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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
46 year old man | Marina del Rey, CA, USA | Seeking women 29-45 within 10 miles
Active within 1 week
My life is about 70% ordinary and 30% extraordinary. Every day starts with a walk on the beach, a bike ride, a swim, or all three. I lift weights 4 times per week but am not a fitness freak. I am attracted to sharp and witty women who I have chemistry with. I am not inhibited. My humor is dry and witty. My friends and family are near and dear to me. I want to meet someone who I can have fun with and treasure as a lover and best friend. I want someone who will improve my life, not add difficulty to it. Games are a waste of time.
Coffee and conversation , Dining out , Hobbies and crafts , Movies/Videos , Exploring new areas , Playing cards , Shopping/Antiques , Travel/Sightseeing , Volunteering
Auto racing / Motorcross, Dancing, Hockey, Other types of exercise, Walking / Hiking, Yoga
I exercise 5 or more times per week.
I have Dogs
I have a cute very little 4 pound dog.
Middle of the Road
I have a love for the West Coast and tropical islands. Big scenes do nothing for me. I love to walk through small towns and villages in and out of the States.
I like going to far away places, swimming, short runs, push ups, designing jewelry, rock, classic, r@b, reggae, and BIG E. smalls . Love, love love movies in bed all cuddled up.
I just started jogging in the deep sand.I love to take mini-vacations. Movies in bed are awesome. backgammon quiet beaches.. I have a great guitar and can only play one song, but well
I have been reading books on philosophy and the Sunday Times
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'2" (187cms)
|5'4" (162cms) to 5'11" (180cms)
|Body type:
|About average
|Athletic and toned
|No preference
|Dark blonde
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde, Platinum
|No Way
|No Way, Occasionally, Yes, but trying to quit
|I'll tell you later
|No preference
|Artistic / Creative / Performance
|Artistic / Creative / Performance, Executive / Management, Financial / Accounting / Real Estate, Legal, Medical / Dental / Veterinary / Fitness, Education / Teacher / Professor, Nonprofit / Volunteer / Activist, Fashion / Model / Beauty
I am a former banker turned artist. I love creating fine jewelry "wearable art" and am proud and honored to have had so much luck in my life.
|No preference
|No preference
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday
|White / Caucasian
|No preference
I am white?
|No preference
Spiritual agnostic.
|English, French, Tagalog
|Bachelors degree
|No preference


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Vermont Village Cinnamon Applesauce
Volunteers felt the texture of Vermont Village Cinnamon was overly spiced and too smooth, comparing it to "baby food." vermnontvillageapplesauce.com.
Reviewed: April 2011
Price When Reviewed: $4.79 for 24 oz.
What do you think?
Your Dark Chocolate Is Lying to You
A new government report found that it might be closer to milk chocolate than you think.
More From Food & Drink

subset: commoncrawl


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- Food & Drink
- 41772 Clarendon St E, Wainfleet, ON
Taco Vino III
Social Media Club Buffalo
Thursday, December 27, 2012 from 5:30 PM to 9:00 PM (EST)
Grab the opportunity to take a break from the holiday hustle and bustle and mingle with some great fellow Buffalonians. Enjoy good wine and endulge in Lloyd's famous tacos and treats! The event will be hosted at Just Vino on Main Street in Buffalo. Taco Vino was nominated this year for Buffalo Niagara Event Professionals (BNEP) People's Choice Outstanding Event Award. Join us again this year and help to keep this a must attend event in Buffalo
The event will take place on Thursday, December 27th from 6:30pm - 9:00pm. Featured wines will be available to pair with your favorite Lloyd's Taco Truck goodies. The fun includes prize drawings throughout the night, $1 off featured wines with a taco purchase and other wine/beer specials.
TACO V.I.P. - This year there will be a V.I.P. hour, from 5:30pm to 6:30pm, where a limited number of guests will have the luxury of little to no lines to get your wine and Lloyd's goodies. There will also be select prizes awarded during the Taco V.I.P. hour before the other guests arrive.
Donate a Canned Good - We will be collecting canned goods and other non-perishable food items for the Food bank of WNY. Everyone who brings a donation will receive additional entries in the prize raffle and will get their picture taken to be posted on the new SMC Instagram account!!
Space is limited, so buy your tickets now!
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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A very solid complete California car with the 289 V8, Auto trans and AC, Also has power brakes and power steering. This car would... More
For sale in our Chicago showroom is this classy example of both sophistication and American power the 1979 Ford Ranchero. This was the... More
1969 Ford Ranchero. Only 59k actual miles and looks it. No rust and very straight. Box is dent free, no rust, newly painted to... More
FORD: 1963 Ranchero, AZ truck, 6-cyl, new exhaust, shocks, radiator, battery and seat, drive now, restore later, $5,000. 520-723-5595.... More
RANCHERO: 1979 GT500, burgundy paint with matching interior, 351 with auto, air, AM/FM, factory Mags, factory dash tach, black tonneau cover, drive anywhere, $6,500... More
FORD: 1967 Ranchero Fairlane 500, auto, V8 289, factory a/c, new paint, interior, tires, brakes, drive anywhere, $7,500. 972-979-2348, TX.... More


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We bring you rules straight from the World Pool Association. They are the only official rules for snooker and pool that exist. Once you have this handy app, you do not have to take others' word for it. Just whip out your phone and find the most relevant rule for 8-ball, ro maybe for 9-ball. In case there is some ball game we have not covered, do drop us a message and we will be sure to include them as well. Till then good luck with your cue and diamond.
Pool & Snooker Rules brings you the WPA standardized rules for the ( 8 Ball & 9 Ball ) & WPBSA standardized rules for Snooker. The WPA is the international governing body of pocket-billiard sports.
** 8 Ball Rules
** 9 Ball Rules
** Snooker Rules
** Detailed description about each rule
** Covers recent changes to game rules
Snooker Scores
- Latest news from the professional snooker world.
- Calendar of 2015/16 World Snooker (WPBSA) organized tournaments, inlcuding ranking, non-ranking, Players Tour Championship, Championship League, etc.
- All 2015/16 tournaments scores.
- Live scores!
- Previous winners of most tournaments.
- Many statistics of current season and all-time (world rankings, matches won, century breaks, etc.)
- Player's personal scores of current and previous seasons.
- Upcoming birthdays of the players.
- All information updated daily.
Pool Drills
Drill included . . .
★ Walk The Line Drill
★ Sniper Drill
★ Safety Run Drill
★ Lucky 7 Drill
★ Kickbacks Drill
★ Gates Drill
★ Derailed Drill
★ Kickbacks Drill
★ Arc Drill
★ 6 Shooter Drill
☆ Pool Tips Preview
Each drill includes . . .
✓ Diagram
✓ Written instructions
✓ Video demo
✓ Voiceover instructions
Snooker Score Counter
Main features:
- digital, extremely easy to use snooker score counter.
o Keep all history of played games
o All mistakes can be fixed by "undo" function
o Supporting all snooker rules with free ball, fouls, multiple reds in one shot, etc.
o Keeping course of match correct with snooker rules. Invalid pots will be shown to user.
- Keep many players profiles on Your device.
- Keep all history of all matches played on device (with statistics).
- Able to continue uncompleted match.
- Match is persisted in every second, so You won't loose Your game in case of phone crash or turn off due to exhausted battery.
- Simple and nice look.
Features expected in next versions:
- Live score sharing over www.
- Sharing match result on social networks (Facebook, Tweeter,…)
- Personal training assistant with charts of progress and common training excersises.
Pool Score
Bring the app to your billiards / pool game of 8 ball pool, or 9 ball pool or even for a game of 'killer'.
Please rate this app on it's simple to use interface. It's for people who just want a no nonsense very simple scoreboard counter.
From the developer of Pool Drills (10 free billiards drills to improve your game) & Pool Tips 1 & Pool Tips 2 (billiards fundamentals lessons)
To input player names: Menu button>Settings>Input names>Save
To reset score & player names: Menu button>Settings>Reset
Updates planned for the future . . .
Absolutely none :) This app is designed to keep life simple, just open up the app, then give your Android phone a few seconds to get it's head around this highly complex app ;) Then either press up or down.
