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Skip to comments.Woman, 63, 'becomes PREGNANT in the mouth' with baby squid after eating calamari
Posted on 06/15/2012 5:01:50 PM PDT by doug from upland
Woman, 63, 'becomes PREGNANT in the mouth' with baby squid after eating calamari
Woman was 'eating when she felt a prickling sensation in her mouth' She was chewing 'sperm sacks', which forcefully shoot sperm Sperm in turn try to embed themselves when they land But have no fear in the West... Squid's internal organs are (generally) removed before serving
By Richard Hartley-parkinson
PUBLISHED: 03:40 EST, 15 June 2012 | UPDATED: 15:06 EST, 15 June 2012
A 63-year-old woman became 'pregnant' with 12 baby squid after eating calamari, according to a claim in a bio-tech report.
The real-life 'octo-mum', from South Korea, was eating a portion of cooked whole squid when she felt a sharp pain in her mouth.
The bizarre claim has been made in a scientific paper from the National Center for Biotechnology Information in Bethesda, Maryland.
Good enough to give birth to? The woman from South Korea was eating cooked squid when she 'was impregnated by the sea creature'
The lady told doctors that she could feel something in her mouth which they described as 'bug-like organisms'.
When examined, the doctors found 'baby cephalopods' attached to her mouth. These are small pods, covered in a cement-like material to make them stick.
(Excerpt) Read more at dailymail.co.uk ...
I didn't read this.
I didn't really read this.
Somebody wake me up, please.
I need to get my wire brush so I can scrub my mind after reading this story. . .
They eat a lot of octopus there (not squid) and there are many dishes where the octopus is barely cooked and in some cases its raw.
I have to say it
The Alien flicks have been running on SyFy lately.
Not something one would want to be remembered for.
Ew, gross. I just made grilled baby octopus for dinner yesterday, too. I'm *so* glad this didn't happen when eating it.
I'm going to get my pressure washer AND wire brush.
Welcome to the National Inquirer.
Didn't I see this headline the last time I was in the supermarket.
Listerine. Spray it in your eyes. Gargle and swish with it. Spray it in your ears and cock your head so it goes to your brain.
Barnacles containing squid instantly attached to her TEETH?
I think I need to go lay down.
Crap. My favorite "eat out" food here in Las Vegas is calamari at Firefly on Paradise. Hmm, hard to know if that's a plug or I'll get a table faster next time....
How do you know it didn't happen? BWAHAHAHAHAHA!
No joke. Reeeeeeally revolting. AFAIC, squid is bait, no matter how sophisticated & elegantly it is named.
"....and I can pull the trigger with my tail"
If I wrote what I was really thinking,I'd be band
File this under "Sentences that have never been written before."
Nothing gets through that...
I appreciate this thread. I had fried catfish tonight. I expect I'll have nightmares in 6 hours or so.
Okay, that's just wrong. Really, reeeeeeeely wrong.
This thread is absolutely hilarious.
apparently no ;)
To quote that coed in "Animal House",
Icky, I-C-K-Y, icky!
Is there anything Asians can't do?
First burgers made out of poop, now oral aquaculture.
they tend to wig under pressure.
You didn't just use that word in THIS THREAD!
Truth be known, it was our Secretary of State, on one of her trips to the Far East. Here is one of the offspring, fully grown. They call it "The Comeback Squid":
Now you see why Texans fry everything? Do you hear alien-hatching stories coming out of even Houston? Of course you don't. Deep fry it, my fellow man.
I'm gonna use a 1,500psi sand blaster!
Parasitized, Zeno-engrafted, etc.
But not Pregnant
Still, way serious icky
Mystery solved folks. I think we've finally found odumbo's real mommy.
What has been read cannot be unread!
We need a demotivational poster with those words with a freaked out cat on it.
Geesh, Doug. Thanks for this nightmare.
"According to Science 2.0, a spermatophore is similar to a cup of semen."
Reminds me of an old joke,,,, which I will not tell,,,, because I like it here!
Oh, Yuck!!
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


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Evelyn Mae Heltne, daughter of Edward Carlgren and Inga Tiller Carlgren, was born May 1, 1919, in the family home near Norway, Kan. She was baptized and confirmed at Our Saviors' Lutheran Church. On Nov. 15, 2013, at the age of 94, she passed away peacefully.
She attended school in Norway, Kan., and enjoyed participating in basketball and tennis. She graduated in 1937. She attended the State Teachers College of Emporia, Kan., and received her teacher's certificate and subsequently taught at Forty-Eight Country School east of Norway, Kan.
In September of 1942, she met the love of her life, Adolph Bernhard (Bernie) Heltne. In 1943, she moved to Minneapolis, Minn., where she worked as a nursing assistant at the Fairview Hospital. On June 7, 1944, Evelyn and Bernie were married at Our Savior's Lutheran Church in Norway, Kan.
She and Bernie lived in Bradley, S.D.; Goodridge, Minn.; and Storla, S.D. In each location, Bernie served as pastor. She was an active partner in the ministry as the pastor's wife, involved especially with the youth groups. She cherished her many lifelong friendships from the parishes they served. Evelyn and Bernie returned to Norway, Kan., in 1978, where she was active in the church and community. She delighted in participating in the Fall Harvest Stand, the annual church Bazaar and the Norway Women's Coffee group. She enjoyed being with her brothers, sisters-in-law, nieces and nephews, grandchildren and friends of her youth. She also enjoyed the many visits from her niece, Kathy Heltne Swanson, and friends from previous parishes.
She was preceded in death by her husband, Adolph Bernhard; her parents, Edward and Inga Carlgren; and brothers Carrol, Ivan, Loren and Joseph, and sister-in-law, Gladys Carlgren. She is survived by son, Carl and wife Brenda, Barnes, Wis.; son, Paul and wife Trish, Scandia, Kan.; daughter, Rebecca and husband Jerry Janulewicz, Grand Island, Neb.; and daughter, Ilah and husband Rodney Gilreath, Golden, Mo. Also surviving Evelyn are eight grandchildren: Tiffany Zitzewitz, Carissa Dobrinski, Briony Bresnahan, Kyle Heltne, Austin Heltne, Nathan Janulewicz, Sarah Rippe and Jenny Tuten; and seven great-grandchildren: Tyler and Maija Zitzewicz, Isabella Dobrinski, Aubrey and Cora Bresnahan, Declan Heltne and Sawyer Rippe. She is also survived by her brother, Charles Carlgren and wife Vivian, and sisters-in-law, Aileen Carlgren, Norma Calrgren and Clarice Pruitt, as well as many nieces and nephews.
Memorials may be given to Family Choice.
Funeral services will be held at 3 p.m., Thursday, Nov. 21, 2013, at Our Savior's Lutheran Church, Norway, Kan., conducted by Pastor Tom Kamprath.
Interment will be made in Norway Cemetery, Norway, Kan.
Friends may call on Tuesday and Wednesday from 9 a.m.-9 p.m. at Bachelor-Surber Funeral Home, Belleville, Kan., where the family will receive friends from 7-8 p.m.
Online condolences and information at www.bachelor-surber.com
Bachelor-Surber Funeral Home, Belleville, Kan., in charge of arrangements.

subset: commoncrawl


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Smile for the camera Chris … you are about to make GH history!
If you are looking for the video game equivalent to Hendrix, you may have found your man. Sixteen-year-old Chris Chike fingers plastic like nobody's business, and in a world seemingly being taken over by video game guitarists, he is the man to beat.
Chris recently demolished the GH3 track "Through the Fire and the Flames" on expert mode en route to a perfect score. Some GH enthusiasts apparently argue that "The Devil Went Down To Georgia" is more difficult than the aforementioned GH3 track. As far as I am concerned, the thought of either on expert mode make my hands cramp up.
I will happily die never knowing what it feels like to score as high as Chris on any of the more difficult songs GH has to offer, because frankly, my fingers will never move that fast unless they have a female attached to them.
In honor of Chris's amazing achievement allow me to quote his reaction upon completion word for word below…
"holy shit holy shit holy shit holy shit no way no way no way no fucking way oh my god oh my god oh my god holy shit 100 fucking % my hands holy shit holy shit holy shit holy shit that just fucking happened."
Of course success in any walk of life breeds doubters, haters, and my personal favorite — jealous haters full of doubt. As I went to clip the video from Youtube, my eyes stumbled upon one comical response to his video that I just couldn't pass up sharing.
"r u fucking retarded, u really think he got a 100 percent on it. he'd be in the world record book." – GearsPlayer999
To answer your question, no we are not, yes we do, and he most certainly is in the Guinness World Record Book. Chris held the previous high score of 880,920 before being dethroned in May of this year by Danny Johnson's 890,971 score. The Guinness World Record Book is aware of Chris's latest video (below) and is in the process of trying to confirm his 987,786 score. You lose. Thanks for playing.


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Jeffrey Loria Continues To Be A Meddlesome TwerpYou don't know shit about baseball, Jeffrey Loria. Sources: Marlins owner Jeffrey Loria personally mandated pitching lineup change [Yahoo] On Tuesday, Loria reportedly gave his players and manager reason to join critics after meddling in the Marlins' star… (more)Source: deadspin.com
More meddling! We've found Miami Marlins owner Jeffrey Loria's to-do listA juicy story out of Miami says Marlins owner Jeffrey Loria is meddling in baseball decisions and infuriating players. Yahoo! Sports' Jeff Passan broke the story, which makes Loria - baseball's most mocked owner - sound like he's been reading the Al DSource: yahoo.com
Report: Marlins owner Loria forced pitching switchReport: Marlins owner Loria forced pitching switchSource: CNNSI.com


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I saw the second half of a film by a Scandinavian (perhaps Dutch) director on a late night PBS foreign film slot, perhaps around 1991.
It had almost no dialogue, and was extremely strange, I'm not even sure what was happening. It was like some weird cross between Kafka and Monty Python. Let me try to describe what I remember.
The main character seemed to be trapped in some sort of empty sanitarium, running around. He wore a suit and was myopic. Cannonballs seemed to fall randomly from the sky. At one point he was being chased by a stuffed animal tied to a string tied to his foot, struggling with it on the ground.
Eventually, he found his cracked eyeglasses, and things seemed better for a short while, before he lost them again. Orderlies came and took him. I think he hung himself in the end.
If this doesn't sound familiar, you haven't seen it! I still can't forget it. I just didn't catch the name of it. Anybody?


