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Generate the recommended pool of lottery balls to play based on the latest trends and patterns.Windows Version 184.108.40.206 ... Added: 03/18/15Total Downloads25,509Last Week156
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Sketchpad for sharing visual ideas.Mac Version 2.7.2 ... Added: 02/28/14Total Downloads21,965Last Week69
Premium turn-by-turn GPS navigation app.Android Version 11.2.2 ... Added: 12/04/11Total Downloads14,350Last Week62
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Slice and dice fruit with a ninja sword.iOS Version 1.7.1 ... Added: 07/21/11Total Downloads198,243Last Week32
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text:
Key things you need to know aboutDr. Garcia-Romeu:
Dr. Garcia-Romeu's Experience
Research training, expertise and qualifications
Dr. Garcia-Romeu's Specialties
- Internal Medicine
- View 1 specialty and 1 board certification
More About Dr. Garcia-Romeu's Background
Hospital Quality
The right hospital is as important as the right doctor
- Medical Center of South Arkansas
- 700 West Grove Street
- El Dorado, AR 71730
9 Facts About Hospital Quality
Patients' risk of dying during a hospital stay for heart bypass surgery could be 85.2% lower, on average, at a hospital rated 5 stars compared to a hospital rated 1 star for that procedure. Choosing the right hospital is critical to your health. View Slideshow
Patient Satisfaction
Patients' feedback on their experience with Dr. Garcia-Romeu
Be the first to evaluate Dr. Garcia-Romeu by taking our Patient Satisfaction Survey.
Your feedback will help other patients make informed decisions, and let the doctor know how he's doing.
8 Facts About Patient Satisfaction Surveys
Patient satisfaction surveys provide a standardized way of reporting about healthcare experiences. But there can be more than meets the eye when reviewing scores. Here's what you should know
Appointments & Offices
Check insurance plans, locations and make an appointment
- South Arkansas Center On Aging
- South Arkansas Center On Aging
- 815 Thompson Ave
- El Dorado, AR 71730
7 Tips to Prepare for Your Appointment
The key to a successful appointment is communication. To get the most out of your visit, prepare ahead of time so you can lead the conversation with your doctor. These 7 tips will get you ready
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subset: commoncrawl


text:
Welcome to the Lunar Rogue Pub!
Friday Night Happy Hour Starts at 4:00pm!
Did you know The Rogue has it's own brew? Ask your server
Premium Whisky of the Week
"Isle of Jura 16 yr"
Blended Whisky of the Week
"Cutty Sark"
Malt Whisky of the Week
"Aberfeldy 12yr"
Cocktail the Week
"Whisky Sour"
Rum of the Week
"PlantationTrinidad Single Cask"
Please enjoy your Spirits Responsibly!
Keep up with all the Rogue News on Facebook and Twitter!
Frank's Whisky Blog
Whisky for the Holidays
Written by The Lunar Rogue Thursday, 18 December 2014 15:13
Whisky For the Holidays
Every year I am asked to recommend a whisky as a gift for a Christmas present. Not as easy as it sounds as everyone has different tastes when it comes to Whisky. Not always knowing if someone appreciates a whisky with smoke, peat, sherry, vanilla, etc can be challenging to make a recommendation they might like in the end. If you have a rought idea of what they like now can be a big help in deciding what to select. The price point you can afford always comes into play for most of us. So that said here are a few of my recommendations and price points. Single Malt under $50.00 in NB It is not easy anymore but I like Aberlour 10, Aberfeldy 12 and Strathisla 12. They are all very good whiskies for the price.


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Breaking Down the Big Ten Bubble Games This WeekPosted by Brendan Brody on March 3rd, 2015
The eyes of not only Big Ten fans but much of the country will be watching this week as there is much still to be determined. As many as eight league teams have legitimate NCAA Tournament aspirations, and there is also a logjam with conference tournament seeding still to be sorted out. Luckily, much will be settled with head-to-head match-ups pitting impacted teams against one another this week. This means that you shouldn't get too comfortable with the standings as they are right now, because things will change rapidly over the next six days. Here's a look at the must-see games for the final week and why they're important.
- Iowa at Indiana, 7:00 EST, ESPN, Tuesday. These two teams haven't played yet this season and the Hoosiers will once again face a team with more size than them. Iowa does a pretty solid job defensively against the three (52nd nationally), so this game should come down to whether the Hoosiers can handle Iowa's pressure and shoot over the top. Troy Williams against either Aaron White or Jarrod Uthoff will be a quality match-up worth watching.
- Ohio State at Penn State, 6:00 EST, BTN, Wednesday. It's worth noting that Penn State beat Ohio State twice last season although they did lose by 20 in Columbus earlier this year. Ohio State really can't afford to slip up here and lose to a Nittany Lions' group that just can't get out of their own way in crunch time. At a minimum, the Buckeyes need to stockpile more wins to move up a seed line or two from where they're projected — another Big Ten road win won't hurt their cause.


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The Ingram Group, a 29-year-old public affairs firm based in Nashville, Thursday issued an announcement regarding their recent move to the Nashville City Center building, 511 Union St., to accommodate their corporate and government consulting practice.
The Group's current client base includes a roster of high profile clients such as Tennessee Governor Bill Haslam, U.S. Senator Lamar Alexander, U.S. Senator Bob Corker, Google, Ryman Properties (formerly Gaylord Entertainment Co.), Pilot Flying J, Eastman, Corrections Corporation of America, and Xerox. The Group has also developed a presence in Chattanooga with clients such as Hamilton County Mayor Jim Coppinger and Memorial Health Care System.
The move to occupy nearly 5,000 square feet at the Nashville City Center doubles the size of the Ingram Group's former space and affords them views of the State Capitol and War Memorial Plaza.
The move comes on the heels of two staff additions. Sam Reed, a former staff attorney to U.S. Congressman Jim Cooper, joined the Group earlier this year as a strategic consultant. Mr. Reed services clients on both the state and federal level, offering counsel on business, legislative, regulatory, and other issues. Additionally, Rachel Albright, who joined the team in early 2011 as a client consultant after serving in U.S. Senate Republican Leader Mitch McConnell's leadership office as his deputy press secretary, will continue to serve clients in both the public and private sectors offering expertise on relationship building, message development, campaign strategy, and media relations.
The Nashville office is one of two, the other being the Ingram Group's Washington, D.C. office which operates as The FIRST Group and includes as partners four prominent former Senate Chiefs of Staff. Coincidentally, both the Ingram Group and the FIRST Group offices are now housed above "Cap Grilles." The FIRST Group sits directly above the famed grille on Pennsylvania Avenue in D.C. and the Ingram Group overlooks the grille at the Hermitage Hotel here in Nashville.
Marcille Durham, the president of the Group who led the company while Ingram was in D.C. as Senator Alexander's chief of staff, said about the firm's growth, "our capacity to provide top-level, strategic advice to our clients – recently boosted by key staff additions – is now further enhanced by next generation office space just steps away from the Capitol."
"For years, the Ingram Group has been the go-to firm for solving problems and maximizing opportunities in the public and private sectors," Tom Ingram said. "We're excited about the next chapter of the Group and look forward to being in the middle of the exciting changes happening here in Nashville."
March 28, 2015
March 26, 2015
The Tennessee Supreme Court has reinstated charges of driving under the influence and violating the open container law against Williamson County resident Mechelle Montgomery in an opinion that ... (click for more)
The Tennessee Supreme Court has affirmed the dismissal of a whistleblower claim by Charles Haynes, a former employee of Formac Stables, Inc., in Obion County. Mr. Haynes worked as a groom ... (click for more)
Roper Corporation, a division of GE, will recruit about 80 manufacturing workers for its LaFayette facility Friday. The recruitment will be held from 10 a.m.-2 p.m. at the Chattooga County Civic ... (click for more)
The Tennessee Supreme Court has reinstated charges of driving under the influence and violating the open container law against Williamson County resident Mechelle Montgomery in an opinion that restores evidence gained during a search. In February of 2012, two officers with the Williamson County Sheriff's Office were dispatched to the residence of a woman named Corey Brown, who ... (click for more)
The Tennessee Supreme Court has affirmed the dismissal of a whistleblower claim by Charles Haynes, a former employee of Formac Stables, Inc., in Obion County. Mr. Haynes worked as a groom at Formac Stables. In April of 2010, hesuffered an injury to the head when kicked by a Tennessee Walking Horse named "Bruce Pearl." Mr. Haynes claimed that Formac's owner refused to allow him ... (click for more)
Temperatures are expected to drop Saturday night to 28 degrees or less. Here is the latest report from the National Weather Service: HARD FREEZE WARNING REMAINS IN EFFECT FROM MIDNIGHT EDT /11 PM CDT/ TONIGHT TO 11 AM EDT /10 AM CDT/ SUNDAY... * EVENT... A HARD FREEZE EXPECTED WITH LOWS FROM THE TEENS NORTH TO THE UPPER 20S SOUTH LATE TONIGHT AND SUNDAY MORNING. ... (click for more)
A man charged with attempted first-degree murder on Sixth Avenue Court on Thursday afternoon was shot himself earlier this month. O'Shae Kadaris Smith, 20, of 2114 E. 12th St., is also charged with reckless endangerment, aggravated assault and possessing a firearm during the commission of a crime. He was one of two people shot on Rawlings Street on March 16. Kendre Allen, ... (click for more)
RE: Finley Stadium Financial Picture Rosy; Beer And Alcohol Sales Approved; AstroTurf Almost Ready and City, County Pitching In $250,000 Each For New Finley Stadium AstroTurf I read a story here five days ago about a rosy future for Finley stadium. It lead me to believe that the stadium was making money and even improvements with donations. Then we get a short ... (click for more)
They say March roars in like a lion and leaves like a lamb. So as you reach for your lambs-wool sweater on this last Sunday of the month, allow me to lighten up a bit with a new edition of my Sunday Funnies. I have a delightful crowd that keeps me supplied in jokes and these are some of the best that have come my way: A WOMAN'S PERFECT BREAKFAST -- She's sitting at the table ... (click for more)


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SugarCRM 5.0 with Ajax and as a Service
After completing the community beta phase, SugarCRM has now released Version 5.0 of its Open Source CRM as a commercial package.
The new features the Web-based application offers include extended visualization options for dashboard data, and an email client with Ajax technology for improved usability.
The new Module Builder looks to facilitate module development for programmers. Modifications to the user interface by the user are now stored separately to enable trouble-free software updates.
The CRM system is available in various versions: besides the Community Edition which is available under the GPLv3 and the "on site" version, there is an on-demand version, which is hosted by vendor on Software-as-a-Service (SaaS) basis. Sugar Professional On-Demand is available for a free 30-day trial subject to registration.
Issue 174/2015
Buy this issue as a PDF
Microsoft Frees Code for .NET Build Engine
MSBuild is now just another GitHub project as Redmond continues its path to the light.
