Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-30 (of 34 Records) |
Query Trace: Hettick JM [original query] |
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Microrna-mediated Krüppel-Like Factor 4 upregulation induces alternatively activated macrophage-associated marker and chemokine transcription in 4,4'-methylene diphenyl diisocyanate exposed macrophages
Lin CC , Law BF , Hettick JM . Xenobiotica 2024 1-22 Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI) is associated with occupational asthma (OA) development. Alveolar macrophage-induced recruitment of immune cells to the lung microenvironment plays an important role during asthma pathogenesis. Previous studies identified that MDI/MDI-glutathione (GSH)-exposure downregulates endogenous hsa-miR-206-3p/hsa-miR-381-3p. Our prior report shows that alternatively activated (M2) macrophage-associated markers/chemokines are induced by MDI/MDI-GSH-mediated Krüppel-Like Factor 4 (KLF4) upregulation in macrophages and stimulates immune cell chemotaxis. However, the underlying molecular mechanism(s) by which MDI/MDI-GSH upregulates KLF4 remain unclear.Following MDI-GSH exposure, microRNA(miR)-inhibitors/mimics or plasmid transfection, endogenous hsa-miR-206-3p/hsa-miR-381-3p, KLF4, or M2 macrophage-associated markers (CD206, TGM2), and chemokines (CCL17, CCL22, CCL24) were measured by either RT-qPCR, western blot, or luciferase assay.MDI-GSH exposure downregulates hsa-miR-206-3p/hsa-miR-381-3p by 1.46- to 9.75-fold whereas upregulates KLF4 by 1.68- to 1.99-fold, respectively. In silico analysis predicts binding between hsa-miR-206-3p/hsa-miR-381-3p and KLF4. Gain- and loss-of-function, luciferase reporter assays and RNA-induced silencing complex-immunoprecipitation (RISC-IP) studies confirm the posttranscriptional regulatory roles of hsa-miR-206-3p/hsa-miR-381-3p and KLF4 in macrophages. Furthermore, hsa-miR-206-3p/hsa-miR-381-3p regulate the expression of M2 macrophage-associated markers and chemokines via KLF4.In conclusion, hsa-miR-206-3p/hsa-miR-381-3p play a major role in regulation of MDI/MDI-GSH-induced M2 macrophage-associated markers and chemokines by targeting the KLF4 transcript, and KLF4-mediated regulation in macrophages. |
4,4'-Methylene diphenyl diisocyanate exposure induces expression of alternatively activated macrophage-associated markers and chemokines partially through Krüppel-like factor 4 mediated signaling in macrophages
Lin CC , Law BF , Hettick JM . Xenobiotica 2023 53 (12) 1-17 Occupational exposure to the most widely used monomeric diisocyanate (dNCO), 4,4'-methylene diphenyl diisocyanate (MDI), may lead to the development of occupational asthma (OA). Alveolar macrophages with alternatively activated (M2) phenotype have been implicated in allergic airway responses and the pathogenesis of asthma. Recent in vivo studies demonstrate that M2 macrophage-associated markers and chemokines are induced by MDI-exposure, however, the underlying molecular mechanism(s) by which this proceeds is unclear.Following MDI exposure (in vivo and in vitro) M2 macrophage-associated transcription factors (TFs), markers, and chemokines were determined by RT-qPCR, western blots, and ELISA.Expression of M2 macrophage-associated TFs and markers including Klf4/KLF4, Cd206/CD206, Tgm2/TGM2, Ccl17/CCL17, Ccl22/CCL22, and CCL24 were induced by MDI/MDI-GSH exposure in bronchoalveolar lavage cells (BALCs)/THP-1 macrophages. The expression of CD206, TGM2, CCL17, CCL22, and CCL24 are upregulated by 3.83-, 7.69-, 6.22-, 6.08-, and 1.90-fold in KLF4-overexpressed macrophages, respectively. Endogenous CD206 and TGM2 were downregulated by 1.65-5.17-fold, and 1.15-1.78-fold, whereas CCL17, CCL22, and CCL24 remain unchanged in KLF4-knockdown macrophages. Finally, MDI-glutathione (GSH) conjugate-treated macrophages show increased chemotactic ability to T-cells and eosinophils, which may be attenuated by KLF4 knockdown.Our data suggest that MDI exposure may induce M2 macrophage-associated markers partially through induction of KLF4. |