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Mike Ceballos for Superintendent of Public Instruction
Recent News
The best way to begin again is to read the results from Tuesday, and accept them. I would have loved the prospects winning this election would have offered us. Wednesday, I rested a bit and then began the return to what I was working on before the campaign began in February.
Thanks, to all of you, for everything. Our family has been humbled by your kindness, enthusiasm, confidence and generosity. I am grateful for so many things. Friendships – renewed and new - a priceless benefit of this campaign which will serve as a continual blessing. The opportunity to listen and learn from so many about our State and the topics we discussed went well beyond student education. We discussed problems and concerns and an equal measure of opportunities and aspirations for our families, communities, and state. It has truly been inspirational and renewed my passion for how we can make a real difference when we get involved. Finally, campaigning encompasses the entire household. Our family came together in a way that reminded me of when our kids were young. With both Samantha and Kelly moving into their adult lives, we recreated that feeling of packing the kids in car and heading off to a new adventure. Experiencing our children, as fully competent and successful adults, made us both proud and extremely happy. Addie and I also found we had some of the same love, beliefs, and differences that brought us together 35 years ago. Now however, I am going to be forced to clean out the garage and begin work on our new kitchen.
So I am back to finishing my doctorate, and will hopefully have my topic, "What is the role of the public in public education" approved. As to the boards, which have been letting me coast, I will return full force. To all my many friends and supporters, I appreciate the handshakes, hugs and expressions of disappointment. Please know that I am a better person because of this experience. Thanks to all of those who have donated time, talent, and money and simply know that we can't find words to adequately express our appreciation. Feel free to call, email, or drop by. I can't wait. I am certain there will be new opportunities resulting from the great campaign we ran.
Please join me in congratulating our next Superintendent of Public Instruction, Jillian Balow. Her success means success for our students and a stronger education system in Wyoming, and I wish her nothing but the best.
About Mike
Mike Ceballos is running for Wyoming Superintendent of Public Instruction because he cares about Wyoming education and wants to bring respect and stability back to the Department of Education. As former president of Qwest Wyoming and a doctoral student at the University of Wyoming, Mike is a leader and a learner with a focused vision for Wyoming's schools.
Mike's Priorities
Cooperation and Collaboration between parents, teachers, businesses, community organizations, the legislature and state officials.
No matter the industry, employers in today's job market are looking for employees with solid educational foundations. But many of our students fail to see how what they are learning in school connects to real life careers and opportunities.
For the link between the classroom and students' futures to be clear, we need to foster collaboration between parents, teachers, businesses, and community organizations. Learn more >
A Common Understanding of the knowledge and skills students must possess for tomorrow's jobs.
Respecting the Teaching Profession by allowing teachers and schools to do what they do best.


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Harrisburg Town Center is made to be convenient, with many services centrally situated. Harrisburg Town Center is home to roughly 520 people who work in services, retail and administrative professions. There are other great apartments in the surrounding area. Even more apartments are available in the adjacent neighborhood of East Charlotte.
Conveniences Around the Apartments for Rent in Harrisburg Town Center, Harrisburg, North Carolina
The neighborhood contains a diverse group of services ranging from misc services to laundries.
Neighborhood Demographics and Costs of Living
Is Harrisburg Town Center the right neighborhood for you? A look at your future neighbors' expenditures might help you make a decision. The cost of living in Harrisburg Town Center is 11% costlier than comparable suburban areas of Harrisburg, such as East Charlotte. On average a household in Harrisburg Town Center spends $59.9K/year in total expenditures. Compare these expenses to those of East Charlotte, which come to around $53.9K/year.
The neighborhoods of Harrisburg Town Center and East Charlotte are identical in terms of households that have children. In both neighborhoods, the percentage of households with children is 41%.
The household median net income in Harrisburg Town Center is higher than that of households in East Charlotte. Its average household income is $78.6K/year. Compared to East Charlotte where the average income is $68.0K, there is a significantly higher concentration of people living comfortable lifestyles in Harrisburg Town Center.
Local Schools and School Districts
The schools near the apartments in Harrisburg Town Center are Carolina International School, C C Griffin Middle School and Harrisburg Elementary School. As school district boundaries are not necessarily congruent with county lines, it's important to confer with local school district authorities to confirm that the apartments for rent you are most interested in are also served by the schools you prefer.
Among Cabarrus County district middle schools, C C Griffin Middle School has the lowest student-teacher ratio at 13-to-1.
Carolina International district has two elementary schools and two middle schools. Carolina International School and Cabarrus County district have comparable student-body sizes, however Cabarrus County district has a more favorable student-teacher ratio of 14-to-1. Cabarrus County district has more blue ribbons and a higher Great Schools score than the Carolina International district.
Eating, Drinking and Recreation
Enjoy delicious food and a thriving atmosphere at destinations along Main St. With a perfect spot in mind, get the full picture with ratings and reviews of apartments in the area. Once you've done your homework on the area, be sure you use our apartment finder tool to complete your hunt for your next apartment in Harrisburg Town Center.
Enjoy a new ethnic food or go gastronomical adventuring in Harrisburg Town Center with quite a few different restaurants in the area. To add to that, easy and delicious grub can be ordered up at Zaxby's and Bellacino's Pizza & Grinders. Both restaurants are in the neighborhood.
For a more inclusive list of places to hang, drink and chill in Harrisburg Town Center, browse the Yelp page.


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6 posts tagged with NPH.
Displaying 1 through 6 of 6. Subscribe:
YOU are the star in NPH's autobiography
An excerpt from Neil Patrick Harris: Choose Your Own Autobiography
We Don't Pee!
The Tony Awards' 2012 Opening Number - What If Life Were More Like Theater? - with Neil Patrick Harris, Patti LuPone, Amanda Seyfried, and Jesse Tyler Ferguson
Jean Valjean Valjean Valjean Valjean
Four Valjeans sing "Bring Him Home". From the encore of the Les Miserables 25th anniversary concert (previously). Another highlight: One Day More from the encore, featuring most of the original cast. The entire show is also available in 12 parts on Youtube: [more inside]
Tomorrow, if anyone asks you what happened at the Tonys, you can say this: Neil Patrick Harris said it all
If you missed the 2011 Tony Awards ceremonies, you can read a live blog or five (WSJ, Backstage.com, Broadway World (read from top to bottom, Twitter-style), Broadway Direct (also Twitter-style)), check out the Wikipedia Page, or just let the evening's presenter Neil Patrick Harris summarize the whole thing in 2 minutes 20 seconds in a spoken word/rap (transcript). [more inside]
Everybody wins!
It all started innocently enough with yet another decade retrospective, this time from AfterElton.com, about the gay and bisexual male celebs of the 2000s. At the end was a poll to pick your own favorite. Things start out quietly, but after it's noted that John Barrowman and Neil Patrick Harris were neck and neck in the voting, both NPH and Barrowman made appeals on their Twitters for votes. Soon, celebrity associates of both were putting in their own two cents. Things snowball from there. [more inside]
Birth of a 'Horrible' Fandom
A brief look at the Big Bang birth of a fandom: the explosion of 'Dr. Horrible' fandom in just 47 days. Quite a lot of "more inside" follows. [more inside]
Page: 1


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"Engineering distortion is a coward act to hide the real intent of destroying. But it is more harmful than attempting destroying because it is deceiving."
Related quotes
"This life's dim windows of the soul
Distorts the heavens from pole to pole
And leads you to believe a lie
When you see with, not through, the eye."
"In a person who is open to experience each stimulus is freely relayed through the nervous system, without being distorted by any process of defensiveness."
"They completely distorted the process. The development agreement process provides for more public input."
"Four justices of the Florida State Supreme Court have distorted the judicial process into nothing more than a mechanism for providing Mr. Gore with the victory he was unable to win November 7. This ruling not only contradicts, but assaults, the most basic principles of jurisprudence,"


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"Since you excel in so many ways—in your faith, your gifted speakers,
your knowledge, your enthusiasm, and your love from us—
I want you to excel also in this gracious act of giving."
—2 Corinthians 8:7 (NLT)
Tithing—giving our first fruits for God's work on earth—shows our faith in His providence. Giving online is a safe, convenient way to honor the Lord with our tithes and gifts, proving through our regular contributions that we are faithful stewards of all He has provided.
We have many members who enjoy the ease of online paying and giving in the privacy of their homes or offices. If you choose to give online (one-time or recurring) you will be directed to a secure site where you may use your credit or debit card or fill out an e-check (ACH) to give to the general budget or make a debt reduction offering. With our online system, you may create a user profile for return visits and set up and personalize recurring contributions.
From the Online giving form, you can manage your scheduled contributions or view your contribution history.
A word about credit card giving
We offer the opportunity to donate by credit card as a convenience to those who manage their finances in a God-honoring way. While the Bible teaches sacrificial and systematic gifts, it also teaches about the dangers of indebtedness. Please make sure the convenience of giving to God via credit card aligns with Biblical teaching regarding debt (if you would like more information about handling finances God's way, our church offers individualized financial coaching.)
About online contribution fees
You will receive credit for every dollar you contribute using a credit card. Some have asked how much credit cards cost FBCL. When you use a credit card, the cost to the church is approximately 2.5% in processor fees. If you are interested in reducing the cost to FBCL, you may want to consider making your contribution using an e-check (ACH). E-checks work much like a debit card, as the amount comes directly out of your bank account, but at a greatly reduced processing fee to the church.
Give now
© 2016 First Baptist Church Lewisville. All rights reserved. MacroMedia Flash Player 7 and Adobe Acrobat are required to view portions of the site.


