Soluble CD93 in allergic asthma

CD93 has been shown critical roles in inflammatory and immune diseases. However, in allergic asthma, the potential roles of soluble CD93 (sCD93) have not been well studied. We conducted house dust mite (HDM) stimulation with Der p 1 in BEAS-2B and U937 cells, followed by treatment with dexamethasone or small interfering RNA against CD93. A HDM-induced murine allergic asthma model was also established. We estimated the power of sCD93 to predict allergic asthma in a retrospective post-hoc analysis containing 96 human samples. HDM-stimulated BEAS-2B cells showed increased mRNA expression levels of IL-6, IL-8, IL-33, TSLP, and CD93. The CD93 level in culture supernatants steadily increased for 24 h after allergen stimulation, which was significantly suppressed by both dexamethasone and CD93 silencing. CD93 silencing increased IL-6 and TSLP, but not IL-33 levels in culture supernatants. HDM-induced asthma mice showed significant airway hyperresponsiveness and inflammation with Th2 cytokine activation, along with decreased CD93 expression in bronchial epithelial cells and lung homogenates but increased serum CD93 levels. The sCD93 level in asthma patients was significantly higher than that in healthy controls and could predict asthma diagnosis with moderate sensitivity (71.4%) and specificity (82.4%) (AUC = 0.787, P < 0.001). The level of sCD93 which has potential role to predict asthma significantly increased after HDM stimulation via IL-6 and TSLP in vitro and in vivo.ope

Novel Sensitive, Two-site ELISA for the Quantification of Der f 1 Using Monoclonal Antibodies

ope

우리나라 집먼지진드기 생태에 관한 고찰 및 표준 조사법 제안

A large number of house dust mites live in homes worldwide and produce the most important allergens which cause allergic diseases to the genetically predisposed individuals perennially. Herewith, the literatures published on the survey of dust mites in Korea were reviewed. In most studies, mites were isolated and investigated from the house dust samples collected by using household vacuum cleaners. Among more than 30 species of house dust mites recorded in Korea, Dermatophagoides farinae was found to be the most predominant species in homes, followed by D. pteronyssinus. House dust mites were most frequently encountered in dust from bedding samples followed by kitchen samples, in which Tyrophagus putrescentiae, a storage mite species, was the most frequently found species. The highest mite density and allergen concentration were shown in autumn. It would be useful if the standard method for the investigation of dust mite density is established for the field survey. Measuring the number of mites per unit area (1 m2) collected in a given time (2 minutes) using a house-hold vacuum cleaners equiped with nonwovens is suggested here as an objective and convenient method for the survey on house dust mites. Mites can be detected directly from fine dust samples using a stereomicroscope or isolated by technique utilizing saturated salt water. In the future, more field surveys and laboratory studies are necessary for the investigation on the relationship between house dust mites in homes and various allergic diseasesope

Allergenicity and Stability of 6 New Korean Bony Fish Extracts

Purpose: Diagnostic tests for allergen sensitization should reflect real exposure. We made 6 new bony fish extracts, which are consumed popularly in Korea, and evaluated their allergenicity and stability. Methods: We manufactured fish extracts from codfish, mackerel, common eel, flounder, cutlass, and catfish. Protein and parvalbumin (PV) were evaluated by Bradford assay, 2-site enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and anti-PV immunoblotting. The immunoglobulin E (IgE) reactivities of the extracts were evaluated with ImmunoCAP and IgE immunoblotting using sera from 24 Korean fish allergy patients, 5 asymptomatic sensitizers, and 11 non-atopic subjects. Stability of the extracts stored in 4 different buffers were evaluated for up to a year. Results: The protein concentrations of commercial SPT fish extracts varied with up to a 7.5-fold difference. SDS-PAGE showed marked differences in the PV concentrations of commercial SPT reagents. Specific IgE measurements for the following investigatory fish extracts-iCodfish, iMackerel, and iEel-were concordant with that of their corresponding Phadia ImmunoCAP measurements. ImmunoCAP results showed marked IgE cross-reactivity among the fish species, and the overall sensitivity of ImmunoCAP with the investigatory fish extracts for identification of culprit fish species was 85.7%. The protein and PV concentrations in the investigatory extracts were highly stable in saline with 0.3% phenol-50% glycerol at 4°C for up to a year. Conclusions: The commercial SPT fish extracts exhibited considerable variation in terms of allergenicity, which may impact on diagnostic accuracy. Our new fish extracts have sufficient allergenicity and stability and may be adequate to various clinical applications.ope

Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing ∼1-2% of all allergic diseases globally; however, their evolutionary origin and diverse lifestyles including reversible parasitism have not been illustrated at the genomic level, which hampers allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the diversification of astigmatic mites. In monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, and then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases. Gene diversification after tandem duplications provides many genetic resources for adaptation to sensing environmental signals, digestion, and detoxification in rapidly changing household environments. Many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UDP glucuronosyltransferases and several important fungal cell wall lytic enzymes, which enable detoxification and digestive functions and provide perfect drug targets for pest control. This comparative study sheds light on the divergent evolution and quick adaptation to human household environments of astigmatic mites and provides insights into the genetic adaptations and even control of human household pests.ope

The effects of a newsletter on bedding control on house dust mite allergen concentrations in childcare centers in Korea

OBJECTIVES: Bedding in childcare centers (CCCs) can hold house dust mite (HDM) allergens. This study examined whether HDM allergen levels can be reduced through the distribution of an educational newsletter on bedding control to parents of CCC children in Korea. METHODS: All 38 CCCs were measured for Der 1 (sum of Der f 1 and Der p 1) concentrations on classroom floors and bedding before the intervention. Educational newsletters on children's bedding control were sent to 21 CCCs by mail, and teachers were asked to distribute the newsletters to the parents of the children (intervention group). The remaining 17 CCCs were not sent newsletters (control group). The measurement of Der 1 concentrations in 38 CCCs was repeated after the intervention. Dust samples were collected with a vacuum cleaner and analyzed using enzyme-linked immunosorbent assay methods. RESULTS: The Der 1 concentrations on the bedding were significantly higher than those on the floors in 38 CCCs at baseline (p<0.05). Although changes of the Der 1 concentrations for the control group (n=17) were not significant, Der 1 concentrations for the intervention group (n=21) decreased significantly from 2077.9 ng/g dust to 963.5 ng/g dust on the floors and from 3683.9 ng/g dust to 610.4 ng/g dust on bedding (p<0.05). CONCLUSIONS: The distribution of educational newsletters on bedding control to parents may be an effective means of controlling HDMs in CCCs.ope

Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.ope

No Difference in Allergenicity Among Small-Sized Dog Breeds Popular in Korea

ope

곰팡이 기능 및 비교 유전체학을 위한 유전체 탐색기와 실험 정보 관리 시스템

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 농업생물공학 전공, 2013. 8. 이용환.효모균 Sacchromyces cerevisiae 의 전체염기서열이 1996년에 발표된 이래, 90종 이상의 진균 염기서열이 공개되었다. 하지만 각각 다른 기관에서 진행하여 보관중인 염기 서열의 불균등성으로 인해 효율적이고 종합적인 비교분석을 위한 데이터의 통합이 방해되고 있었다. Comparative Fungal Genomics Platform 은 단일 표준화된 형식을 통하여 유전자 데이터의 다각적인 통합 분석을 지원하기 위해 개발되었다. 이러한 CFGP 및 관련 데이터베이스의 시스템 구성을 기반으로 종간에 걸친 효율적인 데이터 시각화 및 활용을 촉진하기 위해 새로운 유전체 탐색기가 필요했다. 그에 따라 개발된 SNUGB 는 91종 (129집합) 의 곰팡이, 33 종 (37 집합) 의 식물과 동물의 유전체 염기서열 정보를 담고 있으며 각 유전체의 속성 정보를 시각화, 비교 분석 가능한 플랫폼이다. 저장된 유전체 정보는 도해 다이어그램과 표, 일반 텍스트 방식의 3가지 시각화와 6 가지 표시 선택사항을 통해 탐색할 수 있으며 뷴류학에 기초를 둔 계층구조 방식의 새로운 탐색기를 통하여 각 종의 정보를 빠르게 접근 가능하다. 그리고 SNUGB 내부에 포함된 BLAST annotation 프로그램을 통하여 다른 종과의 비교분석이 가능한 기능도 제공하고 있다. 또한 SNUGB 의 모듈형 설계는 다른 유전체 비교 분석 플랫폼에 쉽게 이식이 가능하고 지속적인 확장이 가능한 장점을 내포하고 있다. 이러한 SNUGB 는 곰팡이계(界) 뿐만이 아니라 다른 계와의 유전체 비교 기능 분석에 강력한 지원 시스템이 될 것이다. 또한 다량의 유전체 염기서열정보와 기능 유전체학이 발전함에 따라 대규모 표현형 검사 실험은 효율적인 데이터 처리라는 문제에 직면하고 있다. 이러한 연유로 여러 모델 생물의 표현형 검사 데이터 관리 및 검색을 지원하기 위해 실험실 정보 관리 시스템 (LIMS) 이 사용되고 있다. 하지만 미생물 표현형 연구에 LIMS 를 적용하려는 많은 노력에도 불구하고, 각각의 실험체의 표현형 정보를 얻기위한 지침이 서로 다르기 때문에 얻어진 데이터 또한 비교 분석이 힘들었다. 전 세계적으로 쌀 재배 지역에 걸쳐 가장 파괴적인 질병인 벼 도열병을 발생시키는 곰팡이인 Magnaporthe oryzae 를 위해 개발된 LIMS 플랫폼 또한 존재 하지 않았다. 이러한 문제를 해결하기 위해 개발된 LIMS for M. oryzae 는 표적 유전자 제거에 대한 통합 관리 시스템뿐만 아니라 실험 과정 관리 시스템과 같은 표현형 분석 및 데이터 수집에 관한 표준화된 지침을 연구자에게 주고 있다. 또한 앞서 개발된 CFGP 플랫폼과의 연동을 통하여 유전체 정보와 표현형 정보의 연결 고리를 제공한다. 이 시스템은 유전자 정보와 그 유전자에 관한 표현형 실험 절차, 표준화된 실험 방법, 관련된 출판 논문을 통합 관리하며 저장된 결과는 다른 연구자와 쉽게 공유 가능하다. 또한 새로운 유전자의 기능 실험 전에 기 저장된 유전자 표현형 실험 결과를 사용자가 원하는 방식의 검색을 통해서 미리 기능 예측이 가능하다. 이러한 모든 기능은 사용자 친화적인 웹 기반의 사용자 중심 인터페이스를 통해 쉽게 접근할 수 있다. 이러한 LIMS for M. oryzae 시스템은 벼 도열병에 대한 중요한 연구 자원이 될 것이며, 도열병뿐만이 아니라 다른 모든 식물병원성 곰팡이의 시스템 수준에서 질병의 기작을 이해하는데 큰 도움을 줄 것이라 예측된다.Since the full genome sequences of Sacchromyces cerevisiae were released in 1996, genome sequences of over 90 fungal species have become publicly available. The heterogeneous formats of genome sequences archived in different sequencing centers hampered the integration of the data for efficient and comprehensive comparative analyses. The Comparative Fungal Genomics Platform (CFGPhttp://cfgp.snu.ac.kr/) was developed to archive these data via a single standardized format that can support multifaceted and integrated analyses of the data. To facilitate efficient data visualization and utilization within and across species based on the architecture of CFGP and associated databases, a new genome browser was needed. So, we developed the SNUGB that integrates various types of genomic information derived from 91 fungal/oomycete (129 datasets) and 33 plant and animal (37 datasets) species, graphically presents germane features and properties of each genome, and supports comparison between genomes. The SNUGB provides three different forms of the data presentation interface, including diagram, table, and text, and six different display options to support visualization and utilization of the stored information. Information for individual species can be quickly accessed via a new tool named the taxonomy browser. In addition, SNUGB offers four useful data annotation/analysis functions, including BLAST annotation. The modular design of SNUGB makes its adoption to support other comparative genomic platforms easy and facilitates continuous expansion. The SNUGB serves as a powerful platform supporting comparative and functional genomics within the fungal kingdom and also across other kingdoms. All data and functions are available at the web site http://genomebrowser.snu.ac.kr/. As the enormous genome reveals and functional genomics becomes more advanced, large-scale phenomics presents challenges encountered in new way for the efficient handling of data. For this reason, laboratory information management system was implemented in support of managing and querying the phenotype screening data for several model organisms. Notwithstanding many efforts of applying LIMS to microbial organism research and these all systems have same disciplines but these commodities are different among the platform because each organism has different guideline for getting information from phenotype assay and there was no previously developed LIMS platform in the field of filamentous fungi which has important pathogenic fungal agents like Magnaporthe oryzae one of the most destructive rice blast diseases throughout the worldwide rice cultivated area. So, we developed LIMS for Magnaporthe oryzae to provide not only integrated management system for targeted knock-out but also standardized method like a guideline of phenotype assay and data acquisition formats like work process management system. In addition, we also produced close linkage that is based on locus number between phenotype data in LIMS system and genotype information in CFGP (http://cfgp.snu.ac.kr), a genome data warehouse. This system will manage knock-out mutant by locus number, phenotype experimental assay by list of tasks, lab standard protocols, related or cited papers and influence on such research field. Sharing and comparing all standardized experimental results between all peer is supported by Global-Standard phenotype assay forms. And more easily speculate unknown genes that supported by ordered pre-existing results in LIMS database. Researchers can simply access these all system using user-friendly web-based LIMS database (http://lims.riceblast.snu.ac.kr). LIMSMO is a valuable resource to serve the community who work on rice blast disease. Furthermore, it will shed light on a model platform for pathogenomics, not only Magnaporthe oryzae but also plant pathogenic fungi, to understand the disease mechanism at the systems level.ABSTRACT i CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii CHAPTER 1. SNUGB: a versatile gnome browser supporting comparative and functional fungal genomics ABSTRACT 2 INTRODUCTION 4 RESULTS 24 I. Data processing via an automated pipeline and the function of Positional Database 24 II. Modular design of SNUGB facilitates its application 26 III. Properties of the fungal/oomycetes genomes archived in SNUGB 27 IV. Comparison of genome sequences of multiple isolates within species 32 V. Update of SNUGB 35 VI. Functions and tools 36 CONCLUSION AND DISCUSSION 50 LITERATURE CITED 51 CHAPTER 2. Laboratory Information Management System for the fungal pathogenomics ABSTRACT 61 INTRODUCTION 62 MATERIALS AND METHODS 66 I. Development of standardized phenotype assays based on published papers 66 II. Server operation 66 III. Database composition 66 IV. External program 67 V. Web system for LIMSMO 67 RESULTS 68 I. Development of standardized protocols 68 II. System structure and overall function of LIMSMO 71 III. Management of experimental data via the workflow-driven experimental design of LIMSMO 80 IV. Case study 85 V. Gold standard for phenotype assays 90 CONCLUSION AND DISCUSSION 94 LITERATURE CITED 97 SUPPLEMENT DATA 102Docto

