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Ladies Overcoats
The Overcoat For Women
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Prom night is reasonably the biggest night in any teenager's life. If you are a plus size girls, then you might need to find that perfect plus size prom dresses. There are plenty of options when shopping for plus size prom dresses, and some stores that specialize in this field. The shopping for plus size prom dresses you will most likely encounter when looking for your gown will be in both specialty
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Fashionable Formal Dresses For Teenagers
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All hotels in Southend-on-Sea, England, United Kingdom
1 room, 2 adults
9-13 Wilson Road, Southend-on-Sea, England, SS1 1HG, United Kingdom, 800-491-6126
The Ilfracombe House, Southend-on-Sea
4-star guesthouse with restaurant, near Southend Pier
- Free full breakfast, free WiFi, and free parking
The property will be renovating from 03 July 2017 to 20 July 2017 (completion date subject to change). The following areas are affected:
Renovation work will only be conducted during business hours on weekdays. Every effort will be made to minimize noise and disturbance.
- Exterior
Renovation work will only be conducted during business hours on weekdays. Every effort will be made to minimize noise and disturbance.
Main amenities
- 20 guestrooms
- Near the beach
- Restaurant and bar/lounge
- 24-hour front desk
- Daily housekeeping
- Laundry service
- Meeting rooms
- Concierge services
- Free newspapers in the lobby
- Luggage storage
- TV in a common area
- Free full breakfast, free WiFi, and free parking
Feel at home
- Refrigerator
- Private bathroom
- Television
- Free toiletries
- Daily housekeeping
- Laundry facilities
What's around
- Heart of Southend-on-Sea
- Southend Pier (0.6 mi / 1 km)
- Westcliff Gardens (0.3 mi / 0.5 km)
- Cliffs Pavilion (0.3 mi / 0.6 km)
- Beecroft Art Gallery (0.3 mi / 0.6 km)
- New Empire Theatre (0.4 mi / 0.6 km)
See all 72 Hotels.com reviews
Value for money
Best value for money
Fantastic service
Great location
Decent vibe
TripAdvisor Traveller Rating: 4
From 271 reviews
The Ilfracombe House, Southend-on-Sea
Hotel Info: 866-539-8117from $77
- Standard Single Room, 1 Twin Bed, Ensuite
- Comfort Apartment, 2 Bedrooms, Kitchen
- Standard Twin Room, 2 Twin Beds, Ensuite
- Standard Double Room, 1 Double Bed, Ensuite
- Luxury Double Room, 1 Double Bed, Ensuite
- Compact Double Room
- Family Room, 1 Bedroom, Ensuite
Want more choice? Check out our other properties in Southend-on-Sea.
More great choices
At a glance
Key facts
Hotel size
- This hotel has 20 rooms
- This hotel is arranged over 3 floors
- Check-in time 2:00 PM-10:30 PM
- Check-out time is 11:00 AM
- Express check-in
Guests planning to arrive outside of normal check-in hours must contact this property in advance to arrange check-in. Please contact the office using the information on the reservation confirmation received after booking.
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- No cribs (infant beds)
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
Nearby Four Star Southend-on-Sea hotels
In the hotel
Food and drink
- Free full breakfast daily
- Restaurant
- Bar/lounge
- Room service (during limited hours)
Working away
- Meeting rooms
- Conference space size (feet) - 140
- Conference space size (meters) - 13
- 24-hour front desk
- Concierge services
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Number of buildings/towers - 1
- Designated smoking areas
- Television in common areas
In the room
Home comforts
- Coffee/tea maker
- Iron/ironing board
Sleep well
- Premium bedding
Freshen up
- Private bathroom
- Free toiletries
- Hair dryer
Be entertained
- Digital TV channels
- DVD player
Stay connected
- Desk
- Free newspaper
- Free WiFi
- Phone
Food and drink
- Refrigerator
- Daily housekeeping
Top Southend-on-Sea hotels
Recently reviewed hotels in Southend-on-Sea
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"Very friendly proprietor, who seemed interested in your enjoyment of your stay. Great breakfast. A true bnb experience. The room was small, but the facilities were nice and…"Jul 22, 2017
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"Wonderful oldworld charm. Rooms were great, and the sitting rooms near the bar area were quaint and cozy."Jul 17, 2017
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"The Delting Coachhouse was very clean and the rooms had been updated with new bathroom. We were greeted warmly and every effort was made to make us feel comfortable. The…"Jul 17, 2017
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"Great place to stay except when hot...no ac makes a little tough...other wise most staff friendly. No evening meals offered. Breakfast great. Great location"Jul 14, 2017
Also popular with travelers from the USA
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subset: commoncrawl


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5 bedroom villa for sale
France - Cabris, Alpes-Maritimes, Provence-Alps-Cote d`Azur
like this property?Call: 03339 871846
Key features:
- Vew
- Intimacy
- Perfect condition
Full description:
Ground floor comprises a living / dining area with a conservatory, fitted kitchen, office and WC.
1st floor comprises 2 bedrooms with ensuite bathrooms.
Lower ground floor comprises a room, laundry, 2 bedrooms with ensuite bathrooms.
Landscaped garden with 8m x 4m swimming pool.
10mn walk to amenities. Cannes and beaches 40mn driving.
Nearest Airports*
- Nice Côte d`Azur (International), 17.7 miles
- Toulon (Le Palyvestre) (International), 52.2 miles
- Provence (International), 84.9 miles
*Distances are straight line measurements. This feature is currently in beta.


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Read the full story here Web Link posted Friday, April 6, 2012, 9:54 AM
Town Square
Palo Alto eyes three new schools at Cubberley
Original post made on Apr 6, 2012
Read the full story here Web Link posted Friday, April 6, 2012, 9:54 AM
Comments (1)
a resident of Fairmeadow
on Apr 6, 2012 at 10:14 am
Please make the small high school and/or the middle school to a math and science academy that we need and other state already has like in Texas. Now we lost gifted education unlike other states.
Don't miss out on the discussion!
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California Democrats seek to revive the Republican Party; Republicans expected to resist
By Douglas Moran | 28 comments | 2,703 views
Chocolate + Tahini Ice Box Pie
By Laura Stec | 0 comments | 1,140 views
Love is a Verb
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Save $5 when you register by Monday, July 24
Registration is now open for the 33rd annual Palo Alto Weekly Moonlight Run and Walk. This family-friendly event which benefits local nonprofits serving kids and families will take place on Friday, Oct. 6 at the Palo Alto Baylands.