Warning: If you end up losing, please do not use your android phone as a missile aimed at your opponent's gleeful grin ;)
MySnookerStats Snooker Scoring
MySnookerStats can track every ball you pot and every shot you play, delivering you up-to-the-second stats like you see on TV. It's so easy-to-use that you'll never want to play another frame without it - you keep score while your opponent is at the table, then swap when it's your turn to clear up.
But the best bit comes when the match is done, when at the touch of a button, you upload the details of your game to your personal account at MySnookerStats.com. On our website you can track your progress and compare your skills with players from all over the world. Re-live your greatest breaks ball-by-ball and see how you stack up against your mates with head-to-head records and ranking tables.
If you're serious about improving your snooker, your progress graphs and match-by-match records will help you to really understand the strengths and weaknesses of your game.
App Features:
- two modes of operation so you can choose how detailed your stats will be: STANDARD (easier to use) or PROFESSIONAL (more detail).
- play matches with handicaps or off levels
- store your name and membership id, and the names, ids and handicaps of the last 50 players you've faced so that setting up for re-matches is a breeze.
- save your last 50 matches on the device, for you to review at your leisure, and so that you can upload them later (perhaps when your internet signal is better).
- 50 levels of undo, just in case you make a mistake in assigning scores
- a ball-by-ball text commentary and a current break bar to give clear feedback and reduce the likelihood of mistakes in the first place.
- the app knows all the rules - respots, foul and a miss, multiple reds in one shot, re-racks, skip the last black when the frame is already won, concede or end frame early - but with the flexibility to record every eventuality.
- brilliant score correction facility for when there's a dispute (or perhaps just in case you missed the fact that a red went in when your opponent fouled earlier on in the frame).
- continue matches over several sessions; in fact, you can exit the app at any time (for comfort breaks and the like!) and simply choose "continue last match" to pick up exactly where you were - even the frame timer is paused!
Get loads more information on our website at www.mysnookerstats.com. MySnookerStats is snooker for the internet generation!
[Keywords: snooker, stats, snookerstats, mysnooker, statistics, scores, scoring, ranking, scoreboard.]
Pool Tips 1
★Pool Tips is packed with dynamic and interesting videos of tips and advice to give you a solid framework of sound fundamental methods.
★ What is unique about Pool Tips is that there is no dull, dogmatic teaching style, tying you down to just one variation of a method. Different methods and styles are discussed for you to build upon and develop your own unique playing style.
☆ Approach
☆ Grip
☆ Bridge
☆ Stance
☆ Stroke
☆ Aiming
☆ Eye Movement
☆ Breaking
☆ Mechanical Bridge
☆ Stroke Drill
☆ Control Drill
☆ Sequence Drill
☆ Pool Tips Preview
★ Pool Tips is ideal for beginners, or intermediate players wishing to check and review the fundamentals of the game.
★ This is the first in a planned series of Pool Tips, going from the fundamentals in this app, then on to intermediate and advanced techniques in future episodes.
★ Each video is packed with information, so please get ready to use the pause and rewind button.
Please note that Pool Tips is a video based application, so there will be a longer download time compared to a game app for example.
✦✦✦✦ This app takes up quite a bit of memory space. Please make sure you have enough memory on your device before downloading this app as it is a video based app with a lot of information ✦✦✦✦
Wishing you fun and success with Pool Tips!
★★★ Pool Tips 2 is now available ★★★
Billiard Rules
* How many Balls must hit the Banks with the opening shot?
* What if the 15th ball is in the rack when playing Straight Pool. How to re-rack?
With "Billiard Rules" you always have the Ruleset for the most common Billiard, Snooker and even Carom Games with you! Once installed, no online syncing is necessary. The rules are always completely on your mobile.
"Billiard Rules" is available in English and in German.
Currently you can view
- 9 detailed Pool Rules
- 4 detailed Snooker Rules and
- 6 detailed Carom Rules
More Games and Disciplines are coming soon...
Snooker Scoreboard
Quick and intuitive user interface. - Just press the ball that was pocketed to add on the points.
Save multiple games and return to them later - Have ongoing games with your friends!
Edit the table - Play a short game with less reds, or adjust a handicap.
Undo function - With history for whole frame.
Try out a demo version at http://snooker.rickels.co.uk/
Pool Scoreboard
» Control the timing of shot (Shot Clock)
» Show score of the match
» Show the names of players
» Show photos of players
» Present the historic of the match
Only in PRO version
» Display various statistics of players
» Customize times of Shot Clock
» Customize the warnings on Shot Clock (sound or voice)
» Customize the warning words on Shot Clock
Version limitations
» Maximum race to 5
» The Shot Clock times can not be changed *
» The Shot Clock warnings can not be changed
» Warnings using only sound effects (no voice effects)
* The Shot Clock times are set according to the recommendations of the World Pool-Billiard Association
Official Billiard Rules
As well as covering the general rules for pocket billiard this application also covers all possible fouls you can encounter during a match.
This application includes an interactive, "Ask a Referee" section that will allow anyone to get an official World-Standardized Rules clarification for any specific situation they may encounter.
Along with the rules and the Q&A section this application will also contain the official equipment specifications for: billiard tables, pool cues, pool balls, pocket openings, and rail angles.
There is also a comprehensive section of guidelines for referees, as well as the World-Standardized Regulations for tournament play including: exceptions to the rules, dress code, and how to protest a ruling.
This application not only includes the rules and regulations of pocket pool but it also provides a general etiquette overview for how to act when around a billiard table and during a match.
The Billiard Congress of America a non-profit trade organization founded 1948 produced this application with the support of the Professional Billiard Instructors Association (PBIA).
The Billiard Congress of America seeks to enhance the success of its members and promote the game of billiards through programs designed to encourage billiards as a lifestyle and make pool everybody's game.
For more information, visit www.bca-pool.com or call 303.243.5070.
Pool Center
Pool Score
The app currently allows you to track the common pool billiard disciplines 14.1 Straight Pool, 8-Ball, 9-Ball and 10-Ball.
Select a discipline, enter player names and match goal and you are ready to rumble! Choose between winner or alternate break or play under pressure by using the shotclock.
Easily find your recent matches by player name, discipline, date or by adding custom keywords like the name of event where the match was played.
And if you had to abort a match you can continue it from here as well.
Snooker Score Calculator
Updates are coming, hopefully it will look a lot better soon.
If you have any ideas for future updates, please contact me.
APA 9-Ball Scorer (Unofficial)
All logo's and trademarks are property of their respective owners. I have no affiliation with the APA except that of a registered player.
RDS Grand Pool
L'application est basée sur les données réelles de la LNH et permet de bâtir un alignement via un repêchage de 25 rondes, compiler des points durant la saison en tentant d'améliorer périodiquement ses sélections via des échanges, consulter facilement sa performance et sa position au classement, et finalement, joindre et créer un groupe afin de pouvoir échanger avec d'autres participants.
L'application vient donc ajouter une dimension multiplateforme au jeu, permettant à l'utilisateur d'y participer dorénavant sur plusieurs appareils : mobile, tablette et ordinateur.
Nouveautés et améliorations pour la saison 2014-15 :
1. La possibilité d'effectuer son repêchage initial à partir de l'application mobile
2. Des outils d'aide à la décision permettant au participant de prendre les meilleures décisions lors du repêchage et des 10 périodes d'échange, par exemple : % de popularité du joueur à l'intérieur de la ronde, indicateur de joueur blessé, fiche détaillée du joueur ou de l'équipe, etc.
3. Création de groupe simplifiée et améliorée : classement du groupe et clavardage.
4. Tableau de bord permet en un clin d'œil de consulter votre performance de la veille et de la période courante ainsi que votre classement général. Également toute l'information pertinente incluant une liste des joueurs blessés ainsi que des notifications de l'activité dans vos groupes.
5. Classement détaillé : permet de consulter le classement général et de voir l'alignement de chacun des participants.
6. Alertes pour ne pas manquer une seule période de transaction.
* Cette version ne supporte pas les mobiles et tablettes sous le système d'exploitation 4,1.*.
* Cette version supporte les tablettes mais est optimisé pour les mobiles.
More from developer
World Atlas
It contains information regarding all the countries of the world, their flags,maps and much more.
Expand your knowledge - with over 200 countries and 200 capitals, this is the most complete atlas app on the market.