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By Greg Knight
November 29, 2012 | Vol. 110, No. 48
The Alaska Housing Finance Corporation is promoting a home energy rebate program meant to see savings – and encourage better energy use – in the homes of Alaska residents.
With no income requirements, Alaskans who live in owner-occupied year-round homes are eligible for up to $10,000 in reimbursable expenses for the renovations.
Butch Schmidt, the energy rater for AHFC in Wrangell, said the program is easy to get started with.
"The program works two ways; by either calling AHFC's program line, or by going online," Schmidt said. "AHFC will ask for your name, address and telephone number. That's all you need to do. After that, they will put you on a list to be seen by me."
The program's aim, Schmidt added, is to make homes more energy efficient. Duplexes are also eligible under the program and will cover both sides of the building. Buildings that are eligible and can only be done once in the lifetime of the homeowner; commercial buildings are not eligible for the program.
"You could do it for your home and if you move, your spouse could do it for another home," Schmidt said. "Once you sign up for the program it takes about two weeks for you to get on the list. Then, I get an email and call to set up an appointment that takes about two hours."
During the appointment Schmidt said he conducts an energy audit that pressurizes the house to simulate a 20-mile-per-hour wind on the wall and studies leaks, wall surfaces, windows, insulation, roofs, ceilings, crawlspaces and attics.
"All this information goes into a program the next day and I'll bring them back a report that indicates what needs to be done as far as new doors, windows, insulation or a new furnace," Schmidt said.
Depending on where the house rates in the study, ranging from a One Star up, improvements will tell the homeowner how many Stars their residence will increase. Points will be awarded from the study and equal one Star for every four points.
"Let's say you're at Two Star-Plus. And you put insulation in the floor and you get four points for it, raising you to Three Star, the first time go up a half Star you get $4,000 for reimbursable receipts," he said. "Every successive step is $1,500, up to a total of $10,000."
The program has been in existence for five years, but after a funding stop two years ago, the program lingered until it was reinvigorated with a budget approved by Governor Sean Parnell's office in the last year.
To sign up for the program, call 1-877-257-3228, or go online at http://www.akrebate.com. For questions about the program, call Schmidt directly at 874-3211.
Wrangell Sentinel | P.O. Box 798, Wrangell, AK 99929
Ph: (907) 874-2301 | email@example.com | www.wrangellsentinel.com Content and information copyright 2016 Pilot Publishing, Inc.
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- Game Details
- News (1)
- Review
- Previews
- Videos (3)
- Images (10)
- Cheats & Walkthroughs (5)
Astro Boy: The Video Game News
Displaying 1–1 of 1 articles
Comic-Con 09: Astroboy Interview
Astroboy, the video game, should delight fans of both gaming and anime -- the movie tie-in 2D side-scroller pulls heavily on classic games of the past, and features a main character who seems almost totally designed for starring in an action game. It will be out for the PS2, Wii, DS and PSP when the movie is released in 2010. We got a chance to...
Articles 1–1 of 1
Astro Boy: The Game Voice Actor Trailer
587 Views | 02:40
Astroboy Video Game Preview From Comic-Con '09
3,036 Views | 03:36
Astroboy E3 2009 Trailer
455 Views | 00:39
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Notes: The current error page you are seeing can be replaced by a custom error page by modifying the "defaultRedirect" attribute of the application's <customErrors> configuration tag to point to a custom error page URL.


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This modern languages resource has 10 recordings of French speech, describing the everyday lives of French teenagers.
The recordings are accompanied by transcripts in French so learners can check their understanding.
Listening exercise one - Thomas and Pierre
Thomas and his friend Pierre go into town to get something to eat and do some shopping. Afterwards, they listen to music at Pierre's house.
Listening exercise two - Pascale at school
Pascale talks about her school, her best friends and the subjects they like and dislike.
Listening exercise three - All about Diane
Diane describes herself, including her appearance, clothes, and favourite leisure activities
Listening exercise four - My village and region
The narrator talks about the village and region where they live, and what activities you can do there.
Listening exercise five - How the boys spend their weekends
Robert, Paul and Jean-Pierre each describe the activities they like to do at the weekend.
Listening exercise six - Survey into young people's leisure time
The narrator describes the result of a survey into how young people like to pass their time, including listening to music, using the computer and doing sport.
Listening exercise seven - Christine's day
The narrator describes Christine's daily routines, including details of school, hobbies and interests.
Listening exercise eight - Phoning to make a reservation
Three callers each leave a message to ask if they can book a hotel room, all with different requirements.
Listening exercise nine - Our jobs
The narrator describes what three young people do for a living, and what their jobs involve.
Listening exercise ten - Le Camping Saint Michel
The narrator describes the Saint Michel campsite - how many places there are, what facilities it offers and what activities you can do there.


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The Bold And The Beautiful Spoiler Video: I Want To Take You Home.
"I will call you if I need you."
Liam offers Steffy some words of comfort and Taylor tries convincing Katie to go home.
Check out this sneak peek at Friday's episode and let Soap Opera Fan Blog know what you think. Read our B&B Spoilers for more of what's ahead.
Jon Bon Jovi got some bad news. Find out what it was in our Celebrity News. And relive a little of the past in the Throwback Thursday B&B Poll: Owen's Jackie M Gig.
Join us on Twitter @soapoperafan and Facebook.
Fun fact! Matthew Purvis wrote this story just for you on November 15th, 2012 |


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It sounds more like a problem with unreal expectations. What frame of reference tells you that the machine should have more power?
All you can do is check the hydraulic pressures and cycle times. I don't have either specification here with me but many times you can check other machines of the same size for a general statement on cycle times. I know for instance that standard cycle time for boom up in larger excavators is around 4 seconds. If your cycle times and pressures are at factory specification there isn't a problem with the machine.
Your machine is pretty old speaking in mini excavator terms. It is also a gray market machine. I haven't looked at one of those in years but as I recall they even had gear pumps.


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The sweepstakes for Masahiro Tanaka officially came to an end on Wednesday, as the Japanese right-hander agreed to a seven-year, $155 million deal with the New York Yankees, as first reported by Ken Rosenthal of Fox Sports.
But now that Tanaka is off the board, who will be the next international player to make the jump directly to the major leagues?
Here's a look at six international prospects that have realistic futures in Major League Baseball.


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Volleyball & Football Matches between Alumni vs School on Dec. 19, 2015.
Posted on 10th November, 2015
MIS celebrates its 59th birthday with Talent Fiesta on 23rd April, 2015
Posted on 9th February, 2015
KITE FLYING FESTIVAL at Holiday Club, Panchsheel Encl., Sept. 7, 2013.
Posted on 22nd July, 2013
Annual Alumni Lunch hosted by 1988 batch, January 26, 2013.
Posted on 31st December, 2012
The MIS Cup on 20th Dec, 2012 - Mother's Blossoms won by 1-0.
Posted on 20th December, 2012
Mother's Blossoms, the alumni of The Mother's International School, was registered as a Society on December 29, 2006. Though the alumni has functioned at an informal level for over 20 years, it was given a formal and legal shape only in December 2006 as a new body, and thus the Mother's Blossoms came into being.
- Updated List of New and Old members.
- Prof. Ashok Rattan, Chairman : Laboratory Medicine, Medanta The Medicity.
- Rupadevi appointed CEO of Power Exchange of India.
Surabhi is a newsletter of Mother's Blossoms, which is a half-yearly publication. Watch out this space for the latest issues for download.Click to see all the issues
Download registration form For Bank Details click here!
7th December 2016
Truth is the foundation of real spirituality and courage is the real soul. He who is faultless does not care for the opinion of others. - Unknown


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Place Names Online was a simple search tool for obtaining information from the State Gazetteer. This has now been replaced by a function within the Property Location Browser.
Place names information from the State Gazetteer and boundaries of suburbs and rural localities can be located via maps and reports in the Property Location Browser.
To access State Gazetteer information from the Property Location Browser follow the steps below:
Note that if you select PDF at step 6 you will get another pop-up asking you to open or save the PDF file.
For more information contact the Geographical Names Unit (GNU)
The State Gazetteer is a database of place names in South Australia. It records any place name that is, or has been, used in South Australia. The gazetteer provides an accurate and definitive list of place names for:
Road names are not recorded in the State Gazetteer.
Each place name is associated with up to 38 fields of information relating to:
The State Gazetteer is free to download from the Department of Planning, Transport and Infrastructure data download portal. The data is licensed under Creative Commons 3.0 licence in accordance with the AusGOAL Framework. You should familiarise yourself with the license conditions before downloading.
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ASUS Silent Knight II
The second cooler from ASUS we are going to talk about today is hardly a new one, even though the cooler we already tested about a year ago, ASUS Silent Knight, got a II in its name. As you remember if you read our review, we were very pleased with that cooling solution, which demonstrated remarkable efficiency. Our today's test session will show how things changed with the launch of the new cooler modification. But before we move on to the tests, let's take a closer look at the product.
Package and Accessories
Radical black color of the box changes to dark gray at the bottom, which then gradually transforms into bright orange:
There is a round window cut out in the center of the package front, which reveals part of the cooler. The back of the box lists all technical specifications and accessories bundled with the cooler. The latter include the following items:
Here everything is exactly the same as by the first cooler modification, so let's move on to the cooler itself.


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WASHINGTON -- The Food Safety and Inspection Service of the U.S. Department of Agriculture re-issued its regulations for package dating following an undercover investigation of supermarket meat and seafood departments by "Dateline NBC." The rules can be found on the agency's Web site, www.fsis.usda.gov. on of such information is voluntary. The summary does note that if sell-by dates are part of the package labeling, and come from a federally inspected facility, then a retailer may not ...
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Today, I got a call from a friend asking why I didn't tell him I was engaged. I'm not, but I wish I was. Rumours about my life seem to be better than the reality. FML
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Word Up! Festival Twitter Stats
@wordupfestival - Tracking Word Up! Festival Twitter profile since September 10, 2011
A burst of books and imagination - London's exciting new children's books & arts festival, Oct 22-24 2011 at Alleyn's School, Townley Road, Dulwich.
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text: There are many online escort guides in London to choose from. London escort guides can vary from low to high end as well as comprise of different races and specialties according to your preference. It is important to be specific if your escort is for a public occasion and will have to play an important social role.


