Correlation of age of pigs and endotoxin in respirable dust in swine barns with pigs’ immune response

Katharine Roque,Yong Heo,Eun Seob Song,Ah Rang Cho,Yun Gyeong Kim,Jae Hee Lee,Ravi Gautam,So Jung Sin,Chang Yeol Kim,Hyung Ah Kim 대한수의학회 2016 대한수의학회 학술대회발표집 Vol.2016 No.-

Association between endotoxin levels in dust from indoor swine housing environments and the immune responses of pigs

Katharine Roque,Kyung-Min Shin,Ji-Hoon Jo,임경동,송은섭,신소정,Ravi Gautam,Jae Hee Lee,김연경,조아랑,김창열,Hyun Ji Kim,Myung Sook Lee,Hyeong-Geu Oh,Byung-Chul Lee,Jung Hee Kim,Hyun Kyu Jeong,김형아,허용,Kwang-Ho Kim 대한수의학회 2018 Journal of Veterinary Science Vol.19 No.3

Indoor animal husbandry environments are inevitably contaminated with endotoxins. Endotoxin exposure is associated with various inflammatory illnesses in animals. This cross-sectional study evaluated the relationship between the degree of endotoxin exposure and the cellular and humoral immune profiles of fattening pigs. Blood samples were taken from the jugular vein of 47 pigs from ten pig farms in Korea. Whole blood cell counts and plasma immunoglobulin (Ig) classes were determined. Peripheral-blood mononuclear cells were stimulated in vitro with concanavalin A for 48 h, and cytokines released into culture supernatants were measured. The barns in which the pigs lived were assessed for endotoxin levels in the total and respirable dust by using the limulus amebocyte lysate kinetic QCL method. Low and high endotoxin exposures were defined as ≤ 30 and > 30 EU/m3, respectively. Compared to pigs with low endotoxin exposure (n = 19), highly exposed pigs (n = 28) had higher circulating neutrophil and lymphocyte (particularly B cells) counts, IgG and IgE levels, interferon-gamma (IFNg) and interleukin (IL)-4 productions, and lower IgA levels and tumor necrosis factor-alpha (TNFa) production. The IL-4, IFN, and TNFa levels significantly correlated with endotoxin level and/or pig age. Constant exposure of pigs to high levels of airborne endotoxins can lead to aberrant immune profiles.

Relationship between chicken cellular immunity and endotoxin levels in dust from chicken housing environments

Katharine Roque,신경민,조지훈,김형아,허용 대한수의학회 2015 Journal of Veterinary Science Vol.16 No.2

Hazardous biochemical agents in animal husbandry indoor environments are known to promote the occurrence of various illnesses among workers and animals. The relationship between endotoxin levels in dust collected from chicken farms and various immunological markers was investigated. Peripheral blood was obtained from 20 broiler chickens and 20 laying hens from four different chicken farms in Korea. Concentrations of total or respirable dust in the inside the chicken farm buildings were measured using a polyvinyl chloride membrane filter and mini volume sampler. Endotoxin levels in the dust were determined by the Limulus Amebocyte Lysate Kinetic method. Interferon-γ production by peripheral blood mononuclear cells stimulated with concanavalin A was significantly lower in broilers or layers from the farms with higher endotoxin concentrations than the chickens from the farms with lower endotoxin levels. An opposite pattern was observed for plasma cortisol concentrations with higher cortisol levels found in chickens from the farms with higher endotoxin levels. When peripheral lymphocytes were examined, the percentage of CD3–Ia+ B cells was lower in layers from farms with higher endotoxin levels than those from locations with lower endotoxin levels. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in chickens.