Get Slim
Get Slim App would help you to get slim by giving you tips on how to reduce weight and stay in shape.
Elements Periodic Table
Spice up your chemistry on Android!
*Complete list of 118 elements
*Search by element name
*Detailed properties of each element
*History, Sources, Isotopes information about each element
*Absolutely free
The app is a mobile periodic table. Which is a tabular arrangement of the chemical elements, organized on the basis of their atomic numbers, electron configurations (electron shell model), and recurring chemical properties. Elements are presented in order of increasing atomic number (the number of protons in the nucleus).
GiveUp also stores your last smoked time, in order to analyze your smoking habits. Moreover the app is completely free, so give it a try.
Why quit smoking now?
No matter how old you are or how long you've smoked, quitting can help you live longer and be healthier. People who stop smoking before age 50 cut their risk of dying in the next 15 years in half compared with those who keep smoking. Ex-smokers enjoy a higher quality of life – they have fewer illnesses like colds and the flu, lower rates of bronchitis and pneumonia, and feel healthier than people who still smoke.


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$6.99Kyla and Jefferson Blake count their 16 years together as a wonderful blessing from God. Their marriage is passionate, their daughter is a joy, Jefferson's medial career is prospering and they have loyal friends.Kyla knows her newly divorced and lifelong friend, Jasmine, is desperately unhappy and no amount of prayer or encouragement seems to help. But what Kyla doesn't know is that Jasmine has a
$6.99Theresa Elaine Hopson, 46, owner of Miss Thang's Holy Ghost Corner and Church Women's Boutique is puzzled. She can't, for the life of her, figure out why even Baby Doll Henderson, despite her false teeth and her navy-blue-socks-with-yellow-jelly-sandals-wearing self, can find a man and she, Theresa, can't. Theresa's been looking all of her life, and it seems like the only thing she finds are
$5.99Surviving life's setbacks through her relationship with a man who helps restore her faith in God, Zoe Clark discovers A LOVA' LIKE NO OTHA'. When thunder and lightning strike on the morning of Zoe Clark's wedding, her seemingly perfect world is turned upside-down as she loses her fiance to a pregnant girlfriend she never knew he had. With her engagement shattered, all her life's plans seem over.
$5.99Theophilus Henry Simmons is young, single, sexy, and just about the finest black man any wishful mother and daughter have ever seen in Charleston, Mississippi. He's also a pastor. And when he starts courting the quiet, homespun Essie Lee Lane-and she accepts his marriage proposal-a few of the church folk decide to teach Essie what it really means to be a pastor's wife, and show her how little she
$9.99Chantell Meyers's father is on his deathbed. Desperate to somehow save him, hantell makes a promise to God that if He allows her father to live, she will become a better person. Now that her father has pulled through, she is determined to keep her word. But soon Chantell is faced with a series of trials, temptations, and revelations that test her faith. After she decides to stop having sex until
$5.99Anya Mitchell feels greatly blessed. The owner of a successful Los Angeles financial services company, she is also prosperous in love, thanks so handsome writer Braxton Vance. True, they have real differences over their future and she must struggle at the same time to show her emotionally wounded cousin Sasha the right way in life. But Anya's faith in God's promise is unshakable...until the night
$9.99Yvonne Fountain Copeland is determined to move on after her husband of fifteen years decides to leave her because their marriage became "boring." Returning to her hometown of Durham, North Carolina, Yvonne is convinced that a change of scenery will help her draw closer to God and find inner peace. Yvonne didn't know that the journey to peace would lead her to sexy, single childless basketball
$6.99A powerful and spiritually satisfying novel from a compelling new voice about three courageous women who must confront the harsh realities of their lives through faith and prayer. Pam Lyons has a husband who places more trust in money and marijuana than in God. Yvonne Hastings is a minister's wife whose husband's infidelity and physical abuse brings their marriage to a crossroads. Taylor Johnson
$12.99Working hard to achieve a promotion in spite of a difficult boss and a budding office romance, talk show producer and Christian Sabrina Bradley accepts an opportunity to produce a television segment involving kept New Year's resolutions, but finds the offer compromised by her own resolution that she would fall in love.
$9.99When child care worker Tamara Britton is asked to temporarily take 14-year-old foster child Sienna Larson into her home, she agrees--but only because it's temporary. Soon, Tamara finds the outspoken, churchgoing teenager to be almost more than she can bear. Yet, despite herself, Tamara begins to open her heart as well as her home. Now, through the bond she forms with Sienna, Tamara begins to
$9.99He's the man if you're lonely, lost, or just want to feel good. He's Jermaine Hill, megahot motivational speaker who inspires people everywhere to go for their dreams and make their lives better. With a top-rated TV show, money beyond his wildest dreams, and women for the asking, he's got everything...except a reason to keep on living. For Jermaine's past taught him that life is about losing


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Originally written on Steelers Depot | Last updated 5/10/13
Free agent tackle Max Starks visited with the San Diego Chargers on Wednesday, but according to Michael Gehlken of The San Diego Union-Tribune, he left town without a contract and remains unsigned as of Friday. Gehlken goes on to state in his reports that just because Starks left without a contract, doesn't necessarily mean that the Chargers won't have interest in signing him at later date. The Chargers reportedly had interest in left tackle Bryant Mckinnie prior to him re-signing back with the Baltimore Ravens earlier this week, so it is not surprising that they wanted to kick the tires Read more [...]
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Rock Stars vs. Rap Stars
Sidestepping the authorities may be something rap and rock has in common, but what happens when the authorities are us? Josh Homme, frontman for Queens of the Stone Age, got upset by Jay Z's tight security measures for his 2013 Made in America Festival and refused to perform if his band got a bag check. "That guy's a kook," Homme said in an interview later. "He has his security frisking the bands on the way in. No one's ever done that. You shouldn't frisk my guys, you should f--k off."
By the end, however, it worked out and the band did perform. This is far from the first time Jay's clashed with rockers. Click on to see other examples of when rappers and rockers engage in some cross-genre beef.
(Photos from left: Brian Ach/Getty Images,Theo Wargo/Getty Images)
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Black Ops Annihilation DLC to land June 28th?
Call of Duty: Black Ops is to receive its third downloadable content pack later this month with the 'Annihilation' map pack to hit Xbox Live on June 28th, leaked in-store advertising has revealed.
Outing four new maps and an additional Zombie level the Call of Duty: Black Ops Annihilation DLC information comes courtesy of leaked GameSpot press materials which follow on from a similar advertising leak earlier this week.
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Following on from the First Strike and Escalation DLCs that have already landed for the latest Call of Duty title, the leaked poster suggests the Annihilation add-on will introduce maps entitled Hangar, Hazard, Silo and Drive-In.
Black Ops' publisher Activision has yet to confirm the existence of any Annihilation downloadable content with the mooted June 28th arrival primed to see the third pack of additional Black Ops content land exclusively for Xbox Live users with PS3 and PC gamers to get the DLC in the following months.
Is Call of Duty: Black Ops the best Call of Duty game to date or will the upcoming Modern Warfare 3 release make it pale in insignificance? Let us know via the T3 Twitter and Facebook feeds.
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text: Warning: Creating default object from empty value in [path]/packages/vbcms/controller/list.php on line 426 Lonestar Airsoft - Obsidian Register Help Remember Me? What's New? Articles The Front Page Documentation Forum Gallery Advanced Search Home The Front Page If this is your first visit, be sure to check out the FAQ by clicking the link above. You may have to register before you can post: click the register link above to proceed. To start viewing messages, select the forum that you want to visit from the selection below. Section Widget Documentation Documentation Categories Widget (Show All) Article (0) CMS (0) Forums (0) Insert Images (0) Promote (0) Recent Articles Obsidian Fun with Hackers by Obsidian View Profile View Forum Posts Private Message Visit Homepage View Articles Published on 03-20-2011 07:32 PM As many have noticed someone apparently had nothing better to do with time on their hands. Thanks to everyone that let ... Read More 8 Comments News from the forum RSS Xcortech X3300W Advanced BB Control System USMC M45 CQBP G&G GMG-42 Tragic deaths in France linked to Airsoft Sentou Youi Type 3 Body armour Vest WE Mauser C96 New TRMR X4 Grenade E&L GBBR AR15 Recent Article Comments Widget -- LSA -- Default Mobile Style Contact Us Lonestar Airsoft Archive Web HostingWeb Hosting Top All times are GMT -5. The time now is 04:29 AM. Powered by vBulletin® Version 4.2.1 Copyright © 2015 vBulletin Solutions, Inc. All rights reserved. Tabs System by vbSoporte - vBulletin en Español All logos and trademarks are property of their respective owners. Comments are property of their posters, all the rest © by Lonestar Airsoft [Output: 23.95 Kb. compressed to 21.83 Kb. by saving 2.12 Kb. (8.87%)]


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Martha Minow
Clerkships, Federal Government, Federal Judges, Feeder Judges, Job Searches, Law Schools, Martha Minow
The Law Clerk Hiring Plan, R.I.P.
