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The Law Firm Of Weiss & Lurie Investigates The Acquisition Of American Medical Systems Holdings, Inc.
Weiss & Lurie, a national class action and shareholder rights law firm with offices in New York City and Los Angeles, is investigating possible breaches of fiduciary duty and other violations of law by the Board of Directors of American Medical Systems Holdings, Inc. (NASDAQ: AMMD) arising from its proposed acquisition by Endo Pharmaceuticals Holdings, Inc. (NASDAQ: ENDP).
Under the terms of the agreement, American Medical stockholders will receive $30 per share. Additionally, on April 10, 2011, American Medical adopted a Retention Bonus Plan, providing certain of its executive officers with a cash bonus in connection with continued employment following the merger.
Weiss & Lurie is investigating whether American Medical's Board acted in the best interests of its shareholders in approving the transaction and whether the Board properly sought to maximize shareholder value.
If you own American Medical shares and would like more information about your rights as a shareholder or additional information concerning our investigation, please contact Michael A. Rogovin either by email at firstname.lastname@example.com or by telephone at (888) 593-4771.
Weiss & Lurie has litigated hundreds of stockholder class and derivative actions for violations of corporate and fiduciary duties. We have recovered over a billion dollars for defrauded institutions and individuals and obtained important corporate governance in these cases. If you have information or would like legal advice concerning possible corporate wrongdoing (such as insider trading, waste of corporate assets, accounting fraud, or issuing materially misleading press releases or SEC filings), consumer fraud (such as false advertising, defective products, or other deceptive business practices), or anti-trust violations, please email us at email@example.com or fill out the form on our website, http://www.weisslurie.com/contact/report_fraud/. Attorney Advertising. Past results do not guarantee a similar outcome.Select the service that is right for you!
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NEW YORK – Toyota on Wednesday said it was likely to slow production at some US plants due to a shortage of parts from quake-hit Japan. "We told team members that production interruption is likely, but we don't know how much, where or for how long," said Toyota US spokesman…
By Katie Halper
Read MoreBy Gary Younge, The Guardian
Read MoreRaw Story is a progressive news site that focuses on stories often ignored in the mainstream media. While giving coverage to the big stories of the day, we also bring our readers' attention to policy, politics, legal and human rights stories that get ignored in an infotainment culture driven solely by pageviews.
Founded in 2004, Raw Story reaches 9 million unique readers per month and serves more than 30 million pageviews.
Copyright © 2004-2013 Raw Story Media, Inc. All rights reserved.

subset: commoncrawl


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Kemba Walker goes from being NCAA champ to locked-out NBA rookie
As Walker took the court in the summer league Clash of the Superstars with his white-shirted teammates Saturday, Goodman League commissioner Miles Rawls continued his usual comedian-style running commentary as the game's announcer.
"Kemba Walker," Rawls called out as Walker connected on a jumper. "He has yet to get a paycheck. He's mad as hell."Also:Walker has been in touch with several of his Bobcats teammates, including Gerald Henderson, Tyrus Thomas, D.J. Augustin and Corey Maggette, and worked out with Thomas and Henderson shortly before coming to D.C. Walker's next stop is Provo, Utah, where he'll play in the Jimmer All-Star Game on Thursday.
Interview — Charlotte Bobcats' Kemba Walker says that NBA lockout feels weird
Interview — Charlotte Bobcats' Bismack Biyombo guarantees he'll play in NBA this season
The game tonite (7 pm MT) will be streamed here:


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Actress Jessica Lange, 64, has fronted a new ad campaign for fashion designer Marc Jacobs's latest beauty line.
Academy Award-winning actress Joan Fontaine, who found stardom playing naive wives in Alfred Hitchcock's "Suspicion" and "Rebecca" and also was featured in films by Billy Wilder, Fritz Lang and Nicholas Ray, died Sunday.
Necessity is the mother of all invention. And evolving concepts of restaurants actually help prove that, yet again.
The DTC sightholders who attend the first sale sight of De Beers at Gabarone on Monday informed that the company has increased prices in all the diamond categories by an average of 5 per cent.
AC Milan defender Mattia De Sciglio could join the club's casualty list after hobbling off injured in a 1-0 Champions League defeat to Atletico Madrid and being taken to hospital on Wednesday.
Roma midfielder Daniel De Rossi was banned for three matches on Monday for punching Inter Milan's Mauro Icardi at the weekend.
Actor Amit Sadh is all geared up to take part in Tour De India 2013 scheduled to take place Sunday.
Swiss team IAM Cycling will ride the Tour de France for the first time as one of four teams to receive wildcard invitations for this year's race, organisers said on Tuesday.
Sanjay Agarwal and Shiv Prakash driving a Gypsy finished as first runner up with cumulative timing of 10.10.35 Hrs while Harpreet Bawa and Parminder Thakur were second runner. Jagmeet Gill and Chandan Sen driving a Gypsy lifted the trophy in the Adventure Trial 4W category with an overall penalty of 0.03.46 Hrs. Subir Roy and Mohammad Mustafa was first runner up with a penalty of 0.03.53 Hrs.
The 15th Raid de Himalaya, the most exciting motorsport event in India, culminated in Leh after travelling over 1800 kms over 6 days which included the highest motorable passes and inhospitable sub zero temperatures. Suresh Rana and Ashwin Naik driving a Grand Vitara were crowned champions in the X-Treme four wheeler (4W) category. Rana has won this coveted trophy for a ninth time, and second time with Ashwin Naik as co-driver. He had an overall timing of 9.46.23 Hrs over 6 days.
The scooter is going places, literally. The Scooter Class (Xtreme) is the new official entry at one of the top ten toughest motorsport rallies of the world -Raid De Himalaya which is held in awe by rallyists in the country and abroad. It will be organized from October 4 to 12. The Raid will begin from Shimla and will traverse some of the highest motorable roads in the world to end at Leh.


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The Museum Café serves a selection of breakfast, lunch and pastries from local bakeries. Open daily 9 a.m. to 4 p.m., Wednesday 9 a.m. to 7 p.m.Museum admission is not required.
Learn More
Cryptogramma acrostichoides
Along Brumley Creek, La Sal Mountains, Utah.
Elevation: 8,858 - 9,842 feet
Collected by: P.A. Rydberg and A.O. Garett
July 12, 1911


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Elfree Stories
Author:Muller, Ann
Publisher: Collins Press, The
Summary: One day while walking in the woods the author came upon an elf who bowed and said his name was Elfree of The Leaf. Many encounters follow as he tells stories of teaching a cat to dance, of storm goblins, frogs, dragonflies, the Wizard of Ravity, and many other tales of woodland and elfin lore. Elfree, always talking in rhyme, also describes how he lives in the forest around the authors' home. Children will enjoy the ...rhyme, the illustrations, the humor, and the story lines. [read more]
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Publisher: Collins Press, The
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- Product Details
Mercedes Benz Series Frame by License 2 Bling®. Featuring Swarovski™ crystals, proprietary construction and jewelry-like craftsmanship, License 2 Bling frames are the choice for celebrities, musicians and car fans everywhere! Also Available TORX Security Screws With Key & Chrome Caps. It is recommended to protect the frame from extreme temperatures and remove it during the winter season. Included caps. Will Fit US Models Only.
- Exclusive Swarovski chaton crystals create a rainbow of color
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In our quest to create the world's most beautiful frame, License 2 Bling chose the world's most beautiful crystal style, the "chaton". Unlike the standard "flat back" crystal, the pointed back chaton has more facets, brilliance and shine. Luxury jewelers use chaton crystals and diamonds exclusively. Nothing else comes close.
An expert's touch is required to beautifully craft products using chatons. Rather than simply gluing a flat back crystal on a frame, each License 2 Bling chaton crystal is hand fitted into the frame with jewelry-like precision. Once placed, the crystals sit flush in the frame, creating a stylish aesthetic. License 2 Bling is the only frame maker in the world to utilize this proprietary embedding process...and it shows.
79.99Brand: License 2 Bling Part Numbers: License 2 Bling® is the world's premier car jewelry manufacturer. License 2 Bling features only the highest grade genuine Swarovski crystals, triple chrome and all-metal construction - no cheap plastic here. License 2 Bling's genuine Swarovski products are, far and away, the finest car license plate frames and car accessories you can buy.
Product Reviews (8)Every Customer can leave a review about our service and the product that was purchased. In order to leave your review for your new product, please follow the link in the email you receive after completing your purchase. You can rate appearance, ease of installation, price/value and product quality.
License 2 Bling® - Mercedes Benz Series FrameAverage Rating: 5 of 5APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYAll License 2 Bling Reviews(5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITY(4)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYHere Kitty, Kitty| Posted by Judith P. | (Hanford, CA)The kitty print chrome plate is made of good quality chrome and comes in a velvet bag. The price is rather high considering that it is for one frame and not two. To some extent, I am still reeling from the cost of this item. I think it is beautiful, and you do get what you pay for, but it is on the pricey side.
Was this review helpful for you? YesNo(5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYMy Wife Loved These Plate Frames| Posted by Michael J. | (Orange, CA)When I presented these frames to my wife, she was floored at the look, but once I had put them on her new car, she was even more impressed (as was I.) It took just minutes to install the frames and secure them with the included hardware. I certainly recommend CARiD to anyone who wants to give that 'up' look to their vehicle.
Was this review helpful for you? YesNo(4.5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYLicense 2 Bling - VIP Series Chrome Frame2012 Honda Pilot
| Posted by Elizabeth M. | (Louisbourg, NS)This plate is absolutely eye-catching, very classy and sparkly, made of strong material as well! It came in a suede bag, which added a nice presentation in receipt of the product.
Was this review helpful for you? YesNo(5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITY(5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYLuxurious2013 Dodge Challenger
| Posted by Chad E. | (Lafayette, LA)I love the look of Swarovski crystals, so this was a must have. I can't stop staring at how great this frame looks on my black car. If you want your car to stand out, this is a must have.
Was this review helpful for you? YesNo(5)APPEARANCEEASE OF INSTALLATIONPRICE/VALUEQUALITYLove, Love, Love It!!!| Posted by Teri V. | (Eureka, CA)Just bought myself a Caddy, and wanted to get something special for it. When I saw Swarovski license plate frames, I thought "nothing's too good for the Caddy "! I wear Swarovski jewelry, now so does my car! It really looks beautiful.
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Last.fm - DStyles23 Xbox Live GT: DStyles23
FUCK DREWBACCA | Dino Spumoni- "Mixing Supreme and Hollister is like having filet mignon with grape soda"
I swear on everything when I leave this Earth, its gon be on both feet, never knees in the dirt.
All I do is smoke weed, drop acid, and listen to MF DOOM http://www.facebook.com/SpacemanNYC http://theinnercitykids.tumblr.com/
RIP - Kids DAC. FREE - MFONE, AGANY, MOOK. Now Serving Felonies.
I want to see the world burn
OP you look like you sip alot of semen.
My style is impetuous. My defense is impregnable, and I'm just ferocious
Last.fm - DStyles23 Xbox Live GT: DStyles23
it's a gucci scarf
it's a gucci scarf
then why is it on your head...?
you will never be as hypebeast as my blood cousin pankeo
you guys need to chill the fuck out.
Chicks barely dancin, glancin every chance they get, like - oh shit, he's so HANSUM
#HANSUMBOYZ / "I mean I just wanted the pussy tbh."- Thad #FREETHAD
I'll start it off:
#HANSUMBOYZ / "I mean I just wanted the pussy tbh."- Thad #FREETHAD


