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At the Nokia Connection event held in Singapore this week, Nokia demonstrated its commitment to harness the power of the internet and mobility to deliver compelling services and solutions to consumers. Nokia announced a number of devices with solutions that can be personalised to connect people in new and better ways.
In a keynote at the opening ceremony of the event, executive vice president and chief development officer at Nokia, Mary McDowell, said: "The Asia Pacific market has been an extremely important region for Nokia over the years and remains hugely important to Nokia. Asian consumers' eagerness to use technology makes this region a great place to introduce new devices and services. Singapore and many of its Southeast Asian neighbours represented here today will be among the first to take advantage of the future solutions Nokia intends to bring as we connect the planet so that everyone can participate."
At the event, Nokia unveiled the new Nokia E72, its latest full QWERTY smartphone which brings a rich mobile email and instant messaging experience right out of the box, making it a great device for people who want to be connected, and productive in both their personal and professional lives.
Building on the formula from the hugely successful Nokia E71, this latest arrival in Nokia's Eseries family maintains the essential elements of its predecessor, whilst still improving its capabilities in a number of areas. The Nokia E72 is expected to begin shipping late in the third quarter of 2009.
Nokia announced the Nokia 5530 XpressMusic, a compact XpressMusic device that uses a touch interface for accessing people and content on the brilliantly clear homescreen. The Nokia 5530 XpressMusic follows in the footsteps of the Nokia 5800 XpressMusic, expanding the touch XpressMusic phone range with a youthful device that has a solid stainless steel frame finish and comes in five vivid colours. The Nokia 5530 XpressMusic is expected to begin shipping in the third quarter of 2009.
Nokia also introduced the Nokia 3710 fold, a mobile device that balances eye catching design with the latest in mobile functionality. The compact Nokia 3710 fold adds a personal touch with an artistic pattern on the cover and comes with an appealing array of features in a renewed, but instantly familiar user interface. The Nokia 3710 fold is expected to be available in the fourth quarter of 2009.


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13 December 2016 introduction 1 min minute read
At Savision, we have created a Service Overview Widget for Live Maps. This widget allows you to create widgets that show a simplified version of the Live Maps Services Overview dashboard and group them under different categories. Check out our short video and learn more about the new Service Overview Widget for Live Maps in less than one minute.

subset: commoncrawl


text:
Have you receive Notices or letters from IRS about your bank account getting levy or wage garnishment? Exercise your right as a taxpayer, and explore the options that are available to you. You won't know unless you call today. CALL 973-200-2807 right now, the clock is ticking.....
We have dedicated our lives in providing services for our clients. We classify all of your business expenses so you don'd miss a deduction on your taxes.
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For the past 10 yrs, we have been developing strategies for both business and individual clients. And we have been working hard and long hours when they need my services as a EA —even after tax season, F.O.K TAX SERVICE Inc. never spend no days OFF. We make sure our clients have access to our service.
Sign up to hear from us.
Copyright © 2018 FOK Tax Services, Inc. - All Rights Reserved.


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Lifestyle store and event space brought to you by Selfish Mother, stocking a thoughtfully curated range of clothing, gifts, books and accessories for the family and home. Hang out and enjoy our giant Lego play area, vintage photo booth, Honesty Café and laid-back vibes … plus, keep an eye on our events calendar for ace happenings. See website Calendar for events 24 and 29 May, 1 June.
Wed 24 May Fmly Store: Crafty lock-in, 6–8pm. Adult-only evening of crafts and catch-ups – make a pompom garland to take home, and enjoy exclusive discounts. BYOB, we'll provide the nibbles. Tickets £7.50. https://events.thefmlystore.com/event/crafty-lock-in/
Mon 29 May Fmly Store: Special Monday opening to celebrate the Bruton Packhorse Fair. Enjoy a free mask-making session with Artist-in-Residence Polly Jeffrey, and real dairy ice cream from Lovingtons.
Thurs 1 June Crafty Thursdays at The FMLY Store. As it's half-term, we're extending our regular morning craft session to last all day – pop in any time before 3pm to make your very own dreamcatcher. FREE.


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Jack Weaver
Williamsport High Williamsport, MD Class of 1979
Memories of Jack Weaver
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Day 225 (Australia – Top End)
There is not really a cycle-friendly way into Darwin city (not one that I could find at least). I stuck to Stuart Highway for as long as I dared before seeking backroads with less traffic. These took me through Bees Creek and Virginia before I returned to the highway for a nightmarish stretch until I could veer into Palmerston. I took Tiger Brennan Drive despite the fact roadworks were in progress. For some of it I could ride on the safe side of orange cones, but for a lot of it I had to brave busy traffic. I eventually made it into an industrial area which took me close to the CBD, by which time I could rely on footpaths. I immediately found the waterfront and didn't hesitate to go for a cleansing swim. Back on the bike, I followed the water's edge to the esplanade, where I shot a quick video before finding my new home. I went to the H Hotel to pick up an envelope from reception containing a set of keys. I then found C2 apartments on the esplanade. I rode into the carpark, got into a lift and travelled up to level 5. I rolled into a pristine apartment overlooking the bay. It is my third-cousin's place and she is away for the weekend. I showered and settled in before going to the Tap bar for some sliders and a beer. I got some groceries at Woolworths, then returned home. I'm too exhausted right now to express how happy I feel with myself for having made it to another Australian capital city. Pretty happy though!
|Cousin's apartment
|Distance ridden today
|Average cycling speed
|Total distance ridden


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Authors /
Aya Ogawa
Aya OgawaAya Ogawa is a Brooklyn-based writer, director, translator and performer. As a playwright and director, her play Serendipity was the winner of the Kennedy Center/American College Theatre Festival and finalist at Humana Festival. Eating Dirt was produced at SoHo Rep, and she directed it for Theaters Against War at HERE. She directed her play A Girl of 16 in its world premiere, hailed by The New York Times for Aya's "stunning visual sense." She wrote and directed oph3lia (nominated for Outstanding Ensemble, New York Innovative Theater Awards) which the New York Times called "Compelling… Harrowing… Great theatre" and Backstage described as "riveting." Her Artifact was presented as a work-in-progress at the PRELUDE '07 Festival as well as in the PERFORMANCE MIX Festival. She is currently developing a new piece with the Foundry Theatre and Adhikaar, a human rights organization dedicated to serving the Nepali community in the U.S. She has been commissioned to translate numerous Japanese plays into English, including works by Pappa TARAHUMARA's Hiroshi Koike; Chong Wishin, Kunio Shimizu,Yoji Sakate, Kobo Abe, Takeshi Kawamura, Keralino Sandorovich and works by the Tokyo-based performance company YUBIWA Hotel. She has translated Five Days in March, Air Conditioner, Enjoy, Free Time and We Are the Undamaged Others by Toshiki Okada. Time Out New York called her translation of Enjoy "effortless, idiomatic translation (surely the process of rabbinical focus)" in its English language premiere production by The Play Company; and her translation of Five Days in March was hailed as "a miracle of transposed idiom" by the Village Voice in the production by Witness Relocation. Aya has performed nationally and internationally. She originated and performed numerous roles with the International WOW Company including JR Oppenheimer in The Bomb and the title role in Alice's Evidence; and Time Out NY hailed her performance as Chomsky in The Loneliness of Noam Chomsky by the Butane Group. International credits include her role in the world premiere of the award-winning Emperor and Kiss with Rinko-Gun Company, Tokyo; she was selected as part of the artist team representing the U.S. at the International Theater Institute's 2006 Congress in Manila, for which she co-created and performed in The Borges Project; and the European premiere of Young Jean Lee's Songs of the Dragon Flying to Heaven at the Vienna Festival. She also worked as a generative artist and performer in Ifdentity, a collaborative project produced by the International Theater Institute for its 2008 Congress in Madrid. In 2005, with her collaborators, she has formed a performance company called knife, inc. whose mission is to create works that explore the multiplicity of experience of living in an international context. The work is designed to investigate the moments of crisis, the collision of the past and the future. The company seeks a modern, international language of art. The company's work is a home for this search. (www.knifeinc.org) She was the recipient of an Artistic Fellowship at New York Theater Workshop (where she is now a Usual Suspect), Van Lier Fellowship at New Dramatists, and HERE Artist Residency. She is also a recipient of the Space Grant at Brooklyn Arts Exchange and two Swing Space grants at the LMCC (Lower Manhattan Cultural Council). She is a 2006 recipient of the Urban Artist Initiative Grant for Individual Artists administered by the Asian American Artist Alliance, a 2008 grantee of the NYSCA Individual Artist Theater Commissioning Grant and Axe-Houghton Foundation.
Perform Aya OgawaView all
The Sonic Life of a Giant Tortoise
Full Length Play, Dramatic Comedy
3m, 2f
Enjoy (Ogawa)
Full Length Play, Comedy
6m, 4f