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- Home
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Trip points to Restless Lady at Hastings
VANCOUVER, British Columbia - A very sharp Restless Lady could be hard to hold off when she tries to run down Brighouse Belles winner Heaven on Hold in the fourth race at Hastings on Sunday. The 6 1/2-furlong second-level allowance race carries a $50,000 claiming option and drew six horses. The eight-race card begins at 1:25 p.m.
Trained by Cindy Krasner, Restless Lady is going for her third straight win and she is coming off of an easy 4 1/4-length win at this level on June 12. With Chad Hoverson aboard, Restless Lady stalked the pace set by Rosada before taking over control of the race on the stretch turn. Rosada,
Yoorock Gal, and Heaven on Hold all have good speed, and if a duel develops Restless Lady will be very tough to beat. Hoverson retains the mount.
Heaven on Hold could end up being the favorite. A 4-year-old Kentucky-bred, Heaven on Hold has won two sprint stakes - the Brighouse Belles here on May 3, and the $50,000 Tiburon at Golden Gate in January of 2008. She is coming off of a third-place finish in the $50,000 Senate Appointee on June 7.
Heaven on Hold, clearly the speed of the speed, will break from the rail with Mario Gutierrez aboard. She is trained by leading trainer Troy Taylor.
Rosada could be coming up to a peak effort in her third start at the meet. Trained by Barb Heads, Rosada was off for exactly one year when she came back to finish fifth as the odds-on favorite in a first-level allowance race for British Columbia-breds on May 17. She took a big step forward when she finished second to Restless Lady on June 12. In her only start in 2008 she finished second to Dancing Allstar in the Emerald Downs Handicap.
Fernando Perez, who rode Heaven on Hold in both the Brighouse Belles and Senate Appointee, retains the mount on Rosada.
Yoorock Gal was favored over Restless Lady and Rosada June 12, but after forcing the early pace she tired to finish a well-beaten third.
Infinite Wealth will be making her first start since she finished fourth in a first-level allowance race on May 2. In her only other start this year she finished second to Destiny's Home in a similar race on April 18. Destiny's Home, fifth after breaking in the air in the Senate Appointee, rounds out the field.


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2015-05-04 06:13:01
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Tekkno Bubbles Party Supplies
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The conversation I had with a fellow teacher today explains why I am here. This fellow teacher discussed the constant bickering at her school. Teachers going against teachers. This shouldn't happen. Teachers are supposed to help each other out. We're not the enemy and neither is administration. This is why SpecialChalk.com is set out to support teachers with the hope of uniting us.
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What brings me here?
January 13, 2011
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Klymaxx Meeting In The Ladies Room
7 ratings
|List Price:
|You Save:
|$1.01 (14% Off)
Available: Usually ships in 1-3 business days
Format: CD
sku: USP 11064
Brand New
Related products:
by S.O.S. Band ~ Icon $7.09
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by Klymaxx ~ The Maxx Is Back ~ $2.98
by Klymaxx ~ The Maxx Is Back ~ $2.40
CD Details
- Released: September 11, 2000
- Originally Released: 2005
- Label: MCA Special Products
- $1.29 on iTunes1.The Men All Pause
- $0.99 on iTunes2.Lock And Key
- $1.29 on iTunes3.I Miss You
- $0.99 on iTunes4.Just Our Luck
- $1.29 on iTunes5.Meeting In The Ladies Room
- $0.99 on iTunes6.Video Kid
- $0.99 on iTunes7.Ask Me No Questions
- $0.99 on iTunes8.Love Bandit
- $0.99 on iTunes9.I Betcha
Song previews provided courtesy of iTunes
Product Description:
Klymaxx enjoyed their biggest success on their debut album for MCA. It got them their biggest hit with "I Miss You," the kind of song that they only hinted at doing before and should have continued updating long afterward. This was the last go-round for the original band, and everyone seemed inspired and energetic. It had an air of brashness and uncertainty, one they unfortunately weren't able to maintain. ~ Ron Wynn
After 7The Best of After 7 (CD)
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Sold OutSave for Later
Music Lovers' Ratings & Reviews:
Customer Rating:
Based on 7 ratings.
Based on 7 ratings.
Be the first Music Lover to write an online review of this product!
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Product Info
- Sales Rank: 20,603
- Shipping Weight: 0.25/lbs (approx)
- International Shipping: 1 item
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Search 321,000 hotels worldwide.
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My wife and I had a wonderful time with our 2 children under the age of 4. The hotel was nice and the staff was tremendous. There was never a time that we had to wait for anything because the staff was so large.
stayed for 2 nights 3 days.... did not expect such a great hotel with friendly staff...breakfast was exceptional. Had dinner there as well. the food was amazing... can't stop talking about the great time I had there. Will definitely go again!!
We stayed at Still Beach House for our week long honeymoon and absolutely loved it. Our room had an amazing view of the Petit Piton and the Soufriere harbor. We had a two room suite with the bedroom being well air-conditioned and the other offering a nice breeze from the balcony. The main room had ...
It's cleanliness, close to the beach, the pool, the staff was excellent for all our needs, including reservations, kayak, etc etc. We got a free upgrade when we arrived, which included a jacuzzi in the bedroom!! The owner of the Villa was very knowledgeable on what to see and what not to see! He ...
I often stay at Coco Palm because it is perfectly located. The staff is amazing as well. The swim up rooms and suites seem to be in better condition than the standard rooms. The downside for me is that there are no kitchens in any of the rooms, and I would prefer to do my own cooking. Overall I love ...
We loved it. The beach and all the water sports were terrific.
Spent five days here with my wife and son and i must say this hotel provided value for money.Our room was nice and spacious with all amenities in place.This hotel's location is perfect with grocery stores,restaurants and even shopping malls just mere footsteps away.The staff there was really attentive ...
great experience there was a lot of activity great service friendly staff and service the night club is lovely there is a lot to do
Will be returning for another carnival experience. I did not try the hotel food but was recommended to a local spot not far from the hotel by a hotel staff member and was pleased. Hotel distance to where all the carnival parties were was about 20 mins away. Rooms and bathroom were spacious wish they ...
Overall was a relaxing stay. All the staff were friendly and welcoming.
You're about to set off on the coolest family vacation, so round up the kids, and start packing. You and your tribe will encounter one of the most popular getaway destinations: St. Lucia. It's here where you'll come across so many things to do and places to see that your family will remember this trip forever. Whether you're making friends with other vacationing families or getting travel advice from the locals, you'll leave town happy.
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Category: Issue Two
Pathfinders Camper Conversions
The word Bespoke is an adjective for anything commissioned to a particular specification. It may...Read More
Camp Oven Bread – Made Easy…
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Issue Two – Spring 2017
by TURAS | Feb 28, 2017 | Issue Two, Magazine Issues, News | 0 |
Welcome to ISSUE 2 of the TURAS Camping and 4WD Adventure emagazine. This edition is action packed with travel stories, articles on preparing your vehicle for your next camping adventure, news on gear, and much much more. Click on the image or the link to access over 200 pages in our 2nd e-magazine.Read More
Issue Two Coming soon!!
Its nearly here! Now that ISSUE ONE has been online for a few months we are now finalising ISSUE...Read More
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1 x 3/4-inch Sterling Silver St. Christopher Medal.
This Crucifix Pendant available from CatholicShop.com features dimensions of 29 X 19 millimeters, approximately 1.14-inches by 3/4-inch.
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A former sixth-grade math teacher, Michael Greller is strong in numbers. Jordan Spieth leaned more on his caddie's words at the British Open. "This is as much mine as it is his," Spieth said as he clutched the silver claret jug. And then he turned to look over at Greller and said, "You deserve all the credit in the world for this major championship." Greller still had to work out some new math at the most critical juncture of the final round at Royal Birkdale. Spieth was hitting his third shot from the driving range and couldn't see any part of the par-4 13th hole. He thought he was about 270 yards away and was planning to hit 3-wood. Greller estimated the yardage closer than that and told him
In the end, what was it that led this summer's most coveted free agent, Gordon Hayward, to finally choose the Celtics? "I don't know if there was one thing," Hayward told ESPN's Adrian Wojnarowski on "The Woj Podcast" released Monday. "I think if I would've stayed in Utah, we would've been really good. It would've been a great situation for me. My family would have been happy. We loved it there. "It was a different feeling in Boston that we had. It was like a gut feeling. When we broke it down, the city, the coaching staff, the players. The feeling of putting on a Boston Celtics uniform and competing for a title outweighed everything." MORE CELTICS Report: Celtics inquire about a potential Kyrie
Ole Miss players were essentially sold a bad bill of goods. If the school actually cares about them, they'll work with the NCAA and give them the choice to leave without penalty.


