Modified Vaccinia Virus Ankara (MVA) as Production Platform for Vaccines against Influenza and Other Viral Respiratory Diseases

Respiratory viruses infections caused by influenza viruses, human parainfluenza virus (hPIV), respiratory syncytial virus (RSV) and coronaviruses are an eminent threat for public health. Currently, there are no licensed vaccines available for hPIV, RSV and coronaviruses, and the available seasonal influenza vaccines have considerable limitations. With regard to pandemic preparedness, it is important that procedures are in place to respond rapidly and produce tailor made vaccines against these respiratory viruses on short notice. Moreover, especially for influenza there is great need for the development of a universal vaccine that induces broad protective immunity against influenza viruses of various subtypes. Modified Vaccinia Virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform. MVA can encode one or more foreign antigens and thus functions as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant capacities and induces humoral and cellular immune responses. However, there are some practical and regulatory issues that need to be addressed in order to develop MVA-based vaccines on short notice at the verge of a pandemic. In this review, we discuss promising novel influenza virus vaccine targets and the use of MVA for vaccine development against various respiratory viruses

Functions of Some Capsular Polysaccharide Biosynthetic Genes in Klebsiella pneumoniae NTUH K-2044

The growing number of Klebsiella pneumoniae infections, commonly acquired in hospitals, has drawn great concern. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide) locus in the NTUH-2044 strain of the pyogenic liver abscess (PLA) K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. Here we report characterization of a group of cps genes and their roles in the pathogenesis of K1 K. pneumoniae. By sequential gene deletion, the cps gene cluster was first re-delimited between genes galF and ugd, which serve as up- and down-stream ends, respectively. Eight gene products were characterized in vitro and in vivo to be involved in the syntheses of UDP-glucose, UDP-glucuronic acid and GDP-fucose building units. Twelve genes were identified as virulence factors based on the observation that their deletion mutants became avirulent or lost K1 antigenicity. Furthermore, deletion of kp3706, kp3709 or kp3712 (ΔwcaI, ΔwcaG or Δatf, respectively), which are all involved in fucose biosynthesis, led to a broad range of transcriptional suppression for 52 upstream genes. The genes suppressed include those coding for unknown regulatory membrane proteins and six multidrug efflux system proteins, as well as proteins required for the K1 CPS biosynthesis. In support of the suppression of multidrug efflux genes, we showed that these three mutants became more sensitive to antibiotics. Taken together, the results suggest that kp3706, kp3709 or kp3712 genes are strongly related to the pathogenesis of K. pneumoniae K1

'Their final blazon' : burial and commemoration among the north midland nobility and gentry, c.1200-1536

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Sequencing of Arabinans from Mycobacterial Cell Wall Arabinogalactan and Lipoarabinomannan by Advanced Tandem Mass Spectrometry

