Last data update: Nov 04, 2024. (Total: 48056 publications since 2009)
Records 1-24 (of 24 Records) |
Query Trace: Nayak AP[original query] |
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Occupational allergies to cannabis
Decuyper II , Green BJ , Sussman GL , Ebo DG , Silvers WS , Pacheco K , King BS , Cohn JR , Zeiger RS , Zeiger JS , Naimi DR , Beezhold DH , Nayak AP . J Allergy Clin Immunol Pract 2020 8 (10) 3331-3338 Within the last decade there has been a significant expansion in access to cannabis for medicinal and adult nonmedical use in the United States and abroad. This has resulted in a rapidly growing and diverse workforce that is involved with the growth, cultivation, handling, and dispensing of the cannabis plant and its products. The objective of this review was to educate physicians on the complexities associated with the health effects of cannabis exposure, the nature of these exposures, and the future practical challenges of managing these in the context of allergic disease. We will detail the biological hazards related to typical modern cannabis industry operations that may potentially drive allergic sensitization in workers. We will highlight the limitations that have hindered the development of objective diagnostic measures that are essential in separating “true” cannabis allergies from nonspecific reactions/irritations that “mimic” allergy-like symptoms. Finally, we will discuss recent advances in the basic and translational scientific research that will aid the development of diagnostic tools and therapeutic standards to serve optimal management of cannabis allergies across the occupational spectrum. |
Cannabis: An emerging occupational allergen
Sussman GL , Beezhold DH , Cohn JR , Silvers WS , Zeiger JS , Nayak AP . Ann Work Expo Health 2020 64 (7) 679-682 Cannabis is the most commonly used psychoactive drug. In recent years, Cannabis access has expanded for both medicinal and non-medicinal has grown. This is also marked with an increasing number of individuals gaining employment in this emerging industry. In this article, we briefly discuss the health hazards associated with Cannabis exposure with an emphasis on the potential for allergic reactions in workers who handle and process Cannabis plant. |
Inhalation of Stachybotrys chartarum fragments induces pulmonary arterial remodeling
Croston TL , Lemons AR , Barnes MA , Goldsmith WT , Orandle MS , Nayak AP , Germolec DR , Green BJ , Beezhold DH . Am J Respir Cell Mol Biol 2019 62 (5) 563-576 Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (Strain A or Strain B), heat-inactivated conidia, or HEPA-filtered air twice a week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments compared to Strain B. Lung tissue, serum and bronchoalveolar lavage fluid (BALF) were collected at 24 and 48 hours following final exposure and processed for histology, flow cytometry, RNA, and proteomic analyses. At 4 weeks post-exposure, a Th2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed following exposure to Strain A, compared to a Th2-mediated response following Strain B exposure. Following exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, Strain A-exposed mice progressed more quickly compared to Strain B-exposed mice. BALF was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing Strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure. |
Endotoxin exposures during harvesting and processing cannabis at an outdoor cannabis farm
Couch JR , Burton NC , Victory KR , Green BJ , Lemons AR , Nayak AP , Beezhold DH . Aerobiologia 2019 35 (2) 367-371 Legalization of medicinal and recreational cannabis use in numerous states within the USA has resulted in the increased commercial cultivation of cannabis. Outdoor cannabis farming operations present a variety of potential physical, chemical, and biological hazards that currently remain uncharacterized. Worker exposures to endotoxins were evaluated at an outdoor US cannabis farm during harvesting and processing activities. Endotoxin area air sample concentrations ranged from below the limit of detection to 15 endotoxin units per cubic meter (EU/m3). Endotoxin breathing zone measurements (2.8–37 EU/m3) were below the Dutch Expert Committee on Occupational Safety occupational exposure limit of 90 /m3. During confidential medical interviews, no adverse health effects were reported by workers while harvesting or processing cannabis. Further endotoxin exposure assessments should be performed especially in larger, indoor cannabis operations where a confined environment may result in higher endotoxin exposures than observed in this outdoor environment. |
Aspergillus fumigatus viability drives allergic responses to inhaled conidia
Nayak AP , Croston TL , Lemons AR , Goldsmith WT , Marshall NB , Kashon ML , Germolec DR , Beezhold DH , Green BJ . Ann Allergy Asthma Immunol 2018 121 (2) 200-210 e2 BACKGROUND: Aspergillus fumigatus induced allergic airway disease has been shown to involve conidial germination in vivo but the immunological mechanisms remain uncharacterized. OBJECTIVE: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or non-culturable A. fumigatus conidia. METHODS: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 x 105 viable or heat inactivated conidia (HIC), twice a week for 13 weeks (26 exposures). Control mice inhaled HEPA-filtered air. The influence of A. fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung mRNA expression, and quantitative proteomics and histopathology of whole lung tissue. RESULTS: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (e.g. Arg1, Chil3 and Retnla) were significantly upregulated in viable A. fumigatus exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-gamma (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult. CONCLUSION: Our studies demonstrate that A. fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease. |
Microbial hazards during harvesting and processing at an outdoor United States cannabis farm.
