Donald Beezhold | West Virginia University (original) (raw)

Papers by Donald Beezhold

Viruses, Jan 27, 2017

Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwid... more Influenza A virus (IAV) infection remains a significant cause of morbidity and mortality worldwide. One key transcription factor that is activated upon IAV infection is nuclear factor Kappa B (NF-κB). NF-κB regulation involves the inhibitor proteins NF-κB inhibitor beta (NFKBIB), (also known as IκB β), which form complexes with NF-κB to sequester it in the cytoplasm. In this study, microarray data showed differential expression of several microRNAs (miRNAs) on exposure to IAV. Target scan analysis revealed that miR-4776, miR-4514 and miR-4742 potentially target NFKBIB messenger RNA (mRNA). Time-course analysis of primary bronchial epithelial cells (HBEpCs) showed that miR-4776 expression is increased within 1 h of infection, followed by its downregulation 4 h post-exposure to IAV. NFKBIB upregulation of miR-4776 correlated with a decrease in NFKBIB expression within 1 h of infection and a subsequent increase in NFKBIB expression 4 h post-infection. In addition, miRNA ago-immunopreci...

Toxicological Sciences, 2000

While less than 1% of the general population is sensitized to latex, the U.S. Occupational Safety... more While less than 1% of the general population is sensitized to latex, the U.S. Occupational Safety and Health Administration estimates that 8-12% of health-care workers are sensitized. The major source of workplace exposure is powdered natural rubber latex (NRL) gloves. NRL is harvested from Hevea brasiliensis trees and ammoniated to prevent coagulation resulting in the hydrolysis of the latex proteins. Prior to use in manufacturing, the latex is formulated by the addition of multiple chemicals. Thus, human exposure is to a mixture of residual chemicals and hydrolyzed latex peptides. Clinical manifestations include irritant contact dermatitis, allergic contact dermatitis (type IV), and type I immediate hypersensitivity response. Type I (IgE-mediated) NRL allergy includes contact urticaria, systemic urticaria, angioedema, rhinitis, conjunctivitis, bronchospasm, and anaphylaxis. Taking an accurate history, including questions on atopic status, food allergy, and possible reactions to latex devices makes diagnosis of type-I latex allergy possible. To confirm a diagnosis, either in vivo skin prick testing (SPT) or in vitro assays for latex-specific IgE are performed. While the SPT is regarded as a primary confirmatory test for IgE-mediated disease, the absence of a U.S. Food and Drug Administration-licensed Hevea brasiliensis latex extract has restricted its use in diagnosis. Serological tests have, therefore, become critically important as alternative diagnostic tests. Three manufacturers currently have FDA clearance for in vitro tests, to detect NRL-specific IgE. The commercially available assays may disagree on the antibody status of an individual serum, which may be due to the assays detecting anti-NRL IgEs to different allergenic NRL proteins. Sensitized individuals produce specific IgE antibody to at least 10 potent Hevea allergens, Hev b 1-Hev b 10, each of which differs in its structure, size, and net charge. The relative content and ratios of Hevs in the final allergen preparation most probably could effect diagnostic accuracy. The Hev proteins have been cloned and expressed as recombinant proteins. Sequencing demonstrates both unique epitopes and sequences commonly found in other plant proteins. Sequence homology helps to explain vitro models can elucidate mechanisms of sensitization and provide an understanding of the role of the exposure route in latex allergy-associated diseases. Together, these efforts can lead to intervention strategies for reducing latex allergy in the workplace.

Viruses, 2021

MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogen... more MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host–virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, wh...

Environ. Sci.: Processes Impacts, 2014

Compared to traditional methods of fungal exposure assessment, molecular methods have provided ne... more Compared to traditional methods of fungal exposure assessment, molecular methods have provided new insight into the richness of fungal communities present in both indoor and outdoor environments. In this study, we describe the diversity of fungi in the homes of asthmatic children located in Kansas City. Fungal diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA derived from fungi collected in air and dust samples from 31 homes participating in the Kansas City Safe and Healthy Homes Program (KCSHHP). Sequencing results were then compared to data obtained using viable and non-viable fungal exposure assessment methods. ITS clone libraries were predominantly derived from the phylum Ascomycota in both air (68%) and dust (92%) samples and followed by the Basidiomycota and Zygomycota. The majority of Ascomycota clones belonged to four orders including the Pleosporales, Eurotiales, Capnodiales, and Dothideales. ITS sequencing revealed the presence of a number of rarely documented fungal species placed in the Pleosporales. Several species placed in the Basidiomycota were detected in ITS clone libraries but not by viable or non-viable methods. The prevalence of organizational taxonomic units (OTUs) was significantly higher in air than in dust samples (p < 0.0001); however, no differences between OTUs in air samples collected in the subjects' room and basement were observed. These sequencing results demonstrate a much broader diversity of Ascomycota and Basidiomycota communities in KCSHHP indoor environments than previously estimated using traditional methods of assessment.