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I have a strong headache problem. What should I have to do for solved this problem as soon as possible. Please reply soon.
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Natural remedy tips for Sexual Debility:
Sex is an important aspect of everyone life, some of the person suffering from
Sexual Dysfunction, some of Home remedies that can help.
Garlic: Garlic work as a great tonic for the people suffering from unable to maintain an erection. According to the great naturopath, Dr. H K Bhakru Garlic can help to relieve sexual debility due to a number of causes like exhaustion, lack of interest ,and even a caused due to sexual over-indulgence.
Method: Crush 1 or 2 cloves of Garlic and eat every day Morning with Cow's Ghee till you get the desired result
Onion: It's also useful in suffering from erectile dysfunction; Also Onion is good for health
Method: Eat raw white onions as a part of a daily meal, added to the salad or simply eat.
Carrot: Best remedy when suffering from fatigue, premature discharge.
Method: Chop 150 Gms of Carrot; add into a half boiled egg with honey. Mix well n use for 2/3 months
Lady's finger: Ayurved advocate that lady's fingers are great for those who suffering from loss of their sexual power.
Ashwagandha : 2 Gms of Ashwagandha Powder Mix with 1 Tsp of Cow's Ghee to Be Taken Twice a Day
Almond Oil : Apply on External Male Genital Organ to Strengthen the Local Nerve
Benefit of Sex:
- Relieves Stress Condition
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Sex is good for you in way you may never have imagined and that the health benefits extended well beyond the bedroom


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When Charles Dickens died having completed only half of his final novel, The Mystery of Edwin Drood, readers worldwide were left with one of the greatest literary mysteries of all time. What happened to Edwin Drood? Did he simply disappear, or was he murdered? Clues abound and evidence points to a vicious attack, but the truth is elusive. The Return of Edwin Drood offers a tantalizing theory. A decade has passed since Drood's disappearance and presumed death. Now he suddenly reappears. He has been living in America where he has gradually overcome amnesia brought on by a savage attack 10 years earlier. His memory now fully restored, Drood returns to his home in England to reclaim his life and property, to rekindle his relationship with Rosa, the woman with whom he was once in love, and to discover the identity of the assailant who stole 10 years of his life. With the help of world-renowned illusionist Madame Robert, Drood sets into motion an elaborate plan to discover the assailant's identity and to force a confession. As Drood closes in on his target, questions arise about the nature of forgiveness and redemption and the relationship between justice and revenge.
In stock
- Type of Show Full-length Play
- Product Code RD9000
- Cast Size 12
- Min. Royalty Rate $80/perf
- Cost $9.95
- Approx. Run Time 100 min
- Target Audience Middle School | High School | College and Adult | Family (all ages) | Senior Adults | Praise Groups
- Performing Group Middle School | High School | College Theatre | Community Theatre | Professional Theatre | Senior Theatre | Touring Group | Praise Group | Dinner Theatre
- Genre Drama


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We booked with Virgin and upgraded to AI with club benefits. From the moment we arrived at the resort we were warmly welcomed with cold towels and a cold fruit juice. We were in our room by 11am a welcomed benefit of the upgrade after a long overnight flight. Our room was by the main family pool where the patio doors led to a garden area by the pool on the ground floor another benefit of the upgrade. We were room 2110 and it was lovely. The aircon worked well and the tea and coffee making facilities were great. Housekeeping are fab and provided a dvd player free on request, this meant that the adults could watch a film whilst my son watched his dvd s on his player. Fiaz was amazing and looked after us very well at the pool. The beach club crew were also fab ensuring that our sunbeds were reserved each day in the spot we asked for. The whole food and beverages team were lovely but our special thanks go to Maarid and the host of waiters and waitresses are amazing. Special mentions go to Saiful, Islam, Bahar, Sabina, Keith, Ali, Parbin, Jonatyne and Joscelyn. The chefs st the resort are very attentive and responsive to special requests, so dont be afraid to ask. Chef Marco and Fatha were great and took interest in ensuring my son was not given food containing or possibly contaminated with egg.
The kids club was a reason I also chose this resort. Thd Kids Paradise was a nice cool retreat for my son, however the large family pool tended to win his vote each day. The kids club appeared clean, well managed and a safe environment for kids. I asked the staff to find me if my son ever wanted to leave so it is important to either take a mobile so they can contact you on the resort on write down where you will be.
The spa was also a source of relaxation and the prices were UK comparative with a 60 minute oriental massage costing approx £70. The steam, sauna and jaccuzi are free to use for guests as is the great gym which looks out over the sea. The gym is adequately kitted out so take a look.
The belly dancer in Espirit was my 4 year old sons highlight, she was there a few times a week between 9-9.30 then we'd head to Behar rooftop for a cocktail, you can also partake in a shisha if that takes your fancy. We went on the courtesy trip to Dubai from Virgin which left at 11am on a Sunday and departed Dubai at 4pm. The trip is approx 1hr 20 mins each way so take a dvd player or toys for little ones a similar time frame from the airport. We booked 2 trips with Virgin for two adults and my 4 year son
1. Dune bashing and evening camp where you could have a henna tattoo and camel ride free. Evening meal which was lovely and very filling with alcolic drinks, soft drinks or water all included.
2. Musamdam day cruise. A few hours drive from resoft to location via Oman border and the passport control. Once there we boarded a dhow boat and set off, dont foget your swimsuits, snorkle and masks are provided for the two stops. Lunch was lovely and soft drinks were plentyfull. Take your suncream as it gets very hot. Take cameras and recorders for the dolphins if you are luck enough to havr them join you!
This was a great two week family holiday, my 4 year old loved it. There is no entertainment for kids so ensure you have stuff for them to do in the evenings, but there is more than enough pools for them and you to enjoy here. If you like this review please rate it, thanks.
- Official Description (provided by the hotel):
- Hilton Ras Al Khaimah Resort & Spa is nestled in a spectacular private bay at the northern tip of the Arabian Peninsula, overlooking the shimmering waters of the Arabian Gulf. Only a 70-minute drive to Dubai International Airport and two hours from Abu Dhabi International Airport. Guestrooms: The Resort consists of 151 guestrooms in villas and 324 guestrooms in the hotel, each with a private balcony and exclusive access to the beautiful 1.5 km sandy beach. All guestrooms are fully equipped with a Plasma TV, minibar, alarm clock, coffee facilities, safe box, iron with board, hair dryer and wired internet (fee). Dining: With 11 restaurants and bars to choose from, guests will be spoilt for choice. Start the day with Hilton Breakfast in the bright and spacious Maarid, then stop by BN Cafe for your favourite coffee, or swim up to Sunset Bar to grab your favourite cocktail. Begin your evening with a traditional Middle Eastern shisha in Al Bahar Rooftop, before contemplating whether to try South American cuisine in Pura Vida or classic Italian fine dining at Piaceri da Gustare. At night, spend great times at the classic X.O. bar serving cognac and vintage ports. Leisure: The Spa features 9 treatment rooms, hamams, Jacuzzis, saunas and steam rooms. For the gym enthusiasts, there is a Hilton fitness by Precor center with aerobics rooms. Or take part in water sports with our on-site diving center offering snorkeling, water skiing, sailing and jet skiing. The perfect end to a day is a swim in one of our six swimming pools; including a salt water pool and a shaded pool for children. An onsite Kid's Club with indoor and outdoor playrooms and a Teens Club will keep the children and teens busy whilst you relax in the best resort in Ras Al Khaimah. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Hilton EMEA, Hotels.com, Priceline, Booking.com, Expedia, Orbitz, Cancelon, getaroom.com, HotelQuickly, Cheap Tickets, TripOnline SA, Travelocity and Hotwire so you can book your Hilton Ras Al Khaimah Resort & Spa reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Hilton Ras Al Khaimah Resort And Spa
- Hilton Ras Al Khaimah Hotel Ras Al Khaimah
- Ras Al Khaimah Hilton