Green BJ , Couch JR , Lemons AR , Burton NC , Victory KR , Nayak AP , Beezhold DH . J Occup Environ Hyg 2018 15 (5) 0 Cannabis cultivation is an emerging industry within the United States. Organic dust derived in part from naturally occurring microorganisms is known to cause byssinosis in the hemp industry. In this pilot study, bacteria and fungi encountered by workers at an outdoor cannabis farm that utilized organic practices were elucidated by 1625% shaded blockS ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) region sequencing, respectively. Area (n = 14) and personal air samples (n = 12) were collected during harvesting and processing activities. 1625% shaded blockS rRNA and ITS regions of extracted bacterial and fungal genomic DNA were amplified and sequenced using Sanger sequencing. Bacterial sequencing resolved 1077 sequences that were clustered into 639 operational taxonomic units (OTUs) and predominantly placed in the phylum, Actinobacteria (46%). Personal air samples revealed higher bacterial and Actinobacteria diversity compared to outdoor area samples collected within the facility (p<0.05). A high degree of dissimilarity between bacteria was identified within and between samples. Fungal sequences (n = 985) were identified and predominantly clustered in the phylum Ascomycota (53%). Of the 216 fungal OTUs elucidated, the cannabis plant pathogenic species, Botrytis cinerea, was the most prevalent and accounted for 34% of all fungal sequences. The relative abundance of B. cinerea was highest in personal air samples (59%) compared to area samples collected in the drying room (19%), greenhouse (18%) and outdoor environment (6%). There was 49% sample similarity between fungi identified within personal air samples, but higher dissimilarity coefficients were observed within and between greenhouse, drying room, and outdoor area air samples. The results of this pilot study suggest that the cannabis farm workers are potentially exposed to Actinobacteria as well as the cannabis plant pathogen, B. cinerea during harvesting, bud stripping, and hand trimming processes. |
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. |
Influence of Aspergillus fumigatus conidia viability on murine pulmonary microRNA and mRNA expression following subchronic inhalation exposure.
