HCV-HIV Coinfection: Pathogenesis of Accelerated Liver ...regist2.virology-education.com › 2015...

HCV-HIV Coinfection: Pathogenesis of Accelerated Liver Disease Progression

Raymond Chung, M.D. Director of Hepatology and Liver Center

Vice Chief, Gastroenterology Kevin and Polly Maroni Research Scholar

Massachusetts General Hospital

Disclosures

• Research Funding (to Institution, clinical trials) – Gilead – Abbvie – Merck – BMS – Mass Biologics

Liver disease is the second leading specific cause of death among HIV(+) persons

D:A:D study n=33,308, 180,000 p/y

Smith C, et al. AIDS. 2010;24:1537-48

Chart1

AIDS

Liver

CVD

Cancer (non-AIDS)

Column1

% of deaths

29.9

13.7

11.6

11.5

Sheet1

Column1

AIDS29.9

Liver13.7

CVD11.6

Cancer (non-AIDS)11.5

To resize chart data range, drag lower right corner of range.

HIV’s adverse effects on HCV

• Higher rates of persistence • Enhanced HCV replication • Accelerated fibrogenesis • Increased frequency of liver decompensation

and death • Until recently, diminished response rates to

antiviral therapy for HCV

Impact of HIV on HCV-related Liver Disease Progression

Systematic review of natural hx studies

Thein H-H et al. AIDS. 2008;22:1979-1991. Thein H-H et al. Hepatology. 2008;48:418-431.

Years

Cirr

hosi

s (%

)

0 5 10 15 20 25 30

RR of cirrhosis vs HCV HCV/HIV 2.1 - No HAART 2.5 - HAART 1.7

Chart1

000

00.1

2.52.5

89.9

1421.8

2535.9

3750

HCV (n = 33,121)

HIV/HCV (n = 3567)

Sheet1

HCV (n = 33,121)HIV/HCV (n = 3567)

000

500.1

102.52.5

1589.9

201421.8

252535.9

303750

Hepatic decompensation is accelerated in HCV-HIV coinfection (VA: 4280 HCV-HIV, 6079 HCV)

Lo Re et al. Ann Intern Med 2014; 160 Lo Re V et al. Annals Int Med 2014; 160:369

CD8+ CD4+

Cytokines (TNF-α, TGF-β, PDGF) Cell killing

Kupffer cell

Hepatocytes

Hepatic stellate cells

TGF-β Activation

FIBROSIS

Death

Hepatitis C disease pathogenesis

CD4+

Pathways to fibrogenesis

Friedman SL. Gastroenterology 2008;134:1655

Cytokines: TGFβ, PDGF, TNFα

HIV pathogenesis and interaction with HCV-related liver disease

Direct and indirect effects of HIV

Chen, Feeney, Chung. Nat Rev Gastro Hep 2014

Determinants of Pathogenesis in HCV-HIV Coinfection

• Effects on HCV-specific T cell responses • T cell independent effects of HIV on HCV

replication • Cooperative effects on

– TGF-β1 secretion

– Hepatic stellate cell activation and fibrogenesis

– Hepatic macrophage polarization

– Hepatocyte apoptosis

• Microbial translocation

HIV Effects on HCV T cell responses

HCV T cell responses in coinfection are blunted compared with HCV

monoinfection

Lauer et al. J Virol 2002 n=17 n=17

CD4+ Proliferative Responses to HCV are dependent on nadir CD4

(n=47)

Kim et al. PLoS Med. 2006

HIV effects on HCV replication

HIV gp120 directly enhances HCV replication in a full-length replicon

(OR6)

Lin et al. Gastroenterology. 2008 Mar;134:803

Chart1

20996.6466666667432084530521553.222277122807.9823419.676666666722204

PBS

CN gp120

SF gp120

Gag

Rev

Tat

Pol

Vif

Renilla Luciferase Unit

Protocol

Effect of HIV compounds on HCV replication and TGF-b1 #4-20075-Mar-07

Two dilutions for each compound: 1 ug/well=10ug/ml, 100 ng/well=1 ug/ml. PEG-IFN: 100 ng/ml and 10 ng/ml

OR6 cell (HCV replicon 1b co-expressing Renilla luciferase) grows in 80 ul G418/10 ml of DMEM 10% FBS for selection.

1. Innoculate 3,000 OR6 cell/well in 96-well plate for two plates on March 05, 2007 (Monday)3,000 cell/well=3,000 cell/100 ul=30,000 cell/ml

Need 0.1 ml X 96 well X 2 plate= 20 ml of 30,000 cell/ml=600,000 cells for 2 plates.

Prepare 30 ml of 30,000 cell/ml =0.9 million cell for each experiment ( 0.9 million cell/30 ml).

2. Aspirate culture supernatant on the next day (March 05,07), Add 100 ul of 10 ug/ml, or 1 ug/ml HIV protein to the appropriate well.

HIV compound stock solution (0.1-1 ug/ul)* Anu from BIDMC used 10 ng/ml (0.01 ng/ul) -1000 ng/ml (1 ng/ul) of gp120 for IL8 study.

Final 10 ug/mlFinal 1 ug/ml

Stock Conc (ug/ml)Stock ulAdd 10% FBS DMEM, ul1.8 ml DMEM +200 ul 10 ug/ml

SF (HIV-1 SF162 gp120, M-tropic)SF110010.91189.1200 ul of 10 ug/ml

Bal (HIV-1 Bal gp120, M-tropic)Bal100012.01188.0200 ul of 10 ug/ml

Gag (HIV-1 IIIB p55 Gag)Gag100012.01188.0200 ul of 10 ug/ml

Rev (HIV-1 Rev (Wild Type))Rev35034.31165.7200 ul of 10 ug/ml

CN (HIV-1 CN54 gp120)CN100012.01188.0200 ul of 10 ug/ml

CM (HIV-1 gp120 CM envelop protein)CM100120.01080.0200 ul of 10 ug/ml

Tat (HIV-1 Tat Protein), dissolved in 25 ul PBS-FBSTat100012.01188.0200 ul of 10 ug/ml

HIV-1HXB2 reverse Transcriptase-Cat#2897Pol100012.01188.0200 ul of 10 ug/ml

HIV-1 vif (Baculovirus)-Cat#11050Vif100012.01188.0200 ul of 10 ug/ml

HIV-196ZM651 gp120-Cat#10080ZM100012.01188.0200 ul of 10 ug/ml

HIV-1 RT (Protease derived)-Cat#3555RT3555100012.01188.0200 ul of 10 ug/ml

HIV-1 vif (E Coli)-Cat#11096V96100012.01188.0200 ul of 10 ug/ml

100 ul /wellOR6OR6OR6OR6OR6OR6OR6OR6OR6OR6OR6OR6

123456789101112

ASF, 1ug/wSF, 1ug/wSF, 1ug/wCN, 1ug/WCN, 1ug/WCN, 1ug/WPol, 1 ug/WPol, 1 ug/WPol, 1 ug/WRT3555, 1 ug/WRT3555, 1 ug/WRT3555, 1 ug/W

BSF, 0.1ug/WSF, 0.1ug/WSF, 0.1ug/WCN, 0.1ug/WCN, 0.1ug/WCN, 0.1ug/WPol, 0.1 ug/WPol, 0.1 ug/WPol, 0.1 ug/WRT3555, 0.1 ug/WRT3555, 0.1 ug/WRT3555, 0.1 ug/W

CBal, 1ug/WBal, 1ug/WBal, 1ug/WCM, 1ug/WCM, 1ug/WCM, 1ug/WVif, 1 ug/WVif, 1 ug/WVif, 1 ug/WV96, 1 ug/WV96, 1 ug/WV96, 1 ug/W

DBal,0.1ug/WBal,0.1ug/WBal,0.1ug/WCM,0.1ug/WCM,0.1ug/WCM,0.1ug/WVif, 0.1 ug/WVif, 0.1 ug/WVif, 0.1 ug/WV96, 0.1 ug/WV96, 0.1 ug/WV96, 0.1 ug/W

EGag, 1ug/WGag, 1ug/WGag, 1ug/WTat, 1ug/WTat, 1ug/WTat,1ug/W1:16 CXCR41:16 CXCR41:16 CXCR41:16 CCR51:16 CCR51:16 CCR5

FGag,0.1ug/WGag,0.1ug/WGag,0.1ug/WTat,0.1ug/WTat,0.1ug/WTat,0.1ug/W1:16 Neg Sup1:16 Neg Sup1:16 Neg SupDME10%FBSDME10%FBSDME10%FBS

GRev,1ug/WRev,1ug/WRev,1ug/W100 ng/ml IFN100 ng/ml IFN100 ng/ml IFNZM, 1ug/WZM, 1ug/WZM, 1ug/WCell onlyCell onlyCell only

HRev,0.1ug/WRev,0.1ug/WRev,0.1ug/W10 ng/ml IFN10 ng/ml IFN10 ng/ml IFNZM, 0.1ug/WZM, 0.1ug/WZM, 0.1ug/WCell onlyCell onlyCell only

Experiment Settings, two plates for 48 hrs-P#3 (Thurs, March 08), and 72 hrs-P#4 (Friday, March 09).

