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This study aims to evaluate COVID-19 cases and vaccine responses among workers in the gaming/entertainment industry.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.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 (P0.001) 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.COVID-19 vaccination was associated with a marked decrease in infections; however, individual humoral responses varied and declined significantly over time.

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 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.

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.

BackgroundTests 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 FindingsConfiguration 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=41 vaccinated individuals and an additional N=14 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 ConclusionsA 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.

Biomonitoring methylene dipheny 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 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 543.29 m/z [M+H](+) ion from MDI exposed mice, but not controls. The 543.29 m/z [M+H](+) ion was detectable within 24 hours of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The 543.29 m/z [M+H](+) ion possessed properties of di-lysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C(27)H(38)N(6)O(6), (b) expected collision induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reverse phase LC-MS identical to synthetic di-lysine-MDI. Further MDI-specific western blot studies suggest albumin (which contains multiple di-lysine sites susceptible to MDI carbamylation) as a possible source for di-lysine-MDI, and the presence of MDI conjugated albumin in urine up to 6 days post respiratory tract exposure. Two additional [M+H](+) ions (558.17 and 863.23 m/z) 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.

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.

Individuals with acute malaria infection generated high levels of antibodies that cross-react with the SARS-CoV-2 Spike protein. Cross-reactive antibodies specifically recognized the sialic acid moiety on N-linked glycans of the Spike protein and do not neutralize in vitro SARS-CoV-2. Sero-surveillance is critical for monitoring and projecting disease burden and risk during the pandemic; however, routine use of Spike protein-based assays may overestimate SARS-CoV-2 exposure and population-level immunity in malaria-endemic countries.

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.

SummaryRecent 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 levelsper se, but rather with the delayed kinetics of NAb production.

BackgroundSeveral serological assays have been developed to detect anti-SARS-CoV-2 IgG antibodies, but evidence about their comparative performance is limited. We sought to assess the sensitivity of four anti-SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISA) in individuals with evidence of prior SARS-CoV-2 infection.MethodsWe obtained sera from 36 individuals with PCR-confirmed SARS-CoV-2 infection between March and May 2020. We evaluated samples collected at around 21 days (±14 days) after their initial PCR test using 3 commercially available ELISA assays, two anti-spike (Ortho- Clinical Diagnostics Vitros, and Euroimmun) and one anti-nucleocapsid (Abbott Architect), and a Yale-developed anti-spike ELISA test. We determined the sensitivity of the tests and compared their results. The Euroimmun and Yale ELISA had an equivocal and indeterminate category, which were considered as both negative and positive.ResultsAmong the 36 individuals with SARS-CoV-2 infection, mean age was 43 (±13) years and 19 (53%) were female. The sensitivities of the tests were not significantly different (Abbott Architect, Ortho Vitros, Euroimmmun, and Yale assays: 86% (95% confidence interval [CI], 71– 95), 94% (95% CI, 81–99), 86% (95% CI, 71–95), and 94% (95% CI, 81–99), respectively; p- value=0.464). The sensitivities of the Euroimmun and Yale ELISA tests increased when the equivocal/indeterminate results were considered positive (97% [95% CI, 85–100] and 100% [95% CI, 90–100], respectively), but were not significantly different from other tests (p=0.082). The cross-correlation coefficient ranged from 0.85–0.98 between three anti-spike protein assays (Ortho Vitros, Euroimmun, Yale) and was 0.58–0.71 between the three anti-spike protein assays and the anti-nucleocapsid assay (Abbott).ConclusionThe sensitivities of four anti-SARS-CoV-2 protein assays did not significantly differ, although the sample size was small. Sensitivity also depended on the interpretation of equivocal and indeterminate results. The strongest correlations were present for the three anti- spike proteins assays. These findings suggest that individual test characteristics and the correlation between different tests should be considered when comparing or aggregating data across different populations studies for serologic surveillance of past SARS-CoV-2 infection.

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

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.

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.

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.