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Kauai's sometimes treacherous beaches will be a little safer thanks to an inexpensive solution dreamed up by resident John Tyler. Lifeguards can't be everywhere, so rescue tubes will be placed at unguarded beaches. (Kauaiworld.com)
Home All News Arts & Leisure Making Kaua'i's Beaches a Little Safer One Tube at a Time


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The FIFA World Cup final between Argentina and Germany could herald a rift in the Catholic church, pitting Argentine-born Pope Francis against German Pope Emeritus Benedict XVI.
Pope Francis has almost surely been secretly rooting for Argentina, despite his pledge to keep his prayers neutral.
But Germany's unbelievable 7-1 rout of Brazil during yesterday's semi-final could make for an awkward final match for the Vatican's holiest football fans.
When Francis first met Benedict XVI in person after his election, he declared, "we are brothers," but family feuds have started over less.
Francis earlier joked of a "war" between him and his protectors, The Swiss Guards, during the Argentina-Switzerland match. But could we now see a holy war between two Popes?
No word from the Vatican as of press time, but fans are already speculating. The jury's out on Jesus, however.


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Send Mother's Day Flowers to Irvine.
Floral Design By Nikki has a beautiful selection of Mother's Day flowers and gifts we can deliver throughout the Irvine area. We can send Mother's Day Roses, brilliant flower bouquets and gift baskets for mom, wherever she lives in the United States and Canada. Order Mother's Day flowers today and avoid the rush!
|We Accept:


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Home :: Fire Alarm Systems :: Smoke Detectors and Bases :: Beam Detectors :: Firebeam Anti-Fog Kit
Specifically developed to overcome the problems of condensation, the Firebeam Anti-Fog Kit will reduce false alarms in environments prone to fogging. Consisting of a retro-fit lens that attaches to the beam face, and a single reflector, both have a special nano-coating.
Condensation can be a problem in some environments where warm air meets a cold surface, just like your windows in the winter. The beam detector cannot identify the difference between this and smoke, and would, therefore, activate the alarm. With the Firebeam Anti-Fog Kit installed, condensation is prevented from forming in the first place on both surfaces, meaning it won't be an issue.
Including just the one nano-coated reflector, you will need to purchase an additional Firebeam Anti-Fog range extending kit to make your standard Firebeam detector even more reliable if needing to monitor a distance greater than 40m.
|Would you like to have one of our certified BAFE service technicians visit your premises or site to commission your new fire extinguishers for the Fire Regulations?
The cost to visit is £25.97 + vat (£31.16 inc vat) with each extinguisher at £6.00 + vat (£7.20 inc vat).
Please note a £10 + vat (£12 inc vat) surcharge will be added for all London postcodes
Optionally, the cost to commission and install (fix to the wall) is £13.00 + vat (£15.60 inc vat). Our technician will place them on stands at no charge if you have purchase these
NOTE - commissioning on-site after delivery is a requirement of British Standard 5306 Part 3, the Fire Safety Order, HMO fire safety guidance, and is commonly required by most insurers. It is not a requirement (but still an option) for a domestic home in single family occupancy
Please select one option
(this message will only appear once and, if you select 'yes', it will automatically add the same for all extinguishers added to cart. You still have the option to remove before submitting your order)
Our Service Coordinator will contact you to arrange a convenient day for the visit


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Free your subjects from their background on full frame with delicious twisty, swirly blur. Twist 60's vignette calls further attention to anything you place within the large central area of focus, for a timeless, vintage look.
True to an 1840 design by Joseph Petzval, this lens reveals striking separation between subjects and their background, a quality that modern lens designs lack.
Twist 60 is a metal-bodied, non-tilting lens that mounts directly to your camera and is recommended for use on full frame cameras only.
Twist 60 Optic, part of the Optic Swap System, is available separately for use in a tilting lens body. For best results, when used in a tilting lens body, keep the lens pointed straight ahead.
To be the first to review this product simply fill out the form to the left and let us know how you feel about this product!
To estimate the freight on this item simply enter the destination postcode and the desired quantity and click the "estimate" button.
Get Free Shipping on all orders! No minimum spend! Items are dispatched from our Melbourne Warehouse within 24 Hours*
Shop with peace of mind knowing all items sold @ C.R.Kennedy & Co are backed by genuine manufacturers warranty.
Place a Click and Collect order online and pickup from your nearest warehouse.
C.R. Kennedy is Australia's leading importer and distributor of photographic equipment. Our head office and main warehouse is located in Melbourne with branch offices and warehouses in Adelaide, Brisbane, Cairns, Perth and Sydney.
All rights reserved.
We pay the store, you take the goods the same day.
Choose how you pay - weekly, fortnightly or monthly.
Up to $1,000 interest free always or up to $1,000 with 6 months interest free.
Accepted here:
*A $6.00 monthly account fee applies. Pay your balance by the due date (the end of next month) and we'll waive the fee. See full Ts & Cs


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Grandfather is the dapper officer right front with the cavalry boots. The rather tall man in the center of the first photo is Baron Pyotr Wrangel, the last White Army general.
Grandfather right front again. Presumably these officers are Baron Wrangel's command staff.
Wounded in combat in the Crimea on the southern front grandfather found himself in a field hospital. There he met a charming White Army nurse named Maria. The rest, as they say, is history.
With the collapse of Baron Wrangel's army in 1920, Vasilly and Maria fled with their baby daughter Vera, my aunt, to Constantinople. As the War for Independence engulfed Turkey they fled again to Belgrade in the new Kingdom of Yugoslavia, where my dad, Vasilly Junior, was born in 1922.


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Newly renovated cottage in centre of white Andalucian village with large private patios and swimming pool. Ideally suited for a couple or small family - one large famly bed room.
Rural village location with amazing views of sea and mountains. Secluded garden, private patios and swimming pool, in centre of traditional white village. 6 guests/annexe 3 more.