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Skip to comments.Five shot at Hattiesburg restaurant, suspect in custody
Posted on 04/02/2012 3:47:16 PM PDT by TigerClaws
Several people were injured after a shooting at a Hattiesburg restaurant along Hwy 49 on Monday.
A single man walked into Cuco's restaurant around 3 p.m. and shot five people. A suspect is in custody. The injured were transported to a local hospital where three are listed in critical condition.
(Excerpt) Read more at m.wafb.com ...
I assume this is Mississippi.
Is Hattieburg in North America somewhere?
Yes. I've been past that restaurant dozens of time when passing through Hattiesburg. Wasn't there a multiple shooting earlier today or yesterday somewhere else in the USA?
Hattiesburg is home to The University of Southern Mississippi.
I am surprised anyone was in the restaurant at 3 A.M.
I'm just a wee bit interested in that particular angle.
Oops, 3 P.M.
Amish fellow?
I will wait for further developments...thanks for the heads up..:) oops :(
Gun free zone?
I think this is the 2nd or 3rd mass/murder/shooting in the U.S. today.
I wonder if the shooters are graduates of the CIA's "The Finders" program.
shun this place and opt for crescent city grill instead
"According to staff reports, a white male walked into Cuco's around 3:30 p.m. and, without saying a word, began firing. Patrons and employees were among those injured. The injured have been hospitalized. Their identities are not known at this time.
The suspect was not an employee of the restaurant."
My goodness, more killings. Is it just me or are these happening with a greater frequency?
Are we sure he wasn't a Hispanic white?
Maybe an African America white guy?
"I assume this is Mississippi."
"Is Hattiesburg in North America somewhere?"
No, its the "OTHER" Hattiesburg, you bunch of D.A.'s. /sarcasm
Or Obama's outside son.
Trivia of the day. Hattiesburg is the site of the only undergound nuclear test east of the Mississippi River in the US.
Or Crechales
Well, if he's an Arab white guy no one will ever know...
Or Crechales
According to staff reports, a white male walked into Cucos around 3:30 p.m. and, without saying a word, began firing. "
Where did you get that quote from? I have read umpteen reports and none of them say that. They all say a single man. Suspect in custody. And they have not identified him or his motive as of 6:59 PM.
Thank God it was a white guy...praise the lord
I don't know about y'all but the guilt and shame I have been enduring with these negatives waves filling me day in day out in the aftemath of the Travon Martin holocaust have been too heavy a cross to bear
Thank God it was a white guy...praise the lord
I don't know about y'all but the guilt and shame I have been enduring with these negatives waves filling me day in day out in the aftemath of the Travon Martin holocaust have been too heavy a cross to bear
A Korean Kids school. The shooter was a 'former' student.
You're probably correct.
Is Mississippi a gun free zone?
It would really be helpful if the poster told us why this is important and why we should care. Without that, he might as well have posted a picture of his garden.
But let's pretend Whitey is the one who kills them all.
Perhaps an NRA member? What do you think?
. . .or perhaps a "white" Hispanic?
Maybe a local Hispanic redneck who actually had a brown neck?
Don't know at this point.
Is Mississippi a blue state?
It was a white guy.
Based on crime stats, his victims are probably whites...but given what is going on with black on white hate crime, I wonder...
A white Hispanic guy?
It would be difficult for a crazed gunman in a public establishment who was really firing randomly not to hit a black person, in Hattiesburg, Mississippi. What would be odd is if all the victims were of one race, while media claim randomness.
It looks like Tyner is a very motivated lefty. His motives and mental state may be problematic for The Narrative.
He didn't toe the liberal line completely. If there was a single hispanic among the injured, watch which angle the media run with.
Anything left of the town after this trivial test?
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


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Kardashian Sibs Wow In See-Through Chain Mail, Plunging Neckline At Dash Miami Beach Opening
Kim, Khloe and Kourtney Kardashian attended the grand opening of their Dash Miami Beach location on Wednesday looking mighty fine.
Kim wore an unusual metallic two-piece ensemble that looked like chain mail. The skirt and cropped top were see through, showing off her black lingerie underneath.
Khloe opted for a simple black jumpsuit with a low scooping v neck, while Kourtney wore a playful white and blue shorts set.
Kourtney was robbed of $50,000 in cash last month, and Khloe is missing around $250,000 worth of jewelry. Police are investigating the film crew from "Keeping Up With The Kardashians" to see if they are behind the recent burglaries.
Meanwhile, Kim is gearing up for her upcoming wedding to rapper Kanye West in Paris, France, and Kourtney is also reportedly preparing to walk down the aisle with her longtime boyfriend Scott Disick.


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Global Temperatures Not Matching Warmist Prognostication
Climate doom? Yeah, not happening
(UK Daily Mail) The Mail on Sunday today presents irrefutable evidence that official predictions of global climate warming have been catastrophically flawed.
The graph on this page blows apart the 'scientific basis' for Britain reshaping its entire economy and spending billions in taxes and subsidies in order to cut emissions of greenhouse gases. These moves have already added :£100 a year to household energy bills.
Let's see that chart
The graph confirms there has been no statistically significant increase in the world's average temperature since January 1997 — as this newspaper first disclosed last year.
And now the climate astrologers will attack the Daily Mail and probably talk about "cherry picked data", Big Oil, etc and so on. Alas
Its source is impeccable. The line showing world temperatures comes from the Met Office 'HadCRUT4' database, which contains readings from more than 30,000 measuring posts. This was added to the 75 and 95 per cent certainty bands to produce the graph by a group that amalgamates the work of 20 climate model centres working for the IPCC. (snip)
The awkward fact is that the earth has warmed just 0.5 degrees over the past 50 years. And Met Office records show that for the past 16 years temperatures have plateaued and, if anything, are going down.
None of this proves that the Earth is not in a warm period. It is. The temp increases could kick back on, or they might dip, like during the time period from the 40's to the 70's. What this does show is that all the Warmist models, usually obtained using computers with little real world data but with notions that the only thing that can cause increased temperatures during this period are due to Mankind, are flawed. And hysterical. Over-the-top. Prognostications from carnival palm readers. And that CO2 (and other GHG) output from Mankind is not the main driver of the global climate.
The works of Mankind surely have an effect on the local climate. This is known as the Urban Heat Island Effect. Globally? Not so much. Someone needs to create a 12 step process to ween Warmists off their narrow dogma.
Crossed at Pirate's Cove. Follow me on Twitter @WilliamTeach.
Also see...
Even though my father, brother, uncles and grandfather were in the military, I seldom handled guns growing up. That's because unlike many of the other people in my family, I've...Read More
William Teach
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And surely teeth nashing from all the little Climate Alarmists (who refuse to walk the talk) (via Tom Nelson) Americans


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I've decided to concentrate on my health and lifestyle. It's taken a few horrible things to have happened to make me want to change something about it, but I'm assured I want to try again to lose a bit more weight.
My first thought was 'Christ, I'm at work a lot of time, I'll come home and be knackered, if I barely have the energy to blog, what makes you think you can go to the gym and eat healthy all the time?!' so I thought how can I combine the two? So here we are.
It doesn't look like a lot right now, but this is what I'll be blogging on for a little while in Summer. Meet my secret alter ego, Chubby Bunny. I thought it sounded cute and not so self-loathing. I don't have a huge amount of followers, so I don't mind you guys knowing that it's me but for some new folk... I quite like the idea of being semi-anonymous. If some of you are interested in fitness, weight loss, or just healthy lifestyle - just pop on over, give me some advice or let's get chatting. I could use all the help I can get really.
If anyone at all is interested in following me on my weight loss journey (however short-lived or long-lived it will be!), just click the buttons below. I'm hoping to post at least twice a week, maybe on a Monday/Tuesday then at the end of the week so Friday/Saturday. And in between all them, I'm gonna be blogging on here every other week... because to be awfully honest, not a lot is going on right now in Chlo world!
I know this community is supportive, strong and all round fabulous people - so let's spread a little love, ey? :)


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Would someone please tell me what method I should use to integrate the following.
I have tried writing the numerator as the derivative of the denominator, but then I'd have an f(x) to compensate for. I thought of using partial fractions, but haven't been able to write this as the sum of two fractions. I'm wondering if I have to use another method, substitution or by parts.
Thank you.
I would tell students, forget rules, just know derivatives.
In indefinite integrable problems of what is it the derivative?
That is the whole and complete point!!
If you can find a function such its derivative is the given function, then you are done! Adding a actually means nothing to those of us who's research is on the integral.
WolfranAlpha. There is even a cell phone app. In fact, I predict that within twenty years calculus textbooks will not have a section on Techniques of Integration. Given the tools available that have been for twenty five years (CAS, calculators, etc), it is hard to see why that is not already true.
Look at to OP. It is sad if one looks at that and does not see it as a derivative form. I find it truly sad that I have seen on this very board some who have to use a substitution to see as an , even without any more effort.


