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Nurses 2 XXX DVDRip XviD Pr0nStarS (Size: 1.37 GB)
Alexis Texas, Jesse Jane, Kayden Kross, Riley Steele, BiBi Jones, Selena Rose, Nikita Von James, Tommy Gunn, Erik Everhard, Manuel Ferrara, Mick Blue, Ben English, James Deen
Hotter, More Intensive Care!
The Naughtiest Nurses Of Coos County General Hospital Are At It Again!
Dr. Ben English has a hard enough time keeping his badly behaved staff in line. The added stress of his sex-crazed daughter BiBi Jones humping everyone in the hospital is too much for his ticker! Luckily Nurse Nikkita Von James knows mouth to mouth. Nurses Kayden Kross and Jesse Jane are locked in a battle of wits vs tits all for the affections of Dr. Manuel Ferrara. Aspiring model Nurse Riley Steele shows off her hottest moves for ultimate poser Erik Everhard, and curvaceous patient Alexis Texas looks to Dr. James Deen to get to the bottom of her swollen booty all while the staff is working 'round the clock to bring Selena Rose out of her coma. An orgy is just what the doctor ordered!
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Neff Brawnee Multi-Color Plaid Boxers
Item # 187637
Take An Additional 25% Off
Stash Points: 98
What is this?This is the number of points you get in The Zumiez Stash for purchasing this item. Stash points are redeemable for exclusive rewards only available to Zumiez Stash members. To redeem your points check out the rewards catalog on thestash.zumiez.com.
The Neff Brawnee boxers in the multi-plaid colorway, featuring a custom Neff elastic waist band, a standard fit made from super comfy cotton, and a Neff logo tag on the hem for extra fun.
|More Details


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Maxalt-MLT - Will Maxalt MLT make me fail a drug test?
- Posted:
- 5 Nov 2011 by luckyladyd
- Topics:
- maxalt, maxalt-mlt, drug test
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Which is more effective in treating migraines, Maxalt or Maxalt-MLT?
2 answers • 16 Aug 2009
Is is safe to take maxalt mlt daily?
i have migraines daily and have been prescribed maxalt mlt and have been taking it sucessfully. I want to know if it is okay to take daily.
3 answers • 1 Oct 2009
Will taking a M. Amphet salts 30 mlgs also known as adderaol make me fail my urine drug?
I go to a liquid methadome recovery center and take the liquid methadome every day. I am drug tested every other week. I also started probation and ...
2 answers • 15 Apr 2010
Will phenegran make me fail a drug test?
I take saouboxen on a daily basis and have been sick latley they prescribed phenegran for it but i need to make sure it doesnt show up in a drug test
1 answer • 14 Dec 2011


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Don't Mess With Texas Barbeque
Barbeque is big business nationwide, but in the Lone Star State, it's a religion.
Barbeque Heaven
Celebrate the Labor Day weekend with the best barbeque.
Fourth of July Recipes: Summer BBQ
Healthy barbeque tips in time for Independence Day.
Man Fed Bear BBQ Before Attack
Jesus Mabalot suffered minor injuries after he was attacked by a black bear.
Stephanie O'Dea's Slow-Cooked BBQ Menu
Summer's almost over so dig into this barbeque menu before it's too late.
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text: The location is good, being about 2-3kms from the Srinagar town in Uttarakhand on the highway itself. Its located near Alaknanda river and gives very nice view from the hotel rooms. The room was of comfortable and the bathroom was clean and tidy as well. Though the WiFi signals were weak in the rooms but good coverage in restaurant and... More


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Alfonso Soriano's Contract is the Worst $100 Million Contract of All Time
You won't find a Chicago Cubs fan who doesn't hate Alfonso Soriano's current deal. Sure, it was exciting to – for once – land the preeminent free agent on the[Read More…]
You won't find a Chicago Cubs fan who doesn't hate Alfonso Soriano's current deal. Sure, it was exciting to – for once – land the preeminent free agent on the[Read More…]
Kerry Wood opened the door ever so slightly this weekend to a return to the Chicago Cubs. At Ryan Dempster's charity even on Friday, Wood told the Chicago Tribune that[Read More…]
According to multiple reports, Cliff Lee is set to sign with the Philadelphia Phillies for something in the neighborhood of 5 years and $120 million. Setting aside for the moment[Read More…]
New Chicago Cubs' first baseman Carlos Pena has some big shoes to fill. Derrek Lee was the man at first for more than half of the 2000s before he was[Read More…]
Like a hot girl who keeps a couple back-up guys at the ready, Brandon Webb is going to take his time and consider all comers before landing on a team.[Read More…]
I'm at least partially kidding. But several Chicago Cubs are already working out in anticipation of the 2011 season at "Camp Colvin," an offseason workout camp in Mesa, Arizona, named[Read More…]
Cliff Lee's impending decision will affect the Chicago Cubs, even though they are not in on the free agent ace. Multiple reports have Lee down to the New York Yankees[Read More…]
Though Matt Garza is the most exciting Cubs' target this offseason, he isn't the one with a Cy Young on his mantle. And that guy, Brandon Webb, is still on[Read More…]
Another source says (1) the Tampa Bay rays are "certain" to trade Matt Garza this winter, and (2) probably to the Chicago Cubs, according to Ed Price. Unfortunately for Cubs fans,[Read More…]