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Rock Climbing Commercial, Real or Special Effects?
UPDATED 8:16 AM EST Jan 06, 2012Are the dizzying effects in a credit card commercial real or special effects?
The views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


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Cheap Flights from Maceio to New York
Did You Know?
- Chicago, IL has the most direct flights to New York.
- Sao Paulo, BR is the most popular connecting city from Maceio, BR to New York.
- The earliest flight from Maceio, BR leaves at 0:25 am.
- The most popular destination from Maceio, BR is Dois De Julho.
- There are 2 connecting cities from Maceio, BR to New York.
- There are 79 carriers flying into New York.


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In Disney's 50th animated feature film, the traditional, "someday my prince will come" princess flick gives way to a more modern fairytale with an "equal partner" twist. And, though Tangled might not fit the classic Disney Princess mold, Mandy Moore would like to think it does, while giving young girls a role model they can emulate while sharing the driver's seat with the usual foxy male leads.
Read more ...
Mandy Moore lets her hair down in Disney's Tangled
Posted Nov 24, 2010 3:28 PM
Redneck comedian Jeff Foxworthy releases his third children's book and talks about his two biggest fans
Posted Nov 24, 2010 3:20 PM
"Redneck" and "New York Times best-selling author" don't usually meet in the same sentence, unless you're comedian Jeff Foxworthy. Known as the best-selling comedy recording artist of all time, Foxworthy recently released Hide!!! his third children's book.
Read more ...
Ferrell vs. Pitt: It's a character face off in DreamWorks' Megamind
Posted Nov 24, 2010 3:19 PM
Saturday Night Live alum, wildly hilarious impressionist and father of three boys, Will Ferrell, never fails to entertain to the extreme, whether he's playing a displaced elf, anchorman underdog or the most brilliant (and unsuccessful) super villain the world has ever known.
Read more ...
Jamie Lee Curtis talks mother love in 'My Mommy Hung the Moon'
Posted Nov 23, 2010 12:05 PM
Jamie Lee Curtis talks about her best-selling picture book, parenting, plus her latest movie role.
Read more ...
Iconic actress Jane Seymour takes a look back at Dr. Quinn Medicine Woman and talks Perfectly Prudence
Posted Nov 23, 2010 12:04 PM
See Jane. See Jane play. See Jane rock an oversexed cougar, a pioneer doctor and a lamé-clad Bond girl. When it comes to iconic actress, Jane Seymour, anything and everything she takes on never fails to make an impression.
Read more ...
Outdoorsy dad Joe Lando takes a look back at Dr. Quinn Medicine Woman and talks Perfectly Prudence
Posted Nov 23, 2010 12:04 PM
He may be best known for his buckskin breeches and brooding, long-maned mountain man character on Emmy award-winning series, Dr. Quinn, Medicine Woman, but there's much more to Joe Lando than the brawny, bearded fellow that shared our living rooms from 1993-1998.
Read more ...
Epic actor Sean Astin takes on parenting three girls and playing Disney's Special Agent Oso
Posted Nov 23, 2010 12:04 PM
Whether you're partial to his official debut in the cult classic, The Goonies, or all about his fantastical character in Lord of the Rings, anything actor, producer, director, and social advocate, Sean Astin, has played, has been nothing short of epic.
Read more ...
'It's just amazing' says NSYNC's Joey Fatone of fatherhood
Posted Nov 23, 2010 12:02 PM
Former Dancing With The Stars runner-up and 'NSYNCer, Joey Fatone, might have once sung "Bye Bye Bye" but the singer, actor, and father is back with a new single, new daughter, and a guest stint on a new episode of Playhouse Disney's hit series, Imagination Movers, to air Saturday, May 22 on Disney Channel.
Read more ...
Playhouse Disney's Nancy Kanter develops preschooler programming that balances entertainment with education
Posted Nov 23, 2010 12:01 PM
Parents are all about embracing and fostering that growing sense of independence in their children, but letting them choose what to watch on television can be difficult. Nancy Kanter, Senior Vice President of Playhouse Disney Worldwide shares her take on what makes Disney programming different.
Read more ...
British explorer Olly Steeds solves history and brings wanderlust to the classroom
Posted Nov 23, 2010 12:01 PM
The iconic Indiana Jones might be firmly rooted in filmlore, but his wry sense of humor, sexy rough-and-tumble appeal, and deep knowledge of ancient civilizations lives on in rogue British reporter, journalist and explorer, Olly Steeds.
Read more ...
More Posts » Page: 1 2 3


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Friday, March 14, 2014
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The New York Knicks defeated the Boston Celtics in a 90-76 win Friday night, taking a 3-0 lead in the series.
It was the Knicks' first road playoff victory in 12 years.
The Knicks' rout was led by Carmelo Anthony, who scored 26 points. Point Guard Raymond Felton contributed a double-double, scoring 15 point and dishing out 10 assists.
But it was defense that won the game for the Knicks, who racked up 14 steals, 5 of which came from Pablo Prigioni.
J.R. Smith was ejected late in the game for elbowing Jason Terry face.
The Celtics were led by Forward Jeff Green who scored 21 points.
Game 4 will be on Sunday at 1 p.m.


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Sketch show set in a strangely familiar Scottish town
The legend of King Arthur springs to life when a man pulls Excalibur out of the pavement.
First broadcast: 17 Sep 2012
The 'quality polis' team of McGregor and Toshan attend a football match.
First broadcast: 10 Sep 2012
Misguided entrepreneur Alex Ciderson invents a new way of packaging beer.
First broadcast: 27 Aug 2012
The local cake shop suffers a decline in sales when a rogue cake decorator is let loose.
First broadcast: 20 Aug 2012
The legend of King Arthur springs to life when a man pulls Excalibur out of the pavement.
BBC © 2014 The BBC is not responsible for the content of external sites. Read more.
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Just Glass was formed in 2003 and comprises a group of both established and emerging glass artists. Members include internationally renowned as well as emerging artists and their work ranges from tiny, intricate pieces to imposing, large-scale works in glass, from domestic vessels to abstract sculpture. Please browse our current artists' pages to see some of their work.
Exhibitions featuring the work of Just Glass members have been mounted in Kew (2003), the Trail of Glass (2005), covering six venues across Richmond, Kew and Twickenham, and Shift (2008),and held at the London Glass Art Gallery, The Leathermarket, London SE1.
During 2013 Just Glass celebrated its tenth anniversary with an exhibition of members' work at The Stables Gallery, Orleans House, Twickenham, Middlesex and a lecture from Just Glass founder, Jera May.
Just Glass is renowned for its biennial Seminars attracting international glass artists to speak on various aspects of British Studio Glass. Speakers have included Stephen Beardsley, Sabrina Cant, Katharine Coleman, MBE, Matt Durran, Frances Federer, Catherine Hough, Richard Jackson, Max Jacquard, Angela Jarman, Kevin Petrie, Colin Reid, Anthony Scala, Angela Thwaites and Richard William Wheater. Our past events pages hold details of all our seminars to date.
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NFL extends TV deals with CBS, Fox, NBC
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(Reuters) - The National Football League reached deals with the CBS, FOX and NBC broadcast networks that will extend television rights for the games through the 2022 season while substantially increasing payments to the league.
The networks agreed to pay annual increases of about 7 percent on average, a person familiar with the deals said. That will lift yearly rights payments to the NFL from the networks to nearly $3.1 billion in 2022, up from about $1.9 billion in 2013, the person said.
The higher payments, which follow a new contract reached with ESPN in September for Monday night games, reflect the importance of sports and particularly the NFL to TV networks. Sports is almost always watched live by viewers, which is of immense importance to advertisers and separates it from most programming such as dramas and comedies, which are often recorded and watched later.
"Live sports, especially football, drives higher audience ratings than anything else on television in the U.S. So NFL is a very important property for these networks," Morningstar analyst Michael Corty said.
Combined with payments from Walt Disney Co's ESPN and satellite TV provider DirecTV, annual payments to the NFL for televising games will reach about $6 billion annually.
In September, the NFL announced an eight-year, $15.2 billion contract extension with Walt Disney Co's ESPN for Monday Night Football. The deal, which includes additional rights beyond just the TV broadcast, represents a roughly 73 percent increase over the previous contract.
Under the new agreement with broadcasters, CBS will continue to televise AFC Sunday afternoon games, and News Corp's FOX broadcast network will keep the NFC's Sunday afternoon games.
NBC, a unit of Comcast Corp, will again carry Sunday night primetime games and the Thursday night NFL season kickoff game. The network will add an annual Thanksgiving primetime game starting in 2012.
The league, which announced the new agreement the broadcasters on Wednesday, said it will expand the number of Thursday night games on its own NFL Network beginning next year, with the exact number not yet determined.
Each network will air three Super Bowls during the nine-year period.
"These agreements underscore the NFL's unique commitment to broadcast television that no other sport has," NFL Commissioner Roger Goodell said in a statement.
Network executives noted the NFL's importance to fans and advertisers in their announcements of the deals.
"No other franchise delivers ratings the way an NFL game does," CBS Chief Executive Leslie Moonves said in a statement.
(Reporting by Soham Chatterjee and Himank Sharma in Bangalore and Lisa Richwine in Los Angeles; Editing by Sriraj Kalluvila and Steve Orlofsky)
- Co-pilot spoke last words heard from missing Malaysian plane |
- U.S., EU impose sanctions after Crimea moves to join Russia |
- Crimeans vote over 90 percent to quit Ukraine for Russia |
- China rejects North Korean crimes report, hits chance of prosecution
- UPDATE 1-Rolls-Royce concurs with Malaysia on missing jet's engine data