Croston TL , Nayak AP , Lemons AR , Goldsmith WT , Gu JK , Germolec DR , Beezhold DH , Green BJ . Clin Exp Allergy 2016 46 (10) 1315-27 BACKGROUND: Personal exposure to fungal bioaerosols derived from contaminated building materials or agricultural commodities may induce or exacerbate a variety of adverse health effects. The genomic mechanisms that underlie pulmonary immune responses to fungal bioaerosols have remained unclear. OBJECTIVE: The impact of fungal viability on the pulmonary microRNA and messenger RNA profiles that regulate murine immune responses was evaluated following subchronic inhalation exposure to Aspergillus fumigatus conidia. METHODS: Three groups of naive B6C3F1/N mice were exposed via nose-only inhalation to A. fumigatus viable conidia, heat-inactivated conidia, or HEPA-filtered air twice a week for 13 weeks. Total RNA was isolated from whole lung 24 and 48 hours post final exposure and was further processed for gene expression and microRNA array analysis. The molecular network pathways between viable and heat-inactivated conidia groups were evaluated. RESULTS: Comparison of datasets revealed increased Il4, Il13, and Il33 expression in mice exposed to viable versus heat-inactivated conidia. Of 415 microRNAs detected, approximately 50% were altered in mice exposed to viable versus heat-inactivated conidia 48 hours post exposure. Significantly downregulated (P < 0.05) miR-29a-3p was predicted to regulate TGF-beta3 and Clec7a, genes involved in innate responses to viable A. fumigatus. Also significantly downregulated (P < 0.05), miR-23b-3p regulates genes involved in pulmonary IL-13 and IL-33 responses and SMAD2, downstream of TGF-beta signaling. Using Ingenuity Pathway Analysis, a novel interaction was identified between viable conidia and SMAD2/3. CONCLUSION AND CLINICAL RELEVANCE: Examination of the pulmonary genetic profiles revealed differentially expressed genes and microRNAs following subchronic inhalation exposure to A. fumigatus. MicroRNAs regulating genes involved in the pulmonary immune responses were those with the greatest fold change. Specifically, germinating A. fumigatus conidia were associated with Clec7a and were predicted to interact with Il13 and Il33. Furthermore, altered microRNAs may serve as potential biomarkers to evaluate fungal exposure. |
Subchronic exposures to fungal bioaerosols promotes allergic pulmonary inflammation in naive mice
Nayak AP , Green BJ , Lemons AR , Marshall NB , Goldsmith WT , Kashon ML , Anderson SE , Germolec DR , Beezhold DH . Clin Exp Allergy 2016 46 (6) 861-70 BACKGROUND: Epidemiological surveys indicate that occupants of mold contaminated environments are at increased risk of respiratory symptoms. The immunological mechanisms associated with these responses require further characterization. OBJECTIVE: The aim of this study was to characterize the immunotoxicological outcomes following repeated inhalation of dry Aspergillus fumigatus spores aerosolized at concentrations potentially encountered in contaminated indoor environments. METHODS: A. fumigatus spores were delivered to the lungs of naive BALB/cJ mice housed in a multi-animal nose-only chamber twice a week for a period of 13 weeks. Mice were evaluated at 24 and 48 hours post-exposure for histopathological changes in lung architecture, recruitment of specific immune cells to the airways, and serum antibody responses. RESULT: Germinating A. fumigatus spores were observed in lungs along with persistent fungal debris in the perivascular regions of the lungs. Repeated exposures promoted pleocellular infiltration with concomitant epithelial mucus hypersecretion, goblet cell metaplasia, subepithelial fibrosis and enhanced airway hyperreactivity. Cellular infiltration in airways was predominated by CD4+ T cells expressing the pro-allergic cytokine IL-13. Furthermore, our studies show that antifungal T cell responses (IFN-gamma+ or IL-17A+ ) co-expressed IL-13, revealing a novel mechanism for the dysregulated immune response to inhaled fungi. Total IgE production was augmented in animals repeatedly exposed to A. fumigatus. CONCLUSIONS & CLINICAL RELEVANCE: Repeated inhalation of fungal aerosols resulted in significant pulmonary pathology mediated by dynamic shifts in specific immune populations and their cytokines. These studies provide novel insights into the immunological mechanisms and targets that govern the health outcomes that result from repeated inhalation of fungal bioaerosols in contaminated environments. This article is protected by copyright. All rights reserved. |
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. |
Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy
Afanou KA , Straumfors A , Skogstad A , Nayak AP , Skaar I , Hjeljord L , Tronsmo A , Eduard W , Green BJ . Appl Environ Microbiol 2015 81 (17) 5794-803 Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-L-lysine coated polycarbonate filters. Ninety percent of submicronic fragments and 1-2 mum fragments, compared to 100% of >2 mum fragments generated from pure freeze dried mycelial fragments of A. versicolor, were positively labeled. In proof of concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabelling technique. Our results indicated that 13% of total collected particles were derived from fungi. This fraction comprises 79% of fragments that were detected by immunolabelling and 21% spore particles that were morphologically identified. The methods reported in this study, enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample. |
