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Remke Industries
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Silicone Cable Connectors are Ideal for Extreme Temperatures
Mini-Link™ Electrical Connectors & Cordsets molded with Silicone are ideally suited for industrial applications for very hot or cold temperatures, extreme temperature ranges and may types of severely harsh environments.(read more)
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Habitat aids international construction
PARKERSBURG – Contributions from Habitat for Humanity in Wood and Washington counties have financed 118 homes around the world, officials said.
The local Habitat affiliates tithe 10 percent of unrestricted funds to the international, which builds homes in other nations.
Wood County since 1990 has tithed $272,000, said Director Alvin Phillips. That's resulted in the construction of 100 homes overseas, he said.
More homes can be constructed overseas where the average cost is about $4,500, Phillips said. It's upward of $80,000 or more in America, he said.
"We can build more homes because the building costs are so much less over there," he said.
According to Habitat for Humanity worldwide, local affiliates tithed more than $13 million in fiscal 2014, July 1, 2013 through June 30.
"Through your tithe, your international colleagues were able to provide safe and secure housing for more than 2,900 families," said Susan Weltner Yow, Affiliate Tithe and Global Engagement director.
Habitat for Humanity in Washington County has tithed $56,762 since 1991, Executive Director Dennis Thomas said. It is a smaller affiliate than Wood County, he said.
The Washington County contributions have financed 18.5 homes, Thomas, citing a breakdown from the international, said.
"All of our tithe money right now goes to Thailand," Thomas said.
Affiliates can request where their contributions go, he said.
Habitat for Humanity, established in 1976, is a nonprofit ministry dedicated to providing safe and decent housing. Recipients work with volunteers in building the homes.
More than 1,500 affiliates are in the United States.
Washington County is building its 19th home. Wood County Habitat hopes to have started construction of its 75th home by end of 2015, Phillips said.
The 69th home in Wood County was dedicated in July.
"We are focused on houses we build here at home, with our own partners, and those are big reasons to celebrate," Phillips said. "But it's important to recognize how far our American dollars go in other, less-developed countries, and remind our supporters that they are a part of a global mission."
The contributions to the international are in keeping with the Christian value of tithing 10 to God's work, Phillips said.
"It is truly a blessing to be part of that bigger effort, and to know our funds just keep working toward our mission of simple, decent housing for people everywhere," Phillips said.

subset: commoncrawl


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Fair Hotel. Great location.
Guest Review Highlights
- Great location
- Room is all right
- Food is all right
- Comfortable
- Service is adequate
- Reception should be more attentive
Wego searches multiple travel sites so you can find and compare the best rates.
Based on 72 reviews from across the web.
Fair Hotel. Great location.
Check-in: 15:00
Check-out: 11:00
Located in the heart of Barcelona, this apartment is within walking distance of Museu de la Xocolata, Palau de la Musica Catalana, and Arc de Triomf. Also nearby are Basilica de Santa Maria del Mar and Barcelona Cathedral.


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Labour promises to help the homeless
Labour is promising "serious action" to end homelessness if it wins next year's election, and the government says that's great news.
Party leader Andrew Little says $60 million would be put into emergency housing over four years, enough to put a roof over the heads of 5100 homeless people a year.
Social Housing Minister Paula Bennett announced in May, ahead of the budget, that $41m was being allocated to pay for about 3000 emergency housing places.
"They've obviously decided to pick up our policy and run with it, I think it's great news," she told reporters on Thursday.
"I'm not sure where they're going to get them from - magic them up from imaginary beds that are sitting empty?"
Mr Little says too many families will spend this winter living in cars, couch surfing or cramped into the lounges and garages of relatives.
"This policy - along with Labour's plans to launch a massive state-backed affordable house building programme and build more state houses rather than sell them off - will help end homelessness in New Zealand."
Mr Little says the policy would provide 1400 beds or 5100 places a year which, along with current provisions, would bring the total emergency housing available to 8100 with 2200 beds at any given time.
"Recent Otago University research estimated there are 42,000 homeless people throughout the country and of those almost 4200 are sleeping rough," he said.
"The homeless are at the sharp end of the government's housing crisis."
Documents released with the announcement show that typically people are homeless for a few months, and there is a considerable flow of people in and out of homelessness.
"This new supply of emergency housing will be enough, over time, to support the homeless population into permanent housing."
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If you're looking for professional, look no further. Krusell's Kalmar FlipWallet for your iPhone 6S or iPhone 6 boasts a beautiful design that adds next-level convenience and functionality.
Made of high quality leather, this FlipWallet Case opens like a book and features a soft microfiber lining on the inside along with a secure magnetic closure that keeps all your personal items safe. You'll find three pockets inside for storing credit cards or business cards, along with a pocket undeath those for cash. This iPhone 6S or iPhone 6 case really is like having a wallet, only better!
On the back of the FlipWallet is one additional storage sleeve that you can keep your daily-use debit or credit card so it's simple to slide out on the fly. Each feature of your iPhone 6S or iPhone 6 is accessible through this case, with the exception of the display which is protected while it's closed. Camera, ports and buttons... no problem.
The precise stitching of Krusell's Kalmar FlipWallet Case only makes it more appealing and great for anyone seeking a professional quality design for their iPhone 6S or iPhone 6.
Please Note: Product images, including color, may differ from actual product appearance.
Works with: iPhone 6S, iPhone 6
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Namtok Phlio National Park
|Namtok Phlio National Park
IUCN category II (national park)
Phlio Waterfall
|Chanthaburi Province, Thailand
|Nearest city
|12°31′31″N 102°10′37″E / 12.52528°N 102.17694°ECoordinates: 12°31′31″N 102°10′37″E / 12.52528°N 102.17694°E
|135 km2 (52 sq mi)
|May 1975
|Governing body
|Department of National Parks, Wildlife and Plant Conservation
Namtok Phlio National Park (Thai: อุทยานแห่งชาติน้ำตกพลิ้ว) is a national park in Chanthaburi Province, Thailand. Home to waterfalls and forests, the park is also the site of a stupa and chedi from the reign of King Rama V.[1]
Namtok Phlio National Park is located 14 kilometres (8.7 mi) south of Chanthaburi town in Mueang, Laem Sing, Khlung and Makham districts. The park's area is 135 square kilometres (52 sq mi). The highest point is Map Wa Krok peak at 925 metres (3,035 ft).[1]
During the reign of King Rama V, the Along Khon chedi was built in 1876. In 1881 a memorial stupa, housing a relic of Princess Sunanta Kumari, was commissioned by King Rama V.[1]
On 2 May 1975, the area was declared a national park as Khao Sa Bap National Park. On 29 September 1982, the park was renamed Namtok Phlio National Park.[1]
The park's main attraction is its namesake waterfall, Phlio, whose pools are home to large numbers of soro brook carp.[2] The King Rama V era chedi and stupa are located near Phlio waterfall. Other park waterfalls include Khlong Narai, Makok and Trok Nong.[1]
Flora and fauna[edit]
Namtok Phlio is covered in tropical rain forest, including such species as Aphanamixis polystachya, Aquilaria crassna, Scaphium scaphigerum, Sandoricum koetjape, Irvingia malayana, Cotylelobium lanceolatum, Alstonia scholaris and Dalbergia oliveri.[1]
Animal species include serow, tiger, bear, barking deer, porcupine, macaque, langur, gibbon, mouse deer, pangolin, mongoose and otter.[1]
The park is host to abundant bird species including heron, brahminy kite, shika, crested serpent eagle, besra, imperial pigeon, red turtle dove, emerald dove, hornbill, barbet, vernal hanging parrot, shrike, woodpecker, drongo, hill myna, bulbul and white-rumped shama.[1]
|Wikimedia Commons has media related to Namtok Phlio National Park.