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Monday, February 21, 2011
The libtards have no clue
by AlexinCT
My favorite was the quote at the end about Walmart od Disney teaching kids. I do not know about Disney, but I bet Walmart would do a better job than these clueless morons.
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Mortgage Website
Budget $750-1500 USD
We are a small start-up mortgage company located in Marin County, CA. We're looking for a web designer / programmer to build a mortgage website. We believe the site could be built on Word Press but we are open to any other platform that will do the best job for this application. Other than the basics (e.g. Home page, About Us, Our Products, Contact Us, etc.), the site should have the following features: 1. Investment Opportunities tab - Where we can post / upload "Investment Opportunities" - consisting of formatted text, photos and/or PDF documents. 1.a. The individual investment opportunities depicted under this tab, should have a "select" check box and link to some sort of a "shopping cart" where the Investor can fill out the amount they'd like to invest. The investors will be able to see some of the details of the investment opportunities on the main listing page, however, will have to register or login before they see all of the details of the individual investment opportunities. Investor registration should be verified by a reply to an automated confirmation email 2. Loan Application Form (on a separate tab and (1st page only, 4-6 fields) on the home page - where applicants can fill out a loan application form. The information captured will be emailed to us and captured in a database. Borrowers will be sent a confirmation email which they'll have to reply to in order to validate their request. Both Investors and borrowers should be able to login to the website and view activities in their accounts. 2.a. After validating the loan application form, applicants will receive the proposed loan rate and terms, which will be automatically calculated based on the information they had submitted on the application form. 3. "Rates & terms" tab which we should be able to update daily. The back end of the rate and term is an engine that requires some simple mathematical formulas (which we can provide) before the rates are posted
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BLAUPUNKT TARRAGONA MP14 Parts Catalog
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iPhone 7 Has Finally Been Released And These Are The Features You Should Know...
#11 The Antenna Design
iPhone 7 is here and we just cannot keep calm. We know how crazy this world can get over any product launch by Apple. iPhone 7 was no different. Here we tell you about all the new features and changes in this rich man's phone.
There are slight changes in the new antenna design. iPhone 7 and iPhone 7 Plus got a minor change of Antenna and Button gaps. Silent buttons and volume rockers are still the same though.
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Be it water or dust! Nothing can spoil the fun of using the new iPhone 7. Take it to the resorts or use it in desert, no harm whatsoever.
Keep reading ahead to see more features in iPhone 7.
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Apple's trademark home button is in a much better condition now. It is re-engineered, solid state - pressure sensitive and working with the new Haptic Engine vibrator.
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#6 Double In iPhone 7 Plus
Just double everything in iPhone7 Plus. It has got optical 2x zoom, and up to 10x digital zoom. It'll also work in the new Portrait camera mode.
#5 Pro Features
In iPhone 7 Plus's camera, you have pro features. It is possibly the best smartphone camera till date.
#4 The Volume
You know how good are the speakers in iPhone 6? Exactly double the quality. Yes, brilliant!
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#2 Battery Life
This is easily the longest battery life ever in any iPhone!
#1 Expected Price
The expected price of iPhone 7 is $649.
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Universal Martial Arts Centers - Palm Desert
KRAV MAGA 6 Week Challenge
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This 6 Week Challenge is an introductory class to Krav Maga, but it focuses more on the fitness aspect of Krav Maga. We teach some striking and basic self-defense techniques during the cycle, but it is mostly geared towards fitness and conditioning. We wanted to create a class that instroduced women to our Self-Defense program without the aggressiveness of such and effective self-defense system, thus eliminating the intimidation factor and creating a cycle of mind building, body strengthening, personal challenge.
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For the Home
Get crates at Michaels, put them together and stain them.
DIY Slide Out Drawers
Would love to do this with kiddos!
The Family Handyman site...good stuff
Old Window: chalkboard paint on 1 side with vinyl lettering ... Maybe photo collage in other side? http://www.walldecorplusmore.com/every-family-has-a-story-to-tell-welcome-to-ours-wall-sticker/
Picture of DIY attic storage assistance http://www.instructables.com/id/DIY-attic-storage-assistance/
All we did, to adhere it to the mirror, was slap some liquid nails on the back and stick it up there, with some more trusty tape, to hold up the frame, till it dried.
Built-in for office space.
Sugar Kettle Water Feature
the tutorial on how to make a custom looking frame around your bigger then life mirror your builder put up in the bathroom.


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Hello people, our latest update coloring sheet that you can have a great time with is The Autobots Transformers Coloring Page, posted on Transformers category. This particullar coloring sheet height & width is around 600 pixel x 436 pixel with approximate file size for around 68.01 Kilobytes.
For even more sheet relevant to the picture right above you, you can check out the next Related Images section on the bottom of the webpage or simply searching by category. You may find many other fascinating coloring picture to obtain. Enjoy the fun!


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Category: On-Screen
Jessie Paege Keeps It Real & Shares About Her New Projects, Her Sock Line & More
Learn why YouTuber Jessie Paege is committed to keeping it real with her fans.Read More
'Descendants 2' Drops July 21 and We Can't Get Enough of the Cast
The cast shares their favorite behind the scenes moments ahead of the premiere!Read More
Chachi Gonzales Gives a Glimpse into 'Chachi's World' Season 3
Chachi Gonzales talks upcoming projects, her boyfriend and more!Read More
'The Thundermans' Celebrates 100 Episodes
'The Thundermans' cast celebrated their 100th episode in serious style!Read More
The Style Evolution of 'Pretty Little Liars'
The finale of PLL is upon us. The show has made us laugh, cry and practically jump out of our...Read More
Isabela Moner Talks 'Transformers' Plus On Set Bloopers, Mark Wahlberg & More
Our June/July cover girl Isabela Moner is about to have a major breakout moment with the release...Read More
The Cast Of Disney's 'Descendants 2' Chooses Cast Mates to Compete in Reality TV Competitions
The cast of Disney's "Descendants 2" keeps things light by playing a game with their castmates!Read More
Meet Juliette Brindak, The Brains Behind YouTube Red's 'Hyperlinked'
Imagine having a television show based on your own life. Who would play you, and who would you...Read More
Hunter March's 'TBH' is A Must-Read for Summer
YouTuber Hunter March is getting real with old and new fans alike in his new book TBH: 51 True...Read More
The Perfect Outfit to Channel Your Inner Wonder Woman
Let's be honest, there isn't a female superhero who has as much girl power as the OG:...Read More