Triclosan induces thymic stromal lymphopoietin in skin promoting Th2 allergic responses
Marshall NB , Lukomska E , Long CM , Kashon ML , Sharpnack DD , Nayak AP , Anderson KL , Meade BJ , Anderson SE . Toxicol Sci 2015 147 (1) 127-39 Triclosan is an antimicrobial chemical incorporated into many personal, medical and household products. 75% of the U.S. population has detectable levels of triclosan in their urine and although it is not typically considered a contact sensitizer, recent studies have begun to link triclosan exposure with augmented allergic disease. We examined the effects of dermal triclosan exposure on the skin and lymph nodes of mice and in a human skin model to identify mechanisms for augmenting allergic responses. Triclosan (0-3%) was applied topically at 24 hour intervals to the ear pinnae of OVA-sensitized BALB/c mice. Skin and draining lymph nodes were evaluated for cellular responses and cytokine expression over time. The effects of triclosan (0-0.75%) on cytokine expression in a human skin tissue model was also examined. Exposure to triclosan increased the expression of TSLP, IL-1beta and TNF-alpha in the skin with concomitant decreases in IL-25, IL-33 and IL-1alpha. Similar changes in TSLP, IL1B and IL33 expression occurred in human skin. Topical application of triclosan also increased draining lymph node cellularity consisting of activated CD86+GL-7+ B cells, CD80+CD86+ dendritic cells, GATA-3+OX-40+IL-4+IL-13+ Th2 cells and IL-17A+ CD4 T cells. In-vivo antibody blockade of TSLP reduced skin irritation, IL-1beta expression, lymph node cellularity, and Th2 responses augmented by triclosan. Repeated dermal exposure to triclosan induces TSLP expression in skin tissue as a potential mechanism for augmenting allergic responses. |
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. |
Aspergillus collagen-like genes (acl): identification, sequence polymorphism, and assessment for PCR-based pathogen detection
Tuntevski K , Durney BC , Snyder AK , Lasala PR , Nayak AP , Green BJ , Beezhold DH , Rio RV , Holland LA , Lukomski S . Appl Environ Microbiol 2013 79 (24) 7882-95 The genus Aspergillus is a burden to public health due to its ubiquitous presence in the environment, its production of allergens, and wide demographic susceptibility among cystic fibrosis, asthmatic, and immunosuppressed patients. Current methods of detection of Aspergillus colonization and infection rely on lengthy morphological characterization or nonstandardized serological assays that are restricted to identifying a fungal etiology. Collagen-like genes have been shown to exhibit species-specific conservation across the noncollagenous regions as well as strain-specific polymorphism in the collagen-like regions. Here we assess the conserved region of the Aspergillus collagen-like (acl) genes and explore the application of PCR amplicon size-based discrimination among the five most common etiologic species of the Aspergillus genus, including Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. Genetic polymorphism and phylogenetic analysis of the aclF1 gene were additionally examined among the available strains. Furthermore, the applicability of the PCR-based assay to identification of these five species in cultures derived from sputum and bronchoalveolar fluid from 19 clinical samples was explored. Application of capillary electrophoresis on nanogels was additionally demonstrated to improve the discrimination between Aspergillus species. Overall, this study demonstrated that Aspergillus acl genes could be used as PCR targets to discriminate between clinically relevant Aspergillus species. Future studies aim to utilize the detection of Aspergillus acl genes in PCR and microfluidic applications to determine the sensitivity and specificity for the identification of Aspergillus colonization and invasive aspergillosis in immunocompromised subjects. |
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. |
Fungal hemolysins
Nayak AP , Green BJ , Beezhold DH . Med Mycol 2012 51 (1) 1-16 Hemolysins are a class of proteins defined by their ability to lyse red cells but have been described to exhibit pleiotropic functions. These proteins have been extensively studied in bacteria and more recently in fungi. Within the last decade, a number of studies have characterized fungal hemolysins and revealed a fascinating yet diverse group of proteins. The purpose of this review is to provide a synopsis of the known fungal hemolysins with an emphasis on those belonging to the aegerolysin protein family. New insight and perspective into fungal hemolysins in biotechnology and health are additionally presented. |
Development of monoclonal antibodies to recombinant terrelysin and characterization of expression in Aspergillus terreus.