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The Box garden bench is made of sturdy polypropylene. This plastic is weatherproof and colorfast.
Product Details
Weights & Dimensions
Other Dimensions
|Product Type: Bench
|Primary Material: Plastic
|Frame Material: Plastic
|Style: Modern
|Hand Painted: No
|Arms Included: Yes
|Back Included: Yes
|Base Type: Four legs
|Cushions Included: No
|Glider: No
|Custom Made: No
|Indoor Use: Yes
|Commercial Use: Yes
|Assembly Required: No
|Product Warranty: 2 years
About the ManufacturerGarden Impressions is the largest garden furniture brand in Europe, and has long been recognized for design and quality at very competitive prices. Whether you are looking for beautiful dining furniture, a lounge suite or parasol/gazebo, Garden Impressions offers you the very best selection!
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If you haven't tried spicing up your go-to smoky-eyed look with a dash of color, it's about time you get in on the trend because it's going to be huge this fall, according to Cosmopolitan magazine.
You may not care what you look like while you work out, but if you consider the gym a legitimate place to meet single guys, you may want to take advantage of a few simple tricks that will make you look hot, not sweaty.
Not everyone was born with luxurious lashes, but luckily, it's easy to recreate a full-bodied look that will make your peepers pop.
Nobody was born perfect, but luckily, there are plenty of tricks that celebrity makeup artists use to fake a flawless face.
Smokey shades look great on everyone, but they're not always appropriate for the office or for daytime events where so much light can make your style look a bit overdone.
If your eyes are on the smaller side or you just want to have a more bright-eyed look, there are plenty of ways to use product and shading techniques to your advantage.
Cat-eye eyeliner is a difficult look to pull off, especially if you tend to second guess your makeup application skills.
Makeup primer is relatively new to the market but is gaining popularity due to the many benefits it provides.
You can style your hair, line your eyes, paint your pout and even bronze your arms and chest, but it'll all be for naught if the only thing you can focus on when you glance in the mirror is that zit on your cheek.
If there's one part of a gal's beauty routine that can be downright tough, it's applying eyeliner without winding up with squiggly, smudged black gunk all over your face.
Fingers, sponges, pads, brushes – which applicator should you use when you're putting on foundation? This handy guide will help you decide which tool is right for you.


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The Embee Sunshade Co., located on Metropolitan Ave near Graham Ave, is one of Williamsburgh's last surviving factories. If you've ever walked past this neighborhood institution, you may have noticed the seemingly random, white plastic letters askew over their entrance, which read "EM EE UNSH."
The missing letters fell off years ago. Regardless, the factory has continued to handcraft high-quality umbrellas at their factory, without fail, since 1940. Their products have become part of the iconic landscape of New York City, providing shade over hot dog carts, lifeguard stands, and NYC Parks Department kiosks.
The City Reliquary Museum, a neighbor nearby on Metropolitan Ave and Havemeyer St, is proud to host an exhibition about their 79-year business history. The show will include Embee promotional materials, parts and materials from the factory, photography by local artist Theo Coulombe of Brooklyn photo Studio, and a backyard filled with dozens of Embee Sunshades. The display will be on view from August 4th through October 28th, 2012.
The opening reception on Saturday August 4th from 5:00 – 9:00 pm will be hosted by the City Reliquary Museum located at 370 Metropolitan Avenue. Refreshments will be provided, including beer generously sponsored by another great Williamsburgh factory, the Brooklyn Brewery.
Transportation for this exhibit is generously provided by Boxart Inc. Fine Art Services.
Located at 370 Metropolitan Avenue in Williamsburgh, Brooklyn, The City Reliquary Museum & Civic Organization provides a wide array of services to the community. As a certified 501(c)3 not-for-profit organization, we are committed to serving the people of New York City – natives, newcomers, and passersby. Originally established as a window-front display only museum in 2002 at the corner of Grand and Havemeyer Sts, it moved into its present location in 2006 and is committed to plan and host public events, which provide neighbors and visitors with a place to meet, exchange ideas, and celebrate the diversity of our community.
The City Reliquary's hours are: Thursday through Sunday: 12pm – 6pm. Admission to the museum is $5 suggested donation. For general information, please visit the Museum's website www.cityreliquary.org or call 718. R U CIVIC.
Photo credit: Flickr
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- Back to School: Better Sleep with Sonno Bed Mattress - August 24, 2016


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In reply to Reid B. Kimball's post on Thu 12 Jan 2012 :
Kickstarter is only for US residents. There is a crowdfunding thing here in Oz too, I've just been avoiding it and trying for production houses and broadcasters.
Sun 15 Jan 2012
In reply to Reid B. Kimball's post on Thu 12 Jan 2012 :
Kickstarter is only for US residents. There is a crowdfunding thing here in Oz too, I've just been avoiding it and trying for production houses and broadcasters.
Wed 18 Jan 2012
Hi all, I'm currently trying to raise funding for a documentary about a network of controversial adolescent drug abuse programs that I was a member of for five years. We just released a pitch trailer and launched an IndieGoGo campaign.
I would love to get some feedback from professionals on what we are doing right and what we could be doing better. This is my first time trying to do something like this and I could use all the help you can offer.
The trailer and IndieGoGo page are here. http://www.indiegogo.com/thegroup
Not asking for donations here, just looking for feedback. Thanks in advance.
Thu 19 Jan 2012
Hi Jacob,
This looks like a really interesting story, especially since you have the inside scoop.
I am wondering if you have any other footage besides the talking heads and pictures. Are there scenes you can shoot? Or can you come up with some creative B-Roll that relates to the feelings that everyone is talking about? I know you are just beginning with telling your story, but I think it is always good to think in these terms. Best of luck!
Thu 19 Jan 2012
Hi Jill,
Thanks for checking it out.
Right now, we have about 13 hours worth of video footage that was shot by members of The Group and more coming. We didn't use any in the trailer because we are still working out the legalities of using it, but we intend to use some of it in the film.
As for B-roll, we plan to get footage of our interview subjects in their daily lives, plus just shooting anything interesting that comes up when we are on location.
Thu 19 Jan 2012
Subject looks compelling so far. I have heard of extreme drug programs (like the right wing christian ones that periodically get shut down by a state gov and then pop up elsewhere under a different name) but this is the first I heard about one totally run by kids. Lots of parallels though in terms of the brainwashing, peer pressure and torture lite techniques. Was there money involved? Anyone profiting?
Fri 20 Jan 2012
In reply to Jill Woodward's post on Fri 20 Jan 2012 :
Hi Jill. There is definitely a profit being made. My family, for example, which is a working-class family, spent around $70,000 in the five years that I was involved. The money is from treatment programs (in-patient and out-patient). They convince parents that without the treatment their child will literally die.
We were very limited in what we could go into with the teaser trailer, but we plan to explore the whole history of the organization in the final film.
The entire operation was founded by one man who is a convicted-felony, former heroin addict and self-professed murderer. There has been a lot written about the organization going back to the 1970s. Here's a link to a little background information, if you're curious.
Thanks for checking it out. If you have any advice, I sure could use it.
Fri 20 Jan 2012
sounds very similar to Kids of Bergen County, where my friend was incarcerated, it changed names and moved around a lot to avoid regulation. From your trailer I almost thought it was the kids organizing themselves, but it sounds very much like there is an adult orchestrating and profiting. Good luck!
Fri 20 Jan 2012
In reply to Jill Woodward's post on Fri 20 Jan 2012 :
It's very similar to the Kids of Bergen County and the Straight program.
Fri 20 Jan 2012
Jacob, nice job on your first film so far, but I do see a couple areas to work on.
Sound quality is inconsistent, some sounded echo-y while others sounded high quality but too soft and quiet, I'm hard of hearing. I like to make sure the sound is loud but not too loud. People can always turn their sound down, but I had maxed the sound on my sys and headphone and still had a hard time hearing a lot of what was said.
Also, be more conscious of where your key lights are falling on the interview subject's face and which direction they are looking in. In one scene, in the bedroom, the key light was behind the person, not in front. So, we see most of their face being dark with the back side of the face lit. Other scenes the subject is to one side of the frame (rule of thirds) but is looking towards the short side, not the long side.
I've made all these mistakes myself. It's a challenge to keep all this conscious when you are trying to setup equipment and think about the interview questions, etc.
Sun 22 Jan 2012
In reply to Reid B. Kimball's post on Sat 21 Jan 2012 :
Reid, thanks for the feedback. We had a lot of difficulties with light in one of the interviews. We were using natural light and had issues with the sun moving behind clouds and also getting dark early. We tried out best to fix it with some color correction, but there was only so much we could do.
Hopefully, if we are able to raise the money, we will be able to budget for lights and have much more control over the finished product.
As for the sound, I confess I don't know much about it, but I did notice that it jumps around from interview to interview.
Thanks for all the feedback. I really appreciate it.