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

1. Alkylating agents are capable of introducing an alkyl group into nucleophilic sites on DNA or RNA through covalent bond. Laromustine is an active member of a relatively new class of sulfonylhydrazine prodrugs under development as antineoplastic alkylating agents, and displays significant single-agent activity. 2. This is the first report of the population pharmacokinetic analysis of laromustine, 106 patients, 66 with hematologic malignancies and 40 with solid tumors, participated in five clinical trials worldwide. Of these, 104 patients were included in the final NONMEM analysis. 3. The population estimates for total clearance (CL) and volume of distribution of the central compartment (V1) were 96.3 L/h and 45.9 L, associated with high inter-patient variability of 52.9% and 79.8% and inter-occasion variability of 26.7% and 49.3%, respectively. The population estimates for Q and V2 were 73.2 L/h and 29.9 L, and inter-patient variability in V2 was 63.1%, respectively. 4. The estimate of Vss (75.8 L) exceeds total body water, indicating that laromustine is distributed to tissues. The half-life is short, less than 1 h, reflecting rapid clearance. Population PK analysis showed laromustine pharmacokinetics to be independent of dose and organ function with no effect on subsequent dosing cycles.

Isocyanates differ from many other xenobiotics in their ability to form S-linked conjugates with glutathione (GSH) through direct nucleophilic addition reactions (e.g. without enzymatic "preactivation" and/or transferase activity), potentially predisposing them to metabolism via the mercapturic acid pathway. In vivo, mono-isocyanates are metabolized via the mercapturic acid pathway and excreted as N-acetylated cysteine conjugates, however, the metabolism of di-isocyanates remains unclear. We assessed the ability of purified human gamma-glutamyl transpeptidase-1 (GGT-1), a primary enzyme of the mercapturic acid pathway, to cleave S-linked GSH conjugates of 4,4'-methylene diphenyl diisocyanate (MDI) and 1,6-hexamethylene diisocyanate (HDI), two widely used industrial chemicals. A combination of liquid chromatography (LC), tandem mass spectrometry (MS/MS) and hydrogen-deuterium exchange studies confirmed GGT-1 mediated formation of the 607.2 and 525.2 m/z (M + H)(+) ions corresponding to bis(cys-gly)-MDI and bis(cys-gly)-HDI, respectively, the cleavage products expected from the corresponding bis(GSH)-diisocyanate conjugates. Additional intermediate metabolites and mono(cys-gly)-conjugates with partially hydrolyzed diisocyanate were also observed. Consistent with GGT enzyme kinetics, metabolism proceeded more rapidly under conditions that favored transpeptidation versus hydrolytic mechanisms of cleavage. Together the data demonstrate the capacity of human GGT-1 to cleave GSH conjugates of both aromatic and aliphatic diisocyanates, suggesting a potential role in their metabolism.

1. Laromustine (VNP40101M, also known as Cloretazine) is a novel sulfonylhydrazine alkylating (anticancer) agent. This article describes the use of quantitative whole-body autoradiography (QWBA) and mass balance to study the tissue distribution, the excretion mass balance and pharmacokinetics after intravenous administration of [(14)C]VNP40101M to rats. A single 10 mg/kg IV bolus dose of [(14)C]VNP40101M was given to rats. 2. The recovery of radioactivity from the Group 1 animals over a 7-day period was an average of 92.1% of the administered dose, which was accounted for in the excreta and carcass. Most of the radioactivity was eliminated within 48 h via urine (48%), with less excreted in feces (5%) and expired air accounted for (11%). The plasma half-life of [(14)C]laromustine was approximately 62 min and the peak plasma concentration (Cmax) averaged 8.3 μg/mL. 3. The QWBA study indicated that the drug-derived radioactivity was widely distributed to tissues through 7 days post-dose after a single 10 mg/kg IV bolus dose of [(14)C]VNP40101M to male pigmented Long-Evans rats. The maximum concentrations were observed at 0.5 or 1 h post-dose for majority tissues (28 of 42). The highest concentrations of radioactivity were found in the small intestine contents at 0.5 h (112.137 µg equiv/g), urinary bladder contents at 3 h (89.636 µg equiv/g) and probably reflect excretion of drug and metabolites. The highest concentrations in specific organs were found in the renal cortex at 1 h (28.582 µg equiv/g), small intestine at 3 h (16.946 µg equiv/g), Harderian gland at 3 h (12.332 µg equiv/g) and pancreas at 3 h (12.635 µg equiv/g). Concentrations in the cerebrum (1.978 µg equiv/g), cerebellum (2.109 µg equiv/g), medulla (1.797 µg equiv/g) and spinal cord (1.510 µg equiv/g) were maximal at 0.5 h post-dose and persisted for 7 days. 4. The predicted total body and target organ exposures for humans given a single 100 µCi IV dose of [(14)C]VNP40101M were well within the medical guidelines for maximum radioactivity exposures in human subjects.