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SONY KV-36XBR250 Owner's Manual
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Hi, The manual is in perfect condition. Thank you very much.. You are doing an excellent job. Thanks again...
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Thank you very much. The best party that I met. The excellent quality. I recommend to all. This is my first purchase in this company. For sure not the last! Cheers from Poland!
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Complete service manual and easy to read,Lots of good info in this one
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Text excerpt from page 24 (click to view)
Using the Wireless Headphones
Setting Up the Headphones
Install the supplied size AA (R6) battery into the headphones.
Using the Headphones
Tip z
For optimal sound reception, do not cover the infrared transmitter on the TV or the infrared sensors on the headphones.
1 Open the battery compartment lid by
pressing and sliding the lid as illustrated.
Infrared sensors
1 Press
. The 2 icon and channel number are displayed.
Infrared transmitter
2 Lift the cover and insert the battery into
the compartment with the positive side up and then close the lid.
2 Turn the power on by placing the
headphones securely on your head.
Power indicator
Notes � To help prevent possible hearing damage due to sudden or prolonged excessive volume, do not set the headphone volume too high while using them. � To prevent possible damage to the infrared transmitter in the television, please press the to turn off the headphone feature when the headphones are not in use.
� When used continuously, the battery will last: � up to 40 hours with alkaline batteries. � up to 20 hours with manganese batteries. � Replace the battery with a new one when the sound becomes weak.
Volume control
If you only want to listen to the sound from the cordless headphones, turn down the TV speaker volume, or press MUTING. To turn off the headphones, remove them from your head and press .
We provide manuals to wide range of devices, including:
Headphones, Woofer, Stereo Double Cassette Deck, Refrigerators, Ckd-products (freestanding Refrigerators), Battery, Freestanding Fridge-freezers Bottom Freezers, Nofrost, Radio Cassette Re, Monitor, Cassette Player With Fm/mw/lw Tuner, Flat Tv Stands, Auxiliary Stoves, Coal, Cd Receivers, Old Radio, Md Receiver, Food Processor, Digital Camcorder, Corded Internet Phones, Body Grooming, Router, Guitar, Logic Analysis System, Dvd/cd Receiver, Freestanding Table Top Freezers < 90 Cm Height, Selector, Dry Vacuum Cleaners, Pocket Computer B, Portable Grand, Mex, Tv Receiver, Dry Shaving
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We can now offer any of the Superhauler trucks models, complete with your choice of name!
As a member of our official club, you already have the opportunity to name a truck - now you can have the model to match!
It's a tradition of ours to give the vehicles in our fleet female names, with only a few exceptions to the rule over the years. However we're throwing the rule book out of the window on this product and allowing you to name your truck anything you like!
Please note messages are limited to 16 characters including spaces and can take an additional 5 working days on standard delivery times.
Please note - the trucks are personalised using a small sticky label with the name printed.


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Category Archives: Uncategorized
Friday Night Flicks: Baikei, Wang Dongling, and Yoshinori Kikizawa
I showed you a Friday Night Flick with some large scale calligraffiti earlier this spring. Tonight's first two flicks are examples of large scale brush work by two Asian masters of calligraphy: Baikei and Wang Dongling. The … Continue reading
Posted in book arts, Uncategorized Tagged book arts, calligraphy, lettering Leave a comment
Friday Night Flicks: More Monotype
It is almost two years since I posted some of Monotype's promotional videos. Today I have two more: a short short video promoting a new type face called SST and a longer one about the development of the updated Johnston … Continue reading
Posted in Design, typography, Uncategorized Tagged design, typography Leave a comment
Friday Night Flicks: The Montreal Signs Project
Last Sunday's post mentioned lettering and calligraphy so this video seems a logical follow-up. Professor Matt Soar, Communications professor at Concordia University, launched the Montreal Signs Project in collaboration with Archivist Emerita Nancy Marrelli. He gives us a short tour of … Continue reading
Friday Night Flicks: 1363, the End of Chivalry
Tonight's short video by Jake Mahaffy has absolutely nothing to do with either books or animation, my usual requirements for inclusion. It did, however, make both David and me laugh, and has won audience awards at several film festivals including Sundance and … Continue reading
Posted in Uncategorized 2 Comments
Almost Done
I am actually off at another CBBAG Saskatchewan Day in Davidson but have scheduled this post so my dedicated readers will not be disappointed. I am in the final stages of my entry for this year's jurying of the Dimensions exhibition. … Continue reading
Friday Night Flicks: Paper Dreams
Paper Dreams was produced by Kenneth Onulak as a Junior Degree Project while he was a student at Rhode Island School of Design. There was an actual piece of paper involved: a photograph of a sheet of paper in a frame was the … Continue reading
Friday Night Flicks: The House Hippo and The Bin Chicken
A number of years ago the Concerned Children's Advertisers produced a public service announcement warning of the dangers of believing everything you see on TV. It was a brilliant parody of the Hinterland Who's Who series on Canadian wildlife. I … Continue reading
Posted in Uncategorized Tagged animation 6 Comments
Bonus Post: The International Edible Book Festival 2017
It's April 1 again, time for The International Edible Book Festival, and once again I have not made an edible book. Instead here is Charlie and The Chocolate Factory as re-imagined by Cake Central. Be sure to check out events … Continue reading
Posted in Uncategorized Tagged edible books 2 Comments
Friday Night Flicks: Rock Band
I was delighted by this recently created musical instrument, and thought the song being played was seasonally appropriate, at least in the Northern Hemisphere. I hope you enjoy Rock Band by Neil Mendoza as much as I did. Perhaps … Continue reading
Posted in Uncategorized Tagged Neil Mendoza, Rube Goldberg Leave a comment
Happy Pi Day 2017
It's once again the day to celebrate π. Last year I gave you a little artist's book (and a recipe for cabbage pie) to make for yourself. This year I found a suitable piece of music on Will Wheaton's Tumblr. If … Continue reading
Posted in Uncategorized Tagged Pi Day 2 Comments


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June 4, 2017
WXMLer Will Dana wins Dean's Medal
WXMLer Will Dana has been awarded the Dean's Medal in the natural sciences. Will worked with Doug Lind and Jim Morrow on understanding Limiting Behavior in Random Graphs. Will Dana will be going to grad school at Michigan next year.
May 30, 2017
WXML Open House
Our Spring 2017 WXML Open House was held Thursday, June 1, from 5-6pm, in Odegaard 136. We had great attendance, as many folks from across campus and beyond came and saw what wonderful research our students have done, ranging from modeling cancer cells to understanding the sounds that various number sequences make. .
May 12, 2017
Crosspost: Matching for Elementary Students
Annie Raymond (from our UW Maths department) gave a beautiful talk to fourth and fifth graders at on the marriage problem. Benjamin Leis was there, and wrote a blog post about it (with pictures!). Benjamin's blog is about his experience running a math club for elementary schools students, and anyone who is interested in his experience, or…