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You can now play Sachin Saga Cricket Champions for PC on a desktop/laptop running Windows XP, Windows 7, Windows 8, Windows 8.1, Windows 10 and MacOS/OS X. This can easily be done with the help of BlueStacks or Andy OS Android emulator.
Get ready to play as the legendary master blaster himself in all his glory! Hit shots the way he did, in iconic matches that he led to victory and in authentic stadiums with real-world conditions. We put you in Sachin's shoes to live and experience the cricketing adventure of god himself.
Choose from over a 100 cricketers and build your dream team. Compete on the leaderboard in timed events and chase the dream to become the ultimate cricket champion and meet Sachin himself.
Exclusive Features:
• Legendary Mode
o Play as the master blaster himself in his most iconic matches from his past in the same conditions and stadiums as he did. Start the journey of a young 16-year-old Sachin that made him a legend and play his glorious career of 24 years.
o Earn Reputation Points [RP] as you progress through Sachin's career. Collect enough RP to compete in different time-limited events.
• Events Mode
o Build your dream team and play a full game as a 11-man squad in up to 50 over matches and compete with real players in the game to fight for the top spot in leaderboards.
o Multiple time-limited events hosted every month that allows you to play your team against other teams.
• Player Cards
o Over a 100 Legendary cricketers to be collected and added to your team.
o Acquire players from recent teams and yesteryears and build your own team.
o Cricketers have varied levels of Rarity ranging from common to legendary.
• Gameplay
o All new revolutionary real-world batting system that emulates real-world playing conditions which result in authentic batting experiences.
o Play the game in its truest form (zone based bat connections) i.e. well-timed connection of the bat with the ball defines the outcome of the shot and a miss timed connection could result in a catch.
o Motion captured batting shots of Sachin himself that allows you to play all his legendary shots the way he does.
o Dynamic camera angles that allow you to relive your shots from the best angles.
o Replay camera – Experience the beauty of the shot in slow motion as you knock one out all the way.
o Watch fielders pull out the ball from thin air to take stunning catches.
o Fielders slide and dive to reach the ball just like in actual games.
• Authentic stadiums
o Play in 11 world famous stadiums from around the globe.
o Gameplay emulates pitch conditions of that stadium.
• Physical rewards & Leaderboards
o Battle friends and other players alike, to win trophies and other in-game rewards.
o Stand a chance to win authentic signed merchandise from Sachin himself by winning the leaderboard events.
o Win other exciting rewards from different time-limited events that you win.
o Win Steak – Win matches in succession to get extra rewards.
How to download and run Sachin Saga Cricket Champions on your PC and Mac
Sachin Saga Cricket Champions For PC can be easily installed and used on a desktop computer or laptop running Windows XP, Windows 7, Windows 8, Windows 8.1, Windows 10 and a Macbook, iMac running Mac OS X. This will be done using an Android emulator. To install Sachin Saga Cricket Champions For PC, we will use BlueStacks app player. The method listed below is set to help you get Sachin Saga Cricket Champions For PC. Go ahead and get it done now.
More Details
Download Links For Sachin Saga Cricket Champions:
Download: Bluestacks Android Emulator
Download: Sachin Saga Cricket Champions (Play Store)
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Step to Step Guide / Sachin Saga Cricket Champions For PC:
- As you have Downloaded and Installed Bluestacks Emulator, from Links which are provided above.
- Now, After the installation, configure it and add your Google account.
- Once everything is done, just open the Market in Play Store and Search for the 'Sachin Saga Cricket Champions'.
- Tap the first result and tap install.
- Once the installation is over, Tap the App icon in Menu to start playing.
- That's all Enjoy!
Also Read: 777 Slots Free For PC / Windows 7/8/10 / Mac – Free Download
That would be all from our side. In case you have got any queries or you have any issue following the guide, do let me know by leaving your thoughts in the comments section below.


text:
Psalm 119:97-104 New International Version (NIV)
מ Mem
97 Oh, how I love your law!
Cross references:
New International Version (NIV)
Holy Bible, New International Version®, NIV® Copyright ©1973, 1978, 1984, 2011 by Biblica, Inc.® Used by permission. All rights reserved worldwide.
Jeremiah 26:1-15 New International Version (NIV)
Jeremiah Threatened With Death
26 Early in the reign of Jehoiakim son of Josiah king of Judah, this word came from the Lord: 2 "This is what the Lord says: Stand in the courtyard of the Lord's house and speak to all the people of the towns of Judah who come to worship in the house of the Lord. Tell them everything I command you; do not omit a word. 3 Perhaps they will listen and each will turn from their evil ways. Then I will relent and not inflict on them the disaster I was planning because of the evil they have done. 4 Say to them, 'This is what the Lord says: If you do not listen to me and follow my law, which I have set before you, 5 and if you do not listen to the words of my servants the prophets, whom I have sent to you again and again (though you have not listened), 6 then I will make this house like Shiloh and this city a curse[a] among all the nations of the earth.'"
7 The priests, the prophets and all the people heard Jeremiah speak these words in the house of the Lord. 8 But as soon as Jeremiah finished telling all the people everything the Lord had commanded him to say, the priests, the prophets and all the people seized him and said, "You must die! 9 Why do you prophesy in the Lord's name that this house will be like Shiloh and this city will be desolate and deserted?" And all the people crowded around Jeremiah in the house of the Lord.
10 When the officials of Judah heard about these things, they went up from the royal palace to the house of the Lord and took their places at the entrance of the New Gate of the Lord's house. 11 Then the priests and the prophets said to the officials and all the people, "This man should be sentenced to death because he has prophesied against this city. You have heard it with your own ears!"
12 Then Jeremiah said to all the officials and all the people: "The Lord sent me to prophesy against this house and this city all the things you have heard. 13 Now reform your ways and your actions and obey the Lord your God. Then the Lord will relent and not bring the disaster he has pronounced against you. 14 As for me, I am in your hands; do with me whatever you think is good and right. 15 Be assured, however, that if you put me to death, you will bring the guilt of innocent blood on yourselves and on this city and on those who live in it, for in truth the Lord has sent me to you to speak all these words in your hearing."
Cross references:
New International Version (NIV)
Holy Bible, New International Version®, NIV® Copyright ©1973, 1978, 1984, 2011 by Biblica, Inc.® Used by permission. All rights reserved worldwide.
Acts 17:22-34 New International Version (NIV)
22 Paul then stood up in the meeting of the Areopagus and said: "People of Athens! I see that in every way you are very religious. 23 For as I walked around and looked carefully at your objects of worship, I even found an altar with this inscription: to an unknown god. So you are ignorant of the very thing you worship—and this is what I am going to proclaim to you.
24 "The God who made the world and everything in it is the Lord of heaven and earth and does not live in temples built by human hands. 25 And he is not served by human hands, as if he needed anything. Rather, he himself gives everyone life and breath and everything else. 26 From one man he made all the nations, that they should inhabit the whole earth; and he marked out their appointed times in history and the boundaries of their lands. 27 God did this so that they would seek him and perhaps reach out for him and find him, though he is not far from any one of us. 28 'For in him we live and move and have our being.'[a] As some of your own poets have said, 'We are his offspring.'[b]
29 "Therefore since we are God's offspring, we should not think that the divine being is like gold or silver or stone—an image made by human design and skill. 30 In the past God overlooked such ignorance, but now he commands all people everywhere to repent. 31 For he has set a day when he will judge the world with justice by the man he has appointed. He has given proof of this to everyone by raising him from the dead."
32 When they heard about the resurrection of the dead, some of them sneered, but others said, "We want to hear you again on this subject." 33 At that, Paul left the Council. 34 Some of the people became followers of Paul and believed. Among them was Dionysius, a member of the Areopagus, also a woman named Damaris, and a number of others.
Cross references:
New International Version (NIV)
Holy Bible, New International Version®, NIV® Copyright ©1973, 1978, 1984, 2011 by Biblica, Inc.® Used by permission. All rights reserved worldwide.
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text:
Reference number : FO-R-12792
This property is exclusive to Ikram Ul Haq and its authenticity has been verified by Forest.ae following Dubai's Laws.
➔ Forest.ae Government Autorisation Number: 12082
Forest is proud to offer this Luxury 1 Bedroom with Full Marina View in Al Habtoor Residential Tower for Rent.
* Full Marina View
* High floor
* 1 Bedroom
* Balcony
* 2 Bathrooms
* BUA: 840 sq. ft.
* 1 Parking space available
* Shared Swimming Pool and Gym
* Located on Marina walk
* Five mints walk from Metro Station
* Close to Shops, Restaurants Supermarkets
For more info contact 800 FOREST
Tel: +971 4 392 7777
Fax: +971 4 392 7111
Email: email@example.com
Suite # 2106, Marina Plaza, Dubai Marina, DUBAI UAE
Dubai Marina
With 180 residential towers built along the perimeter of the iconic marina, one is certainly spoilt for choice when it comes to choosing a unit with the best view of the marina.
Various forms of public transportation available such as the Dubai Metro and the newly launched Dubai Tram makes it easy for residents to commute to their workplace.
With Jumeirah Beach being just a 5 minute walk away, you can definitely enjoy the best of the beach lifestyle and marina lifestyle in this one-of-a-kind location.