Journal of Allergy and Clinical Immunology, 1999

Hev b 5 is an acidic protein (isoelectric point, 3.5) rich in glutamic acid with 9 repeated amino... more Hev b 5 is an acidic protein (isoelectric point, 3.5) rich in glutamic acid with 9 repeated amino acid (AA) sequences of XEEX or XEEEX. Although its function in Hevea brasiliensis is unknown, Hev b 5 has been identified as a major latex allergen. Immunoblot inhibition studies suggest Hev b 5 exists as multiple isoforms or contains a common epitope found in several other proteins. The purpose of this study was to further characterize Hev b 5 and to identify linear IgE-binding epitopes. Octapeptides spanning the entire Hev b 5 protein were synthesized on a derivatized cellulose membrane. The membrane was reacted with sera pooled from health care workers allergic to latex or rabbits immunized with latex proteins. B-cell epitopes were identified by subsequent incubations with the appropriate secondary antibodies and detected by using chemifluorescence. Sera from patients allergic to latex recognized 6 IgE-binding regions located throughout the molecule. Two epitopes (2 and 4) had the common AA sequence of KTEEP. Epitopes 3 and 5 had a similar AA sequence of EEXXA, where X was P, T, or K. Epitopes 1 and 6 appeared to be unrelated to the other epitopes. Database analysis could not identify other proteins with similar sequences. Neither of the XEEEX sequences bound IgE. Control sera failed to react to any peptides. Hev b 5 exists as multiple isoforms, but only small amounts are present in the nonammoniated latex preparations, such as those used for diagnostic tests, and this may help to explain the relatively poor sensitivity of some in vitro tests.

Clinical and Experimental Immunology, 1997

SUMMARY We have previously identified the hevein preprotein as a common allergen for latex allerg... more SUMMARY We have previously identified the hevein preprotein as a common allergen for latex allergic healthcare workers. The B cell epitopes in the hevein protein that are recognized by IgE of latex-allergic individuals have not been identified. In this study, we examined the hevein preprotein using epitope mapping. Overlapping synthetic peptides of 10 amino acids (two aa overlap) were synthesized on a derivatized cellulose membrane using Fmoc chemistry. The peptide spots were probed with pooled sera from 10 latex-allergic patients, and the IgE-reactive peptides identified with anti-IgE MoAbs. We identified six B cell epitopes within the full length hevein preprotein which bound IgE from latex-allergic patients. Two were located in the N-terminal 5-kD hevein domain and four were observed in the 14-kD C-domain. A broad epitope was located between the N-terminal amino acids 13–24. This epitope had nearly complete homology to wheat germ agglutinin (WGA). Immunological cross-reactivity t...

Clinical & Experimental Allergy, 2011

Exposure to soy antigens has been associated with asthma in community outbreaks and in some workp... more 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. To characterize sensitization to soy allergens in SPWs. 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. 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, β-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. 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.

Clinical <html_ent glyph="@amp;" ascii="&"/> Experimental Allergy, 1996

The purpose of this study was to investigate crossreactivity between latex and foods, to identify... more The purpose of this study was to investigate crossreactivity between latex and foods, to identify crossreacting IgE binding proteins, and to assess the clinical significance. Forty-seven latex allergic patients and 46 non-latex allergic patient controls were studied. Allergen sensitization was determined by skin-prick testing (SPT) and allergenic proteins were identified by immunoblot reactivity and amino acid sequence analysis. Immunological reactivity to foods was found to be common, occurring in 33 latex-allergic individuals but in only seven controls (P &amp;lt; 0.000001); 100 of 376 (27%) food skin-prick tests were positive in the latex-allergic subjects. Twenty-seven out of 100 positive food SPTs were associated with clinical symptoms. Seventeen patients manifested a clinical allergy to at least one food including 11 with anaphylaxis, and 14 with local sensitivity reactions. Positive food skin tests occurred most frequently with avocado (53%), potato (40%), banana (38%), tomato (28%), chestnut (28%), and kiwi (17%). Latex-allergic patients (23%) recognize a protein that had sequence homology to a broad class of plant proteins known as patatins. Crossreactivity between latex and several potato proteins was observed by immunoblot inhibition analysis. Sensitization to latex has extensive crossreactivity with certain foods and leads to clinical allergic reactions. Potatoes and tomatoes are newly reported cross-reacting foods. Plant proteins with structural homology to latex proteins may predispose to food allergy.