There was a time – oh, say, July – when the collective thought of Chicago Cubs fans and management, alike, went something like this: "How are we ever going to[Read More…]
The Chicago Cubs were close to a deal for Tampa Bay starter Matt Garza yesterday, and then… nothing happened. Obviously 95% of trades that are discussed never actually happen. But[Read More…]
Even as other deals flounder, change, and ultimately die, there's no doubt about one Chicago Cubs move: they want Brandon Webb. Brandon Webb remains their No. 1 target, and the Cubs[Read More…]
Rarely is a non-event good news in the world of the Chicago Cubs ("why didn't Hendry sign that guy? why didn't he make that trade? why haven't the Cubs won[Read More…]
The Chicago Cubs, ever the fan of the backloaded contract, managed to backload Carlos Pena's one-year deal – it will pay him $5 million this year, and $5 million next[Read More…]
Among the bullpen options the Cubs are considering is a former target, Jason Frasor, who last week accepted the Blue Jays' offer of salary arbitration. He made $2.65 million last year,[Read More…]
This is about a lukewarmy as the Lukewarm Stove gets, but I thought it worth mentioning that, with the Red Sox signing Carl Crawford, the Chicago Cubs' task of moving[Read More…]
The 2010 Winter Meetings will conclude today with the Rule 5 Draft, wherein (generally) players who've been in the minor leagues for five or six years, but haven't yet made[Read More…]
Though the Chicago Cubs are looking at a handful of reclamation project type pitchers, and a trade for Zack Greinke, it looks like it could be Matt Garza that they[Read More…]
The Royals are actively shopping Zack Greinke, as noted on the first day of the Winter Meetings, and Greinke has agreed to waive his no-trade clause in a prospective deal.[Read More…]
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Matt Silas
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I live in Berkeley, CA with my wife, daughter and son. I work in the Camera and Staging department at Pixar. I recently finished my master's in directing/production at UCLA. I have a hard time keeping surfaces clutter free...
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The films of Ozu, Bergman, the brothers Coen, Apatow, and the two Andersons.
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Innogoods introduces the new hosepipe-free ,telescopic handle Zarpax F2 Activebrush for washing Vans,Trucks and RV,s
All the benefits of flowing water without needing a hosepipe? Zarpax makes it possible.
The unique F2 ACTIVEBRUSH™ has a built in aqua chamber which provides an active water flow for gentle cleaning.
The unique,patented valve system fills the chamber quickly (1 second) each time you dip the brush in
bucket of (warm, soapy) water.The water is slowly by the cleaning motion.The F2 ACTIVEBRUSH™ has a telescopic handle making it the perfect brush for cleaning the roofs of SUVs, Vans, RVs, Truck windows and other hard-to-reach places.
Centerforce Simplifies Your Aftermarket T-56 Swap
Many muscle car owners are looking to the aftermarket to upgrade their transmissions and one of the most popular options for this is the compact, lightweight, and strong Tremec T-56. One of the problems with swapping a modern tranny into your old iron has always been getting a variety of components from different manufacturers to line up and work together. Not having everything perfect can kill your clutch or even worse, that expensive new transmission. Centerforce saw a way to help this situation and created their new long-throw clutch slave and release bearing. This helps to compensate for stacked tolerances and allows for full clutch engagement.
First to Market: The 2015+ Ford Mustang EcoBoost Performance Aluminum Radiator
The Mishimoto Ford Mustang EcoBoost Performance Aluminum Radiator is designed and engineered to reduce coolant temperatures substantially, and to protect your 2015 Mustang from overheating during aggressive driving or hot lapping sessions. It is the ideal upgrade to the stock 2015 EcoBoost Mustang radiator, and will outperform even the upgraded Ford performance package radiator. The Mishimoto Mustang EcoBoost radiator features a large three-row core, 100% brazed aluminum, and polished end tanks, enhancing the overall performance of your engine.
The engineers at Mishimoto designed this EcoBoost radiator with a 72.5% increase in coolant capacity over the base-model radiator, and 53.3% increase in coolant capacity over the larger performance package radiator. This 2015 Mustang radiator also has a 22% increase in coolant surface area, and 50% increase in air surface area over the larger performance package radiator, making it an ideal upgrade to keep your EcoBoost running cool!
Big Mike's Performance Parts' STO N SHO Quick Release Front License Plate Bracket Now Available for the 2014-2016 Jaguar F-Type
Big Mike's Performance Parts announces the availability of the STO N SHO™ for the 2014-2016 Jaguar F-Type. The STO N SHO™ is custom fit and consists of a front license plate holder and a base mounting plate that allows for the easy removal of the front license plate when desired and the quick replacement of the front license plate when needed. When the front license plate holder is removed the remaining base plate is virtually undetectable. The STO N SHO™ allows customers to comply with state law while maintaining the beauty of their vehicle. The development of the Jaguar bracket is catapulting the STO N SHO™ into the luxury vehicle marketplace and proving that it isn't just for muscle cars. The STO N SHO™ is not only available for the Jaguar, but many other vehicles as well.
