Protease Allergens Induce the Expression of IL-25 via Erk and p38 MAPK Pathway

유학선,Pornpimon Angkasekwinai,장선희,정연석,Chen Dong 대한의학회 2010 Journal of Korean medical science Vol.25 No.6

Allergic diseases, including asthma, are characterized by T helper type 2 (Th2) cellmediated inflammations, coupled with tissue infiltration by eosinophils. In this study,we demonstrate that multiple protease allergens, including papain and DerP1, efficiently induce interleukin (IL)-25 and thymic stromal lymphopoietin (TSLP) gene expression, and this phenomenon is dependent on the protease activities of these allergens. The IL-25 cytokine level in bronchial alveolar lavage (BAL) was also profoundly and significantly increased after treatment with papain. Additionally, the levels of Th2 cytokines were significantly increased, as compared to those in the OVAonly treatment group. The various protease allergens triggered the expression of IL-25 and TSLP mRNA in mouse lung epithelial cells (MLE12) and primary mouse lung epithelial cells; these effects were inhibited by the deactivation of the protease activity of papain. The allergen papain activates the ErK and p38 MAP pathways;the inhibition of these pathways, but not the NFkB or PI-3 kinase pathways, impairs the induction of IL-25 and TSLP expression by proteases. In this study, we demonstrate that the protease allergens induce IL-25 and TSLP via the MAP kinase signal pathways, and their protease activities are essential to this pathway.

Role of Protease Activated Receptor 2 (PAR2) in Aspergillus Protease Allergen Induces Th2 Related Airway Inflammatory Response

Hak Sun Yu(유학선) 한국생명과학회 2010 생명과학회지 Vol.20 No.4

대부분의 알려진 알러젠들은 단백분해효소의 성격을 가지고 있고 이는 알레르기 반응에서 Th2 면역 반응을 일으키는 데 중요한 역할을 하는 것으로 알려져 있다. 이러한 단백분해효소들과 반응하는 것으로 알려진 protease activated receptor (PAR)는 4가지 종류가 있으며, 이 중 PAR2의 경우 알레르기 질환과 많은 상관관계를 보여 많은 연구가 되고 있다. 본 연구는 Aspergillus protease 알러젠에 의한 초기 및 만성 Th2 면역반응에서 PAR2의 역할을 규명하기 위해 Aspergillus protease 알러젠으로 정상쥐와 PAR2 유전자 결핍쥐 모두 Th2 반응을 유도한 후 면역세포의 침윤 정도 및 Th2 관련 cytokine 및 chemokine 유전자들의 발현 정도를 비교하였다. 그 결과 Aspergillus protease 알러젠으로 비강내로 1회 처리했을 경우 중성구의 침윤이 두드러지는데, 이때 PAR2 결핍 마우스는 이러한 면역세포의 침윤이 유의적으로 감소하였다. 또한, 이와 관련된 IL-25, TSLP, Eotaxin 유전자들의 발현 역시 PAR2 결핍 마우스에 현저히 감소하였다. 한편, Aspergillus protease 알러젠으로 비강내로 6회 처리했을 경우 중성구 대신 호산구의 침윤이 두드러지지만 PAR2 결핍 마우스에서 그 정도가 유의적으로 낮았다. OVA 특이 IgE와 IgGl 농도 역시 현저하게 PAR2 결핍 마우스에서 낮았고, CCL21의 발현이 PAR2 결핍마우스 MEF cells에서 현저히 감소하였다. Th2 초기 면역반응에서 가장 중요한 IL-25의 발현에 MAKP p38 pathway가 관여한다는 것을 이번 연구에서 알 수 있다. 본 연구를 통해 Aspergillus protease 알러젠으로 유도된 알러지성기관지 염증 반응에서 초기 반응뿐만 아니라 만성반응에서도 PAR2가 중요한 것을 알 수 있다. Most allergens have protease activities, suggesting that proteases may be a key link between Th2-type immune reactions in allergic responses. Protease activated receptor (PAR) 2 is activated via the proteolytic cleavage of its N-terminal domain by proteinases. To know the role of PAR2 in Aspergillus protease allergen activated Th2 immune responses in airway epithelial cells, we investigated and compared immune cell recruitment and level of chemokines and cytokines between PAR2 knock out (KO) mice and wild type (WT) mice. There were evident immune cell infiltrations into the bronchial alveolar lavage fluid (BALF) of WT mice, but the infiltrations in PAR2 KO mice were significantly lowered than those of WT mice. The IL-25, TSLP, and eotaxin gene expressions were profoundly increased after Aspergillus protease, but their expression was significantly lowered in PAR2 KO mice in this study. Compared to PAR2 KO mice, OVA specific IgE concentrations in serum of WT mice were quite increased; moreover, the IgE level of PAR2 KO mice was lower than in WT mice. The IL-25 expression by Aspergillus protease stimulation was significantly reduced by p38 specific inhibitor treatment. In this study, we determined that Th2 response was initiated with IL-25 and TSLP mRNA up-regulation in lung epithelial cells via PAR2 after Aspergillus protease allergen treatment.