阿拉伯聚醣 (arabinan) 是結核桿菌細胞壁重要的多聚醣體之一，　分別銜接起半乳聚醣 (galactan)　或是脂化甘露聚醣 (lipomannan) 來形成兩個複雜的大分子- 阿拉伯半乳聚醣　(arabinogalactan, AG) 和脂化阿拉伯甘露聚醣 (lipoarabinomannan, LAM)。 一般結核菌 ( Mycobacterium sp.) 都含有這兩個多醣體。常見的致病性結核菌會引起兩個嚴重的法定傳染病包括了肺結核 (tuberculosis) 以及痲瘋 (leprosy)。 現有的抗結核病用藥中， 胺丁醇（Ethambutol）， 被認為是可抑制阿拉伯醣聚合化酵素 (arabinan transferases) 基因 embCAB。 此基因分別的是去影響阿拉伯醣化的形成 (arabinosylation)。 近年來利用一快速生長的結核桿菌Mycobacterium smegmatis 所進行的一些遺傳和基因缺陷的實驗中， 顯示由 embCAB 所轉錄的基因產物， EmbB/A 和 EmbC 蛋白質， 可分別調控 AG 和 LAM 的阿拉伯醣聚合化。 儘管AG 和LAM 的阿拉伯聚醣結構相似，生化實驗分析結果指示， 相較於AG 的支鏈結構， LAM 明顯的多了直鏈結構式的阿拉伯聚醣鏈末端， 且可能是由 EmbC 蛋白質所調控或轉錄。 從EmbC 缺陷菌種中得到的結果發現它已經不能合成正常菌種的LAM， 取而代之的是類似AG 的結構產生。到目前為止， 也還不能很清楚的了解這種結果發生的原因以及阿拉伯聚醣生合成的分子機制。 進一步的去了解阿拉伯醣聚合化過程是一迫切需要的事， 這有利於我們去對抗此全球性的疾病的蔓延。 本論文將是要朝向去鑑定一個完整的阿拉伯聚醣初始化、 延伸、 分支、 最後終結及末端甘露糖 (Mannose) 修飾等等的機制。 在結構分析上， 首先我們去建立起一關鍵性技術: 有效的應用一內切性阿拉伯聚醣酵素 (endogenous arabinannase) 去水解複雜的阿拉伯聚醣而獲得如同過去所推測的由之22 個阿拉伯糖組成的大分子片斷。 配合各種俱高解析度、精準度且敏感的質譜設備去快速的分析結構與定序。 在此， 分別的去分析來自於各種不同結核桿菌屬像是M. smegmatis、 M. leprae、 M. tuberculosis Rv37、 Emb-resistant M. tuberculosis clinical isolates CSU20等的阿拉伯半乳聚醣　(arabinogalactan, AG) 和脂化阿拉伯甘露聚醣 (lipoarabinomannan, LAM) 結構並做比較。 最後可以將所獲得的結果配合上目前我們對於結核桿菌所的知識做一有系統的整合， 以了解細胞壁的整個結構功能以及生合成之始末原由。 由質譜數據分析證明了來自於 AG 上的阿拉伯聚醣結構是由18 個阿拉伯單糖所組成的且俱有高度分支狀， 符合於過去文獻所推測； 而 LAM 上的阿拉伯聚醣主要是直鏈狀所延伸的結構且變異度高。 在一些慢速生長的且俱致病性的結核菌種中， 半乳糖氨化 (galactosaminylation) 是修飾於AG 上 3,5 鍵結的阿拉伯單糖上面的C-2 位置。 運用基因突變缺陷的技術去建構一可能是影響 LAM 阿拉伯醣聚合化的其中一轉化酵素之轉錄基因末端缺陷的突變株 (embC)。 在基因缺陷實驗所得知的結果中， LAM的阿拉伯聚醣已經形成較為接近 AG 形式的阿拉伯聚醣， 此基因被證明確實影響 LAM 的阿拉伯醣聚合化。 在慢速生長的結核菌種中 (H37Rv、 CSU20 以及leprae)， 皆為有甘露糖 (mannose) 末端修飾種類的阿拉伯聚醣 (ManLAM)， 且各有不同程度的結構複雜度。 我們定義了過去不被報告過的結構及一個更大可能的模型， 且認為雖然這些菌種的阿拉伯聚醣變異性很高， 但應該都是經由一個共通性的結構所組成的。D-arabinofurans are unique polymers present in the mycobacterial cell wall, attached either to a galactofuran or a mannan. Studies on D-arabinofurans from arabinogalactan (AG) and lipoarabinomannan (LAM) have been hampered because of the complexity of the structure, and unavailability of sufficient tools to aid analyses. The main thrust of this thesis work is the development of refined analytical methodology using a newly available endogenous arabinanase to release intact large fragments of arabinan oligomers from mycobacterial AG and LAM for high sensitivity structural motif mapping and sequencing by a combination of low and high collision energy induced dissociation (CID) tandem mass spectrometry. Collectively, the data demonstrated that the arabinans of AG are highly branched and structured into a well defined 18 mers, whereas those of LAM may be further extended with linear chains, giving rise to a highly heterogeneous ensemble. Significantly, evidence was obtained for the first time which validated the linkages and branching pattern of the previously inferred Ara22 structural motif of AG, on which the preferred cleavage sites of the novel arabinanase could be localized. The established linkage-specific MS/MS fragmentation characteristics further led to identification of a galactosamine substituent on the C-2 position of a portion of the internal 3,5-branched Ara residue of the AG of M. tuberculosis, but not that of the nonpathogenic, fast growing M. smegmatis. Similar analysis on LAM arabinan isolated from the genetic mutants of M. smegmatis showed that C-terminal truncation of the EmbC gene encoding for the putative arabinan transferase specific for LAM halts LAM-specific linear extension from an AG-like inner arabinan core structure. Collectively, the data reveal a new model, which implicates an inner branched Ara-(18 –22)-mer core structure as a common structural motif for both LAM and AG. However, direct structural analyses of the