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[lib] List <T> - Basic
List <T> - Basic 1.0b
Forum Thread and demo map
Library allowing to easily create basic/cheap version of List<T> collection types known from .NET
In essence "Dynamic Arrays"
- Features
- Create Int, Real, String List types (can be extended to any DataTable supported type)
- Dynamic = no initial size
- Lists are expanded using 'List <T> Add'
- Easy 'List Values Count' function to get current size
- As additional feature i added GUI-Only 'For Each Value in List' actions/scripts
Note: Although you can create new versions like for example List <dialog item> in galaxy most if not all types are convertible to those 3 basic types. If i'm correct only units are not but for those you have Unit groups already which function same way.
How to use
- Lists New - Initialize new List. (global, same names allowed if type is different)
- List <T> Add - Extend list and stores new value at new index.
- List <T> Set, List <T> Get - Get/Set existing index value.
- List Clear - Clear List data.
Import library
Please use This Thread for questions etc
Posts Quoted:


text: Remember back in kindergarten how you used to mix the paints up to make new colours? Red + blue makes purple. KaraCube uses colour mixing as the basis for this rather original and challenging puzzle game, using beams of light instead of paint. The game has 80s style ambience which might be better suited to tablets/PC as the graphics are quite detailed. Adding a tutorial would be a good addition for those who have forgotten their colour mixing basics. 8/10


text:
Wimbledon Ballot
Wimbledon Ballot
If you are a new member joining Bovingdon and Flaunden Tennis Club you can sign up for a free British Tennis Membership:
Any problems email firstname.lastname@example.com
You can then opt into the Wimbledon draw. The deadline for this is 14 Feb 2020.
All adult members and juniors 11 years and over are eligible.
Centre court:
Weds 03rd July Jill Cohen
Thurs 04th July Angela Morgans
Sat 06th July Chris Sparkes
Mon 08th July Margaret Allen
Tues 09th July Tony Franklin
Sun 14th July Men's finals John Bain
Number 1 court
Mon; 1st July Ruby Fillis
Tues 2nd July David Bird
Thurs 4th July Kevin Bocker
Thurs 04th July Maureen Grant
Mon 8th July Stephen Spikings
Tues 9th July Geoff Allen
Sat 13th July Francesca Myers
Sun 14th July Tim Baldwin
Number 2 court
Weds 3rd July Graham Dore
Thurs 4th July Steve Martell
Sat 6th July John Wareham
Sat 6th July Evan Perkins
You do not need to be present to be entered into this but only those who opted in by February 14th are eligible. Only one pair of tickets per family.
If you would like more information or you any problems logging on to the LTA site, please e-mail:
Rebecca Rose firstname.lastname@example.com


text:
Aircrew Europe Star (France & Germany Clasp)
Defence & War Medals.
1590748. Sgt.(Air Gunner) William KNOX.
158 Squadron.
An exciting Halifax "combat" award won from the Mid-Upper Gun turret on the night of 12/13th August 1944.
The target was BRAUNSCHWEIG (Brunswick) and the aircraft MZ373, NP-K, left RAF Lissett at 21:25. The aircraft was attacked by a JU88 night fighter at 17,500ft on the way to the target.
This was a particularly nasty raid during which 158 Squadron lost two aircraft. The night's total losses were 27 aircraft. Knox's Halifax was also seriously hit by cannon shell and riddled with machine gun fire from the enemy aircraft and holed in the fuel tanks, the starboard elevator and in the tail end of the fuselage. Tail Gunner warned the pilot to initiate an avoiding corkcrew and Mid Upper Gunner (Sgt W Knox) then sighted the enemy aircraft and opened fire at 350yds range. He fired 350 rounds which hit the enemy causing him to break off the attack. (* the enemy aircraft was seen damaged and was probably lost). Sgt W.Baxter (Flight Engineer) made a quick calculation that the fuel and damage situation was such as to allow the aircraft to continue on its mission, which it did, also making a safe return to base. Sgt W.Baxter as also awarded a DFM for this action in the same Gazette as Sgt W. Knox (20th Feb 1945).
The group comes with an extensive file of research on Knox and his fellow crew members with detials of their awards and service. Copy combat reports and a full file of Sgt Knox's sorties copied from the squadron ops' books.
The original squadron recommendation /citation form at first stated that this was an "Immediate" award, but was later altered (probably erroneously) to read "none-immediate" in two out of three places....however, and from long experience in RAF matters we feel that this was indeed an Immediate award in line with most such awards which were made for an individual occurance and 'one off' combat such as this. It could maybe be confirmed by looking at Sgt W.Baxter's papers which probably confirm they were both "immediate" awards!
This DFM was regularly and proudly worn by William Knox and has sustained some edge bumps to the rim and to parts of the naming. ALL the naming is however fully clear and fully readable with no erasure whatever of any characters. The ends of the ribbon bar were superbly and professionally reinforced in silver during the recipients lifetime. It's actually quite nice to see a medal that has shared a proud life with its most worthy winner.
The group comes with a set of RAF sleeve badges, an Air Gunner's
Brevet "AG" Wing and a brass RAF WWII cap badge .
DFMs ...Getting scarcer all the time.....A medal with a great story from WW2.
Sgt Knox bombed Chateaudun on D-Day (6th June 1944) and carried out a total of 39 sorties during 170.36 hrs of operational flying.


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Swine Business Unit Manager (KS-6134)
PLEASE NOTE:? We will not reply if your resume does not meet position requirements.? Please understand your education and experience must match our job listings for you to be considered.?? ? There is NO closing date on this position.? This position will remain open until a suitable candidate is found.?QUALIFICATIONS:- MS or Higher Education in Animal Nutrition, Animal Science, or related Degree- 3-5 years experience in Feed Industry or Animal Health- Extensive Sales and Marketing experience required- Must have solid working knowledge and understanding of current swine production- Knowledge of farm layouts very beneficial- P&L experience- Interpersonal and Communication skills- Strong attention to detail and organizational skills- Computer proficiency, especially with regard to Microsoft Office suite applications- Desire to Travel up to 65%?JOB DESCRIPTION:Established Ag Employer seeking a Business Unit Manager-Swine for their organization.? This position will report directly to the President.? The ideal individual will have recent sales management experience catering to the swine industry and must have solid knowledge of large scale production farms. This organization has a large sales division with many technical sales professionals.? Duties/Responsibilities to include:* Sales, marketing and promotion* Oversight of Sales Professionals, Technical Support, and Service Managers* Developing and implementing marketing strategies as well as pricing strategy tools* Communicating, training and working through multi-cultural channels within organization* Providing key role in supporting product/business development* Actively supporting after-sales services, collections and other sales-related programs and services?**Relocation may not be required, depending on candidates current location. Excellent Compensation and Benefits offered***?ALL INQUIRIES ARE KEPT CONFIDENTIAL. NO FEES TO APPLICANTS.?If you feel that you are not a candidate for this position, but know someone who is, we do offer a Referral Bonus if we place your referral into this position.________________________________________To be considered for this position, please refer to the Job Order # and contact:Ken SmedraHansen Agri-PlacementP.O. Box 1172Grand Island, NE 68802Phone:? email@example.com for this job.