3. Harvest supernatant and transfer to another 96-well plate for storage (-80C) for TGF-b1 ELISA later.

4. Add 100 ul of 1X PBS for wash, gently swirl the plate. Aspirate the PBS.

5. Add 25 ul of 1X Renilla Lysis Buffer to each well, gentle shaking for 30 min at room temperature (need 7 strip X 8 well = 56 well X 20 ul=1120 ul, or 2 ml each time point).

6. 1X Renilla buffer:add 100 ul of 100 X Renilla substrate to 10 ml Renilla Buffer. It is OK to add another 10 ml of 1XPBS to save reagent.

7. Add 100 ul 1X Renilla Assay buffer to each well (12X8 well X 100 ul=9600 ul=9.6 ml for each time point).

8. It is better to add and measure one strip each time to reduce signal difference caused by reader by time delay.

&C&F

&R&N

Raw-D2

1st and 2nd readHIV proteins on HCV replication Day 28-Mar-07

123456789101112

A347351702819656182941355014587179601267914810149642053012613Lum

B30230202391581918473977358841044992931523282851285710584Lum

C167552152112953228914962831161969799129191538898036223Lum

D1634413601854610127147289110480613361107891038186075805Lum

E1322017264872611372102708198183531133213436626854807963Lum

F17426121206956160509666137541086614005117771342761905885Lum

G10794106819219143255326524091654800305873648736176Lum

H110311141712316162114241126071003011298183734393226495Lum

3rd read -whole plate

123456789101112

A1500580838870874657006669779654556521728883836802Lum

B1493497397482902646212425470643266206367258525294Lum

C79909330594511828331301778739196519725841013214Lum

D862469014587479962884407232474954997458832253060Lum

E6909947557766635628246841030966747667445536164186Lum

F926779354449915059488846689280756876826336543632Lum

G595973805417244138286254849693154173442066823216Lum

H70626734737713392201698564976920110572657615329Lum

4th read whole plate

123456789101112

A1284265746619705450775232661049315237576169925588Lum

B1053083865765640832972060366933704907291248293887Lum

C66617188400610287201075541234995172472529002552Lum

D560549093658366757402891163451374147368334422333Lum

E515472433140466749213718678248385071320922473405Lum

F710452622715674136016431445362594866547125713248Lum

G44534474418094235169161637382066127721608916300Lum

H5478534355961431066055884871526874571958910915Lum


123456789101112









D2Edit

1st and 2nd readHIV proteins on HCV replication Day 28-Mar-07

123Ave% Change456Ave% Change789Ave% Change101112Ave% Change

ASF, 1ug/w34735170281965623806180.0CN, 1ug/W1829413550145871547782.1Pol, 1 ug/W1796012679148101515078.2RT3555, 1 ug/W1496420530126131603688.6

BSF, 0.1ug/W30230202391581922096159.9CN, 0.1ug/W18473977358841137733.8Pol, 0.1 ug/W104499293152321165837.1RT3555, 0.1 ug/W828512857105841057524.4

CBal, 1ug/W16755215211295317076100.9CM, 1ug/W2289149628312205-74.1Vif, 1 ug/W161969799129191297152.6V96, 1 ug/W15388980362231047123.2

DBal,0.1ug/W163441360185461283050.9CM,0.1ug/W101271472891101132233.2Vif, 0.1 ug/W48061336110789965213.5V96, 0.1 ug/W10381860758058264-2.8

EGag, 1ug/W132201726487261307053.7Tat, 1ug/W11372102708198994717.01:16 CXCR41835311332134361437469.11:16 CCR56268548079636570-22.7

FGag,0.1ug/W174261212069561216743.1Tat,0.1ug/W160509666137541315754.81:16 Neg Sup1086614005117771221643.7DME10%FBS13427619058858501-0.0

GRev,1ug/W107941068192191023120.4100 ng/ml IFN143255326241-97.2ZM, 1ug/W5240916548006402-24.7Cell only30587364873617634417304.9

HRev,0.1ug/W1103111417123161158836.310 ng/ml IFN162114241172-98.0ZM, 0.1ug/W1260710030112981131233.1Cell only18373439322649529600248.2

The OR6 cells condition were not in the best condition, convert to neg signal at 21023

AveAve% ChangeSTDP

SF, 1ug/w17595172281765617493105.8SF, 1ug/w43460425534361043208106.3

Bal, 1ug/W16755215211295317076100.9Bal, 1ug/W41385531573199442179101.4

Gag, 1ug/W132201726487261307053.7Gag, 1ug/W326534264221553215533.2

Rev,1ug/W107941068192191023120.4Rev,1ug/W266612638222771227719.0

CN, 1ug/W18294175501742717757108.9CN, 1ug/W45186433494304543197106.3

CM,0.1ug/W101271472891101132233.2CM,0.1ug/W2501436378225022375813.7

Tat, 1ug/W11372102708198994717.0Tat, 1ug/W280892536720249228089.1

Pol, 1 ug/W1796012679148101515078.2Pol, 1 ug/W4436131317365813742078.7

Vif, 1 ug/W161969799129191297152.6Vif, 1 ug/W400042420431910222046.3

DME10%FBS13427619058858501-0.0DME10%FBS331651528914536209970.5




BSF, 0.1ug/W149349739748210718106.8CN, 0.1ug/W90264621242553573.4Pol, 0.1 ug/W4706432662065079-2.0RT3555, 0.1 ug/W3672585252944939-4.7

CBal, 1ug/W799093305945775549.6CM, 1ug/W118283313011105-78.7Vif, 1 ug/W778739196519607517.2V96, 1 ug/W7258410132144858-6.3

DBal,0.1ug/W862469014587670429.3CM,0.1ug/W4799628844075165-0.4Vif, 0.1 ug/W2324749549974939-4.7V96, 0.1 ug/W4588322530603624-30.1


FGag,0.1ug/W926779354449721739.2Tat,0.1ug/W915059488846798154.01:16 Neg Sup689280756876728140.5DME10%FBS82633654363251830.0



4th read -whole plate



BSF, 0.1ug/W10530838657658227118.6CN, 0.1ug/W64083297206039224.2Pol, 0.1 ug/W36693370490739825.8RT3555, 0.1 ug/W29124829388738763.0


DBal,0.1ug/W560549093658472425.5CM,0.1ug/W36675740289140998.9Vif, 0.1 ug/W1634513741473639-3.3V96, 0.1 ug/W3683344223333153-16.2


FGag,0.1ug/W710452622715502733.6Tat,0.1ug/W674136016431559148.61:16 Neg Sup445362594866519338.0DME10%FBS54712571324837630.0