The Law Clerk Hiring Plan appears to be dead. At least one top law school has openly abandoned it….
Commencement, Eric Holder, Law Schools, Martha Minow
Commencement Speakers: Coming To A Law School Near You
Will a commencement speaker this year cause a controversy?
Success Tips For New Associates
Succeeding as a new associate is a juggling act that will involve balancing your evolving legal expertise with managing your workload, creating relationships with partners, fellow associates and support staff at your firm, and building strong business relationships. Here are a few tips to help you thrive during this pivotal time in your legal career. […]
Biglaw, Job Searches, Law School Deans, Law Schools, Martha Minow
Quote of the Day: HARVARD LAW SECURE
While no one was immune from the economic downturn, over the past two years, graduate employment figures for Harvard law students have matched those over the prior twenty years. — Martha Minow, dean of Harvard Law School, in an email sent to the HLS class of 2012 (reprinted in full after the jump).
Commencement, Guido Calabresi, Law Schools, Martha Minow, Ruth Bader Ginsburg
Even Harvard and Yale Couldn't Pull Off Flawless Commencements
Here are a couple of things I learned last week: 1) Above the Law readers love commencement train wreck stories. 2) Emory Law students feel picked on. Armed with this new information, I bring you stories of commencement ridiculousness at schools with student bodies mature enough to take a little scrutiny. Graduation has come and […]
Biglaw, Labor / Employment, Martha Minow, Minority Issues, Partner Issues, Racism
(Potential) Lawsuit of the Day: More From John Ray About Ropes & Gray
On Friday, we discussed the discrimination claims made against Ropes & Gray by John H. Ray III. Ray, a 2000 graduate of Harvard Law School and an African-American man, claimed that he was discriminated against and passed over for partner on account of his race. At the time of our prior post, Ray did not […]
Biglaw, Labor / Employment, Martha Minow, Minority Issues, Partner Issues, Racism
(Potential) Lawsuit of the Day: Another Discrimination Claim Against Ropes & Gray
When I worked in private practice, I once had a case opposite Ropes & Gray. The Ropes lawyers made a highly positive impression on me. They were very talented advocates (and they continue to be talented advocates; note the firm's recent, high-profile victory in the defense of an in-house lawyer for a drug company). Of […]
Grade Reform, Law Schools, Martha Minow
Harvard Law School to Stealth Grade Reform
Last year, Harvard Law School abandoned letter grading and went to a High Pass/Pass/Low Pass/Fail system. The news was greeted with much fanfare, as it seemed like HLS was trying to become a kinder, gentler academic environment — one that wouldn't be dominated by cutthroat competition to beat the curve. You know, something like a […]
Elena Kagan, Martha Minow, Politics, SCOTUS, Senate Judiciary Committee, Solicitor General's Office, Sonia Sotomayor, Supreme Court
Congratulations to Associate Justice Elena Kagan!
This should not come as a huge surprise, but Solicitor General Elena Kagan was just confirmed by the Senate as to be the 112th justice of the United States Supreme Court. Kagan, the first woman to serve as Solicitor General, is the fourth woman ever to serve on the Court. CORRECTION: I replaced "as" with […]
Law Schools, Martha Minow, Rankings
Can Stanford Overtake Harvard and Yale and Become the #1 Law School?
Hello, West Coast readers! How's it hangin' out there past the Rockies? Here at Above the Law, we try to overcome any suggestion of East Coast bias by consistently publishing a post later in the day for our readers in the Pacific time zone. And we try to be generally aware of West Coast firms […]
Kinney's Evan Jowers Now Permanently Based In Hong Kong
After a decade of 60+ trips to Hong Kong from his former Miami home, our Evan Jowers has finally taken the plunge and moved to Hong Kong on a permanent basis. Since '06, Evan has been head of Kinney's Asia recruiting and over that time Kinney has easily placed more US associates, counsels and partners at top tier US and UK firms than any other recruiting firm (we have also made many in-house placements). (…)
Email Scandals, Harvard Law Review, Martha Minow, Racism
The Harvard Law School 'Racist' Email Controversy:
Dean Martha Minow Weighs In
A replacement for Elena Kagan as dean of Harvard Law School has been named. It's the well-liked law school professor Martha Minow. Harvard University President Drew Faust announced the news to HLS students, today: I am delighted to let you know that Martha Minow has agreed to serve as the next Dean of Harvard Law […]


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We and numerous others have long used sheep as a predictive model system in which to explore stem cell transplantation. Unfortunately, while numerous markers are available to identify and isolate human HSC, no reagents exist that allow HSC/progenitors from sheep to be identified or purified, greatly impeding the application of this well-established large animal model to the study of autologous or allogeneic HSC transplantation. The current studies were undertaken to create a monoclonal antibody (MoAb) to sheep CD34 that would enable isolation and study of sheep HSC/progenitors.
A partial cDNA to the extracellular domain of the sheep CD34 antigen was PCR cloned, characterized, and used to genetically immunize mice and create hybridomas.
The resultant MoAb to sheep CD34 allows flow cytometric detection of sheep HSC/progenitors present within bone marrow, cord blood, and mobilized peripheral blood. Moreover, this antibody can be used to enrich for HSC/progenitors with enhanced in vitro colony-forming potential, and also identifies endothelial cells in situ within paraffin-embedded tissue sections, in similarity to antibodies to human CD34.
The availability of this monoclonal antibody recognizing the stem cell antigen CD34 in sheep will greatly facilitate the study of autologous and allogeneic HSC transplantation using this clinically relevant large animal model.
Sheep have long been used as a predictive model system in which to study development, disease, and physiology [1-10]. As a result of this physiologic similarity, since 1979, we and others have used the sheep model to explore stem cell transplantation [3, 10-28]. The large size and long life span of the sheep make it well-suited for the study of stem cell transplantation, since they allow evaluation of donor cell activity in the same animal for years after transplant and enable the investigator to obtain sufficient donor cells from the primary recipients to perform serial transplantation. Furthermore, by transplanting early in gestation, prior to immune maturation, it is possible to study enriched populations of putative human hematopoietic stem cells (HSC) in a healthy physiologically normal environment. Indeed, successful engraftment and multilineage differentiation of human HSC derived from fetal liver, fetal bone marrow, cord blood, adult bone marrow, and mobilized adult peripheral blood has now been observed in primary, secondary, and tertiary recipients using this model system [12, 15, 29-32].
However, while this model is ideal for studying the potential and behavior of human stem cells, as a xenogeneic model, events observed may not entirely reproduce what would be seen in a clinical setting. Unfortunately, while numerous markers are available to identify and isolate primitive human HSC, no reagents exist that identify or purify HSC/progenitors from sheep for transplantation studies, greatly impeding the application of this large animal model system to the study of autologous or allogeneic HSC transplantation.
Numerous markers are present on human HSC, but to date, CD34 has been the most widely used for HSC identification and isolation. CD34 is an integral membrane glycoprotein whose precise function is largely unknown [33, 34]. CD34 was first identified using the early human myeloblastic cell line KG-1a [35, 36], and CD34+ cells represent roughly 1-3% of bone marrow mononuclear cells (BMMNC) in a normal adult [33, 34]. Recent studies have now demonstrated that CD34 expression by HSC is a reversible process influenced by cell activation, and that some of the most primitive quiescent HSC may in fact be CD34- [37-41]. Nevertheless, the demonstration that autologous BM CD34+ were able to durably engraft baboons [42], led to the testing of human CD34+ cells for both autologous and allogeneic transplantations. This enriched cell population has produced durable hematopoietic reconstitution in both settings, providing evidence that CD34 is expressed on at least some of the most primitive long-term engrafting HSC, and establishing the rationale for widespread use of CD34+ cells for clinical transplantations.