Centerforce Gives Older Ford Trucks New Life
Early Ford trucks are known for being trusty workhorses but nothing can last forever and thats especially true of a vehicle's clutch. Many owners choose to replace their clutch with a stock-style clutch, not knowing that there is a better option available to them and its because of this that Centerforce introduced their Centerforce I model clutch for early Ford trucks. The CF I functions just like a stock clutch only one that's made better and will last longer. There is no sacrifice in drivability or increase in noise. It's the perfect replacement clutch for your old workhorse.
Help Your Classic Chevy Truck, Van, or SUV Keep Putting Power to the Ground
Finally there is a modern clutch option for owners of 1962-1995 Chevy and GMC trucks, vans, and SUVs thanks to Centerforce and their Dual Friction clutch. The Dual Friction features a full facing on one side and a puck style facing on the other giving the user the best of both designs. The full face offers smooth engagement and long life while the puck side gives greatly increased holding power. The Centerforce Dual Friction kit includes a new pressure plate, Dual Friction disc, throwout bearing, pilot bearing, alignment tool and even new clutch bolts.
Ford's Newest Pony Car Deserves a Serious Clutch
The 2015 Mustang has taken the performance car market by storm. Centerforce knows that people are going to go power crazy on these things and as such decided to introduce Dual Friction, DFX, and DYAD multi-disc clutches for the 2015 Mustang GT. The Dual Friction is perfect for the owner with light to moderate modifications who refuses to sacrifice drivability. The DFX is perfect for those cars that will spend a significant portion of their life on track. The DYAD multi-disc clutch is ideal for those Mustang owners who plan to go power crazy thanks to its ability to reliably hold over 1300 lb-ft of torque without any of the noise or drivability compromises that were typically associated with multi-disc clutch setups.
New this year from Centerforce is a Dual Friction clutch kit and lightweight SFI approved flywheel for the Subaru BRZ and Scion FR-S. The Dual Friction clutch when combined with the lightweight flywheel drastically change the personality of the Toyobaru twins by helping to improve the response of its unique 2.0L flat four. The Dual Friction also opens the possibility of power increases to owners without having to worry about damaging the stock clutch or transmission. The Dual Friction clutch, lightweight flywheel, and Subaru BRZ/Scion FR-S are a perfect match!
Centerforce Debuts Clutches for 6-Cylinder Nissan/Infiniti
Centerforce is pleased to introduce several new clutches for Nissan and Infiniti cars equipped with either the VG or VQ36 engines. These include a Centerforce I model for those expecting to keep their power and torque levels as stock and simply want a better engineered and longer lasting replacement. They also introduced a Dual Friction model for those owners with mild to moderate modifications who want to dramatically increase clamp load and life without sacrificing daily drivability. Lastly they introduced a DFX clutch for those hard core track rats that wouldn't necessarily mind a couple of drivability sacrifices in the name of ultimate performance.
Weapons Grade Fabrication Profile Racks are designed to create a powerful and rugged appearance while looking like a factory option. In addition to the appearance, the racks are engineered for durability and longevity.
Each rack is vehicle specific offering a matching contour of the truck's cab and bed to give it a factory like appearance.
Weapons Grade racks offer a modular design which are available in a variety of configurations, from a front 2 corner ladder rack/headache rack to a full 4 corner bed utility/roof rack system with integration for lights and accessories.
For more information, visit us at SEMA 2015 booth #34320 or online at www.wgfab.com.


text:
White Box Testing
Also known as glass box, structural, clear box and open box testing. A software testing technique whereby explicit knowledge of the internal workings of the item being tested are used to select the test data. Unlike black box testing, white box testing uses specific knowledge of programming code to examine outputs. The test is accurate only if the tester knows what the program is supposed to do. He or she can then see if the program diverges from its intended goal. White box testing does not account for errors caused by omission, and all visible code must also be readable.
For a complete software examination, both white box and black box tests are required.
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From wacky alarm clocks to lecture hall tools and after class entertainment, these Android apps are a good fit for a student's life and budget. Read More »Sharing Threat Intelligence
A growing number of startups make the sharing of threat intelligence a key part of their solutions. Read More »Smiley Faces and Symbols
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Glary Utilities Portable
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- License:
- Free
- Language:
- English
- Windows 98
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- Latest version:
- 220.127.116.118 07/06/12
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Glary Utilities Portable is fully compatible with:
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Glary Utilities Portable is also compatible with
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Handy pocket toolkit for PC maintenance
After testing the desktop version of Glary Utilities a while ago, I was pleased to see that the developers of this great PC toolkit have now released a portable edition.
With Glary Utilities Portable you can now take all the program's tools anywhere with you, and use them to optimize, repair, tweak and improve any computer without leaving any usage traces on the host computer. Glary Utilities Portable doesn't require installation and can be run directly from any USB device.
Now, don't let yourself be fooled by the "portable" tag of this program. Glary Utilities Portable is by no means a stripped-down version of the desktop program. It does include all the system utilities you need for all sorts of PC maintenance tasks, and they're all neatly organized into several categories so you can easily find what you're looking for: disk cleaner, startup manager, tracks eraser, duplicate file finder and many more.