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MPSC Forest Service Answer Key 2017 – Get here answer keys of Maharashtra Forest Service Exam 2017. Maharashtra Public Service Commission is issued the Forest Service answer keys 2017. It contains correct answer option of all questions asked for all question paper sets. In the event that any question is found to be incorrect, it is cancelled and same is indicated in official MPSC Forest Service Answer Key 2017.
Test takers can use keys to know answers and estimate marks scored. Final keys are used to evaluate papers to declare result of MPSC Forest Service 2017 for preliminary and mains.
MPSC Forest Service Answer Key 2017
When we talk about the uses of Answer Key of MPSC Forest Service 2017, there are many important ones that can be considered. The answer key is a document showing correct answers to the questions that were asked in the exam paper. These are released so that candidates can refer to them to know about the correct answers to all the questions they could not understand.
- With the help of answer key, the candidates can come to know about the correct answers to all the questions.
- By using the answer key, candidates can calculate an estimated score that they are most likely to obtain in the final result of the exam.
- Using this estimate, the candidates can have an idea of where they stand in the vast competition.
- Also, the candidates can plan about further selection round or can have an idea of where they are going to be posted, when they have a rough idea of the score.
MPSC Forest Service Answer Key 2017 (Prelims)
The prelims exam was held on 04 Jun 2017. The official answer key has been released on 05 Jun 2017. Answer Key of MPSC 2017 Forest Service is given below.
Check MPSC Pre Answer Key 2017 here – Click here to check answer key of Maharashtra MPSC Forest Service (Pre) Examination 2017.
After checking answer keys, go through details of MPSC Forest Service Result 2017.
Answer Key Notice
Maharashtra MPSC Forest Service Examination 2017 Answer Key
The MPSC Forest Service Answer Key 2017 will be published by the exam officials after the exam has been conducted. Since the exam is held in two phases i.e. the prelims and the mains, the answer key for both of them are released separately.
Get here more information about the MPSC Forest Service Answer Key 2017 for prelims and mains.
MPSC Forest Service Answer Key 2016
Check MPSC Mains Answer Key 2016
- Click here to check the revised Paper I Answer Key
- Click here to check the revised Paper II Answer Key
Check MPSC Prelims Answer Key 2016