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.

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 (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.

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.

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 quasistable 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 (10 μM-10 mM), 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.

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 secreting mAbs that may be developed into useful diagnostic reagents.

Introduction Airway fluid glutathione (GSH) reactivity with inhaled vapors of diisocyanate, a common occupational allergen, is postulated to be a key step in exposure-induced asthma pathogenesis. Methods A mixed (vapor/liquid) phase exposure system was used to model the in vivo reactivity of inhaled HDI vapor with GSH in the airway fluid. HDI–GSH reaction products, and their capacity to transfer HDI to human albumin, were characterized through mass spectrometry and serologic assays, using HDI-specific polyclonal rabbit serum. Results HDI vapor exposure of 10 mM GSH solutions resulted in primarily S-linked, bis(GSH)–HDI reaction products. In contrast, lower GSH concentrations (100 μM) resulted in mainly mono(GSH)–HDI conjugates, with varying degrees of HDI hydrolysis, dimerization and/or intra-molecular cyclization, depending upon the presence/absence of H 2 PO 4 − /HPO 4 2− and Na + /Cl − ions. The ion composition and GSH concentration of the fluid phase, during HDI vapor exposure, strongly influenced the transfer of HDI from GSH to albumin, as did the pH and duration of the carbamoylating reaction. When carbamoylation was performed overnight at pH 7, 25 of albumin’s lysines were identified as potential sites of conjugation with partially hydrolyzed HDI. When carbamoylation was performed at pH 9, more rapid (within 3 h) and extensive modification was observed, including additional lysine sites, intra-molecular cross-linkage with HDI, and novel HDI–GSH conjugation. Conclusions The data define potential mechanisms by which the levels of GSH, H 2 PO 4 − /HPO 4 2− , and/or other ions (e.g. H + /OH − , Na + , Cl − ) affect the reactivity of HDI vapor with self-molecules in solution (e.g. airway fluid), and thus, might influence the clinical response to HDI respiratory tract exposure.

1. Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown. 2. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to an aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 and 24 h post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to the protein content of the cellular fraction of the lavage fluid. 3. Following MDI aerosol exposure, expression of the number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin. 4. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin.

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.

Methylene diphenyl diisocyanate (MDI) is an important industrial chemical and asthmagenic respiratory sensitizer, however its metabolism remains unclear. In this study we used LC-MS and LC-MS/MS to identify novel reaction products of MDI with oxidized glutathione (GSSG), including an 837 m/z [M+H]+ ion corresponding to GSSG bound (via one of its N-termini) to partially hydrolyzed MDI, and an 863 m/z [M+H]+ ion corresponding to GSSG cross-linked by MDI (via its two γ-glutamate N-termini). Further studies with heavy isotope labeled and native reduced glutathione (GSH) identified an [M+H]+ ion corresponding to previously described mono(GSH)-MDI, and evidence for “oligomeric” GSH-MDI conjugates. This study also investigated transformational changes in MDI after incubation with an S9 fraction prepared from murine liver. LC-MS analyses of the S9 reaction products revealed the formation of [M+H]+ ions with m/z’s and retention times identical to the newly described GSSG-MDI (837 and 863) conjugates and the previously described mono(GSH)-MDI conjugates. Together the data identify novel biological transformations of MDI, which could have implications for exposure-related health effects, and may help target future in vivo studies of metabolism.