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CosplayFU's Blog » Cosplay Events » Trunks Cosplay from Dragon Ball, Italy
February 5th, 2013 by adminComment
Actually that is Trunks. The purple hair is a huge give away, and Android 18 is a female.
d(^_^o) Hello Kurosaki-kun, yes, you are correct, I just corrected the character name, thanks a lot for your comment~~


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Bonefish Grill7415 Corporate Blvd, Baton Rouge, LA 70809http:/
(225) 216-1191
Your local seafood restaurant in Baton Rouge, LA specializing in market-fresh seafood & fish cooked over a wood-burning grill. Join us today & try one of our great apps, martinis or cocktails! Seafood restaurant, fresh fish, fish restaurant, specialty cocktails, innovative recipes, fine dining, grilled fish
Are You the Business Owner?Claim your free business listing on Superpages.com and add important information about your business online. The more reviews and additional information you provide about your business, the easier it will be for customers to find you online.
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This Oneida utility knife is a must-have for every kitchen. This handy knife is ideal for a number of food preparation and other household tasks. You'll love the versatility of this Oneida utility knife.
- Durable ceramic construction ensures years of use.
- Rubber-coated ergonomic handle provides a comfortable, easily controlled grip.
- Details:
- Includes protective knife cover
- 4 1/2-in. blade
- Hand wash
- Manufacturer's lifetime limited warranty
- Model no. 55175
Promotional offers available online at Kohls.com may differ from those offered in Kohl's stores.


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Topic: Spyglass Entertainment Group LLC
All Content
Bankruptcy pushes MGM toward Spyglass deal
Bankruptcy: MGM favors a plan calling for Spyglass Entertainment chiefs Gary Barber and Roger Birnbaum to take over a significantly slimmed down MGM following a prepackaged bankruptcy.
10/26/2010 10:59 am -
MGM bankruptcy plan announced. What does it mean for James Bond and 'The Hobbit'?
MGM bankruptcy: Privately held production company Spyglass Entertainment, a co-financier of such recent films as "Star Trek" and "G.I. Joe: Rise of Cobra," will contribute some assets and merge two subsidiaries into MGM for a 4.69 percent stake in the company, according to the plan.
10/08/2010 01:02 pm


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Most Popular Tags: illustration menu cocktail hawaii polynesian tiki california hotel palm trees mugs bar photo postcard restaurant interior restaurant photo written description
List of All 179 Tags
Beachbum Berry
Eye of the Goof
Forbidden Island
Humu Kon Tiki
Lopaka Sound
Smuggler's Cove
The Hukilau
Tiki Central
Tin Can Mailman
Arkiva Tropika is the online archive of the collection of Mimi Payne. This large collection includes a wide array of mostly pre-1970 menus, postcards, matchbooks, and paper ephemera souvenir items, mainly from Polynesian and other Exotic themed restaurants, hotels, nightclubs, bars, and the like. Mimi can be contacted at aloha@ arkivatropika.com.
Mimi can be contacted at aloha@ arkivatropika.com.
Both the dinner menu and also the cocktail menu from this Bali Hai are loaded with tropical island imagery and greetings beckoning its patrons to their "Isle of Enchantment". Interesting, though, that neither menu has an address or any mention of where this dining paradise was located !
From the inside of this 1962 dinner menu:
"Welcome to Bali Hai - the Isle of Enchantment. A land of throbbing rhythms, strange imagery, soft breeze and softer song. An island where life floats along, indolent, languorous, with the thrum of ukuleles or the thudding beat of a gourd, swaying palms, the stillness of a lagoon. From this isle, we will start your voyage to the many out-flung islands in the wondrous Pacific. We hope you will feel the unhurried tempo of the soft trade-winds and enjoy your visit to the Bali Hai with pleasure and relaxation.
Your own personal Island Vahine awaits your beckoning to serve your slightest whims. Starting at your leisure, she sets before you a multitude of beautiful Polynesian tidbits."


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Milwaukee's number #1 select team is having tryouts on the following days.
8U - Aug 12th & 19th at 8am - McCarty Park, West Allis
9U - TBD (interviewing for coach
10U - Aug 12th & 19th at 10am - McCarty Park, West Allis
11U - Aug 12th & 19th at Noon - McCarty Park, West Allis
13U - TBD (interviewing for coach)
14U - Aug 8 at 5:30pm & Aug 11th at 10am - Stormonth Field, Fox Point
Visit http://www.WisconsinRockhoundsBaseball.com and Pre-Register for our 2013 select Youth baseball tryouts.
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- Wauwatosa Recreation and Summer School New Registration Procedures
- Milwaukee Montessori School Welcomes Catherine Brys as Director of Child Care Program
- Holiday Food Drive For Hunger Task Force to be Featured in St. Patrick's Day Parades
- CigarMKE SPRING FLING EVENT - FEBRUARY 21ST
- Free Sound Reinforcement & Live Sound Classes
- Historian John Gurda To Speak at Elm Grove Playhouse
- Announcing the Wauwatosa Distinguished Citizen for 2013: Maureen Connors Badding
- McKinley Lodge Fish Fry


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Jose Theodore and Saves for Kids
In today's Washington Post, Tarik El-Bashir was finally able to share the story of Washington Capitals goaltender Jose Theodore and his life since August, when he lost his infant son Chace who died in August from respiratory complications related to a premature birth. While we at Rock The Red do not have children, we have all lost someone close to us - and cannot even begin to imagine the intensity Jose felt, and continues to feel, while under the pressure of playing professional hockey. We fully support his creation of the Saves For Kids program, to which Theodore will donate $2 for each save, $100 for every win and $500 for each shutout to benefit the Neonatal Intensive Care Unit at Children's National Medical Center.
We wish to support Theo in his cause, and are donating all profits from our store from now until the Capitals play their final seconds of hockey this season. So whether you're interested in being Ovechkin's homeboy, partying with Poti, getting free burritos, or inquiring about the powers behind Langway's moustache- or any of the other shirts we offer - you will be supporting Jose Theodore and Children's National Medical Center. We realize that this won't amount to much, but every penny counts when you're dealing with something of this importance.
If you are interested in making your own donation, send a check made payable to Washington Capitals Charities, earmarked for Saves for Kids, to:


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Oct 12, 2010, 10:18 #1
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What are the security pros and cons of using a Framework vs. CMS vs. Custom Built?
I'm up in the air in terms of going the route of a Framework, a CMS, or a custom built solution. I can use each route and accomplish the goal, though I want to make sure that it's secure.
Here's what my understanding is:
Custom Built Solution Pros:
- Custom code, not publically known as to how the system is built
- If you know how to build something secure, you can keep up to date and fix on the fly
Custom Built Solution Cons:
- If you don't know how to build something secure, you're probably going to have some vulnerabilities
Framework Built Solution Pros:
- The framework could have methods/functions that do common things, of which where these methods/functions would be built securely
- The framework, if a vulnerability was found, could be updated promptly by the community
Custom Built Solution Cons:
- The framework code is publically known
- If you don't know how to build something secure, you're probably still going to have some vulnerabilities
And I believe the CMS and Framework pros/cons are quite simmilar. Am I on track with this? Is what I am saying completely wrong? Please help me out!
Oct 13, 2010, 02:39 #2
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And I hope you remembered to handle multibyte strings everywhere, and made some really good design decisions - or never let anybody work on that code.
Just look at CodeIgniter - the 'Form helpers' library (which is a 'core' component) has stagnated like the rest of it and is still full of XSS holes. You can't generalize, there are hundreds of 'frameworks' ranging from utter crap to nearing zen-like perfection.
The more people actively using it usually contributes to more secure code, to the extent that I'd prefer a framework with a thousand developers using it professionally than a one-man job.
Nov 19, 2010, 03:47 #3
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I know this is a dated thread, but Harry, I've written a good thing explaining why NOT to use Frameworks, http://blog.8thstudio.com/?p=51
Of course, it depends on the person and their beliefs, but in the long run, Frameworks will bottle neck and well become unsupported by their developers.Phase 8 Facebook Applications and Web Development
Nov 19, 2010, 11:54 #4
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aalicki - while your argument is valid - I think it does not address web application security too well.
Security is difficult, getting it right is difficult, keeping up to date with all new attack vectors is difficult and expensive.
There are benefits of not using frameworks and there are benefits to do so.
The odds are that if I invent framework of my own (be it oop or procedural - it does not matter), I will not get things like data validation/sanitation right.
The good part is that there is benefit (although you should not rely on that) of security by obscurity, the bad part, well you are on your own to figure out where the vulnerabilities are and how they are being exploited.
Securosis has rather good article series.