Although we and others have used the fetal sheep model extensively to study the potential and behavior of human HSC, there are no antibodies which allow identification or purification of sheep HSC/progenitors, hindering the development of experimental HSC transplantation strategies in this model. Therefore, in the present studies, we developed monoclonal antibodies to ovine CD34. We PCR cloned and sequenced an 858bp cDNA corresponding to the extracellular domain of sheep CD34, genetically immunized mice, and created monoclonal antibodies. One antibody (8D11) was selected for all subsequent studies. Using flow cytometry, 8D11 identified a small, discrete population of CD45+ cells within sheep BM and cord blood (CB). This population comprised 1.1±0.4% of the total sheep BMMNC and 3.7±0.4% in CB, proportions in close accord with the incidence of CD34+ cells in human BM and CB. The ability of 8D11 to enrich for sheep hematopoietic progenitors was demonstrated by magnetically sorting 8D11+ cells and showing that these CD34+ cells were roughly 100-fold enriched for colony-forming potential (CFU) and 10-fold for CAFC as compared with BMMNC, whereas CD34-negative cells were devoid of progenitors with colony-forming potential. Further evidence of the utility of 8D11 as a marker of primitive hematopoietic cells in the sheep model came from studies in which gene-marked HSC/progenitors were identified in vivo with 8D11 2.5 years after in utero gene transfer, and studies which showed that G-CSF mobilization resulted in a 56-fold increase in the absolute levels of circulating CD34+ cells on day 2 of mobilization. In addition to its ability to identify sheep HSC/progenitors, 8D11 also robustly labeled the lining of blood vessels in sheep tissues, further extending the utility of this antibody.
In conclusion, this first successful generation of a monoclonal antibody to sheep CD34 will greatly facilitate using the sheep as a large animal model to study allogeneic HSC transplantation both in utero and in post-natal recipients using BM, CB, and mobilized PB as cell sources.
To obtain a cDNA clone for the extracellular domain of the sheep CD34 molecule, RNA purified from freshly isolated sheep dermal fibroblasts was provided by Dr. Paul Simmons and used to synthesize cDNA using the SuperScript® II First-Strand Synthesis System (Invitrogen, Carlsbad, CA). These resultant cDNAs were employed as templates to amplify an 858bp fragment of the extracellular domain of sheep CD34 by PCR (30 cycles) using the following primers: sense primer: 5′-atgctgggccgcaggggcgcg-3′; antisense primer: 5′-ggtcttccgggaatagctctggtg -3′. Since the sheep CD34 gene had not been sequenced, these primers were designed based on the NCBI sequence for bovine CD34. The resultant PCR product was analyzed on a 0.8% agarose gel to confirm that the correct size product had been obtained. The 858bp band corresponding to the extracellular domain of the sheep CD34 gene was excised from the gel, purified using the Qiaex II kit (Qiagen, Inc., Valencia, CA) and cloned into the pGEM-T Easy vector (Promega, San Luis Obispo, CA), according to manufacturer's instructions. The recombinant plasmid was propagated in MAX Efficiency® DH5α™ Competent Cells (Invitrogen, Carslbad, CA) and purified using the QiaPrep Midiprep system (Qiagen). Sequencing was performed using SP6 and T7 sequencing primers at the Nevada Genomics Center. Once the DNA sequence had been analyzed and aligned with bovine and goat sequences for CD34 to confirm its identity, the recombinant pGEM-T Easy plasmid was provided to Genovac AG (Freiburg, Germany).
The recombinant sheep CD34 plasmid (pGEM-T Easy) was employed by Genovac AG (Freiburg, Germany) for the commercial production of custom monoclonal antibodies using a proprietary procedure. Briefly, the sheep CD34 cDNA was subcloned into a proprietary expression vector and used to genetically immunize mice by repeated intra-dermal injections, thus stimulating an immune response. Lymphocytes harvested from the mice were fused to mouse myeloma cells to establish CD34-specific hybridomas. The hybridomas were screened at Genovac using proprietary recombinant cells transfected in vitro with a sheep CD34-expression vector. We then tested positive hybridoma supernatants on primary sheep hematopoietic cells and tissue sections. All sheep were a Rambouillet/Merino cross. One clone (8D11) was chosen for subsequent subcloning, yielding a pure IgG1-producing hybridoma, the supernatant of which was used for all subsequent studies.
BM was aspirated into heparinized syringes from the posterior iliac crest of healthy control adult sheep following the procedure detailed in an IACUC-approved protocol, and BMMNC isolated by Ficoll-Hypaque (Sigma Chemicals) density gradient centrifugation, washed twice and resuspended in IMDM, 10% FCS.
Cord blood was aspirated into heparinized syringes from the umbilical vein of healthy control sheep during delivery following the procedure detailed in an IACUC-approved protocol. CBMNC were isolated as detailed above for BMMNC.
BMMNC were cultured at a concentration of 5×105/ml in methylcellulose (CFU-Mix, CFU-GM, BFU-E) using erythropoietin–containing MethoCult 4330 supplemented with recombinant ovine IL-3 (100U/ml), GM-CSF (100U/ml), SCF (1000U/mL) and sheep leukocyte-derived PHA-stimulated leukocyte conditioned medium (PHA-LCM) (5% vol/vol), as previously described [13, 43]. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 9-12 days. Hematopoietic colonies were then enumerated in situ [13, 43]. In the case of magnetically sorted CD34+ BM cells, cells obtained after MiniMacs sorting were cultured at 1×105cells/ml.
CAFC assays were performed essentially as previously described for mouse [44-46] with minor modifications. In short, a sheep stromal layer was grown in 96-well microtiter plates (Costar, Cambridge, MA) in IMDM supplemented with 10% fetal calf serum, 5% horse serum, 10-5M hydrocortisone, 3.3mM L-glutamine, 80U/ml penicillin, 80μg/ml streptomycin, and 10-4M β–mercaptoethanol. These layers were then grow, to confluence, treated with Mitomycin C, and seeded with either whole unfractionated sheep BM or 8D11-selected BM cells at 20000, 10000, 5000, 2500, 1250, 625, 312, 156, 78, and 39 cells per well. The cells were maintained at 37°C and 5% CO2. Half of the medium was replaced weekly, and wells were evaluated at days 28 and 35 for cobblestone areas growing underneath the stromal layer, since it is well established that day 28 and 35 CAFC represent the most primitive HSC with long-term repopulating ability.
1×106 sheep PB, CB, or BM MNC were transferred to separate tubes, pelleted, and resuspended in PBS, 0.1% sodium azide. Cells were fixed in 2% paraformaldehyde, blocked for 15 minutes with 10% normal goat serum in TBS, and stained for 30 minutes at room temperature with a 1:10 dilution of supernatant containing monoclonal anti-sheep CD34. CD34 staining was then visualized with a PE-labeled anti-mouse IgG secondary antibody (Southern Biotech, Birmingham, AL) diluted 1:20, washed, and the cells stained with a 1:10 dilution of FITC-labeled antibody to sheep CD45 (AbD Serotec, Raleigh, NC). Following a final wash, cells were analyzed on a FACScan (Becton Dickinson Immuno-Systems, San Jose, CA). Negative controls consisted of PB, CB, and BM MNC stained with IgG1 control antibody and the identical PE-labeled secondary antibody used for CD34 visualization.
Human recombinant granulocyte–colony-stimulating factor (G-CSF) Neupogen (Amgen, Inc., Thousand Oaks, CA) was administered for 4 days once a day subcutaneously in the morning, weighing animals before injection, and using a dose of 4.8-5.7μg/kg. PB was drawn daily prior to injection and analyzed by flow cytometry to evaluate the total white blood cell (WBC) count and CD34+ progenitor cell content within the circulation.