Researchers Demonstrate Hack by Heat
Malware could pass data and commands between disconnected computers without leaving a trace on the network.
Kernel Developers Adopt Code of Conflict
New rules emphasize collegiality in coding.
Ubuntu Switches to systemd
Upstart lands in the dust bin as a new era begins for Linux.
Old Vulnerabilities Are Kept Alive Through Bad Configuration
HP's annual Cyber Risk report offers a bleak look at the state of IT.
Linus Torvalds Announces Linux 4.0
But what do the big numbers really mean?
Microsoft Frees CoreCLR
.NET Core execution engine is the basis for cross-platform .NET implementations.
New Trojan Attacks Linux Servers
The Xnote trojan hides itself on the target system and will launch a variety of attacks on command.
Cisco Releases Annual Security Report
Spammers go low-volume, and 90% of IE browsers are unpatched.
Zero Day Exploits Target Flash
Adobe scrambles to release patches for vulnerable Flash Player.


text:
5 foods for healthy hair
Healthy hair doesn't stop with the kind of shampoo you use or how often you wash your hair. It also includes your diet. Here are five foods that will help keep your hair healthy:
1. Blueberries
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'Jasmine' world premiere selfie! http://t.co/3UJpzyTZQe
Spent this week working it, trying to get our names up in lights #Filmart http://t.co/6kGouWW9dk
Cheers! A celebratory drink with Trevor before the #jasminemovie world premiere #HKIFF #Filmart http://t.cn/RALDec8
@Vikki_Inspired cool! Hope you get to see it. Was good times watching it. Brought back good memories of when I first moved to HK 😄
Good to see so many old friends and familiar faces at the 'Jasmine' premiere and afterparty. Partying l http://t.cn/RALv1U5
RT @Aaronmichelson2: Congrats to my buddy @BOFharry and @3CThirdCulture for the today's @SCMP_News. Proud to be a #thirdculture kid http:/…
'Jasmine' made front cover of The Hollywood Reporter. Congrats to my homies in the cast and crew. This http://t.cn/RA2fJ2k
Last night at the HKIFF opening Gala. Me and Louis Koo's cardboard cut out. https://t.co/0EUfIH8x2b
Producer team at Filmart pimping our film Love Stalk 愛‧打卡 http://t.co/vQCHiGfoGx
Actor, screenwriter, director from New York and now bringing my flavor to Hong Kong :) http://www.facebook.com/joefiorelloartist http://www.weibo.com/joefio ...Read more
|Actor, writer, host, director
|Favorite Books
|Harry Potter
|Favorite Faves
|If I'm not V.I.P. I'm outta here!
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The Manor House Hotel
The Manor House Rooms and Suites
History, charm and bags of character, this beautiful 19th century country house has it all.
Think sweeping down the grand wooden staircase, collapsing onto a romantic four-poster bed, taking Sunday afternoon tea in Hedleys dining room, or drinks before dinner in the grand Manor Lounge. The Manor House hotel offers historic country house charm, with all the luxurious facilities of a world-class resort.
Our recently refurbished Manor House accommodation has been given a modern, stylish new look with beautiful bedrooms and luxurious bathrooms.
Newly refurbished featuring chic and stylish décor, queen-size bed and luxurious en-suite with bath and shower.
Newly refurbished, offering the same stylish décor and features as the Manor Double but with two single beds.
Newly refurbished and offering extra space for families, this spacious stylish room with king-size bed and luxurious en-suite has an adjoining lounge area with space for two extra beds, comfortably accommodating two adults and two children plus a cot.
Situated within the original 19th century Manor House itself, these atmospheric rooms are rich in character and historic period features, complemented by traditional country house décor.
Offering the same features as the Manor Period Double but with a larger king-size bed.
These rooms boast a queen-size wooden four-poster bed. Each one echoes the romantic charm of The Manor House with traditional décor and are ideal rooms to celebrate a special night away.
All the historic character of our other Manor Period rooms but with two queen-size beds, accommodating two adults and up to two children plus a cot.
Check in from 4.00pm, check out by 11.00am
Breakfast served in Le Patio 7.00am - 10.30am
Please note breakfast will be served in The Olive Tree Tues-Fri.
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The Celtic Manor Resort is a world-class destination for golf, with three championship courses, The Twenty Ten, Roman Road and The Montgomerie, plus state of the art Golf Academy and two luxury clubhouses.
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TheCelticManorRT @TomFelton: What an amazing weekend at @TheCelticManor 4 boys, 3 golf courses, 2 dozen peronis and 1 hunter lodge = perfection #thecelt…
Executive Head Chef Mike Bates treated members of the media to a cookery masterclass in the Hunter Lodges.
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text:
Many thanks for your help.
chimney liner for new furnace?
I just received two estimates for a new 90% AFUE gas furnace for my 2300 sq. ft. house. We have a chimney that is steel on the inside. One guy (A), who actually estimated for a 93% AFUE, says we still should get a chimney liner; the other (B) says we don't need one (because of the steel). Both are from reputable companies, though 'A' is from a company that specializes in HVAC (and seems to be well thought of in that regard), while 'B' is from a large all purpose company that hasn't been reviewed here with regard to furnaces. 'B' estimate also came in significantly lower, even when you account for the liner. So, (1) any thoughts on the liner issue, and (2) is it the 93% for 'A' versus '90%' for 'B', or am I missing something, in that, factoring out the liner for 'A' for the moment, 'A' is around $2,100 and 'B' is around $1,400? Very large difference that has me uneasy.
Many thanks for your help.
Many thanks for your help.
If the furnace is using PVC pipe, the chimney cannot be used at all, but if it is vented with galv. pipe......and if the flue runs up the center of the house, an inside wall,.. then no liner is required. You probably have a double wall chimney "B" vent going up to the roof. Price diff. is rather large. I would stay with the HVAC man, but if the other bidd is well thought out, then go with the lower one.
Condensation from gas furnace (American Standard - 80)
New Furnace Comparisons
Adding a new furnace
how to choose size of new furnace/ac
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- Watch These 10 Home Trends Take Off in 2015
- 11 Things Never to Keep in Your Bedroom
- 12 Places You Never Clean—But Should!


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Page 1
The Journal of Immunology
Histone Deacetylase Inhibitor LAQ824 Augments
Inflammatory Responses in Macrophages through
Transcriptional Regulation of IL-10
Hongwei Wang,* Fengdong Cheng,* Karrune Woan,* Eva Sahakian,* Oscar Merino,*
Jennifer Rock-Klotz,* Ildefonso Vicente-Suarez,* Javier Pinilla-Ibarz,* Kenneth L. Wright,*
Edward Seto,†Kapil Bhalla,‡Alejandro Villagra,* and Eduardo M. Sotomayor*
APCs are important in the initiation of productive Ag-specific T cell responses and the induction of T cell anergy. The inflammatory
statusof theAPC atthe timeofencounter withAg-specific T cells playsa centralroleindetermining such divergent T cell outcomes.
A better understanding of the regulation of proinflammatory and anti-inflammatory genes in its natural setting, the chromatin
substrate, might provide novel insights to overcome anergic mechanisms mediated by APCs. In this study, we show for the first
time, to our knowledge, that treatment of BALB/c murine macrophages with the histone deacetylase inhibitor LAQ824 induces
chromatin changes at the level of the IL-10 gene promoter that lead to enhanced recruitment of the transcriptional repressors
HDAC11 and PU.1. Such an effect is associated with diminished IL-10 production and induction of inflammatory cells able of
priming naive Ag-specific T cells, but more importantly, capable of restoring the responsiveness of anergized Ag-specific CD4+
T cells. The Journal of Immunology, 2011, 186: 3986–3996.
such factors as the particular APC type as well as the context,
inflammatory versus non-inflammatory, in which the APC acquires
the Ag for processing and presentation to Ag-specific T cells (1, 2).
Not surprisingly, APCs isolated from a noninflammatory tumor
microenvironment are relatively inefficient at priming protective
responses, inducing instead T cell anergy (3–5).
During the past several years, numerous studies in experimental
models as well as in humans have provided sufficient evidence
supporting the conclusion that the induction of T cell anergy to tu-
mor Ags represents a significant barrier to harness antitumor immu-
nity (5–9). Importantlessonslearnedfromthesestudies point toma-
nipulation of the inflammatory status of the APC as an enticing
strategy to overcome anergic mechanisms in cancer (10–13). A bet-
ter understanding of the molecular/signaling mechanism(s) regulat-
and might unveil novel targets to overcome anergy to tumor Ags.
Recently, a significant effort has been devoted to better un-
derstand the regulation of pro- and anti-inflammatory genes in their
he potency of an immune response is dictated in large part
by the potency of the APC and its ability to optimally
prime the T cell response. This, in turn, is influenced by
natural setting, the chromatin substrate (14). Chromatin modifi-
cation by acetylation/deacetylation of histone tails is an important
mechanism of regulation of gene transcription, including genes
involved in the inflammatory response (15). In general, histone
acetylation mediated by histone acetyl transferases results in
transcriptionally active chromatin. In contrast, histone deacetyla-
tion mediated by histone deacetylases (HDACs) leads to an in-
active chromatin and gene repression (16).
HDACs exist as large multimeric complexes and are recruited to
gene promoters by corepressors or by multiprotein transcriptional
complexes. Eighteen HDACs have been identified, and they have
been grouped into four principal classes (17, 18). HDACs are the
molecular target of several structurally diverse compounds known
as histone deacetylase inhibitors (HDIs). Existing HDIs inhibit
proliferation of malignant cells in vitro by inducing cell cycle
arrest and apoptosis, and some of them have already demonstrated
significant antitumor activity in cancer patients (19, 20). In con-
trast to their well-known effects upon cancer cells, little is still
known about the immunological effects of HDIs. Although some
studies have shown that HDIs have anti-inflammatory properties
(21, 22), promote the expression of the suppressive factor IDO in
dendritic cells (DCs) (23), and diminish the morbidity and mor-
tality of graft-versus-host disease (24), others have highlighted
the proinflammatory effects of these compounds. For instance,
Tomasi's group (25, 26) has shown that treatment of melanoma
cells with HDIs augments their Ag-presenting capabilities, leading
to activation of IFN-g–secreting T cells via the class I pathway. Vo
et al. (27) have recently demonstrated that in vivo treatment of
tumor-bearing mice with the hydroxamic acid analog pan-HDI
LAQ824 significantly enhances the antitumor activity of adop-
tively transferred Ag-specific T cells. Needless to say, the un-
derlying molecular mechanism(s) by which HDIs influence in-
flammatory responses remain to be fully elucidated.