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When I visited a foreign country fifteen years ago, I ran into a very different approach to children who bullied: "ignore them."
I was told that bullying was a way in which children who did not "fit in" or represent societal norms were actually driven from school, usually in grade school. It seemed to be the way in which a whole country accepted the necessity to relieve themselves of individuals who did not march to their common drum.
I've thought a lot about bullying and how bullies are created and sustained. There are some common aspects of bullies, whether they are eight or sixty-eight.
- We must realize that bullies are NOT born, they are Created.
- People who allow themselves to bully, deny their empathy towards their victims.
- Bullies practice learning how to blame others for their own actions.
- Bullies feel entitled to do whatever it takes to get their needs met.
- Bullies are always FEARFUL–unable to face their own fear, they act out on others.
A) In our foreign country, bullies were created at home and sustained by schools. The easiest way to create a bully is to accede to the child, trying to please every wish, demand, outburst, and raging behavior. The more a parent allows the child to set the rules at home, the more the child learns to control others through intimidation, fear and out right physical attack. For Harry Potter fans, think of Dudley: a boy was indulged constantly by his doting parents.
B) To be able to inflict emotional or physical pain on someone else, usually we humans have to feel like we are in the act of survival–for our sense of self or for our physical body. For a Bully, inflicting pain upon others is a wish to control them. To see the reactions to fear and pain induce the Bully to believe that he/she has reached supremacy. And, at the same time, the Bully is able to mask his or her own secret sense of inadequacy.
C) When children no longer have to accept responsibility for their actions, they learn to practice how to blame others for their actions. Somebody else always "made me do it." Parents who accept that their child is blameless, teach their children to look to others for explanation of reactionary behavior. The easiest way to teach children about self-responsibility when there are more than one child in the house, is to hold each child responsible for his or her behavior. When children learn that there are consequences for reactionary behavior, they will learn how to abstain from the instant thought and actions of "he made me do it."
D) When children don't have to earn their rewards, they learn to expect gifts without expending any effort. A reward can be simply a walk to the park, or a glass of chocolate milk, or as great as getting a pet or having their favorite meal. This does not mean that parents are never to give their children gifts, but it does mean that it behooves the parent and child for the parent to remind the child that through their good choices or behavior that they are going to the park, etc. As rewarding as it is to see our children's eyes light up with our thoughtfulness–these are indulgences. Too many indulgences and we are not teaching our children how to EARN our regard. (Note, I didn't say "affection," regard and respect have to be earned to have value.)
E) When respect is not earned at home, but is freely given as a GIFT, a child learns to expect others to give her/him the same gifts, without understanding the need to EARN them. A child who learned and practiced the internal self-deceit of outward respect, FEARS that others may find out that he or she is not WORTHY of their actual regard. And thus, to protect his own ego from the painful acknowledgment of being "less than"–the bully does everything he or she can to protect their fragile ego. FEAR is a huge driving factor in bullies.
What to do about a bully? Have it be common knowledge that Bullies are Fearful of being found out–that they have been unable to build a respected self–that they are torturing others for their own lack of courage. School programs would do well to promote an educational understanding about bullies–and a follow-up with a mediation process that gives the bully every opportunity to explore his or her own fears and personal failings.
(We adults have our own issues with accepting adult bullies that we tend to ignore. By ignoring grown up bullies, we are teaching the watching eyes of our children that they, too, must ignore bullies. Remember the foreign country that ignored school bullies? It is one of the most "class conscious" countries in the world.)
Be gentle with yourself, your children and give the gift of earned rewards.