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It's session day again. The first Friday of every month for a whopping 58 months now beer bloggers around the world have written on the same topic. This month's edition was chosen by Beersay:
"A Christmas Carol". The idea for me was based loosely around the visits of three ghosts to Ebenezer Scrooge, but relayed in a post about the beers of Christmas past, present and future. What did you drink during Christmas holidays of old, have you plans for anything exciting this year and is there something you'd really like to do one day, perhaps when the kids have flown the nest? Do you have your own interpretation, was Scrooge perhaps a beer geek? Or maybe it's all one day. What will you drink Christmas morning, Christmas afternoon and what will you top off the holiday with that evening?
A great topic. One in which you can move your elbows about. It inspires me to think about where the blog has been, is and will be. I now have 23 posts under one of those categories to the lower left of winter and Christmas beer. Have I actually made sense of what that heading means?
Christmas past. Going back to the beginning, in 2004 and again in 2005 I wrote post about collections of strong, winter and Christmas beers. Highlights seem to have been Harvey's Elizabethan Ale and La Choulette De Noël. Can you believe Rouge Santa's Private Reserve Ale was $3.79 USD for a 22 oz bomber? Beers past were pretty damn good as far as I can tell. But was I? Not so sure about this sentence: "[a]ll I get is one recessed biscuity note which sits oddly, leaving an impression of a big bitter arugula salad with one animal cracker crumbled on top." Not so bad. A little sarcastic yet accurate referencing non-beer words to describe beer flavours. Yet a little precious, no? And did I drink all that to write that on post on those Yuletide evenings? Intemperance abounded at Christmas past.
Christmas present A very fine Christmas present is the state of the Christmas present this year. If I lost you with that punnery, I will let you know that the photo above was taken today. When I asked for a sample of Narragansett's porter, I got two. When I shamelessly asked for a few more, I got twelve. This beer is incredibly good. Mr. B stated on Facebook recently:
It occurs to me that if I could get a regular supply of the Narragansett Brewing Company Porter, it would become a staple in my beer fridge this winter. The slightly jarring burnt note I found in the finish last year seems entirely absent in this year's version.
I couldn't agree more. I love this stuff. I do. I loooooooove it. Which means I should be concerned that it has brought out Scrooge like feelings within me this Yuletide. These are mine. Understand. Me. Me. Me. I am not sure if you can covet your own stuff but these twelve cans of porter are making me sense it might be possible.
Christmas future. What would I wish for myself, for you all? Well, the Christmas photo contest closes on Sunday 10 December which means in the near future more little beer nerds will get more little beer presents under the tree and in their stockings? I wish there were more for you, more entries and more prizes. My Christmas future would have a prize for all. What else would my Christmas future have? A case of La Choulette De Noël perhaps? Or maybe another 250,000 words on the Oxford Companion to Beer wiki proving both that it is a great foundation for any exploration of beer but also that there is so much more to be explored and yet to be discovered. I would wish for that. I would also wish for Albany ale. The real stuff that 96 year old Charles H. Haswell in 1899 looked back upon in his youth and thought was a mighty good drink. We are working on it but it already may be having an effect, hunting out as much of the actual as you can. If I am coveting more this year, I also see in my future less and less patience for questionable claims to authenticity. If you are going to hold yourself out as something, please do the prep that such claims require.
There you have it. Insobriety, covetousness and impatience. Past, present and future. Exactly what Christmas brings out in all of us.


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Settlement Reached In Green Hill Cemetery Case
Attorney General Spitzer and Secretary of State Randy A. Daniels today announced the settlement of a lawsuit with the former trustees and officers of Green Hill Cemetery Association, located in Amsterdam, New York.
The matter was referred to the Attorney General's office by the New York State Cemetery Board. The Attorney General's lawsuit was filed in 1999 against Donald Castor and his wife, Katherine. At the time, the Castors were the Cemetery's officers and its only board members.
The Attorney General's complaint alleged that the Castors had breached their fiduciary duties to the Cemetery by improperly withdrawing funds from the Cemetery's trust funds without the required court order. The Complaint further alleged that the Castors used the improperly withdrawn funds to make payments to themselves and their company, Cemetery Services, Inc. The lawsuit sought restitution from the Castors and their removal from the Cemetery's board of trustees.
In the spring of 2000, Green Hill's lot owners undertook a voluntary effort to clean up the 40 acre cemetery. In November 2000, the lot owners elected a new board of trustees, which excluded the Castors.
Under the settlement, the Castors will pay $40,000 in restitution that will be deposited into Green Hill's Permanent Maintenance Fund. The Castors are also prohibited from serving, in the future, as directors or officers of any New York not-for-profit corporation.


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Why join the Knights of Columbus: http://iowakofc.org/page/membership
Q & A for prospective members: https://www.kofc.org/un/en/membership/join/prospective.html
TANGIBLE REWARDS ($$$, insurance, and other benefits) for you and your family through membership in the Knights of Columbus: http://1drv.ms/1MsIYmR
FREE member/family insurance benefits simply by virtue of membership in the Knights of Columbus: http://1drv.ms/1IE5Znc
Great convention, Isaac! Thanks.
Isaac, please contact me. I'd like to know if there will be space and time for me at the State Convention to introduce the use of the IowaKofC.org platform as a host for COUNCIL websites. I have also left you voice mail. Thanks for your consideration.
Welcome to the IowaKofC.org website. I hope you'll take a look at the Council 4208 website; all you need to do is go to DecorahKnights.tk or http://iowakofc.org/profile/decorahknights and just scroll up and down to see all the information about the Decorah Knights of Columbus; it's that simple. If it hasn't already been done, I hope you'll take the time to set up a website (aka profile) for your Council on the IowaKofC.org website. Website training is at http://iowakofc.org/page/website-training Feel free to use DecorahKnights.tk as a model. And please don't forget to give us feedback on our website in the comment box at the very bottom of DecorahKnights.tk, by sending us a message via the IowaKofC.org message system (click on the "Inbox" link near the top right of your profile), or by sending email to firstname.lastname@example.com
And please add us as a "Friend" if this isn't already the case. :-)
The Council 4208 webmaster
Welcome to
Iowa Knights of Columbus
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Come And Check Out Oativa For Yourself
An Oativa demonstration on Saturday 19th June in Clown Conservatory, taking 2 hours (11am to 1pm) the demonstration includes a light lunch, there's only 10 places available, only £22.50 per person. Call 01246 201041 to book your place.
Click here to return to the previous page


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On Second Thought is a one-hour, daily news talk show that airs at 9 a.m. ET weekdays.
A profile of "Gone With the Wind" author Margaret Mitchell. Includes re-creations based on her letters, and explores how racial issues affected her life. Tuesday, December 6 at 9 PM
High School Football is Back, and so is Football Fridays on GPB!
The long wait is finally over. Yes, I went to some spring practices. Sure I checked out some 7 on 7 action this summer. Of course I... MORE
GPB Passport is the new member benefit that provides you with extended access to an on-demand library of quality local and national public television programming, including tens of thousands of hours of your favorite programs.
House Session 10:00am
Senate Session 10:00am
Part-time Lawmakers and Ethical Concerns in the Georgia State Legislature


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MMA Fighting is reporting that UFC middleweight contender Chris Weidman has injured his shoulder and been forced to withdraw from his upcoming bout with Tim Boetsch.
Replacing Weidman at UFC 155 will be Costa Philippou, who was scheduled to fight Nick Ring at UFC 154 before the fight was cancelled when Ring was unable to get cleared by the commission.
The report indicates that Weidman may have a torn labrum with a possible shoulder dislocation or separation. That means Weidman could be sidelined from anywhere between three-to-six months. The injury is the latest piece to what has been a turbulent November for the Weidman family. A few weeks ago, Weidman lost his house to Hurricane Sandy as it ravaged the East Coast.
Continue reading "Weidman Injures Shoulder, Withdraws From UFC 155 Bout"
MMA Fighting's Ariel Helwani is reporting that Nick Ring vs. Constantinos Philippou has been scratched from tonight's UFC 154 card. According to Helwani's tweet, Ring was not feeling well and the doctor refused to clear him.
Philippou's manager, Lex McMahon, told Helwani that the UFC will not be seeking a last minute replacement for Ring.
The removal means at Mark Bocek vs. Rafael Dos Anjos will be moved to the main card and Antonio Carvalho vs. Rodrigo Damnn will now fight on the FX card.
All of the participants for the UFC 154: St. Pierre vs. Condit card successfully made weight in preparation for their bouts Saturday at the Bell Centre in Montreal, Quebec.
The event will be headlined by a UFC welterweight title unification bout featuring current champion Georges St. Pierre and interim champion Carlos Condit. The co-main event will feature a number one contender bout between Martin Kampmann and Johny Hendricks.
The offical weights can be found after the jump.
Continue reading "UFC 154: St. Pierre vs. Condit Weigh-In Results"


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View Rule
|View EO 12866 Meetings
|Printer-Friendly Version Download RIN Data in XML
|Publication ID: 2012
|Title: Requirements Pertaining to Third Party Conformity Assessment Bodies
|Abstract: Section 14(a) of the Consumer Product Safety Act (CPSA), as amended by the Consumer Product Safety Improvement Act of 2008, requires the Commission to publish requirements for third party conformity assessment bodies to assess conformity with a children's product safety rule, and to maintain on its Web site an up-to-date list of Commission-accepted third party conformity assessment bodies. Section 14(e) of the CPSA provides that the Commission may withdraw or suspend its acceptance of a third party conformity assessment body. The Commission will be considering a rule that would (1) codify the requirements and procedures that a third party conformance body must meet to become a Commission-accepted third party conformity assessment body; (2) address how a third party conformity assessment body may voluntarily discontinue participation in the Commission's children's product assessment program; and (3) describe the grounds and procedures for withdrawal or suspension of Commission acceptance of a third party conformity assessment body. On March 14, 2012, staff submitted for Commission consideration a briefing package on a proposed rule. On April 27, 2012, the Commission voted unanimously to approve publication of the proposed rule in the Federal Register. The proposed rule published on May 24, 2012. The Comment period ended on August 7, 2012. Following review of the comments, staff will send to the Commission a briefing package on the final rule.
|Agency: Consumer Product Safety Commission(CPSC)
|Priority: Substantive, Nonsignificant
|RIN Status: Previously published in the Unified Agenda
|Agenda Stage of Rulemaking: Final Rule Stage
|Major: Undetermined
|Unfunded Mandates: No
|CFR Citation: 16 CFR 1112 and 1118
|Legal Authority: 15 USC 2063
Legal Deadline:
|Regulatory Flexibility Analysis Required: No
|Government Levels Affected: None
|Federalism: No
|Included in the Regulatory Plan: No
|International Impacts: This regulatory action will be likely to have international trade and investment effects, or otherwise be of international interest.
|RIN Data Printed in the FR: No
Agency Contact:
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Consumer Product Safety Commission
4330 East West Highway,
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Phone:301 504-7562
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Unstuffed PeppersMarch 2011
This two-step version of stuffed peppersdelivers that familiar flavor but cuts the cooking timein half. We love how reader Corinne Strebig usesflavorful sausage instead of ground meat and includesearthy wild rice instead of plain white.
Makes: 4 servings
- 1 cup long-grain rice
- 1 pound mild italian sausage, casings discarded
- 2 green bell peppers, chopped
- 1 10 ounce package white mushrooms, sliced
- 1 red onion, chopped
- In a large saucepan, bring 2 cups water, the rice and 1/2 teaspoon salt to a boil. Cover the pan, lower the heat and simmer until tender, about 40 minutes. Remove from the heat and let stand, covered, for 10 minutes; fluff with a fork. Meanwhile, heat a large skillet over medium heat. Add 1 tablespoon extra-virgin olive oil and the sausage and cook, breaking up the meat, until browned, about 5 minutes. Using a slotted spoon, transfer the sausage to the rice. Return the skillet to the heat and add 1 more tablespoon olive oil, the bell peppers, mushrooms and onion. Season with salt and pepper and cook until the vegetables are crisp-tender, about 5 minutes. Add the veggies to the sausage and rice and toss.