BMMNC were washed twice with MiniMacs (Miltenyi Biotec, Inc., Auburn, CA) wash buffer, counted, and brought to a concentration of 108 cells per 300μl of buffer. 300μl of cell suspension was stained for 30min at 4°C with 50μl of monoclonal anti-ovine CD34. Samples were washed with MiniMacs buffer and the cell pellet resuspended in 300ul of MiniMacs buffer and incubated with 50μl of Goat anti-mouse IgG microbeads for 15min at room temperature. Samples were again washed and resuspended in 500μl of MiniMacs buffer. CD34+ cells were then separated on a MiniMacs magnetic column as per the manufacturer's instructions (Miltenyi Biotec, Inc.).
Paraffin-embedded sections were de-waxed in xylene and rehydrated through a graded ethanol series to diH2O. Sections were blocked for 15-minutes with serum-free Protein Block (Dako, Carpentaria, CA) and incubated at room temperature for 1 hour with a 1:10 dilution of 8D11 supernatant. Residual unbound primary antibody was removed by three washes in TBS, containing 0.05% Tween 20. Sections were then incubated for 30 minutes at room temperature in the dark with PE-conjugated anti-mouse IgG secondary antibody (Southern Biotech) diluted 1:20. After three 1 minute washes, sections were counterstained with DAPI/Antifade solution (Vector Laboratories, Burlingame, CA) and coverslipped. Slides were viewed on an Olympus BX60 microscope. Sections processed in the absence of primary antibody served as controls.
To characterize the hematopoietic protein recognized by clone 8D11, sheep BMMNC were magnetically sorted with clone 8D11. The resultant cell pellet was resuspended in 200μl of PBS and 200μl of 2X protein sample buffer (0.5M Tris-HCl pH 6.8, 5% glycerol, 2% SDS and 100 mM DTT). Samples were boiled for 5 minutes and centrifuged at 16,000g for 15 min to remove insoluble material. Samples were loaded onto a pre-cast 7.5% Ready gel with a 4.5% stacker (BioRad Laboratories, Hercules, CA) and run for 1 hour at 200 volts. After electrophoresis, the proteins were transferred to nitrocellulose membranes in a Bio-Rad Electroblotter Apparatus. Membranes were blocked in TBST (TBS, 0.05% Tween 20) containing 5% milk for 1 h at room temperature and then reacted overnight at 4°C with 8D11 diluted 1:50 in TBST-milk. After three washes in TBST, the membranes were treated with affinity-purified horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega, Madison, WI) diluted 1:1,000. Membranes were washed three times in TBST, and then developed with a DAB-based HRP detection kit (Vector Laboratories, Burlingame, CA).
All images were captured with an Olympus DP70 CCD camera attached to an Olympus BX60 microscope, using Olympus DP Controller Software version 126.96.36.199 (Olympus America, Melville, NY, USA). Images were then subjected to minimal global processing, such as brightness and contrast adjustment and color balance in Adobe Photoshop CS.
RNA isolated from sheep dermal fibroblasts was reverse transcribed to cDNA and subjected to PCR with primers designed to amplify the extracellular domain of the sheep CD34 molecule, as described in Materials and Methods. Since the sheep sequence was unknown at the time of initiating these studies, we designed the primers using the bovine CD34 sequence based on the rationale that, as a general rule, sheep and cow homology at the nucleotide level is greater than 90% within the coding regions. PCR amplification yielded an 858bp product (Figure 1). The full nucleotide sequence of this cDNA is shown in Figure 2A. This sequence was then subjected to BLAST sequence alignment search against the cow and human databases at NCBI, which revealed that the extracellular domain of the sheep CD34 molecule shares roughly 92% homology with the bovine counterpart. When aligned to the human CD34 sequence, sheep CD34 shared 90% homology for the first 84 nucleotides and roughly 78% homology from nucleotides 453-858, while exhibiting minimal homology between nucleotides 85 and 452. We next analyzed the amplified sequence using NCBI's ORF Finder and confirmed that we had obtained a single open reading frame, the first three nucleotides of which were the ATG start codon. The resultant amino acid sequence for the extracellular domain of sheep CD34 appears in Figure 2B. When aligned to the cow and human protein sequences using NCBI's Protein Blast alignment tool, we found that the extracellular portion of sheep CD34 was roughly 85% homologous to bovine CD34 and 53% homologous to human CD34 at the amino acid level.
The recombinant pGEM-T Easy plasmid clone harboring the sheep CD34 extracellular domain was employed for the commercial production of custom monoclonal antibodies at Genovac AG (please see Materials and Methods for details). The production of CD34-specific hybridomas is described in detail in the Materials and Methods. The hybridoma supernatants were initially screened at Genovac by flow cytometry using cells transfected in vitro either with the CD34-expression vector, which was used to immunize the mice or with the identical "empty" vector containing no CD34 insert. Using this procedure, 6 potential clones, producing ovine CD34-specific monoclonal antibodies, were identified. As can be seen in Figure 3A, which is a representative FACS plot of these six most promising clones, this procedure resulted in the generation of hybridomas that yielded good specificity for ovine CD34. Subsequently, the hybridoma supernatants from these positive clones were further tested on primary sheep hematopoietic cells and sheep tissue sections at the University of Nevada, Reno, as detailed below.
Having established that the 6 hybridoma clones potentially recognized the extracellular domain of ovine CD34, based on the screening employing transiently transfected cells (see above), we next attempted to determine whether these antibodies were capable of identifying and ultimately enriching for hematopoietic stem/progenitor cells. To accomplish this, we collected cord blood from a panel of normal healthy sheep at birth (n=12) and bone marrow aspirates from a panel of normal healthy adult sheep (n=13), and mononuclear cells were obtained by Ficoll-Hypaque density gradient centrifugation. These cells were then stained with anti-ovine CD45-FITC and each of the six candidate anti-ovine CD34 monoclonal antibodies. The anti-ovine CD34 antibodies were then detected with anti-mouse IgG-PE and the cells analyzed by flow cytometry. Each of these clones was able to detect a small, discrete population of CD45+CD34+ cells within the cord blood and bone marrow of each of the sheep. However, clone 8D11 consistently gave the best results with all 10 sheep in the panel, for this reason, clone 8D11 was used for the remaining studies in this manuscript. Figure 4A shows a representative FACS plot obtained when BMMNC from one of the 13 control sheep were stained with clone 8D11, and Figure 4B shows a representative FACS plot obtained when CBMNC from one of the 12 control sheep were stained with clone 8D11. The CD45+CD34+ population identified with this clone comprised 1.1±0.4% of the total marrow nucleated cells (n=13) and 3.7±0.4% of the total cord blood nucleated cells (n=12). These percentages are in agreement with the levels of CD34 cells found in human cord blood and adult human bone marrow, suggesting that the antibodies were identifying the appropriate cell population. To confirm this experimentally, we used clone 8D11 to perform magnetic cell sorting (MiniMacs) of either fresh (n=4) or frozen (n=3) adult sheep bone marrow mononuclear cells and assessed the methylcellulose colony-forming potential of the cells prior to and following magnetic sorting to assess the ability of this antibody to enrich for primitive sheep stem/progenitor cells. As can be seen in Figure 5A, which displays the results obtained with fresh sheep bone marrow, magnetic selection with clone 8D11 resulted in a roughly 100-fold enrichment for cells with colony-forming potential (p<0.001, 2-tailed t-test), since the CD34+ fraction generated 134±10 colonies per 103 cells plated, while the unfractionated BM cells generated only 1.36±0.06 colonies per 103 cells plated. Moreover, when the CD34- flow through fraction from the MiniMacs column was plated in methylcellulose, this population only generated 0.1±0.08 colonies/103 plated cells, strongly supporting the conclusion that the primitive stem/progenitor cells with colony-forming potential were found almost exclusively within the CD34+ fraction identified with clone 8D11. Three additional experiments were then performed with frozen sheep bone marrow, again assessing the ability of the 8D11 clone to identify and enrich for cells with primitive colony-forming potential. As we saw with the fresh bone marrow, essentially all cells with colony-forming potential were present within the CD34+ cell fraction producing an average of 177±138, 98±17, and 223±39 colonies per 103 cells plated in each experiment, respectively. In contrast, the CD34- fraction did not produce any colonies in any of the three experiments conducted with frozen sheep BM, even if plated at a 50-fold higher cell number (5×104 cells).