Down regulation of cellular immunity in pigs exposed to high endotoxin level in dusts

Katharine Roque,Gyeong Dong Lim,Ravi Gautam,Chanyeol Kim,Hyung Ah Kim,Yong Heo 한국실험동물학회 2015 한국실험동물학회 학술발표대회 논문집 Vol.2015 No.8

Epizootiological characteristics of viable bacteria and fungi in indoor air from porcine, chicken, or bovine husbandry confinement buildings

Katharine Roque,임경동,Ji-Hoon Jo,Kyung-Min Shin,송은섭,Ravi Gautam,김창열,Kyungsuk Lee,신승원,유한상,허용,김형아 대한수의학회 2016 Journal of Veterinary Science Vol.17 No.4

Microorganisms found in bioaerosols from animal confinement buildings not only foster the risk of spreading diseases among livestockbuildings, but also pose health hazards to farm workers and nearby residents. This study identified the various microorganisms present in theair of swine, chicken, and cattle farms with different kinds of ventilation conditions in Korea. Microbial air samples were collected onto Petridishes with bacterial or fungal growth media using a cascade impactor. Endotoxin levels in total dust were determined by the limulus amebocytelysate kinetic QCL method. Prevalent Gram-positive bacteria were Staphylococcus (S.) lentus, S. chromogenes, Bacillus (B.) cereus, B. licheniformis, and Enterococcus faecalis, while the dominant fungi and Gram-negative bacteria were Candida albicans and Sphingomonaspaucimobilis, respectively. Considering no significant relationship between the indoor dust endotoxin levels and the isolation ofGram-negative bacteria from the indoor air, monitoring the indoor airborne endotoxin level was found to be also critical for risk assessmenton health for animals or workers. The present study confirms the importance of microbiological monitoring and control on animal husbandryindoor air to ensure animal and worker welfare.

Association of bovine cellular immunity with endotoxin level in dust from Korean beef cattle housing environments

Katharine Roque,임경동,송은섭,Ravi Gautam,이재희,김연경,조아랑,신소정,김창열,김형아,허용 계명대학교 자연과학연구소 2016 Quantitative Bio-Science Vol.35 No.2

Hazardous biochemical agents in indoor animal husbandry environments promote the occurrence of various illnesses among husbandry workers and industrial animals. The relationship between endotoxin levels in dust collected from Korean beef cattle farms and markers of bovine cellular immunity was investigated. Peripheral blood was obtained from 25 cattle from five cattle farms in Korea. Endotoxin levels present in total or respirable dust were determined by the Limulus Amebocyte Lysate Kinetic method. Cytokine production was evaluated following the stimulation of peripheral mononuclear cells with concanavalin A for 72 h in a 5% CO2 incubator. Production of both interleukin (IL)-4 and interferon (IFN)γ was significantly higher in the high endotoxin exposure group (100.9±70.6 EU/m3) compared with the low endotoxin exposure group (17.7±18.6 EU/m3), with a lower IFNγ/IL-4 ratio in animals from the high endotoxin farms, indicating immunity skewed toward a type-2 response. The proportion of γδ T lymphocyte, important bovine immune cells involved in protection against microbial infection, was lower in cattle from the high endotoxin farms than in those from the low endotoxin farms. The numbers of white blood cells, red blood cells, lymphocytes, eosinophils, and basophils were significantly downregulated in cattle from the high endotoxin farms. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in Korean beef cattle.

Downregulation of WC1+ gamma delta T cells in cattle exposed to higher level of airborne endotoxin

katharine roque,Jaehee Lee,Gyeongdong Lim,Eunsob Song,Ahrang Jo,Eungyong Kim,Sojung Sin,Changyeol Kim,Hyung Ah Kim,Yong Heo 한국실험동물학회 2016 한국실험동물학회 학술발표대회 논문집 Vol.2016 No.08

Association of Chicken Immunologic Status with Dust or Endotoxin Level in Husbandry Indoor Environment

Yong Heo,Katharine Roque,Kyung Min Shin,Ji Hoon Jo,Hyoung Ah Kim,Kyung Dong Lim 한국실험동물학회 2014 한국실험동물학회 학술발표대회 논문집 Vol.2014 No.2

국내 개발 인공피부의 친염증성 싸이토카인 발현 평가

조지훈 ( Ji Hoon Jo ),( Katharine Roque ),신소정 ( So Jung Sin ),조아랑 ( Ah Rang Cho ),허용 ( Yong Heo ) 한국동물실험대체법학회 2013 동물실험대체법학회지 Vol.7 No.1