In this study, we show that the pan-HDI LAQ824 induces several
chromatin changes in macrophages that resulted in enhanced
recruitment of the transcriptional repressors HDAC11 and PU.1
*Department of Immunology and Malignant Hematology, H. Lee Moffitt Cancer
Center, Tampa, FL 33613;
Cancer Center and Research Institute, Tampa, FL 33613; and‡University of Kansas
Medical Center, Kansas City, KS 66160
†Department of Molecular Oncology, H. Lee Moffitt
Received for publication April 15, 2010. Accepted for publication January 29, 2011.
This work was supported by Public Health Service Grants CA87583 and CA134807
(to E.M.S.) and by a grant from the Donald A. Adam Comprehensive Melanoma
Research Center.
Address correspondence and reprint requests to Dr. Eduardo M. Sotomayor, H. Lee
Moffit Cancer Center and Research Institute, 12902 Magnolia Drive, FOB-3 Room
5.3125, Tampa, FL 33613. E-mail address: email@example.com
Abbreviations used in this article: ChIP, chromatin immunoprecipitation; DC, den-
dritic cell; HA, hemagglutinin; HDAC, histone deacetylase; HDI, histone deacetylase
inhibitor; PEM, peritoneal elicited macrophage.
Page 2
to the IL-10 gene promoter. Such an effect is associated with in-
hibition of IL-10 production and induction of cells able of priming
naive Ag-specific T cells and capable of restoring the responsive-
ness of anergized CD4+T cells.
Materials and Methods
Male BALB/c mice (6–8 wk old) were obtained from the National Insti-
tutes of Health (Frederick, MD). TCR-transgenic mice expressing an ab
TCR specific for aa 110–120 from influenza hemagglutinin (HA) presented
by I-Edwere a generous gift of H. von Boehmer (28). All experiments
involving the use of mice were performed in accordance with protocols
approved by the Animal Care and Use Committee of the University of
South Florida College of Medicine.
Cell lines
the B-cell lymphoma cell line A20 was obtained from the American Type
Culture Collection. A20HA was generated by electroporation-mediated
plasmid transfection, and transfected cells were selected as previously
reported (3, 6). Cells were cultured in vitro in RPMI 1640 media, supple-
mented with 10% FBS, penicillin/streptomycin (50 U/ml), L-glutamine (2
mM), and 2-ME (50 mM) (complete media), and grown at 37˚C and 5%
CO2. A20 mediawas also supplemented with additionalsodiumpyruvate (1
mM) and nonessential amino acids (13; Mediatech, Manassas, VA).
Isolation of peritoneal elicited macrophages
BALB/c mice were injected i.p. with 1 ml thioglycollate (DIFCO Labo-
ratories, Detroit, MI). Four days later, peritoneal elicited macrophages
(PEM) were isolated by peritoneal lavage as previously described (30).
Isolation of splenic macrophages
BALB/c mice were injected i.v. with 1 3 106A20 lymphoma cells. Three
weeks later, animals were sacrificed, and their spleens were removed.
Splenocytes were isolated, washed with PBS, and then suspended in
complete RPMI 1640 medium. Cells were then cultured at 37˚C and 5%
CO2for 2 h to allow macrophages to adhere to the plate. Nonadherent cells
were then removed with two washes of cold PBS. Splenic macrophages
were then incubated for 1 additional h, and the cells were treated as in-
LPS (Escherichia coli 055:B5, catalog number L-2880) was purchased
from Sigma-Aldrich (St. Louis, MO). The HDAC inhibitors LAQ824 and
LBH589 were provided by Dr. K. Bhalla (University of Kansas Medical
Center). TSA was obtained from Sigma-Aldrich, and MS-275 is from
Calbiochem (San Diego, CA). HDIs were first reconstituted in DMSO for
stock preparation (10 mM). Solutions were then diluted in RPMI 1640 for
in vitro use in cell cultures.
Abs and immunoblottings
Histone H3 (D1H2) mAb (4499), histone H4 (Lys8) polyclonal Ab (2592),
acetyl-histone H3 (Lys9) polyclonal Ab (9671), and acetyl-histone H4
(Lys8) polyclonal Ab (2594) were purchased from Cell Signaling Tech-
nology (Danvers, MA) for Western blotting. The GAPDH (FL-335, sc-
25778), PolII (H-224, sc-9001), and PU.1 (Spi-1/T-21, sc-352) Abs were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Hyper-
acetylated H3 (06-599), hyperacetylated H4 (06-598), Sp1 (07-645), and
STAT3 (06-596) polyclonal Abs were purchased from Millipore (Billerica,
MA). Two different polyclonal Abs against HDAC11 were used: ab47036
from Abcam (Cambridge, MA) for immunoblots and Ab H4539 from
Sigma-Aldrich was used for chromatin immunoprecipitation (ChIP) anal-
R&D Systems (Minneapolis, MN). Recombinant mouse IL-10 (550070)
was purchased from BD Biosciences (San Diego, CA).
Phenotypic and functional analysis of macrophages
The expression of B7.2 in PEMs was determined by staining with biotin-
conjugated anti-CD86 (GL1 Ab; BD Pharmingen, San Jose, CA) and
followed by strepavidin-PE (Caltag Laboratories, Carlsbad, CA) Abs. Ten
thousand gated events were collected on an FACScan (BD Biosciences) and
analyzed using FlowJo software (Tree Star). In a parallel plate, PEMs were
treated with HDI alone, LPS alone, or a combination thereof. Then super-
natants were collected, and production of IL-10, IL-12p70, IL-1a, IL-1b,
IL-6, RANTES, TNF-a and GM-CSF was determined by ELISA (R&D
Ag-presentation studies
PEMs or splenic macrophages (1 3 105/well) were treated with LPS (2 mg/
ml) or with LPS plus HDI for 24 h. Cells were then washed, and 5 3 104
purified naive Ag-specific CD4+T cells (isolated from the spleen of HA
TCR-transgenic mice) or a similar number of anergized Ag-specific CD4+
T cells (isolated from the spleen of A20HA lymphoma-bearing mice) were
(HApeptide110–120SFERFEIFPKE). After48h, supernatants were collected
and stored at 220˚C until assayed for IL-2 and IFN-g production by ELISA.
Valuesfor Tcellsculturedinmediaalone areusually.10%ofthevaluesfor
Ag-stimulated T cells. The amount of cytokine production is expressed as
picograms per milliliter per 100 clonotype-positive CD4+T cells.
Real-time PCR analysis
Macrophage cell line or primary murine macrophages were plated at 2 3 106
cells per 35-mm well and cultured under conditions detailed for each exper-
and cDNA obtained with the iScript cDNA synthesis kit (Bio-Rad, Hercules,
CA). Target mRNA was quantified using MyIQ single-color real-time PCR
detection system(Bio-Rad) and iQSYBRgreen Supermix (Bio-Rad). Mouse
IL-12p40 primers (left, 59-GCAACGTTGGAAAGGAAAGA-39; right, 59-
AAAGCCAACCAAGCAGAAGA-39) and mouse IL-10 primers (left, 59-
CAGGGATCTTAGCTAACGGAAA-39; right, 59-GCTCAGTGAATAAAT-
AGAATGGGAAC-39) were used for PCR amplification (cycling parameters:
∼100% in all of the experiments performed. Quantification is expressed in
arbitrary units, and target mRNA levels were normalized to GAPDH ex-
pression using the method of Pfaffl (31).
ChIP assays
ChIP studies were performed as previously described (32). The primers
used were: IL-10 (proximal region) sense, 59-GGAGGAGGAGCCT-
GAATAAC-39 and antisense, 59-CTGTTCTTGGTCCCCCTTTT-39; IL-10
(distal region) sense, 59-AACTCAGCCTGGAACTGACC-39 and antisense,
59-GCCTCTCCTCCTGACACTCTT-39; and IL-12 sense, 59-GTGGAGC-
CAAACAGGGAGGTA-39 and antisense, 59-GACGTCGAAATCCCAGTT-
TA-39. All samples and inputs were quantified using MyIQ single-color
real-time PCR detection system (Bio-Rad) and iQ SYBR green Supermix
(Bio-Rad). Single-product amplification was confirmed by melt curve
analysis, and primer efficiency was ∼100% in all experiments performed.
Target sequence levels were normalized to the input signal using the
method of Pfaffl (31). Quantification is expressed in arbitrary units as the
ratio between treated and untreated cells. All ChIP experiments were re-
peated three times, and final quantitative real-time PCR was done in trip-
T cell anergy model
We used a well-established experimental model of tumor-induced Ag-
specific CD4+T cell anergy (3, 6). Briefly, 2.5 3 106naive CD4+trans-
genic T cells specific for an MHC class II epitope of influenza HA were
injected i.v. into A20HA lymphoma-bearing mice. Twenty-one days after
T cell transfer, animals were sacrificed, and T cells were reisolated from
their spleens. Cytokine production by clonotypic CD4+T cells in response
to HA peptide110–120presented by untreated, LPS-treated, or PEM-treated
with LPS plus LAQ824 was then determined as described under Ag pre-
sentation studies.
Statistical analysis
Unpaired t tests were performed using Microsoft Excel (Microsoft) with
significance at p , 0.05.
Phenotypic and functional changes in macrophages treated
with the HDI LAQ824
First, we asked whether treatment of macrophages with the hy-
droxamic acid analog pan-HDI LAQ824 would result in changes
in histone acetylation and the induction or not of inflammatory
cells. As shown in Fig. 1A, treatment of PEM with LAQ824 was
associated with increased acetylation of histones H3 and H4
The Journal of Immunology 3987
Page 3
relative to their acetylated status in untreated PEM (None). No
changes in the expression of MHC molecules or costimulatory
molecules were observed in PEM treated with LAQ824 alone
(data not shown). However, higher expression of B7.2 was ob-
served in PEMs treated with LPS plus LAQ824 as compared with
cells treated with LPS alone (Fig. 1B). Furthermore, treatment of
PEMs with LPS in the presence of increasing concentrations of
LAQ824 resulted in a dose-dependent inhibition of the anti-
inflammatory cytokine IL-10 (Fig. 1C). Of note, this inhibitory
effect was accompanied by an increased production of the pro-
inflammatory cytokine IL-12 (Fig. 1D). It should be pointed out
that LAQ824 was not toxic to PEMs because these cells were
viable (as determined by trypan blue exclusion) and capable of
producing IL-12 even when they were exposed to the highest
concentration of HDI (100 nM) used in our experiments.
To further confirm the proinflammatory effects of LAQ824, we
determined next the production of cytokines/chemokines by PEMs
treated with HDI. As shown in Fig. 2, PEMs treated with LPS and
LAQ824 produced higher levels of IL-1a, IL-1b, IL-6, RANTES,
TNF-a, and GM-CSF as compared with PEMs treated with LPS
alone. Of note, LAQ824 also inhibits the production of IL-10 by
macrophages isolated from the spleen of tumor-bearing mice (Fig.