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by John Savage
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Perspective ARTICLE
PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders
- Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, Netherlands
The vast majority of patients suffering from a primary immunodeficiency (PID) have defects in their T- and/or B-cell compartments. Despite advances in molecular diagnostics, in many patients no underlying genetic defect has been identified. B- and T-lymphocytes are unique in their ability to create a receptor by genomic rearrangement of their antigen receptor genes via V(D)J recombination. During this process, stable circular excision products are formed that do not replicate when the cell proliferates. Excision circles can be reliably quantified using real-time quantitative (RQ-)PCR techniques. Frequently occurring δREC–ψJα T-cell receptor excision circles (TRECs) have been used to assess thymic output and intronRSS–Kde recombination excision circles (KREC) to quantify B-cell replication history. In this perspective, we describe how TRECs and KRECs are formed during precursor – T- and B-cell differentiation, respectively. Furthermore, we discuss new insights obtained with TRECs and KRECs and specifically how these excision circles can be applied to support therapy monitoring, patient classification and newborn screening of PID.
Primary Immunodeficiencies of the Lymphoid System
Primary immunodeficiencies (PID) are rare inherited disorders of the immune system. Mutations in a single gene can affect one or more immunological components of the individual, resulting in increased susceptibility to infections, often accompanied by immunoregulatory defects (Notarangelo, 2010).
The vast majority of PID patients have impaired T- and/or B-cell function. T- and B-cells have the ability to create a unique receptor to recognize antigen. Furthermore, upon encounter with their specific antigen, they generate immunological memory by means of long-lived antigen-specific memory cells and antibody-producing plasma cells. Thus, defects in these cells grossly impair immunity. T-cell defects result in combined immunodeficiency (CID), affecting both cellular and humoral immunity. These patients suffer from opportunistic infections. The most severe CID variant (SCID) is fatal in the first months of life unless stem cell transplantation is provided. These patients lack T-cells, often combined with lack of B-cells, due to early arrests in precursor lymphocyte differentiation (Figure 1). B-cell defects lead to antibody deficiency. This defect in humoral immunity results in increased susceptibility to encapsulated bacteria. A subgroup of patients has agammaglobulinemia, due to a B-cell differentiation arrest in bone marrow and the consequent absence of mature B-cells and serum immunoglobulins (Figure 1). Over 30% of patients develop irreversible organ damage, mainly in the lungs, in childhood or early adulthood (Quinti et al., 2007). Early treatment with intravenous immunoglobulins and prophylactic antibiotics may prevent this (Lucas et al., 2010).
Figure 1. Human B- and T-cell differentiation stages including V(D)J recombination bars (Dik et al., 2005; van Zelm et al., 2005) and genetic defects underlying PID that result in impaired precursor differentiation. Blocks in B-cell development are derived from immunophenotyping studies on bone marrow samples from agammaglobulinemia and SCID patients. Developmental blocks in the T-cell lineage largely rely on data from targeted mutation studies in the mouse.
Whereas many genetic defects have been identified (Notarangelo et al., 2009), a molecular diagnosis has not been made in the vast majority of patients with a primary antibody deficiency (van der Burg et al., 2011). Furthermore, mild genetic variants in known disease-causing genes can give rise to a broad clinical spectrum of diseases (Schuetz et al., 2008; Conley et al., 2009; IJspeert et al., 2011). Thus, new assays are required to establish an early identification of disease and proper diagnosis with estimation of future complication risks. Over the past 10 years, many studies have addressed immunological defects to define subgroups of CID or antibody deficient patients. One of the newly explored methods involves quantification of circular DNA products that are generated in early lymphocyte development during the creation of unique B- and T-cell receptors. In this perspective, we will first address how recombination excision circles are formed and how these products can be used to study the mature B- and T-cell compartments. Subsequently, we will discuss recent insights obtained by quantification of these excision circles and potential applications to support early diagnosis, classification, therapy monitoring and newborn screening of PID.
V(D)J Recombination and Formation of Excision Circles
The gene complexes encoding the T- and B-cell receptor components do not contain a functional first exon. Instead, they contain multiple variable (V), diversity (D), and joining (J) genes. During antigen-independent differentiation in bone marrow (B-cells) and thymus (T-cells), stepwise rearrangements are introduced in the genome to couple one of each segment together to form a functional first exon (Figure 1).
Precursor-T cells in thymus first start to rearrange their T-cell receptor (TCR) D and TCRG genes (Figure 1). When this leads to a functional receptor, the cell exits the thymus as TCRγδ+ T-cell. Most cells, however, do not form a functional γδ TcR and start rearranging their TCRB and TCRA genes. Upon successful completion, these cells exit the thymus with an αβ TcR. In TCRD and TCRB loci, D to J gene rearrangements are induced prior to V to DJ rearrangements. TCRA and TCRG lack D gene segments and directly couple a V to a J gene segment.
Precursor B-cells start with D to J gene rearrangements in the Ig heavy chain locus (IGH), followed by V to DJ gene rearrangements (Figure 1). Upon successful completion, Ig light chain rearrangements are induced in the IGK locus, leading to direct V to J coupling. When IGK light chain rearrangements are not successful, V to J gene rearrangements are induced in the IGL light chain locus. Thus, mature B-cells can exit the bone marrow expressing an antigen receptor with either an Igκ or an Igλ isotype.
Double strand DNA breaks are introduced by the recombination activating gene products RAG1 and RAG2 on the borders of two gene segments and the flanking recombination signal sequences (RSS). The resulting coding ends at the side of the gene segments are ligated together forming a coding joint. The intervening DNA is excised and circularized by ligating the blunt DNA signal ends, thereby forming a signal joint. While multiple of these episomal recombination excision circles are formed in every developing B- and T-cell, they seem to have no specific function. They are stable products, but cannot replicate in the cell, do not duplicate during mitosis, and consequently are diluted during cell division, while the corresponding rearrangement on genomic DNA (coding joint) is inherited by all daughter cells (Livak and Schatz, 1996; Breit et al., 1997; van Zelm et al., 2007a). Thus, quantification of coding joints and signal joints from a specific gene rearrangement can be used to study cellular turnover and proliferation (van Zelm et al., 2007a).
Generation of TRECs and KRECs in Lymphoid Cells
When a B- or T-cell has completed a gene rearrangement and starts to proliferate, only one of the daughter cells will contain the corresponding excision circle. Thus, it is not possible to study turnover or proliferation of single cells: each cell either contains a circle or not. In fact, measurement of turnover or proliferation via excision circles is performed at the level of a cell population. By comparing the number of detected signal joints on excision circles to the total number of coding joints in the genome, the average proliferation is established. The condition to detect signal joints at the population level requires the need to select a common rearrangement. However, V(D)J recombination of Ig and TCR loci is optimized to generate a large repertoire of antigen receptors: in each B- and T-cell unique combinations of V, D, and J gene segments are formed. Therefore, most rearrangements are formed to generate a functional B- or T-cell receptor and will consequently not be useful as targets to study proliferation.
Fortunately, most B- and T-cells initiate a single-step gene rearrangement to render a specific locus non-functional. This concerns the δREC–ψJα rearrangement that deletes the entire TCRD locus in T-cells and the intronRSS–Kde rearrangement that deletes the constant region of IGK in B-cells (Figure 2). The δREC–ψJα rearrangement is initiated following unsuccessful generation of a γδ TcR and is estimated to have occurred on 70–80% of TCRD alleles in mature αβ+ T-cells (Verschuren et al., 1997). Nearly all newly formed αβ+ T-cells will have a T-cell receptor excision circle (TREC) with the δREC–ψJα signal joint (Figure 2A). However, the coding joint of this rearrangement is not stably present in the genome. As the TCRD locus is located within the TCRA locus, subsequent Vα–Jα rearrangements will remove the δREC–ψJα coding joint from the locus and place it on a novel circular excision product (Breit et al., 1997; Hazenberg et al., 2000).
Figure 2. V(D)J recombination and the formation of excision circles. (A) Sequential rearrangements in the TCRAD locus. Following V(D)J recombination of TCRD, the whole locus is then preferentially deleted by δREC–ψJα rearrangements in the αβ+ T-cell lineage. This gives rise to a δREC–ψJα signal joint on an excision circle (TREC) and a δREC–ψJα coding joint in the genome. The coding joint is deleted from the genome by TCRA (Vα-Jα) rearrangements and is then located on a TREC as well. (B) V(D)J recombination on the IGK locus results in a VJκ coding joint. Subsequent rearrangement between the intronRSS and the Kde elements can render the IGK allele non-functional by deleting the Cκ exons and the enhancers. Consequently, the coding joint precludes any further rearrangements in the IGK locus and therefore remains present in the genome, whereas an intronRSS–Kde signal joint is formed on an excision circle (KREC). (C) Oligonucleotide characteristics for TREC, KREC, and control gene quantification (Pongers-Willemse et al., 1998; Hazenberg et al., 2000; Dik et al., 2005; van Zelm et al., 2007a).