123456789101112









D2-Graph

Effect of HIV compounds on HCV replication

48 hoursHIV concentration 1 ug/well = 10 ug/ml

%Ave% ChangeSTDp

13.6PBS20997-02856

12.8CN gp120432081065531

14.9SF gp120453051166750

12.5Gag2155332694

11.7Rev2277182664

14.3Tat2280893262

12.8Pol23420122998

14.2Vif2220463153

D2-Graph

Relative Luciferase Activity

HIV gp120 increase HCV replication in OR6 cells

Raw-D3

PBS

CN gp120

SF gp120

Gag

Rev

Tat

Pol

Vif


D3Edit

PBS

CN gp120

SF gp120

Gag

Rev

Tat

Pol

Vif


1st and 2nd readHIV protein on HCV replication Day 39-Mar-07

123456789101112

A713174674758185383024471961766393385480656895332453766826205Lum

B627323608735070233292408919954259482478933121200211467914475Lum

C372071766121899146417582057117081817935312205971676712844Lum

D461882442531477140112619413559207531025322072215373252214461Lum

E296072045518135220781589315224197593083227434174281494022164Lum

F396801987516058179592078217256320911928713843112462526922938Lum

G418544426222783468229466161921275514801621036409656978Lum

H283963981150036482217253262185293935715737468011344632Lum

3rd read whole plate

123456789101112

A347282365827934181642317130874189802596627797168111919911947Lum

B29919170441759311542118219815134441297215473968376066876Lum

C1871593911108689995711575811965918119982790965934Lum

D224001332016799782313463670296494906995710525151847413Lum

E1856714443109541359892981041811805184711706511549919513811Lum

F268391176398231207712267112921894011516882371891496015883Lum

G2551127143138362383453141131479818122365994003336997Lum

H183852372933675364164295165973171821160443384822926938Lum

4th read whole plate

123456789101112

A28090176512273515227160162291513405201482077813230137498973Lum

B24811131141332488209673790596201015112570719449815407Lum

C15316756585049847319104188731313697836168454550Lum

D16283111931263354051118554058024414777158540124745361Lum

E1351694297435934370616401810913246116258184703810091Lum

F1869279526728881594398498129157906605347131104110046Lum

G167781956710197373326268754954536392286772797826724Lum

H132331791422457310167219103872116315176309893363520304Lum

1st and 2nd readHIV protein on HCV replication Day 39-Mar-07



BSF, 0.1ug/W62732360873507044630125.2CN, 0.1ug/W2332924089199542245713.3Pol, 0.1 ug/W2594824789331212795341.0RT3555, 0.1 ug/W20021146791447516392-17.3


DBal,0.1ug/W4618824425314773403071.7CM,0.1ug/W14011261941355917921-9.6Vif, 0.1 ug/W20753102532207217693-10.7V96, 0.1 ug/W2153732522144612284015.2

EGag, 1ug/W2960720455181352273214.7Tat, 1ug/W22078158931522417732-10.51:16 CXCR41975930832274342600831.21:16 CCR517428149402216418177-8.3

FGag,0.1ug/W3968019875160582520427.2Tat,0.1ug/W17959207821725618666-5.81:16 Neg Sup320911928713843217409.7DME10%FBS11246252692293819818-0.0





ASF, 1ug/w34728236582793428773CN, 1ug/W18164231713087424070Pol, 1 ug/W18980259662779724248RT3555, 1 ug/W16811191991194715986

BSF, 0.1ug/W29919170441759321519CN, 0.1ug/W1154211821981511059Pol, 0.1 ug/W13444129721547313963RT3555, 0.1 ug/W9683760668768055

CBal, 1ug/W1871593911108613064CM, 1ug/W89995711571004Vif, 1 ug/W581196591811911196V96, 1 ug/W9827909659348286

DBal,0.1ug/W22400133201679917506CM,0.1ug/W78231346367029329Vif, 0.1 ug/W9649490699578171V96, 0.1 ug/W1052515184741311041

EGag, 1ug/W18567144431095414655Tat, 1ug/W13598929810418111051:16 CXCR4118051847117065157801:16 CCR51154991951381111518

FGag,0.1ug/W2683911763982316142Tat,0.1ug/W120771226711292118791:16 Neg Sup1894011516882313093DME10%FBS7189149601588312677

GRev,1ug/W25511271431383622163100 ng/ml IFN238345314299ZM, 1ug/W11314798181229139Cell only36599400333699737876

HRev,0.1ug/W1838523729336752526310 ng/ml IFN364164295274ZM, 0.1ug/W16597317182116023158Cell only44338482292693839835

4th read -whole plate


ASF, 1ug/w28090176512273522825CN, 1ug/W15227160162291518053Pol, 1 ug/W13405201482077818110RT3555, 1 ug/W1323013749897311984

BSF, 0.1ug/W24811131141332417083CN, 0.1ug/W8820967379058799Pol, 0.1 ug/W9620101511257010780RT3555, 0.1 ug/W7194498154075861

CBal, 1ug/W153167565850410462CM, 1ug/W984731910875Vif, 1 ug/W41887313136978399V96, 1 ug/W8361684545506585

DBal,0.1ug/W16283111931263313370CM,0.1ug/W54051118554057332Vif, 0.1 ug/W8024414777156629V96, 0.1 ug/W85401247453618792

EGag, 1ug/W135169429743510127Tat, 1ug/W93437061640176021:16 CXCR481091324611625109931:16 CCR581847038100918438

FGag,0.1ug/W186927952672811124Tat,0.1ug/W88159439849889171:16 Neg Sup12915790660538958DME10%FBS471311041100468600

GRev,1ug/W16778195671019715514100 ng/ml IFN373326268322ZM, 1ug/W7549545363926465Cell only28677279782672427793

HRev,0.1ug/W1323317914224571786810 ng/ml IFN310167219232ZM, 0.1ug/W10387211631517615575Cell only30989336352030428309


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HCV directly induces profibrogenic cytokines

Huh7.5.1

ROS Fluorescence

JFH1

ROS Fluorescence

HCV induces ROS formation and TGFβ in hepatocytes

Lin et al. Gastroenterology. 2010;138:2509-18

RO

S/C

ell V

iabi

lity

Arb

itrar

y U

nit

0

1

2

3

Huh7.5.1 JFH1

TGFβ

1/G

AP

DH

mR

NA

Arb

itrar

y U

nit

0

1

2

3

4

Huh7.5.1 JFH1

HCV induction of TGF-β1 is ROS-dependent and partially dependent on p38 MAPK, ERK and JNK


RO

S/C

ell V

iabi

lity

Arb

itrar

y U

nit

#

- + + + + - + + JFH1 HCV

0

1

2

3

DM

SO

DM

SO

DPI

SB –p38

SP- JNK

U0126-ER

K

LY – PI3K

TGF β

1/G

AP

DH

mR

NA

A

rbitr

ary

Uni

t β

1/G

AP

DH

mR

NA

A

rbitr

ary

Uni

t - + + + + - + + JFH1 HCV

0

1

2

3

4

DM

SO

DM

SO

DPI

SB – p38

SP-JNK

U0126 - ER

K

LY- PI3K

* * *

JNK

NFκB

p38KMAPK ERK

ROS

p38KMAPK

P P

JNK

P

ERK

P

NFκB

Nucleus

TGFβ1

P

NFκB

HCV

JNK

NFκBNFκB

p38KMAPK ERK

ROS

p38KMAPK

P

p38KMAPK

P P

JNK

P

JNK

P

ERK

P

ERK

P

NFκB

P

NFκBNFκB

Nucleus

TGFβ1TGFβ1

P

NFκB

P

NFκBNFκB

HCVHCV

Model of events underlying HCV induction of TGF-β1 production in hepatocytes


HCV and HIV exert cooperative effects on TGF-β1 secretion

HIV or gp120 increase TGF-β secretion in JFH1-infected or uninfected Huh7.5.1 cells

TGF-

β1 C

once

ntra

tion

(pg/

ml)

Lin et al. Gastroenterology. 2008;134:803

Chart1

Serum FreeSerum Free

CXCR4 tropic HIVCXCR4 tropic HIV

CCR5 tropic HIVCCR5 tropic HIV

Negative HIVNegative HIV

SF gp120SF gp120

CN gp120CN gp120

GagGag

RevRev

TatTat

PolPol

VifVif

Huh7.5.1

JFH1

397.6

1593.2

985.6

2996

1002.4

2231.6

546

1626.8

1013.6

2996

1078

2965.2

372.4

1520.4

316.4

1570.8

324.8

1503.6

358.4

1464.4

302.4

1517.6

HIV protein &virus

Effect of HIV compounds on HCV replication and TGF-b1 #5-200716-Apr-07

One dilutions for each compound: 1 ug/well=10ug/ml, two for HIV virus.Use Serum Free medium for TGF-b1 Study

OR6 cell (HCV replicon 1b co-expressing Renilla luciferase) grows in 80 ul G418/10 ml of Serum Free medium for selection.

Inoculate JFH1 to Huh7.5.1 April 12, 07, grow JFH1and Hu7.5.1 in serum free medium

1. Innoculate 6,000 cell/well OR6, Cured OR6, Huh7.5.1(3000/well), or JFH1 (D4) cells in 96-well plate for two plates on April 16, 2007 (Monday)

Need 0.1 ml X 96 well X 0.5 plate= 5 ml of 60,000 cell/ml=300,000 cells for half plate/each cell.6,000 cell/well=6,000 cell/100 ul=60,000 cell/ml


2. Aspirate culture supernatant on the next day (April 17,07), Add 100 ul of 10 ug/ml HIV protein or HIV supernatant to the appropriate well.