3A). No changes in IL-12 production were observed in tumor-
bearing macrophages treated with HDI plus LPS as compared
with macrophages treated with LPS alone (Fig. 3B).
Pan-HDIs LBH589 and TSA, but not the class I selective HDI
MS-275, inhibit IL-10 production by macrophages
HDIs represent a family of structurally diverse compounds that
inhibit the enzymatic function of HDACs. LAQ824 as well as
LBH589, TSA, and SAHA are pan-HDIs that belong to the
hydroxamic acid family of compounds. To determine whether the
other members of this family share the proinflammatory effects of
LAQ824, we treated PEMs with either LBH589 or TSA. Remi-
in a dose-dependent manner the production of IL-10 by PEMs in
response to LPS (Fig. 4A, 4B, left panel). Such an inhibition was
associated with increased production of IL-12 (Fig. 4A, 4B, right
panel). Similar changes, although of a lesser magnitude, were
observed in PEMs treated with SAHA (data not shown).
In contrast to the above results, treatment of PEMs with a more
selective HDI, compound MS-275, which inhibits the enzymatic
function of class I HDACs (HDAC 1, 2, 3, and 8) did not result in
inhibition of IL-10 or augmentation of IL-12 production by treated
cells (Fig. 4C). These findings argue against a role for class I
HDACs in regulating IL-10 production in PEMs and point to class
II and/or class IV HDACs as potential molecular targets involved
in the inhibitory effect on IL-10 mediated by pan-HDIs.
LAQ824-treated PEMs efficiently prime naive Ag-specific
T cells and restore the responsiveness of anergized T cells
Next, we determined whether the acquisition of inflammatory
properties by LAQ824-treated macrophages renders these cells
better activators of Ag-specific CD4+T cells. PEMs were therefore
treated with LAQ824, LPS, or a combination of LPS plus LAQ824
for 24 h. Following this treatment, naive CD4+T cells specific for
an MHC class II-restricted epitope of influenza HA were added to
the PEM monolayer and stimulated, or not, with cognate HA
peptide. First, clonotypic T cells encountering cognate peptide on
untreated PEM (None) or on PEM treated with LAQ824 (HDI
alone) produce similar levels of IL-2 (Fig. 5A, top panel). A
slightly increased production of IL-2 was observed in T cells
encountering Ag in LPS-treated PEMs (Fig. 5A, top panel, LPS
alone). IL-2 production was further enhanced in CD4+T cells that
encountered HA peptide in PEMs treated with LPS plus increas-
ing concentrations of LAQ824 (Fig. 5A, top panel, LAQ+LPS).
LAQ824-treated PEMs also triggered an enhanced effector func-
tion of clonotypic CD4+T cells, as determined by their capacity to
produce higher levels of IFN-g in response to cognate peptide
(Fig. 5A, bottom panel).
Using a TCR-transgenic model, we have previously demon-
strated that CD4+T cells specific for an MHC class II epitope of
influenza HA are rendered anergic during the growth of a B cell
lymphoma expressing HA as a model tumor Ag (A20HA). Iso-
tional changes in macrophages treated
with the HDI LAQ824. A, PEMs were
treated or not with LAQ824 (12.5 nM)
for 24 h. Then, cell lysates were ob-
tained, and acetylation of histones H3
and H4 was determined by immuno-
blotting using anti-acetyl H3 and H4
Abs, respectively. B, PEMs were treated
with LPS (2 mg/ml) or with LPS plus
LAQ824 (12.5 nM) for 24 h. Cells were
then stained with an anti-B7.2 Ab or an
isotype control. In a parallel experiment,
PEMs were treated with LPS alone (2
mg/ml) or LPS plus increasing concen-
trations of LAQ824 as indicated. After
24 h, supernatants were collected and the
levels of IL-10 (C) and IL-12 (D) were
determined by ELISA. Shown is a rep-
resentative experiment of five experi-
ments with similar results. *p , 0.05,
**p , 0.01.
Phenotypic and func-
Page 4
lation of these clonotype-positive T cells from tumor-bearing mice
followed by their in vitro restimulation with HA peptide plus
APCs demonstrated that these cells were anergic given their
inability to produce IL-2 or IFN-g (3, 6). However, in vitro
incubation of these same anergic T cells with LAQ824-treated
PEMs resulted in restoration of T cell responsiveness (Fig. 5B).
Indeed, anergized Ag-specific CD4+T cells encountering cognate
Ag on PEMs treated with LAQ824 and LPS regained their ability
to produce IL-2 (Fig. 5B, top panel) and IFN-g (Fig. 5B, bottom
panel). In contrast, anergic T cells encountering cognate Ag on
untreated (None), HDI alone, or LPS-treated PEM were unable to
produce IL-2 (Fig. 5B, top panel) and produced minimal amounts
of IFN-g (Fig. 5B, bottom panel). Therefore, LAQ824-treated
PEMs effectively prime naive Ag-specific CD4+T cells and re-
store the responsiveness of anergic CD4+T cells.
To better define the role of IL-10 inhibition induced by LAQ824
in the augmentation of the APC function of macrophages, we
cultured PEMs with naive Ag-specific CD4+T cells in the presence
of recombinant murine IL-10. As shown in Fig. 5C, T cells pro-
duced less IFN-g in response to cognate Ag presented by LPS-
treated macrophages when rIL-10 was added (Fig. 5C, LPS alone,
black bar versus gray bar). Similarly, the enhanced APC function
displayed by macrophages treated with 12.5 nM LAQ824+LPS
(Fig. 5C, LAQ+LPS, gray bar) was abrogated when rIL-10 was
added back to the cultures (Fig. 5C, LAQ+LPS, black bar). This
result was not surprising given the well-known ability of IL-10 to
inhibit Th1-type responses (33). Given that the inhibition of IL-10
induced by LAQ824 at the dose of 12.5 nM is incomplete (Fig.
1C), we asked next whether neutralization of the remaining se-
creted IL-10 with anti–IL-10 Abs could further augment the APC
duction by macrophages from tumor-bearing
mice. A, Splenic macrophages from A20
bearing mice were treated with LPS (2 mg/
ml), LAQ824 alone (12.5 nM), or LPS (2 mg/
ml) plus LAQ824 for 24 h. The supernatants
were collected and production of IL-10 (A)
and IL-12 (B) were determined by ELISA.
Shown is a representative experiment of two
experiments with similar results. **p , 0.01.
LAQ824 inhibits IL-10 pro-
(12.5 nM), or LPS (2 mg/ml) plus increasing concentrations of LAQ824 as indicated. After 24 h, supernatants were collected, and cytokine/chemokine
levels were determined by ELISA. Shown is a representative experiment of three experiments with similar results. *p , 0.05, **p , 0.01.
Production of proinflammatory mediators by macrophages treated with HDI LAQ824. PEMs were treated with LPS (2 mg/ml), LAQ824
The Journal of Immunology3989
Page 5
function of LAQ824-treated macrophages. As shown in Fig. 5D,
IL-10 blockade was insufficient to enhance the APC function of
LPS-treated macrophages to augment IFN-g production by CD4+
T cells (Fig. 5D, LPS alone, black bar versus gray bar), and it was
not able to significantly enhance the effect of LAQ824 treatment
(Fig. 5D, LAQ+LPS, black bar versus gray bar). Taken together,
these data point to a contributory role of IL-10 inhibition in the
enhanced APC function displayed by LAQ824-treated macro-
phages because this effect was reversed when rIL-10 was added
back to the cultures. However, neutralizing the remaining IL-10
protein that could have still been produced by LAQ824-treated
macrophages did not result in further enhancement of their APC
IL-10 mRNA expression and histone acetylation of the IL-10
gene promoter in PEMs treated with LAQ824
IL-10 is an anti-inflammatory cytokine that plays an important role
intheestablishmentandmaintenance ofTcell anergy(34,35).One
of the most striking and consistent effects of pan-HDIs was their
ability to inhibit IL-10 production (Figs. 1C, 3, 4). To better un-
derstand the mechanism(s) underlying this inhibitory effect, we
evaluated next the kinetics of IL-10 mRNA expression in mac-
rophages treated with LPS, LAQ824, or LPS plus LAQ824. In
response to LPS stimulation, a rapid increase in IL-10 mRNAwas
observed at 1 h, followed by a rapid decline, and, after 6 h, the IL-
10 mRNA levels were back to baseline (Fig. 6A, open triangles).
In PEMs treated with LPS plus LAQ824, a decreased induction of
IL-10 mRNA was observed at 1 h (Fig. 6A, closed circles). This
initial response was followed by a progressive decline, and, by 6 h,
IL-10 mRNA levels were back to baseline levels. No changes in
IL-10 mRNA levels were observed in PEMs treated with LAQ824
alone (Fig. 6A, open squares). Therefore, treatment of PEMs with
LAQ824 resulted in decreased IL-10 gene transcriptional activity
in response to LPS stimulation.
Next, we determined the acetylation status of histones at the
level of the IL-10 promoter. First, by using a ChIP assay, we as-
sessed the kinetics of acetylation of histones H3 and H4 in PEMs
treated with LPS alone. As seen in Fig. 6B, acetylation changes
occurred within a particular time window, with a peak acetylation
occurring at 60 min. This was followed by a progressive decline,
and by 4 h, H3 and H4 acetylation levels were almost back to
baseline. Given that the peak acetylation was observed 1 h after
LPS stimulation, in the next set of experiments, PEMs were pre-
treated, or not, with LAQ824 for 24 h and then stimulated or not
with LPS and replenished LAQ824 as appropriate to maintain
pretreatment conditions for an additional 1 h. By ChIP assay, we
treated with LPS (2 mg/ml), HDI alone, or LPS (2 mg/ml) plus increasing concentrations of the HDI LBH589 (A), TSA (B), or MS-275 (C) as indicated.
After 24 h, supernatants were collected, and production of IL-10 (left panel) and IL-12 (right panel) was determined by ELISA. Shown is a representative
experiment of two experiments with similar results. *p , 0.05, **p , 0.01.
Production of IL-10 and IL-12 by PEMs treated with the pan-HDIs LBH589 or TSA or with the class I selective HDI MS-275. PEMs were
Page 6
found that in response to LPS, histones H3 and H4 display in-
creased acetylation (Fig. 6C, Control LPS) relative to untreated
PEM (Fig. 6C, Control w/o LPS). When PEMs were treated with
HDI alone, no significant changes in acetylation of H3 and H4
were observed (Fig. 6C, LAQ w/o LPS). However, treatment of
PEMs with LPS in the presence of HDI resulted in decreased H3
and H4 acetylation (Fig. 6C, LAQ LPS). This effect was specific
for IL-10 because a similar analysis of H3 and H4 acetylation at
the level of the IL-12 promoter revealed no differences between
PEMs treated with LPS alone and PEMs treated with LPS plus
HDI (Fig. 6D, Control LPS versus LAQ/LPS). Taken together,
treatment of PEMs with LAQ824 resulted in decreased IL-10 gene
transcriptional activity and diminished H3 and H4 acetylation at
the level of the IL-10 gene promoter.