Methylene diphenyl diisocyanate (MDI) is an important industrial chemical and asthmagenic respiratory sensitizer, however its metabolism remains unclear. In this study we used LC-MS and LC-MS/MS to identify novel reaction products of MDI with oxidized glutathione (GSSG), including an 837 m/z [M + H] + ion corresponding to GSSG bound (via one of its N-termini) to partially hydrolyzed MDI, and an 863 m/z [M + H] + ion corresponding to GSSG cross - linked by MDI (via its two γ-glutamine N-termini). Further studies with heavy isotope labeled and native reduced glutathione (GSH) identified an [M + H] + ion corresponding to previously described mono(GSH)-MDI, and evidence for “oligomeric” GSH-MDI conjugates. This study also investigated transformational changes in MDI after incubation with an S9 fraction prepared from murine liver. LC-MS analyses of the S9 reaction products revealed the formation of [M + H] + ions with m/z 's and retention times identical to the newly described GSSG-MDI (837 and 863) conjugates and the previously described mono(GSH)-MDI conjugate. Together the data identify novel biological transformations of MDI, which could have implications for exposure-related health effects, and may help target future in vivo studies of metabolism.

Metabolomics is a relatively new and rapidly growing area of post-genomic biological research. As use of metabolomics technology grows throughout the spectrum of drug discovery and development, and its applications broaden, its impact is expanding dramatically. This review seeks to provide the reader with a brief history of the development of metabolomics, its significance and strategies for conducting metabolomics studies. The most widely used analytical tools for metabolomics: NMR, LC-MS and GC-MS, are discussed along with considerations for their use. Herein, we will show how metabolomics can assist in pharmaceutical research studies, such as pharmacology and toxicology, and discuss some examples of the importance of metabolomics analysis in research and development.

Since 1970, the invention of flow cytometry FACS has become widely used for single-cell measurements in medicine and biology [1]. In the last 30 years FACS has become the work horse to provide fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. A recent breakthrough, cytometry time-of-flight (CyTOF, mass cytometry), builds on flow cytometry and adds MS for unprecedented marker detection in biological samples [2,3]. CyTOF is a novel and exciting technology for real-time analysis of single cells using inductively coupled plasma TOF-MS. Current CyTOF technique allows the detection of 40 parameters at the single-cell level; this capacity will increase as more isotopes become available. CyTOF can resolve multiple metal conjugated antibodies for biomarkers detection on cells with minimal signal overlap, which maximizes the information obtained from each individual sample. To make the best use of mass cytometry to produce high-quality quantitative data from clinical samples, we must understand the experimental variables and how to minimize them. The ability to analyze multiplexed assays is of great importance for clinical diagnostic and other analytical applications. Mass cytometry is a significant advance for studies in medical fields including immunology, hematology and oncology. CyTOF has the ability to perform complex characterization at single-cell level both phenotypically and functionally from normal and diseased states. Single cell technologies have allowed researchers to measure the effects of a drug and better understand its mechanism of action. While research in drug companies is focused on the mechanism of drug–target interactions and its resulting pharmacology, CyTOF techniques may become a driving tool for biomarker discovery. There is increasing interest from a variety of disciplines for single-cell experimentation methods, such as cancer research, cardiovascular research, embryonic stem cells and development, gene expression profiling, hematopoietic stem cells and progenitors, immunity/ infectious disease, induced pluripotent stem cells, neural research and RNA sequencing [4–9]. The advantages of CyTOF are clear, and investigators employing best practices may achieve robust discovery in a routine bioanalytical setting.

Glutathione has previously been identified as a reaction target for toluene diisocyanate (TDI) in vitro and in vivo, and has been suggested to contribute to toxic and allergic reactions to exposure. In this study, the reactivity of reduced glutathione (GSH) with TDI in vitro was further investigated using a mixed phase (vapor/liquid) exposure system to model the in vivo biophysics of exposure in the lower respiratory tract. HPLC/MS/MS was used to characterize the observed reaction products. Under the conditions tested, the major reaction products between TDI vapor and GSH were S-linked bis(GSH)-TDI and to a lesser extent mono(GSH)-TDI conjugates (with one N═C═O hydrolyzed). The vapor-phase-generated GSH-TDI conjugates were capable of transcarbamoylating human albumin in a pH-dependent manner, resulting in changes in the self-protein's conformation/charge, on the basis of electrophoretic mobility under native conditions. Specific sites of human albumin-TDI conjugation, mediated by GSH-TDI, were identified (Lys(73), Lys(159), Lys(190), Lys(199), Lys(212), Lys(351), Lys(136/137), Lys(413/414), and Lys(524/525)) along with overlap with those susceptible to direct conjugation by TDI. Together, the data extend the proof-of-principle for GSH to act as a "shuttle" for a reactive form of TDI, which could contribute to clinical responses to exposure.