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Murie Center
Rating: 0 stars 0 0 review 49
Po Box 399 Moose WY 83012 USA
Bringing people together to inspire action that preserves nature.
Outreach & public interpretation planned tours: the murie center has created strong relationship with many organizations that like to bring many groups of visitors to the murie ranch. These organizations will bring new groups of people on a frequent basis e. G. , tauck tours is a tour guide that brings participants through yellowstone and grand teton national park. While at the murie center we show the group through mardys house and the homestead, and point out and talk about the other cabins. Tours also include an overview of the muries achievements and how the murie center continues their legacy today. Teton science schools is another very strong partner of the murie center and frequently brings out groups of young students as well as, graduate students. Drop-in guests: at the murie center we have two types of drop-in guests, those who are looking for the murie center and those who wander down the road, unknowing where they will end up. Both type of drop-in visitor need different types of tours. With those who have previous murie knowledge and are interested in knowing specific information we may take them into the studio to see some of the murie archives and artifacts. For those who dont know where they are, we need to seize the opportunity to engage new visitors and supporters of the murie center. These people are usually interested in the history of the physical the ranch and knowing more about who the muries were.
conservation leadership we effect change in the conservation world by: engaging new conservationists: in order to ensure that wild places arereserved and humans and wildlife find a harmonious balance on the land they share, we will need more conservationists from all walks of life. One of our goals at the murie center is to reach out to these inactive conservationists who are waiting for the opportunity to contribute to an effort that has excluded them, or simply eluded them. Inspiring and empowering people:the murie center has an opportunity and an obligation to use its voice and its resources effectively as the muries did in countless forums, bringing bright minds together with the biggest of ideas to make the world a better place. We work to continue to inspire conservation leadership into the 21st century. Advocating for large landscape conservation and citizen action: the muries knew how valuable intact ecosystems were to sustaining healthy and diverse populations of wildlife and foremost among their accomplishments were the establishment of the arctic national wildlife refuge and the passage of the wilderness act. Their holistic ecological philosophy guides our work to provide tools and resources for citizen action for a better world.
preservation & maintenance the murie center, in agreement with our landlord grand teton national park, works to ensure that all assets on the murie ranch (a national historic landmark) are kept in good condition. This applies equally to, maintaining in good condition or better, and performing, regularly scheduled maintenance, preventative maintenance, and life-cycle maintenance on the land, buildings, infrastructure, and other improvements and assets used by the organization at the murie ranch.
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Smile A4 Print by Miniwilla
Brand Miniwilla
Smile print by Miniwilla. Designed and printed in Sweden.
A4 Size.
Colour - Black and White.
Printed on 250gsm matte art paper.
We love this bold monochrome print by Miniwilla.
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Adele Dress with Pebbles Print by Soft Gallery
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When you add some personality to your envelopes, people can't wait to see what's inside!
Remember the dearly departed with a Day of the Dead event. Here's how to turn your get-together into a stunning celebration with Avery.
With Avery, there's more than one way to address your envelopes. We've got labels in a variety of shapes, sizes and materials, and free designs to decorate them on Avery Design & Print.
See why the fun part isn't the unwrapping of the gift.
Add a fun, festive topper to topiaries, decorations, cupcakes and more with a personalized Avery Round Label.
When Thanksgiving food takes time to make, serve up some grub to shore up restless appetites. Create custom labels with free templates on Avery Design & Print to identify your munchies or place settings, then ship out the supplies to the hungry crew.
Get into the Halloween spirit with these haunting projects—from party invites and favors to give you goose bumps, to wicked stickers and iron-on templates to dress up your ghoulish goodie bags.
Feeling the holiday pressure? These tips can help you tackle the Thanksgiving hustle and bustle.
Treat your tricksters to party favor bags filled with bone-crunching treats!
Spread some fresh and minty holiday cheer to those near and dear with these boxes of Winter Breath mints decorated with fun designs on square labels.


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THIS IS A DIRECTORY PAGE. Britannica does not currently have an article on this topic.
Learn about this topic in these articles:
use of lipogram
a written text deliberately composed of words not having a certain letter (such as the Odyssey of Tryphiodorus, which had no alpha in the first book, no beta in the second, and so on). The French writer Georges Perec composed his novel La Disparition (1969; A Void) entirely without using the letter e; his English translator, Gilbert Adair, succeeded in avoiding that...


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Clinton TV ad hits Trump hard; he promises an all-out effort
CLEVELAND (AP) ? Hillary Clinton has unveiled an emotional television ad criticizing Donald Trump that features the parents of a slain Muslim American Army captain. Trump is assuring supporters he will have no regrets if he loses the presidential election because he is going all out in the final weeks of the campaign.
LEADING OFF: Cubs face Kershaw, try to end Series drought
A look at what's happening all around the majors today:
U.S Defense Secretary Carter arrives in IraqBAGHDAD (AP) ? U.S. Defense Secretary Ash Carter arrived in Iraq Saturday to meet with his commanders and assess the progress in the opening days of the operation to retake the northern city of Mosul from Islamic State militants.
The Latest: Iraqi army launches new push on town near Mosul
KIRKUK, Iraq (AP) ? The Latest on developments in Iraq where Iraqi forces and their allies launched a major offensive this week to retake Mosul, the country's second-largest city from the Islamic State group (all times local):
India launches phone app to monitor New Delhi's pollutionNEW DELHI (AP) ? The Indian capital, laboring under the label of being the world's most polluted city, is trying something new to help clean up its air.
IS assault on Iraq's Kirkuk ends after 24-hour battle
KIRKUK, Iraq (AP) ? A massive Islamic State assault on targets in and around the northern Iraqi city of Kirkuk came to an end Saturday after a day and night of heavy clashes, as Iraqi forces launched a new advance southeast of the IS-held city of Mosul.
Suspicious envelope found at Clinton campaign office: police
An envelope containing a white powdery substance was sent to one of White House hopeful Hillary Clinton's campaign offices in New York, though police initially ruled out any danger. The envelope was first delivered to Clinton's offices in Manhattan, where campaign workers then transferred it to her Brooklyn headquarters, New York Police Department Lieutenant Thomas Antonetti told AFP. "The preliminary investigation determined that it was negative in terms of containing a hazardous substance," he added, noting the Department of Health was conducting further evaluation to determine the nature of the substance.
Montana judge sparks outrage with no prison time for incestHELENA, Mont. (AP) ? A judge's decision not to order prison time for a Montana man who raped his 12-year-old daughter has sparked outrage from afar and calls closer to home to toughen the state's law, which allows such lenience in certain circumstances.
U.S. Defense Secretary Ash Carter arrives in Iraq to assess progress of the operation to retake Mosul from IS militantsBAGHDAD (AP) ? U.S. Defense Secretary Ash Carter arrives in Iraq to assess progress of the operation to retake Mosul from IS militants.
EU parliament chief, Canada minister in bid to save trade deal
EU parliament head Martin Schulz and Canada's trade minister Chrystia Freeland were meeting Saturday, saying they hoped to revive a trade deal threatened by the refusal of a Belgian region to sign on. Schulz wrote on Twitter he would also meet with Wallonia's socialist government head Paul Magnette, who has moved to stop the bloc's 28 nations from signing the accord. The meetings in Brussels are aimed at "reviving CETA talks.


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Most broadcasters in the Czech Republic are now either testing or have adopted HbbTV.
New DTH/IoT combo box from Antik
Antik Technology has developed a unique interactive set-top box with OTT content and smart home/IoT gateway.
Abox42 announces new UHD smart STBs
Abox42 GmbH unveils at IBC 2016 in Amsterdam its new Smart STB platform M50, developed for advanced operator projects worldwide.
TV Nova starts HbbTV test
The Czech Republic's leading broadcaster TV Nova, which is owned by CME, has begun trialling HbbTV.
CanalDigitaal launches linear channels on hybrid STB
M7 Group's Dutch DTH platform CanalDigitaal is adding two RTL channels, RTL Crime and RTL Telekids, as linear channels on its new hybrid satellite receivers.
Timvision launches hybrid Android TV set-top
Google's Android TV is now coming to Italy with a hybrid set top-box delivered by Timvision, the TV platform of telecom operator Telecom Italia (TIM).
Zattoo and ABOX42 launch DVB-C/IP multiscreen TV solution
Swiss IPTV company Zattoo and German set-top-box provider ABOX42 demonstrate a new white label DVB-C/IP hybrid multiscreen TV solution for cable operators at ANGA COM 2016.
First HbbTV app for TV Markiza
Slovakia's leading broadcaster TV Markiza has launched its first HbbTV app in co-operation with the Czech development company Mautilus.
Key HbbTV ad test in Czech Republic
The Czech Republic's Atmedia is preparing to launch its first campaign using the HbbTV platform.
HbbTV: a Polish success story
HbbTV has been a resounding success for the Polish public broadcaster TVP in the last four years.


