Collaborative Projects between KMUH and NICR

General

Fluidigm Access Array + MySeq

–YouTube 1: https://www.youtube.com/watch?v=s9HUhuCbbhU

–YouTube 2: https://www.youtube.com/watch?v=WXpNjcRwdj8

–AA_Illumina_ug_100-3770 (pdf)

–RecoverAll Total Nucleic Acid Isolation Kit for FFPE (pdf)

  台大郭頌鑫醫師抽取DNA and RNA from tiny endoscopic biopsy samples使用。

–陳華鍵老師他們新開的公司行動基因,用的是類似方法、只是不同機器(Ion Torrent PGM/Proton)

–Archer: Archer™ FusionPlex™ FGFR Panel (USD 495 / 8 rxn)

–Life: Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel

–與台大合作部分，詳見HPV and p16 in HNSCC. (按我連結) 

–ds-cDNA protocal from Yi-Mi and Dan (03/05/2015). (按我下載)

–ds-cDNA protocal from 黃醫師Lab 智娟 (2015/04/19). (按我下載)

Gene Fusion Panel

–16個receptor tyrosine kinases (pdf 3815)+ RSPO2/3 (pdf 3594) 

ALK, AXL, FGFR1, FGFR2, FGFR3, NTRK1, NTRK3, PDGFRA, RET, ROS1, MUSK, EGFR,INSR,INSRR, MET, NTRK2, RSPO2, RSPO3

–黃醫師 final list on 2015/04/10

ALK, ABL1, FGFR1, FGFR2, FGFR3, NTRK1, NTRK3, PDGFRA, PDGFRB, RET, ROS1, FLT3

(4 x 96 primers)

Cholangiocarcinoma Panel

ARAF, ARID1A, ARID2, AXIN1, BAP1, BRAF, CDH1, CDKN2A, CTNNB1, FBXW7, FLT4, IDH1, IDH2, KRAS, MLL3, NRAS, PBRM1, PI3K3CA, PTEN, SMAD4, TERT, TP53, VEGFC, VEGFR3, 

hTERTp -124, and hTERTp -126 (526 amplicons)

2015/04/20 Line messages from Dr. Huang: (1) 50 ng for one panel, 2 panels for each TLCN sample. (2) 2 PCRs to increase specificity (3)  if possible, ship 500 ng / 10 ul / sample will be the best. 

TK Panel

Dear Prof Lou,

We will order your primer tomorrow. Beside those oral pharyngeal CA panel, we already have some TK panel, which include: FGFR1, FGFR2, FGFR3, ALK, ROS1, NTRK1, NTRK3, RET, PDGFRA, PDGFRB, FLT3. More TK genes will be added later. We can run your samples in your cancer panel and current TK panel if you are interested. (2015/04/29 email)

sgRNA construction

關於CRISPR sgRNA的設計跟構築

除了委託廠商代工外

現在常用的方法

是利用外切 restriction enzyme去製造linear plasmid

再利用合成stick end 互補股引子的方式去 ligation


常用的restriction enzyme會是類似BbsI這類特別的酵素

因為他們的切點在辨識序列的外側 (5' or 3')來行成stick end

所以只要在合成primer的時後先預留正確的extra base

將回溶/reannealing/PNK 處理過的片段作ligation

就能得到sgRNA vector，

要在經定序後確認，即可得到sgRNA。


目前sgRNA的確認只能用定序的，因為塞入的片段太小(23bp)，

加上sgRNA設計的時候沒有在原始被移除的序列中加上vector其他部位已經有的切位

所以需要這部，或是可以在製造linear vector的時後check 不會有 uncut vector存在


addgene提供的 空sgRNA vector 多數是用這個方法(Humanized sgRNA是例外，我被暗算過)
empty sgRNAhttp://www.addgene.org/crispr/empty-grna-vectors/