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.

Summary Background Isocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized. Objective The aim of the study was to identify and characterize the cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI). Methods Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI–albumin conjugates or control antigen, and changes in phenotype, gene and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI–albumin conjugates prepared with fluorescently labelled albumin. In vivo, human HDI exposure was performed via a specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR. Results Human monocytes take up HDI–albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (P-values 0.007–0.05) changes in mircoarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP-1) and pattern-recognition receptors that bind chitin (chitinases) and oxidized low-density lipoprotein (CD68) were also increased following isocyanate–albumin exposure. In vivo, HDI-exposed subjects exhibited a drastic increase in the percentage of PBMCs with the same HDI–albumin responsive phenotype characterized in vitro (HLA-DR+/CD11c+ with altered light scatter properties). An exposure-dependent decrease (46±11%; P

BACKGROUND The structural characteristics of diisocyanate chemical protein antigens vary depending upon the methods of production, and may influence diisocyanate antigen immunoassays. The impact of different antigen preparation methods on immunoassay sensitivity, specificity, and predictive value for identifying workers with diisocyanate asthma (DA) has not been systematically evaluated. OBJECTIVE Evaluate the influence of preparation methodology of hexamethylene diisocyanate human serum albumin (HDI-HSA) conjugates on the performance of specific antibody assays for identifying workers with confirmed HDI asthma. METHODS Asthmatic reactions to HDI exposure were assessed in 80 autobody shop workers by specific inhalation challenge (SIC). HDI-specific IgE and IgG in serum were measured by RAST and ELISA with seven different HDI-HSA conjugates prepared in liquid phase with monomeric or polymeric HDI, or vapour-phase monomeric HDI. The HDI : HSA substitution ratios were determined by mass spectrometry. RESULTS DA was confirmed by SIC in 23 subjects. The maximal sensitivity for detecting specific IgE among workers with positive SIC results was higher with RAST and with polymeric vs. monomeric HDI-albumin conjugates (21.7% vs. 8.7%) with a generally high specificity (>or=95%). HDI-HSA specific IgG antibody was also detected in 22-43% of HDI asthmatics depending upon the conjugate used. The specificity of specific IgG varied from 88% to 96%, and it was higher for monomeric (vs. polymeric) HDI-albumin conjugates with low (vs. high) substitution ratios. CONCLUSION The test performance of specific IgE and IgG immunoassays for identifying a positive SIC response varied with different HDI-HSA conjugates. Standard test antigens and common immunoassays must be used to minimize inter-laboratory variability.

Cytometry by time-of-flight (CyTOF) is a novel technology for the real-time analysis of single cells. CyTOF is a significant advance in fields including immunology, hematology, and oncology. It resolves multiple metal-conjugated probes per cell with minimal signal overlap, which maximizes the information obtained from each individual sample. CyTOF provides the ability to phenotypically and functionally profile cells from normal and diseased states. Single cell technologies enable researchers to measure the effects of a drug at the single cell level and better understand its mechanism of action. Here, we discuss novel instruments for the analysis of individual biological cells, the impact of recent innovations in support of drug development, and the important roles of CyTOF in drug profiling.

Leptospirosis is a zoonotic disease associated with a changing global epidemiology. Recently, an increased incidence of canine leptospirosis in the northeastern United States and Canada has been associated with increasing rates of infection among reservoir hosts, such as skunks, raccoons, and squirrels, that are common in suburban settings. We describe a case of leptospirosis that provides new insight into the epidemiology, diagnosis, and pathogenesis of this disease acquired in the suburban setting. Atypical lymphocytosis corresponded to an expansion of γ-δ T cells in peripheral blood.