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Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory Molecule
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Background— T-cell–mediated immunity contributes to the pathogenesis of atherosclerosis, but little is known about how these responses are regulated. We explored the influence of the inducible costimulatory molecule (ICOS) on atherosclerosis and associated immune responses.
Methods and Results— Bone morrow chimeras were generated by transplanting ICOS-deficient or wild-type bone marrow into irradiated atherosclerosis-prone, LDR receptor–deficient mice, and the chimeric mice were fed a high-cholesterol diet for 8 weeks. Compared with controls, mice transplanted with ICOS-deficient marrow had a 43% increase in the atherosclerotic burden, and importantly, their lesions had a 3-fold increase in CD4+ T cells, as well as increased macrophage, smooth muscle cell, and collagen content. CD4+ T cells from ICOS-deficient chimeras proliferated more and secreted more interferon-γ and tumor necrosis factor-α than T cells from control mice, which suggests a lack of regulation. FoxP3+ regulatory T cells (Treg) were found to constitutively express high ICOS levels, which suggests a role for ICOS in Treg function. ICOS-deficient mice had decreased numbers of FoxP3+ Treg and impaired in vitro Treg suppressive function compared with control mice.
Conclusions— ICOS has a key role in regulation of atherosclerosis, through its effect on regulatory T-cell responses.
Received April 10, 2006; revision received August 2, 2006; accepted September 8, 2006.
Atherosclerosis is characterized by the accumulation of lipids and chronic inflammatory cells in arterial vessel walls.1 Although there is ample evidence that immune responses contribute to the pathogenesis of this common disease, there are many unanswered questions about how those immune responses are regulated and how they may be targeted for therapeutic intervention.2 CD4+ T-helper (Th) cells are evident in the atherosclerotic plaque and have been shown to be important in the development of atherosclerosis.3,4 Immune responses mediated by CD4+ Th1 cells specific for plaque antigens such as oxidized LDL (oxLDL) or heat shock protein 60/65 may have an important role in the propagation of the inflammatory process.5 CD4+ T-cell responses to antigen are modulated by costimulatory signals, delivered by B7 family molecules on antigen-presenting cells that bind to CD28 family molecules on the T cells. We have shown that the absence of B7-1 and B7-2 significantly reduces the progression of atherosclerosis.6 Inducible costimulatory molecule (ICOS) and its ligand (ICOS ligand) are CD28 and B7 family members, respectively, that also have an important role in modulating T-cell activation. ICOS is expressed on activated T cells after antigen stimulation7 and may be important in secondary activation of differentiated T cells. ICOS appears to support T-cell proliferation and Th2 differentiation.8–10 In addition, ICOS has been implicated in regulatory T-cell (Treg) function.11,12 ICOS-deficient mice are more susceptible to experimental autoimmune encephalitis.7 ICOS has also been shown to be important in antibody isotype switching.7,13,14
Clinical Perspective p 2055
Recent evidence suggests that the local balance between effector and natural regulatory T cells influences the development of atherosclerosis in animal models.15,16 Furthermore, Th1 proinflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-18, and IL-12 may have a key role in promoting atherogenesis,17,18 whereas T-cell responsiveness to transforming growth factor-β (TGF-β) has been shown to limit atherosclerosis.19 In addition, natural antibodies specific for oxidized phospholipids may have a protective effect on the development of atherosclerotic lesions.20,21 Because ICOS is reported to influence effector T-cell, antibody, and regulatory T-cell responses, we hypothesized that the ICOS pathway regulates immune responses to plaque antigens and thereby influences development of arterial disease. To test this hypothesis, we used a bone marrow chimeric approach to examine the influence of ICOS deficiency on atherosclerosis in LDL receptor (LDLR)–deficient mice. Our findings show that ICOS deficiency in bone marrow–derived cells results in enhanced atherosclerosis and associated immune responses due to the requirement of ICOS for development and function of atheroprotective natural Treg.
LDLR−/− mice, backcrossed 10 times onto a C57BL/6 background, and wild-type C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me). ICOS−/− mice were derived as described elsewhere14 and were backcrossed 10 generations onto a C57BL/6 background. All mice were housed and bred in accordance with the institutional guidelines of Brigham and Women's Hospital and Harvard Medical School.
Bone Marrow Transplantation Protocol
At 8 weeks of age, LDLR−/− mice were lethally irradiated with a cesium source (total dose 1300 rad, split into 2 doses 4 hours apart). Donor bone marrow cells were injected after the second irradiation. Eight-week-old ICOS−/− or wild-type donor mice were euthanized by CO2 asphyxiation, and femurs, tibias, and humeri were harvested by a clean technique. Bone marrow cells were extracted, and 20 million cells were injected via tail vein into sex-matched irradiated LDLR−/− recipient mice. Recipient mice received normal chow and water containing sulfamethoxazole (4.14 mg/mL) and trimethoprim (0.83 mg/mL) for 4 weeks.
Induction of Atherosclerosis
Four weeks after the transplantation, the mice were begun on a semipurified cholate-free diet (No. D12108; Research Diets Inc, New Brunswick, NJ) containing 40% kcal lipid, 1.25% cholesterol22 ad libitum (6 to 8 males and 6 to 8 females per group). After 8 weeks on this diet, the mice were fasted overnight and killed by CO2 asphyxiation. Blood was collected by vena cava nicking, and the arterial tree was perfused with Dulbecco's phosphate-buffered saline (Gibco BRL, Gaithersburg, Md). The heart and the whole perfused aorta were dissected from the aortic valve to the iliac bifurcation. The heart base, including the aortic sinus with the proximal aortic arch and the aortic branches, was cut and separated from the remaining aorta, then rapidly frozen in optimal cutting temperature embedding medium (OCT, ProSciTech, Thuringowa, Australia); the remaining thoracic and abdominal aorta (descending aorta) from each mouse was fixed in 10% buffered formalin.
Aortic Atherosclerotic Lesion Analysis
The aortic sinus was analyzed as described previously.6 Alternate 5-μm cryosections of ≈200 μm of the aortic sinus were prepared. From the area in which 3 aortic valve cusps are clearly seen, alternate sections were collected for quantification and stained with oil red O according to the method of Paigen et al.23 Section images were captured digitally (SV Micro, Zeiss, Jena, Germany) and quantified with Image-Oro Plus software (Media Cybernetics, Silver Spring, Md). Plaque lesion area and percent of the total cross-sectional vessel wall area were quantified by an independent operator, blinded to the experimental protocol, and the results were expressed as the average of 6 sections per mouse.
Serial cryostat sections of aortic sinus adjacent to the oil red O–stained sections were stained with the respective molecule-specific antibodies, as described previously.24 Antibodies included rat anti-mouse Mac-3 for macrophage identification (1:1000, BD Pharmingen, San Diego, Calif) and anti-CD4+ for T cells (1:120, BD Pharmingen). For mouse smooth muscle cell actin staining, primary antibody (fluorescein isothiocyanate [FITC]-conjugated α-actin, 1:500, Sigma, St Louis, Mo) was applied, followed by anti-FITC biotin-conjugated secondary antibody (1:400, Sigma). Proliferating cells were stained by rabbit antinuclear antigen Ki67 antibody (Novocastra Laboratories, Newcastle upon Tyne, United Kingdom). For FoxP3 staining, we used a goat anti-mouse/rat FoxP3 antibody FJK-16s (eBioscience, San Diego, Calif).
Quantitative analysis of lesional content of macrophages, smooth muscle cells, and collagen was determined by computer-assisted image analysis6 and expressed as percentage of intimal area to normalize for overall differences between the study groups. Quantification of CD4, Ki67, and FoxP3 staining was done by counting individual positively stained lesional cells, which are easily resolved, in the aortic sinus sections.
Polymerase Chain Reaction Analysis for Engraftment
To assess engraftment, the presence of the LDLR DNA gene product (383 bp) was analyzed by polymerase chain reaction (PCR) in peripheral blood leukocytes (PBLs) sampled from the recipient mice 4 weeks after bone marrow transplantation (before initiation of high-cholesterol diet) and in CD4+ splenocytes obtained when mice were euthanized as previously described6 with a GFX genomic blood DNA purification kit (GE Healthcare Bio-Sciences, Piscataway, NJ).
Serum Cholesterol Analysis
Overnight fasting serum samples were collected after 8 weeks of cholesterol-enriched diet. Total serum cholesterol levels, triglycerides, and plasma lipoproteins were analyzed by an online dual-enzymatic method for simultaneous quantification of cholesterol and triglycerides by high-performance liquid chromatography at Skylight Biotech Inc (Akita, Japan) according to the procedure described by Usui et al25 and were expressed in milligrams per deciliter (mg/dL).
Serum Immunoglobulin Analysis
IgM, IgG1, IgG2c, and IgG3 antibodies specific for malondialdehyde LDL and copper-oxidized (Cu-Ox) LDL were detected by isotype-specific ELISA as described previously.17 IgG2c was detected with an IgG2a/c-specific reagent that may underestimate IgG2c levels. We also determined titers in serum of IgM autoantibodies to oxLDL with the T15/EO6 idiotype as described previously.26
Ex Vivo Assays of CD4+ Proliferation and Cytokine Secretion