A comparison of performance metrics for cloth face masks as source control devices for simulated cough and exhalation aerosols (preprint)
Lindsley WG , Blachere FM , Beezhold DH , Law BF , Derk RC , Hettick JM , Woodfork K , Goldsmith WT , Harris JR , Duling MG , Boutin B , Nurkiewicz T , Noti JD . medRxiv 2021 Universal mask wearing is recommended by the Centers for Disease Control and Prevention to help control the spread of COVID-19. Masks reduce the expulsion of respiratory aerosols (called source control) and offer some protection to the wearer. However, masks vary greatly in their designs and construction materials, and it is not clear which are most effective. Our study tested 15 reusable cloth masks (which included face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators as source control devices for aerosols ≤ 7 µm produced during simulated coughing and exhalation. These measurements were compared with the mask filtration efficiencies, airflow resistances, and fit factors. The source control collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. The filtration efficiencies of the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on an elastomeric manikin headform and 1.0 to 4.0 on human test subjects. The correlation coefficients between the source control efficacies and the other performance metrics ranged from 0.31 to 0.66 and were significant in all but one case. However, none of the alternative metrics were strong predictors of the source control performance of cloth masks. Our results suggest that a better understanding of the relationships between source control performance and metrics like filtration efficiency, airflow resistance, and fit factor are needed to develop simple methods to estimate the effectiveness of masks as source control devices for respiratory aerosols. |
MicroRNA-Mediated Calcineurin Signaling Activation Induces CCL2, CCL3, CCL5, IL8 and Chemotactic Activities in 4,4'-Methylene Diphenyl Diisocyanate Exposed Macrophages
Lin CC , Law BF , Hettick JM . Xenobiotica 2021 51 (12) 1-20 Occupational exposure to 4,4'-methylene diphenyl diisocyanate (MDI), the most widely used monomeric diisocyanate, is one of the leading causes of occupational asthma (OA). Previously, we identified microRNA (miR)-206-3p/miR-381-3p-mediated PPP3CA/calcineurin signaling regulated iNOS transcription in macrophages and bronchoalveolar lavage cells (BALCs) after acute MDI exposure; however, whether PPP3CA/calcineurin signaling participates in regulation of other asthma-associated mediators secreted by macrophages/BALCs after MDI exposure is unknown.Several asthma-associated, macrophage-secreted mediator mRNAs from MDI exposed murine BALCs and MDI-glutathione (GSH) conjugate treated differentiated THP-1 macrophages were analyzed using RT-qPCR.Endogenous IL1B, TNF, CCL2, CCL3, CCL5, and TGFB1 were upregulated in MDI or MDI-GSH conjugate exposed BALCs and macrophages, respectively. Calcineurin inhibitor tacrolimus (FK506) attenuated the MDI-GSH conjugate-mediated induction of CCL2, CCL3, CCL5, and CXCL8/IL8 but not others. Transfection of either miR-inhibitor-206-3p or miR-inhibitor-381-3p in macrophages induced chemokine CCL2, CCL3, CCL5, and CXCL8 transcription, whereas FK506 attenuated the miR-inhibitor-206-3p or miR-inhibitor-381-3p-mediated effects. Finally, MDI-GSH conjugate treated macrophages showed increased chemotactic ability to various immune cells, which may be attenuated by FK506.In conclusion, these results indicate that MDI exposure to macrophages/BALCs may recruit immune cells into the airway via induction of chemokines by miR-206-3p and miR-381-3p-mediated calcineurin signaling activation. |
A comparison of performance metrics for cloth masks as source control devices for simulated cough and exhalation aerosols.