Chromatin changes in the IL-10 gene promoter induced by the
Previously, we have reported the sequence of chromatin mod-
ifications that occur at the level of the IL-10 gene promoter in
macrophages stimulated with LPS (32). Briefly, phosphorylation
of Ser10on H3 is an early event that occurs 30 min after LPS
stimulation. This is followed by increased acetylation of H3 and
H4 at 60 min, subsequent recruitment to the IL-10 gene promoter
of the transcriptional activators Sp1 and STAT3, and, ultimately,
IL-10 gene expression. This sequence of events was affected in
RAW264.7 macrophage cells pretreated with LAQ824 for 24 h
and then stimulated with LPS with replenished LAQ824 (Fig. 7).
First, phosphorylation of Ser10on H3 at the IL-10 promoter has
been shown necessary for transcriptional activation (36). No dif-
ferences in the kinetics of phosphorylation of H3 at the Ser10
position were observed among macrophages treated with LPS
alone (Fig. 7A, solid lines) or cells treated with LAQ824 and LPS
(Fig. 7A, dashed lines). Second, in macrophages treated with LPS,
we observed a peak acetylation of H3 (Fig. 7B) and H4 (Fig. 7C)
at 1 h, which was followed by a progressive decline, and, by 3 h,
H3 and H4 acetylation levels were back to baseline. In sharp
contrast, a diminished H3 and H4 acetylation was observed at all
evaluated time points in cells treated with LAQ824 plus LPS (Fig.
7B, 7C, dashed lines). The effect of LAQ824 upon the global
transcriptional activity of the IL-10 promoter was evaluated next
were treated with LPS alone (2 mg/ml), LAQ824 alone (12.5 nM), or LPS plus LAQ824 as indicated for 24 h. Then, cells were washed with RPMI 1640,
and 5 3 104naive anti-HA transgenic CD4+T cells (A) or 5 3 104anergized CD4+T cells isolated from A20HA-bearing mice (B) were added to the PEMs
in the presence of cognate HA peptide (12.5 mg/ml). After 48 h, supernatants were collected, and IL-2 and IFN-g production was measured by ELISA. The
amount of cytokine production is expressed as picograms per milliliter per 100 clonotype-positive CD4+T cells. Shown is a representative experiment of
three experiments with similar results. PEMs (1 3 105/well) were treated with LPS alone (2 mg/ml) or LPS plus LAQ824 (12.5 nM) in the presence or not
of recombinant murine IL-10 (C) or neutralizing anti-mouse IL-10 Ab (D) for 24 h. Then, cells were washed, with warmed RPMI 1640 and 5 3 104naive
anti-HA transgenic CD4+T cells were added to the PEMs in the presence of cognate HA peptide (12.5 mg/ml). Recombinant murine IL-10 (C) or
neutralizing anti-mouse IL-10 Ab (D) were replenished, or not, in the macrophages/T cell cultures to maintain the respective pretreated conditions, but
without LPS. After 48 h, supernatants were collected, and IFN-g production was measured by ELISA. *p , 0.05, **p , 0.01.
PEMs treated with LAQ824 effectively activate Ag-specific CD4+T cells and restore the function of anergized T cells. PEMs (1 3 105/well)
The Journal of Immunology3991
Page 7
by determining the binding of PolII to the IL-10 gene promoter.
Unlike macrophages treated with LPS alone, only a minimal and
transient increase in PolII binding was observed in macrophages
treated with LAQ824 plus LPS (Fig. 7D).
Sp1 and STAT3 are known IL-10 transcriptional activators that
are recruited to the proximal region of the IL-10 gene promoter in
macrophages stimulated with LPS (37–39). As shown in Fig. 7E,
recruitment of Sp1 peaks at 1 h after LPS stimulation and is then
followed by a rapid decline at 2 h. Similarly, STAT3 binding to the
IL-10 promoter is evident within 60 min of LPS stimulation. This
is followed by a progressive decline to basal levels by 3 h (Fig.
7F). No such kinetics of recruitment of either Sp1 or STAT3 was
observed in cells treated with LAQ824 and LPS (Fig. 7E, 7F,
dashed lines).
PU.1 is a transcriptional repressor that interacts with the distal
IL-10 gene promoter region (40, 41). Recently, we have demon-
treated with 2 mg/ml LPS (open triangles), 12.5 nM LAQ824 (open squares), or the combination thereof (black circles). Cell aliquots were obtained at 1, 6,
and 12 h, and total RNAwas isolated. IL-10 mRNA relative to GAPHD mRNAwas determined by quantitative real-time PCR. Data are depicted as the ratio
of treated cells versus untreated cells. Shown is a representative experiment of three experiments with similar results. B, Kinetics of H3 and H4 acetylation
of the IL-10 gene promoter in response to LPS stimulation. PEMs were treated with LPS (1 mg/ml) for different periods of time (minutes) as indicated.
Nuclear extracts were then obtained, and ChIP using anti-hyperacetylated H3 or anti-hyperacetylated H4 Abs was performed. Shown is a representative
experiment of two experiments performed with similar results. C and D, PEMs were treated or not with LAQ824 (20 nM) for 24 h. Then LAQ824 was
replenished as appropriate to maintain pretreatment conditions, and LPS (1 mg/ml) was added or not to PEM cultures for an additional 60 min. Cells were
then harvested, and nuclear extracts were obtained. ChIP, using anti-hyperacetylated H3 or anti-hyperacetylated H4 Abs, was then performed, and samples
were subject to PCR analysis using primers specific for the IL-10 (C) or IL-12 (D) promoters. IgG and Input (10%) controls are also shown. Signal in-
tensities obtained from ethidium bromide-stained gels were quantified using the ImageQuant 5.2 software (Molecular Dynamics). Shown is a representative
experiment of three performed with similar results.
Changes in IL-10 mRNA expression and histone acetylation of the IL-10 gene promoter in PEMs treated with LAQ824. A, PEMs were
Page 8
strated that HDAC11, by interacting with the distal segment of
the IL-10 gene promoter, negatively regulates the expression of
this cytokine in murine and human APCs (32). As shown in Fig.
7G and 7H, ChIP analysis of the IL-10 gene distal promoter in
macrophages treated with LAQ824 revealed some interesting
findings. Recruitment of PU.1 to the IL-10 gene promoter in cells
treated with LPS reached a peak within 2 h and remained elevated
for the duration of the analysis (Fig. 7G, solid line). In macro-
phages treated with LAQ824, the binding of PU.1 was already
elevated at time zero and increased further in response to LPS
stimulation (Fig. 7G, dashed line). A similar pattern was observed
when we evaluated the kinetics of HDAC11 recruitment to the IL-
10 gene promoter. First, in macrophages treated with LPS alone,
HDAC11 binding was detectable after 2 h, the change was modest
in magnitude, and it remained slightly elevated at 3 h (Fig. 7H,
solid line). In macrophages treated with LAQ824, recruitment of
HDAC11 reached a higher peak (relative to macrophages treated
with LPS alone) within 2 h, and it was followed by a rapid return
to baseline levels at 3 h after LPS stimulation (Fig. 7H, dashed
Therefore, treatment of macrophages with LAQ824 is associated
with several chromatin changes at the level of the IL-10 gene
promoter, among them an enhanced recruitment of the transcrip-
tional repressors PU.1 and HDAC11.
In this study, we have demonstrated that the pan-HDI LAQ824, by
inhibiting IL-10 and increasing the expression of B7.2 and the
production of several proinflammatory mediators, induced inflam-
matory macrophages that effectively activate Ag-specific CD4+
T cells and restore the responsiveness of anergic T cells.
cells were treated with LAQ824 (20 nM) for
24 h. Cells were then washed and treated
or not with LPS (1 mg/ml) and replenished
LAQ824 or not to maintain pretreatment
conditions. Cells were collected at base-
line (time 0) or at 30, 60, 120, or 180 min
after LPS treatment and then subjected to
ChIP analysis using: an Ab to phosphory-
lated Ser10of H3 (A), anti-hyperacety-
lated H3 (B), anti-hyperacetylated H4 (C),
anti-polymerase II (D), anti-Sp1 (E), anti-
STAT3 (F), anti-PU.1 (G), or anti-HDAC11
(H). Quantitative real-time PCR analysis of
the IL-10 gene promoter was performed.
Values were obtained with the Pfaffl method
and are presented relative to input before
immunoprecipitation. Arbitrary units are
presented as percentage relative to no-treat-
ment condition. Error bars represent SD
from triplicates.
Chromatin modifications in
The Journal of Immunology 3993
Page 9
Among the above changes, the most striking effect of LAQ824
was its ability to inhibit the production of the immunosuppressive
cytokine IL-10. Such an effect was also displayed by other mem-
bers of the hydroxamic acid family like LBH589, TSA, and
SAHA, but not by the more specific HDI MS-275, which mainly
targetclassI HDACs.The central role ofIL-10 inthe establishment
and maintenance of T cell anergy (34, 35, 42–44) prompted us to
further investigate the underlying mechanism(s) by which these
particular HDIs inhibit IL-10 production in macrophages.
The dynamic production of pro- and anti-inflammatory medi-
ators at the site of Ag encounter has been shown to shape the
10 plays a key role in negatively regulating these dynamic changes
to prevent self-tissue damage that might otherwise occur if an
ongoing immune response is not kept in check (39). This pro-
tective property of IL-10 imposes, however, a significant barrier to
our efforts to effectively harness antitumor immune responses.
Indeed, production of this cytokine at the tumor site by tumor cells
themselves or infiltrating cells, such as APCs or regulatory T cells,
creates a microenvironment that is conducive to T cell anergy
rather than T cell activation (5). A better understanding of the
genetic and/or epigenetic mechanisms regulating IL-10 production
therefore has important implications to manipulate the inflam-
matory status of APCs and their intrinsic ability to prime, or not,
Ag-specific T cell responses.
Recent studies have shown that IL-10 production is regulated at
the chromatin level by changes in the acetylation status of the gene
promoter (46, 47). For instance, changes in the chromatin struc-
ture of the IL-10 promoter in T cells differentiated into the Th1 or
Th2 phenotype closely regulate IL-10 expression (48). In mac-
rophages, increased acetylation of the IL-10 promoter has been
associated with enhanced transcriptional activity (46). Conversely,
we have recently demonstrated that decreased acetylation of the
IL-10 promoter is associated with decreased IL-10 transcriptional
activity in murine and human APCs (32). Given the above, we
expected that treatment of macrophages with HDIs would result in
increased histone acetylation and increased IL-10 production.