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Translator's Preface [to Repertory] by J. Walker McSpadden
Introduction [to Repertory] by Paul Bourget
Saintsbury Introduction, Volume XVIII – Part II
Saintsbury Introduction, Volume XVIII – Part I
Introduction [to Analytical Studies] by J. Walker McSpadden:
The Physiology of Marriage (Physiologie du Mariage)
Petty Troubles of Married Life (Petites Misères de la Vie Conjugale)
Saintsbury Introduction, Volume XVII – Part II
Introduction [to Balzac's Drama] by J. Walker McSpadden:
The Resources of Quinola (Les Ressources de Quinola)
Pamela Giraud
The Stepmother (La Marâtre)
The greatest fame of Balzac will rest in the future, as in the past, upon his novels and short stories. These comprise the bulk of his work and his most noteworthy effort – an effort so pronounced as to hide all side-excursions. For this reason his chief side-excursion – into the realms of drama – has been almost entirely overlooked. Indeed, many of his readers are unaware that he ever wrote plays, while others have passed them by with the idea that they were slight, devoid of interest, and to be classified with the Works of Youth. Complete editions – so-called – of Balzac's works have fostered this belief by omitting the dramas; and it has remained for the present edition to include, for the first time, this valuable material, not alone for its own sake, but also in order to show the many-sided author as he was, in all his efficiencies and occasional deficiencies. (more…)
Saintsbury Introduction, Volume XVII – Part I
Balzac as a Dramatist by Epiphanius Wilson:
The Resources of Quinola (Les Ressources de Quinola)
Pamela Giraud
The Stepmother (La Marâtre)
Honoré de Balzac is known to the world in general as a novel-writer, a producer of romances, in which begin the reign of realism in French fiction. His Comédie Humaine is a description of French society, as it existed from the time of the Revolution to that of the Restoration. In this series of stories we find the author engaged in analyzing the manners, motives and external life of the French man and woman in all grades of society. When we open these volumes, we enter a gallery of striking and varied pictures, which glow with all the color, chiaroscuro and life-like detail of a Dutch panel. (more…)
Saintsbury Introduction, Volume XVI – Part II
The Seamy Side of History (L'Envers de l'Histoire Contemporaine):
Madame de la Chanterie (Madame de la Chanterie)
The Initiate (L'Initié)
A Prince of Bohemia (Un Prince de la Bohème)
A Man of Business (Un Homme d'Affaires)
Gaudissart II
Sarrasine (Sarrasine)
Facino Cane (Facino Cane)
Z. Marcas (Z. Marcas)
An Episode Under the Terror (Un Episode sous la Terreur)
It would be difficult to find another book, composed of two parts by the same author, which offers more remarkable variations and contrasts than the volume which contains L'Envers de l'Histoire Contemporaine and Z Marcas. And in certain respects it must be said that the contrast of the longer and later story with the earlier and shorter one is not such as to inspire us with any great certainty that, had Balzac's comparatively short life been prolonged, we should have had many more masterpieces. (more…)
Saintsbury Introduction, Volume XVI – Part I
The Member for Arcis (Le Député d'Arcis)
Le Député d'Arcis, like the still less generally known Les Petits Bourgeois, stands on a rather different footing from the rest of Balzac's work. Both were posthumous, and both, having been left unfinished, were completed by the author's friend, Charles Rabou. (more…)
Saintsbury Introduction, Volume XV – Part II
The Gondreville Mystery (Une Ténébreuse Affaire)
Parisians in the Country: The Muse of the Department (Les Parisiens en province: La Muse du Département)
While I was engaged in preparing these Introductions, I saw in an English newspaper, of some literary as well as other repute, remarks on Balzac as compared with some writers of crime and detective stories in the present day. (more…)
Saintsbury Introduction, Volume XV – Part I
The Chouans (Les Chouans)
A Passion in the Desert (Une Passion dans le désert)
When, many years after its original publication, Balzac reprinted Les Chouans as a part of the Comédie Humaine, he spoke of it in the dedication to his old friend M. Théodore Dablin as "perhaps better than its reputation." He probably referred to the long time which had passed without a fresh demand for it; for, as has been pointed out in the General Introduction to this Series of translations, it first made his fame, and with it he first emerged from the purgatory of anonymous hack-writing. (more…)
Saintsbury Introduction, Volume XIV – Part II
The Middle Classes (Les Petits Bourgeois)
A MAIN – I should myself be disposed to say the main – interest of Les Petits Bourgeois arises from the fact that it was not only the last published, except scraps, of Balzac's works, but was actually never included in the various editions of the Comédie Humaine till the appearance of the so-called édition définitive a few years ago. (more…)
Saintsbury Introduction, Volume XIV – Part I
The Rise and Fall of Cesar Birotteau (Grandeur et Décadence de César Birotteau)
The Secrets of a Princess (Les Secrets de la princesse de Cadignan)
Few books of Balzac's have been the subject of more diverse judgment than Cesar Birotteau. From the opinion of the unnamed solicitor, who told Madame Serville that it was an invaluable work to consult on bankruptcy, to that of M. Paul Lacroix (beloved of many as the Bibliophile Jacob), that it might be forgiven for the sake of Le Père Goriot and the Peau de Chagrin, there is not perhaps quite so great a distance as may appear; but other expressions, opposed not merely in form, but in fact, might probably be collected. (more…)