Phylogeny of Mite Taxa (Acari : Sarcoptiformes) Based on Small Subunit Ribosomal RNA Sequences

이근희,유학선,박상균,이선주,이경아,김선미,옥미선,정해진,Lee Keun Hee,Yu Hak Sun,Park Sang Kyun,Lee Sun Joo,Lee Kyeong Ah,Kim Sun Mee,Ock Mee Sun,Jeong Hae Jin Korean Society of Life Science 2006 생명과학회지 Vol.16 No.1

We analyzed the phylogenic relationships of 23 partial 18S rDNA sequences of 22 species (1 species has 2 strains) belonging to Sarcorptiforms include 4 new sequences, using several tools. Although geographic distributions are quite far from, sequence similarity of two strains of Dermatophygoides pteronyssinus isolated from Japan and New Zealand were very high. This result suggests that mite migration by animals including human occurred in the two continents. We investigated the Endeostigmata taxonomic relationship between the Prostigmata and Oribatida subgroups using small fragments (340-400 bp) of their 185 rDNA sequences. But Endeostigmata was not grouped with Oribatida or Prostigmata. In conclusion, it is first reported phylogenic relationship for classified mites included in Sarcoptiformes using 185 rDNA sequence analysis and its system is a very powerful tool for classification of mites.

콘택트렌즈 소독제 및 PHMB의 3종 가시아메바 포낭에 대한 살충능력 평가

신종원,유학선,공현희,정동일 慶北大學校 醫科大學 1996 慶北醫大誌 Vol.37 No.3

목적 : 국내에 시판되고 있는 콘택트렌즈 보존 및 소독액, 그리고 아메바성 각막염의 치료제로서 인정받고 있는 polyhexamethylene biguanide (PHMB)의 가시아메바 포낭에 대한 살충효과를 평가하고자 한다. 대상 및 방법 : 카스텔라니가시아메바 Ma주, 대식가시아메바 Nagington주 및 담수가시아메바 KA/LI주에 대한 소독액의 살충효과의 평가는 한천 plate위에서 수행하였고, 최저살충농도(minimal cysticidal concentration : MCC)의 결정은 microplate를 이용하였다. 7일간 관찰 후, 탈낭하지 않는 가시아메바 포낭은 사멸한 것으로 판정하였다. Chlorhexidine 및 PHMB의 MCC를 결정하기 위해 8시간 및 48시간 처리 실험을 분리하여 시행하였다. 결과 : 시판 소독액 중 chlorhexidine과 thimerosal를 함유하는 용액 C만이 가시아메바 포낭에 대한 살충효과가 있었고, 나머지 3종의 소독액은 살충효과가 없었다. 0.02% PHMB로 4시간 이상 처리한 가시아메바 포낭은 충주에 관계없이 사멸되었다. Chlorhexidine 과 PHMB, 두 약제 모두 8시간 처리 MCC가 48시간 처리 MCC보다 다소 높았다. PHMB에 대한 감수성은 가시아메바 충주에 따라 차이가 격심하였다. 결론 : 3종 가시아메바 포낭에 대한 PHMB의 MCC (0.29∼9.38㎍/㎖)가 chlorhexidine의 그것(2.73∼18.75㎍/㎖)보다 낮아 PHMB는 아메바성 각막염의 치료제 및 콘택트렌즈 소독제로 사용 될 수 있을 것으로 사료되며 화학소독액에 PHMB를 10㎍/㎖ 첨가하는 것이 요망된다. In order to evaluate the cysticidal effects of contact lens disinfectants and polyhexamethylene biguanide(PHMB) against 3 clinical isolates of Acanthamoeba. in vitro cysticidal assay was performed on agar plates covered with heat-inactivated Escherichia coli. Three clinical isolates of Acanthamoeba were A. castellanii Ma strain. A. polyphaga Nagington strain, and A. lugdunensis KA/LI strain. After treatment of Acanthamoeba cysts with the disinfectants or PHMB, the cysts were cultivated for 7 or more days. Cysts which were not excysted in 7 days after treatment were recognized killed. Among commercial disinfectants, the solution C. which contains chlorhexidine and thimerosal. was the only one with cysticidal effects on Acanthamoeba cysts. PHMB of 0.02% concentration was also cysticidal. Sensitivity to PHMB varied profoundly from isolate to isolate. Minimal cysticidal concentration (MCC) of chorhexidine and PHMB were determined by cultivation of cysts treated for 8 or 48 hours with serially diluted chlorhexidine and PHMB in wells of a 96-well microplate containing axenic culture media for amoebae. MCC of 48-hour treatment with both chemicals was lower than that of 8-hour treatment. MCC of PHMB against amoebic cysts was lower than that of chlorhexidine. In the light of these results. PHMB could be a contact lens disinfectant as well as a chemotherapeutic agent of amoebic keratitis.