In contrast, the intronRSS–Kde rearrangement does generate a stable coding joint in the IGK locus of B-cells and cannot be removed by subsequent gene rearrangements, because all enhancers are removed from the allele (Figure 2B). Moreover, this rearrangement is one of the last to occur on 35–40% of IGK alleles prior to migration of B-cells to the periphery and is not followed by proliferation in bone marrow (van Zelm et al., 2005). Thus, most B-cells will have a kappa-deletion recombination excision circle (KREC) with the intronRSS–Kde signal joint. The coding joint to signal joint ratio of this rearrangement can be used to quantify B-cell proliferation in secondary lymphoid organs (van Zelm et al., 2007a). In fact, the intronRSS–Kde fulfills all criteria to be a robust target for replication history studies. (a) it is a frequently occurring gene rearrangement; (b) it is one of the last Ig gene rearrangements in bone marrow–derived B cells before obtaining a functional Ig molecule, ensuring that the corresponding KRECs are abundantly present in naive B lymphocytes; (c) it is a single-step rearrangement, which allows easy design of RQ-PCR primers and probes for accurate detection of the coding joints and signal joints; and (d) it is an end-stage rearrangement, precluding further rearrangements.
The δREC–ψJα rearrangement fulfills all but the last criterion. Therefore, the δREC–ψJα TRECs cannot be straightforwardly used to quantify T-cell proliferation. Still, TREC quantification is suitable for many applications without need for the corresponding coding joint.
How to Quantify and Interpret Recombination Excision Circles
V(D)J recombination excision circles are formed by blunt end ligation of signal ends after RAG cleavage. Thus, a signal joint is formed between two pieces of DNA that were not in close proximity before and is an ideal target for a specific PCR for excision circle detection. δREC–ψJα TRECs can be reliably detected with TaqMan-based real-time quantitative (RQ-) PCR (Figure 2; Table 1). For exact quantification, several additional steps need to be undertaken. First, a standard curve for quantification is required. This is commonly done on serial dilutions of a signal joint construct cloned in a bacterial plasmid. Thus, the exact number of TRECs can be quantified for a given DNA sample (TRECs/μg DNA). Furthermore, the number of TRECs per 106 cells can be estimated based on the theoretical recovery of 1 μg DNA from approximately 150,000 cells (Hazenberg et al., 2002; Bains et al., 2009). Alternatively, more accurate TREC contents can be obtained when quantification is performed relative to a control gene, such as albumin or the TCRA constant region (Hazenberg et al., 2000; Zubakov et al., 2010). This would require a parallel RQ-PCR on the same sample and quantification using a dilution series of a bacterial plasmid containing the target gene. Because such a control gene is biallelically present in the genome, the number of TRECs per 106 cells can be calculated as follows (Sottini et al., 2010):
Table 1. Application of TREC and/or KREC analysis in newborn screening to identify immunodeficient patients with various types of genetic defects that result in extremely low total T- and/or B-cell numbers.
δREC–ψJα rearrangements are formed in nearly all αβ+ T-cells and subsequently, most thymic emigrants contain δREC–ψJα TRECs. Since TRECs cannot replicate, the TREC content is diluted when T-cells undergo homeostatic or antigen-induced proliferation. Still, quantification of mature T-cell proliferation is complicated, since the exact frequency of TRECs per cell of new thymic emigrants is unknown. Unfortunately, the δREC–ψJα coding joint cannot be used as marker, since it is also removed from the genome by subsequent Vα–Jα rearrangements (Figure 2). Furthermore, TREC measurements do not allow direct quantitative statements on thymic output. TRECs are highly abundant in recent thymic emigrants, but they are stable structures and they can remain present in "old" thymic emigrants (Verschuren et al., 1997; Douek et al., 1998; Hazenberg et al., 2000, 2001; Sodora et al., 2000). Therefore TRECs are not by definition markers of recent thymic emigrants.
Recently, we have introduced the recombination excision circle of the intronRSS–Kde rearrangement (KREC) to study the replication history of B-cells (van Zelm et al., 2007a). Since the intronRSS–Kde rearrangement is one of the last rearrangements to occur in bone marrow and the coding joint remains stably present in the genome, it allowed us to reliably quantify cell divisions. To further enable straightforward quantification of proliferation, we created a cell line that contains one intronRSS–Kde coding joint and one signal joint per genome. DNA from this cell line, U698-DB01, can be used to correct for differences in signal joint and coding joint PCR efficiencies. When PCRs are performed on similar DNA quantities of both the cell line and the samples of interest, the average number of cell divisions of the sample can be quantified as follows:
Furthermore, the frequency of B-cells that contain an intronRSS–Kde coding joint can be determined when a control gene is quantified in both the sample and the control cell line. The frequency of IGK alleles contain the intronRSS–Kde coding joint can be calculated by:
The above described approaches for quantification of excision circles are very important to take into account for correct interpretation of the obtained data. Still, in a few settings, straightforward quantification of excision circles with respect to a control gene can be sufficient to address a specific question. First, since TRECs and KRECs are specifically formed in developing lymphocytes, they can be used to determine the presence of T- and B-cells, respectively. This has been exploited in recent years to develop newborn screening tests for SCID and agammaglobulinemia (see below). Furthermore, very recent studies demonstrated a linear correlation between the frequency of TRECs in blood with human individual age (Zubakov et al., 2010). These analyses only require the difference in CT value of the TREC signal joint PCR as compared with the CT value of the control gene PCR (Zubakov et al., 2010). The strong linear decline in TRECs with age is very intriguing. Since TRECs were determined from full blood, the decrease is likely to result from both a decrease in blood T-cell numbers in young children and increased proliferation of mature T-cells in later childhood and adulthood (Comans-Bitter et al., 1997). The TREC decline in an aging immune system in normal individuals can be used as a reference for studies on premature or early immunosenescence under particular disease conditions (Betjes et al., 2011). Interestingly, the decline in KRECs was much less pronounced (Menno C. van Zelm, unpublished observations). A potential explanation is the more stable generation of new B-cells from bone marrow in older individuals as compared with new T-cell from thymus. Thus, the KREC content remains much higher since the dilution from additional proliferation is limited.
Newborn Screening of SCID and Agammaglobulinemia
Early diagnosis is critical for patients suffering from PID to prevent irreversible organ damage or early death. This is especially true for SCID patients, in whom infections become quickly fatal in the first months of life. Ideally, patients should be identified in a newborn screening protocol prior to developing disease symptoms to allow successful stem cell replacement therapy (Buckley, 2004; Puck, 2007). This would require a straightforward screening method that can be applied on dried heel-stick blood spots on Guthrie cards from neonates.
In 2005, Chan and Puck (2005) introduced a newborn screening method based on RQ-PCR detection of δREC–ψJα TRECs. This method proved to be very reliable in detecting T-cells in healthy neonates. Furthermore, TRECs were consistently very low to absent in blood from SCID patients suffering from various gene defects Table 1; Chan and Puck, 2005; Morinishi et al., 2009; Routes et al., 2009; Gerstel-Thompson et al., 2010; Hale et al., 2010). Based on these promising results, state-wide newborn screening is implemented in Wisconsin and assay validation has started in Massachusetts (Routes et al., 2009; Gerstel-Thompson et al., 2010).
Besides the easy detection with RQ-PCR and the specific generation of TRECs in developing T-cells, the success of SCID newborn screening with TRECs benefited from the additional phenomena of TREC dilution in adults. Young children have high TREC numbers, which provide a good signal to noise ratio. Furthermore, the presence of maternal T-cells or residual T-cells in "leaky" SCID patients thus hardly affects TREC numbers due to their extensive replication history.
Analogous to SCID, efforts have been initiated to set-up KREC-based screening for B-cell maturation defects (Nakagawa et al., 2011). These initial results revealed the presence of >200 KREC copies/μg DNA in healthy children, whereas no KRECs were detected in 30 XLA patients and 5 non-BTK agammaglobulinemia patients.
Recent Insights and New Directions for Analysis of TRECs and KRECs in PID
Hematopoietic Stem Cell Treatment Evaluation
The only corrective treatment for patients with PID used to be hematopoietic stem cell replacement therapies. Stem cell replacement therapy is the first treatment choice for patients with a severe T-cell defect. Over the past decades, enormous advances have been made with stem cell transplantation and the success rate is very high in young patients and with good donor matches. Still, the limited availability of properly matched donors requires the need for alternatives. Supported by the advances in molecular diagnostics of PID, gene therapy protocols have been developed, in which the patients' own hematopoietic stem cells are corrected with a wild type copy of the defective gene ("gene therapy"). Despite setbacks due to adverse insertional mutagenesis (Hacein-Bey-Abina et al., 2003), many gene therapy protocols have been restarted in the last years (Aiuti et al., 2009).
Quantification of δREC–ψJα TRECs has been informative for assessment of thymic T-cell neogenesis (Douek et al., 1998; Hazenberg et al., 2000). Initial insights have been obtained from HIV patients, in whom a low TREC content was demonstrated that could be reverted upon antiretroviral treatment (Douek et al., 1998; Lewin et al., 2002; Di Mascio et al., 2006). Furthermore, low TREC levels following hematopoietic stem cell transplantation have been shown to correlate with clinical morbidity and mortality from infection (Douek et al., 2000; Hazenberg et al., 2002; Lewin et al., 2002; Brown et al., 2010).
Recently, B- and T-cell recovery of PID patients following stem cell transplantation have been studied in a multiplex setting for KREC and TREC detection (Sottini et al., 2010). Such evaluations can be very informative to obtain early insight into B- and T-cell neogenesis, and a valuable addition to flow cytometric and donor/acceptor chimerism analysis.
Classification of CVID Patients