Stability of extracts from pollens of allergenic importance in Korea

BACKGROUND/AIMS: Accurate diagnosis and the effects of allergen-specific immunotherapy for pollinosis are greatly dependent on the potency and stability of the extract. This study aimed to examine factors, such as temperature and storage buffer composition, that affect the stability of allergen extracts from pollens of allergenic importance in Korea. METHODS: We prepared four pollen allergen extracts from ragweed, mugwort, Japanese hop, and sawtooth oak, which are the most important causes of seasonal rhinitis in Korea. Changes of protein and major allergen concentration were measured over 1 year by Bradford assay, two-site enzyme-linked immunosorbent assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reconstitution of the lyophilized allergen extract in various buffers and stored at room temperature (RT, 18°C to 26°C) or refrigerated (4°C). RESULTS: More than 90% of the original protein concentration in all four extracts examined was detected over 1 year when 50% glycerol was added and refrigerated, whereas 57.9% to 94.5% remained in the extracts at RT. The addition of 50% glycerol to the storage buffer was found to prevent protein degradation at RT. Amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at RT when reconstituted in a buffer without 50% glycerol. However, 55.6% to 92.8% of Amb a 1 content was detected after 1 year of incubation at 4°C in all buffer conditions except 0.3% phenol. CONCLUSION: Addition of 50% glycerol as well as refrigeration was found to be important in increasing the shelf-life of allergen extracts from pollens of allergenic importance.ope