폐흡충의 이배체와 삼배체 vasa 유전자 분석 및 특징

이근희,유학선,허재원,유성숙,최선희,박상균,이선주,정동일,공현희,옥미선,정해진,Lee, Keun-Hee,Yu, Hak-Sun,Hur, Jae-Won,Yu, Sung-Suk,Choi, Sun-Hee,Park, Sang-Kyun,Lee, Sun-Joo,Chung, Dong-Il,Kong, Hyun-Hee,Ock, Mee-Sun,Jeong, Hae-Jin Korean Society of Life Science 2007 생명과학회지 Vol.17 No.4

폐흡충은 국내를 비롯한 아시아에서 폐흡충증을 일으키는 중요한 기생충이다. 이러한 폐흡충은 이배체 와 삼배체가 국내에 알려져 있다. 이배체 폐흡충은 양성생식을 하고 삼배체 폐흡충은 단위생식을 하는 것으로 알려져 있다. 그러나 이러한 원인에 대하여서는 알려진 바가 없다. 생식유전자 중 초파리에서 최초 분리되었고 포유동물에서도 그 기능이 밝혀진 vasa 유전자가 가장 유명하다. 이 유전자는 생식세포의 분화에 관여하며 종에 따라서는 정자생성에도 관여하는 것으로 알려져 있다. 이번 연구는 삼배체 폐흡충의 단위생식과 vasa 유전자와의 관계를 규명해 보고자 하였다. 폐흡충의 이배체와 삼배체 성충으로부터 vasa 유전자 전체의 염기서열을 얻을 수 있었다. 두 염기서열의 경우 8개의 ATP-binding domain이 관찰되었고 helicase가 결합할 것으로 예상되는 RGG motif도 관찰되었다. 총 622아미노산 서열로 구성될 것으로 보이며 이배체는 69.018 kDa, 삼배체는 68.930 kDa 크기의 단백질을 만들 것으로 예상되었다. Vasa 재조합 단백질은 GST와 fusion되어 93 kDa 크기에서 관찰되었다. mRNA의 발현은 이배체에 비해 삼배체가 다소 높았다. Anti-Pw-VASA항체를 이용한 면역조직화학법을 수행한 결과 이배체와 삼배체는 다른 기관에서는 면역반응력을 보이지 않고 고환에서만 면역반응력을 볼 수 있었다. 결과를 종합해 보면 vasa 유전자는 이배체 삼배체 모두 가지고 있었으며 정상적으로 발현되었다. In this study, we isolated, characterized, and compared the vasa homologous genes of diploid and triploid Paragonimus westermani and localized VASA homologous proteins in both lung fluke types. Open reading frames of Pw-vasa-2n and Pw-vasa-3n were of 1812 bp, and encoded deduced proteins of 622 amino acids with calculated molecular weights of 69.0 kDa and 68.9 kDa and pI's of 9.11 and 9.03, respectively. A comparison of these two VASA deduced protein sequences showed that only 6 of the 622 amino acids differed. The deduced sequences of Pw-VASA-2n and Pw-VASA-3n contained eight consensus sequences characteristic of the DEAD-box protein family and their N-terminal regions contained four arginine-glycine-glycine (RGG) motifs. These two lung fluke VASA-like proteins were more similar to those of other VASA proteins than to those of other DEAD-family proteins isolated from several organisms (planarian, zebra fish, mouse, and human). vasa homologous gene transcription and VASA protein expressions in triploid type lung flukes was slightly stronger than in the diploid type. Immunostaining showed that testes and a portion of the ovaries of both diploid and triploid lung flukes reacted strongly to anti-Pw-VASA antibody.