裡面也有提供類似冷泉港的單一vector。


利用這個方式建構的sgRNA成本會遠低於委託廠商代工

主要花的錢包含vector購買(NT 3000)/ primer 合成 (~50 bp per clone)/
PNK 跟 restriction enzyme  (各約莫三千元左右，可多次使用)



以下在附上我常用的sgRNA設計網站
sgRNA design
http://www.e-crisp.org/E-CRISP/


最後附上 sgRNA構築的protocol

sgRNA construct

二期癌症缺口計畫2015

2015.01.15(W4) 11:00 am­ 口腔癌缺口計畫會議

總計畫中文: 針對台灣口腔癌前病變惡性轉化開發快速診斷與有效化學預防之整合研究

總計畫英文: Rapid diagnosis and effective chemoprevention for malignant transformation of oral potentially malignant disorders in Taiwan  

  

計畫編號: MOHW103-TDU-212-114005

三月中旬舉行第二次meeting, 討論期中報告繳交．

#1由 sufang 在 五, 05/29/2015 - 13:22 發表。

100例病人葉酸濃度紀錄 謝謝高醫、謝謝奕淇

2015/05 (n=20)

#2由 mmiiee 在 二, 04/14/2015 - 09:09 發表。 口腔癌缺口計劃2015/4/8會議記錄 口腔癌缺口計劃2015/4/8會議記錄 日期: 2015年4月8日 (星期三) 時間: 10:00-12:30 am 地點: 竹南院區 研究大樓2樓 R2-2031會議室 出席人員: (出席人員以螢光筆標示之) 江士昇 助研究員、李岳倫 助研究員、林佩瑩 副管理師、林素芳 副研究員、 夏興國 助研究員、張書銘 副研究員、郭靜娟 副研究員、陳奕淇 計劃助理、 陳雅雯 助研究員、陳嬿如 博士後研究員、張憶壽 研究員、劉柯俊 副研究員、 劉滄梧 副所長 (依姓名筆畫排序)

癌症個人化醫療前瞻論壇 @ 台北喜來登 星月廳 (B2F)

20150111_癌症個人化醫療前瞻論壇 @ 台北喜來登 星月廳 (B2F)
由 sufang 在 日, 01/11/2015 - 20:41 發表 Pre-published Meeting Oral Cancer

Opening remarks: 賴瓊慧教授 (副研發長) 主持人: 王子豪醫師 (國外廠商contact him about STIP1)

閻紫宸教授: 台灣口腔癌病患新穎癒後生物標誌的發現與臨床意義

–Why OSCC: Taiwan has the oral cancer incidence in men worldwide

–Oral cavity cancer: incidence, survival and treatment

–廖俊達教授is the head of CGH HNSCC group leader (pastic surgeon is very important)

–OSCC 2-yr survival: CGMHLK vs all centers

–Clinical path of OSCC

    –全世界一樣的biologic phenotype: ECS positive (survival rate 30%, how to pick them up? )and LN meta

–Unanswered questions: (a) how unique is Taiwan's OSCC? (b) Which genetic alterations can be used as the molecular markers for disease recurrence, metastasiss survival and second primary timor? (c) which targeted therapy can be used for OSCC patients?

–2000年以後是 molecular era, 2010 以後 是 immunotherapy era

–OSCC --LN meta 40% (positive) --? ECS positive (60%) --> 24%

–OSCC --LN meta 40% (positive) --? ECS negative (40%) --> 16%

–OSCC --LN meta 60% (negative) –Comparison 印度 （檳榔子 and no alcohol, 我們的是檳榔汁

–Outline of the study

Stage I: exploration (345 FFPE: ABI 45 core genes targeted based as deep as possible, at least 5-yr follow-up on 345 FFPE samples ; 其實發現妥善處理 應該到1996年的檢體都可用)

Stage II: verification

ultra-deep sequencing of all coding exns of 409 cancer genes using an ndependent cohort (currently, n=300