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van Baarle, Debbiea,b,*; Kostense, Stefana,*; Hovenkamp, Egberta; Ogg, Grahamc; Nanlohy, Neninga; Callan, Margaret FCc; Dukers, Nicole HTMd; McMichael, Andrew Jc; van Oers, Marinus HJb; Miedema, Franka,e
From the aDepartment of Clinical Viro-Immunology, CLB/Sanquin & Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, the bDepartment of Hematology, Academic Medical Center, Amsterdam, The Netherlands, the cMolecular Immunology Group, Institute of Molecular Medicine, Oxford, UK, the dDepartment of Public Health, Municipal Health Service, Amsterdam and the eDepartment of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Note: *These authors contributed equally to the presented work.
Correspondence to D van Baarle, Dept. Clinical Viro-Immunology, CLB, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Tel: +31 20 5123314; fax: +31 20 5123310; email: firstname.lastname@example.com
Received: 5 October 2001; revised: 24 May 2002; accepted: 10 June 2002.
Objective: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein–Barr virus (EBV)-specific CD8+ T cells was performed.
Design: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally.
Methods: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells.
Results: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27− T cells, in contrast to rising numbers of EBV-specific CD27− CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27− CD8+ T cells. Higher numbers of HIV-specific CD27− CD8+ T cells were associated with delayed disease progression. Virus-specific CD27− T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function.
Conclusions: Taken together, our data indicate that virus-specific CD27− T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27− effector T cells may lead to progression of viral disease.
CD8+ MHC class I-restricted cytotoxic T-lymphocyte (CTL) responses are essential in controlling virus infections [1–3]. After exposure to virus, CD8+ T cells are activated to kill virus-infected cells or inhibit viral replication, which is achieved through diverse effector functions, such as cytolysis and cytokine release, most notably interferon gamma (IFNγ) [4,5].
In human viral infections such as HIV (the causative agent of AIDS), Epstein–Barr virus (EBV, a widespread persistent gamma herpesvirus), and cytomegalovirus (CMV, a widespread beta-herpesvirus), high levels of antigen-specific CD8+ T cells are found [6–9]. Most chronic infections are adequately controlled by virus-specific T cells. Although apparently HIV-specific T cells initially control viral replication, HIV-specific T-cell responses are lost in individuals progressing to AIDS [10]. In addition, HIV-infected individuals are at high risk for EBV-positive AIDS-related non- Hodgkin's lymphomas (AIDS-NHL) [11], which are thought to develop because of progressive loss of EBV-specific T-cell immunity [12].
Recently primed virus-specific T cells are mostly of the CD45RO+CD27+ phenotype with cytolytic activity only after restimulation [13]. These CD27+ T cells irreversibly switch off CD27 expression when stimulated for prolonged periods [14] and eventually revert to CD45RA+ T cells [15–18]. The readily detectable effector functions of CD27− T cells, like high granzyme B expression and direct cytolytic activity, would predict these cells to be critical in control of virus replication [19,20].
Significant numbers of CMV- [20,21], EBV [22] and hepatitis C virus-specific T cells [23] were reported to be of the CD27− phenotype. In contrast, in HIV-infected individuals the majority of HIV-specific T cells are of the CD27+ memory phenotype, whereas only minor populations of CD27− cells are found [7,21,24]. To define the role of CD8+ CD27−, so-called effector T cells in human viral infections in relation to clinical outcome, we investigated the differentiation status of HIV- and EBV-specific T cells in the course of HIV-1 infection. We studied the kinetics of virus-specific CD8+ T lymphocytes using MHC class I-peptide tetrameric complexes [25], and assessed their differentiation status applying the CD45RO and CD27 surface markers in different groups of HIV-infected individuals in the course of infection. In addition, we studied the functional consequences of differentiation towards the CD27− phenotype in these individuals by measuring IFNγ production in virus-specific T cells. Furthermore, in a cross-sectional analysis, the impact of higher percentages of HIV-specific CD27− T cells was examined in relation to clinical outcome and HIV RNA viral load.
This study was performed on samples from participants of the Amsterdam Cohort studies on AIDS and HIV-1 infection and was approved by the ethical committee of the Academic Medical Center. Peripheral blood mononuclear cells (PBMC) were cryopreserved according to a standard computerized freezing protocol. HIV-seropositive male individuals were selected according to duration of follow-up, availability of samples and HLA-type (HLA-A2 and/or B8).
In a cross-sectional analysis, 33 HIV-1-infected individuals with a broad range of viral load were studied for the presence and phenotype of HIV-specific CD8+ T cells. Furthermore, we analysed longitudinal PBMC samples from nine HIV-1-infected individuals for HIV-specific CD8+ T cells and 11 HIV-1-infected individuals for EBV-specific CD8+ T cells (all during the natural course of HIV-infection), including four individuals who were studied for both EBV- and HIV-specific T cells. Five of the 11 individuals were selected because they progressed to AIDS-related diffuse large cell NHL, an EBV-related disease and therefore particularly interesting to study in the context of EBV-specific immunity. In addition, three individuals were selected who progressed to AIDS (classification of the Centers for Disease Control 1993) with opportunistic infections (AIDS-OI) and three who remained long-term asymptomatic (LTA) with CD4+ T-cell counts > 500 × 106 cells/l during more than 8 years of asymptomatic follow-up. The earliest time point studied was between 1 and 2 years after HIV-seroconversion or study entry. The latest time point studied for progressors was around AIDS-diagnosis and for LTA on average between 6 and 8 years after HIV-seroconversion or study entry. No time points were included at which the individuals were on anti-retroviral therapy. Clinical and laboratory findings of these HIV-1 infected individuals have been in part published elsewhere [7,26] and are summarized in Table 1.
MHC class I tetramers complexed with EBV and HIV-peptides were produced as previously described [25]. The peptides used were two immunodominant epitopes from EBV lytic cycle proteins, the HLA A2-restricted epitope GLCTLVAML (A2-GLC) from BMLF-1 and the HLA B8-restricted epitope RAKFKQLL (B8-RAK) from BZLF-1, and one immunodominant epitope from the EBV latent antigen EBNA-3A, the HLA B8-restricted epitope FLRGRAYGL (B8-FLR). From HIV p17Gag and Pol proteins, SLYNTVATL and ILKEPVHGV were complexed with HLA-A2; p24Gag and Nef peptides EIYKRWII and FLKEKGGL were refolded in HLA-B8 proteins.
Four colour fluorescence analysis was performed. Briefly, PBMC were thawed and 1.5 × 106 cells were stained with appropriate MHC class I tetramers and fluorochrome-conjugated antibodies anti-CD8, anti-CD45RO, anti-CD27, for subset distribution analysis; anti-CD8, CD27 and anti-CD28 to further subdivide the CD27− population. To determine granzyme B (GB) content, GB antibodies were used (CLB). After staining, the cells were washed with Phosphate-buffered saline (PBS) containing bovine serum albumin (BSA) and fixed in PBS/1% paraformaldehyde (PFA), and at least 250000 events were acquired using a FACSCalibur flow cytometer (Becton Dickinson, San José, California, USA). Lymphocytes were gated by forward and sideward scatter. Data were analysed using the software program CELL Quest (Becton Dickinson).
Two million PBMC/ml were stimulated with 1μg EBV-peptide (RAK), used in the tetrameric complexes, or PMA/ionomycin (positive control) or not stimulated (medium alone as negative control) at 37°C for 4 h in the presence of 3 μM monensin [27]. After incubation, cells were washed and stained in PBS supplemented with 0.5% (v/v) BSA for 15 min with HLA-B8-RAK tetramers (PE) in case no intracellular IFNγ was stained. For phenotypic analysis, cells were also stained with PerCP conjugated monoclonal antibody (MAb) CD8 (Becton Dickinson), anti-CD27 (fluorescein isiothiocyanate, CLB) and anti-CD45RO (APC) (Becton Dickinson). After membrane staining, cells were washed with PBA and fixed with 4% paraformaldehyde, permeabilized (Permeabilization kit; Becton Dickinson) and stained intracellularly with IFNγ-PE (Becton Dickinson) for 30 min at 4°C. At least 200 000 events in the lymphogate were acquired using a FACSCalibur flow cytometer (Becton Dickinson). Negative control stimulation did not induce IFNγ production and PMA/ionomycin stimulation resulted in high levels of IFNγ production in both CD8+ and CD4+ T cells (data not shown) [27].
IFNγ producing antigen-specific T cells were enumerated using IFNγ-specific ELIspot assays as previously described [28] using 96-well nylon-backed plates (Nunc, Roskilde, Denmark) and MAb from MABTECH (Stockholm, Sweden). PBMC were incubated overnight at 37°C in triplicate wells at 1 × 105 cells/well in case of HLA B8-restriced responses or 2 × 105 cells/well in case of HLA A2-restricted responses in the absence or presence of 2 μM peptide. As a positive control to test the capacity of PBMC to produce IFNγ in general, phytohemoagglutinin (PHA) (Murex Diagnostics, Dartford, UK) was added. Individual cytokine-producing cells were detected as dark purple spots which were counted after computerized visualization by a scanner (Hewlett Packard Company, Baise, Idaho, USA). The number of specific T-cell responders per 106 PBMC was calculated after subtracting negative control values. Because the percentage of dead cells and the percentage of CD8+ T cells was assessed in the same samples, the number of specific T-cell responders/106 living CD8+ T cells could be calculated.