Final 10 ug/mlOR6 cell+G418, 400 ul needAll other cells, 1100 ul

Stock Conc (ug/ml)Stock ulAdd Serum Free medium, ulStock ulAdd SerumFree medium,ul

SF (HIV-1 SF162 gp120, M-tropic)SF11003.6396.410.01090.0

Bal (HIV-1 Bal gp120, M-tropic)Bal10004.0396.011.01089.0

Gag (HIV-1 IIIB p55 Gag)Gag10004.0396.011.01089.0

Rev (HIV-1 Rev (Wild Type))Rev35011.4388.631.41068.6

CN (HIV-1 CN54 gp120)CN10004.0396.011.01089.0

Tat (HIV-1 Tat Protein), dissolved in 25 ul PBS-FBSTat10004.0396.011.01089.0

HIV-1HXB2 reverse Transcriptase-Cat#2897Pol10004.0396.011.01089.0

HIV-1 vif (Baculovirus)-Cat#11050Vif10004.0396.011.01089.0

1:16 HIV Supernatant: 125 ul HIV + 1875 ul Serum Free; 1:64 HIV: 31.25 HIV + 1968.75 ul Serum FreeOR6:25 ul +375 ul G418 (1:16); 6.25 ul + 393.75 G418(1:64)

Plate #1OR6OR6OR6OR6OR6OR6Cured OR6Cured OR6Cured OR6Cured OR6Cured OR6Cured OR6

Plate #2JFH1JFH1JFH1JFH1JFH1JFH1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1

123456789101112

ASF, 1ug/wSF, 1ug/wSF, 1ug/w1:16 CXCR41:16 CXCR41:16 CXCR4SF, 1ug/wSF, 1ug/wSF, 1ug/w1:16 CXCR41:16 CXCR41:16 CXCR4

BBal, 1ug/WBal, 1ug/WBal, 1ug/W1:64 CXCR41:64 CXCR41:64 CXCR4Bal, 1ug/WBal, 1ug/WBal, 1ug/W1:64 CXCR41:64 CXCR41:64 CXCR4

CGag, 1ug/WGag, 1ug/WGag, 1ug/W1:16 CCR51:16 CCR51:16 CCR5Gag, 1ug/WGag, 1ug/WGag, 1ug/W1:16 CCR51:16 CCR51:16 CCR5

DRev,1ug/WRev,1ug/WRev,1ug/W1:64 CCR51:64 CCR51:64 CCR5Rev,1ug/WRev,1ug/WRev,1ug/W1:64 CCR51:64 CCR51:64 CCR5

ECN, 1ug/WCN, 1ug/WCN, 1ug/W1:16 Neg Sup1:16 Neg Sup1:16 Neg SupCN, 1ug/WCN, 1ug/WCN, 1ug/W1:16 Neg Sup1:16 Neg Sup1:16 Neg Sup

FTat, 1ug/WTat, 1ug/WTat,1ug/WDME10%FBSDME10%FBSDME10%FBSTat, 1ug/WTat, 1ug/WTat,1ug/WDME10%FBSDME10%FBSDME10%FBS

GPol, 1 ug/WPol, 1 ug/WPol, 1 ug/WSerum FreeSerum FreeSerum FreePol, 1 ug/WPol, 1 ug/WPol, 1 ug/WSerum FreeSerum FreeSerum Free

HVif, 1 ug/WVif, 1 ug/WVif, 1 ug/WCell onlyCell onlyCell onlyVif, 1 ug/WVif, 1 ug/WVif, 1 ug/WCell onlyCell onlyCell only

Experiment Settings, two plates for 48 hrs- OR6 (Thurs, April 19), and 72 hrs-JFH1 cells (Day3).



5. For Plate #1, OR6 cells, Add 25 ul of 1X Renilla Lysis Buffer to each well, gentle shaking for 30 min at room temperature

(need 7 strip X 8 well = 56 well X 20 ul=1120 ul, or 2 ml each time point).




9. For plate #1 cured OR6 cells and Plate #2, harvest protein lysates 100 ul/well for HCV core ELISA or STAT1 Western.

Renilla Data

Serum Free OR6 with HIV virus gp120 protein- 6000 OR6 cell/well

Day2 after incubation, 04/19/2007adjust to 21318 for 3000 ce// Well for 2 days.

Ave

123SF, 1ug/wAdjusted% increase4561:16 CXCR4Adjusted% increase

A15092412825014748414920431930995561348367154614483630995

Bal, 1ug/W1:64 CXCR4

B158808121613530641402113000512102410638213496013496028881

Gag, 1ug/W1:16 CCR5

C1318211040959830810120221657706931095168193912951627716

Rev,1ug/W1:64 CCR5

D1222268546067082915891960011294694157859449768220904

CN, 1ug/W1:16 Neg Sup

E1748351186251573101502573215592329174708882119027019318

Tat, 1ug/WDME10%FBS

F1262416182367292851191821562867106215965819658120668

Pol, 1 ug/WSerum FreeAdjusted factor

G1519676326311736690315193271387179974116092199741213180

0.214

Vif, 1 ug/WCell only

H1234391694671901381034392213630204028563534984031250566876

Inactivated HIV virus and gp120 greatly increase HCV replication in OR6 cells in serum free medium

Serum FreeCXCR4 tropiv HIVCCR5 tropic HIVNeg HIV supernatantSF gp120CN gp120GagRevTatPolVif

2131830995277161931831930321552165719600182151932721636

Renilla Data

Reinilla Relative Unit

ELISA Setting

23-Apr-07TGF-b1 ELISA#3 -Quantikine Assay SettingsWenyu

O.D. at 450 nm, with the correction wavelength set at 540 nm or 570 nm

123456789101112

AST8SF, OR61:16 CX4, OR6SF, CuredOR61:16 CX4,Cured6SF, JFH11:16 CX4,JFH1SF, Hu7.5.11:16 CX4,Huh7SerFreMedi

BST7Bal, OR61:64 CX4,OR6Bal, Cured1:64 CX4,Cured6Bal,JFH11:64 CX4,JFH1Bal, Huh7.5.11:64 CX4,Huh7Petri-OR6

CST6Gag, OR61:16 CR5,OR6Gag, CuredR61:16 CR5,Cured6Gag, JFH11:16 CR5,JFH1Gag, Huh7.5.11:16 CR5,Huh7Petri-CuredOR6

DST5Rev, OR61:64 CR5,OR6Rev, CuredR61:64CR5,Cured6Rev, JFH11:64 CR5,JFH1Rev, Huh7.5.11:64 CR5,Huh7Petri-JFh1,D11

EST4CN, OR61:16 Neg Sup,OR6CN, CuredR610% FBS/Neg S CuredCN, JFH11:16 Neg Sup,JFH1CN, Huh7.5.11:16NegSup,HuhPetri-Huh7

FST3Tat, OR610%FBS, OR6Tat, CuredR6Ser Free,Cured6Tat,JFH110%FBS,JFH1Tat, Huh7.5.110%FBS,Huh7old JFH1-D11

GST2Pol, OR6Ser Free,OR6Pol, CuredR6100 ul 5E mixPol, JFH1Ser Free,JFH1Pol, Huh7.5.1Ser Free,Huh7Filter JFH1-D8

HST1Vif, OR6Cell only,OR6Vif, CuredR6Cell only,Cured6Vif, JFH1Cell only,JFH1Vif, Huh7.5.1Cell only,Huh7less 10%FBS Cured

To make 8 ml of 1X Diluent RD5-26: 2 ml of RD5-26 Concentrate + 6 ml of dH2O.

1. TGFb1-Stock-ST1: add 2 ml of 1X RD5-26 to TGF-b1 Standard to make 2000 pg/ml. Mix to ensure complete reconstitution and sit for 5 min.

StandardST1-StockST2ST3ST4ST5ST6ST7ST8make 100 ul/test, use 50 ul

pg/ml2000100050025012562.531.250

Diluted RD5-26 (1X), ul0100100100100100100100

100 ul ofn/aST1-StockST2ST3ST4ST5ST6ST7

2. For TGF-b1 activation:

For Supernatant: 60 ul of supernate + 12 ul of 1 N HCL incubate 10 min at room temperature, then add 12 ul of 1.2 N NaOH/0.5 M Hepes for neutralization (84 ul total).