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Copyright © 2012 The Authors All rights reserved.
Cell Reports, Volume 2, Issue 4, 914-926, 18 October 2012
Endogenous Control of Immunity against Infection: Tenascin-C Regulates TLR4-Mediated Inflammation via MicroRNA-155
Anna M. Piccinini1 and Kim S. Midwood1, ,
Corresponding authorGraphical Abstract
► Endogenous danger signals mediate effective immunity against pathogenic infection ► Tenascin-C is required to generate a proinflammatory response to LPS in vivo ► Tenascin-C post-transcriptionally regulates TNF-α production by macrophages ► Tenascin-C controls TNF-α translation by driving microRNA-155 expression
- Endogenous molecules generated upon pathogen invasion or tissue damage serve as danger signals that activate host defense; however, their precise immunological role remains unclear. Tenascin-C is an extracellular matrix glycoprotein that is specifically induced upon injury and infection. Here, we show that its expression is required to generate an effective immune response to bacterial lipopolysaccharide (LPS) during experimental sepsis in vivo. Tenascin-C enables macrophage translation of proinflammatory cytokines upon LPS activation of toll-like receptor 4 (TLR4) and suppresses the synthesis of anti-inflammatory cytokines. It mediates posttranscriptional control of a specific subset of inflammatory mediators via induction of the microRNA miR-155. Thus, tenascin-C plays a key role in regulating the inflammatory axis during pathogenic activation of TLR signaling.
The danger theory of immunity changed the way we think about host defense; defining the immune system as the primary response not only against infection but also against sterile tissue injury (Matzinger, 1994). The identification of evolutionarily conserved "pattern-recognition" receptors (PRRs) and their ability to sense "danger signals" generated upon infection (pathogen-associated molecular patterns [PAMPs]) and tissue injury (damage-associated molecular patterns [DAMPs]), and in response to activate inflammatory signaling pathways, has further delineated the molecular machinery that drives immunity (Beutler, 2007; Gordon, 2002; Medzhitov and Janeway, 2002).
DAMPs have been defined as endogenous molecules specifically generated upon tissue damage. They comprise a variety of proteins, proteoglycans, and fatty acids, including intracellular molecules released from necrotic cells, extracellular matrix (ECM) fragments created by tissue destruction, and ECM molecules whose expression is specifically induced upon tissue injury (Bianchi, 2007; Gordon, 2002). Physiologically committed to alerting the body to damage and initiating tissue repair, even in the absence of microbial invasion, DAMPs can directly induce an inflammatory response via activation of PRRs (Chen and Nuñez, 2010). They do so in a manner distinct to PAMP-mediated activation of PRRs in order to drive unique cellular responses (Midwood et al., 2009; Osterloh et al., 2007; Silva et al., 2007).
However, DAMPs are also generated upon microbial infection. An emerging feature of infectious disease is that levels of endogenous danger signals typically associated with tissue injury are also rapidly elevated upon pathogen invasion. For example, the nuclear DNA-binding protein HMGB1 is promptly released from infected cells (Lotze and Tracey, 2005), and expression of the glycoprotein tenascin-C is induced upon infection, often appearing before any obvious sign of tissue damage (Midwood and Orend, 2009). Although levels of DAMPs are tightly regulated during acute inflammatory responses to infection and are cleared after the microbial threat has been eliminated, persistently high levels occur in patients with sepsis (Piccinini and Midwood, 2010; van Zoelen et al., 2009; Yang et al., 2004), the clinical manifestation of a dysregulated immune response to infection, which accounts for ∼9% of the overall annual mortality in the USA (Angus et al., 2001). Consistent with these data, PAMPs can directly induce the expression of endogenous danger signals in vitro. For instance, respiratory syncytial virus infection induces intracellular Hsp72 secretion in airway epithelial cells (Wheeler et al., 2009). Furthermore, tenascin-C, S100A8/S100A9, biglycan, and HMGB1 are secreted by macrophages following stimulation with Gram-negative bacterial lipopolysaccharide (LPS) (Ehrchen et al., 2009; Goh et al., 2010; Schaefer et al., 2005; Wang et al., 1999). These data imply a role for DAMPs not only in mediating tissue repair upon injury but also driving inflammation during pathogenic infection. During microbial invasion, these molecules may amplify inflammatory signals by directly activating PRRs (El Mezayen et al., 2007; van der Poll and Opal, 2008); however, their role in infection-induced inflammatory signaling remains largely undefined.
Here, we identify a unique role for the danger signal tenascin-C in regulating immunity mediated by toll-like receptor 4 (TLR4) in response to pathogenic infection. Tenascin-C is an ECM glycoprotein that is absent in most healthy adult tissues but that is specifically expressed upon tissue injury (Chiquet-Ehrismann and Chiquet, 2003) as well as during infection (Kaarteenaho-Wiik et al., 2000; Päällysaho et al., 1993). Levels are high in patients with sepsis (Schenk et al., 1995). In vitro, LPS activation of TLR4 transiently induces tenascin-C expression in human myeloid cells (Goh et al., 2010). Our data show that tenascin-C is required for the proinflammatory response to LPS in vivo. Targeted deletion of tenascin-C impairs TNF-α production and subsequent downstream cytokine synthesis, in response to LPS in septic mice and bone marrow-derived macrophages (BMDMs). We also provide mechanistic insights into tenascin-C-dependent signaling downstream of LPS activation of TLR4 and demonstrate that tenascin-C operates posttranscriptionally, modulating levels of TNF-α translation. Moreover, we found that tenascin-C mediates this control through the induction of miR-155, a microRNA (miRNA) whose expression is strongly induced by LPS in macrophages (O'Connell et al., 2007; Tili et al., 2007), and that positively regulates the production of TNF-α (Bala et al., 2011; Tili et al., 2007). Our study identifies tenascin-C as a regulator of the early immune response and shows that an effective and coordinated inflammatory response to pathogenic activation of TLRs requires the participation of endogenous danger signals.
Tenascin-C Is Required for the Proinflammatory Response to LPS In Vivo
To examine the role of tenascin-C in host defense against pathogenic infection, we induced sepsis with a sublethal dose of LPS in tnc+/+ and tnc−/− mice. The major clinical symptoms of sepsis, including weight loss, reduced mobility, uveitis, ruffled fur, and diarrhea, were evident in tnc+/+ mice 1.5 hr after injection of LPS, in contrast to tnc−/− mice, which showed no symptoms by 1.5 hr. Although symptoms became progressively more severe in tnc+/+ mice 4 and 8 hr after injection, tnc−/− mice exhibited significantly less weight loss (p < 0.0001 at 4 hr; p = 0.0006 at 8 hr), less mobility impairment (p < 0.0001 at 4 and 8 hr), and less fur ruffling (p < 0.0001 at 4 and 8 hr) than tnc+/+ mice, with mild uveitis and diarrhea (Figures 1A, 1B, S1A, and S1B).
On the molecular level, sepsis is driven by a "cytokine storm" comprising elevated circulating levels of proinflammatory cytokines induced in response to LPS. Analysis of plasma levels of key inflammatory mediators of sepsis showed that tnc−/− mice synthesize significantly less TNF-α (p < 0.0001) and IL-6 (p < 0.0001) compared to tnc+/+ mice 1.5 hr after LPS injection, consistent with the lack of clinical symptoms in the absence of tenascin-C. By 4 hr, there was significantly less TNF-α (p = 0.001) in tnc−/− mice compared to tnc+/+ mice. Plasma TNF-α and IL-6 dropped by 8 hr similarly in both tnc−/− and tnc+/+ mice. Furthermore, tnc−/− mice fail to induce release of HMGB1, a late mediator essential for organ damage in sepsis (Wang et al., 1999), which was significantly increased in tnc+/+ mice 8 hr after LPS injection (Figure 1C).
Tissue damage following systemic LPS injection results from immune cell recruitment to susceptible sites, most notably, rapid neutrophil infiltration to lungs and liver. Immunohistochemical staining of neutrophil elastase in lungs and liver from tnc+/+ mice 1.5 hr after LPS injection demonstrated substantial neutrophil infiltration, which was significantly reduced in tnc−/− mice, an effect also evident at 4 and 8 hr after injection (Figures 1D, S1C, and S1D).
To determine whether tenascin-C expression is elevated in mice with LPS-induced sepsis, we quantified plasma tenascin-C in tnc+/+ mice after LPS injection. Immunoblot analysis and ELISA revealed increased tenascin-C in mice upon administration of LPS. Notably, the kinetics of tenascin-C expression was similar to the early proinflammatory cytokines TNF-α and IL-6, peaking 1.5 hr after LPS injection and decreasing over time (Figures 1E–1G). Together, these results indicate that tenascin-C expression is required for the proinflammatory response to LPS in vivo.
Bone Marrow Engraftment Rescues TNF-α Synthesis in Tnc−/− Mice
TNF-α was the cytokine most affected by tenascin-C ablation. To determine if the reduced plasma levels of TNF-α observed in tnc−/− mice are mediated by defects in their hematopoietic cell population, we performed allogeneic bone marrow transplantation. Whole bone marrow cells isolated from tnc+/+ or tnc−/− male mice were injected into sublethally irradiated tnc+/+ or tnc−/− female recipient mice. Tnc+/+/+/+, tnc+/+/−/−, tnc−/−/−/−, and tnc−/−/+/+ chimeric mice were generated. All mice displayed equally high donor engraftment as shown by quantitative RT-PCR analysis of the mouse testis-specific Y-encoded protein (TSPY) gene in erythrocyte-depleted peripheral blood cells from recipient mice (Figure 2A). These data indicate that the majority of bone marrow cells are from the donor.
Analysis of tenascin-C mRNA in peripheral blood cells from tnc+/+/−/− mice 1.5 hr after LPS injection showed significantly reduced expression of tenascin-C compared to tnc+/+/+/+ mice. The tenascin-C mRNA observed in tnc+/+/−/− mice is likely produced by residual bone marrow cells of the tnc+/+ recipient. Replenishment of tnc−/− mice with tnc+/+ bone marrow resulted in significantly more tenascin-C expression compared to tnc−/−/−/− mice (Figure 2B).
TNF-α levels in plasma from tnc+/+/−/− chimeric mice were significantly lower (p = 0.0033) than those from tnc+/+/+/+ chimeric mice 1.5 hr after LPS injection (Figure 2C). Accordingly, significantly more TNF-α (p < 0.0001) was found in plasma from tnc−/−/+/+ chimeric mice compared to tnc−/−/−/− chimeric mice (Figure 2D). Eight hours after LPS injection, TNF-α was reduced almost to basal levels in each group. Collectively, these data indicate that tenascin-C derived from bone marrow-derived cells drives TNF-α synthesis during LPS-induced sepsis and that bone marrow-derived cells from tnc−/− mice exhibit defects in TNF-α synthesis in vivo.
LPS-Activated BMDMs Express Tenascin-C
We next investigated whether primary BMDMs obtained from tnc+/+ mice express tenascin-C in response to LPS over time. Nonstimulated cells showed low basal levels of tenascin-C expression, which significantly increased upon stimulation with LPS. Tenascin-C mRNA started to increase at 1 hr and peaked between 4 and 8 hr, returning to basal levels by 24 hr (Figure 3A), as observed in human myeloid cells (Goh et al., 2010). Increased tenascin-C mRNA correlated with tenascin-C protein synthesis. Nonstimulated BMDMs produced no detectable tenascin-C; however, cell-associated tenascin-C significantly increased after LPS activation, peaking 8 hr after stimulation (Figure 3B). Tenascin-C was also secreted into the medium where its levels significantly increased 24 hr after stimulation (Figure 3C). These data demonstrate that tenascin-C expression in BMDMs is transiently induced by LPS.
To verify that tenascin-C ablation does not affect macrophage development in vitro and in vivo, we assayed tnc+/+ and tnc−/− BMDMs or freshly isolated peritoneal macrophages for CD11b, F4/80, and CD68 expression. Each maturation marker was equally highly expressed in tnc+/+ and tnc−/− cells (Figures S2A–S2O).
Tenascin-C Posttranscriptionally Regulates LPS-Mediated Synthesis of Specific Cytokine Subsets in BMDMs
To further examine the mechanism behind impaired proinflammatory cytokine synthesis in tnc−/− mice upon LPS treatment, we investigated the effects of tenascin-C on cytokine synthesis in BMDMs. There was no significant difference in IL-1β, IL-12, and TGF-β1 secretion between tnc+/+ and tnc−/− BMDMs. However, BMDMs obtained from tnc−/− mice secreted significantly less TNF-α, IL-6, and CXCL1, and significantly more IL-10, upon stimulation with LPS than tnc+/+ cells (Figure 4A).
The expression of TNF-α, IL-6, CXCL1, and IL-10 mRNA was induced in tnc+/+ BMDMs by LPS stimulation and was not significantly affected by tenascin-C ablation (Figure 4B). No difference in IL-12 and TGF-β1 mRNA expression was observed in tnc+/+ and tnc−/− BMDMs (data not shown). These results suggest that regulation of cytokine synthesis by tenascin-C occurs at a posttranscriptional level.
To ensure that the effect of tenascin-C on cytokine synthesis is not due to abnormal macrophage activation in vitro or in vivo, we analyzed the expression of activation markers in tnc+/+ and tnc−/− BMDMs or freshly isolated peritoneal macrophages. Equal expression of MHC II, CD40, or CD86 and the LPS receptor TLR4/MD2 was observed in tnc+/+ and tnc−/− BMDMs (Figures S3A–S3J) or peritoneal macrophages (data not shown).
Tenascin-C Promotes Translation of Proinflammatory Cytokines
Impaired proinflammatory cytokine production without any reduction in cytokine mRNA levels in tnc−/− cells led us to investigate whether tnc−/− BMDMs exhibit abnormal intracellular cytokine levels as a consequence of deficient secretion. There was no significant difference in cellular TNF-α, IL-6, and CXCL1 levels in LPS-stimulated tnc+/+ and tnc−/− BMDMs (Figure 5A). Cytokine levels in the cellular fraction did not account for the defective cytokine secretion by tnc−/− BMDMs, as shown by the sum of the amounts of cellular and secreted protein fractions (Figure 5B). These data demonstrate that proinflammatory cytokine cellular trafficking occurs normally in tenascin-C null BMDMs.
