24 IU/ml). The workplace had recently introduced MDI into the foundry as part of a new process, but MDI air levels had not been measured. Respirators were not required. In summary, post-mortem findings support the diagnosis of diisocyanate asthma and a severe asthma attack at work as the cause of death in a foundry worker.

Immunogenic amino acid motifs and linear epitopes of COVID-19 mRNA vaccines.

Reverse vaccinology is an evolving approach for improving vaccine effectiveness and minimizing adverse responses by limiting immunizations to critical epitopes. Towards this goal, we sought to identify immunogenic amino acid motifs and linear epitopes of the SARS-CoV-2 spike protein that elicit IgG in COVID-19 mRNA vaccine recipients. Paired pre/post vaccination samples from N = 20 healthy adults, and post-vaccine samples from an additional N = 13 individuals were used to immunoprecipitate IgG targets expressed by a bacterial display random peptide library, and preferentially recognized peptides were mapped to the spike primary sequence. The data identify several distinct amino acid motifs recognized by vaccine-induced IgG, a subset of those targeted by IgG from natural infection, which may mimic 3-dimensional conformation (mimotopes). Dominant linear epitopes were identified in the C-terminal domains of the S1 and S2 subunits (aa 558-569, 627-638, and 1148-1159) which have been previously associated with SARS-CoV-2 neutralization in vitro and demonstrate identity to bat coronavirus and SARS-CoV, but limited homology to non-pathogenic human coronavirus. The identified COVID-19 mRNA vaccine epitopes should be considered in the context of variants, immune escape and vaccine and therapy design moving forward.

Associations of SARS-CoV-2 serum IgG with occupation and demographics of military personnel.

BACKGROUND: Countries across the globe have mobilized their armed forces in response to COVID-19, placing them at increased risk for viral exposure. Humoral responses to SARS-CoV-2 among military personnel serve as biomarkers of infection and provide a basis for disease surveillance and recognition of work-related risk factors. METHODS: Enzyme-linked immunosorbent assays (ELISA) were used to measure SARS-CoV-2 spike antigen-specific IgG in serum obtained from N = 988 US National Guard soldiers between April-June 2020. Occupational information, e.g. military operating specialty (MOS) codes, and demographic data were obtained via questionnaire. Plaque assays with live SARS-CoV-2 were used to assess serum neutralizing capacity for limited subjects (N = 12). RESULTS: The SARS-CoV-2 IgG seropositivity rate among the study population was 10.3% and significantly associated with occupation and demographics. Odds ratios were highest for those working in MOS 2T-Transportation (3.6; 95% CI 0.7-18) and 92F-Fuel specialist/ground and aircraft (6.8; 95% CI 1.5-30), as well as black race (2.2; 95% CI 1.2-4.1), household size ≥6 (2.5; 95% CI 1.3-4.6) and known COVID-19 exposure (2.0; 95% CI 1.2-3.3). Seropositivity tracked along major interstate highways and clustered near the international airport and the New York City border. SARS-CoV-2 spike IgG+ serum exhibited low to moderate SARS-CoV-2 neutralizing capacity with IC50s ranging from 1:15 to 1:280. In limited follow-up testing SARS-CoV-2 serum IgG levels remained elevated up to 7 months. CONCLUSIONS: The data highlight increased SARS-CoV-2 seroprevalence among National Guard vs. the local civilian population in association with transportation-related occupations and specific demographics.

Human IgG and IgA responses to COVID-19 mRNA vaccines.

SARS-CoV-2 spike antigen-specific IgG and IgA elicited by infection mediate viral neutralization and are likely an important component of natural immunity, however, limited information exists on vaccine induced responses. We measured COVID-19 mRNA vaccine induced IgG and IgA in serum serially, up to 145 days post vaccination in 4 subjects. Spike antigen-specific IgG levels rose exponentially and plateaued 21 days after the initial vaccine dose. After the second vaccine dose IgG levels increased further, reaching a maximum approximately 7-10 days later, and remained elevated (average of 58% peak levels) during the additional >100 day follow up period. COVID-19 mRNA vaccination elicited spike antigen-specific IgA with similar kinetics of induction and time to peak levels, but more rapid decline in serum levels following both the 1st and 2nd vaccine doses (<18% peak levels within 100 days of the 2nd shot). The data demonstrate COVID-19 mRNA vaccines effectively induce spike antigen specific IgG and IgA and highlight marked differences in their persistence in serum.