For group comparisons Mann–Whitney tests were performed, using the software program SPSS 7.5 (SPSS Inc., Chicago, Illinois, USA). Cross-sectional data were analysed by Spearman correlation tests and analysis of variance stepwise regression analysis. Repeated measurements analyses were performed on longitudinal data after cube root transformation of all variables to obtain a normal distribution, which was necessary for the analyses. In the analyses, we corrected for dependency between observations within a person assuming a compound symmetry (CS) structure using the software program SAS/STAT (SAS Institute Inc. Cary, North Carolina, USA).
To study HIV- and EBV-specific T cells in the course of HIV-infection, PBMC from HIV-infected individuals collected at several time points in the course of HIV-infection were stained using HLA-HIV-peptide and HLA-EBV-peptide tetrameric complexes (Fig. 1b, c; left panel). As shown in Figure 2, high numbers of HIV- (range 0.1–2.7% of CD8+ T cells) and EBV-specific T cells (range 0.1–3.7% of CD8+ T cells) were present. Overall, no change in the number of both HIV- (P = 0.97, Wilcoxon test) and EBV-specific T cells (P = 0.97, Wilcoxon test) was observed in the course of HIV-infection (Fig. 2), suggesting that the well-established immunodominant HIV- and EBV-peptides used in the tetrameric complexes were still recognized by specific CD8+ T cells after several years of HIV-positive follow-up.
To investigate the differentiation stage of HIV- versus EBV-specific T cells in individuals who experienced relatively long periods of high HIV-viraemia, we applied the CD27 and CD45RO T-cell antigen expression patterns, revealing distinct subsets of T cells (Fig. 1a). Many HIV-specific CD8+ T cells in a long-term asymptomatic (LTA) HIV carrier were directed against Gag. (Fig. 1c; left panel) Despite persistent active viral-replication (+/– 100 000 viral RNA copies/ml) which one would expect to drive HIV-specific T cells to the CD27− phenotype [14,29], costaining with CD8, CD45RO and CD27 revealed that almost all the HIV (B8-Gag)-specific CD8+ T cells were of the CD27+ memory (CD45RO+CD27+) phenotype (Fig. 1c; right panel). Most EBV-specific CD8+ T cells in a HLA-B8+ long-term HIV-EBV carrier were directed against the lytic epitope RAK and the majority was of the CD27+ memory phenotype (Fig. 1b; left panel). However, in contrast to HIV-specific T cells, a substantial proportion of the EBV-specific CD8+ T cells could also be found in the CD45RO+CD27− subpopulation. (Fig. 1b; right panel)
To investigate the development of HIV- and EBV-specific CD8+ T cells over time, a longitudinal study was performed in HIV-infected individuals. As shown in Fig. 3a for one long-term asymptomatic individual, early in HIV infection (within 3 years after study entry or HIV-seroconversion) the majority of the HIV- (left panel) and EBV-specific CD8+ T cells (middle panel) had a CD45RO+CD27+ phenotype. In contrast to the HIV-specific T cells, a substantial proportion (30–50%) of the EBV-specific T cells was of the CD27− phenotype already early in HIV- infection. In the course of HIV-1 infection, the HIV-specific CD8+ T cells remained of the memory phenotype, whereas the proportion CD27− EBV-specific T cells increased.
As a measure of the extent to which virus-specific T cells had differentiated to the CD27− stage, the ratio of CD27−/CD27+ memory virus-specific tetramer-binding T cells was determined for all individuals. (Fig. 3c) By doing so the fraction of naive cells, which can vary between individuals, was excluded. Early in HIV infection (within 3 years after study entry or HIV-seroconversion) the majority of the HIV- (n = 9, left panel) and EBV-specific CD8+ T cells (n = 11, right panel) had a low ratio of CD27− /CD27+ T cells, due to the low percentage of CD27− T cells. (Fig. 3c) In the course of HIV-1 infection, both the percentage of CD27− (data not shown) as well as the CD27−/CD27+ ratio of HIV-specific CD8+ T cells remained stable, whereas in most individuals, the percentage of CD27− and the CD27− /CD27+ ratio of EBV-specific T cells increased, indicating that the majority of EBV-specific CD8+ T cells had differentiated to and increasingly accumulated at the CD27−stage (Fig. 3c). Late in HIV-1 infection, between 6 and 12 years after study entry or HIV-seroconversion, both the percentage of CD27− as well as the ratio CD27−/ CD27+ was significantly higher (median = 1.1) for EBV-specific CD8+ T cells than for HIV-specific CD8+ T cells (median = 0.3) (P < 0.014, Mann–Whitney test). In addition, co-staining with CD28 revealed that both CD27− and CD27+ EBV- and HIV-specific T cells were CD28− (data not shown).
Although in HIV-infected individuals the median ratio of EBV-specific CD27−/CD27+ was increased, in a small group of individuals the EBV-specific CD8+ T cells maintained CD27 expression throughout follow up. To study whether this could be related to clinical outcome, we compared the ratio CD27−/CD27+ for AIDS-NHL patients (n = 5), who were likely to have defective EBV-specific immunity [12], with HIV-1-infected individuals who either progressed to AIDS with opportunistic infections (PROG/AIDS-OI, n = 3) or remained clinically stable (LTA, n = 3). As shown in Figure 3d (and Fig. 3b), in AIDS-NHL patients EBV-specific T cells predominantly were of the CD27+ phenotype (low CD27−/CD27+ ratio), whereas an increase in the CD27−/CD27+ ratio was observed for all EBV-epitope-specific T cells in the LTA group, due to a higher percentage of CD27− T cells. Later in HIV-infection, both the percentage of CD27− and the CD27−/CD27+ ratio in LTA was significantly higher (median = 1.48) than in AIDS-NHL patients (median = 0.43) (P < 0.034, Mann–Whitney test). Progressors to opportunistic infections (AIDS-OI) also showed a trend towards higher percentages of EBV-specific CD27− T cells (n = 3) (median ratio CD27−/CD27+ = 0.80). The maintenance of the CD27-expression in AIDS-NHL patients occurred despite high levels of EBV load in the PBMC, which was comparable with the EBV load in LTA and other progressors to AIDS (Table 1).
Thus, in AIDS-NHL patients, who are known to have failing immune control of EBV infection, we observed the same lack of differentiation to the CD27− effector phenotype for EBV-specific T cells as for HIV-specific T cells, which also fail to control HIV-infection.
Although in the majority of HIV-infected persons most HIV-specific T cells expressed CD27, in some HIV-infected individuals a higher percentage of CD27− within HIV-specific T cells was observed. To investigate the effect of the percentage of CD27− within HIV-specific T cells on disease progression we correlated the percentage of HIV-tetramer+ T cells lacking CD27 expression with viral load and months of AIDS-free follow up in HLA-A2 or HLA-B8 positive participants of the Amsterdam Cohort. In samples drawn between 1 and 3 years after seroconversion (n = 11) an inverse correlation between HIV viral load and months of AIDS-free survival was found (Spearman, R = 0.78, P = 0.005) (Fig. 4a) as described before [30]. For the same samples an inverse correlation was observed between the percentage of CD27− within HIV-specific T cells and viral load (R = 0.79, P = 0.004). (Fig. 4b) Interestingly, high percentages of HIV-specific CD27− cells detected between 1 and 3 years after seroconversion (n = 11) and in samples randomly drawn during HIV-infection (n = 33) correlated with delayed disease progression (Spearman: R = 0.77, P = 0.005 and R = 0.46, P = 0.006, respectively (Fig. 4c and d, respectively), whereas the total number of tetramer+ T cells did not correlate with protection from disease (R = 0.22, P = 0.49)(data not shown). In a multivariate stepwise analysis, the percentage of CD27− within HIV-specific T cells was predictive for progression rate independent of viral load (CD27−: β = 0.82, P = 0.002; viral load: β = 0.112, P = 0.75).
These data suggest that CD27− CD8+ T cells may be more efficient than HIV-specific T cells with other phenotypes, in particular CD27+ memory T cells, in controlling HIV-infection and delaying disease progression.
Although it is known that CD27− CD8+ T cells (total CD8+) express higher levels of granzymes and perforin and exert direct cytolytic activity, we have no understanding of the effect of CD27− differentiation on anti-viral CD8+ T-cell function. Therefore, we tested the relation between the percentage of virus-specific CD27− T cells and – as a read-out for effector function – the number of IFNγ-producing T cells. We measured the number of IFNγ-producing CD8+ T cells after stimulation with EBV-peptides for all individuals. To perform regression analysis we selected those HIV-1-infected individuals in whom a significant accumulation of CD27− within EBV-specific T cells was observed (three progressors to OI and three LTA). Using regression analyses (mixed linear model) the percentage of EBV-specific CD27− T cells measured at all time points (n = 34) was positively correlated with the number of EBV-specific CD8+ T cells producing IFNγ (β = 1.2), which was highly significant in multivariate (P < 0.001) analyses controlling for EBV load and the number of CD4+ T cells. (Fig. 4e)
To investigate in detail whether the overall increase in IFNγ-producing antigen-specific T cells was indeed caused by an increase in the percentage of virus-specific CD27− T cells, we compared simultaneously the distribution over the different T-cell subsets of EBV-specific T cells defined by tetramer-staining and by IFNγ production, using intracellular IFNγ staining after stimulation with the specific EBV peptide. In a long-term asymptomatic HIV-infected individual (LTA 0036) early in HIV-infection (left panels), tetramer+ T cells resided for 58% in the CD45RO+CD27+ memory and for 33% in the CD27− subset (Fig. 5a). In contrast only 28% of the IFNγ+ EBV-specific T cells, which were lower in number than the tetramer+ T cells, resided in the CD45RO+CD27+ memory and 60% in the CD27− subset (Fig. 5c), showing that IFNγ-producing cells were enriched in the CD27− subset compared with tetramer binding cells. Similar distribution patterns were observed for another LTA individual (LTA1160, Fig. 5d). Moreover, at the later time point the percentage of tetramer+ T cells (LTA0036) had not increased in the CD8+ T-cell population, but EBV-specific T cells became two-fold more enriched in the CD27− fraction (right panels (Fig. 5a). Concomittantly, the percentage of IFNγ-producing T cells had doubled from 0.16 to 0.30% and accumulated in the CD27− fraction (Fig. 5c). The other LTA individual (LTA1160) did show an increase in the number of tetramer-binding cells. (Fig. 5d) At the same time the EBV-specific cells became more enriched in the CD27− fraction (right panels (Fig. 5d). Concomittantly, the percentage of IFNγ-producing T cells had increased from 0.14 to 0.47% and accumulated mainly in the CD27− fraction (Fig. 5f).