All the tools included in Glary Utilities Portable are generally very easy to use, so don't be afraid of trying them out. Also, in the case where any of them make important changes to the system – such as the Registry Cleaner – Glary Utilities Portable creates a backup copy first. The only weird thing is that the appearance of these tools is completely different from Glary Utilities Portable's main interface, but that's no big deal.
Glary Utilities Portable is a light yet powerful maintenance toolkit that you can take anywhere to repair, clean, optimize and improve any computer.
- Easy to use
- No installation required, can be run from a USB drive
- Impressive selection of maintenance tools
- Minor design discrepancies
"awesome program"
The best of the rest, portable, freeware!
Save before remove files... -awesome
- remove unused/temporary/unwanted files
- better then ccleaner registry cleaner
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- Date:
- 03 Jul 2014
Top Downloads: General Optimization
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Giveaway: Advanced System Optimizer
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Optimization and customization in Windows XP
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free download Microsoft office 2014
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Cannot save as the PPTX document as pdf in office 2013
Download corrupted
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text:
|Project Name
|Price (USD)
|Project Name
|Price (USD)
|Extract Email address from Singapore Job Sites
|Extract email addresses from Job sites: 1) Go to these 4 job sites i) http://www.stjobs.sg/ ii)...jobstreet.com.sg/ 2) Search for the jobs: i) Web Designer ii) Graphic Designer iii) Marketing Executive ...3) Extract for EACH job posting for the above. i) Position for Hire ii) Hiring person's email address
|Data Mining, Internet Research
|May 12, 2015
|May 12, 2015Ended
|Extract - Followersinformation 1014-1
|...for a freelancer to search and export photographers emails/addresses/website all found in web platform...notice that there is not limit on this job, this is is an open job hirings. You will use an account we...to follow followers of these photographers and extract data. Extractions must be only photographers
|Data Entry, Excel
|Oct 9, 2014
|Oct 9, 2014Ended
|Need freelancer to extract 1000 samples from a web search and web site
|Need a Freelancer to go to a website and collect the following information - Label Name - Label URL ...URL - Contact name - Email address - Address (if there) - Country It is very important that every...every sample has email. Please bid on the project and let me know how much you charge per 1000 samples
|Data Processing, Data Entry, Excel, Web Scraping, Web Search
|Jul 11, 2012
|Jul 11, 2012Ended
|Extract email
|Hi, I need extract email from web site. Can you help me? Thx Gabriel
|Apr 11, 2013
|Apr 11, 2013Ended
|Email Extract
|Hi, I want to extract all the email database from www.magicbricks.com I need all the Email database, I...want someone to extract all the data from there and first let me know what approx Email List we can generate...scrappers to extract the email list from the url, Please only bid here if you can do this job Thank You
|Internet Marketing, Marketing, Bulk Marketing, Advertising, Email Marketing
|Feb 21, 2013
|Feb 21, 2013Ended
|Extract email addresses from websites
|...to load 100 URLs into a listbox and then extract the email addresses from these websites. It should be...explore everything. Obviously, I do not expect to extract from embedded Flash movies/images, for example...com/contact.htm http://contacthelp.com/directory/Internet/Web+Sites/Craigslist?ListingID=332 as a test to start
|Engineering, Software Architecture, Software Testing
|Nov 29, 2011
|Nov 29, 2011Ended
|freelancer job
|Web designing&graphic desining
|Oct 26, 2014
|Oct 26, 2014Ended
|Extract 5,000+ names and email addresses
|(REPOSTED) This is a quick simple project job. You will be provided with a URL string containing...will take you to a web page where you will click on a link to obtain a name and email address from our...super simple - first column 'Name', second column 'Email address'. Then you will alter part of the URL string
|Data Processing, Data Entry, Excel
|Aug 29, 2015
|Aug 29, 2015Ended
|Email web extract server
|...looking for an expert in web scraping that can help up set up an email extractor web service (server solution)...solution) based on scrapy or similar Core web service is to allow registered users (local businesses)...search engine ; google.fr google.in etc.) - extract emails from webpage related - export list
|PHP, Python, Bulk Marketing, Web Scraping
|Feb 19, 2012
|Feb 19, 2012Ended
|Extract 5,000+ names and email addresses
|(REPOSTED) This is a quick simple project job. You will be provided with a URL string containing...will take you to a web page where you will click on a link to obtain a name and email address from our...super simple - first column 'Name', second column 'Email address'. Then you will alter part of the URL string
|Data Processing, Data Entry, Excel
|Aug 30, 2015
|Aug 30, 2015Ended
|web scraping------extract list from website
|we need extract the shop list from the page of "Shop Finder" one website . In this page of "Shop Finder"...the name of the shop, address, phone #, fax #, email, website, contact person. We need all the information...list. We just need the freelancer who has strong exprience and take this job very seriously and carefully
|Excel, Web Scraping, Data Mining
|Feb 7, 2012
|Feb 7, 2012Ended
|JOB freelancer
|...Website design , programming ,web dev solutions. WHY ME :- 1:- 7 Years SEO/Web Design experience 2-500
|Apr 3, 2014
|Apr 3, 2014Ended