mannose-capped LAMs from M. tuberculosis and M. leprae showed them to be distinct from that of the fast growing, non-pathogenic species, a feature which was not previously recognized. Man4Ara10 and Man6Ara13 were identified as distinct terminal motifs whereas Man2Ara7 probably represents an internal repeat motif variably found in various strains of M. tuberculosis and M. leprae. The new insights on the arabinan structure provided by the current analysis lead us to a broader perspective on the assembly and biogenesis of the mycobacterial arabinan never before realized and addressed.Chapter 1 Introduction ……………………….…………………………………1~26 1.1 Tuberculosis and immunity …………….…………………………………………1 1.2 Classification …...…………………………...……………………….…………....2 1.3 D-Arabinofurans structure ………………………………………………………...5 1.4 Mycobacterial cell wall …………………………………………………………...6 (1) Peptidoglycan structure ……….………..…………………………...………...7 (2) Mycolic acid structure ……...……………………………………...…….…....7 (3) Arabinogalactan (AG) structure ……………………………………...………..8 (4) Lipoarabinomannan (LAM) structure ………………………………………..10 1.5 The immunomodulatory activities of LAM …………………………………..…13 1.6 Biosynthesis of mycobacterial D-arabinofurans ……………………………...…14 1.7 D-Arabinanase enzyme ………………………………………………………….17 1.8 Strategy for structural analysis of polysaccharides …………………………….18 1.9 Mass spectrometry ……………………………………………………………….19 1.10 History of structural analysis of AG and LAM arabinan ………………………20 Chapter 2 Objective ……………………………………………………..……..27~29 Chapter 3 Materials and instruments ………………………………………30~31 3.1 Reagent ….……………………………………………………………………….30 3.2 Strains ……………………………………………………………………………30 3.3 Instruments and apparatus ….……………………………………………………30 Chapter 4 Methods ………………………………………………………..……32~39 4.1 Preparation of soluble arabinogalactan and lipoarabinomannan ………………32 4.2 Purification of endogenous arabinanase …………………………………………33 4.3 Arabinanase activity assay and arabinan digestion ……………………………33 4.4 Fractionation and desalting ……………………………………………………34 4.5 Digestion with α-mannosidase …………………………………………………35 4.6 Digestion with Endoarabinanase ………………………………………………35 4.7 Chemical derivatization ………………………………………………………… 35 (1) Permethylation ……………………………………………………….………35 (2) Peracetylation .……...………………………………………………..………36 (3) Reduction …………………………………………………………………….36 4.8 Sep-Pak C18 ……………………………………………………………..………36 4.9 Zip-Tip C18 ………………………………………………………………...……37 4.10 Phenol-sulfuric acid assay ……………………………………………...………37 4.11 Sugar composition analysis …………………………………………………….37 4.12 Gel electrophoresis ……………………………………………………………..38 4.13 Mass spectrometry analysis …………………………………………………….38 (1) MALDI-TOF and MALDI-Q-TOF ………………………………………….38 (2) MALDI-TOF/TOF ……………………………………………………….…..39 Chapter 5 Preparation of arabinan and determination of its high-energy CID MS/MS fragmentation pattern ………………………………………..……40~54 5.1 General workup strategy ……………………………………………………….40 5.2 Msm-arabinanase ………………………………………………………………41 5.3 Separation of arabinan …………………………………………………………...42 (1) Size exclusion HPLC …………………………………………………...……42 (2) Bio-Gel P-10 column ……………………………………………………...…43 (3) PGC (porous graphitized carbon) column …………………………...………43 5.4 Rule of fragmentation in high-energy CID MS/MS ……………………………..43 5.5 Discussion ………………………………………………………….……………46 Chapter 6 Mycobacterium smegmatis AG ………………………….………55~71 6.1 Mass spectra of Mycobacterium smegmatis AG-arabinan ………………………55 6.2 Characterization of arabinan in low-energy CID ……………………..