Further evidence for the ability of clone 8D11 to identify and allow isolation of sheep hematopoietic cells with primitive colony-forming potential came from studies in which we compared the frequency of cobblestone are-forming cells (CAFC) in unfractionated sheep bone marrow to that in the cells isolated based on 8D11 positivity. The results of these analyses (n=3) can be seen in Figure 5B. As shown in this figure, which is data from experiments in which 10,000 cells were plated per well, magnetic sorting with the 8D11 clone resulted in a roughly 10-fold enrichment for cells with CAFC potential (p<0.001, 2-tailed t-test). As we progressed down the limiting dilution curve, CAFC continued to be observed in the majority of wells with 8D11-selected cells down to our lowest dilution of only 39 cells per well. In contrast, when looking at the unfractionated marrow, CAFC were only observed in a fraction of the wells that had 312 cells, and were not observed in any of the wells in which fewer than 312 cells were plated. These data thus demonstrate that 8D11 labels hematopoietic cells with very primitive in vitro colony-forming potential, strongly suggesting it identifies HSC/progenitors present within sheep bone marrow.
To confirm the specificity of clone 8D11 for the CD34 antigen expressed on ovine HSC/progenitors, bone marrow mononuclear cells from a control sheep were subjected to magnetic cell sorting with clone 8D11. Protein extracts from the resultant CD34+ cells were then analyzed by Western blotting as detailed in the materials and methods, using 8D11 as the primary antibody. As can be seen in Figure 5C, this antibody recognizes a single protein near the anticipated molecular weight of roughly 100-110kDa[47], thus confirming its specificity for the ovine CD34 antigen.
To further test the ability of 8D11 to identify primitive HSC and establish whether the identified cells possessed in vivo functionality, we used this antibody in other ongoing in utero gene transfer (IUGT) studies in our laboratory in which retroviral vectors were injected into the peritoneal cavity of early gestational sheep with the goal of achieving transduction of long-lived HSC in vivo [48-50]. These animals have exhibited long-term transduction within their hematopoietic system, some for over 5 years after in utero treatment, and we have previously reported that transplantation of unfractionated marrow from these sheep into secondary fetal sheep recipients results in long-term transgene presence/expression within the hematopoietic system of these secondary recipients [48-50]. In the present study, we used 8D11 to sort CD34+ cells from the bone marrow of several of the sheep from these studies to assess the presence of vector DNA within the HSC/progenitor compartment. As shown in Figure 6, which is a representative PCR from 2 of these animals (amplified as we have previously described [48-50]), CD34+ cells sorted from the BM of these animals at over 2.5 years after IUGT were still positive for the vector-encoded NeoR gene, confirming that the long-term persistence of transduced cells within the hematopoietic system of these sheep was indeed due to transduction of primitive CD34+ HSC/progenitors, and providing compelling, albeit somewhat indirect, evidence that the 8D11 antibody allows identification of cells with long-term repopulating ability in vivo.
We have previously demonstrated that administration of human G-CSF (Neupogen) to sheep results in effective mobilization, as evidenced by a rapid rise on white blood cell count [12]. In the present studies, we wished to assess whether administration of G-CSF would mobilize HSC/progenitors identifiable with 8D11. As in our prior studies, administration of G-CSF resulted in a rapid rise in total white blood cell count from 1.4×106 cells/ml at day 0 to 12.8×106 cells/ml by day 2. While low levels of CD45+CD34+ cells (as identified by 8D11) were observed in the peripheral blood at day 0 (2.24%), by day 2 of mobilization, the levels of CD45+CD34+ cells had risen 6.2-fold to 13.83%. Even more striking was the rise in absolute numbers of CD45+CD34+ cells in the circulation as a result of G-CSF mobilization. After taking into account the rise in total white blood cell count, the levels of CD45+CD34+ cells in circulation increased from 31×104 cells/ml at day 0 to 1.77×106 cells/ml by day 2 of mobilization, representing a 56-fold increase in the absolute numbers of CD45+CD34+ cells in circulation. By day 4, levels had decreased back to 4.79% which, due to the increased levels of total white blood cells, still represented roughly 27-fold higher absolute numbers of CD34+ cells/ml of PB than at day 0 prior to mobilization. In similarity to what is seen in clinical patients undergoing mobilization with G-CSF, we also observed a modest 1.7-fold increase in the percentage of CD34+ cells within the bone marrow of sheep receiving G-CSF, with this rise occurring at day 4. These studies thus demonstrate that G-CSF efficiently mobilizes CD34+ HSC/progenitors in sheep, and provides further confirmation that clone 8D11 allows identification of primitive HSC/progenitors in vivo. Figure 4C shows a representative dot plot obtained after staining normal sheep peripheral blood with clone 8D11 prior to and at two days following mobilization with G-CSF.
In addition to hematopoietic stem/progenitor cells, microvascular endothelial cells are one of the main cell types within the body that express significant levels of the CD34 antigen[51]. Having demonstrated that the anti-ovine CD34 monoclonal antibodies were able to identify and allow selection of hematopoietic stem/progenitor cells, we next investigated whether these antibodies could also identify microvascular endothelial cells within formalin-fixed, paraffin-embedded sheep tissue blocks. To accomplish this objective, tissue sections were prepared from the liver of normal healthy adult sheep and stained with one of the anti-ovine CD34 antibodies (clone 8D11). The signal was then detected by incubating the sections with anti-mouse IgG-PE to assess whether this antibody could also be used to identify endothelial cells within this context. As can be seen in Figure 7, this antibody robustly labeled the lining of the hepatic vessels, and some hematopoietic cells within the vessels as well, providing compelling evidence that the epitope recognized by this clone is preserved upon formalin fixation and paraffin embedding, and further extending the utility of this antibody to include analysis of paraffin-embedded tissue sections.
Sheep possess several characteristics that make them well-suited as a model system in which to explore stem cell transplantation including their large size, their relatively long life span, the fact that they are outbred, allowing far more genetic variation, much like what is seen within human populations, and the fact that they share many important physiological and developmental characteristics with humans [1-10], [12, 15, 29, 30, 32, 39]. Unfortunately, while numerous markers are available to identify and isolate primitive human HSC, no reagents exist that allow hematopoietic stem and progenitor cells from sheep to be identified or purified for transplantation studies. This dearth of HSC/progenitor reagents has greatly impeded the application of this valuable large animal model system to the study of autologous or allogeneic HSC transplantation. In the present studies, we used PCR to clone a portion of the extracellular domain of the ovine CD34 molecule, using primers based on the publicly available bovine sequence, given the lack of sequence data on the sheep homolog. Sequence analysis at the nucleotide and protein level revealed a fair degree of homology to the human CD34 molecule at both levels, but, not surprisingly, a much greater level of homology to the bovine molecule.
Having established the degree of homology at the sequence level, we next determined whether the ovine CD34 molecule possessed homology to its human counterpart at the functional level. Our in vitro colony-forming assays revealed that sorting for the CD34+ fraction with the 8D11 clone resulted in a substantial enrichment for primitive hematopoietic cells with colony-forming potential at both the CFU-GM level and at the level of much more primitive CAFC. Furthermore, these analyses demonstrated that essentially all of the cells with colony-forming potential were contained within the fraction of cells labeling positively with 8D11, since the negative flow-through fraction obtained from either fresh or frozen sheep BM was incapable of forming colonies in vitro.
Further confirmation of the ability of this clone to identify functional HSC/progenitors came from our studies in which this antibody was used to sort CD34+ cells from the bone marrow of animals that had received in utero gene transfer by direct intraperitoneal vector injection prior to commencing the present studies [48-50]. These animals had consistently exhibited transgene positivity within the bone marrow and peripheral blood throughout the course of these other ongoing studies. In the present study, we used 8D11 to sort CD34+ cells from these sheep and analyze this population of cells for the presence of the vector-encoded NeoR gene by PCR. These studies demonstrated that the two animals analyzed both contained NeoR+ cells within the CD34+ population recognized by 8D11, thus providing evidence that this clone does recognize HSC/progenitors with long-term in vivo engraftment capability. This was further confirmed by studies in which we showed that G-CSF mobilization of sheep resulted in a 56-fold increase in the absolute levels of circulating CD34+ cells by day 2 of mobilization. It is important to note that the true gold standard for proving that our CD34 antibody recognizes true long-term engrafting HSC in sheep would be to perform experiments in which CD34+ cells are transplanted into sheep recipients and assessed for their ability to provide durable hematopoietic repopulation. Unfortunately, while experiments of this nature are simple and can be performed quickly in small animal models such as mice, in the sheep model, in similarity to other large animals or humans, long-term repopulation experiments would likely require an additional 8-12 months. Thus, for this initial report on the creation and characterization of the antibody to sheep CD34, we used the retroviral marking study as an indirect means of assessing the ability of this antibody to identify HSC, or at least very long-lived progenitors in vivo.