Despite recent advances in genetics of PID, in many PID patients no molecular diagnosis is made. The number of patients with a humoral immunodeficiency without molecular diagnosis is especially high. Most of these patients are diagnosed as having common variable immunodeficiency (CVID). As apparent from the name, these patients have a highly diverse clinical phenotype, likely in part resulting from genetic variation. In the past decade the "Freiburg" and "Paris" CVID classifications have been developed based on the composition of the peripheral B-cell compartment (Warnatz et al., 2002; Piqueras et al., 2003). Several patient subgroups display distinct abnormalities in B-cell subsets of which the cause remains unknown. Quantification of the replication history (with KRECs) of sorted B-cell subsets on top of flow cytometric analysis could help to explain aberrancies in the B-cell compartments of these patients. In absence of a molecular analysis, such immunological parameters will be important to define subgroups of patients and prediction of future complications.
Studies on Normal and Aberrant Lymphoid Maturation
Memory B cells in blood have been classically divided into CD27+IgD+ and CD27+IgD− subsets. The presence of CD27+IgD+ cells in patients with CD40L deficiency who lack functional germinal centers has lead to the conclusion that these can be generated independently of T cell help (Agematsu et al., 1998; Weller et al., 2001, 2004). Furthermore, they strongly resemble splenic marginal zone B cells (Weller et al., 2004). However, these conclusions have been challenged by the facts that natural effector cells showed molecular footprints of the GC reaction and that similar IGH gene rearrangements were found in natural effector B cells and in CD27+IgG+ B cells (Seifert and Kuppers, 2009). Furthermore, the absolute numbers of CD27+IgD+ B cells are clearly reduced in CD40L-deficient patients (Agematsu et al., 1998; Weller et al., 2001, 2004). Thus, this B-cell subset in healthy individuals is likely to be a mixed population of GC-derived and splenic marginal zone-derived "natural effector" memory B cells. By using the intronRSS–Kde coding joint / signal joint ratio, we quantified homeostatic and antigen-induced B-cell proliferation in purified B-cell subsets of healthy individuals (van Zelm et al., 2007a,b). The replication history of CD27+IgD+ B-cells was consistently lower than CD27+IgD− B-cells, both in childhood tonsil (4 vs 8 cell cycles) and in adult blood (6 vs 11 cell cycles; van Zelm et al., 2007a). Interestingly, germinal center B cells from childhood tonsil already had a replication history of eight cell cycles (van Zelm et al., 2007a). The replication history of the total subset of CD27+IgD+ B cells was clearly less than B cells undergoing a T-cell dependent response in the germinal center and supports the idea that at least part of the CD27+IgD+ B cells are derived from T-cell dependent responses (Weller et al., 2001). Thus, the use of KRECs to quantify the replication history can be of added value to identify the origin and maturation pathways of memory B cells. This is especially interesting, since CD27+IgD− memory B cells are in fact a mixture of IgM-only, IgG+ and IgA+ B cells. Furthermore, recent studies have identified CD27−IgA+ and CD27−IgG+ memory B cells (Fecteau et al., 2006; Wei et al., 2007; Cagigi et al., 2009). Quantification of their replication history in combination with additional molecular and phenotypic analysis could help to delineate their origin and maturation pathways.
The replication history of normal B-cell subsets has provided a template for analysis of the aberrant population in B-cells of patients with immunological disease (Moir et al., 2008; Rakhmanov et al., 2009; van der Burg et al., 2010). Interestingly, both in patients suffering from HIV and in a subgroup of CVID patients, an expanded CD21low B-cell population has been observed (Warnatz et al., 2002; Moir et al., 2008). These cells showed a replication history of ∼4 cell cycles, which was increased as compared with naive mature B-cells (two divisions), but decreased as compared with memory B-cells (6–10 cell cycles). It is currently debated whether these cells are naive anergic B-cells or tissue-like memory-B cells (Moir et al., 2008; Isnardi et al., 2010; Rakhmanov et al., 2009). Further analysis of CD27 and Ig isotype expression could help to resolve this discussion.
Recently, we analyzed the replication history of B-cells in patients with Nijmegen breakage syndrome (NBS; repair syndrome; van der Burg et al., 2010). NBS patients have a DNA repair defect and reduced B- and T-cell numbers resulting from inefficient V(D)J recombination. Still, both coding joints and signal joints can be robustly formed in absence of functional NBN protein and show normal characteristics (Saidi et al., 2010; van der Burg et al., 2010). Interestingly, the replication history of NBS patients' naive mature B-cells was ∼4 cell cycles, which was similar to the CD21low B-cells in HIV and CVID patients that were described above. The increased proliferation in NBS most likely resulted from decreased output of new B-cell emigrants from bone marrow and increased homeostatic proliferation to compensate the total peripheral B-cell number (van der Burg et al., 2010). The decreased KREC levels thus reflect a form of premature senescence of the immune system in NBS patients.
The above described observations clearly illustrate how KRECs can be used to support studies on the B-cell compartment of patients suffering from a PID. Unfortunately, analogous analysis of the T-cell compartment is hampered by the lack of a good internal control for quantification with δREC–ψJα TRECs. The corresponding coding joint is removed from the genome by subsequent Vα–Jα rearrangements. Therefore, it remains unclear how many TRECs are formed in thymus. Furthermore, following δREC–ψJα rearrangements, thymocytes undergo several rounds of proliferation. Still, these disadvantages as compared to the B-cell compartment can be overcome. First, the frequency of δREC–ψJα coding joints and signal joints can be quantified in thymocyte subsets and the proliferation in thymus can be calculated for CD4+ and CD8+ single positive thymocytes. Based on these data, the TREC frequencies in peripheral T-cell subsets can be corrected to calculate peripheral T-cell proliferation (K. van der Weerd et al. submitted for publication). Additionally, TRECs from other TCR rearrangements can be used to calculate T-cell proliferation. These would have to be single-step rearrangements, relatively frequent, and the coding joint should remain stably present in the genome. We are currently evaluating the potential of such TRECs in other TCR loci.
Newborn Screening for SCID and Agammaglobulinemia
With the introduction of TRECs to detect patients with SCID, all major criteria were met for inclusion in newborn screening protocols. The estimated incidence of SCID is >1:100,000 live births (Chan and Puck, 2005) and life-saving therapies exist. Furthermore, it has become clear that early diagnosis and early treatment initiation increases successful outcome rates. Using RQ-PCR techniques, the absence of blood T-cells can be robustly determined from DNA of dried blood spots. It should be possible to detect all SCID patients, independently of the underlying genetic defect. This is supported by multiple studies in which patients with various identified and unidentified genetic defects have been detected (Table 1).
The successful detection of agammaglobulinemia patients with KRECs now opens the way to inclusion of this technique for detection of agammaglobulinemia in newborn screening. The clinical phenotype of agammaglobulinemia is milder than SCID. Still, the disease is underdiagnosed in the first years of life and patients can develop serious organ damage before a diagnosis of immunodeficiency has been made. Thus, early identification by newborn screening can contribute to quality of life and reduction of health care costs.
Finally, RQ-PCR-based newborn screening has the advantage that tests can be combined in a multiplex setting. This has been successfully tested for detection of TRECs and an internal control gene (Gerstel-Thompson et al., 2010). Theoretically, it would be possible to also include KREC detection in this assay. Thus, a single assay could be developed to screen for both agammaglobulinemia and SCID with a combined estimated incidence of 1:30,000–1:50,000 births. Furthermore, T-cell deficient cases can be further separated in B-cell positive and B-cell negative subgroups, guiding additional diagnostics toward either T−B+ or T−B− SCID (Table 1). Consequently, such a combined test should keep the costs low, while increasing the benefits with regard to disease incidence and guiding of further diagnostics.
In conclusion, we here provide an overview of how V(D)J recombination excision circles can be used to obtain insights into the normal and aberrant lymphoid immune system. We are convinced that KREC and TREC analysis in combination with flow cytometric evaluation of T- and B-cell subpopulations of PID patients will provide new insights into their disease and may contribute to the identification of new genetic defects. Especially, combined quantification of TRECs and KRECs could support early diagnosis in newborn screening protocols, patient subgroup classification and evaluation of stem cell replacement therapy.
Conflict of Interest Statement
Jacques J. M. van Dongen is inventor of the KREC assay, which has been patented (PCT/NL 2005/000761; priority date 25 Oct 2004) and licensed to InVivoScribe Technologies, San Diego, CA; revenues of the patent go to Erasmus MC. The other authors have declared that no conflict of interest exists.
We thank Dr. K. Imai (National Defense Medical College, Saitama, Japan), and K. van der Weerd (Erasmus MC, Rotterdam, Netherlands) for sharing unpublished data, and Mrs. S. de Bruin-Versteeg for assistance in preparing the figures. Menno C. van Zelm is supported by fellowships from the Erasmus University Rotterdam (EUR-Fellowship) and the Erasmus MC, and by Veni grant 916.110.90 from ZonMW/NWO. Mirjam van der Burg is supported by a grant from the foundation "Sophia Kinderziekenhuis Fonds" (SKF; grant 589).
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Keywords: V(D)J recombination, KREC, TREC, T-cell, B-cell, primary immunodeficiencies, SCID, argammaglobulinemia
Citation: van Zelm MC, van der Burg M, Langerak AW and van Dongen JJM (2011) PID comes full circle: applications of V(D)J recombination excision circles in research, diagnostics and newborn screening of primary immunodeficiency disorders. Front. Immun. 2:12. doi: 10.3389/fimmu.2011.00012
Received: 04 February 2011; Accepted: 20 April 2011;
Published online: 04 May 2011.
Edited by:Stuart G. Tangye, Garvan Institute of Medical Research, Australia
Reviewed by:Klaus Warnatz, University of Freiburg, Germany
Stuart G. Tangye, Garvan Institute of Medical Research, Australia
Tim Niehues, HELIOS, Germany
Copyright: © 2011 Zelm, Burg, Langerak and Dongen. This is an open-access article subject to an on-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.
*Correspondence: Menno C. van Zelm, Molecular Immunology Unit, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, Netherlands. e-mail: email@example.com