Similar results were obtained when HIV-specific CD8+ T cells were studied after initiation of HAART, which led to increased percentages of B8-Nef-specific CD27− T cells and percentage of IFNγ production (data not shown). Thus, the overall increase in IFNγ-producing antigen-specific T cells was indeed related to an increase in the percentage of virus-specific CD27− T cells (Fig. 5)
To confirm that the observed higher percentage of IFNγ in CD27− T cells and the overall increase in IFNγ in the course of HIV-infection could indeed be token as a sign for effector function, we also stained tetramer+ T cells with GB antibodies to determine the GB content of the EBV-specific T cells of these two LTA individuals. As shown in Fig. 5b and e, the number of GB-positive CD8+ tetramer+ T cells increased in the course of infection from 18 to 26% in LTA0036 (Fig. 5b) and from 43 to 66% (Fig. 5e) in LTA1160. Furthermore, CD27− T cells consistently had higher GB content than CD27+ T cells in these two LTA individuals (LTA0036: 30% early and 37% late/LTA1160: 70% early and 74% late in CD27− versus LTA0036: 12% early and 10% late/LTA1160: 36% early and 58% late in CD27+ T cells). (Fig. 5b/e).
In this study, we investigated the differentiation status of HIV- and EBV-specific CD8+ T cells in the course of HIV-1 infection to evaluate the role of virus-specific CD27− CD8+ T cells in human viral infections in relation to clinical outcome. Our data indicate that differentiation of HIV- or EBV-specific T cells in the CD27− stage was associated with slower progression to AIDS or protection against AIDS-NHL, respectively. In AIDS-NHL patients, EBV-specific CD8+ T cells remained of the less differentiated CD27+ phenotype in the course of HIV-1 infection, despite high levels of EBV load in PBMC. For HIV-specific T cells the percentage of CD27− T cells appeared to be predictive of disease progression, independent of HIV serum load. Finally, we observed that virus-specific CD27− T cells showed elevated IFNγ production compared with CD27+ T cells in response to viral peptides in vitro, which is indicative of the strong effector function of these cells.
The observed defective maturation of HIV-specific T cells is compatible with the reported lower perforin levels in CD27+ T cells [21]. However, we show here that this defective maturation does not seem to be specific for HIV-specific T cells, since EBV-specific T cells in AIDS-NHL patients were also shown to be mainly of the CD27+ phenotype. We further show that high numbers of fully differentiated CD8+ T cells specific for HIV or EBV are associated with protection from progression to AIDS or AIDS-NHL, respectively. The accumulation of virus-specific CTL in the CD27− CD8+ population in these clinical conditions suggests that these T cells may be critical for controlling viral infections that are chronically active as reflected by active viral replication. This is compatible with our finding that these T cells have augmented antiviral effector function compared to CD27+ T cells, as reflected by the correlation between the percentage CD27− T cells and high IFNγ production by virus-specific CD8+ T cells. In addition, CD27− T cells have also been shown to contain more GB and perforin and exert a stronger direct cytolytic activity compared to CD27+ cells [19,20]. Moreover, in a previous study we have shown that HIV-peptide-specific T cells which contain higher percentages of CD27− T cells, also have a higher capacity to produce IFNγ after peptide stimulation [27].
Although IFNγ production does not infer direct cytolytic activity, it has been shown that effector functions such as cytolytic capacity and IFNγ production largely coincide [31]. In addition, the observed state of T-cell effector dysfunction in several clinical conditions has been shown to occur both at the level of IFNγ production and cytolytic activity [32,33]. Furthermore, in healthy individuals there is a good correlation between IFNγ-producing T cells and CTLprecursor (CTLp) frequencies [34]. Moreover, our own observations indicate that the number of EBV-specific CTLp [12] and IFNγ-producing T cells correlate as they both decrease in the course of HIV-infection in AIDS-NHL patients [12,26]. Interestingly, parallel analysis of IFNγ-producing EBV-specific T cells and GB-containing EBV-specific T cells showed that both increase in the course of HIV-infection and both are higher in the CD27− population of T cells.
One could argue that the tetramer-staining and IFNγ-producing CD8+ cells after peptide-stimulation might be CD8+ natural killer (NK) cells. However, cells that stain with HLA-peptide tetramers are T-cell receptor (TCR) alpha-beta postive cells (thus CD8+ T lymphocytes) and not NK cells. In addition, cells that respond with IFNγ production upon 8–9 aminoacid long peptides, recognize with their TCR these peptides in the context of MHC class I, an interaction specific for CD8+ T cells and not NK cells. Furthermore, CD8+ NK cells are mostly found not to be CD8 bright but CD8 dim cells. Tetramer-staining shows that the CD8-bright cells stain with the tetramer, and not the CD8 dim cells. Finally, all techniques to analyse virus-specific CD8+ T cells are highly validated using depletion and purification of certain cell populations and co-stainings with other CD-markers.
A large dissociation between tetramer+ and IFNγ+ EBV-specific T cells was observed in this study. The proportion of IFNγ-producing T cells within tetramer+ T cells has been shown to vary both in HIV-infected individuals as well as in healthy individuals and is on average 25% [26]. In HIV-infected individuals, we have shown that this percentage can be somewhat lower early in HIV-infection and further decreases in patients developing EBV-related disease, which is suggestive of dysfunction [26].
Since development of functional CTL is known to be dependent on CD4+ T-cell help [35,36] and HIV-specific CD4+ T cells are believed to be irreversibly lost during acute infection [35], HIV-specific CD8+ T cells may not be able to efficiently differentiate to CD27− T cells despite high HIV viraemia. Indeed, we observed significant numbers of CD27− cells only in LTA individuals that experience a relatively stable clinical course, have modest viral loads and are known to still have measurable HIV-specific CD4+ T-helper activity [37]. In contrast to HIV-specific helper T cells, it is most likely that EBV-specific CD4+ T cells were present before HIV seroconversion, and this CD4+ T-cell help may decrease more gradually. When EBV-specific CD4+ T- cell help is eventually lost, due to immune activation-induced increased EBV reactivation, the functional differentiation to EBV-specific CD27− T cells will also be hampered and the individuals become at high risk of developing NHL.
In addition, as shown previously, CD4+ T-cell numbers indeed correlated with the number of EBV-specific IFNγ-producing T cells, suggesting a role for CD4+ T cells in maintaining functional capacity of CD8+ T cells [26]. Furthermore, the expression of CD27 has been shown to decrease after interaction with its specific ligand, CD70 [38] and CD70 expression is upregulated by T helper 1 cytokines [39]. Therefore, it could be that helper T cells, directly or indirectly, contribute to this differentiation process of CD8+ T cells.
Despite the fact that high percentages of EBV-specific CD27− T cells seem to be important in controlling EBV, healthy EBV carriers are heterogeneous in the expression of T-cell markers such as CD27 and CD28 and usually have low levels of these so-called 'effector' T cells. A possible explanation for this phenomenon is that during HIV-infection there is a continuous antigenic trigger caused by a chronic high level of viral load, continuously driving EBV-specific T cells into the CD27− compartment and thereby inducing and sustaining a high percentage of CD27− T cells. In immunocompetent individuals, acute EBV-infection leads to a peak in viral load [40]. The initiation of a CTL response, also inducing CD28− and CD27− T cells [22], leads to a reduction in EBV load and subsequent vanishing of the 'effector' T cells.
In conclusion, we show that fully differentiated CD8+ T cells that are specific for HIV but also EBV are associated with protection from progression to AIDS or AIDS-NHL, respectively. Thus, our data indicate that impaired maturation of CD8+ T cells is not specific for HIV, but may well be a general phenomenon in other conditions with failing anti-viral or anti-tumour immune control. Since virus-specific CD27− T cells may be critical for the control of chronic active viral infections, our data support the notion that phenotypic analyses of virus or tumour-specific CD8+ T cells based on CD27 expression may be informative for protective immunity to virus-induced disease and tumour development.
This study was part of the Amsterdam Cohort Studies on AIDS and HIV-1 infection, a collaboration of the Municipal Health Service, the Academic Medical Centre and CLB. We thank Dr.M.Th.L. Roos and collaborators for T lymphocyte immunophenotyping and processing of patient samples. We thank Dr. D. Hamann and Dr. R.A.W. van Lier for critical reading of the manuscript.
Sponsorship: This study was financially supported by the Dutch Cancer Society (grant number 96-1168) and the Dutch AIDS Fund (grant number 1007 and 2461).
CD27; Epstein–Barr virus; HIV; CD8+ T cells; disease progression
© 2002 Lippincott Williams & Wilkins, Inc.
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