Lindsley WG , Blachere FM , Beezhold DH , Law BF , Derk RC , Hettick JM , Woodfork K , Goldsmith WT , Harris JR , Duling MG , Boutin B , Nurkiewicz T , Boots T , Coyle J , Noti JD . Aerosol Sci Technol 2021 55 (10) 1125-1142 Universal mask wearing is recommended to help control the spread of COVID-19. Masks reduce the expulsion of aerosols of respiratory fluids into the environment (called source control) and offer some protection to the wearer. Masks are often characterized using filtration efficiency, airflow resistance, and manikin or human fit factors, which are standard metrics used for personal protective devices. However, none of these metrics are direct measurements of how effectively a mask blocks coughed and exhaled aerosols. We studied the source control performance of 15 cloth masks (face masks, neck gaiters, and bandanas), two medical masks, and two N95 filtering facepiece respirators by measuring their ability to block aerosols ≤7 µm expelled during simulated coughing and exhalation (called source control collection efficiency). These measurements were compared with filtration efficiencies, airflow resistances, and fit factors measured on manikin headforms and humans. Collection efficiencies for the cloth masks ranged from 17% to 71% for coughing and 35% to 66% for exhalation. Filtration efficiencies for the cloth masks ranged from 1.4% to 98%, while the fit factors were 1.3 to 7.4 on headforms and 1.0 to 4.0 on human subjects. The Spearman’s rank correlation coefficients between the source control collection efficiencies and the standard metrics ranged from 0.03 to 0.68 and were significant in all but two cases. However, none of the standard metrics were strongly correlated with source control performance. A better understanding of the relationships between source control collection efficiency, filtration efficiency, airflow resistance, and fit factor is needed. ©, This work was authored as part of the Contributor's official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 USC. 105, no copyright protection is available for such works under US Law. |
Acute 4,4'-methylene diphenyl diisocyanate exposure-mediated downregulation of miR-206-3p and miR-381-3p activates inducible nitric oxide synthase transcription by targeting calcineurin/NFAT signaling in macrophages
Lin CC , Law BF , Hettick JM . Toxicol Sci 2019 173 (1) 100-113 Exposure to 4,4'-methylene diphenyl diisocyanate (MDI) in the occupational setting may lead to development of occupational asthma (OA), and the underlying molecular mechanisms of MDI-induced disease pathogenesis remain an active area of research. Using a nose-only mouse inhalation model, we find that circulating microRNA (miR)-206-3p and miR-381-3p are downregulated after MDI exposure; however, cellular miR-206-3p and miR-381-3p responses after MDI aerosol exposure and their pathophysiological roles in MDI-OA are unknown. We hypothesize that miR-206-3p and miR-381-3p-regulated mechanisms cause increased expression of the inducible nitric oxide synthase (iNOS) after MDI aerosol exposure. We examined cellular miR-206-3p and miR-381-3p, calcineurins, nuclear factors of activated T-cells (NFATs) and iNOS levels from both nose-only exposed murine bronchoalveolar lavage cells (BALCs) and differentiated THP-1 macrophages treated with MDI-glutathione (GSH) conjugates. Both in vivo murine MDI aerosol exposure and in vitro MDI-GSH exposures in THP-1 macrophages results in downregulation of endogenous miR-206-3p and miR-381-3p and upregulation of PPP3CA and iNOS expression. Transfection of THP-1 macrophages with miR-inhibitor-206-3p and miR-inhibitor-381-3p resulted in the upregulation of PPP3CA and iNOS. Using RNA-induced silencing complex immunoprecipitation (RISC-IP) and translational reporter assays, we verified that PPP3CA, but not iNOS, is directly targeted by both miR-206-3p and miR-381-3p. Downregulation of miR-206-3p and miR-381-3p following by MDI exposure induces Calcineurin/NFAT signaling-mediated iNOS transcription in macrophages and BALCs. |
Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4'-methylene diphenyl diisocyanate exposure
Lin CC , Law BF , Siegel PD , Hettick JM . Biomarkers 2018 24 (1) 1-45 BACKGROUND: Occupational exposure to the most widely used diisocyanate, 4,4'-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA. OBJECTIVE: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA. MATERIALS AND METHODS: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580 +/- 1497 mug/m(3) MDI-aerosol for 60 minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY miRs qPCR Pro fi ling Service was used to profile circulating miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan(R) miR assays. RESULTS: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens. DISCUSSION AND CONCLUSION: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations. |
Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol
Hettick JM , Law BF , Lin CC , Wisnewski AV , Siegel PD . Xenobiotica 2017 48 (6) 1-32 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. 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 aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 hours and 24 hours post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to protein content of the cellular fraction of the lavage fluid. Following MDI aerosol exposure, expression of a 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. 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. |
Topical application of the anti-microbial chemical triclosan induces immunomodulatory responses through the S100A8/A9-TLR4 pathway
Marshall NB , Lukomska E , Nayak AP , Long CM , Hettick JM , Anderson SE . J Immunotoxicol 2017 14 (1) 50-59 The anti-microbial compound triclosan is incorporated into numerous consumer products and is detectable in the urine of 75% of the general United States population. Recent epidemiological studies report positive associations with urinary triclosan levels and allergic disease. Although not sensitizing, earlier studies previously found that repeated topical application of triclosan augments the allergic response to ovalbumin (OVA) though a thymic stromal lymphopoietin (TSLP) pathway in mice. In the present study, early immunological effects following triclosan exposure were further evaluated following topical application in a murine model. These investigations revealed abundant expression of S100A8/A9, which reportedly acts as an endogenous ligand for Toll-like Receptor 4 (TLR4), in skin tissues and in infiltrating leukocytes during topical application of 0.75-3.0% triclosan. Expression of Tlr4 along with Tlr1, Tlr2 and Tlr6 increased in skin tissues over time with triclosan exposure; high levels of TLR4 were expressed on skin-infiltrating leukocytes. In vivo antibody blockade of the TLR4/MD-2 receptor complex impaired local inflammatory responses after four days, as evidenced by decreased Il6, Tnfalpha, S100a8, S100a9, Tlr1, Tlr2, Tlr4 and Tlr6 expression in the skin and decreased lymph node cellularity and production of IL-4 and IL-13 by lymph node T-cells. After nine days of triclosan exposure with TLR4/MD-2 blockade, impaired T-helper cell type 2 (TH2) cytokine responses were sustained, but other early effects on skin and lymph node cellularity were lost; this suggested alternative ligands/receptors compensated for the loss of TLR4 signaling. Taken together, these data suggest the S100A8/A9-TLR4 pathway plays an early role in augmenting immunomodulatory responses with triclosan exposure and support a role for the innate immune system in chemical adjuvancy. |
Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility
Miller MR , Miller KA , Bian J , James ME , Zhang S , Lynch MJ , Callery PS , Hettick JM , Cockburn A , Liu J , Li C , Crane BR , Charon NW . Nat Microbiol 2016 (10) 16134 Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola) 1. These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space 2-11. The universal joint or hook that links the rotary motor to the filament is composed of ∼120-130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex 9,12-17. In other bacteria, the hook can be readily dissociated by treatments such as heat 18. In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking 12,13,17. Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes. |
The Influence of Diisocyanate Antigen Preparation Methodology on Monoclonal and Serum Antibody Recognition
Hagerman LM , Law BF , Bledsoe TA , Hettick JM , Kashon ML , Lemons AR , Wisnewski AV , Siegel PD . J Occup Environ Hyg 2016 13 (11) 829-39 Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences. |
Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin
Mhike M , Hettick JM , Chipinda I , Law BF , Bledsoe TA , Lemons AR , Nayak AP , Green BJ , Beezhold DH , Simoyi RH , Siegel PD . J Immunol Methods 2016 431 38-44 Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity. |
Production of a Chaetomium globosum enolase monoclonal antibody
Green BJ , Nayak AP , Lemons AR , Rittenour WR , Hettick JM , Beezhold DH . Monoclon Antib Immunodiagn Immunother 2014 33 (6) 428-37 Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species. |
A murine inhalation model to characterize pulmonary exposure to dry Aspergillus fumigatus conidia
Buskirk AD , Green BJ , Lemons AR , Nayak AP , Goldsmith WT , Kashon ML , Anderson SE , Hettick JM , Templeton SP , Germolec DR , Beezhold DH . PLoS One 2014 9 (10) e109855 Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Deltaalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1x105 twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8+ IL17+ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Deltaalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants. |
Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles
Nayak AP , Hettick JM , Siegel PD , Anderson SE , Long CM , Green BJ , Beezhold DH . Toxicol Sci 2014 140 (2) 327-37 Diisocyanates (dNCOs) are potent chemical allergens utilized in various industries. It has been proposed that skin exposure to dNCOs produces immune sensitization leading to work-related asthma and allergic disease. We examined dNCOs sensitization by using a dermal murine model of toluene diisocyanate (TDI) exposure to characterize the disposition of TDI in the skin, identify the predominant haptenated proteins, and discern the associated antigen uptake by dendritic cells. Ears of BALB/c mice were dosed once with TDI (0.1% or 4% v/v acetone). Ears and draining lymph nodes (DLNs) were excised at selected time points between 1 h and 15 days post-exposure and were processed for histological, immunohistochemical, and proteomic analyses. Monoclonal antibodies specific for TDI-haptenated protein (TDI-hp) and antibodies to various cell markers were utilized with confocal microscopy to determine co-localization patterns. Histopathological changes were observed following exposure in ear tissue of mice dosed with 4% TDI/acetone. Immunohistochemical staining demonstrated TDI-hp localization in the stratum corneum, hair follicles, and sebaceous glands. TDI-hp were co-localized with CD11b+ (integrin alphaM/Mac-1), CD207+ (langerin), and CD103+ (integrin alphaE) cells in the hair follicles and in sebaceous glands. TDI-hp were also identified in the DLN 1 h post-exposure. Cytoskeletal and cuticular keratins along with mouse serum albumin were identified as major haptenated species in the skin. The results of this study demonstrate that the stratum corneum, hair follicles, and associated sebaceous glands in mice are dendritic cell accessible reservoirs for TDI-hp and thus identify a mechanism for immune recognition following epicutaneous exposure to TDI. |
A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates
Lemons AR , Siegel PD , Mhike M , Law BF , Hettick JM , Bledsoe TA , Nayak AP , Beezhold DH , Green BJ . J Occup Environ Hyg 2014 11 (2) 101-10 Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 mug of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers. |
Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures
Buskirk AD , Templeton SP , Nayak AP , Hettick JM , Law BF , Green BJ , Beezhold DH . J Immunotoxicol 2013 11 (2) 180-9 Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Deltaarp2 (tan) or Deltaalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Deltaalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8+ IL-17+ (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia. |
Characterization of Cannabis sativa allergens
Nayak AP , Green BJ , Sussman G , Berlin N , Lata H , Chandra S , Elsohly MA , Hettick JM , Beezhold DH . Ann Allergy Asthma Immunol 2013 111 (1) 32-37 e4 BACKGROUND: Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa. OBJECTIVE: To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients. METHODS: Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified. RESULTS: Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity. CONCLUSION: Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species. |
Characterization of methylene diphenyl diisocyanate haptenated human serum albumin and hemoglobin
Mhike M , Chipinda I , Hettick JM , Simoyi RH , Lemons A , Green BJ , Siegel PD . Anal Biochem 2013 440 (2) 197-204 Protein haptenation by polyurethane industrial intermediate methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers' sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent, electrophilic nature of dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI: protein. Adducts were characterized by (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity. |
Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin
Wisnewski AV , Mhike M , Hettick JM , Liu J , Siegel PD . Toxicol In Vitro 2012 27 (2) 662-71 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 10mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100muM) 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 3h) 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. |
Comparative analysis of aromatic diisocyanate conjugation to human albumin utilizing multiplexed tandem mass spectrometry
Hettick JM , Siegel PD . Int J Mass Spectrom 2012 309 168-175 Diisocyanates are industrially important chemicals that serve as polymerizing agents in a variety of polyurethane products. In addition to their many industrial uses, diisocyanates have been implicated as causative agents of occupational allergic respiratory disease, although the specific mechanism(s) by which these diseases occur remains unknown. In this study the sites of conjugation of the two most industrially important monomeric diisocyanates, methylene diphenyl diisocyanate (MDI) and toluene diisocyanate (TDI) on human serum albumin are identified utilizing multiplexed tandem mass spectrometry on a quadrupole time-of-flight mass spectrometer. Analysis of human albumin reacted with MDI and TDI over the range of 1:1-40:1 (isocyanate:protein) mol ratio reveals that MDI and TDI react with a maximum of 20 and 37 residues, respectively. Conjugation of diisocyanates to albumin