|Job Freelancer
|...the experience to program web-based PHP applications in college. i need job freelance
|PHP, Javascript, Wordpress, eCommerce, jQuery / Prototype,
|Feb 20, 2015
|Feb 20, 2015Ended
|Extract Email Addresses from One Website - Simple Job w/ Software
|...simple job for someone who already has such web-scraping software. I require you to extract every email...email address displayed within a website. The website has over 20,000 pages (directory-styled website)...have email addresses, some don't. I need you to scrape the entire site and extract the email addresses
|Data Entry, Excel, Web Scraping, Web Search, Email Marketing
|Nov 8, 2013
|Nov 8, 2013Ended
|81245 email mod for freelancer scrpt
|Looking for someone that can make the freelancer script send html format emails out. Presently using...someone that can do the needed mods so that all email clients can see them as html and not source code...scriptalliance.com job complete terms: must be able to get emails on the various common email clients such
|Perl, Anything Goes
|Nov 16, 2002
|Nov 16, 2002Ended
|Freelancer Web Developer Required
|...of our business friends are looking for a skilled web programmer / developer who can bring fresh new ideas...contact details i.e.. telephone / mobile number, email address, skype etc…. Skill set required … PHP...on whether you have the right experience for the job All the best
|PHP, Adobe Flash, Website Design, MySQL, HTML
|Mar 31, 2012
|Mar 31, 2012Ended
|Extract email addresses and details out of set of websites
|Dear Freelancer, we are a family business in the music area and we are preparing an email campaign...Bars but not with all the information. Now the job is to complete the draft list we have and fill-in...fill-in missing information. Especially the email address or addresses (in case a club lists several) we are
|Data Processing, Data Entry, Excel
|Aug 7, 2009
|Aug 7, 2009Ended
|Web Designer Freelancer - USA
|...over-flow for current projects we have booked. The job will mainly involve website updates, new page creation...Must be an independent individual contractor/ freelancer (not a team, group or firm) - Must be as local...starting with NYC/ NJ Metro area and outward - USA freelancer only (sorry, for time-sensitivity and efficiency
|Website Design, Wordpress, CSS, HTML
|Mar 5, 2012
|Mar 5, 2012Ended
|extract emails from a site
|I need someone to extract all the emails from a certain web forum. I am unsure of how many emails there...is a simple job and I will pay $30 as I know most will just run the site through a email harvest program
|Data Processing, Data Entry, Excel
|May 14, 2012
|May 14, 2012Ended
|Extract 10,00 email addresses from URL string
|This is a quick simple project job. You will be provided with a URL strong containing some variables...will take you to a web page where you will click on a link to obtain a name and email address from our...super simple - first column 'Name', second column 'Email address'. Then you will alter part of the URL
|Data Processing, Data Entry, Excel
|Aug 29, 2015
|Aug 29, 2015Ended


text:
Find It! Books, Music, Movies & More!
Welcome to the Fort Saskatchewan Public Library! Read up on our Library News!
Application forms are available on the City of Fort Saskatchewan's website - or click here
FSPL booklet: The Library Board needs you!
"The library is looking for board members! I have two young children and I chose to become a board member last year for them. I think literacy and knowledge are so important. It's critical that we continue making sure we have a cutting edge library that provides resources to all age groups. This is a wonderful opportunity to volunteer, it's not a huge time commitment but it can make an incredible impact. If you would like to know more about what we do please don't hesitate to ask. Applications due October 16." Morgan N.
If you missed Paula Simon's visit on Saturday, September 26, you can now watch the video on YouTube!
Did you know that the Fort Saskatchewan Public Library has an email mailing list? Send your request to be added to this list to email@example.com and you'll receive our weekly New Book List and other timely announcements.


text:
its okey
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All Comments (139)
its okey
I like them
watching "normal" people fuck is probably more fun than watching the porn stars
I came more than once just watching all that, wow! The poor guy never did get off. It must be dead with not coming after all of her pussy action. Jesus, what does it take to cum for this guy?? If I was her, I would have went through the roof!
Does anyone know her name?
Wendy somer and friends vol 7, but i can't find the cast
that girl wants him so bad, its hot!!!
tolles girl; sehr natürlich - der boy ist etwas lahm, würde bei dem girl nen prallen Dauerständer bekommen
She's gorgeous and really enjoying herself. Nice to see a real couple enjoy each other so much.
came and came watching this. hot.
Dman i would like to fuck her so much. I would suck her tits if i had to i would give all my money on her.
umm i like her pussy
What an incredibly hot couple! I want both of them
Sie geht scho recht leidenschaftlich zu Werke. Lecker!
Love the kissing, passion, touching... hot
seems as if both of them have problems getting an orgasm.
Perfect body. Not so slim. Very nice natural hairy pussy. I love it.
All time favourite! It looks very natural and they are clearly enjoying themselves.
One of the hottest clips on this site. She was so hot making herself cum like that, but what a set of brass balls that guy had. He got fucked and sucked every which way and kept his shit.
Please, anyone know her name ?
sie ist sehr natürlich und sexy. ich finde sie geil. vor allem wie SIE auf ihm kommt. das hat was.