본 연구를 통하여 대표적인 친염증성 싸이토카인인 IL-1α, IL-8, IL-18이 국내 회사에서 개발한 인공 피부인 Keraskin™에서 발현되고 있음을 알 수 있었다. IL-1α는 각질세포이외에도 탐식세포, T 림프구, 호중구 등에서도 분비되는 것으로 알려져 있으며 염증 반응 유도에 관계할 뿐만 아니라 T 림프구의 TH17 세포로의 분화에 기여하는 것으로 알려져 있다 (Akdis et al., 2011). IL-8은 주로 탐식세포, 단핵구에서 생성되지만 각질세포, 림프구, 호중구, 간세포에서도 합성되는 것으로 보고되고 있다. 류마티스성 관절염 발병시, 세균 또는 바이러스 감염시 합성이 증가하는 것으로 알려져 있으며 염증 부위로 항원공여세포나 T림프구의 이동을 촉진하는 것으로 알려져 있다(Commins et al., 2010). IL-18은 주로 탐식세포에서 생성되지만 각질세포, 성교세포 (astrocytes), 수지상세포 등에서도 분비되며 IL-12의 도움을 받아 interferon-gamma의 합성을 촉진하는 것으로 알려져 있고 각종 염증성 질환에 합성이 증가되는 것으로 알려져 있다 (Akdis et al., 2011; Commins et al., 2010; Akdis et al., 2011). 이들 싸이토카인 중에서 IL-18은 사람 각질세포주인 NCTC2544 세포주에서 피부감작물질을 첨가하였을 때에 비감작물질을 첨가한 경우에 비해 생성 수준이 증가됨이 보고되었다(Galbiati et al., 2011). 또한 본 연구진은 마우스의 각질세포주인 HEL-30 세포주를 사용하여 피부 비감 작성물질에 비해 감작성물질 노출시 IL-1α 생성이 증가된다는 보고를 최근에 한 바 있다 (Son et al., 2013). 본 연구에서 IL-6 수준은 sandwich ELISA 방법으로 검출한계 (10 pg/㎖) 이하였다. IL-6는 탐식세포가 주 생성원이지만 각질세포, 림프구, 간세포 등에서도 분비되는 것으로 알려져 있다 (Commins et al., 2010). 본 연구진의 과거 연구에서도 사람 각질세포 주인 HaCaT 세포주에서 IL-6가 분비됨을 확인한 바 있다 (김 등, 2011 & 2012). 이에 본 연구진이 사용한 IL-6 sandwich ELISA 체계의 문제점은 아님을 알 수 있다. 본 연구가 한 롯트 (lot)의 Keraskin™ 조직만을 이용하였기 때문에 현재 시점에서는 Keraskin™ 조직 자체가 IL-6를 발현하지 않는다는 결론을 낼 수는 없을 것이다. 추가적인 롯트의 Keraskin™ 조직을 이용하여 실험함으로써 최종적인 판단을 할 수 있을 것이다. A reconstructed human epidermis, Keraskin™ developed in Korea, was evaluated for basal production of pro-inflammatory cytokines, interleukin (IL)-1α, IL-6, IL-8, and IL-18 in the absence of stimulators. Keraskin™ tissues in culture plate insert were incubated in 5% CO2 incubator in the presence of DMEM culture media containing 10% fetal bovine serum for 24, 48, 72, and 96 hours. Cutlure supernatants and Keraskin™ tissue lysates were obtained at the each incubation period. Level of each cytokine was determined using a sandwich ELISA method. Comparing production of IL-1α and IL-8 in culture supernatants after 24 hour incubation, level of these cytokines was significantly augmented at 48, 72, and 96 hours without statistical difference among these incubation periods. IL-18 production level in culture supernatants was the highest after 48 hour incubation. Levels of IL-1α, IL-8, and IL-18 in cell lysates were similar at the all culture periods. Level of IL-6 in both samples was below detection limit, which requires further investigation. Overall, Keraskin™ could be a valuable reconstructed human epidermis for alternative test for skin sensitization screening.