…………56 (1) Characterization of Ara7 by low-energy CID MS/MS ……………………….57 (2) Characterization of Ara8 by low-energy CID MS/MS …………….…………57 (3) Characterization of Ara11,12 by low-energy CID MS/MS ……………………58 (4) Characterization of Ara18 by low-energy CID MS/MS ……………………58 6.3 High-energy CID in MALDI TOF-TOF..…………………………………...……59 (1) Characterization of Ara18 by high-energy CID MS/MS ……………………..59 (2) Characterization of Ara7 and Ara12 by high-energy CID MS/MS …………...60 6.4 Characterization of Ara19/Ara20 by low and high-energy CID MS/MS …………61 6.5 Discussion ………………………………………………………………….……61 Chapter 7 M. tuberculosis CSU20AG and Leprae AG …………………..…72 ~91 7.1 Mass spectra of M. tuberculosis CSU20AG-arabinan ………………………..72 7.2 Characterization of GalN by high-energy CID MS/MS………………………….75 (1) Characterization of Ara13-GalN (m/z 2374) …………………………………75 (2) Characterization of Ara6-GalN (m/z 1238) …………………………………..76 (3) Characterization of Ara18,20-GalN (m/z 3174, 3494) in high-energy CID …...77 7.3 Mass spectra of M. Leprae AG-arabinan ………………………………...………78 7.4 Discussion ……………………………………………………………………….79 Chapter 8 LAM from M. smegmatis and EmbC mutants ………….………92~110 8.1 Mass spectra of M. smegmatis LAM-arabinan …………………………………..92 8.2 Characterization of LAM-Ara18 by high-energy CID ………………………...…92 8.3 EmbC and C-terminal mutation LAM-arabinan ………………………..….……93 8.4 Mass Spectra of LAM-arabinan derived from EmbC C-terminal mutants ……...94 8.5 Characterization of Msm- and EmbCΔ358c –LAM arabinans by low-energy CID…………………………………………………………………………………...96 8.6 Discussion …………………………………………………………….…………99 Chapter 9 M. tuberculosis and M. leprae LAM ………………………….…111~131 9.1 The evidence of SDS-PAGE in LAM ……………………………….…………111 9.2 Mass Spectra of M. tuberculosis CSU20LAM-arabinan ……………………….111 9.3 Characterization of CSU20LAM-arabinan by high-energy CID MS/MS …...…113 9.4 Mass spectra of M. tuberculosis RvLAM-arabinan …………………………….114 9.5 Characterization of RvLAM by high-energy CID MS/MS …………………….115 9.6 Mass spectra of LepLAM-arabinan …………………………………………….116 9.7 Characterization of LepLAM-arabinan by high-energy CID ………..…………117 9.8 MS/MS comparison of LAM-Ara10, 13 …………………………………………118 9.9 Discussion ………………………………………………………………...……119 Chapter 10 Discussion……………………………………………………..…132~147 References…………………………………..………………………….…..…148~158 Publications …..……………………………...……………………………….159~185 List of Figures and Supplementary I. Figures Figure 1.1 A schematic diagram of bacterial cell wall ………………………………23 Figure 1.2 A chemical model of the mycobaterial cell wall …………………………23 Figure 1.3 Structure of arabinogalactan (AG) ……………………………………….24 Figure 1.4 Structural mode of AG-arabinan …………………………………………25 Figure 1.5 Structural model of mycobacterial LAM ……………………...…………26 Figure 1.6 A cartoon representing how the Emb proteins function ………………….26 Figure 5.1 A flow chart for the characterization of arabinan structure……………….49 Figure 5.2 Purification of crude Msm-arabinanase and examination of enzymatic activity ……………………………………………………………………………….50 Figure 5.3 Separation of the arabinan oligomers by Superdex peptide HPLC ……...51 Figure 5.4 Fractionation of AG digestion by Bio-gel ……………….…………….....52 Figure 5.5 High-energy MALDI CID MS/MS spectrum of Ara4 standard ……….…53 Figure 5.6 High-energy MALDI CID MS/MS spectrum of Ara5 standard ………….54 Figure 5.7 High-energy MALDI CID MS/MS spectrum of Ara6 standard ………….54 Figure 6.1 MALDI-MS spectra of Msm-arabinanase digested MsmAG-arabinan .