We next investigated whether tenascin-C affects proinflammatory cytokine translation. Brefeldin A, an inhibitor of ER-to-Golgi protein transport, resulted in intracellular accumulation of TNF-α, IL-6, and CXCL1 in tnc+/+ cells at 24 hr. In contrast, intracellular accumulation of TNF-α was abrogated in tnc−/− BMDMs. Significantly less IL-6 and CXCL1 also accumulated within the Golgi in tnc−/− BMDMs (Figure 5C). Together, these results provided the first clue that tenascin-C may be required for effective translation of LPS-induced proinflammatory cytokines in BMDMs.
Tenascin-C Drives LPS-Induced miR-155 Expression
Protein translation is regulated on several levels, among which miRNAs are key regulators of protein synthesis. Recent data have highlighted the importance of miRNA-mediated control of LPS-induced inflammatory signaling pathways (O'Neill et al., 2011); therefore, we investigated whether tenascin-C affects miRNA activity. miRNA expression in tnc+/+ and tnc−/− BMDMs stimulated with LPS for 8 hr was analyzed with an array containing 375 miRNAs. We found 35 miRNAs that were reproducibly and significantly up- or downregulated by LPS (Figure 6A). A number of miRNAs have emerged as important regulators of TLR signaling, including those induced by LPS activation of TLR4 (O'Neill et al., 2011). Among these, we found expression of early-response genes such as miR-155, miR-125b, and let-7i (Figure 6B) as well as late-response genes, including miR-146a, miR-132, and miR-21 (Figure 6C). In tnc+/+ BMDMs, LPS strongly induced miR-155 expression, which was significantly inhibited in tnc−/− cells (Figures 6A and 6B). Using quantitative RT-PCR, we confirmed these results and showed that miR-155 expression was rapidly induced by LPS, peaking at 24 hr and progressively declining in tnc+/+ BMDMs. Conversely, LPS-induced miR-155 expression in tnc−/− BMDMs was inhibited and did not recover (Figure 6D). Furthermore, analysis of the primary transcript pri-miR-155 in the same samples revealed no significant difference between tnc−/− and tnc+/+ BMDMs, which suggests that tenascin-C posttranscriptionally regulates LPS-induced miR-155 expression. In line with studies showing that miR-155 expression is induced immediately after LPS stimulation (O'Connell et al., 2007) and contributes to TNF-α production (Bala et al., 2011; Tili et al., 2007), changes in TNF-α levels and miR-155 expression significantly correlated in LPS-stimulated tnc+/+ BMDMs. Accordingly, low TNF-α levels correlated with low miR-155 expression in tnc−/− BMDMs (Figure 6E). This effect was specific because IL-6, CXCL1, and IL-10 synthesis did not correlate with miR155 expression (p = 0.233; data not shown). To determine whether these data are relevant in vivo, we examined the expression of LPS-induced miR-155 in the spleen of tnc+/+ and tnc−/− mice upon LPS injection. Tnc−/− mice expressed significantly less miR-155 than tnc+/+ mice (Figure 6F). Taken together, these data suggest that tenascin-C is required for the induction of miR-155 by LPS in BMDMs and in septic mice.
Tenascin-C Controls TNF-α Release via the Induction of miR-155
To determine whether reconstituting miR-155 expression rescues LPS-induced TNF-α production in the absence of tenascin-C, we overexpressed the mature miRNA in tnc−/− BMDMs. Cells were transiently transfected with a negative control precursor or miR-155 precursor, which mimics the endogenous precursor miR-155. Transfection of miR-155 precursor, but not control precursor, resulted in increased expression of mature miR-155. Stimulation of transfected tnc−/− BMDMs with LPS for 8 hr led to increased miR-155 expression, at a level similar to that induced by LPS in control tnc+/+ BMDMs (Figures 7A and S4A). We then analyzed the effect of miR-155 overexpression on cytokine release in tnc−/− BMDMs upon LPS stimulation. In the absence of LPS, low-basal cytokine synthesis was detected in untransfected and transfected cells. TNF-α production in tnc−/− BMDMs transfected with miR-155 precursor and stimulated with LPS for 8 hr was significantly higher than in LPS-activated tnc−/− cells transfected with control precursor and was equal to that of control tnc+/+ BMDMs (Figures 7B and S4B). Thus, miR-155 overexpression rescued TNF-α release in tnc−/− BMDMs in response to LPS. Together, these data provide evidence that tenascin-C posttranscriptionally controls the production of TNF-α in response to LPS via miR-155.
Since 1994, Polly Matzinger's danger model, which suggested that the immune system is designed to combat danger mediated by infection and injury, rather than to merely recognize nonself, has revolutionized our understanding of how the innate immune system works (Matzinger, 1994). Both PAMPs and DAMPs act as danger signals that trigger inflammation through activation of PPRs, including TLRs (Gordon, 2002). Established drivers of sterile inflammation, DAMPs have also been reported to be specifically induced upon infection (Ehrchen et al., 2009; Merline et al., 2011; Piccinini and Midwood, 2010; Wheeler et al., 2009). However, little is known about their role in infection-induced TLR signaling. This study identified how control of LPS-mediated TLR4 signaling is exerted by the proinflammatory ECM glycoprotein tenascin-C. In particular, we show that tenascin-C is an early LPS-responsive gene that is required for the proinflammatory response to LPS in experimental sepsis. Furthermore, we demonstrate that tenascin-C derived from bone marrow-derived cells posttranscriptionally controls TNF-α levels via regulation of miR-155 expression.
The induction of tenascin-C expression upon infection in vivo (Päällysaho et al., 1993; Schenk et al., 1995; Tiitta et al., 1992) and its transcriptional regulation by LPS in human myeloid cells (Goh et al., 2010) prompted us to investigate whether this DAMP plays a role in pathogenic inflammation. Using an experimental model of LPS-induced systemic inflammation, we showed that circulating tenascin-C increased rapidly in septic tnc+/+ mice, peaking 90 min after LPS injection, long before the induction of HMGB1 release, an established tissue injury marker in sepsis (Wang et al., 1999). These data support specific tenascin-C upregulation by LPS-induced TLR4 signaling rather than tissue damage-driven release of tenascin-C. Indeed, we demonstrated that tenascin-C is expressed at both mRNA and protein level in LPS-activated BMDMs, the major cellular players in sepsis. In response to bacterial endotoxins, macrophages promptly release proinflammatory cytokines, including TNF-α, which is both crucial for effective innate immunity and a key pathologic contributor to sepsis (Tracey et al., 1987). However, septic tnc−/− mice had significantly less circulating TNF-α, a phenomenon that was reversed by allogeneic transplantation of bone marrow from tnc+/+ mice. Consistent with low-circulating TNF-α, less plasma IL-6 and reduced neutrophil infiltration to lungs and liver were observed in tnc−/− mice after LPS injection compared to tnc+/+ mice. Furthermore, tnc−/− mice failed to induce release of HMGB1, a late mediator of sepsis that is released by activated macrophages partly through a TNF-α-dependent mechanism (Chen et al., 2004). These data suggest that tenascin-C is an early-response gene that regulates LPS-induced TLR4-mediated inflammation in vivo operating upstream of TNF-α and HMGB1. This is further supported by our in vitro findings that tnc−/− BMDMs secreted significantly less TNF-α, IL-6, and CXCL1, upon stimulation with LPS than tnc−/− cells. This effect was specific because it was not observed in other cytokine subsets, including IL-1β, IL-12, or TGFβ1. Furthermore, tenascin-C appears to specifically control the switch from anti- to proinflammatory cytokine programs downstream of TLR4 activation. In the absence of tenascin-C, not only do BMDMs induce submaximal TNF-α levels, but they also secreted more IL-10, an anti-inflammatory cytokine that is key to dampening the inflammatory response to infection induced by TLR signaling. Thus, transient tenascin-C expression promotes an initial proinflammatory response that is later suppressed by IL-10 synthesis.
The ability to express biologically active cytokines on demand is regulated at the transcriptional, translational, and posttranscriptional level. We have demonstrated here that tenascin-C controls the synthesis of specific cytokine subsets in BMDMs at the posttranscriptional level. Neither the extent of induction nor the kinetics of expression of TNF-α, IL-6, CXCL1, and IL-10 mRNA was significantly affected by tenascin-C. This provides evidence that the impaired synthesis of proinflammatory cytokines is not at a gene transcription level. However, exocytosis of cytokines to their site of action, which is mediated predominantly by classic secretory pathways in activated macrophages (Lacy and Stow, 2011), can also be the focus of a regulatory event. By quantifying cytokines in the cellular fraction, we found no significant difference in the intracellular cytokine levels between LPS-activated tnc+/+ and tnc−/− BMDMs. Thus, cellular trafficking of cytokines does not account for reduced cytokine release in the absence of tenascin-C. However, when protein transport from the ER to the Golgi was prevented by brefeldin A during stimulation of tnc−/− BMDMs with LPS, intracellular accumulation of TNF-α was abrogated. IL-6 and CXCL1 accumulation within the Golgi in tnc−/− BMDMs was significantly reduced when compared to tnc+/+ BMDMs. Together, these data suggest that tenascin-C is required for effective translation of LPS-induced proinflammatory cytokines in BMDMs.
A key finding of our study is the observation that tenascin-C mediates an initial proinflammatory response to infection by posttranscriptionally modulating the expression of the early TLR-induced miRNA miR-155. This miRNA is processed from the nonprotein-coding transcript of the bic gene, which is extremely well conserved between humans and mice and is highly expressed in lymphoid organs (Tam, 2001). Required for the innate immune response to bacterial and viral infection, miR-155 is strongly induced by LPS and type I interferons in macrophages (O'Connell et al., 2007; Thai et al., 2007; Tili et al., 2007). In line with this, by means of a miRNA screen and validation analysis, we found that expression of both primary transcript pri-miR155 and mature miR-155 was induced in tnc+/+ BMDMs immediately after LPS stimulation. However, the induction of mature miR-155 in tnc−/− BMDMs was significantly inhibited. This is underscored by the significantly lower miR-155 expression detected in vivo in the spleen of septic tnc−/− mice. Fundamental to the posttranscriptional control of gene expression, miRNAs have emerged as key regulators that fine-tune TLR signaling (O'Neill et al., 2011). Emerging evidence points to a proinflammatory role for miR-155 during TLR-mediated inflammation. It suppresses the domain-containing inositol-5′-phosphatase 1 (SHIP1), a negative regulator of TLR-induced responses, allowing the initial propagation of a proinflammatory response (O'Connell et al., 2009). Moreover, miR-155 has been proposed to increase the translation of TNF-α transcripts either by acting at the 3′ UTR of TNF-α mRNA to release its self-inhibitory effects (Tili et al., 2007) or by increasing TNF-α mRNA stability (Bala et al., 2011). In line with this, miR-155 overexpression in macrophages results in increased TNF-α production, and miR-155 downregulation results in lower TNF-α levels (Bala et al., 2011). Further evidence is provided by in vivo studies; miR-155 transgenic mice have elevated serum TNF-α and increased susceptibility to septic shock, whereas miR-155-deficient B cells fail to produce TNF-α (Thai et al., 2007; Tili et al., 2007). Consistent with this, we found a positive correlation between miR-155 expression and TNF-α production in tnc+/+ BMDMs upon LPS stimulation. Interestingly, in the absence of tenascin-C, impaired TNF-α synthesis was parallel to impaired miR-155 expression. Overexpressing miR-155 in tnc−/− BMDMs fully restored their ability to release TNF-α in response to LPS. These data suggest that tenascin-C induced by LPS in BMDMs uses miR-155 to modulate TNF-α levels. The synthesis of IL-6, CXCL1, and IL-10 did not correlate with miR-155 expression, nor was the release of these cytokines rescued in tnc−/− BMDMs by miR-155 overexpression. This is in line with data that show that the effect of miR-155 on the expression of cytokines other than TNF-α is likely to be indirect, for example mediated via suppression of negative regulators of cytokine-production suppressor of cytokine signaling 1 (SOCS1) (Androulidaki et al., 2009) and/or SHIP1 (Kurowska-Stolarska et al., 2011; O'Connell et al., 2009), which are both validated targets of miR-155, or as a result of impaired production of TNF-α, which itself induces the expression of both IL-6 and CXCL1. Finally, tenascin-C appears to specifically control the induction of miR-155 without affecting the expression of other LPS-responsive miRNAs that have been functionally linked to TLR signaling, including miR-146a and miR-21 (Sheedy et al., 2010; Taganov et al., 2006).