After 8 weeks of diet, splenic CD4+ T cells were isolated by anti-CD4 microbeads (Miltenyi Biotec, Auburn, Calif). The cells were stimulated in 96-well cultures (2.5×105/well) with plate-bound anti-CD3ε (145-2C11, BD Pharmingen) or human copper-oxidized LDL (Biomedical Technologies Inc, Stoughton, Mass) plus irradiated spleen cells (2.5×106/well). Ovalbumin (10 μg/mL) and medium alone were used as controls. Culture supernatants were removed at 48 hours and analyzed by flow cytometry–based cytokine bead assays (BD Pharmingen) of culture supernatants for IFN-γ, IL-2, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-10.27 Cultures were assayed for proliferation after 64 hours by uptake of [3H]thymidine (1 μCi/well), added 16 hours before harvest. Data are expressed as mean proliferation indices of triplicates calculated from the ratios of incorporated radioactive counts per minute in the presence or absence of antigen.
Real-Time PCR
CD4+ spleen cells stimulated with anti-CD3 were analyzed for cytokine production by TaqMan real-time PCR (RT-PCR). Total RNA was isolated from 5×106 million purified T cells, reverse transcribed, and analyzed by quantitative RT-PCR with SYBR green PCR mix (Bio-Rad Laboratories, Hercules, Calif).24,28 All real-time reactions were performed on the iCycler iQ real-time PCR detection system (Bio-Rad), and analysis was done with the accompanying software. The presence of single amplicons resulting from RT-PCR was verified by dissociation curve analysis. Levels of specific gene expression in the samples are presented relative to endogenous levels of β-actin housekeeping gene expression in the same sample.
Flow Cytometry
Lymphocytes were stained with BD Biosciences phycoerythrin (PE)- or allophycocyanin-conjugated anti-CD25 (PC61), PE- or FITC- or PE-Cy5–conjugated anti-CD4 (L3T4), FITC-conjugated anti-CD3, and PE-conjugated anti-ICOS (7E.17G9). For intracellular staining of FoxP3, a FoxP3-allophycocyanin antibody (FJK-16s) and Foxp3 staining buffer set from eBioscience were used according to the manufacturer's recommended protocol. All samples were analyzed using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences) as described previously.24
Regulatory T-Cell Isolation and Functional Assays
Tregs were isolated from spleen cell suspensions by the CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec), which depletes samples of non-CD4+ T cells followed by positive selection of CD4+CD25+ cells. The purity of the isolated CD4+CD25+ and CD4+CD25− populations was >95% by fluorescence-activated cell sorter analysis. CD4+CD25− responder cells (5×104/well) were cultured in 96-well plates (0.2 mL) with soluble anti-CD3ε (1 μg/mL) plus irradiated spleen cells (5×104/well) and increasing numbers of CD4+CD25+ regulatory T cells (responder to suppressor ratio of 1:16 to 1:1) for 72 hours. Cultures were pulsed with [3H]thymidine for the last 16 hours of culture. Culture supernatants were removed at 48 hours and analyzed for cytokines, as described above. Anti-TGF-β–blocking antibody (R&D Systems Inc, Minneapolis, Minn) was added to some suppression assays at 25 μg/mL.
Statistical Analysis
All statistical analyses were performed with Prism software (GraphPad Software, Inc, San Diego, Calif). Differences between groups of mice were analyzed by the Student t test or the Mann-Whitney test (for data that did not pass the Kolmogorov-Smirnov normality test), and data are expressed as mean±SEM.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Bone Marrow Engraftment, Weights, and Serum Lipids
To confirm engraftment, mice were bled at 4 weeks after transplantation, and PBLs were counted. There was no difference in total cell number between the groups (7.7±1.2×106/mL versus 9.1±0.8×106/mL in wild-type and ICOS-null cell recipients, respectively, P=0.35). To further assess engraftment, we looked for the presence of wild-type and mutant LDLR genes by PCR in PBLs and splenocytes of the recipient mice. The wild-type product was equally abundant in recipients of ICOS-null or wild-type marrow (Figure 1A). Importantly, there was also no ICOS protein expression on PBLs sampled at 4 weeks or on CD4+ splenocytes obtained at the time of euthanasia from recipients of ICOS−/− bone marrow (Figure 1B).
There was no significant difference in male or female weights between the groups (Table 1). The analysis of total serum cholesterol levels, as determined by enzymatic assay, revealed no statistically significant differences between the 2 groups (Table 2).
Quantitative and Phenotypic Differences in Atherosclerotic Lesions in ICOS-Deficient and Control Mice
After 8 weeks of high-cholesterol diet, mice transplanted with ICOS−/− cells had significantly more atherosclerosis in the aortic sinus than control mice transplanted with wild-type cells (Figure 2). In mice transplanted with ICOS−/− cells, average lesion area was increased from 309 000±42 000 μm2 (n=10) to 474 000±37 000 μm2 (n=15; P<0.05; Figure 2C). Fractional lesion area was increased from 37.4±2.2% to 53.6±2.2% (P<0.0001), a 43% increase in the atherosclerotic burden (Figure 2D). The increase in atherosclerosis in recipients of ICOS−/− cells was significant when atherosclerosis was evaluated according to sex. For wild-type versus ICOS−/− males, the respective fractional areas were 34.5±1.8% (n=6) versus 50.2±3.1% (n=7; P<0.005). For wild-type versus ICOS−/− females, the respective fractional areas were 41.7±4.3% (n=4) versus 56.6±2.8% (n=8; P<0.01). We found minimal and unquantifiable atherosclerotic lesions in the aortic arch and descending aorta, consistent with previous reports of irradiated LDLR−/− bone marrow recipients.29
After 8 weeks of proatherogenic diet, there was a 3-fold increase in CD4+ T cells in the intima of mice transplanted with ICOS−/− cells (Figures 3A, 3B, and 3K). In addition, there was increased smooth muscle cell content (Figures 3E, 3F, and 3M) and collagen deposition (Figures 3G, 3H, and 3N) in these mice. These differences between wild-type and ICOS−/− groups were significant when both sexes were evaluated together and when each sex was evaluated separately. There also were more macrophages in the ICOS−/− group than the control group (Figures 3C, 3D, and 3L), but the difference was not statistically significant. The present immunohistochemical data suggest that there is some heterogeneity within the ICOS−/− group, most evident in the smooth muscle cell content, with a distinct subgroup that does not show an increase compared with the control group. We found an increased presence of proliferating cells, stained by a Ki67-specific antibody, in mice transplanted with ICOS−/− cells compared with control mice (Figures 3I, 3J, and 3O). Taken together, these immunohistochemical data suggest that mice transplanted with ICOS−/− cells have more active atherosclerotic lesions with more T-cell infiltration; more local proliferation of T cells, macrophages, and/or smooth muscle cells; and more synthesis of extracellular matrix.
Enhanced Immunologic Responses in ICOS-Deficient Mice
To examine whether ICOS deficiency altered T-cell function, we isolated CD4+ T cells from mice that received ICOS−/− or wild-type bone marrow and compared their responses to polyclonal and antigen-specific stimuli. CD4+ T cells isolated from mice transplanted with ICOS−/− cells had a significantly greater proliferative response to anti-CD3 or oxLDL than control mice (Figure 4A). Although the magnitude of the oxLDL-specific response was low, the differences were consistent and statistically significant when T cells were stimulated with 10 μg/mL oxLDL (Figure 4B).
CD4+ T cells isolated from mice transplanted with ICOS−/− bone marrow secreted more Th1 cytokines (IL-2, IFN-γ, and TNF-α) and more Th2 cytokines (IL-10, IL-4, and IL-5) on stimulation with anti-CD3 (Figures 4C and 4D) than CD4+ T cells isolated from controls. In contrast, the CD4+ T cells from ICOS−/− bone marrow recipients produced significantly less antiinflammatory cytokine TGF-β than CD4+ T from control mice (Figure 4E). Quantitative RT-PCR analysis of the cytokine mRNA expression of the stimulated CD4+ T cells was concordant with the cytokine protein assays, with the exception of IL-10, which did not differ significantly between the experimental groups (Figures 4F through 4H).
We found a significant increase in serum monocyte chemotactic protein-1 and a trend for increased TNF-α in mice transplanted with ICOS−/− cells compared with controls (Figure 4I). There were no significant differences in IFN-γ, IL-10, IL-12, and IL-6. These results are consistent with an enhanced systemic inflammatory response in the mice with ICOS-deficient marrow.
Antibody responses to oxLDL may modulate atherosclerotic disease, and ICOS may influence production of these antibodies.20,21 We therefore measured titers of serum antibodies specific for different forms of oxLDL (Figure 5). There were statistically significant reductions in anti-malondialdehyde-LDL IgG1 and anti-oxLDL IgG3 in the ICOS-deficient group, which may reflect impaired isotype switching to these isotypes in these mice due to ICOS deficiency.30 However, the data do not support a global defect in isotype switching in the mice transplanted with ICOS-deficient bone marrow.30 There were no differences in the phosphorylcholine-specific IgM natural antibody identified by the EO6 antibodies idiotype, which has been shown to be protective in atherosclerosis.26 Overall, the data do not support a role for changes in antibody titers as an explanation for the difference in atherosclerosis phenotype between groups.
Relationship Between ICOS and Regulatory T-Cell Numbers and Function
In light of published data that suggest that ICOS may be important in Treg function and the present data indicating enhanced immune responses in the absence of ICOS, we evaluated the relationship between ICOS and Treg. Using 2 specific molecular markers of these cells, CD25 and intracellular FoxP3, we found that the frequency of ICOS-expressing cells was 3-fold higher in the CD4+ Treg subset than in the CD4+ non-Treg subset (Figure 6A). These data suggest that ICOS has a role in the development or function of this T-cell subset. We therefore examined Treg counts and function in ICOS−/− mice (unirradiated, LDLR+/+). We found a 30% reduction in Treg counts as evidenced by FoxP3 staining in the spleen of ICOS−/− mice on regular or high-cholesterol diet compared with control mice (Figure 6B). There was no significant difference in total spleen CD4+ T-cell counts between the groups. We also quantified Treg in LDLR−/− mice transplanted with ICOS−/− versus wild-type bone marrow on high-cholesterol diet for 8 weeks. For this analysis, we included iliac lymph nodes, which drain the atherosclerotic descending aorta. Treg counts were reduced by 40% in the iliac lymph nodes and the spleen in mice transplanted with ICOS−/− cells compared with control mice (Figure 6C). These data support the hypothesis that ICOS positively influences the development or maintenance of Treg.