proceeds in a concentration-dependant manner with MDI and TDI reacting at a preferred subset of 5 and 10 residues, respectively, in the limiting case of a 1:1 mol ratio. MDI reacts at fewer residues than does TDI, and is not observed to react with any residues exclusive of TDI. These results cannot be explained on the basis of simple sterics or hydrophobicity, but rather on the basis of increased reactivity of one TDI isocyanate moiety due to electron withdrawing character of the second isocyanate moiety. Furthermore, reaction of diisocyanates with albumin in a phosphate buffered saline (PBS) solution provides three additional reactive sites that are not observed in ammonium bicarbonate buffer. Two lysine residues, Lys(199) and Lys(525), are observed to be reactive to both diisocyanates at all concentrations and in all solvent systems employed in this study. This study presents a comprehensive conjugation map of MDI and TDI on human albumin. The results suggest that several albumin residues are reactive toward both MDI and TDI and may be useful for biomonitoring of diisocyanate exposures. (Published by Elsevier B.V.) |
Vapor conjugation of toluene diisocyanate to specific lysines of human albumin
Hettick JM , Siegel PD , Green BJ , Liu J , Wisnewski AV . Anal Biochem 2011 421 (2) 706-11 Exposure to toluene diisocyanate (TDI), an industrially important crosslinking agent used in the production of polyurethane products, can cause asthma in sensitive workers. Albumin has been identified as a major reaction target for TDI in vivo, and TDI-albumin reaction products have been proposed to serve as exposure biomarkers and to act as asthmagens, yet they remain incompletely characterized. In the current study, we used a multiplexed tandem mass spectrometry (MS/MS) approach to identify the sites of albumin conjugation by TDI vapors, modeling the air/liquid interface of the lung. Vapor phase TDI was found to react with human albumin in a dose-dependent manner, with up to 18 potential sites of conjugation, the most susceptible being Lys351 and the dilysine site Lys413-414. Sites of vapor TDI conjugation to albumin were quantitatively limited compared with those recently described for liquid phase TDI, especially in domains IIA and IIIB of albumin. We hypothesize that the orientation of albumin at the air/liquid interface plays an important role in vapor TDI conjugation and, thus, could influence biological responses to exposure and the development of in vitro assays for exposure and immune sensitivity. |
Toluene diisocyanate reactivity with glutathione across a vapor/liquid interface and subsequent transcarbamoylation of human albumin
Wisnewski AV , Hettick JM , Siegel PD . Chem Res Toxicol 2011 24 (10) 1686-93 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 horizontal lineC horizontal lineO 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. |
Tissue binding affects the kinetics of theophylline diffusion through the stratum corneum barrier layer of skin
Frasch HF , Barbero AM , Hettick JM , Nitsche JM . J Pharm Sci 2011 100 (7) 2989-95 New data sets on both (i) equilibrium theophylline (TH) partitioning/binding in stratum corneum and (ii) transient TH diffusion through human epidermis are explained by an extended partition-diffusion model with reversible binding. Data conform to a linear binding isotherm within the tested concentration range (0-2000 mcg/mL) with an equilibrium ratio of bound-to-free solute of approximately 1.4. The permeability coefficient for TH is 4.86 x 10(-5) cm/h, and the lag time is 20.1 h. Binding occurs as a slow process, significantly affecting the kinetics of dermal penetration. |
Production, characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins
Ruwona TB , Johnson VJ , Hettick JM , Schmechel D , Beezhold D , Wang W , Simoyi RH , Siegel PD . J Immunol Methods 2011 373 127-35 Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring. |
Haptenation: chemical reactivity and protein binding
Chipinda I , Hettick JM , Siegel PD . J Allergy (Cairo) 2011 2011 839682 Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed. |
Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen, Aspergillus terreus
Nayak AP , Green BJ , Janotka E , Hettick JM , Friend S , Vesper SJ , Schmechel D , Beezhold DH . Clin Vaccine Immunol 2011 18 (9) 1568-76 A. terreus has been difficult to identify in cases of aspergillosis and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (mAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG(1) isotype murine mAbs were developed and tested for cross reactivity against hyphal extracts of 54 fungal species. Sixteen mAbs were shown to be specific for A. terreus. HEA antigens were detected in conidia, hyphae and in CSN of A. terreus. HEA antigens were expressed in high levels in the hyphae during early stages of A. terreus growth at 37 degrees C, whereas at room temperature, the expression of HEA antigens peaked by day 4-5. Expression kinetics of HEA antigens in CSN showed a lag, with peak levels at later time points at RT and 37 degrees C compared to hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA antigen epitopes by mAb ELISA. Immunoprecipitation and proteomic analysis demonstrated that mAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while mAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using Confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures, while dipeptidyl-peptidase V was mostly confined to the cytoplasm. |