제주도 토양에서 분리한 가시아메바의 riboprinting에 의한 동정

김현동,전자호,유학선,공현희 慶北大學校 醫科大學 1996 慶北醫大誌 Vol.37 No.4

목적 : 가시아메바의 국내 분리주를 확보하고 국내 분포하는 가시아메바의 종류를 파악하며 그 분리주들의 병원성을 간접적으로 평가하기 위해 제주도의 토양에서 분리한 가시아메바 분리주를 riboprinting을 이용하여 종 동정을 하고자 한다. 재료 및 방법 : 제주도의 서로 다른 일곱 장소에서 무균 튜브에 흙을 담아와서 agar plate에서 배양한 후 서로 다른 형태적 특징을 보이는 포낭을 cloning하여 무균배양으로 키웠다. 각각의 핵 DNA를 분리하여 small subunit ribosomal RNA coding DNA 중 일부를 증폭하여 나온 PCR 산물을 8가지 제한효소로 소화시켜 그 RFLP양상을 16비교주와 비교한 후 Nei & Li의 공식에 대입한 후 UPGMA program로 유전적 거리를 구하였다. 결과 : 포낭의 형태는 KA/CS8주를 제외한 7주는 모두 Pussard 및 Pons의 분류에 따른 제 2군에 속하였고 KA/CS8주는 제 3군에 속하였다. 가시아메바 KA/CS1주와 KA/CS2주는 동일한 RELP양상을 나타내었으며 이들은 카스텔라니 가시아메바 Castellani주와 가장 유전적으로 가까왔고, KA/CS3주와 KA/CS6주는 A. triangularis SH621주와, KA/CS4주는 대식가시아메바 Jones주와, KA/CS5주는 대식가시아메바 P23주와. KA/CS7주는 카스텔라니 가시아메바 Neff주와 각각 유전적으로 가장 가까왔고, KA/CS8주는 비교주 중 어느 종과도 유전적으로 가깝지 않았다. 결론 : Acanthamoeba KA/CS1, KA/CS2, KA/CS3, KA/CS4, KA/CS6분리주들은 카스텔라니 가시아메바 complex에 속하였고, KA/CS5, KA/CS7분리주들은 대식가시아메바 complex에 속하였다. In order to collect and identify Acanthamoeba Korean strains. Acanthamoeba strains were isolated from soil of Cheju-do and were identified by comparison of the riboprints with the reference strains. Seven out of eight isolates from soil of Cheju-do belonged to the morphological group Ⅱ (Pussard and Pons, 1977) and the other one belonged to group Ⅲ by cyst morphology. The riboprints of seven isolates belonging to group Ⅱ were compared to those of Acanthamoeba reference strains (11 species 16 strains). KA/CS1, KA/CS2, KA/CS3, KA/CS4 and KA/CS6 strains belonged to A. castellanii complex. KA/CS1 and KA/CS2 strains showed identical riboprints to each other and were the most closely related to A. castellanii Castellani strain. KA/CS3 and KA/CS6 strains were the most closely related to A. triangularis SH621 strain, and KA/CS4 strain to A. polyphaga Jones strain. KA/CS5 and KA/CS7 strains belonged to A. polyphaga complex. KA/CS5 strain was the most closely related to A. polyphaga P23 strain and KA/CS7 strain to A. castellanii Neff strain. KA/CS8 strain belonging to group Ⅲ showed more than 20% estimated sequence divergence from the reference strains (5 species) and made a distinct division in a phenogram reconstructed based on the riboprints.