Although an increased global acetylation of histones H3 and H4
was observed in cells treated with LAQ824 (Fig. 1A), the opposite
outcome was observed at the level of the IL-10 gene promoter of
HDI-treated cells. To our surprise, diminished H3 and H4 acety-
lation at the IL-10 promoter was observed at all evaluated time
points in treated macrophages (Fig. 7B, 7C). This decrease in
histone acetylation, which occurs early, might explain the de-
creased recruitment of the transcriptional activators STAT3 and
Sp1 to the IL-10 gene promoter (Fig. 7E, 7F). It is plausible that
a more compacted chromatin due to diminished histone acet-
ylation in the IL-10 promoter region might block the access of
these transcriptional activators to the promoter region resulting in
the decreased IL-10 gene transcriptional activity observed in
LAQ824-treated cells.
However, we are left to explain how HDIs induce decreased
histone acetylation at the level of the IL-10 gene promoter in the
first place. Kinetic analysis of H3 and H4 acetylation provided
some hints. In macrophages pretreated with LAQ824 and then
stimulated with LPS and replenished LAQ824, we observed an
initial acetylation of H3 and H4 that reaches its peak at 1 h
poststimulation. However, the magnitude of these changes was
significantly lower than in macrophages treated with LPS alone.
Following this peak acetylation, we observed a rapid abrogation of
such a response in cells treated with HDI, suggesting either a lack
of stimuli to support H3 and H4 acetylation and/or the induction of
counterregulatory mechanism(s) that attenuated the degree of H3
and H4 acetylation observed in cells treated with LPS alone.
Of particular interest is the finding that following stimulation,
enhanced recruitment of two transcriptional repressors to the distal
promoter of IL-10 was observed, a process that occurs faster for
PU.1 than for HDAC11 (Fig. 7G, 7H). It is plausible therefore that
recruitment of PU.1 and HDAC11 to the IL-10 gene promoter
might represent a counterregulatory mechanism triggered by HDIs
to diminish H3 and H4 acetylation and block the sequence of
events that lead to IL-10 gene transcriptional activation. Sup-
porting the above, we have recently demonstrated that over-
expression of HDAC11 in the macrophage cell line RAW264.7
resulted in decreased H3 and H4 acetylation of the IL-10 gene
promoter and inhibition of IL-10 gene transcriptional activity (32).
Interestingly, Bradbury et al. (49) have found that treatment of
myeloid leukemic blasts with TSA, a member of the hydroxamic
acid family of HDIs, resulted in a 60–200-fold induction of
HDAC11 mRNA expression. Similarly, we have also found that
RAW264.7 cells treated with either TSA or LAQ824 display in-
creased HDAC11 mRNA and protein expression (data not shown).
Such an effect of HDIs upon HDAC11 expression might explain,
at least in part, the increased recruitment of this particular HDAC
to the IL-10 gene promoter in cells treated with LAQ824. The
mechanism(s) by which HDI increases the expression of HDAC11
in macrophages, however, remains to be elucidated.
An additional observation in macrophages treated with LAQ824
is their enhanced expression of B7.2 and increased production
of several proinflammatory mediators, in particular IL-12. It is
plausible that these effects are secondary to the inhibition of IL-10,
a cytokine that has been shown to downregulate the expression
of costimulatory molecules and the production of IL-12 in APCs
(50, 51). This switch from an IL-10– to an IL-12–producing and
B7.2-expression state is critical in polarizing T cells toward Th1
cell-mediated immunity. In our system, the demonstration that H3
and H4 acetylation in the IL-12 promoter was neither inhibited
nor enhanced in cells treated with LAQ824 (Fig. 6D) suggests
that the enhanced production of this proinflammatory mediator in
treated macrophages might be secondary to the abrogation of IL-10
production by LAQ824. Our experiments using rIL-10 also point
to a contributory role of IL-10 inhibition (induced by LAQ824) in
the enhanced APC function displayed by treated macrophages
because this effect was reversed when rIL-10 was added back to
the cultures. It remains to be demonstrated whether the upregu-
lation of B7.2 is a direct or indirect effect of LAQ824. It is
noteworthy to mention that B7.2 was preferentially upregulated in
treated macrophages, whereas no changes were observed in vari-
ous other costimulatory molecules (data not shown). Although the
exact mechanism for this selectivity has yet to be elucidated, our
results are consistent with observations in human DCs whereby
IL-10 mediated downregulation of B7.2 but not B7.1 (50).
Our findings are at odds with the studies by Reddy et al. (23),
who recently found that the HDI SAHA actually attenuated in-
flammatory responses in DCs through IDO-dependent mecha-
nisms. Injection of DCs treated ex vivo with SAHA also decreased
the severity of graft-versus-host disease in their murine allogeneic
bone marrow transplantation model. Several differences between
Reddy's study (23) and ours may explain these seemingly con-
flicting data. First, in our in vitro system measuring cytokine se-
cretion, we used macrophages that were treated with HDI and LPS
given at the same time. In their study, DCs primarily, but in one
experiment macrophages, were pretreated with SAHA prior to
stimulation with TLR agonist. Second, they found that SAHA
treatment did not induce significant changes in the production of
IL-10 by DCs. Supporting their observation, we have also found
that among all of the members of the hydroxamic family of HDI,
SAHA is the weakest inhibitor of IL-10 production in macro-
Page 10
phages (data not shown). It is plausible therefore that differences
among members of the same family of HDI, with LAQ824 and
LBH589 being more potent inhibitors of IL-10 production than
SAHA, might explain at least in part the divergent effects of these
HDI upon the inflammatory status of DCs and/or macrophages. Of
note, the overall potencies of HDIs must also be reconciled with
the relative potencies, as pan-HDIs do not inhibit all members of
the HDAC family at equimolar concentrations, which may explain
the differing effects on a single target such as IL-10 (52). Our
findings are, however, consistent with reports by others indicating
that HDIs can potentiate inflammatory and antitumor responses
in vitro and in vivo (25–27). Among the latter, Vo et al. (27) has
demonstrated an enhanced T cell-mediated immunity in tumor-
bearing mice treated with LAQ824. Whether the preferential ex-
pansion of adoptively transferred Ag-specific T cells observed in
this model is the result of T cell interaction with inflammatory
APCs induced by in vivo treatment with LAQ824 treatment re-
mains to be explored.
Taken together, LAQ824-treated macrophages cannot only more
potently activate naive T cells, but also restore the reactivity of
anergized T cells from tumor-bearing mice. LAQ824 by inducing
inflammatory cells can potentially tip the balance toward T cell
activation rather than T cell anergy, an effect that holds promise for
the use of HDIs in the adjuvant setting to ultimately improve the
efficacy of cancer immunotherapy.
The authors have no financial conflicts of interest.
1. Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, and S. Amigorena. 2002.
Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev.
Immunol. 20: 621–667.
2. Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic
dendritic cells. Annu. Rev. Immunol. 21: 685–711.
3. Sotomayor, E. M., I. Borrello, F. M. Rattis, A. G. Cuenca, J. Abrams,
K. Staveley-O'Carroll, and H. I. Levitsky. 2001. Cross-presentation of tumor
antigens by bone marrow-derived antigen-presenting cells is the dominant
mechanism in the induction of T-cell tolerance during B-cell lymphoma pro-
gression. Blood 98: 1070–1077.
4. Cuenca, A., F. Cheng, H. Wang, J. Brayer, P. Horna, L. Gu, H. Bien,
I. M. Borrello, H. I. Levitsky, and E. M. Sotomayor. 2003. Extra-lymphatic solid
tumor growth is not immunologically ignored and results in early induction of
antigen-specific T-cell anergy: dominant role of cross-tolerance to tumor anti-
gens. Cancer Res. 63: 9007–9015.
5. Rabinovich, G. A., D. Gabrilovich, and E. M. Sotomayor. 2007. Immunosup-
pressive Strategies that are Mediated by Tumor Cells. Annu. Rev. Immunol. 25:
6. Staveley-O' Carroll, K., E. Sotomayor, J. Montgomery, I. Borrello, L. Hwang,
S. Fein, D. Pardoll, and H. Levitsky. 1998. Induction of antigen-specific T cell
anergy: An early event in the course of tumor progression. Proc. Natl. Acad. Sci.
7. Bogen, B., L. Munthe, A. Sollien, P. Hofgaard, H. Omholt, F. Dagnaes,
Z. Dembic, and G. F. Lauritzsen. 1995. Naive CD4+ T cells confer idiotype-
specific tumor resistance in the absence of antibodies. Eur. J. Immunol. 25:
8. Pardoll, D. 2003. Does the immune system see tumors as foreign or self? Annu.
Rev. Immunol. 21: 807–839.
9. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin,
B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, et al. 2002. Potential reg-
ulatory function of human dendritic cells expressing indoleamine 2,3-dioxyge-
nase. Science 297: 1867–1870.
10. Cheng, F., H. W. Wang, A. Cuenca, M. Huang, T. Ghansah, J. Brayer,
W. G. Kerr, K. Takeda, S. Akira, S. P. Schoenberger, et al. 2003. A critical role
for Stat3 signaling in immune tolerance. Immunity 19: 425–436.
11. Evel-Kabler, K., X. T. Song, M. Aldrich, X. F. Huang, and S. Y. Chen. 2006.
SOCS1 restricts dendritic cells' ability to break self tolerance and induce anti-
tumor immunity by regulating IL-12 production and signaling. J. Clin. Invest.
116: 90–100.
12. Horna, P., and E. M. Sotomayor. 2007. Cellular and molecular mechanisms
of tumor-induced T-cell tolerance. Curr. Cancer Drug Targets 7: 41–53.
13. Song, X. T., K. Evel-Kabler, L. Shen, L. Rollins, X. F. Huang, and S. Y. Chen.
2008. A20 is an antigen presentation attenuator, and its inhibition overcomes
regulatory T cell-mediated suppression. Nat. Med. 14: 258–265.
14. Georgopoulos, K. 2009. From immunity to tolerance through HDAC. Nat.
Immunol. 10: 13–14.
15. Foster, S. L., D. C. Hargreaves, and R. Medzhitov. 2007. Gene-specific control of
inflammation by TLR-induced chromatin modifications. Nature 447: 972–978.
16. Glozak, M. A., and E. Seto. 2007. Histone deacetylases and cancer. Oncogene
26: 5420–5432.
17. Yang, X. J., and E. Seto. 2003. Collaborative spirit of histone deacetylases in
regulating chromatin structure and gene expression. Curr. Opin. Genet. Dev. 13:
18. Yang, X. J., and E. Seto. 2008. The Rpd3/Hda1 family of lysine deacetylases:
from bacteria and yeast to mice and men. Nat. Rev. Mol. Cell Biol. 9: 206–218.