Das Girl geht meiner echten Leidenschaft zu Werke - das sieht man selten in Porno-Filmen. Sie scheint geradezu gierig nach Berührung, Küssen, Fingern, Poppen in jeder Lage zu sein... Wer ist das Girl? Gibt es noch mehr Material von Ihr (außer dem Video, welches unten aufgelistet ist)?
geile titten hat die kleine
his hair is faaaabulous!!
she is my dream, someone please find out her name
sexiest girl i've seen for ages! my thick dick was hard during the first foreplay and my balls swollen and full by the time she came for the first time! wld love her to do that stuff to me. shot a load just before her second cum tho - just had to!
got dvd wendy somers and friends vol7 to see more of this beauty but unfortunately the seen she was in was horrible
Very real clip. Loved how she had trouble taking off her shirt. They showed incredible passion for each other. Can't believe how much she gushed from her pussy!!
Very real clip. Loved how she had trouble taking off her shirt and pants. They showed incredible passion for each other. Her body is as close to perfect as you can get, with a beautiful hairy pussy. And I absolutely loved the way she drenched her partner's crotch around 23:00. One of my all-time favorites here!
All this fucking and sucking and NO cumshot! What alimp dick this guy is.
she didnt even wax for him, thts just lazy!
OMG, what a beautiful cock! I'd love to bounce on that cock (like she was at 18:00). I'd grind my clit into him, made him cum all over himself. I felt sorry for him, he obviously wanted to cum so bad and she couldn't get him to. What a shame to not be able to see that beautiful cock shoot cum all over her.
This guy just can't keep it up! Notice how nearly every position they do, he goes limp and she has to stop and blow him to get it up again?
aku juga mau
nice big natural floppy titties! she is a nice size too
Wearing a crucifix in a porno... ah, irony.
to have my gf shoot her load over my cock and going up her hairy pussy with no condom on, i dont know how i would be able not to cum. the guy in this vid should easily be shooting gallons of thick cum
i would do anything just to fuck her
i love everything about her gorgeous face, beautiful tits and ass, perfect body i'm in love!
Yes, she really want him! Nice video
Can someone identify her ? her name ?
his cock is well big!
I'd kill to last 40 minutes without cumming, but this guy's gotta be miserable--- they try everything but he stays bone dry, not a drop from those big balls. At least she gets off several times. This film would be so much sexier and more satisfying if he could finally blow his load at the end.
I like her, she is naughty!! Instead of him I would be better in this movie... and the movie would take maximum 20 min!
very hot and horny couple. super tits and wet pussy, expoding in an enormous female cumshot
super horny superrrrrrr nice tits and good fucking
fucking so sexy. so, so much better and more arousing than watching freakish porn stars. that guy is so hot, i want to see him cum tho
Snow day today. Stayed home from work and decided to watch some porn and have a couple orgasms. Had to watch this one again, because it really does the trick. He has an amazing cock and I love the size of his balls. I like a guy with big balls to suck and play with and to feel slapping against me as he is fucking me hard from behind. I only wish she could have made him cum so I could see that gorgeous cock shoot some nice warm cum all over her belly or boobs. Guess I will have to find another one for that.
I would like to fuck her in all three holes. I would also like him to fuck me with that long cock of his.
this is the one couple I could actually stand to watch over and over, i've watched this vid about 7 times more than any other one porn ive ever seen, shes so freaking hot, to spite his issues they look like such a real couple thats really into each other
this young lady is HOT. Do more with her. She loves sex and does a super job.
Dieses Video gab es frueher in einem Format, dass man es mit normalem WindowsXP herunterladen konnte. Wer dieses Video noch hat, wird gebeten, es, eventuell unter anderem Namen, hochzuladen.
what a kisser!
wow he is like the hottest mofo i have ever seen good lord
basang basa na ako habang pinapanood ko. grabe
That girl was so horny for him. LOL
This woman is amazing, and the guy should be a cock-n-balls model.
wonderful natural full breasts - there's no copying Mother Nature
I can't keep my hand out of my panties just thinking about him inside me. He's gorgeous,
this girl reminds me of my ex's friend i fucked on her bed. good stuff missy
She's slightly chubby, something I quite like, especially the belly. She's got a great pair of tits too. Very fuckable!
Can you rate this with more than 5, 5++. Super, two times squirting over his dig. I wish I was there to see it real cumming out of her.
I love that she is a squirter 23:00 and 38:00. However she did have to achieve it on her own.
Her hairy box is perfect, you just don't see it enough on these sites. Need lots more!!
Her name is Extremelara.
see also http://www.youporn.com/watch/244487/cute-girl-gets-her-hairy-asshole-stuffed-with-hard-cock-part-1/?from=related3&al=2&from_id=246618&pos=1
I love watching couples instead of porn stars, there so hot
what is wrong with this guy? talk about limpdick. and sometimes i like a hairy pussy.. but not hers, for some reason. the only girls who can get away with it are hairy japanese girls...
my cum was dripping fast while watching this vid...need some cock to slide on mine...who wants?
she looks very horny
She is hot as hell and if he can't keep it up with her he might as well give it up.He should have taken a Viagra. I would rather have my smaller model that is working than his young big one that don't.
i would absolutely demolish this lass, she's fucking stunning and she's good at sex fuck yes
Appetite love play is more exiting than hard core porn movie
Herry potter and his pet dog
this kind of sex
I'd fuck her like that but if she shaves her pussy I'd lick her untill she cums all over my face
He does not read her very well, or is just selfish. (which I guess is the same thing) Several times she seemed about to come, and he switches gears. Good thing she can take matters into her own hands when needed.
nice ass vs big boobs.