Actually used: (60/84) x 50 ul=35 ul used

3. Add 50 ul of Assay Diluent RD1-21 to each well.

4. Add 50 ul of ST8-1, and activated sample to the appropriate well, cover and incubate at Room temp for 2 hours.

5. Aspirate each well and wash for 4 times, invert the plate and blot it against clean paper towels.

6. Add 100 ul of TGF-b1 Conjugate to each well, cover and incubate at room temp for 2 hours.

7. Wash for 4 times.

8. Add 100 ul of Substrate Solution to each well, incubate for 30 min at room temperature. Protect from light.

9. Add 100 ul of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.

10. Read the OD within 30 min. Reader setting: 450 nm, correction set to 540. If not correction subtraction OD at (450 nm) by OD at 540 nm or 570 nm.

OD

123456789101112

A0.2370.430.620.4760.6260.4380.5610.320.5270.201??????????Corrected [450]

B0.3840.5910.4260.5380.4260.4650.3190.3410.3190.29??????????Corrected [450]

C0.5040.4030.5590.3940.590.3240.4870.2540.4970.548??????????Corrected [450]

D0.7640.390.40.4530.540.3280.3120.2290.3341.206??????????Corrected [450]

E1.1980.5380.5340.3792.0190.440.440.3430.5361.544??????????Corrected [450]

F1.8520.3871.9780.320.4020.3452.0280.2572.1011.815??????????Corrected [450]

G3.1360.4070.370.2891.0020.3330.2880.2530.3011.848??????????Corrected [450]

H4.550.4190.7180.4961.0310.3910.5420.40.4961.469??????????Corrected [450]

123456789101112

A0.1130.2030.2930.2250.2990.2140.2650.1530.2520.09600450

B0.1790.3150.2010.2510.2010.2170.1490.1590.1490.13700450

C0.2350.1860.2610.1860.2720.1530.230.120.2320.25900450

D0.3610.1840.1890.2140.2580.1550.1490.1080.1560.55600450

E0.5590.2570.2490.1790.8750.2080.210.1620.2530.71200450

F0.8540.1850.9010.1510.190.1630.9240.120.9570.83700450

G1.4460.1920.1770.1380.4730.1590.1390.1210.1440.85200450

H2.0220.20.3390.2340.4810.1870.2530.180.2340.66900450

123456789101112

A0.0390.040.0420.0410.0420.0430.0410.040.0410.03900540

B0.040.1320.040.0470.040.0490.0390.040.0410.0400540

C0.040.040.0430.040.0410.040.0410.0380.0410.04100540

D0.0430.0440.0410.0410.0610.040.0410.0390.040.04400540

E0.0460.0420.0440.0420.0650.0410.040.0390.040.04700540

F0.0490.0420.060.040.040.040.060.0420.0490.04700540

G0.0560.0410.0410.0390.0430.0440.0390.040.0390.04800540

H0.0640.040.0430.0420.0440.0440.0490.0770.0520.04600540

ED Data

29-Jan-07TGF-b1 ELISA -Quantikine AssayWenyu


123456789101112

A0.2370.430.620.4760.6260.4380.5610.320.5270.201

B0.3840.5910.4260.5380.4260.4650.3190.3410.3190.29

C0.5040.4030.5590.3940.590.3240.4870.2540.4970.548

D0.7640.390.40.4530.540.3280.3120.2290.3341.206

E1.1980.5380.5340.3792.0190.440.440.3430.5361.544

F1.8520.3871.9780.320.4020.3452.0280.2572.1011.815

G3.1360.4070.370.2891.0020.3330.2880.2530.3011.848

H4.550.4190.7180.4961.0310.3910.5420.40.4961.469

OD 450 nmpg/ml

ST800

ST70.14731.25

ST60.26762.5

ST50.527125

ST40.961250

ST31.615500

ST22.8991000

ST14.3132000

The test sample TGF-b1 Concentration: pg/ml

123456789101112

A5.68891.410175.800111.842178.46594.964149.59542.553134.494-10.302-99.578-99.578

B70.979162.92089.634139.37989.634106.95642.10951.88042.10929.228-99.578-99.578

C124.27879.418148.70775.421162.47644.330116.72713.239121.169143.821-99.578-99.578

D239.75973.64478.086101.626140.26846.10639.0002.13548.771436.077-99.578-99.578

E432.524139.379137.60368.758797.17795.85295.85252.769138.491586.202-99.578-99.578

F723.00372.312778.96742.55378.97453.657801.17514.571833.598706.569-99.578-99.578

G1293.30281.19564.76128.784345.46948.32728.34012.79434.114721.226-99.578-99.578

H1921.34186.525219.328120.725358.34974.088141.15678.086120.725552.890-99.578-99.578

12345678910


5.791.4175.8111.8178.595.0149.642.6134.5-10.3


71.0162.989.6139.489.6107.042.151.942.129.2


124.379.4148.775.4162.544.3116.713.2121.2143.8


239.873.678.1101.6140.346.139.02.148.8436.1


432.5139.4137.668.8797.295.995.952.8138.5586.2


723.072.3779.042.679.053.7801.214.6833.6706.6


1293.381.264.828.8345.548.328.312.834.1721.2


1921.386.5219.3120.7358.374.1141.278.1120.7552.9

&F

Page &P

ED Data

TGFb1-Standard Curve

Analy

TGF-b1 ELISA -Quantikine AssayTGF-b1 concentration: pg/ml

Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV replicon in serum free medium


OR664.8129.698.167.6132.9139.479.473.672.371.272.5

Cured OR641.279.669.145.364.268.845.444.639.241.645.2

% OR6/Cured57.362.842.049.2107.0102.674.965.084.471.260.4

Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV replicon


OR664.8129.698.167.6132.9139.479.473.672.371.272.5

% to serum free Cured OR657.3214.6138.164.1222.6238.392.778.675.572.876.0

Inactivated HIV virus and gp120 moderately increase TGF-b1 expression in cured OR6 cells


Cured OR641.279.669.145.364.268.845.444.639.241.645.2

% to serum free Cured OR60.093.267.710.055.867.010.28.3-4.91.09.7


Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV JFH1 cells in serum free medium

Serum FreeCXCR4 tropiv HIVCCR5 tropic HIVNegative HIVSF gp120CN gp120GagRevTatPolVif

JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

Huh7.5.134.155.245.839.552.653.832.331.330.631.834.2

% of JFH1/Hu7.5.166.993.874.047.1103.496.868.179.275.564.558.5

Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV JFH1 cells


JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

% to serum free Huh7.5.166.9213.8133.770.4213.8210.659.264.557.553.458.9

Inactivated HIV virus and gp120 moderately increase TGF-b1 expression in Hu7.5.1 cells


Huh7.5.134.155.245.839.552.653.832.331.330.631.834.2

% to serum free Huh7.5.10.061.934.315.854.357.8-5.3-8.2-10.3-6.70.3Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV JFH1 cells in serum free medium

35 ul of TGFb1 convert to 1000 ul =1000/35.7=28

Analy

OR6

Cured OR6

TGF-b1 (pg/ml)

Convert

JFH1

Huh7.5.1

Serum Free

CXCR4 tropiv HIV

CCR5 tropic HIV

Negative HIV

SF gp120

CN gp120

Gag

Rev

Tat

Pol

Vif

TGF-b1 ELISA -Quantikine Assay

35 ul of TGFb1 convert to 1000 ul =1000/35.7=28TGF-b1 concentration: pg/ml



Cured OR612.249.646.115.344.248.813.514.614.512.910.5

OR664.8129.698.167.6132.9139.479.473.672.371.272.5

% OR6/Cured431.1161.3112.8341.8200.7185.7488.1404.1398.6451.9590.5

Converted to * 28


Serum FreeCXCR4 tropic HIVCCR5 tropic HIVNeg HIV supernatantSF gp120CN gp120GagRevTatPolVif

Cured OR6341.61388.81290.8428.41237.61366.4378.0408.8406.0361.2294.0

OR61814.43628.82746.81892.83721.23903.22223.22060.82024.41993.62030.0

Cured OR6 STD ±17.787.486.421.889.098.420.421.520.921.719.6

OR6 STD ±114.3268.4170.2115.5266.1287.6128.9130.2132.7118.7125.6




Huh7.5.114.235.235.819.536.238.513.311.311.612.810.8

JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

% of JFH1/Hu7.5.1300.7204.0122.6197.9195.6175.1308.3396.5362.9308.6401.9

Converted *28

Serum FreeCXCR4 tropic HIVCCR5 tropic HIVNegative HIVSF gp120CN gp120GagRevTatPolVif