19. O'Connor, O. A., M. L. Heaney, L. Schwartz, S. Richardson, R. Willim,
B. MacGregor-Cortelli, T. Curly, C. Moskowitz, C. Portlock, S. Horwitz, et al.
2006. Clinical experience with intravenous and oral formulations of the novel
histone deacetylase inhibitor suberoylanilide hydroxamic acid in patients with
advanced hematologic malignancies. J. Clin. Oncol. 24: 166–173.
20. Marks, P. A. 2007. Discovery and development of SAHA as an anticancer agent.
Oncogene 26: 1351–1356.
21. Camelo, S., A. H. Iglesias, D. Hwang, B. Due, H. Ryu, K. Smith, S. G. Gray,
J. Imitola, G. Duran, B. Assaf, et al. 2005. Transcriptional therapy with the
histone deacetylase inhibitor trichostatin A ameliorates experimental autoim-
mune encephalomyelitis. J. Neuroimmunol. 164: 10–21.
22. Glauben, R., A. Batra, I. Fedke, M. Zeitz, H. A. Lehr, F. Leoni, P. Mascagni,
G. Fantuzzi, C. A. Dinarello, and B. Siegmund. 2006. Histone hyperacetylation
is associated with amelioration of experimental colitis in mice. J. Immunol. 176:
23. Reddy, P., Y. Sun, T. Toubai, R. Duran-Struuck, S. G. Clouthier, E. Weisiger,
Y. Maeda, I. Tawara, O. Krijanovski, E. Gatza, et al. 2008. Histone deacetylase
inhibition modulates indoleamine 2,3-dioxygenase-dependent DC functions and
regulates experimental graft-versus-host disease in mice. J. Clin. Invest. 118:
24. Reddy, P., Y. Maeda, K. Hotary, C. Liu, L. L. Reznikov, C. A. Dinarello, and
J. L. Ferrara. 2004. Histone deacetylase inhibitor suberoylanilide hydroxamic
acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia
effect. Proc. Natl. Acad. Sci. USA 101: 3921–3926.
25. Tomasi, T. B., W. J. Magner, and A. N. Khan. 2006. Epigenetic regulation of
immune escape genes in cancer. Cancer Immunol. Immunother. 55: 1159–1184.
26. Khan, A. N., and T. B. Tomasi. 2008. Histone deacetylase regulation of immune
gene expression in tumor cells. Immunol. Res. 40: 164–178.
27. Vo, D. D., R. M. Prins, J. L. Begley, T. R. Donahue, L. F. Morris, K. W. Bruhn,
P. de la Rocha, M. Y. Yang, S. Mok, H. J. Garban, et al. 2009. Enhanced anti-
tumor activity induced by adoptive T-cell transfer and adjunctive use of the
histone deacetylase inhibitor LAQ824. Cancer Res. 69: 8693–8699.
28. Kirberg, J., A. Baron, S. Jakob, A. Rolink, K. Karjalainen, and H. von Boehmer.
1994. Thymic selection of CD8+ single positive cells with a class II major
histocompatibility complex-restricted receptor. J. Exp. Med. 180: 25–34.
29. Vicente-Suarez, I., Y. Takahashi, F. Cheng, P. Horna, H. W. Wang, H. G. Wang,
and E. M. Sotomayor. 2007. Identification of a novel negative role of flagellin in
regulating IL-10 production. Eur. J. Immunol. 37: 3164–3175.
30. Sotomayor, E. M., Y. X. Fu, M. Lopez-Cepero, L. Herbert, J. J. Jimenez,
C. Albarracin, and D. M. Lopez. 1991. Role of tumor-derived cytokines on the
immune system of mice bearing a mammary adenocarcinoma. II. Down-
regulation of macrophage-mediated cytotoxicity by tumor-derived granulocyte-
macrophage colony-stimulating factor. J. Immunol. 147: 2816–2823.
31. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-
time RT-PCR. Nucleic Acids Res. 29: e45.
32. Villagra, A., F. Cheng, H. W. Wang, I. Suarez, M. Glozak, M. Maurin,
D. Nguyen, K. L. Wright, P. W. Atadja, K. Bhalla, et al. 2009. The histone
deacetylase HDAC11 regulates the expression of interleukin 10 and immune
tolerance. Nat. Immunol. 10: 92–100.
33. Shurin, M. R., L. Lu, P. Kalinski, A. M. Stewart-Akers, and M. T. Lotze. 1999.
Th1/Th2 balance in cancer, transplantation and pregnancy. Springer Semin.
Immunopathol. 21: 339–359.
34. Groux, H., M. Bigler, J. E. de Vries, and M. G. Roncarolo. 1996. Interleukin-10
induces a long-term antigen-specific anergic state in human CD4+ T cells. J.
Exp. Med. 184: 19–29.
35. Schwartz, R. H. 2003. T cell anergy. Annu. Rev. Immunol. 21: 305–334.
36. Lucas, M., X. Zhang, V. Prasanna, and D. M. Mosser. 2005. ERK activation
following macrophage FcgammaR ligation leads to chromatin modifications at
the IL-10 locus. J. Immunol. 175: 469–477.
37. Benkhart, E. M., M. Siedlar, A. Wedel, T. Werner, and H. W. Ziegler-Heitbrock.
2000. Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression. J.
Immunol. 165: 1612–1617.
38. Tone, M., M. J. Powell, Y. Tone, S. A. Thompson, and H. Waldmann. 2000. IL-
10 gene expression is controlled by the transcription factors Sp1 and Sp3. J.
Immunol. 165: 286–291.
39. Saraiva, M., and A. O'Garra. 2010. The regulation of IL-10 production by im-
mune cells. Nat. Rev. Immunol. 10: 170–181.
40. Reuss, E., R. Fimmers, A. Kruger, C. Becker, C. Rittner, and T. Ho ¨hler. 2002.
Differential regulation of interleukin-10 production by genetic and environ-
mental factors - a twin study. Genes Immun. 3: 407–413.
41. Suzuki, M., T. Yamada, F. Kihara-Negishi, T. Sakurai, and T. Oikawa. 2003. Direct
association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for
PU.1-mediated transcriptional repression. Oncogene 22: 8688–8698.
42. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O'Garra. 2001.
Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19: 683–
The Journal of Immunology3995
Page 11
43. Li, M. O., and R. A. Flavell. 2008. Contextual regulation of inflammation:
a duet by transforming growth factor-beta and interleukin-10. Immunity 28: 468–
44. Rubtsov, Y. P., J. P. Rasmussen, E. Y. Chi, J. Fontenot, L. Castelli, X. Ye,
P. Treuting, L. Siewe, A. Roers, W. R. Henderson, Jr. et al. 2008. Regulatory
T cell-derived interleukin-10 limits inflammation at environmental interfaces.
Immunity 28: 546–558.
45. Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, and A. Lanzavecchia. 2005.
Selected Toll-like receptor agonist combinations synergistically trigger a T
helper type 1-polarizing program in dendritic cells. Nat. Immunol. 6: 769–776.
46. Zhang, X., J. P. Edwards, and D. M. Mosser. 2006. Dynamic and transient
remodeling of the macrophage IL-10 promoter during transcription. J. Immunol.
177: 1282–1288.
47. Yao, Y., W. Li, M. H. Kaplan, and C. H. Chang. 2005. Interleukin (IL)-4 inhibits
IL-10 to promote IL-12 production by dendritic cells. J. Exp. Med. 201: 1899–
48. Im, S. H., A. Hueber, S. Monticelli, K. H. Kang, and A. Rao. 2004. Chromatin-
level regulation of the IL10 gene in T cells. J. Biol. Chem. 279: 46818–46825.
49. Bradbury, C. A., F. L. Khanim, R. Hayden, C. M. Bunce, D. A. White,
M. T. Drayson, C. Craddock, and B. M. Turner. 2005. Histone deacetylases in
acute myeloid leukaemia show a distinctive pattern of expression that changes
selectively in response to deacetylase inhibitors. Leukemia 19: 1751–1759.
50. Buelens, C., V. Verhasselt, D. De Groote, K. Thielemans, M. Goldman, and
F. Willems. 1997. Interleukin-10 prevents the generation of dendritic cells from
human peripheral blood mononuclear cells cultured with interleukin-4 and
granulocyte/macrophage-colony-stimulating factor. Eur. J. Immunol. 27: 756–
51. Gerlini, G., A. Tun-Kyi, C. Dudli, G. Burg, N. Pimpinelli, and F. O. Nestle. 2004.
Metastatic melanoma secreted IL-10 down-regulates CD1 molecules on den-
dritic cells in metastatic tumor lesions. Am. J. Pathol. 165: 1853–1863.
52. Atadja, P. 2009. Development of the pan-DAC inhibitor panobinostat (LBH589):
successes and challenges. Cancer Lett. 280: 233–241.


text:
Music video used to build computer vision algorithm
By Ben Coxworth
May 19, 2011
Although already incorporated into devices such as the Microsoft Kinect gaming console, the ability of computer vision systems to recognize specific body poses is still very much a work in progress. One of the big challenges involves the chaos that such systems encounter in real-world use - while it's one thing to initially train a computer to recognize a given person standing and pointing against a neutral background, for instance, it's quite another to expect it to recognize that same stance in visual data where variables such as background, clothing and body type are constantly changing. A new interactive music video from Dutch electronic band C-Mon & Kypski, however, may help address that problem.
The band is inviting people to go to the One Frame of Fame website, where they will be presented with a variety of freeze frames of band members from the video for the song Less is More. Participants can then use their webcam to take a picture of themselves, in the same pose as the band member, and submit it to the project. Those snapshots are edited into the video, taking the place of the original frame that they copied, with a newly-updated version of the complete video going up once an hour.
Any one of the original "seed" frames displays a band member against a blank background, while the multiple submitted imitations of that seed display a variety of people in a variety of settings - usually with chaotic backgrounds. The only thing that the seeds and their various imitations have in common is the pose of the human subjects.
A team at New York University's Courant Institute of Mathematical Sciences is using this visual database to develop a "pose estimation" algorithm for use in computer vision systems. Having an initial clean base image of each pose, combined with a variety of images of that same pose shot under different conditions, is ideal for the purpose.
"This turned out to be the perfect data source for developing an algorithm that learns to compute similarity based on pose," said NYU researcher Graham Taylor. "Armed with the band's data and a few machine learning tricks up our sleeves, we built a system that is highly effective at matching people in similar pose but under widely different settings."Share
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text:
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text:
New Film Renders Screen Reflection Almost Non-Existent 112
from the flat-screen dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
SONY has been on my "do not patronize" list for years and will remain so for the foreseeable future.
SONY has been on my "do not patronize" list for years and will remain so for the foreseeable future.
Cue the people telling us to just get over the root-kit or whatever else Sony has done.
Sony long ago declared war against the consumer, and much like an aged convict in prison, it does not matter how goody two-shoes they are today. They still murdered somebody. Parole Denied. Let them turn to dust.