Allergy and Asthma Proceedings, 1996

atex, natural rubber latex, and natural latex rubber are terms used to denote the processed plant... more atex, natural rubber latex, and natural latex rubber are terms used to denote the processed plant cytosol from the tree hevea braziliensis. Natural rubber latex (NRL) is produced by specialized laticifer cells and composed of polyisoprene, lipids, phospholipids, and proteins. Ammonia or other preservatives are immediately added to NRL to prevent degradation. Other rubber chemical additives including accelerators (MBT, thiurams, carbamates) and antioxidants (phenylenediamine) are used to allow rubber to attain its desired properties. I Allergic reactions occur most commonly to NRL disposable gloves? Clinical reactions include local contact hand urticaria and dermatitis. Many of these reactions are irritantinduced or caused by delayed hypersensitivity responses. However, severe IgE mediated allergy, including NRLinduced occupational asthma, has necessitated a small minority of highly trained allergic health care workers to discontinue their vocation of choice. 3 The cost of one such health care worker can be $215,000, of which 90% is paid by the employer. 4 Life-threatening anaphylactic reactions and deaths have been reported from exposure to NRL during medical and surgical procedures. 3. 5 Clearly it is important that health care workers know the exact material composition and risks of the gloves they use. New materials and formulations are appearing on the market, making it no longer easy to identify materials by casual inspection. Some new vinyl formulations look and feel like NRL. Other new synthetic rubbers are composed of hydrocarbon polymers or elastomers.

Clinical & Experimental Immunology, 2008

SUMMARY Latex allergy is an occupational hazard for health care workers. Extractable latex protei... more SUMMARY Latex allergy is an occupational hazard for health care workers. Extractable latex proteins are known to be allergenic, but most latex allergens have not been specifically identified. The purpose of this study was to characterize the IgE response of latex-allergic patients to latex proteins and to identify common protein allergens. Serum was obtained from 40 individuals who were skin test-positive to latex; 85% were health care workers. Western blots for IgE reactivity were performed using both ammoniated (AL) and non-ammoniated (NAL) latex proteins and IgE-reactive NAL proteins were analysed by microsequence analysis. The patients were grouped according to common patterns of reactivity. Pattern 1, the most common pattern of reactivity (9/40 patients) recognized two protein bands in both NAL and AL at 46 and 110kD. A second, heterogeneous pattern of reactivity (pattern 2) recognized a diffuse pattern of polypeptides in the AL preparation. The n-terminal amino acid sequences ...

Journal of Allergy and Clinical Immunology

B lymphocytes produce IgA that interacts with commensal bacteria, but the dialogue between them i... more B lymphocytes produce IgA that interacts with commensal bacteria, but the dialogue between them is not well understood. These interactions may contribute to autoimmune disease, as both antibiotics and disrupted B cell signaling prevent spontaneous arthritis in K/BxN mice. Bruton's tyrosine kinase (Btk) supports germinal center interactions, but its role in mucosal immunity was not known. METHODS: Intestinal B cells, IgA, and commensal microbes from Btkdeficient, disease-protected K/BxN mice were compared with arthritisprone littermates. Bacterial IgA coating was also studied. Relevant bacteria were introduced into antibiotic-treated K/BxN mice. RESULTS: Peyer's patch B cells, IgA class-switch, intestinal IgA, and bacterial coating were dramatically reduced in btk-deficient K/BxN mice. The IgA-coated community was shifted, including reduced P. distasonis. Re-introduction of P. distasonis to antibiotic-treated K/BxN abrogated disease-protection. CONCLUSIONS: B cell receptor signaling supports the interplay between B lymphocytes, IgA and commensal microbes. BTK-inhibitors in clinical trials for autoimmune and allergic diseases may may have unforeseen effects on the microbiome that influence disease outcome.

Journal of occupational and environmental hygiene, Jan 25, 2018

Cannabis cultivation is an emerging industry within the United States. Organic dust derived in pa... more 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 16░S 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. 16░S 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 deg...