…64 Figure 6.2 MALDI MS/MS of permethylated Ara7, Ara8, Ara11, and Ara12 from MsmAG …………...…………………………………………………………………65 Figure 6. 3 MALDI MS/MS spectrum of permethylated Ara18 ………………….…..66 Figure 6.4 Expanded high-energy CID MS/MS spectrum of permethylated Ara18 …67 Figure 6.5 A complete assignment of the high-energy CID MS/MS fragment ions given by the Ara18 structure ………………………………………………………….68 Figure 6.6 MALDI MS/MS spectra of permethylated Ara7 and Ara12 ………………69 Figure 6.7 MALDI MS/MS analysis of permethylated Ara19, and Ara20 ……………70 Figure 6.8 The major components of MsmAG-arabinan released by Msm-arabinanase ……………………………………………………………………71 Figure 7.1 MALDI MS spectra of CSU20AG-arabinan …………………………….82 Figure 7.2 Mass spectra of permethylated CSU20 AG-arabinan after sub-fractionation ……………………………………………………………………..83 Figure 7.3 Mass spectra of permethylated, reduced arabinan in HPLC fractions ...…84 Figure 7.4 MALDI MS spectra of the permethyl derivatives of HPLC fraction D after further digestion by endoarabinanase …………..……………………………………85 Figure 7.5 MALDI MS/MS spectrum of permethylated Ara13-GalN and Ara6-GalN .86 Figure 7.6 MALDI MS/MS spectrum of permethylated Ara18, 20-GalN ……….……87 Figure 7.7 MALDI MS spectrum of the permethyl derivatives of LepAG-arabinan ..88 Figure 7.8 MALDI MS/MS spectrum of permethylated reduced Ara7 from LepAG-arabinan ……………………………..………………………………………89 Figure 7.9 MALDI MS/MS spectrum of permethylated reduced Ara18 from LepAG-arabinan …………..…………………………………………………………90 Figure 7.10 GalN-containing arabinan of Msm-arabinanase digestion product from CSU20AG …………………………………………………………………………...91 Figure 8.1 MALDI MS spectrum of permethylated derivatives of MsmLAM-arabinan …………………………………………………………...……101 Figure 8.2 High-energy CID MS/MS spectrum of permethylated reduced Ara18 from MsmLAM ……………………………………………………………………..……102 Figure 8.3 Predicted TM-topology of MsmEmbC ………………………………....103 Figure 8.4 Analysis of LAM and LM of Msm wild-type and the mutants by SDS-PAGE …………………………………………………………………………104 Figure 8.5 MALDI-MS spectra of permethyl derivatives of arabinan from EmbC C-terminal mutants of Msm ……………………………………………………..…105 Figure 8.6 Low-energy CID MALDI-MS/MS analysis of arabinosyl oligomers derived from Msm-arabinanase digestion ………………………………….………106 Figure 8.7 Low energy CID MALDI MS/MS analysis of arabinosyl oligomers derived from Msm-arabinanase digestion …………………………………………………..107 Figure 8.8 Low-energy CID MALDI-MS/MS analysis of arabinosyl oligomers derived from Msm-arabinanase digestion ………………………………………… 108 Figure 8.9 Arabinan structure deduced from low energy CID MS/MS analysis ..…109 Figure 8.10 High-energy CID MS/MS spectrum of permethylated reduced Ara18 derived from EmbCΔ358cLAM ……………………………………………………110 Figure 9.1 Evaluation of the degree of LAM digestion by gel electrophoresis ……121 Figure 9.2 Evaluation of Microspin efficiency …………………………………….121 Figure 9.3 MALDI MS spectra for the permethyl derivatives of CSU20LAM-arabinan …………………………………………………………...…122 Figure 9.4 MALDI MS/MS spectrum of permethyl derivatives of CSU20LAM Man4Ara10 ……..……………………………………………………………………123 Figure 9.5 MALDI MS/MS spectrum of permethyl derivatives of CSU20LAM Man6Ara13 …………………………………………………………………………..124 Figure 9.6 MALDI MS spectra of the permethyl derivatives of RvLAM-arabinan .125 Figure 9.7 MALDI MS/MS spectrum of permethyl derivatives of H37RvLAM Man2Ara7 ……………………………………………………………………….