加入 miRNAs

conclusion: (1)80% found non-synmous mutations and 80% of them are on the top 

potential targetable genes

Stage II 90% found non-synmous mutations NOTCH1增加了

TP53 and NOTCH 與 TCGA相當

Pathwayss affected in OSCC (II)

--110 人 as base , 並以 image (free software abailable)DNA repair 

 

 

tage III: –compare to TCGA data, PI3KCA and TP53 是相當相似的 Notch1 似乎過低 Genetic alterations on prognosis: after 2004 we improve OSCC survival by "team combined care" 

–2015年: proton therapy 10 genes with mutation frequency > 2% were used to construct the OSCC prognostic gene panel with clinical outcomes --> 可以清清楚楚地把345個病人 (1996-2012 ) 分成兩群

–Internal validatio of OSCC prognostic gene panel: either by years (2004 as cutoff) or randomization

Q0: treated 病人還會有多少人嚼檳榔

Q1: CDKN2A (p16)  婁培人醫師: Taiwan p16 status: more mutations than what have been reporter in the Caucasians. (存疑)

                                廖仲達醫師: E6 E7 expressions are related to metastasis of oral cancer. 已發表蠻多篇相關papers

Q2: validation by TCGA data or Indian data 每一年3% 的survival patients 又會長出OSCC related: field cancerization NGS gene panel for developing primary tumor Stage 3: prognostic markers? 409 cancer genes in important signaling pathways cover 10 cancer genes and more than wp essential cancer pathways 

---

胡松年博士: 消化性結腸炎癌病患者的基因變異圖譜分析

003-2011年來台多次 UC的病人後來成CRC的機率很高

whole genome : HiSeq 2000 and ion proton

數據庫: GWAS, CLINVAER, COSMIC, HGMD_

1 case clinical sample Exom-seq 100X germ-line 突變很高 -> DNA repair發生問題? 但四個數據庫裡頭都有的只有0個; 後改成任兩個有的則多落在germ-line突變

IBD 與 CRC 都有的 germlline mutations are similar to that reported by others DNA repair point mutations

CRC mutational landscape 1000 Genome

CHS (南方漢族人) vs CHB (北京漢族人)

208 WES and low coverage WGS --低頻mutation in DNA repair system -- 7 DNA repair system 均有 -- 106 genes

---

陳淑貞: personalized medicine

​

–Cancer is a disease of genetic alteration

–Genetics for precision cancer medicine (diagnosis, therapeutics and prognosis)

–NGS and clinical oncology (NGS technology advancement; NGS for clinical oncology) 2013 FDA approves 2 important platform and 2014 FDA improves another platform.

–High accuracy error rate ~0.1%;high sensitivity : 1-3%; high specificity; cane detece all tpes of genetic relationship associated with cancers

–Eric Green 2011 年 presented: from base pairs to bedside Nature 2011 (pdf 2868) (ENCODE, TCGA, 

NCI+NHGRI initiated on 2006 (3-types) --> 2009 (20 cancer types)

ICGC project maps 

Pan-cancer analysis  (pdf 4139, 4140)

-A CUP patient harbors 16 copies of MET -? crizotinib cure for >19 month

-Genetically matched therapy are more effective (on TN-breast cancer)

-based on these results NCI-MATCH (plan to perform on 180 patients, molecular analysis for therapy choice trial)

-Outlier patient obtained better treatment than other patients 

1. 2012 Science: Everolimus sensitivity marker on bladder cancer: NF2 TSC1 "exome-sequencing of ...)