We previously found high tenascin-C levels in inflamed rheumatoid joints, where it is specifically expressed by synovial fibroblasts and myeloid cells (Goh et al., 2010). We showed that tenascin-C induces cytokine synthesis in both human and murine macrophages and in rheumatoid arthritis (RA) synovial fibroblasts via activation of TLR4 (Midwood et al., 2009). Tenascin-C also induces cytokine synthesis in human chondrocytes in a TLR4-dependent manner (Patel et al., 2011). Other studies report that murine myeloid cells require α9-integrin expression to be activated by tenascin-C (Kanayama et al., 2009, Kanayama et al., 2011). These data suggest that a number of receptors can play a role in tenascin-C-driven inflammation. We found that the fibrinogen-like globe of tenascin-C activates TLR4 (Midwood et al., 2009), whereas α9-integrin was activated by the third fibronectin type III-like repeat of tenascin-C (Kanayama et al., 2011). Data from these studies, carried out in the absence of cell-activating levels of LPS, imply that within one tenascin-C molecule exist at least two proinflammatory domains with distinct mechanisms. Data presented here show that upon cell stimulation with LPS, tenascin-C sustains TNF-α synthesis by controlling miR-155 expression. It is not yet known how tenascin-C regulates miR-155 levels. Secreted tenascin-C may activate TLR4, α9-integrin, or other cell surface receptors. However, addition of soluble tenascin-C at a concentration comparable to that made by LPS-stimulated tnc+/+ BMDMs (<10 ng/ml) did not rescue defects in tnc−/− BMDM cytokine synthesis. Moreover, high concentrations of soluble tenascin-C (up to 1 μg/ml) inhibited cytokine synthesis (Figure S4C). These data indicate that, rather than amplifying activation of TLR4 by LPS, adding high concentrations of exogenous tenascin-C prevents LPS activation of TLR4. This may reflect competition for the same receptor by the two stimuli. Together, these results indicate that addition of soluble tenascin-C is not sufficient to rescue LPS-mediated cytokine synthesis in BMDMs derived from tenascin-C-deficient mice. Thus, ligation of a receptor by soluble tenascin-C at the cell surface may not be responsible for stimulating miR-155 expression and sustaining cytokine production during LPS activation. It may be that receptor activation requires tenascin-C assembly into a cell-associated matrix; this may function as a scaffold essential for receptor ligation or for recruitment of potential coreceptor(s). Alternatively, in light of emerging evidence highlighting a role for the 3′ UTR of matrix molecule mRNAs in controlling miRNA function (Jeyapalan and Yang, 2012; Lee et al., 2009), noncoding regions of the tenascin-C gene may drive miRNA biogenesis.
These data raise a number of questions, foremost of which is why tenascin-C should exhibit multiple proinflammatory modes of action. The answer may lie in the role of tenascin-C in driving both pathogenic and sterile inflammation. During acute inflammatory responses to LPS, tenascin-C acts swiftly to sustain cytokine synthesis at the posttranscriptional level, enabling a robust, immediate response to microbial threat. Upon sterile tissue injury, however, the ability of tenascin-C to drive de novo cytokine synthesis, via activation of TLR4 or integrins, ensures effective tissue repair but that sterile inflammation also contributes to chronicity in diseases such as RA.
Both tenascin-C and miR-155 come into play during infection, promoting innate immune responses that culminate in the synthesis of proinflammatory cytokines. Physiologically low and specifically regulated, their expression is transiently induced by LPS in innate immune cells in a NF-κB-dependent manner (Bala et al., 2011; Goh et al., 2010; Rai et al., 2008). Their dysregulation, however, can lead to both cancerous phenotypes (Calin and Croce, 2006; Chiquet-Ehrismann and Chiquet, 2003) and inflammatory and autoimmune diseases, such as sepsis (Schenk et al., 1995; Tili et al., 2007) and RA (Midwood et al., 2009; Stanczyk et al., 2008). Pharmacological inhibition of LPS-induced miR-155 expression by glucocorticoids in macrophages has been reported by Zheng et al., 2012. Notably, tenascin-C expression was also inhibited by glucocorticoids in bone marrow stromal cells and fibroblasts (Ekblom et al., 1993). Furthermore, both tenascin-C and miR-155 have been shown to affect the ability of the innate immune system to prime adaptive immune responses. In particular, they promote dendritic cell-mediated polarization of Th17 lymphocytes during inflammation (O'Connell et al., 2010; Ruhmann et al., 2012).
In conclusion, our study has identified how tenascin-C control of miR-155 levels regulates the inflammatory axis upon pathogenic activation of TLR4. These data highlight how endogenous danger signals are required for optimal cytokine synthesis in response to PAMP activation of TLRs. This finding provides insight into the positive regulation of TLR4 signaling and DAMP function, which goes beyond sterile inflammation and defines DAMPs additionally as key controllers of nonsterile inflammation. Understanding which molecular inputs are required for optimal induction of cytokines upon TLR activation may refine our strategies to manipulate excessive inflammation.
Experimental Procedures
Littermate tnc+/+ and tnc−/− mice were from heterozygous breeding pairs on a 129/SvJ background (Saga et al., 1992). All mice were male aged 8–12 weeks, except for bone marrow transplantation, which were female aged 6–8 weeks. For the generation of BMDMs, bone marrow was flushed from tibias and femurs of tnc+/+ and tnc−/− mice, and erythrocytes were lysed with Red Blood Cell Lysis Buffer (Sigma-Aldrich). The resulting cells were cultured in DMEM (PAA) supplemented with 20% FCS (GIBCO), 1% Antibiotic-Antimycotic solution (PAA), and 50 μM β-mercaptoethanol (Invitrogen) containing 100 ng/ml recombinant murine M-CSF (PeproTech). After 7 days, adherent cells were washed, replated, and stimulated with LPS alone (100 ng/ml; Enzo Life Sciences) or with brefeldin A (1 μg/ml; Sigma-Aldrich). To avoid cell toxicity, we treated BMDMs with brefeldin A for the last 8 hr of incubation with LPS. For the generation of thioglycollate-elicited peritoneal macrophages, tnc+/+ and tnc−/− mice were injected intraperitoneally with 1 ml thioglycollate (3% w/v; Sigma-Aldrich). Peritoneal exudates were collected 5 days after with PBS, and cells were analyzed by FACS. For analysis of LPS-induced sepsis, tnc+/+ and tnc−/− mice were injected intraperitoneally with LPS from E. coli 055:B5 (8 mg/kg; Sigma-Aldrich). Mice were observed for 8 hr. Body weight, mobility, uveitis, respiration, fur ruffling, and diarrhea were recorded at 0, 1.5, 4, and 8 hr after LPS injection. Mobility impairment was graded using an arbitrary scale where 0 = none, 1 = mild (reduced and slower movement), 2 = moderate (movement upon stimulation), and 3 = severe (no movement). Mice were killed after 1.5, 4, or 8 hr, and plasma and tissues were collected. All procedures were approved by the UK Home Office.
ELISAs for TNF-α, IL-6, IL-8 (R&D), IL-1β (eBioscience), IL-12, TGF-β1, IL-10, (BD Biosciences), HMGB1, and TN-C (IBL) were used to quantify secreted and cell-associated proinflammatory mediators in plasma, cell supernatants, and total protein extracts, respectively. Extracts were prepared by lysing cells (1 × 106) with 0.1 ml RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich).
Immunohistochemical Analysis
Formalin-fixed tissues from tnc+/+ and tnc−/− mice were paraffin embedded, cut into 4 μm sections, and stained for neutrophil elastase (MCA7771G; AbD Serotec). Immunostaining specificity was confirmed by omitting the primary antibody or using a nonimmune rat IgG2a (MCA1212; AbD Serotec). Neutrophil elastase+ cell number was determined by counting cells in five different areas of the total tissue section.
Plasma samples from tnc+/+ and tnc−/− mice were resolved by SDS-PAGE and analyzed by western blotting using goat anti-tenascin-C polyclonal Ab (AF3358; R&D Systems).
Bone Marrow Transplantation
Each tnc−/− and tnc+/+ female recipient mouse was exposed to a single dose of 9 Gy at 80 cGy/min radiation in a γ irradiator (Nordion) and, 3 hr later, intravenously injected with whole bone marrow cells isolated from one tnc−/− or tnc+/+ male mouse resuspended in 0.2 ml PBS. Transplanted mice received antibiotic orally for 11 weeks (2.5% Baytril; Bayer). The degree of donor engraftment was assessed as described previously by Wang et al., 2002. Genomic DNA was extracted from peripheral blood cells of female recipient and control mice using a QIAamp DNA Blood Mini kit (QIAGEN). DNA samples were analyzed for the presence of the Y chromosome by two-standard curve method based on quantitative real-time PCR in a Corbett Rotor-gene 6000 machine (Corbett Research) with TaqMan primers and probe designed to detect the TSPY gene (forward 5′-TCCTTGGGCTCTTCATTATTCTTAAC-3′; reverse 5′-GAGAACCACGTTGGTTTGAGATG-3′; probe 6FAM-TCCTGGATCAGAGTGGCTTACCCAGG-TAMRA; Applied Biosystems). Standard curves were obtained by mixing male and female DNA from nontreated control mice. Samples were standardized against a mouse GAPDH genomic primer/probe set (Applied Biosystems). Samples were calibrated against DNA from male-nontreated control mouse.
RNA Extraction and Quantitative Real-Time PCR
Total RNA was extracted from cells (1 × 106) using a RNeasy Mini Kit (QIAGEN). cDNA was synthesized from equivalent amounts of RNA with AffinityScript reverse transcriptase and oligo(dT) primer (Stratagene). Quantitative real-time PCR was performed in a Corbett Rotor-gene 6000 machine with TaqMan primer sets for mouse tenascin-C (Mm00495662_m1), TNF-α (Mm99999068_m1), IL-6 (Mm00446190_m1), CXCL1 (Mm00433859_m1), IL-10 (Mm00439614_m1), CD11b (Mm00434455_m1), CD68 (Mm00839636_g1), F4/80 (Mm00802529_m1), and HPRT1 (Mm00446968_m1) (Applied Biosystems). For miRNA analysis, total RNA including small RNA was extracted from cells (2 × 106) using TRIzol (Invitrogen). For mature miRNA detection, RNA was reverse transcribed using the TaqMan miRNA reverse-transcription kit, including the miRNA-specific primers, followed by real-time PCR with individual miRNA TaqMan assays for the endogenous reference RNA RNU6B, miR-155, miR-146a, and miR-21 (Applied Biosystems). For pri-miRNA detection, RNA was reverse transcribed with AffinityScript reverse transcriptase and random primers (Stratagene), followed by real-time PCR with TaqMan primer sets for mouse pri-miR155 (Mm03306395_pri) and HPRT1 (Applied Biosystems). Changes in expression were calculated by the change-in-threshold (ΔΔCT) method with HPRT1 and RNU6B as endogenous controls for gene expression and miRNA analysis, respectively, and were normalized to results obtained with untreated cells.
miRNA Array
The expression profile of 375 miRNAs was analyzed with TaqMan Low Density Arrays in an ABI 7900HT (Applied Biosystems). Total RNA was extracted from cells with TRIzol. RNA integrity was analyzed using an Agilent RNA 6000 Nano kit and an Agilent 2100 bioanalyzer (Agilent Technologies). cDNA was synthesized with a TaqMan miRNA reverse-transcription kit and Megaplex RT primers and was followed by real-time PCR with a TaqMan universal PCR master mix and TaqMan array rodent miRNA cards (Applied Biosystems). Data were analyzed with SDS software (Applied Biosystems).
Transient Transfection
For transfection of miRNA, 2 × 106tnc−/− BMDMs were transfected with 5 nM negative control precursor or miR-155 precursors with siPORT NeoFX transfection agent (Ambion) for 8 hr. Cells were allowed to recover for 24 hr before treatment with LPS for 8 hr.
Statistical Analysis
Statistical analysis was performed using Student's t test, one-way ANOVA with Dunnett's multiple comparison posttest, or two-way ANOVA with Bonferroni posttest where appropriate; correlation was assessed with Spearman's rho test (Prism 5; GraphPad Software).
Clinical Evaluation of Septic Mice
Mice were observed during a period of 8 hr upon LPS injection. Pictures of mice were taken to record uveitis at 0, 4 and 8 hr after LPS injection. Uveitis was considered mild when eyes were watery, moderate when one or two eyes displayed pus and severe when one or two eyes were closed. Fur ruffling was recorded at 0, 1.5, 4 and 8 hr after LPS injection and was scored as 0, 1, 2 or 3 for absent, mild, moderate or severe, respectively.
Flow Cytometry
For surface staining, peritoneal macrophages and BMDMs were stained for 20 min at 4°C with anti-F4/80-PE (Caltag Medsystems), anti-CD11b-APC, anti-MHC II (I-A/I-E)-PE, anti-CD40-APC, anti-CD80-PE, anti-CD86-FITC, anti-TLR4/MD-2-APC and appropriate isotype controls (eBioscience). For intracellular staining, cells were fixed with 4% paraformaldehyde in PBS for 20 min, were made permeable with PBS containing 2% (vol/vol) BSA, 2mM EDTA, 0.02% NaN3 and 0.05% (vol/vol) saponin and were stained with anti-CD68-APC (AbD Serotec). Samples were analyzed on a FACSCanto II (BD Bioscience) and data were analyzed with FlowJo software (TreeStar).
Treatment of BMDMs with Soluble Tenascin-C
To assess whether addition of exogenous tenascin-C to tnc−/− BMDMs can rescue defects in cytokine synthesis in response to LPS, recombinant human tenascin-C, was synthesized and purified as described previously (Midwood et al., 2009), or commercially purified human tenascin-C (IBL, Japan) was used. 1x105tnc+/+ and tnc−/− BMDMs were stimulated for 24 hr with 100ng/ml LPS without or with 1 ng/ml to 1 μg/ml recombinant purified tenascin-C. Cell supernatants were analyzed by ELISA to quantify secreted TNF-α and IL-6 (R&D). Viability of the cells throughout the experimental time period was examined by the MTT cell viability assay (Sigma-Aldrich). Cell viability was not affected by treatment with tenascin-C. Addition of tenascin-C to tnc−/− and tnc+/+ BMDMs in the absence of LPS stimulated cytokine synthesis (data not shown).
Statistical Analysis
Statistical analysis was performed using Student's t test or two-way ANOVA with Bonferroni posttest where appropriate; (Prism 5; GraphPad Software).
We thank A. Hazlehurst for support with animal work, D. Essex for immunohistochemical assistance, F. McCann for advice on FACS, A. Martinez-Sanchez for advice on miRNA array, and M. Feldmann for reading the manuscript. This work was supported by Arthritis Research UK and Medical Research Council New Investigators Research Grant (G070010881726 to K.S.M.).
Accession Numbers
The Gene Expression Omnibus (GEO) accession number for the microarray data reported in this paper is GSE41584.
Supplemental Information
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Publication Information
Received: April 18, 2012
Revised: July 30, 2012
Accepted: September 7, 2012
Published online: October 18, 2012