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B cell receptor
Protein phosphatase 2A
Ulex europaeus agglutinin-1
T cell-specific gene targeting reveals that α4 is required for early T cell development
α4-mediated signaling is involved in a variety of functions in mammalian cells. To determine whether this is true for immunocompetent cells, we generated mutant (Lck-α4–) micein which the α4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice showed impaired early T cell development. Thymi at most ages were small and their architecturewas disorganized. This defect was not due to increased thymocyte apoptosis but to decreased cell proliferation. T cell development was found to be severely arrested at the CD4/CD8 double-negative 3stage and the thymus contained very few double-positive or single-positive (SP) mature thymocytes. The mutant thymocytes showed impaired proliferative responses to anti-CD3 monoclonal antibody (mAb) stimulation or to the cytokines IL-2, IL-1 or TNF. In the spleen, the numbers of mature SP T cells were decreased and their proliferative responses to anti-CD3 plus IL-2 or to anti-CD28 mAb were impaired. A severe impairment of CD3-induced expression of CD25 was also observed. These data suggest that α4 plays a critical role in the proliferation of thymocytes, which is necessary for early T cell development.
1 Introduction
α4 was initially identified as a signal transduction molecule that associates with the Igα component of the B cell receptor (BCR) complex 1, 2. Thatα4 transiently associates with a tyrosine-phosphorylated molecule after BCR cross-linking suggested its involvement in BCR-mediated signal transduction. Alpha4 associates with the catalytic subunit of protein phosphatase 2A (PP2A) and enhances its catalytic activity 3. This association was disrupted by treating cells with the immunosuppressant rapamycin, which inhibits the proliferation of lymphocytes by decreasing PP2A activity. Alpha4 homologues are highly conserved in evolution in yeast as well as among mammalian species 3–6.Mutation of the yeast homologue Tap42 is lethal due to impaired signal transduction through the rapamycin-sensitive Tor pathway, and the same is true for α4 in the mouse.
For complete understanding the mTor/α4-associated pathway, it is necessary to determine the role of α4 in immune-competent cells. Global deletion of α4 gene in all tissues revealedthat α4 is essential for mouse development. Initially, we deleted the α4 gene specifically in B cells using the Cre/loxP system 7. CD19-α4– mice had reduced numbers of splenic B cells and bone marrow pre-B cells. The proliferative response of α4-deficient B cells to different stimuli including BCR was impaired and rapamycin sensitivity was decreased. These results demonstrated that α4 is a pivotal signal transduction molecule involved in BCR-mediated signaling and that it is involved in the rapamycin-sensitive signal transduction pathway.
While the Ag recognition mechanism of T cells is clearly different from that of B cells (because T cells recognize peptides from degraded proteins which are bound to MHC molecules), T and B cells do show certain similarities in Ag receptor signal transduction. The CD3 complex is necessary for TCR expression as are CD79α and CD79β for the BCR 8–11]. Dynamics of T and B cell responses are regulated by the similar cellular mechanisms yielding intermediates, effectors, and memory cells [12–15. Similar molecules are involved in signal transduction through the Ag receptor in both types of cells; for example, adaptor molecules such as SLP76 and BLINK, and src- and Syk-type tyrosine kinases 16–19.
However, the immunosuppressant rapamycin apparently exerts different effects on the different cell types 20. In particular, rapamycin was initially reported to block T cell proliferation rather by inhibiting IL-2-induced signaling 21, 22. However, according to data from α4-deficient B cells, in which primarily the BCR-mediated signal transduction was impaired, we hypothesized that α4 plays an important role in TCR-mediated signal transduction. To determine the role of α4 in T cells, we established a mutant mouse in which the α4 gene was specifically deleted only in T cells (Lck-α4– mice). Lck-α4– mice had reduced numbers of thymocytes and splenic T cells. The number of immature double-negative (DN) thymocytes was reduced and the proliferative response to CD3 stimulation was also impaired. These results show that the α4-mediated signal transduction plays an important role in early T cell development as well as for mature T cell function.
2 Results
2.1 Lck-Cre/flox-α4 mice
Mice with α4 conditionally targeted in T cells only (Lck-α4– mice) were created by mating α4-floxed females with male transgenics expressing Cre under the control of the mouse Lck promoter 23. The mutant mice showed normal development but marked impairment of thymocyte production (Table 1). The number of thymocytes was less than two million per mouse, averaging only 4.3% of the number in wild-type (WT) littermates. Expression of α4 was not detected by Western blot analysis of thymocytes from Lck-α4– mice (Fig. 1a), and this effect was extended to the peripheral spleen T cells in comparison with B cells of both WT and mutant mice (Fig. 1b). The extent of α4 deletion was shown (as estimated less than 5% remained) by reverse transcription (RT)-PCR analysis of DN3 thymocytes in comparison with WT littermates (Fig. 1c). The results demonstrate that T cell development in the thymus is severely impaired in Lck-α4– mice. However, a substantial number of T cells could be found in the spleen of Lck-α4– mice (30% of the number in WT littermates).
2.2 T cell development in the thymus
The thymi of Lck-α4– mice contained a higher percentage (but reduced numbers) of immature DN cells (92.3%) compared to control littermates (2.5%) (Fig. 2a). The numbers and percentages of double-positive (DP) and single-positive (SP) thymocytes were markedly reduced in Lck-α4– mice (1.2% versus 86.7% of WT littermates for DP and 6.5% versus 10.8% for SP cells). These results suggest that T cell differentiation from DN to DP was impaired in Lck-α4– mice. To determine more precisely at which stage T cell development was impaired in the thymus of Lck-α4– mice, DN thymocytes were analyzed for expression of CD25 and CD44, which define distinct immature thymocyte subsets 24, 25. The earliest subset is CD44+CD25– (DN1). Subsequent subsets are defined as CD44+CD25+ (DN2), CD44–CD25+ (DN3), and CD44–CD25– (DN4). In Lck-α4– mice, the proportion of the DN3 population was increased (78.3% versus 55.6% of WT mice) and the DN4 population was decreased (14.1% versus 29.6% of WT mice), indicating that DN differentiation is blocked at the DN3 stage (Fig. 2a). Regardless of this impairment of thymus cell differentiation, there were some mature CD3+ T cells in the spleen (5.9% versus 22.5% of WT mice; Fig. 2b), with the comparable ratio of CD4 and CD8 SP cells.
We further investigated T cell development in Lck-α4– mice by immunohistochemical analysis of the thymus. Hematoxylin/eosin (H/E) staining of control littermate thymi revealed a clear demarcation between the cortex and the medulla (Fig. 3a). However, the thymi of Lck-α4– mice maintained no clear distinction between the cortex and the medulla. Because the fucose-binding lectin Ulex europaeus agglutinin-1 (UEA-1) reacts with thymic medullary epithelial cells, thymic sections were double-stained with TCRVβ and UEA-1 to visualize the cortico-medullary junction (Fig. 3b). Thymi of control littermates showed a clear distinction between TCRVβ-positive and UEA-1-positive regions. Thymi from Lck-α4– mice possessed a few UEA-1-positive or TCRVβ-positive cells.
CD4/CD8 double staining of thymic sections showed that the cortex contained mostly DP cells in the control littermates, while the thymus of Lck-α4– animals contained almost no DP cells (Fig. 3c). This is in agreement with the flow cytometric analysis, which indicated the presence of a small number of DP thymocyte in Lck-α4– mice (Fig. 2a). Next, the DN thymocyte population was analyzed by CD25/CD117 double staining. CD117 is normally expressed on DN1 and DN2 cells in much the same way as CD44. CD117-positive and CD25-positive cells were detected brightly at the thymic periphery in control littermate thymi (Fig. 3d), but this population was not detected in Lck-α4– mice.
2.3 Proliferative capacity of α4-deficient thymocytes
There are two possibilities to explain the reduced number of thymocytes in Lck-α4– mice: decreased proliferation and increased apoptosis. We first examined the proliferation of thymocytes by the BrdU method (Fig. 4a). In the thymus from control littermates, most proliferating cells were DP, CD25-negative, BrdU-positive cortical cells. In contrast, in Lck-α4– mice, only a small number of BrdU-positive cells were observed. Thymocyte apoptosis was visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method (Fig. 4b). Similar numbers of TUNEL-positive cells were found in Lck-α4– mice and littermate controls, indicating that apoptosis was not enhanced in the thymus of the Lck-α4– animals. These results suggest that impairment of proliferation rather than increased apoptosis was responsible for the decreased number of thymocytes in Lck-α4– mice.
To further investigate the proliferative response of α4-deficient thymocytes, we cultured total thymocytes in the presence of different stimuli (Fig. 5a). Thymocytes from Lck-α4– mice showed severe impairment of proliferation induced by anti-CD3 mAb or concanavalin A, presumably due to the fewer SP thymocytes in the mutant mice. The DN cells of the mutant mice, containing γ δ T cells (14.1% versus 8.1% of WT mice; Fig. 5b), showed a decrease of the proliferation after stimulation with IL-2, IL-2 + IL-1β, and IL-2 + TNF (Fig. 5c). These results confirmed that impairment of proliferation was a major contributor to disrupted T cell development.
2.4 Splenic T cells in Lck-α4– mice show impaired CD3 signaling