2. Cancer Discovery: Metastatic urothelial carcinoma : mTOR everolimus sensitivity 

3. NEJM: Anaplastic thyroid cancer : everolimus  TSC2 mutation -> 14-month

 

Cetuximab resistance in mCRC patients

-32 (responders): 20 (non-responder) Genetic alterations asspciated awith Cetuximan resistance 

–只sequence EGFR-RAS-MAPK 

–Non-responder K-RAS exon 2 在TCGA data裡頭已被filter掉

—NRAS 

—BRAF V600E is druggable

—ACT genomics: turn genomics into action

Start: 01/11/2015

2014-01-20 冷泉港 CRISPR 說明會

大家好，

謹訂 1/20 下午1：30 在 R1-1041 舉辦 CRISPR 說明會，

由冷泉港的技術專員 陳博士與李小姐 負責解說，

內容包括 CRISPR 原理與應用、冷泉港 CRISPR 產品介紹與操作方式，

還有示範如何設計 guide RNA

如有其他問題或需求，歡迎現場提問，

請大家務必準時到場，謝謝

另外，當日的 Journal Club 順延一次

Nature. 2014 Dec 18;516(7531):423-7. doi: 10.1038/nature13902. Epub 2014 Oct 22.

In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.

Maddalo D1, Manchado E1, Concepcion CP2, Bonetti C1, Vidigal JA1, Han YC1, Ogrodowski P1, Crippa A3, Rekhtman N4, de Stanchina E5, Lowe SW6, Ventura A1.

Author information

Abstract

Chromosomal rearrangements have a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions. A recently discovered example is a fusion between the genes echinoderm microtubule-associated protein like 4 (EML4) and anaplastic lymphoma kinase (ALK), generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC) and is clinically relevant because it confers sensitivity to ALK inhibitors. Despite their importance, modelling such genetic events in mice has proven challenging and requires complex manipulation of the germ line. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumours invariably harbour the Eml4-Alk inversion, express the Eml4-Alk fusion gene, display histopathological and molecular features typical of ALK(+) human NSCLCs, and respond to treatment with ALK inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms.

PMID: 25337876

帥呆了! 要來做做看嗎?

2014-01-06 Journal club paper

Dear all:

這是我下週要報告的paper

選自今年(2014)的Cell Cycle 

題目為: RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation.

敬請多多指教

謝謝

    嬿如 敬上

2014-12-16 Journal Club Paper

大家好12/16我要報的 Paper

選自 Cell Death and Differentiation (2014) 21, 797–810

Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

請查收!!!   謝謝大家!!!

與IBPR開會小記

由 sufang 在 五, 12/05/2014 - 10:12 發表 Pre-published IBPR Meeting

杏國Syncore (DBPR204, DBPR 104)

健亞Genovat (DBPR108, T2DM)

中天(Microbio)

 

cKIT, IDO1, IDH1, 大分子

 

COE of New Drug Discovery

1. Success in clinical trials and regulation approval
2. Drug discovery engine
3. Team of excellent investigators
4. Mentor/coaching/networking

Triangular relationship (Target, Drug, Disease)

1. PK/PD correlation
2. Biomarkers
3. Translational model

TNFa 關節炎 Huber??

DBPR110: NS5A/HCV infection

DBPR211: 夏克山、明秀 CB1/T2DM antigonist (peripheral) 2015Q4

DBPR112: EGFR (irreversable)/NSCLC 2015Q4

DBPR114: Multi-target kinase, PanCA, Liver, Gastric Ca 2017 Q2 (此藥剛倍NRPB approved? 興邦)

---

Work-in-progress (WIP)

c-Kit/Flt3 drug resistant GIST/AML

Zn-DPA-SN38: small molecule, PS會翻出來, solide tumor CRC (鄒倫)

IDO1: Solid tumor/CRC (伍素瑩、明秀、夏克山、翁紹華)

Multiple kinase (PO) DBPR114 之延伸, 目標為口服. Solid tumor.