The guy did finish. Toward the end it skips ahead. He may have finished early or something and so they were going to try to proceed as if he hadn't.. but it was difficult for him to fully get it up the 2nd time. So what? They both finished and it was damn hot!
anon, I think you are correct.......He had a climax and it wasn't fully hard, as much and hard as she tried to get it rock hard.....My wife(with my full OK and green light) met her former bf/basketball coach from her 12th grade for lunch at his hotel...We all knew it wasn't fro lunch..She was lunch for his dick.....Anyway they went up to his room and kissed and played for an hour or more and then got naked and got in th ebed and he fucked her....Wel she said in 3 minutes he pulled it out and shot his load on the bed.....I'm not sure I believed that, but he did climax...She said then they turned around in the bed to get out of the wet spot and lay there and kissed and touched and she said all of a sudden it was hard again...I think she sucked it hard but she never admitted to doing it....So with it hard he fucked her a long time the second time..I wish I knew just how long it was as all of this was and still is a big turn on for me....But she said they fucke da long time and this guy was 50 years old then........Let's talk about that if you like and other things, Dan
I want to fuck that Cunt
Hot! She has a great natural body, and obviously loves to suck cock and fuck. Wow!
bestes Video
damn - bjork fuks like a pro!!!
That actually happens to me. It takes me up to three hours to finally come.
If she is gonna be satisfied with that, I would definitely not fuck her.
best vid on youporn!
PuSsY GAME ..... I love it MUAAAA
She is nicee,,esta pa darle asta por el apellido
I hope this guy knows how lucky he is. She's cute, hot, sexy, sensual, and NASTY. He's like a dial tone with a limp dick. I would make her work less and come more. What a natural beauty.
ma è il trota! il figlio di Bossi!
Love how at 19h42 she teases him by licking his asshole! Hot!


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Super 8 Suffolk Tidewater
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
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Travel Blogs from Suffolk
After over 4,000 miles, 13 different rooms/hotels and visiting over 45 Timmons and 11 Wall family members, NOT TO MENTION Rosie's mom, day and multiple uncles, aunts and of course Theresa, Rich & Ellie Goiffon we are home.
My back and left arm are killing me (Rosie;s favorite driving position.
All in all, a great time.
Thanks for following our travels.
Fighting Generals and Napping Butterflies
... which is where George Washington defeated British General Charles Cornwallis and won final victory in the Revolutionary War. The National Park Service Visitors' Center at the site has Washington's actual headquarters tent on display, and while it is protected behind glass in a special room, a tunnel allows you to walk inside and look at his personal effects. It is truly impressive. Though it was still ...
Chademo Charging, Commodore Theatre, and an i8
... to stop in for a movie and dinner while charging. What was a bit of an afterthought turned into the most fun of the day as the owner (who holds a reservation for the 17,000 and something Tesla Model X (their soon to be released SUV model) to come off the line). To give back he installed the chargers and offers free charging to Tesla owners and we are much appreciative. The theater, which opened in 1945 and was reopened in ...
Almost Home!
... to thank GOD for our safe travels. It was awesome to see how HE has touched this earth with so much beauty. (as a Camp Merrie-Woode lyric states) " We can't do anything without Gods help." Finally, I would like to thank my parents for providing so much love and joy in my life. I'm grateful my mom planned this adventure through the west. It was interesting to meet new people in ...
T minus 2 days, 8 hours and 30 minutes "ish"
... and dryer!
Today I completed my first ever "solo" camper mod that involved power tools. I successfully installed magnetic locks on our kitchen cabinet doors without any blood or tears shed! There was remarkably little damage to the camper as well, which I consider a major bonus. Now as we travel down the highway on Friday I won't have to worry about all of our dishes or canned goods flying around the camper and crashing through the ...


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For many years, the internet has been hailed as the death of print. In the periodicals area of the library, we know that's not true, because we see hundreds of students and visitors going though our magazines every semester.
Just so you know, we update our list of subscriptions all the time. We're trying to always have the newest, most interesting sources for fashion, art, and design. Here are some newer titles you might not have seen yet.
Acne Paper began as the print sidekick of the Acne Studios in Stockholm. It expresses the studio's point of view on art, fashion, and current culture, with offices in Stockholm, London, and Paris. It's grown to be an influential source for fashion ideas.
You can take a look at the work of Acne Studios:
Fat Magazine comes from similar interests in art, fashion, and daily life expressed through designed things. Published in Helsinki, the capital city of Finland, it picks up the thread of challenge common to art and brings it into current-day fashion layout, graphic design, and photography.
http://www.fat.fi/ and also
The magazine Hi Fructose is focused on illustration, not fashion. The magazine is filled with graphic design, drawing, sculpture, and other contemporary arts aimed at inspiring artists of all sorts.