Huh7.5.1397.6985.61002.4546.01013.61078.0372.4316.4324.8358.4302.4

JFH11593.22996.02231.61626.82996.02965.21520.41570.81503.61464.41517.6

Huh7.5.1 STD ±21.460.069.133.258.756.421.419.820.724.321.9

JFH1 STD ±105.2209.7165.1110.6193.8189.3103.4112.8123.5101.5118.7



Huh7.5.1

JFH1

Huh7.5.1

JFH1

Cured OR6

OR6

HIV or gp120 increase TGFβ secretion in JFH1-infected or uninfected Huh7.5.1 cells

TGF-

β1 C

once

ntra

tion

(pg/

ml)

Lin et al. Gastroenterology. 2008;134:803

This enhancement of TGFβ secretion is also ROS-dependent

Chart1

Serum FreeSerum Free

CXCR4 tropic HIVCXCR4 tropic HIV

CCR5 tropic HIVCCR5 tropic HIV

Negative HIVNegative HIV

SF gp120SF gp120

CN gp120CN gp120

GagGag

RevRev

TatTat

PolPol

VifVif

Huh7.5.1

JFH1

397.6

1593.2

985.6

2996

1002.4

2231.6

546

1626.8

1013.6

2996

1078

2965.2

372.4

1520.4

316.4

1570.8

324.8

1503.6

358.4

1464.4

302.4

1517.6

HIV protein &virus

Effect of HIV compounds on HCV replication and TGF-b1 #5-200716-Apr-07

One dilutions for each compound: 1 ug/well=10ug/ml, two for HIV virus.Use Serum Free medium for TGF-b1 Study

OR6 cell (HCV replicon 1b co-expressing Renilla luciferase) grows in 80 ul G418/10 ml of Serum Free medium for selection.

Inoculate JFH1 to Huh7.5.1 April 12, 07, grow JFH1and Hu7.5.1 in serum free medium

1. Innoculate 6,000 cell/well OR6, Cured OR6, Huh7.5.1(3000/well), or JFH1 (D4) cells in 96-well plate for two plates on April 16, 2007 (Monday)

Need 0.1 ml X 96 well X 0.5 plate= 5 ml of 60,000 cell/ml=300,000 cells for half plate/each cell.6,000 cell/well=6,000 cell/100 ul=60,000 cell/ml


2. Aspirate culture supernatant on the next day (April 17,07), Add 100 ul of 10 ug/ml HIV protein or HIV supernatant to the appropriate well.

Final 10 ug/mlOR6 cell+G418, 400 ul needAll other cells, 1100 ul

Stock Conc (ug/ml)Stock ulAdd Serum Free medium, ulStock ulAdd SerumFree medium,ul

SF (HIV-1 SF162 gp120, M-tropic)SF11003.6396.410.01090.0

Bal (HIV-1 Bal gp120, M-tropic)Bal10004.0396.011.01089.0

Gag (HIV-1 IIIB p55 Gag)Gag10004.0396.011.01089.0

Rev (HIV-1 Rev (Wild Type))Rev35011.4388.631.41068.6

CN (HIV-1 CN54 gp120)CN10004.0396.011.01089.0

Tat (HIV-1 Tat Protein), dissolved in 25 ul PBS-FBSTat10004.0396.011.01089.0

HIV-1HXB2 reverse Transcriptase-Cat#2897Pol10004.0396.011.01089.0

HIV-1 vif (Baculovirus)-Cat#11050Vif10004.0396.011.01089.0

1:16 HIV Supernatant: 125 ul HIV + 1875 ul Serum Free; 1:64 HIV: 31.25 HIV + 1968.75 ul Serum FreeOR6:25 ul +375 ul G418 (1:16); 6.25 ul + 393.75 G418(1:64)

Plate #1OR6OR6OR6OR6OR6OR6Cured OR6Cured OR6Cured OR6Cured OR6Cured OR6Cured OR6

Plate #2JFH1JFH1JFH1JFH1JFH1JFH1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1Huh7.5.1

123456789101112

ASF, 1ug/wSF, 1ug/wSF, 1ug/w1:16 CXCR41:16 CXCR41:16 CXCR4SF, 1ug/wSF, 1ug/wSF, 1ug/w1:16 CXCR41:16 CXCR41:16 CXCR4

BBal, 1ug/WBal, 1ug/WBal, 1ug/W1:64 CXCR41:64 CXCR41:64 CXCR4Bal, 1ug/WBal, 1ug/WBal, 1ug/W1:64 CXCR41:64 CXCR41:64 CXCR4

CGag, 1ug/WGag, 1ug/WGag, 1ug/W1:16 CCR51:16 CCR51:16 CCR5Gag, 1ug/WGag, 1ug/WGag, 1ug/W1:16 CCR51:16 CCR51:16 CCR5

DRev,1ug/WRev,1ug/WRev,1ug/W1:64 CCR51:64 CCR51:64 CCR5Rev,1ug/WRev,1ug/WRev,1ug/W1:64 CCR51:64 CCR51:64 CCR5

ECN, 1ug/WCN, 1ug/WCN, 1ug/W1:16 Neg Sup1:16 Neg Sup1:16 Neg SupCN, 1ug/WCN, 1ug/WCN, 1ug/W1:16 Neg Sup1:16 Neg Sup1:16 Neg Sup

FTat, 1ug/WTat, 1ug/WTat,1ug/WDME10%FBSDME10%FBSDME10%FBSTat, 1ug/WTat, 1ug/WTat,1ug/WDME10%FBSDME10%FBSDME10%FBS

GPol, 1 ug/WPol, 1 ug/WPol, 1 ug/WSerum FreeSerum FreeSerum FreePol, 1 ug/WPol, 1 ug/WPol, 1 ug/WSerum FreeSerum FreeSerum Free

HVif, 1 ug/WVif, 1 ug/WVif, 1 ug/WCell onlyCell onlyCell onlyVif, 1 ug/WVif, 1 ug/WVif, 1 ug/WCell onlyCell onlyCell only

Experiment Settings, two plates for 48 hrs- OR6 (Thurs, April 19), and 72 hrs-JFH1 cells (Day3).



5. For Plate #1, OR6 cells, Add 25 ul of 1X Renilla Lysis Buffer to each well, gentle shaking for 30 min at room temperature

(need 7 strip X 8 well = 56 well X 20 ul=1120 ul, or 2 ml each time point).




9. For plate #1 cured OR6 cells and Plate #2, harvest protein lysates 100 ul/well for HCV core ELISA or STAT1 Western.

Renilla Data

Serum Free OR6 with HIV virus gp120 protein- 6000 OR6 cell/well

Day2 after incubation, 04/19/2007adjust to 21318 for 3000 ce// Well for 2 days.

Ave

123SF, 1ug/wAdjusted% increase4561:16 CXCR4Adjusted% increase

A15092412825014748414920431930995561348367154614483630995

Bal, 1ug/W1:64 CXCR4

B158808121613530641402113000512102410638213496013496028881

Gag, 1ug/W1:16 CCR5

C1318211040959830810120221657706931095168193912951627716

Rev,1ug/W1:64 CCR5

D1222268546067082915891960011294694157859449768220904

CN, 1ug/W1:16 Neg Sup

E1748351186251573101502573215592329174708882119027019318

Tat, 1ug/WDME10%FBS

F1262416182367292851191821562867106215965819658120668

Pol, 1 ug/WSerum FreeAdjusted factor

G1519676326311736690315193271387179974116092199741213180

0.214

Vif, 1 ug/WCell only

H1234391694671901381034392213630204028563534984031250566876

Inactivated HIV virus and gp120 greatly increase HCV replication in OR6 cells in serum free medium


2131830995277161931831930321552165719600182151932721636

Renilla Data

Reinilla Relative Unit

ELISA Setting

23-Apr-07TGF-b1 ELISA#3 -Quantikine Assay SettingsWenyu


123456789101112









To make 8 ml of 1X Diluent RD5-26: 2 ml of RD5-26 Concentrate + 6 ml of dH2O.

1. TGFb1-Stock-ST1: add 2 ml of 1X RD5-26 to TGF-b1 Standard to make 2000 pg/ml. Mix to ensure complete reconstitution and sit for 5 min.