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$2 million bond for Windsor votersBy Christian Avard
Staff Writer | March 02,2013WINDSOR Windsor Town Manager Tom Marsh said the wastewater treatment facility is a critical part of the towns infrastructure. Now voters will have to decide whether its worth investing in an aging facility in need of renovations.
According to Marsh, the answer is yes.
Theres been a high level of care to the facility over the years. But time has taken its toll and without these repairs wed be looking at a critical failure, Marsh said.
The town of Windsor has before it a big-ticket item on Town Meeting Day. Its a $2 million bond for renovations to the towns wastewater treatment facility. The article will be voted on by Australian ballot.
Marsh said the state suggests Vermont cities and towns conduct an analysis of wastewater treatment facilities every 20 years. The town of Windsors treatment plant is about 20 years old, and the analysis concluded it needed a complete overhaul.
The estimates came back at approximately $12 million to $13 million, but a project like that was out of the question, Marsh said. The town did another review and identified the plants most critical needs.
According to the study, the town needs a new digester, a section of the plant that takes sewage and breaks it down to generate methane gas, and storage tanks which Marsh estimates are 40 years old. The $2 million bond would go toward pipe upgrades, replacements and new containers.
Marsh said pending approval, the town will rebuild the clarifiers first and the digester next. He estimates that taxpayers will see an increase between 3 and 4 percent a year over the next few years to pay off the bond.
Its going to take 18 months to two years from where we are today to complete this stage, Marsh said. The second stage where were doing repairs out of our capital plan wont impact ratepayers as much.
Approximately 1,600 sewer customers are responsible for paying the bond, but the entire town votes on the bond question.
Windsor residents will vote on money matters at 7 p.m. Monday in the White Auditorium at Windsor High School. Voting at the polls, including the $2 million bond, will be held from 9 a.m. to 7 p.m. Tuesday at the Windsor Municipal Building at 29 Union St.
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