The presence of ICOS on Treg, together with the increased number of T cells and enhanced lesion formation in mice with ICOS-deficient bone marrow, suggested that Treg may control the proatherogenic effector T cells and influence the inflammatory burden in the atherosclerotic plaque. We therefore looked for the presence of Tregs in the mouse atherosclerotic lesions by immunohistochemical staining for FoxP3. We found that mice transplanted with either wild-type or ICOS−/− bone marrow had readily detectable Treg in the atherosclerotic lesions (Figure 6D); however, we could not demonstrate significant quantitative differences between the groups. The total CD4+ T-cell counts in the lesions were more than 3-fold higher in the mice transplanted with ICOS−/− cells compared with control mice (Figure 3), which suggests that the Tregs that are present in the lesions of these mice are insufficient in number or function to suppress the local effector T-cell response.
To evaluate whether regulatory T-cell function, in addition to numbers, is influenced by ICOS expression, we compared the suppressive function of the Treg isolated from ICOS−/− and wild-type mice. Indeed, the ability of Treg from ICOS−/− mice to suppress proliferation of wild-type or ICOS−/− cell responders was reduced compared with Treg from wild-type mice (Figure 6E). Reduced suppression by ICOS−/− Treg was also clearly evident by analysis of interferon-γ and TNF-α in the supernatants of these assay cultures (Figures 6F and 6G). To address whether TGF-β has a role in Treg function in atherosclerotic mice, we used a TGF-β–blocking antibody in the suppression assay. We found that blocking TGF-β caused reduced suppression by Treg from LDLR−/− mice transplanted with wild-type bone marrow (Figure 6H). Anti-TGF-β did not cause significant effects on the already reduced suppression seen with Treg from LDLR−/− mice transplanted with wild-type ICOS−/− bone marrow.
In this study, we found that ICOS deficiency on bone marrow–derived cells increases the atherosclerotic burden in LDLR−/− mice. Importantly, the increase in lesions is associated with increased infiltration of CD4+ T cells, which indicates that plaque-based T-cell responses are more active in the absence of ICOS. In addition, the lesions in the ICOS-deficient mice had more smooth muscle cell and collagen deposition. The increased atherosclerotic burden in ICOS−/− bone marrow–recipient mice correlated with increased ex vivo T-cell responsiveness to polyclonal and antigen-specific stimuli and in monocyte chemotactic protein serum levels. Taken together, these data imply that ICOS deficiency leads to an enhanced T-cell response to hypercholesterolemia and plaque antigens and increased atherosclerosis.
Similar to the present data, ICOS deficiency has been shown to enhance helper T-cell responses in models of autoimmune diseases, including experimental autoimmune encephalitis7 and insulitis.11 Experimental evidence clearly show that ICOS is a positive costimulatory molecule for CD4+ T cells.7 Therefore, it is paradoxical that ICOS deficiency increases immune responses in vivo. A possible explanation is the role of ICOS in the development or function of Th2 cells, which could downregulate Th1 responses.8 However, recent studies show that ICOS is also involved in Th1 differentiation.31,32 Consistent with this, we saw an increase in both Th1 and Th2 cytokine production by CD4+ T cells isolated from ICOS-deficient hypercholesterolemic mice ex vivo. Therefore, we do not think a change in Th1:Th2 balance explains the effect of ICOS deficiency in the present study. The present data also do not support the idea that the effect of ICOS deficiency is due to changes in antibody responses to oxidatively modified LDL or phosphorylcholine, which are known to modulate atherosclerosis.20,26
The best-characterized subset of regulatory T cells (Tregs) are the "natural" CD4+CD25+ Tregs, which constitute ≈10% of peripheral CD4+ T cells.33 Most natural Tregs express the transcription factor FoxP3.34 We considered that impaired natural Treg activity may be the mechanism underlying the increased immune response and atherosclerotic burden in the setting of ICOS deficiency. Indeed, we found that ICOS plays an important role in induction or maintenance, as well as function, of natural Treg. There was a 40% reduction in the number of Tregs in the absence of ICOS as determined by FoxP3 expression. This difference is especially significant given the ability of small numbers of Tregs to suppress T-cell responses. Furthermore, we found a reduction in Treg suppressive function in ICOS−/− mice assessed in vitro. This is consistent with recent studies showing that ICOS is crucial to Treg-dependent self-tolerance.11,12,35,36 Therefore, ICOS deficiency in the setting of hypercholesterolemia would be predicted to enhance proatherogenic T-cell responses and exacerbate lesion development, as we saw in the present study. A recent article reported that apolipoprotein E–deficient mice immunized with an ICOS-Ig fusion protein had increased early lesion development,37 but that study did not clarify whether and how the immunization altered the ICOS pathway in vivo, nor did it provide a mechanism by which the ICOS pathway might influence atherosclerosis. The present data are in accordance with a study demonstrating the importance of Treg in inhibiting development of atherosclerosis in apolipoprotein E mice.16
Interestingly, the only cytokine that we analyzed whose production was decreased in mice receiving ICOS−/− cells versus control mice was TGF-β. In addition, an in vitro suppressive assay from these mice suggests that TGF-β plays a role in Treg function. This cytokine is a potent regulator of effector T-cell differentiation, and it inhibits the acquisition of specific Th1 or Th2 cell functions.38 Conversely, TGF-β promotes the development of FoxP3-expressing Tregs.39,40 Therefore, reduced TGF-β may contribute to the increased immune responses seen in the ICOS−/− mice in the present study. Overall, the present data support the notion that Tregs inhibit atherosclerosis through TGF-β secretion.16,19
In summary, our results indicate that ICOS is important for the regulation of proatherogenic T-cell responses in hypercholesterolemic animals. Enhancement of Treg responses, through manipulation of the ICOS pathway, has potential as a therapeutic strategy for atherosclerotic disease.
Sources of Funding
This work was supported by National Institutes of Health grants P50HL56985 (Dr Lichtman), R01AI3831O (Dr Sharpe), and R01HL67249 (Dr Sukhova).
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Wan YY, Flavell RA. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc Natl Acad Sci U S A. 2005; 102: 5126–5131.
CD4+ T-lymphocyte–mediated adaptive immune responses contribute significantly to the inflammatory process that occurs in atherosclerotic lesions. Costimulatory pathways that involve B7 and CD28 family molecules are essential for the initiation of most CD4+ T-cell responses to antigens and therefore may promote arterial disease. However, recent studies have shown that the B7-CD28 pathway is also essential for regulatory T-cell development and function and that regulatory T-cell populations may serve to suppress proatherogenic T cells. Inducible costimulatory molecule (ICOS) is a CD28 family member for which in vivo functions are incompletely understood. This study tested the hypothesis that ICOS modulates proatherogenic T-cell responses. The data indicate that a deficiency in ICOS in mouse bone marrow–derived cells results in a marked increase in T-cell responses to hypercholesterolemia, associated with more T-cell infiltrates in lesions and more lesion development. The effects of ICOS deficiency are related to a reduction in numbers and function of regulatory T cells. These data suggest that pharmacological blockade of costimulatory pathways as a potential therapeutic strategy for atherosclerosis must be refined to avoid impairment of the beneficial effects of regulatory T cells.
Guest Editor for this article was Roberto Bolli, MD.
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- Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory MoleculeIsrael Gotsman, Nir Grabie, Rajat Gupta, Rosa Dacosta, Malcolm MacConmara, James Lederer, Galina Sukhova, Joseph L. Witztum, Arlene H. Sharpe and Andrew H. LichtmanCirculation. 2006;114:2047-2055, originally published November 6, 2006http://dx.doi.org/10.1161/CIRCULATIONAHA.106.633263
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- Impaired Regulatory T-Cell Response and Enhanced Atherosclerosis in the Absence of Inducible Costimulatory MoleculeIsrael Gotsman, Nir Grabie, Rajat Gupta, Rosa Dacosta, Malcolm MacConmara, James Lederer, Galina Sukhova, Joseph L. Witztum, Arlene H. Sharpe and Andrew H. LichtmanCirculation. 2006;114:2047-2055, originally published November 6, 2006http://dx.doi.org/10.1161/CIRCULATIONAHA.106.633263
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LOGYx is a software program for X-ray imaging assessment and image analysis training. It offers a wide range of real-life situations to which screening security officers are exposed, simulating a fully-featured X-ray imaging system. LOGYx is already extensively used at training centers and airports to assess the proficiency of screening personnel and raise their skills through its utilization.
Welcome to the new site LOGYx
Take advantage right now of the new version !
- To find out more click here.
Enac, our educational partner
The French National School of Civil Aviation (ENAC) has accompanied the development of LOGYx since its inception. ENAC has contributed its instructional expertise and enabled us to take advantage of the experience accumulated by the School's security training staff.