StandardST1-StockST2ST3ST4ST5ST6ST7ST8make 100 ul/test, use 50 ul

pg/ml2000100050025012562.531.250

Diluted RD5-26 (1X), ul0100100100100100100100

100 ul ofn/aST1-StockST2ST3ST4ST5ST6ST7

2. For TGF-b1 activation:

For Supernatant: 60 ul of supernate + 12 ul of 1 N HCL incubate 10 min at room temperature, then add 12 ul of 1.2 N NaOH/0.5 M Hepes for neutralization (84 ul total).

Actually used: (60/84) x 50 ul=35 ul used

3. Add 50 ul of Assay Diluent RD1-21 to each well.

4. Add 50 ul of ST8-1, and activated sample to the appropriate well, cover and incubate at Room temp for 2 hours.

5. Aspirate each well and wash for 4 times, invert the plate and blot it against clean paper towels.

6. Add 100 ul of TGF-b1 Conjugate to each well, cover and incubate at room temp for 2 hours.

7. Wash for 4 times.

8. Add 100 ul of Substrate Solution to each well, incubate for 30 min at room temperature. Protect from light.

9. Add 100 ul of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.

10. Read the OD within 30 min. Reader setting: 450 nm, correction set to 540. If not correction subtraction OD at (450 nm) by OD at 540 nm or 570 nm.

OD

123456789101112

A0.2370.430.620.4760.6260.4380.5610.320.5270.201??????????Corrected [450]

B0.3840.5910.4260.5380.4260.4650.3190.3410.3190.29??????????Corrected [450]

C0.5040.4030.5590.3940.590.3240.4870.2540.4970.548??????????Corrected [450]

D0.7640.390.40.4530.540.3280.3120.2290.3341.206??????????Corrected [450]

E1.1980.5380.5340.3792.0190.440.440.3430.5361.544??????????Corrected [450]

F1.8520.3871.9780.320.4020.3452.0280.2572.1011.815??????????Corrected [450]

G3.1360.4070.370.2891.0020.3330.2880.2530.3011.848??????????Corrected [450]

H4.550.4190.7180.4961.0310.3910.5420.40.4961.469??????????Corrected [450]

123456789101112

A0.1130.2030.2930.2250.2990.2140.2650.1530.2520.09600450

B0.1790.3150.2010.2510.2010.2170.1490.1590.1490.13700450

C0.2350.1860.2610.1860.2720.1530.230.120.2320.25900450

D0.3610.1840.1890.2140.2580.1550.1490.1080.1560.55600450

E0.5590.2570.2490.1790.8750.2080.210.1620.2530.71200450

F0.8540.1850.9010.1510.190.1630.9240.120.9570.83700450

G1.4460.1920.1770.1380.4730.1590.1390.1210.1440.85200450

H2.0220.20.3390.2340.4810.1870.2530.180.2340.66900450

123456789101112

A0.0390.040.0420.0410.0420.0430.0410.040.0410.03900540

B0.040.1320.040.0470.040.0490.0390.040.0410.0400540

C0.040.040.0430.040.0410.040.0410.0380.0410.04100540

D0.0430.0440.0410.0410.0610.040.0410.0390.040.04400540

E0.0460.0420.0440.0420.0650.0410.040.0390.040.04700540

F0.0490.0420.060.040.040.040.060.0420.0490.04700540

G0.0560.0410.0410.0390.0430.0440.0390.040.0390.04800540

H0.0640.040.0430.0420.0440.0440.0490.0770.0520.04600540

ED Data

29-Jan-07TGF-b1 ELISA -Quantikine AssayWenyu


123456789101112

A0.2370.430.620.4760.6260.4380.5610.320.5270.201

B0.3840.5910.4260.5380.4260.4650.3190.3410.3190.29

C0.5040.4030.5590.3940.590.3240.4870.2540.4970.548

D0.7640.390.40.4530.540.3280.3120.2290.3341.206

E1.1980.5380.5340.3792.0190.440.440.3430.5361.544

F1.8520.3871.9780.320.4020.3452.0280.2572.1011.815

G3.1360.4070.370.2891.0020.3330.2880.2530.3011.848

H4.550.4190.7180.4961.0310.3910.5420.40.4961.469

OD 450 nmpg/ml

ST800

ST70.14731.25

ST60.26762.5

ST50.527125

ST40.961250

ST31.615500

ST22.8991000

ST14.3132000

The test sample TGF-b1 Concentration: pg/ml

123456789101112

A5.68891.410175.800111.842178.46594.964149.59542.553134.494-10.302-99.578-99.578

B70.979162.92089.634139.37989.634106.95642.10951.88042.10929.228-99.578-99.578

C124.27879.418148.70775.421162.47644.330116.72713.239121.169143.821-99.578-99.578

D239.75973.64478.086101.626140.26846.10639.0002.13548.771436.077-99.578-99.578

E432.524139.379137.60368.758797.17795.85295.85252.769138.491586.202-99.578-99.578

F723.00372.312778.96742.55378.97453.657801.17514.571833.598706.569-99.578-99.578

G1293.30281.19564.76128.784345.46948.32728.34012.79434.114721.226-99.578-99.578

H1921.34186.525219.328120.725358.34974.088141.15678.086120.725552.890-99.578-99.578

12345678910


5.791.4175.8111.8178.595.0149.642.6134.5-10.3


71.0162.989.6139.489.6107.042.151.942.129.2


124.379.4148.775.4162.544.3116.713.2121.2143.8


239.873.678.1101.6140.346.139.02.148.8436.1


432.5139.4137.668.8797.295.995.952.8138.5586.2


723.072.3779.042.679.053.7801.214.6833.6706.6


1293.381.264.828.8345.548.328.312.834.1721.2


1921.386.5219.3120.7358.374.1141.278.1120.7552.9

&F

Page &P

ED Data

TGFb1-Standard Curve

Analy

TGF-b1 ELISA -Quantikine AssayTGF-b1 concentration: pg/ml



OR664.8129.698.167.6132.9139.479.473.672.371.272.5

Cured OR641.279.669.145.364.268.845.444.639.241.645.2

% OR6/Cured57.362.842.049.2107.0102.674.965.084.471.260.4

Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV replicon


OR664.8129.698.167.6132.9139.479.473.672.371.272.5

% to serum free Cured OR657.3214.6138.164.1222.6238.392.778.675.572.876.0



Cured OR641.279.669.145.364.268.845.444.639.241.645.2

% to serum free Cured OR60.093.267.710.055.867.010.28.3-4.91.09.7




JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

Huh7.5.134.155.245.839.552.653.832.331.330.631.834.2

% of JFH1/Hu7.5.166.993.874.047.1103.496.868.179.275.564.558.5

Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV JFH1 cells


JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

% to serum free Huh7.5.166.9213.8133.770.4213.8210.659.264.557.553.458.9

Inactivated HIV virus and gp120 moderately increase TGF-b1 expression in Hu7.5.1 cells


Huh7.5.134.155.245.839.552.653.832.331.330.631.834.2

% to serum free Huh7.5.10.061.934.315.854.357.8-5.3-8.2-10.3-6.70.3Inactivated HIV virus and gp120 greatly increase TGF-b1 expression in HCV JFH1 cells in serum free medium


Analy

OR6

Cured OR6

TGF-b1 (pg/ml)

Convert

JFH1

Huh7.5.1

Serum Free

CXCR4 tropiv HIV

CCR5 tropic HIV

Negative HIV

SF gp120

CN gp120

Gag

Rev

Tat

Pol

Vif

TGF-b1 ELISA -Quantikine Assay

35 ul of TGFb1 convert to 1000 ul =1000/35.7=28TGF-b1 concentration: pg/ml



Cured OR612.249.646.115.344.248.813.514.614.512.910.5

OR664.8129.698.167.6132.9139.479.473.672.371.272.5

% OR6/Cured431.1161.3112.8341.8200.7185.7488.1404.1398.6451.9590.5

Converted to * 28


Serum FreeCXCR4 tropic HIVCCR5 tropic HIVNeg HIV supernatantSF gp120CN gp120GagRevTatPolVif

Cured OR6341.61388.81290.8428.41237.61366.4378.0408.8406.0361.2294.0

OR61814.43628.82746.81892.83721.23903.22223.22060.82024.41993.62030.0

Cured OR6 STD ±17.787.486.421.889.098.420.421.520.921.719.6

OR6 STD ±114.3268.4170.2115.5266.1287.6128.9130.2132.7118.7125.6




Huh7.5.114.235.235.819.536.238.513.311.311.612.810.8

JFH156.9107.079.758.1107.0105.954.356.153.752.354.2

% of JFH1/Hu7.5.1300.7204.0122.6197.9195.6175.1308.3396.5362.9308.6401.9

Converted *28

Serum FreeCXCR4 tropic HIVCCR5 tropic HIVNegative HIVSF gp120CN gp120GagRevTatPolVif