Only the complete destruction of Sony will assuage they deep and intense desire for justice against Sony and their evil deeds.
I don't care if they release the fucking cure for cancer tomorrow. The execution is still going to proceed on schedule.
The key to the low reflectance is the formation of an uneven surface, which consists of both concave and convex structures (tiny bumps) that cover the entire film.
From that description it sounds like Sony has reinvented screens with matte finish. Surely there is more to this.
Research?! On teh Internetz?! You crazy, crazy man!
If a 6600 used paper tape instead of core memory, it would use up tape at about 30 miles/second. -- Grishman, Assembly Language Programming


text:
|After winning at Texas, Greg Biffle has surged to the top of the Sprint Cup standings. (US Presswire)
Updated April 16
Jimmie Johnson led a race-high 156 laps in Saturday night's Samsung Mobile 500 at Texas Motor Speedway and turned in another solid performance in the Lone Star State.
But it wasn't enough for the five-time Sprint Cup champion.
"Yeah, man, second sucks," Johnson said after his runner-up finish to Greg Biffle.
You can understand Johnson's frustration. Despite running well since his opening week problems at Daytona, the Hendrick Motorsports driver has yet to find Victory Lane.
In fact, you have to go back to the fall to find the last time the No. 48 Chevrolet crossed the finish line first.
|More on NASCAR
|Related links
He's come close. Saturday night was heading in the right direction until Biffle ran him down. Two weeks ago Johnson appeared to be in great shape at Martinsville until some late race fireworks swept him up in an accident with teammate Jeff Gordon.
Although he was disappointed about not sealing the deal in Texas, the Martinsville turn of events really frustrated Johnson.
"Martinsville, because we're much closer to the end," Johnson said comparing the two. "But they're both frustrating. The racer in me wants to win. We've been really close over the last five or six months. You look through last year even through different points, leading a lot of races and didn't execute at the end for whatever reason. It's nice to be running well. It's nice to be in this position. But I want to get back to our winning ways really soon."
If he does that before teammates Gordon, Dale Earnhardt Jr. and Kasey Kahne, Johnson will bring team owner Rick Hendrick his long-awaited 200th career Sprint Cup win.
However, that potential honor is not weighing on Johnson's mind.
"To be honest it hasn't played much on my mind because I just want to win and I know winning is what my job is about, winning is what Hendrick Motorsports is about, and if we win we'll take care of the 200th," he said. "I seem to think about the 200th after the fact and think, man, Martinsville could have been an amazing one, two, three, this weekend, previous weekends."
It hasn't happened yet, but there's always the next weekend for Johnson and Hendrick, which happens to take place at Kansas Speedway in a few days.
|Brad Keselowski
|Took over the Chase lead by 20 points thanks to a sixth place finish and Jimmie Johnson?s misfortune. This is now the Penske Racing driver?s championship to lose.
|Jimmie Johnson
|Saw his seven point lead evaporate thanks to a cut tire and trip into the wall on Sunday. Johnson now must hope a similar such fate happens to Keselowski next week at Homestead to have any shot at a sixth title.
|Kyle Busch
|Nearly ran the table in Phoenix by leading all practices and taking the pole had he been able to win the race. But a poor decision on lane choice for a late restart cost Busch a shot at Victory Lane.
|Kasey Kahne
|Ending the year on a positive note but wasn?t able to match his Phoenix victory of last November. Still a solid day with a fourth place performance.
|Clint Bowyer
|Was the victim of Jeff Gordon?s revenge that completely eliminated Bowyer from championship contention. He wasn?t an angel in the turn of events but Bowyer definitely got the worst of the situation.
|Denny Hamlin
|Hamlin?s second place finish in Phoenix a welcome rebound from two weeks of disappointment for the Joe Gibbs Racing driver. He?ll be a factor for a win next Sunday in Homestead.
|Kevin Harvick
|Snapped a 44-race winless drought with Sunday?s win ironically coming on the weekend when reports of his evacuation from Richard Childress Racing to Stewart-Haas Racing in 2014 broke out.
|Greg Biffle
|A decent day for ?The Biff? in Phoenix, who was able to dodge all the last lap craziness to bring home his Roush Fenway Racing Ford seventh.
|Joey Logano
|Minding his own business until getting swept up in the Gordon-Bowyer fight. Logano was on his way to a nice effort to follow up his ninth Nationwide Series win on Saturday.
|Ryan Newman
|Rough way to earn a fifth place finish as Newman was one of the unfortunate souls to get caught in the last lap crash on the main straightaway.
|Tony Stewart
|Not a god day at all for Smoke who spun late after making contact racing for position. A 19th place finish for Stewart.
|Matt Kenseth
|Will make the final Sprint Cup start of his career for Roush Fenway Racing next week at Homestead on the heels of a 14th place effort in Phoenix.
|Martin Truex Jr.
|His race was over nearly before it started with a blown engine. After a stellar regular season Truex Jr. has suffered mightily throughout the Chase.
|Carl Edwards
|An 11th place finish for Edwards as he brings to a close one of the most disappointing seasons in his career.


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Description from the owner
- 2 bedrooms
- 1 bathrooms
- Sleeps 7
- Apartment
Bedrooms: 2
Bathrooms: 1
Sleeps: 7
Type: Apt. / CondoBrand new
Description from the owner
The apartment "Poppy" is surrounded by the nature of the countryside, but at the same time it is close to the most beautiful Tuscan towns – Lucca, Pisa, Pistoia, Vinci, Firenze and so on - famous for their art, history, vineyards and cuisine. The apartment "Poppy" belongs to the Corte Tommasi Residence in the small Tuscan village of Orentano - below you can find the description of the Residence.
The apartment is brand new, built and furnished in the typical Tuscan style with high quality materials. In the bedrooms there is air-conditioning and in every room there is heating.
The apartment "Poppy/Papavero" gives out onto the panoramic ...Read more
About the owner
About the owner
Helen R.
Helen R.
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Calendar updated 4 months ago
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Brand new
Orentano, Tuscany, Italy
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Stewardship brings Casino Royale theme back for TrailfestJames Wilson
Staff Writer
Those yearning for a Monte Carlo-type casino experience without the hassle and expense of a trans-Atlantic flight are in luck.
The Sierra Buttes Trail Stewardship plans to bring back its "Casino Royale" theme to its third annual Trailfest this Saturday night at Nakoma Golf Resort and Spa. Doors will open at 5 p.m.
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The Dish
Tag: The Four Seasons
The ultimate burger? Four Seasons' chef Darryl Moiles combines cravings for burger he'll serve at high-profile fest event
- 7:25 pm Friday, November 9th, 2012 by M.M. Cloutier
For an upcoming beachside burger fest bringing five high-profile visiting chefs and TV personalities to Palm Beach at the Four Seasons, the resort's executive chef Darryl Moiles has been experimenting with burger combinations and has struck upon what could amount to a crave-attack finalist: a burger topped with, among other things, a "secret" sauce and lobster salad.
OK, the Burgers by the Beach event in the Dec. 7-11 Palm Beach Food & Wine Festival lineup isn't a competition, but Moiles' beef-and-lobster burger should stack up nicely against signature burgers served at the affair by The Food Network's Robert Irvine, Marc Summers and Jeff [More]
PB's Four Seasons Is Firing Up the Grill With Beachfront Seafood Bakes
- 6:47 pm Wednesday, September 26th, 2012 by M.M. Cloutier
"The Perfect Storm" is coming to Palm Beach's Four Seasons; alas, not with George Clooney, but with clams, lobster meat, chicken, Italian sausage, potatoes and corn on the cob.
"The Perfect Storm" is a dish on the docket for the Four Seasons' monthly series of alfresco, beachfront "seafood bakes" at the resort's Atlantic Bar & Grill, where the evening scene unfolds amid twinkling tree lights, lantern-illuminated tables and music.
The family-style "bakes" start Friday (Sept. 28) and continue, with one 7 p.m. seating, on the last Friday of each month through December.
Each main course will be prepared, in view, by Four Seasons chefs at the restaurant's outdoor grill.
The monthly changing menu might [More]
2 PB Resorts Among Dining Venues Jazzed About Florida Lobster's Harvesting-Season Kickoff Today; Specials to Include Spiny Lobster at $8.40/Pound
- 5:23 pm Monday, August 6th, 2012 by M.M. Cloutier
This Wednesday (Aug. 8), The Four Seasons plans to have 100 pounds of Florida's spiny lobster on hand in what amounts to a one-day blowout sale—and if years past are any indication, diners at the resort will consume every bit of it at the price of $8.40 a pound before the day is through.
Throughout the day at the resort's alfresco, oceanfront Atlantic Bar & Grill, Florida lobster—now commercially available since the spiny lobster season began today and runs through March—will be served grilled (the tails) with cilantro-lime butter and a choice of a-la-carte accompaniments such as grilled Florida corn-on-the-cob ($2 [More]
Food For Thought: Chavigny's Peach Melba, Ocean Bistro's `Category 5′ Dog
- 5:09 pm Monday, June 25th, 2012 by M.M. Cloutier
Over the years, scads of chefs have riffed on Escoffier's 1890s-invented Peach Melba, which originally featured peaches and spun sugar-laced vanilla ice cream, and later, raspberry sauce, too.
But beer sorbet as a component? That's en element in Cafe Boulud Pastry Chef Arnaud Chavigny's "modern version of the old classic."
Chavigny's Peach Melba (shown right) features such layers as a raspberry jam-filled raspberry financier, fresh poached peach, and a quenelle of raspberry lambic and the aforementioned beer sorbet, plus fresh shaved peach, roasted slivered almonds, white peach sauce and rosettes of vanilla chantilly.
Meanwhile, the Four Seasons has introduced a number of new [More]
PB's Cafe Boulud Has New Canadian Sibling
- 7:16 pm Wednesday, June 13th, 2012 by M.M. Cloutier
The newest restaurant by internationally known chef/restaurateur Daniel Boulud—the mastermind behind Palm Beach's Cafe Boulud, several New York restaurants and others as far afield as Beijing and Singapore—has opened at Canada's Ritz-Carlton Montreal, which recently debuted a $100-plus-million renovation.
It has been a very Canadian season for 57-year-old Boulud—a longtime Montreal visitor who counts a few Montreal chefs among his good friends—as the New Yorker and Lyon, France native is opening another restaurant at the new Four Seasons in Toronto in late September.
Boulud's Ritz-Carlton Montreal restaurant, Maison Boulud, features an interior designed by the Japanese firm Super Potato—many of the furnishings were [More]
Stone Crab Claws: Get 'Em While You Can; Some PB Restaurants Still Serve The Delicacies, Others Wind Down Toward May 15 Harvesting-Season End
- 6:28 pm Monday, May 7th, 2012 by M.M. Cloutier