Browsing by Author "Kim, Sungwon"
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- Development and validation of a house finch interleukin-1β (HfIL-1β) ELISA systemKim, Sungwon; Park, Myeongseon; Leon, Ariel E.; Adelman, James S.; Hawley, Dana M.; Dalloul, Rami A. (2017-08-30)Background A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody. Methods A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. Results A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches. Conclusions Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches.
- Functional Characterization of the Avian Inflammatory Mediators Nod1, MIF and IL-22Kim, Sungwon (Virginia Tech, 2011-09-16)Inflammation can be initiated by an innate immune sensor, followed by activation of a signal mediator, resulting in control of immune response by a signal regulator. Mammalian nucleotide-binding oligomerization domain protein 1 (Nod1) and Nod2 initiate host innate immune response by recognition of specific bacterial molecules, resulting in the production of pro-inflammatory cytokines, chemokines, and anti-microbial peptides. A candidate sequence of chicken Nod1 (ChNod1) was identified with no current evidence of ChNod2. Stimulation of transiently overexpressed ChNod1 and its mutants with mammalian Nod-specific ligands was not conclusive of the function of ChNod1 most likely due to self-activation of ChNod1. In vitro studies showed no significant difference in expression of Nod1, its signaling molecules and pro-inflammatory cytokines in stimulated chicken mononuclear cells with synthetic ligands for mammalian Nod1 or Nod2. A signal mediator, macrophage migration inhibitory factor (MIF) inhibits the random migration of macrophages. Chemotaxis assay using recombinant ChMIF (rChMIF) revealed a substantial decrease in migration of macrophages. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1β and iNOS during monocytes stimulation with LPS. Additionally, Con A-stimulated lymphocytes exhibited enhanced IFN-γ and IL-2 transcripts in the presence of rChMIF. IL-22, which may act as a signal regulator, is an important effector of activated Th1 and Th17 as well as natural killer cells during inflammation. Recombinant ChIL-22 alone did not have an impact on chicken embryo kidney epithelial cells (CKECs); however, co-stimulation of CKECs with LPS and rChIL-22 enhanced the production of pro-inflammatory cytokines and anti-microbial peptides. Furthermore, rChIL-22 alone stimulated acute phase reactants in chicken embryo liver cells. These effects of rChIL-22 were abolished by addition of rChIL22 binding protein. Taken together, these results indicate an important role of ChIL-22 on epithelial cells and hepatocytes during inflammation. In this project, we identified and characterized the avian inflammatory mediators ChNod1, ChMIF, and ChIL-22. Studying each of their biological function in avian inflammation, especially under pathogenic challenges in epithelial tissues will provide a foundation for understanding the role of these inflammatory mediators in mucosal immunity.
- Functional characterization of the turkey macrophage migration inhibitory factorPark, Myeongseon; Kim, Sungwon; Fetterer, Raymond H.; Dalloul, Rami A. (2016-08)Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in Escherichia coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-gamma and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive.
- Identification and expression analysis of duck interleukin-17D in Riemerella anatipestifer infectionDiaz, Joyce Anne R.; Kim, Woo H.; Fernandez, Cherry P.; Jeong, Jipseol; Afrin, Fahmida; Lillehoj, Hyun S.; Kim, Suk; Kim, Sungwon; Dalloul, Rami A.; Min, Wongi (2016-08)Interleukin (IL)-17D is a proinfiammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. dull-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinfiammatory cytokines such as IL-6, IL-8, and IL-1 beta in duck embryo fibroblast cells. Very low levels of dull-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The dull-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species.
- Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and AnalysisDalloul, Rami A.; Long, Julie A.; Zimin, Aleksey V.; Aslam, Luqman; Beal, Kathryn; Blomberg, Le Ann; Bouffard, Pascal; Burt, David W.; Crasta, Oswald; Crooijmans, Richard P. M. A.; Cooper, Kristal; Coulombe, Roger A.; De, Supriyo; Delany, Mary E.; Dodgson, Jerry B.; Dong, Jennifer J.; Evans, Clive; Frederickson, Karin M.; Flicek, Paul; Florea, Liliana; Folkerts, Otto; Groenen, Martien A. M.; Harkins, Tim T.; Herrero, Javier; Hoffmann, Steve; Megens, Hendrik-Jan; Jiang, Andrew; de Jong, Pieter; Kaiser, Pete; Kim, Heebal; Kim, Kyu-Won; Kim, Sungwon; Langenberger, David; Lee, Mi-Kyung; Lee, Taeheon; Mane, Shrinivasrao P.; Marcais, Guillaume; Marz, Manja; McElroy, Audrey P.; Modise, Thero; Nefedov, Mikhail; Notredame, Cédric; Paton, Ian R.; Payne, William S.; Pertea, Geo; Prickett, Dennis; Puiu, Daniela; Qioa, Dan; Raineri, Emanuele; Ruffier, Magali; Salzberg, Steven L.; Schatz, Michael C.; Scheuring, Chantel; Schmidt, Carl J.; Schroeder, Steven; Searle, Stephen M. J.; Smith, Edward J.; Smith, Jacqueline; Sonstegard, Tad S.; Stadler, Peter F.; Tafer, Hakim; Tu, Zhijian Jake; Van Tassell, Curtis P.; Vilella, Albert J.; Williams, Kelly P.; Yorke, James A.; Zhang, Liqing; Zhang, Hong-Bin; Zhang, Xiaojun; Zhang, Yang; Reed, Kent M. (PLOS, 2010-09-01)A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (,1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.