Early Detection of Active Tuberculosis in Cases of Latent Tuberculosis Infection by Inflammasome Response after Ex-Vivo Stimulation with Mycobacterium Tuberculosis-Specific Antigens - Esat-6 and Cfp-10

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Project title/計畫英文名

Project Number/計畫編號

MOST103-2314-B002-137-MY3

Translated Name/計畫中文名

藉由偵測結核菌特異性抗原 – ESAT-6和CFP-10體外刺激後發炎體反應之不同，由潛伏結核感染者當中早期診斷活動性結核病

Project Principal Investigator/計畫主持人

JANN-YUAN WANG

Funding Organization

National Science and Technology Council

Website

link

Start date/計畫起

01-08-2014

Expected Completion/計畫迄

12-07-2015

Description

Abstract

摘要：結核病個案，都來自於潛伏感染，如果能夠預測哪些人會進展為活動性疾病，就能夠早期投予抗結核藥物以避免進一步散播；甚至可以使用預防性治療根除潛伏感染，讓結核菌根本沒有發病的機會。近年來藉由體外結核菌特異性抗原刺激免疫細胞後所產生的丙型干擾素反應，對於診斷潛伏結核感染有較高的準確度，這種方法稱為丙型干擾素釋放試驗。然而，丙型干擾素釋放試驗陽性的個案當中，兩年內也大約只有 10-15%會真正發生活動性結核病。最近的研究顯示結核菌感染時，經由結核菌的蛋白，特別是 ESAT-6，會活化宿主體內的發炎體（inflammasome），而發炎體活化的程度，與宿主的預後有關。但相關的研究仍然十分不足。因此，我們設計了這個研究，針對代表結核病自然史中三個不同階段的族群，包括結核病人（代表發病）、潛伏結核感染者（代表感染）、以及健康人（代表未感染），抽取週邊血液後，與結核菌之特異性抗原 – ESAT-6和 CFP-10 – 進行隔夜刺激，之後萃取出單核細胞 RNA進行發炎體的相關研究，同時取出培養皿中的上清液進行各種免疫發炎標記的分析。針對結核病人，我們將持續追蹤，於抗結核藥物治療兩個月時再次抽血檢測。針對潛伏結核感染者，我們將追蹤兩年或追蹤至產生結核病，期望能了解個案一系列的免疫變化以供比較。藉此，我們希望可以找到更多活動性結核病的相關因子，增加對結核病的預測準確度。研究假說：偵測週邊血液單核細胞受到結核菌特異性抗原刺激後發炎體與免疫發炎標記的變化，可以準確的預測宿主將由潛伏結核感染演變為活動性結核病。首要目標：找出週邊血液單核細胞受結核菌特異性抗原刺激後發炎體和免疫發炎標記的變化，以區分活動性結核病和潛伏結核感染。次要目標：（1）針對活動性結核病人、潛伏結核感染者、以及健康人，比較週邊血液單核細胞受結核菌特異性抗原刺激前與刺激後發炎體與免疫發炎標記有何不同；（2）規則記錄潛伏結核感染者演變為活動性結核病的過程中，週邊血液單核細胞受結核菌特異性抗原刺激前後發炎體與免疫發炎標記一系列的變化。
Abstract: All tuberculosis (TB) cases come from latent infection. If we can predict who will progress to active TB, we would be able to prevent further transmission by early anti-tuberculous treatment, and moreover, to terminate the transmission process by preventive therapy for latent TB infection (LTBI). Numerous studies demonstrate that measuring the interferon-gamma response after ex-vivo stimulation of peripheral blood mononuclear cells by Mycobacterium tuberculosis-specific antigens – Interferon-gamma release assay (IGRA) - has a higher accuracy than TST in the diagnosis of LTBI. However, only 10-15% of the LTBI cases diagnosed by IGRA will progress to active TB within 2 years. Some recent studies revealed that during M. tuberculosis infection, mycobacterial proteins, especially Early Secretory Antigenic Target (ESAT)-6, can activate host inflammasome. Furthermore, the extent of activation of inflammasome seems correlate host outcome. However, study regarding to inflammasome in the pathogenesis of TB is still lacking. Therefore, in this study, the peripheral blood mononuclear cells (PBMCs) will be collected from three different populations representing each status of the natural course of TB, i.e. active TB patients (representing disease status), LTBI cases (representing infection status), and healthy subjects (representing un-infection status). The PBMCs will be stimulated by M. tuberculosis-specific antigens – ESAT-6 and Culture Filtrate Protein (CFP)-10 for 16-24 hours. We will then extract RNA from both unstimulated and stimulated PBMCs and measure inflammasome response. We will also collected the culture supernatant to assay inflammatory markers. We will follow active TB patients and collect their PBMC for repeat assay and stimulation after 2 months of anti-TB treatment. Participants with LTBI will be followed for 2 years or until the development of active TB, to trace the serial change in host immune response during the disease process. We hope we can identify more immune markers to improve the prediction of active TB. Research Hypothesis: Inflammasome/inflammatory markers of PBMCs after stimulation by M. tuberculosis-specific antigens can provide a good diagnostic aid to discriminate active TB from latent M. tuberculosis infection. Primary Endpoints: identifying the change in inflammasome response and inflammatory markers of PBMCs that are useful in differentiating active TB from latent infection by M. tuberculosis. Secondary Endpoints: (1) to compare the differences in TB-specific immune responses of inflammasome and inflammatory markers in stimulated and unstimulated PBMCs from active TB patients (disease group), latent M. tuberculosis infection cases (infection group), and healthy subjects (health group); and (2) to follow the dynamic change in TB-specific immune responses of inflammasome and inflammatory markers in stimulated and unstimulated PBMCs from latent infection status to active disease status by following latent infection cases.

Keyword(s)

CFP-10蛋白
ESAT-6蛋白
發炎體
潛伏結核感染
週邊血液單核細胞
結核病
CFP-10
ESAT-6
inflammasome
latent tuberculosis infection
peripheral blood mononuclear cell
tuberculosis