Antibodies to the human CD34 antigen have been classified into class I, II and III antibodies based on CD34 glycosylation. Unfortunately, we do not yet know whether this same class system will apply to the sheep CD34 molecule and, if so, into which class our antibody will fall, although we do know that our antibody does not recognize the human CD34 antigen (data not shown). This lack of species cross-reactivity is perhaps not surprising, given that commercially available antibodies to human CD34 do not recognize the ovine counterpart. With respect to classification of the anti-ovine CD34 antibody, we presume that since the cDNA was injected into mice, it is most likely that our antibody would fall into class III, since glycosylation in mouse is likely different from sheep. However, to answer this question definitively, future studies will be required in which the ovine CD34 antigen is deglycosylated with various enzymes and the mAb tested for retention or loss of reactivity.
In conclusion, this first successful generation of a monoclonal antibody to the sheep CD34 antigen and proof that this antibody labels primitive HSC/progenitors in vivo engrafting potential will greatly facilitate the use of the well established sheep model to study allogeneic HSC transplantation both in utero and in post-natal recipients using bone marrow, mobilized peripheral blood, and cord blood as cell sources.
This work was supported by Grant Nos. HD043038, HL52955 and HL49042 from the National Institutes of Health.
Author Contributions:
CDP: Conception and design, financial support, collection, analysis, and interpretation of data, and writing of manuscript
DDH-F and PJS: Provision of study materials and collection/interpretation of data
CS and VV: Collection and assembly of data
DT: Other: Provided veterinary care for sheep
GA-P: Conception and design, financial support, collection, analysis, and interpretation of data
EDZ: Financial support, administrative support, final approval of manuscript
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Author has written 11 stories for Young Adult, Supernatural, and Sci-Fi.
WELCOME To Darwin's Domain
26 June, 2015 - Update
Hello and welcome!
End of an Era is officially complete! Thank you so much for all the reviews and hits the story got! Remember there will be a sequel story to it, I'm hoping to start posting it mid to late July.
Reposting Rise of the Crimson Death! I'm tickled with the numbers that have stopped by to read the story! Remember I'm offering a give one, get one on reviews! So if you review and have a story I'm going to give you one in return! :D
Michael is still slated to be removed from the profile mid July so I can revise/edit the story for print!! (After rereading some of it, it can be rather pretentious at times. O_o)
What else have I been up to:
Busy busy! LOL!
TOHS stories strictly by timeline: (updated 17 May, 2015)
End of an Era (Complete)
White Out (Complete)
Sink or Swim (Complete)
Rise of the Crimson Death (In Process)
Nathan's Tale (Complete)
All Legends Die (Complete)
New Kid in Town (Complete!)
Paroled (Complete)
Bestowed (Complete)
Ah there we go... Now if you were looking for a game plan to read the stories, you're all set! Please feel free to leave a review for me - good, bad, needs improvement, errors you caught etc!
(Updated 26 June, 2015)
Gemutations: Plague Unfortunate Circumstance Tom wakes up in a strange environment with no knowledge of how he got there. He's soon about to find out, that escape may be impossible.
The Only Half Saga Invasion is nearing a close. Meirakat's army parks itself on the doorstep of the Arturo residence and it's up to Cabal and Company to get them out of the mess Garrett's gotten them into. But at what cost?
BOOKS FOR PURCHASE(Updated 17 May, 2015)
The Only Half Saga: Before the Fall Cabal was born to a vampire mother - his father was human, but turned the night of his conception. That has made Cabal a being wanted by neither human nor vampire societies. With only his mother for company, he's found that there are things even she cannot help him with about his differences. Things are made doubly difficult for them when the bombs fall - and they find they have to struggle through the detritus left behind.
The Only Half Saga: Redemption After 150 years the Earth is finally showing signs of bouncing back from nuclear devastation. Cabal is still tracking his father through the remnants of North America. A lead brings him to the town of Redemption. Though his intention is to gather information and move on, he finds himself remaining. Cabal never thought he would ever find acceptance much less love. In Redemption he manages to find both.
Tales from the Dead Zone(Vol1): A collection of short stories filling the missing years between the Only Half novels.
The Brothers Martin: Forgotten Ties Dr. Tom Martin is brought the worst case of his medical career. Standard medical techniques will not save Ms. Stevenson's life and he is forced to use experimental cybernetic techniques to bring her back from the brink. His efforts to make the process as minimally invasive as possible keep getting thwarted - which leads him to believe there is something much more sinister about the course her recovery is taking. This case is threatening to expose things about Tom's past he would rather remain secret.
Gemutations: Mercenaries and Angels, the 10th Anniversary, 2nd Edition is now available for purchase! Tigershark is a mercenary working for the CEO of a major Conglomerate Corporation. His current assignment is an information saboteur, hunting her he comes to find out things aren't always what they seem on the surface.
Gemutations: Plague Six years after the events of Mercenaries and Angels, David has taken on a new role in society, an advocate for gemue rights. Already facing some of the worst prejudice in recent history, gemues' lives only stand to get worse when creatures appear that can spread their mutation like a common cold. It is up to David and Angelina with the unexpected help of Dr. Tom Martin and his brother Steve to stop the virus before it becomes a plague.


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RUSH - R40 Live Tour 2015
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Robert Plant Acts in Indie Movie [WATCH]
If Robert Plant came to your door wanting to repair your driveway, would you buy it? The former Led Zeppelin front man appears in the upcoming British indie film, Doreen: The Movie - and you can see the clip below, in which Plant's character owns a tarmac business. The best part? Check out Plant's business ...
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Robert Plant Acts in Indie Movie [WATCH]
Rolling Stones and Ed Sheeran Perform ?Beast of Burden'
Ed Sheeran joined the Rolling Stones on stage during the band's Zip Code tour tour in Kansas City this past weekend, performing the song "Beast of Burden". Watch the fan-filmed footage below. Sheeran also opened the show, which took place at Arrowhead Stadium on Saturday. "Beast of Burden" appeared on the Stones' 1978 album, Some ...
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Rolling Stones and Ed Sheeran Perform 'Beast of Burden'
Chris Squire's Wife Releases Statement
Scotland Squire, wife of Grammy-winning bassist, vocalist and founding member of Yes, released a statement Wednesday regarding Squire's death and cremation. The statement reads, "My best friend and the love of my life Chris Squire will be cremated today in Scottsdale, Arizona at 3:00 pm. ¬Ý ¬Ý "However, knowing Chris, he might be running a ...
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Chris Squire's Wife Releases Statement
8 Great Canadian Rock Anthems
In honor of Canada Day and our friends to the North, here are eight great Canadian rock anthems. 1. Tom Cochrane - "Life Is a Highway" 2. Neil Young - "Rockin' in the Free World" 3. The Guess Who - "American Woman" 4. Bachman Turner Overdrive - "Taking Care of Business" Listen: Randy Bachman Explains ...
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8 Great Canadian Rock Anthems
AC/DC's Catalog Being Released on Streaming Services
When AC/DC's catalog was finally released digitally on iTunes in 2012, it was with much anticipation. It took a full 10 years from the time iTunes launched to the band's foray into the digital world. And in the last few¬Ý years, as music consumers moved towards streaming services, AC/DC held back again. But the band's ...
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AC/DC's Catalog Being Released on Streaming Services
Lyric Video: Delta Deep - "Down In The Delta"
Delta Deep - the new band featuring Def Leppard guitarist Phil Collen and Stone Temple Pilots bassist Robert DeLeo - have released a lyric video for their song "Down In the Delta." The track is the lead single from the band's debut album, released last week.¬Ý The album, which features appearances by Joe Elliott (Def ...
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Lyric Video: Delta Deep - "Down In The Delta"


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