…..126 Figure 9.8 MALDI-MS spectrum of permethylated, reduced LepLAM-arabinan …127 Figure 9.9 MALDI MS/MS spectrum of permethylated, reduced Man2Ara10 from LepLAM and CSU20LAM ……………………………………………………...…128 Figure 9.10 MALDI MS/MS spectrum of permethylated reduced Ara10 of LAM …129 Figure 9.11 MALDI MS/MS spectrum of permethylated reduced Ara13 of LAM ....130 Figure 9.12 Summary of ManLAM arabinan motifs ………………………………131 Figure 10.1 Models for assembly of AG ……...……………………………………144 Figure 10.2 Model for assembly of LAM ………………………………………….145 Figure 10.3 Model for assembly of ManLAM ……………………………………..146 Figure 10.4 Summary of arabinan structures, model and arabinosylation …………147 II. Supplementary Table1 Table of nominal, monoisotopic, and average mass of arabinofuranose …...18

The Montana Postural Care Project: A pilot study implementing posture care management in a rural, low-resource region

Background: Mobility impairment limits control of posture and body alignment. This leads to altered body shapes, co-occurring problems with pain and sleep, cardiopulmonary concerns, digestive health issues, and emergent health outcomes, which further complicate functions of daily living. 24-hour posture care management was developed to remedy these challenges by restoring body symmetry. Objective: To determine the feasibility of introducing posture care management to a rural-based, medically complex patient population, evaluate response of body symmetry, and examine its impact on pain and sleep quality. Methods: This pilot study employed a longitudinal, quasi-experimental study design from March 2016 to September 2018. The posture care management intervention introduced positioning support for use when lying down, a personalized training workshop for caregiver teams, and in-home initial and follow-up assessments to provide materials and collaboratively develop a personalized care plan. Participants were followed pre-post for 6–9 months. Results: A total of 73 participants enrolled in the study; 55 (75 %) completed. The majority were male (55 %) with a median age of 11. Most caregivers were immediate family members, and most participants had 1+ diagnosis characterized as a neurodevelopmental disorder. A majority of participants improved body symmetry (56–76 %), and 53 % with comparable information saw improvement in body symmetry with no worsening of pain or sleep quality. Conclusion: This study established the feasibility of administering posture care management in North America. These findings provide preliminary evidence of improvements in body symmetry and address concerns that posture care management can interfere with pain and sleep. Future research should consider levels of caregiver engagement and explore remote-monitored options of a posture care management intervention

Physicochemical Characteristics and Anti-Inflammatory Activities of Antrodan, a Novel Glycoprotein Isolated from Antrodia cinnamomea Mycelia

Antrodia cinnamomea (AC) is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named “antrodan” was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6 ± 0.8 mg/g). The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%), and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which β-glucan (14.20%) was prominently higher than α-glucan (1.45%). Its FTIR confirmed the presence of β-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and β-glucans, which had (1→ 4)-linked α-Glcp and (1→ 3)-linked β-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 μg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 μg/mL