Toxicology and Applied Pharmacology

Chemosphere, 2017

Nanocellulose (NC) is emerging as a highly promising nanomaterial for a wide range of application... more Nanocellulose (NC) is emerging as a highly promising nanomaterial for a wide range of applications. Moreover, many types of NC are produced, each exhibiting a slightly different shape, size, and chemistry. The main objective of this study was to compare cytotoxic effects of cellulose nanocrystals (CNC) and nanofibrillated cellulose (NCF). The human lung epithelial cells (A549) were exposed for 24 h and 72 h to five different NC particles to determine how variations in properties contribute to cellular outcomes, including cytotoxicity, oxidative stress, and cytokine secretion. Our results showed that NCF were more toxic compared to CNC particles with respect to cytotoxicity and oxidative stress responses. However, exposure to CNC caused an inflammatory response with significantly elevated inflammatory cytokines/chemokines compared to NCF. Interestingly, cellulose staining indicated that CNC particles, but not NCF, were taken up by the cells. Furthermore, clustering analysis of the in...

Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, Jan 30, 2016

Personal exposure to fungal bioaerosols derived from contaminated building materials or agricultu... more 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. 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. Three groups of naïve 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. Comparison of datasets revealed increased Il4, Il13, and Il33 expression i...

Virology, Jan 27, 2016

Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strain... more Influenza antiviral drugs that use protein inhibitors can lose their efficacy as resistant strains emerge. As an alternative strategy, we investigated the use of small interfering RNA molecules (siRNAs) by characterizing three siRNAs (M747, M776 and M832) targeting the influenza matrix 2 gene and three (NS570, NS595 and NS615) targeting the nonstructural protein 1 and 2 genes. We also re-examined two previously reported siRNAs, M331 and M950, which target the matrix 1 and 2 genes. Treatment with M331-, M776-, M832-, and M950-siRNAs attenuated influenza titer. M776-siRNA treated cells had 29.8% less infectious virus than cells treated with the previously characterized siRNA, M950. NS570-, NS595- and NS615-siRNAs reduced nonstructural protein 1 and 2 expression and enhanced type I interferon expression by 50%. Combination siRNA treatment attenuated 20.9% more infectious virus than single siRNA treatment. Our results suggest a potential use for these siRNAs as an effective anti-influen...

The Journal of Allergy and Clinical Immunology, Feb 1, 2015

Journal of Allergy and Clinical Immunology, Dec 31, 1996

Background: Natural rubber latex (NRL) gloves are the major source of proteins that cause latex a... more Background: Natural rubber latex (NRL) gloves are the major source of proteins that cause latex allergic reactions in sensitized health care workers and patients. Objective: This study evaluated the effect of manufacturing changes on reducing protein, antigen, and allergen levels of latex medical gloves. Methods: Three types of NRL gloves were manufactured with a common batch of compounded latex. The NRL gloves were analyzed for total protein by using the American Society for Testing and Materials D5712-95 LoWry method, and specifically for latex proteins by immunoassay. Allergen levels in the extracts were determined by end-point titration skin prick tests (SPTs) on patients allergic to NRL. Results: Extracts from regular powdered gloves had detectable levels of latex proteins and allergens (62% SPT positive), whereas the powder-free gloves were low in protein content and allergenicity (5% to 8% SPT positive). No significant difference in SPT reactivity was observed between the chlorinated powder-free gloves and the polymer-coated gloves. Although the protein levels determined by the Lowry assay correlated with SPT reactivity (r = 0.95), the test was restricted by a high detection limit (9.3 I~g/ml). Fifty-eight percent of patients allergic to latex reacted at the 50 Ixg/gm detection limit allowed by the Food and Drug Administration. The ELISA had a good correlation with SPT reactivity (r = 0.93), and because of the greater sensitivity, gloves testing below the ELISA reporting limit (0.06 I~g/ml) have a significantly lower potential for eliciting reactions in patients allergic to latex. Conclusions: Results of protein assays are acceptable criteria with which to rate the potential allergenicity of gloves; however, the American Society for Testing and Materials D5712-95 assay may lack the sensitivity to provide clinically relevant data.

Biomedical Instrumentation Technology, 1995

Antigenic protein levels in commercially available latex medical gloves were studied. For compari... more Antigenic protein levels in commercially available latex medical gloves were studied. For comparison, the gloves were divided into four main groups; powdered examination, powder-free examination, powdered surgical, and powder-free surgical. Residual protein levels of 91 different glove brands were determined using the LEAP (latex ELISA for antigenic proteins) assay and found to be extremely variable. The possible sources of variability were investigated. Lot-to-lot variability was determined by testing multiple lots of five different brands of surgical gloves. The authors also determined glove-to-glove variability, variability within an individual glove, and test-method variability. It was observed that lot-to-lot variability (25-61% relative standard deviation--RSD) and glove-to-glove variability (51% RSD) were of similar magnitude and accounted for most of the variability found within a given brand of gloves. However, the greatest source of variability identified was brand-to-brand variability (143-283% RSD).