Occupational sensitization to soy allergens in workers at a processing facility
Green BJ , Cummings KJ , Rittenour WR , Hettick JM , Bledsoe TA , Blachere FM , Siegel PD , Gaughan DM , Kullman GJ , Kreiss K , Cox-Ganser J , Beezhold DH . Clin Exp Allergy 2011 41 (7) 1022-30 BACKGROUND: Exposure to soy antigens has been associated with asthma in community outbreaks and in some workplaces. Recently, 135 soy flake processing workers (SPWs) in a Tennessee facility were evaluated for immune reactivity to soy. Allergic sensitization to soy was common and was five times more prevalent than in health care worker controls (HCWs) with no known soy exposure. OBJECTIVE: To characterize sensitization to soy allergens in SPWs. Methods Sera that were positive to soy ImmunoCAP (n=27) were tested in IgE immunoblots. Wild-type (WT) and transgenic (TG) antigens were sequenced using nanoscale Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (nanoUPLC MS/MS). IgE reactivity towards 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSP), a protein found in TG soy, was additionally investigated. De-identified sera from 50 HCWs were used as a control. Results Immunoblotting of WT and TG soy flake extracts revealed IgE against multiple soy antigens with reactivity towards 48, 54, and 62 kDa bands being the most common. The prominent proteins that bound SPW IgE were identified by nanoUPLC MS/MS analysis to be the high molecular weight soybean storage proteins, beta-conglycinin (Gly m 5), and Glycinin (Gly m 6). No specific IgE reactivity could be detected to lower molecular weight soy allergens, Gly m 1 and Gly m 2, in soybean hull (SH) extracts. IgE reactivity was comparable between WT and TG extracts; however, IgE antibodies to CP4-EPSP could not be detected. CONCLUSIONS AND CLINICAL RELEVANCE: SPWs with specific IgE to soy reacted most commonly with higher molecular weight soybean storage proteins compared with the lower molecular weight SH allergens identified in community asthma studies. IgE reactivity was comparable between WT and TG soy extracts, while no IgE reactivity to CP4-EPSP was observed. High molecular weight soybean storage allergens, Gly m 5 and Gly m 6, may be respiratory sensitizers in occupational exposed SPWs. |
Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry
Hettick JM , Siegel PD . Anal Biochem 2011 414 (2) 232-8 Diisocyanates are highly reactive chemical compounds widely used in the manufacture of polyurethanes. Although diisocyanates have been identified as causative agents of allergic respiratory diseases, the specific mechanism by which these diseases occur is largely unknown. To better understand the chemical species produced when diisocyanates react with protein, tandem mass spectrometry was employed to unambiguously identify the binding sites of the industrially important isomers, 2,4- and 2,6-toluene diisocyanate on human serum albumin at varying diisocyanate:protein ratios. The 2,4- isomer results in approximately two-fold higher conjugation product ion abundances than does the 2,6- isomer, suggesting the 2,4- isomer has a higher reactivity towards albumin. Both isomers preferentially react with the N-terminal amine of the protein and the epsilon-NH(2) of lysine. At a low (1:2) diisocyanate:protein ratio, five binding sites are identified, whereas at a high 40:1 ratio, near-stoichiometric conjugation is observed with a maximum of thirty-seven binding sites identified. Binding sites observed at the lowest conjugation ratios are conserved at higher binding ratios suggesting a subset of five to ten preferential binding sites on albumin. Diisocyanate-protein conjugation results in a variety of different reaction products, including intra- and inter-molecular crosslinking, diisocyanate self-polymerization, and diisocyanate hydrolysis. |
Fungal pigments inhibit the MALDI-TOF mass spectrometry analysis of darkly pigmented fungi
Buskirk AD , Hettick JM , Chipinda I , Law BF , Siegel PD , Slaven JE , Green BJ , Beezhold DH . Anal Biochem 2010 411 (1) 122-8 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate monilaceous fungal species, however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF MS. Signal suppression was observed in samples containing greater than 25 ng/muL pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, non-pigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. Addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less-melanized fungi that may be analyzed by MALDI-TOF MS. |
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