GA (glutaminase 2? GLS2?) MM, NHL (李秀珠)

Opioid AAM/chronic pain

CXCR4 (克山) Hematopoietic stem cell mobilization

MRSA drug resistant SA infection (2016)

---

Future direction:

–SM (small molecules) drug discovery

–Biologics drug discovery

  *Therapeutic antibodies

  *Antibody drug conjugates

–Novel therapeutic paltforms

  *SBDD (structure based drug design)/computational/informatics

  *PDX (translational pharmacology models)

  *protein engineering platform

–Actively seeking internal/external collaborations

  NICR, IPHS, NIIDV

–大份子

• R-Spondin 3/CRC 徐祖安 Competitive Intelligence
• VEGFR2/Solid tumor NICR
• NTRS1 -> EGFR link; NSCLC (105年政策額度)
• Adipopectin (日本人有一篇paper) 這是和侯明峰一樣的東西嗎?/TZDM

#1由 sufang 在 五, 12/12/2014 - 10:43 發表。
2014/05 與石泉所長雙邊會議

Notes from (1) 石泉所長雙邊會議 (05/2014) (2) chat with 素菁、靜娟

•鄒倫 李季珠: GA, Glutaminase inhibitor

•葉燈光: DMPK

•翁紹華: 化學合成、IDO1 inhibitor

•靜娟:

•Tki: EGFR, AURKA, AURKB, IDO1, FLT3, c-Kit

•DBPR114

•老鼠model <-> clinical

•Alimta

•預配方, pre-formulation

•success rate: phase 1,2,3 (50% x 50% x70%=17.5%)

•藍始榮、龔一泰

•Prof 楊宗德

•Immune therapy: PL-D1/CD274, IDO1

•glycoprotein

•PDX mouse

•stromal cells: immortalized fibroblast, MSC, PDMC

•HOK+carcinogen vs HOK+stromal cells+carcinoge vs HOK+cancer cells+carcinogen

•transwell to collect paracrine

•IDO1, Kyneurine assays (DiO, DiI?)

•CXCL1, 2, 3 (=Gro-a, b, g)

•MLR: mixed-lymphocyte reaction

•J.I 2013, Hsu, Su-Ching paper

•363, 442

2014-12-02 Journal Club Paper

您好 這是我要報的paper.請查收. 祝 研安 ehsing 敬上
____________________
2014 Mo Cell
Mutant p53 Reprograms TNF Signaling in Cancer Cells through Interaction with the Tumor Suppressor DAB2IP 

Fusion gene detection

由 sufang 在 六, 11/22/2014 - 09:01 發表 Cholangiocarcinoma cholangiocarcinoma FFPE Gene Fusion

Nov 5 2014 

Daw-yang Hwang 黃道揚 to LTCHEN, yngmiin 

Dear Prof Lin, Dr. Chiang, and SF:

I designed primers for ALK, ROS-1 FGFR2, FGFR3, and I can add primers for RET.
In my research part (bladder and upper urinary tract cancer), I am collaborating with urologists in KMUH.  Hopefully I can get cDNA samples from then late this month. I can run the above panel for cholangiocarcinoma, oral cancer, or any cancer samples you are interested. I am currently testing some FFPE samples.
Brief, the protocol:
1. Samples (fresh or FFPE)
2. Extract RNA
3. RT-PCR for single-starnded cDNA
4. second cDNA systhesis (double-stranded cDNA)
5. Nextera XT kit
6. Specific PCR amplification
7. Barcode PCR (up to 288 different samples in one MiSeq run)
8. Purification
8. MiSeq run   

Maybe we can meet sometimes to have a discussion, for:
A) where to run MiSeq
B) discuss whether we want to include potential fusion on both ends or run known fusion-direction.

Kindly regards,
Daw-yang

 

A company developed similar kits this year:
http://www.enzymatics.com/archer/

 

---

Nov 5 2014

Su-Fang Lin  to 陳所長

 

Dear 所長: 

那我就向黃秀芬醫師申請TLCN的cholangioncarcinoma sample cohort做:

1) FGFR2 IHC 染色 (我們剛剛買了一些commercial的tissue array, 在測試抗體條件)

2) cDNA for 道揚's RT-PCR test.

相請教您的是，用您的名字申請? 還是用我的?
 