Delayed production of neutralizing antibodies correlates with fatal COVID-19.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.

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.

Analysis of Lung Gene Expression Reveals a Role for Cl- Channels in Diisocyanate-induced Airway Eosinophilia in a Mouse Model of Asthma Pathology.

Diisocyanates are well-recognized causes of asthma. However, sensitized workers frequently lack diisocyanate-specific IgE, which complicates diagnosis and suggests the disease involves IgE-independent mechanisms. We used a mouse model of methylene diphenyl diisocyanate (MDI) asthma to identify biological pathways that may contribute to asthma pathogenesis. MDI sensitization and respiratory tract exposure were performed in Balb/c, transgenic B-cell (e.g., IgE)-deficient mice and a genetic background (C57BL/6)-matched strain. Eosinophils in airway fluid were quantitated by flow cytometry. Lung tissue gene expression was assessed using whole-genome mRNA microarrays. Informatic software was used to identify biological pathways affected by respiratory tract exposure and potential targets for disease intervention. Airway eosinophilia and changes (>1.5-fold; P value < 0.05) in expression of 192 genes occurred in all three mouse strains tested, with enrichment in chemokines and a pattern associated with alternatively activated monocytes/macrophages. CLCA1 (calcium-activated chloride channel regulator 1) was the most upregulated gene transcript (>100-fold) in all exposed mouse lungs versus controls, followed closely by SLC26A4, another transcript involved in Cl- conductance. Crofelemer, a U.S. Food and Drug Administration-approved Cl- channel inhibitor, reduced MDI exposure induction of airway eosinophilia, mucus, CLCA1, and other asthma-associated gene transcripts. Expression changes in a core set of genes occurs independent of IgE in a mouse model of chemical-induced airway eosinophilia. In addition to chemokines and alternatively activated monocytes/macrophages, the data suggest a crucial role for Cl- channels in diisocyanate asthma pathology and as a possible target for intervention.

Kinetics of antibody responses dictate COVID-19 outcome.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

LC-UV-MS and MS/MS Characterize Glutathione Reactivity with Different Isomers (2,2' and 2,4' vs. 4,4') of Methylene Diphenyl-Diisocyanate.

Methylene diphenyl diisocyanate (MDI), the most abundantly produced diisocyanate worldwide, is among the best recognized chemical causes of occupational asthma. The bulk of synthesized MDI, the 4,4' isomer, has been the focus of most biochemical research to date. The biological reactivity of other MDI isomers (2,2' and 2,4'), present at concentrations approaching 50% in some commercial products, remains less clear. We hypothesized 2,2' and 2,4' MDI react with glutathione (GSH), a major anti-oxidant of the lower airways, similarly to 4,4' MDI, and that the products could be characterized using a combination of LC-UV-MS and MS/MS. Purified 2,2' and 2,4' MDI isomers were mixed with GSH in pH-buffered aqueous phase at 37°C and reaction products were analyzed at varying time points. Within minutes, S-linked bis(GSH)-MDI conjugates were detectable as the dominant [M+H]+ ion, with an 865.25 m/z and more intense [M+2H]2+ ions of the same nominal mass. Upon longer reaction, [M+H]+ ions with greater retention times and the 558.17 m/z expected for mono(GSH)-MDI reaction products were observed, and exhibited MS/MS collision-induced dissociation (CID)-fragmentation patterns consistent with cyclized structures. Compared with 4,4' MDI, 2,2' and 2,4' isomers exhibit similar rapid reactivity with GSH and formation of bis(GSH)-MDI conjugates, but greater formation of cyclized mono(GSH) conjugates following extended reaction times (10 minutes to 2 hours). Further translational studies will be required to determine if the present in vitro findings extend to the complex lower airway microenvironment in vivo.

Dilysine-Methylene Diphenyl Diisocyanate (MDI), a Urine Biomarker of MDI Exposure?

Biomonitoring of methylene diphenyl diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention and in understanding pathways by which the internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique m/ z 543.29 [M + H]+ ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H]+ ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H]+ ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H]+ ions ( m/ z 558.17 and 863.23) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates, respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.

Development and Validation of LC-MS-MS Assay for the Determination of the Emerging Alkylating Agent Laromustine and Its Active Metabolite in Human Plasma.