Trichinella Infection Ameliorated Vincristine-Induced Neuroinflammation in Mice

조영래,박환태,유학선,공현희 대한기생충학ㆍ열대의학회 2022 The Korean Journal of Parasitology Vol.60 No.4

Vincristine (VCR) is a chemotherapeutic agent widely used in treatment of malignancies. However, VCR has a limitation in use since it commonly causes a painful neuropathy (VCR-induced peripheral neuropathy, VIPN). Inflammatory cytokines secreted by immune cells such as macrophages can exacerbate allodynia and hyperalgesia, because inhibiting the inflammatory response is a treatment target for VIPN. In this study, we investigated whether Trichinella spiralis, a widely studied helminth for its immunomodulatory abilities, can alleviate VCR-induced allodynia. Von Frey test showed that T. spiralis infection improved mechanical allodynia at 10 days after VCR injection. We further observed whether the difference was due to mitigated axon degeneration, but no significant difference between the groups in axonal degeneration in sciatic nerves and intra-epidermal nerve fibers was found. Conversely, we observed that number of infiltrated macrophages was decreased in the sciatic nerves of the T. spiralis infected mice. Moreover, treatment of T. spiralis excretory-secretory products caused peritoneal macrophages to secrete decreased level of IL-1β. This study suggests that T. spiralis can relieve VCR-induced mechanical allodynia by suppressing neuroinflammation and that application of controllable degree of helminth may prove beneficial for VIPN treatment.

Phylogenetic Characteristics of Fasciola hepatica Isolated from a Korean Patient

정미진,박재균,유학선 대한기생충학ㆍ열대의학회 2022 The Korean Journal of Parasitology Vol.60 No.5

Fascioliasis is a parasitic infection caused by liver flukes. Although several cases have been reported in Korea, phylogenetic analysis of isolates is lacking. In this study, a 66-year-old woman with right upper quadrant (RUQ) abdominal pain was diagnosed as fascioliasis involving abdominal muscle by imaging study. She received praziquantel treatment, but symptoms were not improved. Lateral movement of the abscess lesion was followed. Trematode parasite was surgically removed from the patient’s rectus abdominis muscle. The fluke was identified as Fasciola hepatica based on sequence analysis of 18S rDNA. To determine the phylogenetic position of this Fasciola strain (named Korean Fasciola 1; KF1), the cox1 gene (273 bp) was analyzed and compared with the genes of 17 F. hepatica strains isolated from cows, sheep, goats, and humans from various countries. Phylogenetic analysis showed that KF1 was closely related with the isolates from China goat.

각막굴절교정학 콘택트렌즈와 연관된 아칸트아메바 각막염

이지은,한태원,유학선,이종수,Ji Eun Lee,Tae Won Hahn,Hak Sun Yu,Jong Soo Lee 대한안과학회 2007 대한안과학회지 Vol.48 No.2

Purpose: To report 4 cases of Acanthamoeba keratitis related to orthokeratology lens overnight wear. Methods: Four patients had histories of overnight orthkeratology lens wear of 10 months to 3 years when they presented with corneal ulcers. Results: The organism isolated by corneal scraping was Aanthamoeba. The patients were treated with polyhexamethylene biguanide (PHMB), and chlorhexidine, resulting in a resolution of ocular inflammation. Conclusion: The risk of Acanthamoeba keratitis as a potential complication of overnight orthkeratology should be considered.

콘택트렌즈 소독액의 아메바 포낭에 대한 살충능력 평가

한영상,박재성,이종수,유학선,이지은 부산대학교 병원 암연구소 2007 부산대병원학술지 Vol.- No.21

Purpose: To evaluate the cysticidal effect of 5 kinds of commercially available contact lens disinfectants against 2 clinical is이ates of Acanthamoeba. Subject and Methods: Five kinds of commercially available contact lens disinfectants were soaked with cysts of Acanthamoeba ludgdunesis and castellanii at the concentration of 10³,10⁴ and lO^(S) cells/ml for land 4 or 6 hours. Cysts which were not excysted in 7 days after treatment were recognized to be killed. Morphologic changes were evaluated by electron microscopic observation. Results: Contact lens disinfectants which contain myristamidopropyl dimethylamine (MAPD) showed the best cysticidal effect. These disinfectants demonstrated a cysticidal effect on both Acanthamoeba species of all concentrations in 6-hour treatment. Contact lens disinfectants which contain polyhexamethylene biguanide (PHMB) did not demonstrate cysticidal effect, except for Acanthamoeba castellanii at the concentration of 10³ cells/ml, in either 4- or 6-hour treatment. Separation of plasma membrane from endocyst and damage of organelles were prominent in cases showing a cysticidal effect. Conclusions: Contact lens disinfectant which contains MAPD may be helpful in preventing the Acanthamoeba keratitis. A higher concentration of PHMB is required to be effective in preventing Acanthamoeba keratitis.