There are substantial numbers of T cells (30% of control littermates) in the spleen of Lck-α4– mice (Table 1 and Fig. 2b). To examine the molecular mechanism of impaired thymocyte proliferation, splenic T cells were stimulated with anti-CD3 mAb in vitro (because only the spleen contained a sufficient amount of cells to analyze) (Fig. 6). Alpha4-deficient B cells showed severe impairment of cell proliferation induced by anti-BCR cross-linking 7. However, the α4-deficient T cells responded well to anti-CD3 cross-linking, albeit slightly reduced in comparison with the response of control littermates. Addition of IL-2 or anti-CD28 mAb as costimulators, together with anti-CD3 cross-linking, did not result in an increase of T cell proliferation to the level seen in control littermates. Because higher doses of IL-2 failed to enhance anti-CD3-induced proliferation, we investigated whether the IL-2 receptor was up-regulated after anti-CD3 stimulation of splenic T cells. As shown in Fig. 7, anti-CD3 did not induce IL-2R expression by splenic T cells from Lck-α4– mice. On the other hand, anti-CD3 stimulation did induce CD28 expression, although also to a lesser degree than in control littermates. These results suggest that CD3 signal transduction pathways leading to IL-2 receptor expression are impaired in Lck-α4– mice and therefore T cells cannot be optimally activated.
3 Discussion
We developed mutant mice in which the α4 gene was deleted in a T cell-specific manner using the Cre/loxP system. These mice had impaired T cell development in the thymus and impaired CD3 signal transduction, demonstrating that α4 plays an important role in T cell immunity.
In Lck-α4– mice, the proliferative response to different stimuli was compromised, as was also observed previously in CD19-α4– mice. However, the pattern of defects was not identical in the two types of mutant mice. In Lck-α4– mice, CD3-induced proliferation of splenic T cells was not greatly impaired, but in contrast, BCR-induced proliferation of splenic B cells from CD19-α4– mice was dramatically reduced. In CD19-α4– mice, α4-deficient B cells showed a less drastic impairment of the response to anti-CD40, LPS or IL-4 costimulation. In Lck-α4– mice, however, anti-CD28 or IL-2 second-signal-induced proliferation was completely abrogated. This defect was due to impaired CD3-induced CD25 expressionrather than compromised second-signal transduction. The anti-CD28-induced proliferation signal was possibly also compromised via the failure of CD25 up-regulation. Therefore, α4 is required in CD3-induced TCR signaling as is also the case for BCR-induced signaling, indicating a critical role of α4 in Ag receptor signaling. The results obtained with thymocytes indicate that cytokine-induced signaling is also compromised in Lck-α4– mice. However, it does not follow that α4 is involved only in general proliferation mechanisms, because CD3-induced events, such as CD25 up-regulation, were impaired, but CD28 up-regulation or thymidine uptake was not.
In the peripheral lymphoid organs, the decrease in number of T cells was not as drastic as in the thymus and there were substantial numbers of T cells in the spleen. Because γ/δ-positive T cells can develop extrathymically 26, we tested whether these splenic T cells were γ/δ-positive. However, most of the T cells in the spleen were α β-positive (data not shown); this is in accordance with the finding that these T cells were CD4 or CD8 SP cells.
Defined cortical and medullary zones were absent and most of the cellular components of the thymus, i.e. DP and SP cells, were also drastically decreased in Lck-α4– mice.Thymic epithelial cells in the medulla defined by UEA-1 positivity were essentially unidentifiable. Therefore, factors produced by these thymocytes and the interdependent nonlymphoid thymic stroma are missing in the Lck-α4– thymus. Intercellular interactions between precursor cells and thymic stroma are critical for the proliferation and differentiation of thymocytes in the normal thymus 27. It is therefore possible that gross disruption of thymic architecture and composition results in secondary effects on the proliferation of residual thymocytes. We speculate that accumulation of T cells that have survived the thymic developmental stage could be maintained in the splenic environment and account for recovery of the peripheral T cell number.
The Lck-α4– mice maintained a markedly reduced number of T cells during thymocyte development, which is quite different from the case of CD19-α4– mice. CD19-α4– mice had a decreased number of cells both in the spleen and bone marrow, but the decrease was less severe in the latter. The CD19 promoter becomes active in the pre-B cell stage in the bone marrow and its activity increases later in development, which is most pronounced in the spleen 28. On the other hand, the Lck proximal promoter becomes active between the DN2 and DN3 stage and is most active in the thymus 23. This may be one reason why the α4 deficiency phenotype was manifested at an earlier stage in the T cell-specific as opposed to the B cell-specific gene targeting system.
Another possibility is that α4 is involved in the pre-TCR signal transduction. The signal transduction pathways of pre-TCR and mature TCR share many similarities 29, 30. Genetic inactivation of many different genes has been shown to cause defects in T cell development, especially at the DN3 stage, characterized by a CD44-negative and CD25-positive phenotype. It is at this stage that the pre-TCR complex is first expressed, and is believed to exert its function in a subset of DN3 cells 31. The DN3-to-DN4 transition requires signals from the pre-TCR complex. Targeted disruption of genes such as TCRβ, RAG1 and RAG2, CD3 subunits, Lck or CD45 resulted in blockade of T cell development at this stage 32. In the case of our mice, α4 gene deletion is regulated by the Lck proximal promoter, which is activated at an early stage of T cell development, between the DN2 and DN4 stages 23. The DN3-to-DN4 transition is impaired in Lck-α4– mice, which might suggest that α4 is required for pre-TCR signal transduction.
4 Materials and methods
4.1 Antibodies and reagents
Anti-mouse α4 Ab was prepared as previously described 2. Anti-PP2Ac Ab was purchased from Upstate Biotechnology Inc (Lake Placid, NY). Phycoerythrin (PE)-B220, PE-CD8, PE-CD25, PE-anti-TCRδ, biotin-CD3, biotin-CD44, fluorescein isothiocyanate (FITC)-CD4 and FITC-CD28 Ab were purchased from BD PharMingen (San Diego, CA) for flow cytometric analysis. FITC-TCRV β, FITC-CD8, FITC-CD25, biotin-UEA-1, biotin-CD4 and biotin-CD117 were purchased from BD PharMingen for immunohistochemistry. Streptavidin-Cy3 was purchased from Molecular Probes (Eugene, OR). CD3mAb and CD28 mAb were purchased from BD PharMingen for proliferation assays. IL-2 was a kind gift from Dr. Hamuro (Ajinomoto, Japan). IL-1β and TNF were purchased from Sigma Aldrich Japan (Tokyo, Japan).
4.2 RT-PCR analysis
To determine the extent of α4 deletion at various thymocyte stages, we carried out RT-PCR analysis using specific primers for α4. Poly(A) RNA from sorted DN3 thymocytes was isolated using the MicroPoly (A) Pure from Ambion (Austin, TX). The cDNA was synthesized with the GeneAmp RNA PCR kit (Applied Biosystems Japan Ltd., Tokyo, Japan). The primers for α4 are 5′-ATGGCAGCGTCTGAAGAC-3′ and 5′-TGATAGCAATGACACTGA-3′; and for β–actin 5′-GTTGCTATCCAGGCTGTGCT-3′ and 5′-CGGATGTCCACGTCACACTT-3′. Each PCR cycle consisted of denaturation for 1 min at 94°C, annealing for 1 min at 60°C and extension for 3 min at 72°C. After 35 cycles of amplification, PCR products were analyzed by electrophoresis on 1.5% agarose gels stained with ethidium bromide.
4.3 Western blot analysis
Cells were lysed in lysis buffer containing 1% Nonidet-P40, 150 mM NaCl, 10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 0.05% NaN3, 100 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, and10 μg/ml aprotinin. The lysates were centrifuged for 5 min at 12,000×g at 4°C to remove nuclei. Proteins were separated by SDS-PAGE and were transferred onto nitrocellulose filters by electroblotting. PP2Ac and α4 were detected by anti-PP2Ac and anti-α4 Ab at a dilution of 1:1,000. The blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer's protocol.
4.4 Flow cytometric analysis and sorting
Cell surface markers were analyzed with mAb by flow cytometry. Single-cell suspensions of lymphoid tissues were incubated with a saturating amount of each mAb conjugated either to fluorochromeor to biotin. Two-color analyses were performed with the fluorochromes FITC and PE or with Red670 and PE. Lymphoid cell populations were analyzed after gating by forward and side scatter on a FACScan using CellQuest software (BD Biosciences, San Jose, CA). For analysis of DN cell population, thymocytes were first purified with the mixed beads conjugated with anti-CD4, anti-CD8 and anti-B220 mAb (Dynal, Oslo, Norway). DN3 cell acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences).
4.5 Mice
Mice carrying three identically-oriented loxP sites in the α4 gene (α4-flox) have previously been reported 7. Lck-Cre transgenic mice, in which the Cre gene is underthe control of the p56lck proximal promoter, were as described 23.
4.6 In-situ proliferation and apoptosis assays on thymocytes
Mice were injected intraperitoneally with BrdU (1 mg in 200 μl of PBS), and thymi were taken 6 h after injection. Thymus tissue sections (6 mm thick) were prepared and immunohistochemical analysis was performed as previously described 7. BrdU staining was done with anti-5–2′-deoxyuridine mAb (Amersham Pharmacia Biotech) and counterstained with Alexa Fluor 546(Molecular Probes) and FITC-CD25. Double-stained sections were analyzed using a confocal laser scanning microscope (FV300, Olympus, Japan). TUNEL assays in the thymus were carried out using an in-situ cell death detection kit (Roche, Indianapolis, IN). Thymus tissues were fixed in 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.1% Triton X-100, 0.1% sodium citrate for 2 min at 4°C. The tissues were then incubated with the solution containing TdT enzyme and FITC-labeled dUTP. After incubation for 60 min at 37°C, the samples were analyzed using confocal laser scanning microscope as above.
4.7 In-vitro cell proliferation assay
Single-cell suspensions of splenocytes were treated with 0.1 M ammonium chloride solution to lyse red blood cells and then incubated at 37°C for 1 h to allow macrophages to adhere. Nonadherentcells were recovered and incubated with Dynabeads anti-mouse B220 (Dynal) to remove B cells by magnetic sorting. More than 90% of the purified cells were positive for the CD3 marker. For proliferation assays, purified T cells were cultured at 1×105/well in 96-well microtiter plates with or without stimulation in 200 μl of RPMI 1640 culture medium containing 10% heat-inactivated fetal calf serum, 2 mM LL-glutamine, and 5×10–5 M 2-mercaptoethanol. After 2 days, cells were pulsed with 0.5 μCi=18.5 kBq of [3H]thymidine/well (Amersham Pharmacia Biotech) for the last 16 h. Cells were harvested onto glass-fiber filters, and the incorporated radioactivity was measured.
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