The objective of this study was to validate a method for the determination of laromustine (VNP40101M) and short-lived its active metabolite (VNP4090CE) that has a half-life in human blood of <90 s in human plasma by liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection. We overcome the stability dilemma by acidified the human plasma with citric acid. Laromustine "breaks" down on the source of mass spectrometry to give m/z 249 which is the same m/z for VNP4090CE. Because VNP4090CE and laromustine elute at approximate retention time of 1.93 and 2.94 min, respectively, we were able to quantify both of them in one method. VNP40101M, VNP4090CE and the internal standards were extracted from human plasma by liquid-liquid extraction into ethyl ether. The ethyl ether layer was evaporated, reconstituted and analyzed using LC with MS/MS detection. Validation parameters such as selectivity, limit of quantitation, linearity, precision, accuracy, recovery, autosampler viability, freeze-thaw cycles and compounds stability are evaluated for this method. Results were calculated using peak area ratios, and calibration curves were generated using a weighted (1/x2) linear least-squares regression. Calibration curves for VNP40101M and VNP4090CE in human plasma ranged from 1.00 to 1,000 ng/mL. In this study, both intra- and inter-assay results demonstrated a relative standard deviation for calibration standards (inter-assay) and quality control samples (intra- and inter-assay) to be ≤15.0%. In this method, there is ~1.79% isotopic interference of VNP40101M to VNP40101M-IS, and ~3.76% isotopic interference of VNP4090CE to VNP4090CE-IS. It was concluded that there was no significant carryover.

Reaction products of hexamethylene diisocyanate vapors with "self" molecules in the airways of rabbits exposed via tracheostomy.

1. Hexamethylenediisocyanate (HDI) is a widely used aliphatic diisocyanate and a well-recognized cause of occupational asthma. 2. "Self" molecules (peptides/proteins) in the lower airways, susceptible to chemical reactivity with HDI, have been hypothesized to play a role in asthma pathogenesis and/or chemical metabolism, but remain poorly characterized. 3. This study employed unique approaches to identify and characterize "self" targets of HDI reactivity in the lower airways. Anesthetized rabbits free breathed through a tracheostomy tube connected to chambers containing either, O2, or O2 plus ∼200 ppb HDI vapors. Following 60 minutes of exposure, the airways were lavaged and the fluid was analyzed by LC-MS and LC-MS/MS. 4. The low-molecular weight (<3 kDa) fraction of HDI exposed, but not control rabbit bronchoalveolar lavage (BAL) fluid identified 783.26 and 476.18 m/z [M+H]+ ions with high energy collision-induced dissociation (HCD) fragmentation patterns consistent with bis glutathione (GSH)-HDI and mono(GSH)-HDI. Proteomic analyses of the high molecular weight (>3 kDa) fraction of exposed rabbit BAL fluid identified HDI modification of specific lysines in uteroglobin (aka clara cell protein) and albumin. 5. In summary, this study utilized a unique approach to chemical vapor exposure in rabbits, to identify HDI reaction products with "self" molecules in the lower airways.

Polymerization of hexamethylene diisocyanate in solution and a 260.23 m/z [M+H]+ ion in exposed human cells.

Hexamethylene diisocyanate (HDI) is an important industrial chemical that can cause asthma, however pathogenic mechanisms remain unclear. Upon entry into the respiratory tract, HDI's N=C=O groups may undergo nucleophilic addition (conjugate) to host molecules (e.g. proteins), or instead react with water (hydrolyze), releasing CO2 and leaving a primary amine in place of the original N=C=O. We hypothesized that (primary amine groups present on) hydrolyzed or partially hydrolyzed HDI may compete with proteins and water as a reaction target for HDI in solution, resulting in polymers that could be identified and characterized using LC-MS and LC-MS/MS. Analysis of the reaction products formed when HDI was mixed with a pH buffered, isotonic, protein containing solution identified multiple [M+H]+ ions with m/z's and collision-induced dissociation (CID) fragmentation patterns consistent with those expected for dimers (259.25/285.23 m/z), and trimers (401.36/427.35 m/z) of partially hydrolyzed HDI (e.g. ureas/oligoureas). Human peripheral blood mononuclear cells (PBMCs) and monocyte-like U937, but not airway epithelial NCI-H292 cell lines cultured with these HDI ureas contained a novel 260.23 m/z [M+H]+ ion. LC-MS/MS analysis of the 260.23 m/z [M+H]+ ion suggest the formula C13H29N3O2 and a structure containing partially hydrolyzed HDI, however definitive characterization will require further orthogonal analyses.