Toluene diisocyanate (TDI), a widely used aromatic diisocyanate with the potential to cause asthma, reacts with albumin in the airway fluid, which acts as a carrier protein for chemical presentation to the immune system. Structural elucidation of TDI-albumin conjugates is crucial to understanding the human immune response to TDI exposure.Investigate the dependence of TDI's antigenicity on the biophysics of exposure and its association with TDI asthma.Toluene diisocyanate-albumin conjugates were generated by exposing albumin to TDI in liquid or vapor phase (liquid or vapor TDI-albumin, respectively). Conjugates were characterized by native gel electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry, and used as antigens in ELISA assays for serum specific-IgE and IgG.The physical phase of TDI (vapor vs liquid) affects the formation of TDI-albumin conjugates, with measurable differences in the amount of TDI per albumin molecule, migration in native gels, matrix-assisted laser desorption/ionization-mass spectrometry mass/charge spectra, and antigenicity. Vapor TDI-albumin conjugates were recognized by IgE from 44% of subjects with TDI asthma, whereas liquid TDI-albumin conjugates are recognized by IgE from only 17% of these patients. A significant (P.05) association between TDI exposure and vapor TDI-albumin specific serum IgG was also observed.Biophysics of TDI exposure substantially affects formation of TDI-albumin conjugates recognized by the immune system in association with exposure and asthma.The data suggest that serology may help identify TDI asthmatics and exposed workers if the appropriate form of TDI is used as the antigenic basis for analysis.

BACKGROUND Glutathione (GSH), one of the major anti-oxidants of the lung, has been linked to the human response to isocyanate exposure. However, the ability of GSH to modulate key chemical reactions, thought to be central to the development of human isocyanate allergy, has not been directly analyzed under biologic exposure conditions. OBJECTIVE To better understand the potential role of GSH in the response to occupational isocyanate exposure, we evaluated its effects on two processes thought to be involved in the development of isocyanate allergy, isocyanate-protein conjugation and epithelial cell toxicity. METHODS The effects of GSH on (1) isocyanate conjugation with albumin, its major target in the airway fluid and (2) isocyanate-induced toxicity to human airway epithelial cell lines, A549 and NCI-H292, were tested using two different in vitro models. For protein conjugation studies, a newly described vapour exposure system was used to model the air/liquid interface at the surface of the epithelial fluid in the airways. Epithelial cell exposures were performed in fluid phase to mimic the in vivo exposure of airway cells covered by epithelial lining fluid. RESULTS Reduced GSH prevented hexamethylene diisocyanate (HDI) conjugation to albumin in a dose-dependent manner, while oxidized GSH (GSSG) conversely increased conjugation rates. GSH levels equivalent to those found in normal human airway fluid (100 microm) provided >90% protection against HDI-protein conjugation when albumin was exposed to HDI vapour levels 10-fold above permissible occupational limits. Physiologic levels of GSH, but not GSSG, also reduced HDI toxicity to human airway epithelial cells in a dose-dependent manner, when present extracellularly, however, drugs that modulate intra-cellular GSH levels did not significantly alter isocyanate toxicity. CONCLUSIONS Together with previously reported genetic and toxicity studies, the data suggest that airway GSH plays an important role in protection against HDI exposure and may help prevent the development of allergic sensitization and asthma.

Background The human immune response to isocyanate, a leading cause of occupational asthma, remains incompletely characterized, including the cell types involved and the form of the chemical that acts as an antigen. Objective The purpose of this investigation was to characterize human T cells that respond to hexamethylene diisocyanate (HDI), an aliphatic isocyanate routinely used in the automobile body industry. Methods Human T-cell lines were generated and characterized from peripheral blood of HDI-exposed and HDI-unexposed subjects, using two different HDI antigens, HDI-conjugated albumin and HDI-exposed human airway epithelial cells (NCI-H292). Flow cytometry was used to characterize the phenotype of HDI-responsive T cells. ELISA and intracellular staining techniques were used to evaluate HDI-induced cytokine production. DNA sequence analysis of T-cell receptors was used to further define clonal populations of HDI-responsive T cells. Results HDI antigen preparations but not "mock exposed" control antigens lead to increased proliferation of specific cell types, CD3 + CD4 − CD8 dim and/or CD3 + CD4 − CD8 − cells, from HDI-exposed but not from HDI-unexposed subjects. These HDI-responsive T cells expressed unique oligoclonal γ/δ rather than α/β T-cell receptors, with characteristics suggestive of antigen-mediated selection and specificity. The HDI-stimulated γ/δ T cells were associated with T H 1-like cytokines and produce IFN-γ but not IL-5 or IL-13. Conclusions These data are the first to demonstrate that HDI can selectively stimulate γ/δ T cells with the potential to modulate the human immune response to exposure.