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Bharara Orders Banker's Basquiat Back to Brazil
Hannibal, a 1981 work by Brooklyn artist Jean-Michel Basquiat, was repatriated to Brazil after a long legal battle.
Credit: U.S. Attorney Southern District of New York
The crate that arrived at JFK airport would not have caught anyone's eye. According to the shipping label, it contained an unnamed painting valued at $100.
Turns out that valuation was off by about $7,999,800.
Far from some amateur's daubing, the canvas inside, Hannibal, was actually a 1981 work by Brooklyn-born artist Jean-Michel Basquiat. Last week, U.S. Attorney for the Southern District of New York Preet Bharara said that the painting was on its way to Brazil, where authorities for nearly a decade have said it belongs.
By the time the crate turned up at JFK in 2007, authorities on three continents had been searching for the painting, along with a number of other works by masters like Roy Lichtenstein and Serge Poliakoff. The paintings had once been part of the collection of Edemar Cid Ferreira, a wealthy Brazilian banker. Ferreira had been arrested in a major embezzling and money laundering scheme, and had bought the paintings to hide his ill-gotten millions.
Though Ferreira had been convicted in 2006, the Brazilian authorities were still trying to track down his art collection, which was scattered around the world. All told, Ferreira had more than $20 million worth of paintings, all of which rightly belonged to the Brazilian government in compensation for his crimes. Authorities in Brazil enlisted Interpol and the U.S. Department of Homeland Security to locate the works, and the agencies have been tracking them down, one at a time, over the years. The Lichtenstein was recovered and repatriated in 2010, and a work by Poliakoff was returned in 2014.
The Basquiat was determined to be fair game for seizure in the U.S. because it had been imported in violation of customs laws. Recovered by Homeland Security agents in New Haven, Connecticut, it was then confiscated under civil forfeiture laws.
As the New York Times reported in 2013, Ferreira's use of valuable artwork to launder his money is an increasingly popular choice for criminals. In an industry that still often operates with handshakes, and with few regulations governing the sale of works worth many millions of dollars, it's an attractive place to shield assets from government eyes.
The art world also attracts other kinds of shady dealings. We wrote last year about Brian Ramnarine, a forger based in Long Island City, who capitalized on the informality of the trade to churn out perhaps millions of dollars in unauthorized sculptures by artists like Jasper Johns and Kenny Scharf. He was able to get away with his scheme for decades before finally ending up in prison.
"Art and antiquities have special value and meaning that cannot readily be quantified," Bharara said in a press release. "As a result, they have long been the subject of theft and deception, as well as a means to launder illicit proceeds. Art should serve to inspire the mind and nourish the soul, and not be allowed to become a conduit for crime."
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Lectures and Special Events
Exploring the Museum Archives: New York Archives Week
October 5, 2014
In honor of New York Archives Week, come to the Library to discover the museum's rich history of scientific exploration from around the world. Rarely seen collections of field notes, films, photography, artwork, and memorabilia will be on display to tell the hidden stories behind the museum's world-famous dioramas and exhibitions. Watch early moving-image footage from historic Central Asiatic Expeditions to Mongolia, in which a team led by Roy Chapman Andrews discovers the first dinosaur eggs, or browse the original landscape studies painted in the field during Carl Akeley's perilous expeditions to Africa. The Library staff will explain how these one-of-a-kind objects are cared for and give hands-on demonstrations of the new Digital Special Collections, an online endeavor to make the Library's extensive image collection available for research and reference.
This event is part of the New York Archives Week, which runs October 6–12, 2014, an annual celebration aimed at informing the general public about the diverse array of archival materials available in the metropolitan New York region.
More in this Series:
The Remarkable Nature of Edward Lear
November 7, 2016
Robert Peck, author of the new, illustrated book The Natural History of Edward Lear, discusses the remarkable life and natural history paintings of this beloved children's writer, and why he abruptly and mysteriously abandoned his scientific work.
Yes, Humans Are Causing Earthquakes
November 10, 2016
Join Dr. Justin Rubinstein, deputy chief of the Induced Seismicity Project at the United States Geological Survey, as he discusses this new breed of human-caused earthquakes.
SeismoDome: Sights and Sounds of Earthquakes and Global Seismology
November 19, 2016
Earthquakes happen frequently—but what causes them? Why are they unpredictable? What do they tell us about Earth's deep interior?
Hackathon: Hack the Stacks
November 20, 2016
Discover how programmers, librarians, and archivists worked together to mine the Museum's library data, unlocking new ways to visualize and understand the Museum Library's collections. Hear first-hand accounts from the data trenches, and find out what hackers can really create in 24 hours.
Hayden Special Event: Winter Telescope Party
December 21, 2016