Best,

Su-Fang

---

Nov 6 2014

Su-Fang Lin  to Daw-yang Hwang., LTCHEN 

 

Dear Daw-Yang:
 

Congratulations to your 104 NHRI grant!!!   我先押寶FGFR3 gene fusions in your bladder and upper uinary tract unrothelial cancer! (by the way, we have just completed FGFR3 IHC staining for some commercial tissue arrays, in case you need it someday, please let me know :)
 

Su-Fang 敬賀

---

Nov 6 2014 

Daw-yang Hwang 黃道揚 to me 

 

Dear SF,

Thank you for your great help, I plan to run several tyrosine kinase fusion genes together in the bladder cancer samples.  Which organ origins are those commercial samples?
Let me know if you want to test your oral cancer (or other cancers) samples.  I will run in-house designed oncogene panel for my bladder cancer, using Fluidigm-MiSeq plateform.

Talk to you soon,
Daw-yang

 

---

Nov 6 2014

Su-Fang Lin  to Daw-yang Hwang

 

I remember we did include some bladder cancer tissues , please check this post
http://eln.nhri.org.tw/lims/?q=node/1798  (還記得你的user name/password嗎? DYH/DYH123)
 

I will call you to discuss more about FGFR3 gene fusions in oral cancer. 你今天何時比較有空?  11點OK嗎? 或是下午、或是明天你可以的時間! 謝謝。 (對了，請再給我一次你office電話)

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Nov 6 2014 

Daw-yang Hwang 黃道揚 to me 

 

I will check my user name in the NHRI system.

You can call me at 11AM, 07-3121101 x 2266 (this is the lab, I will be there at 11AM)
(Cellular phone is 0932351464 or 0975356113 if case you need them)

Talk to you soon,
Daw-yang 

 

---

Nov 6 2014

Su-Fang Lin  to Daw-yang Hwang

OK, I will call you at 11AM (lab phone 我比較會講話)
剛剛那個post 連結請用 DYH (user name), DYH123 (password) 登入。與NHRI account 無關喔。
 

Best,

Su-Fang

---

Nov 6 2014

Daw-yang Hwang 黃道揚 to me 

 

Dear SF,
Below is the estimated cost of my fusion gene analysis:

A. The cost for known FGFR2 fusion gene detection is minimal:
1. primer (some cost,  < NTD 5000)
2. RNA extraction (no cost in NHRI)
3. RT-PCR (some cost)
4. Run gel for positive band
5. Sanger sequencing band in 4. (some cost)
Total cost should be less than NTD 10000

For unknwon TK fusion (including FGFR2-fusion, and all other TK candidates) (48 samples)
1. primer (no cost, since I am going to order these primers anyway)
2. RNA extraction (no cost in NHRI)
3. RT-double stranded cDNA (second stranded cDNA kit 10000)
4. Nextera XT library kit (~20000)
5. fusion specific PCR (Fluidigm plate 20 000)
6. Barcode PCR (free from my previous lab)
7. MiSeq (NTD 60 000)
Total cost for run 48 samples in all possible fusion genes is NTD 110,000, 96 samples will be NTD 160,000 (if still use one MiSeq kit)

For your references, the cost for RNA-Seq  (rRNA-depletion, minimal 10M read; we get 15M reads) is ~NTD 28000 (威健 OR 基米 has similar prices). 

Regards,
Daw-yang
 

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Nov 6 2014 

Su-Fang Lin  to Daw-yang Hwang. 

Thanks a lot for the valuable info! And sorry I had been in a meeting whole afternoon, in case you had called me. 