Huh7.5.1397.6985.61002.4546.01013.61078.0372.4316.4324.8358.4302.4

JFH11593.22996.02231.61626.82996.02965.21520.41570.81503.61464.41517.6

Huh7.5.1 STD ±21.460.069.133.258.756.421.419.820.724.321.9

JFH1 STD ±105.2209.7165.1110.6193.8189.3103.4112.8123.5101.5118.7



Huh7.5.1

JFH1

Huh7.5.1

JFH1

Cured OR6

OR6

HIV and HCV cooperative effects on hepatic fibrogenesis

HIV-1 and HCV cooperatively increase type I collagen secretion in hepatic stellate cells

Lin et al., J Biol Chem. 2011;286(4):2665-74

LX-2 cell lysates

CoL

1A/G

AP

DH

mR

NA

(A

rbitr

ary

Uni

ts)

Arb

itrar

y U

nit

* ** ** **

0

1

2

3

LX-2 Supernatants

CoL

1A1

prot

ein

(µg/

ml)

**

# #

# # #

0

2

4

6

8

10

CoL

1A1

prot

ein

(µg/

ml)

HIV-1 increases TIMP-1 expression and secretion in HSCs

LX-2 Cell Lysates

TIM

P1/

GA

PD

H m

RN

A

Arb

itrar

y U

nit * *

** **

0

1

2

3

4

LX-2 Supernatants

TIM

P1 p

rote

in (n

g/m

l)

* *

** **

0

40

80

120

160

200

Lin et al., J Biol Chem. 2011;286(4):2665-74

ROS inhibition blocks HIV-1 and HCV-induced TIMP-1 and collagen mRNA expression in HSCs

Lin et al., J Biol Chem. 2011;286(4):2665-74.

TIMP-1

TIM

P1/

GA

PD

H A

rbitr

ary

Uni

t

* * ** **

0

1

2

3

4 HSC

HSC+DPI

Col1A1

* ** ** **

0

1

2

3

4 HSC

HSC+DPI

CoL

1A/G

AP

DH

Arb

itrar

y U

nit

Model of HIV/HCV regulation of hepatic fibrogenesis in HSCs and hepatocytes

NF κ B

HCV

ROS

P

NF κ B TGF β 1 CoL1A TIMP1

P

NF κ B

HIV

HSC Hepatocyte

NF κ B NF κ B

HCV HCV

ROS

P

NF κ B

P

NF κ B NF κ B

Nucleus

TGF β 1 CoL1A TIMP1

TGF β 1 CoL1A1 TIMP1

P

NF κ B

P

NF κ B NF κ B

HIV HIV

HSC Hepatocyte Hepatocyte

Cooperative effects of HCV and HIV on the hepatic macrophage

population

• Macrophages important reservoirs for HIV and contribute to chronic liver disease (KCs)

• Activated tissue macrophages polarize into • pro-inflammatory (M1): CD86, CD80 • pro-repair/fibrotic (M2): CD163, Arg1, CD206

• M2 polarized macrophages in turn activate HSCs • Peripheral levels of soluble CD163 associated with

• liver fibrosis in HCV, HBV, and NASH • HIV infection

The contribution of the macrophage to HCV-HIV liver disease

Kazankov K, Hepatology. 2014 60:521-30 Kazankov K, J Gastroenterol Hepatol 2015 Feeney E et al, AASLD Nov 2014 Chew K et al, OFID 2014 Dec 3

Cooperative effects of HCV and HIV on hepatocyte apoptosis

FADD

Caspase 8

↓ Caspase 8 Activation tBid

Mitochondria

Active Caspase 9

Caspase 3,6,7 Activation

Cell death

Cytochrome c

Intrinsic Pathway

Extrinsic Pathway

Death Receptor

Death Ligand

Phagocytosis by HSCs activation

HCV and HIV induce caspase 3/7 activity in Huh 7.5.1 cells C

aspa

se3/

7/C

ell

p = 0.004

p = 0.01

p = 0.002

p = 0.004

0

1

2

3

4

5

6

7

Jang JY et al, J Hepatol 2011; 54:612-20

Cas

pase

3/7/

Cel

l

HCV and HIV-mediated hepatocyte apoptosis is blocked by caspase inhibitors

0

1

2

3

4

5

6

7

no Caspase Inhibitor

Pan-caspase Inhibitor

Caspase 9 Inhibitor

Caspase 8 Inhibitor

Jang JY et al, J Hepatol 2011; 54:612-20

FADD

Caspase 8

↓ Caspase 8 Activation tBid

Mitochondria

Active Caspase 9

Caspase 3,6,7 Activation

Cell death

Cytochrome c

Intrinsic Pathway

Extrinsic Pathway

↑DR4, DR5

↑ TRAIL (in HIV)

Phagocytosis by HSCs activation

Microbial Translocation and Pathogenesis

• Evidence for MT with HIV CD4 depletion enteropathy • Translocated LPS

– Triggers TNF-α secretion by KCs – TLR4 ligand HSCs enhanced TGF-β signaling

• Stimulates fibrogenesis – Triggers chemokine secretion – Enhances HSC senstivity to TGF-β signaling

• Enteropathy is not fully reversed by ART • ?role for clearance/alteration of microflora

– probiosis – antibiosis

Seki E et al. Nature Med 2007; 13(11):1324 Balagopal A et al. Gastro 2008;135:226-33

Summary • HIV cooperatively interacts with HCV to accelerate liver

disease through several mechanisms, both direct and indirect – increased HCV replication, oxidative stress, TGF-β, M2

polarization, hepatocyte apoptosis, microbial translocation

• Multiple cell types affected by both HCV, HIV cooperatively interact to accelerate fibrosis

• Effective HIV suppression still incompletely removes the profibrogenic environment

• These findings provide a framework for understanding HIV and its contribution to acceleration of other liver diseases (HBV, NASH)

Acknowledgments

MGH • Wenyu Lin • Shadi Salloum • Guoyang Wu • Ethan Weinberg • Hong Zhao • Andrew Tai • Jae Young Jang • Kattareeya Kumthip • Nadia Alatrakchi • Shadi Salloum • Anna Lidofsky • Annie Kruger Ragon Institute • Mark Brockman

UCSF • Ma Somsouk • Steve Deeks • Peter Hunt Collaborators • Detlef Schuppan, Beth Israel

Deaconess Medical Center • Scott Friedman, Mt. Sinai (LX-2

cells) • Nobuyuki Kato, Okayama (OR6)

NIH AIDS Research and Reference Reagent Program

Funding • NIAID, NIDDK, NIDA

HCV-HIV Coinfection: Pathogenesis of Accelerated Liver Disease ProgressionDisclosuresLiver disease is the second leading specific cause of death among HIV(+) persons�D:A:D study �n=33,308, 180,000 p/yHIV’s adverse effects on HCVImpact of HIV on HCV-related Liver Disease Progression�Systematic review of natural hx studiesHepatic decompensation is accelerated in HCV-HIV coinfection�(VA: 4280 HCV-HIV, 6079 HCV)Slide Number 7Pathways to fibrogenesisSlide Number 9Determinants of Pathogenesis in HCV-HIV CoinfectionHIV Effects on HCV T cell responsesHCV T cell responses in coinfection are blunted compared with HCV monoinfection CD4+ Proliferative Responses to HCV are dependent on nadir CD4�(n=47)HIV effects on HCV replicationHIV gp120 directly enhances HCV replication in a full-length replicon (OR6)HCV directly induces profibrogenic cytokinesSlide Number 17Slide Number 18Model of events underlying HCV induction of TGF-b1 production in hepatocytesHCV and HIV exert cooperative effects on TGF-b1 secretion HIV or gp120 increase TGF- secretion in JFH1-infected or uninfected Huh7.5.1 cellsHIV or gp120 increase TGF secretion in JFH1-infected or uninfected Huh7.5.1 cellsHIV and HCV cooperative effects on hepatic fibrogenesis�Slide Number 24Slide Number 25Slide Number 26Slide Number 27Cooperative effects of HCV and HIV on the hepatic macrophage populationSlide Number 29Cooperative effects of HCV and HIV on hepatocyte apoptosisSlide Number 31Slide Number 32Slide Number 33Slide Number 34Microbial Translocation and PathogenesisSummaryAcknowledgments

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