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Via Sempione N.12, Malpensa 28838, Milán (Italia) Ver mapa
White coloured building, dates back to an original building of the 60's, but renovated and well kept.
Medium in size, while single rooms are rather small. They are furnished in a classic style, elegant and sober, with pastel decor. Bathrooms are as well in very good conditions, mostly in marble finishing.
Actividades y ocio
Cómo llegar
Situated directly on the Lake and surrounded by a beautiful park, the hotel remains very accessible from the main road that borders the Lake. Baveno is just 3 kms away from more famous Stresa but offers the same enchanting views on the Lake and close facilities to Milan and Malpensa airport.


text: I am building a message board and the response area is written in a textarea like the one I am writing in right now. How then do they format the data, when pulling it from (or putting it in) the database. Mainly what I am after is a CrLf. Otherwise the variable just writes on one line forever off the page. My first thought was to write a function to add a CrLf after a certain amount of bytes. Is there something easier? I can't find anything.


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Ocean City Fish Co Early Bird Monday-Friday 4 P.M.-5:30 P.M.
Visit the The Original Greene Turtle Sports Bar & Grille for their Great Happy Hour. there is fun for everyone with a Game Room, Keno, Wifi, 50 Flat Screen & HD TVs, DJs with a Dance floor!! Burgers, Wings, Homemade Crab Cakes, Salads and MORE!
Weekday Dine-In Special all-you-can-eat HOT STEAMED CRABS just $1.50 each * MONDAY -FRIDAY 11- 3 pm ONLY
My husband and I have never been disappointed with our meal here. The food is fresh and delicious. I would highly rec...Linda
Every time we visit this place, they just keep getting better and better. Food is very tasty and service has been so...Dave
We used to go to the 25th street location, but they are closed now. They had good food and drinks all around. Cheeses...Joseph Price
Will be returning for the third time in last year. Best grilled chicken sandwich. The lemon wedge desert is by far th...Tom Garbo


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Oinkari Aurresku WorkshopTwo families from the Basque Country are coming to Boise this summer to teach Basque dance. They will be teaching an Aurresku workshop in Boise the week before San Inazio, but need a place to stay. If anyone is available to host a family of 4 for the week before San Inazio (preferably living close to downtown), please use the contact info below.
Contact: 208-557-1960 (email@example.com)
New Boise Basque FlagRelease Date: 9/23/2016
Basque Charities - Basque FoundationRelease Date: 9/23/2016
San Inazio: Thank You to AllRelease Date: 8/31/2016
2016 Legacy Award WinnersRelease Date: 7/8/2016
News from Aiztan ArteanRelease Date: 7/8/2016
Monthly Dinner Host FamiliesRelease Date: 7/8/2016
Legacy AwardRelease Date: 4/12/2016
First Aid Kit & Defibrillator at the Basque CenterRelease Date: 3/1/2016
Reminder: 2016 Membership DuesRelease Date: 2/10/2016
General Meeting & Election RecapRelease Date: 2/10/2016
Sheepherders Ball 2015Release Date: 2/10/2016


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2 Bedroom End of Terrace House For Sale
Upper Bridge Street, Wye, Ashford, TN25
Guide price: 170,000 - 190,000.Two bedroom terrace house which offers downstairs living. this property is situated in the sought after village of Wye which has its own Train Station, doctors, dentist and high street with independent shops. This property is also on a...
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Au Petit Chevrot - Gressan
Au Petit Chevrot offers centrally-located apartment accommodation.
Apartment accommodations come with desks and safes. Practical amenities include kitchens, stovetops, refrigerators and cookware/dishes/utensils. Accommodations are also individually furnished as well as having free Wi-Fi. In addition, the apartment accommodation features a garden.
Au Petit Chevrot - Gressan
Hotel Info: 1-877-214-4288from $69
- Standard Apartment, 1 Bedroom (4 people)
- Standard Apartment, 1 Bedroom (3 people)
- Standard Apartment, 2 Bedrooms (5 people)
- Standard Apartment, 2 Bedrooms (4 people)
- Standard Apartment, 2 Bedrooms (6 people)
- Standard Apartment, 1 Bedroom (2 people)
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 12 rooms
- This hotel is arranged over 3 floors
- Check-in time 2 PM-7 PM
- Check-out time is 10 AM
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed (dogs and cats only) *
- Up to 10 kg
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
In the hotel
- Front desk (limited hours)
- Concierge services
- Tours/ticket assistance
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 2
- Garden
In the room
- Individually furnished
- Separate living room
- Separate sitting area
- Private bathroom
- Shower only
- Satellite TV channels
- Desk
- Free WiFi
- Refrigerator
- Kitchen
- Stovetop
- Cookware, dishware, and utensils
- Weekly housekeeping provided
- In-room safe
- Access via exterior corridors
Special features
On Site
- Bicycle rentals on site
- Free bicycles on site
- Hiking/biking trails nearby
- Mountain biking nearby
- Ski lessons available nearby
- Ski equipment rental shops nearby
- Cross-country skiing nearby
- Downhill skiing nearby
- Snowboarding nearby
Au Petit Chevrot - Gressan's small print
Also known as
- Au Petit Chevrot
- Au Petit Chevrot Apartment
- Au Petit Chevrot Apartment Gressan
- Au Petit Chevrot Gressan
- Petit Chevrot
- Au Petit Chevrot Italy/Gressan - Aosta
All guests, including children, must be present at check-in and show their government-issued photo ID card or passport.
Cash transactions at this property cannot exceed EUR 2999.99, due to national regulations. For further details, please contact the property using information in the booking confirmation.
This property does not have elevators.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- City/local tax: EUR 1
Optional extras
Cribs/Infant beds are available for EUR 10 per night
Pets are allowed for an extra charge of EUR 10 per pet, per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Recent reviews
Excellent 4.5 /5 from 20 reviews
Very pretty little apartments. Great for families.


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This study of Hong Kong's new towns covers the historical and conceptual origins of new towns and satellite towns worldwide, as well as development procedures and controls, aspects of design, design problems, and the role of government and the private sector in catering to the public need. Hong Kong's physical size and rapid population growth provide unique material for this volume, which will prove useful to town planners and students in the field of community planning.It has been the practice to apply territory- wide standards set out in the Hong Kong Outline Plan [the 1980s up-date of the ... especially with the general introduction of the additional ten volumes of the Transport Planning and Design Manual, anbsp;...
|Hong Kong's new towns
|M. Roger Bristow
|Oxford University Press, USA - 1989-12


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Special Events
Throughout the year, the Virginia Symphony holds a number of special events for donors and other friends of the VSO. From galas to cocktail parties, all of these events feature exclusive performances by VSO musicians as well as fun and festivity with good friends and symphony supporters. We've finished our 2015-2016 season, but check back soon for updates on next season's special events!


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This November, overcome stress and anxiety with thankfulness—sign up for our "Give Thanks" devotional.
6:5–8 Slaves follow the example of Christ through obedient submission (Phil. 2:1–11). All believers are called to share Christ's humiliation and sufferings in this age, in order to be exalted and glorified with Him in the next (Rom. 8:17). Paul is not interested in making anyone's share of suffering greater than it has to be (1 Cor. 7:21). He also does not pretend there is an easy route around it. As slaves serve the exalted Christ, and not merely an earthly master, they do so in the new reality inaugurated by Christ (2 Cor. 5:17). Paul would insist that being owned by Christ makes all other definitions of our personhood irrelevant: "You were bought with a price; do not become slaves of men" (1 Cor. 7:23). By rendering ungrudging service to their true heavenly owner, slaves can work not for their value in the marketplace, but for their value to the One who poured out His own life for them.
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The Romance of an Old Playhouse
Pyper, George D.
Place Published: Salt Lake City
Publisher: The Deseret News Press
Date Published: 1937
Edition: Revised edition
Binding: Hardcover
Book Id: 24334
SIGNED. 405pp. Octavo [23 cm] Red cloth covered boards with gilt stamped titles and a gilt stamped vignette on the front cover. Near fine. The edges of the covers are very lightly bumped and rubbed.
Inscribed by Pyper on the front free endsheet. The history of the Salt Lake Theatre. Colored frontis of the Salt Lake Theatre taken from the original painting by Utah artist Lee Greene Richards.