Best,
Su-Fang

Start: 11/05/2014

CTCF and HOXs

由 sufang 在 四, 11/20/2014 - 12:53 發表 EBV and KSHV CTCF

Mol Cell Biol. 2014 Oct;34(20):3867-79. doi: 10.1128/MCB.00567-14. Epub 2014 Aug 18.
CTCF controls HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure in NT2/D1 cells.
Xu M1, Zhao GN1, Lv X1, Liu G1, Wang LY2, Hao DL1, Wang J3, Liu DP4, Liang CC1.
Author information
Abstract

HOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5' HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites

由 sufang 在 四, 11/20/2014 - 13:20 發表 Pre-published IDO1 KYN

Remarkable Role of Indoleamine 2,3-Dioxygenase and Tryptophan Metabolites in Infectious Diseases: Potential Role in Macrophage-Mediated Inflammatory Diseases (PubMed) (pdf 4235)

Figure 1. Schematic overview of the kynurenine pathway. It is estimated that only 1% of dietary tryptophan (TRP) can be converted into serotonin (5-HT). The remaining 99% of TRP is metabolized via the kynurenine (KYN) pathway. Tryptophan hydroxylase (TH), 5-hydroxy TRP (5-HTP), N-acetylserotonin (NAS), indoleamine 2,3-dioxygenase 1 and 2 (IDO1/2), tryptophan 2,3-dioxygenase (TDO), kynurenine 3-monooxygenase (KMO), kynurenine aminotransferase (KAT I, II, III), kynurenic acid (KA), anthranilic acid (AA), 3-hydroxykynurenine (3-HK), xanthurenic acid (XA), 3-hydroxyanthranilic acid (3-HAA), 3-hydroxyanthranilic acid oxidase (3-HAO), quinolinic acid (QUIN), quinolinic-acid phosphoribosyl transferase (QPRT), nicotinic acid mononucleotide (NaMN), nicotinamide mononucleotide adenylyltransferase (NMNAT), nicotinamide adenine dinucleotide (NAD).

Figure 2: T-cell immune regulation by IDO1. (a) IDO1 is induced by IFN-γ-dependent and/or -independent signal pathways, depending on the variety of immune stimuli by macrophages and dendritic cells (DCs) [28, 83]. IDO1 activity is suppressed by the formation of NO or the competitive enzyme inhibitor, 1MT. Marked increases in IDO1 suppress immune responses by locally depleting L-TRP and preventing T-cell proliferation [44]. Expression of IDO1 has been observed in certain types of activated macrophages and DCs. IDO1-expressing cells deplete TRP from the extracellular milieu and secrete TRP metabolites, including KYN, 3-HK, 3-HAA, and QUIN, which induce T-cell apoptosis and suppress immune responses in vitro. (b) CD19+ plasmacytoid DCs (pDCs) express high levels of IDO1, which can activate mature regulatory T (Treg) cells via activation of the protein kinase general control nonderepressing-2 (GCN2) pathway of protein synthesis inhibition [84]. pDC-produced IDO1 and activated Treg can convert naïve T cells into new Treg. IDO1 acts in an autocrine manner to suppress pDC production of IL-6, which prevents the conversion of Treg into IL-17-producing Th17 proinflammatory cells [79]. IDO1 also downregulates type I IFN (IFN-α) production by pDC [80].

Figure 3: IDO1 induction and proinflammatory cytokines. IFN-γ drives intense enzymatic IDO1 activity, resulting in TRP depletion and high-level production of immunoregulatory TRP metabolites, KYNs, which may foster Tregs expansion. Induced Tregs use TGF-β to maintain an IDO1-dependent regulatory environment, with IDO1 mostly functioning as a signaling molecule. Both mechanisms are interrupted by IL-6, which drives IDO1 degradation as potent inflammatory stimuli enter the local environment [85].

Figure 4: The mechanism of IDO1 regulation on viral infectious diseases. Innate defense occurs when pathogens contact or invade host cells and elicit the production of cytokines and chemokines, which in turn induce an influx of immune cells that affect pathogen clearance. Type I IFNs are critical mediators of innate immunity and limit disease caused by many viruses [73, 86]. The enhancement of TRP breakdown by IDO1 regulates the signal pathway for IFNs production, and TRP metabolites might contribute to the function of IFNs producing cells.