Nayak AP , Green BJ , Friend S , Beezhold DH . J Med Microbiol 2011 61 489-499 Aspergillus terreus is an emerging pathogen that mostly affects immunocompromised patients with infections that are often difficult to manage therapeutically. Current diagnostic strategies are limited to the detection of fungal growth using radiological methods or biopsy that often does not enable species-specific identification. As a result, there is a critical need for diagnostic techniques to enable early and specific identification of the causative agent. In this study, we describe monoclonal antibodies (mAbs) developed to a previously described recombinant terrelysin. Sixteen hybridomas of various IgG isotypes were generated to the recombinant protein, of which seven demonstrated reactivity to the native protein in hyphal extracts. Cross-reactivity analysis using hyphal extracts from 29 fungal species, including 12 Aspergillus species and 5 different strains of A. terreus showed that 3 mAbs (13G10, 15B5 and 10G4) were A. terreus-specific. Epitope analysis demonstrated mAbs 13G10 and 10G4 recognize the same epitope 'PSNEFE', while mAb 15B5 recognized the epitope 'LYEGQFHS'. Time course studies showed that terrelysin expression was highest during early hyphal growth and dramatically reduced after mycelial expansion. Immunolocalization studies demonstrated that terrelysin is localized not only within the cytoplasm of hyphae but appeared to be more abundant at the hyphal tip. These findings were confirmed in cultures grown at room temperature as well as at 37 degrees C. Additionally, terrelysin was detected in the supernatant of A. terreus cultures. These observations suggest terrelysin may be a candidate biomarker for A. terreus infection. |
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. |
Production and characterization of IgM monoclonal antibodies against hyphal antigens of Stachybotrys species
Nayak AP , Green BJ , Janotka E , Blachere FM , Vesper SJ , Beezhold DH , Schmechel D . Hybridoma (Larchmt) 2011 30 (1) 29-36 Stachybotrys is a hydrophilic fungal genus that is well known for its ability to colonize water-damaged building materials in indoor environments. Personal exposure to Stachybotrys chartarum allergens, mycotoxins, cytolytic peptides, and other immunostimulatory macromolecules has been proposed to exacerbate respiratory morbidity. To date, advances in Stachybotrys detection have focused on the identification of unique biomarkers that can be detected in human serum; however, the availability of immunodiagnostic reagents to Stachybotrys species have been limited. In this study, we report the initial characterization of monoclonal antibodies (MAbs) against a semi-purified cytolytic S. chlorohalonata preparation (cScp) derived from hyphae. BALB/c mice were immunized with cScp and hybridomas were screened against the cScp using an antigen-mediated indirect ELISA. Eight immunoglobulin M MAbs were produced and four were specifically identified in the capture ELISA to react with the cScp. Cross-reactivity of the MAbs was tested against crude hyphal extracts derived from 15 Stachybotrys isolates representing nine Stachybotrys species as well as 39 other environmentally abundant fungi using a capture ELISA. MAb reactivity to spore and hyphal antigens was also tested by a capture ELISA and by fluorescent halogen immunoassay (fHIA). ELISA analysis demonstrated that all MAbs strongly reacted with extracts of S. chartarum but not with extracts of 39 other fungi. However, four MAbs showed cross-reactivity to the phylogenetically related genus Memnoniella. fHIA analysis confirmed that greatest MAb reactivity was ultrastructurally localized in hyphae and phialides. The results of this study further demonstrate the feasibility of specific MAb-based immunoassays for the detection of S. chartarum. |
Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus.
Nayak AP , Blachere FM , Hettick JM , Lukomski S , Schmechel D , Beezhold DH . Mycopathologia 2010 171 (1) 23-34 Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly beta-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the beta-sheet confirmation up to 65 degrees C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus. |
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