연구논문 : 국내 개발 인공피부의 친염증성 싸이토카인 발현 평가

조지훈 ( Ji Hoon Jo ),( Katharine Roque ),신소정 ( So Jung Sin ),조아랑 ( Ah Rang Cho ),허용 ( Yong Heo ) 한국동물실험대체법학회 2013 동물실험대체법학회지 Vol.7 No.1

본 연구를 통하여 대표적인 친염증성 싸이토카인인 IL-1α, IL-8, IL-18이 국내 회사에서 개발한 인공 피부인 Keraskin™에서 발현되고 있음을 알 수 있었다. IL-1α는 각질세포이외에도 탐식세포, T 림프구, 호중구 등에서도 분비되는 것으로 알려져 있으며 염증 반응 유도에 관계할 뿐만 아니라 T 림프구의 TH17 세포로의 분화에 기여하는 것으로 알려져 있다 (Akdis et al., 2011). IL-8은 주로 탐식세포, 단핵구에서 생성되지만 각질세포, 림프구, 호중구, 간세포에서도 합성되는 것으로 보고되고 있다. 류마티스성 관절염 발병시, 세균 또는 바이러스 감염시 합성이 증가하는 것으로 알려져 있으며 염증 부위로 항원공여세포나 T림프구의 이동을 촉진하는 것으로 알려져 있다(Commins et al., 2010). IL-18은 주로 탐식세포에서 생성되지만 각질세포, 성교세포 (astrocytes), 수지상세포 등에서도 분비되며 IL-12의 도움을 받아 interferon-gamma의 합성을 촉진하는 것으로 알려져 있고 각종 염증성 질환에 합성이 증가되는 것으로 알려져 있다 (Akdis et al., 2011; Commins et al., 2010; Akdis et al., 2011). 이들 싸이토카인 중에서 IL-18은 사람 각질세포주인 NCTC2544 세포주에서 피부감작물질을 첨가하였을 때에 비감작물질을 첨가한 경우에 비해 생성 수준이 증가됨이 보고되었다(Galbiati et al., 2011). 또한 본 연구진은 마우스의 각질세포주인 HEL-30 세포주를 사용하여 피부 비감 작성물질에 비해 감작성물질 노출시 IL-1α 생성이 증가된다는 보고를 최근에 한 바 있다 (Son et al., 2013). 본 연구에서 IL-6 수준은 sandwich ELISA 방법으로 검출한계 (10 pg/㎖) 이하였다. IL-6는 탐식세포가 주 생성원이지만 각질세포, 림프구, 간세포 등에서도 분비되는 것으로 알려져 있다 (Commins et al., 2010). 본 연구진의 과거 연구에서도 사람 각질세포 주인 HaCaT 세포주에서 IL-6가 분비됨을 확인한 바 있다 (김 등, 2011 & 2012). 이에 본 연구진이 사용한 IL-6 sandwich ELISA 체계의 문제점은 아님을 알 수 있다. 본 연구가 한 롯트 (lot)의 Keraskin™ 조직만을 이용하였기 때문에 현재 시점에서는 Keraskin™ 조직 자체가 IL-6를 발현하지 않는다는 결론을 낼 수는 없을 것이다. 추가적인 롯트의 Keraskin™ 조직을 이용하여 실험함으로써 최종적인 판단을 할 수 있을 것이다. A reconstructed human epidermis, Keraskin™ developed in Korea, was evaluated for basal production of pro-inflammatory cytokines, interleukin (IL)-1α, IL-6, IL-8, and IL-18 in the absence of stimulators. Keraskin™ tissues in culture plate insert were incubated in 5% CO2 incubator in the presence of DMEM culture media containing 10% fetal bovine serum for 24, 48, 72, and 96 hours. Cutlure supernatants and Keraskin™ tissue lysates were obtained at the each incubation period. Level of each cytokine was determined using a sandwich ELISA method. Comparing production of IL-1α and IL-8 in culture supernatants after 24 hour incubation, level of these cytokines was significantly augmented at 48, 72, and 96 hours without statistical difference among these incubation periods. IL-18 production level in culture supernatants was the highest after 48 hour incubation. Levels of IL-1α, IL-8, and IL-18 in cell lysates were similar at the all culture periods. Level of IL-6 in both samples was below detection limit, which requires further investigation. Overall, Keraskin™ could be a valuable reconstructed human epidermis for alternative test for skin sensitization screening.