You can look at more about the magazine here:
The next title, Oh Comely, comes from gentler impulses, advertising itself as a compilation that "makes people smile, full of quiet moments and stories." This magazine, published in London, is designed to be pretty and soothing, rather than challenging and thought provoking. "Read it with a cup of tea or a toddy," declares the publicity text.
And of course it comes with/from a blog…
You can find the current issues of all these titles on Display at the Periodicals Desk on the 6th floor. For the most recent additions to our list of titles, you can look at the "Newly Added Titles" tab on the Periodicals by Subject List.


text:
- 58Promote
Tablet Cases
Shineby Allan Redd
Protect your iPad with an impact resistant hard shell case featuring an extremely slim profile.
thats really beautiful!
thanks do much Daily Rorschach!!
Very cool!!
thank you Bob! i'm really glad you like it :)
I love it!
This has a lovely fresh feeling, Allan.
thank you Rebecca! :)
cool! interesting design it kind of reminds me of some sort of magical map, or the one where you have to draw a line to find you way out :)) really cool! I would look great with some line drawing on the top too! A+
thanks for the detailed feedback Vasare Nar! i'm so glad you see something in this!
Like it...
With the desire to send more customers to your S6 store, I've posted this image at: http://pinterest.com/pixel404/s6-random-favorites/ Here's another link that might be of interest: http://blog.shareaholic.com/2012/01/p...
l-traffic/ The data in the shareaholic article is from January... Pinterest has grown considerably since then! Cheers!
thanks amigo!
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Devotions for Couples
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Product Description
Product Information
|Format: Hardcover
Number of Pages: 256
Vendor: Zondervan
Publication Date: 1998
Dimensions: 8 X 5.18 (inches)
Availability: Expected to ship on or about 10/25/15.
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Publisher's Description
Is your marriage the one you pictured when you said "I do"? Is the passion still hot, the friendship still warm, and the relationship still meaningful? If not, you owe yourselves this book, delivered to you in the vulnerable style that has become the author's trademark. Written by Patrick Morley, the award-winning author and pioneer men's leader, Devotions for Couples invites you to come together and let God's Word do some searching, encouraging, and strengthening of your hearts . . . and your marriage. Both profound and practical, these 120 devotional readings will help you apply biblical truths to deepen your relationship, communicate better, understand each other, learn about what pulls you apart and how to stay together, raise your children, and share a future filled with grace, faith, growth, and appreciation of one another.
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Bob and PatriciaColorado5 Stars Out Of 5An Incredible Journey!November 14, 2010Bob and PatriciaColoradoQuality: 5Value: 5Meets Expectations: 5After 45 years of marriage and many books read, and devotionals shared, this book opened doors that were never fully open. It was an incredible journey! Our marriage is better today because of this book and we continue to share it as a gift to many.
Jim Neal5 Stars Out Of 5August 11, 2009Jim NealWe have been married over 37 years and continue to guard our marriage with good sound Bible study supplemented by Christ-centered devotionals. This devotional has been one of the best we have ever used.
Rev. Terry Lippstreu5 Stars Out Of 5January 20, 2009Rev. Terry LippstreuBest couple devotional I've found. This has been my wedding gift to every couple I've married for the past four years. All have been very pleased.
Chuck & Chri Najimy5 Stars Out Of 5November 28, 2007Chuck & Chri NajimyThis is the best couples devotional we have used. It has helped us improve our communication, caused us to look deeply into our spiritual life together and to recognize and improve on our weaknesses. It has helped us improve our already great marriage. We have given several as gifts, and are now ordering more for gifts. We highly recommend this book for every married couple!
Jennifer Borden5 Stars Out Of 5October 26, 2007Jennifer BordenEven though some of the topics aren't relative to us at all times, this book gets you to talk to one another about the issues in your relationship and keep communication lines clear. It has deepened the way that we relate to each other and has helped us have special times together.
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December 17, 2013 - Industry News
A team driver based in Washington state has asked for an exemption to the hours-of-service rule for himself and his co-driver.
Tags: HOS, Federal Register
We offer e-newsletters that deliver targeted news and information for the entire fleet industry.
The expert, Mark Betner from CITGO, will answer your questions
The expert, Rence Oliphant from Hendrickson, will answer your questions
Relay Bypass Kit Monitors Amperage Draw
Set of Tools Cleans Male Pins on Terminals
SecureFuel Provides Transparent Fuel Payments
McLeod Offers 'Branded' Driver App for Fleets


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Rebecca Kargle Duffer, 60, of Bowling Green, formerly of Hodgenville, died Tuesday, Sept. 29, 2009. The funeral is at 10 a.m. CDT today at J.C. Kirby & Son Lovers Lane Chapel in Bowling Green with burial at 2 p.m. EDT in Red Hill Cemetery in Hodgenville. Visitation continues at 9 a.m. CDT today at the funeral home.
By DAVE JARRETT
Guest columnist
I continue to be amazed at the outstanding support given to the military, veterans and their dependents in Hardin County. We have a large number of veterans' organizations that provide support not only to veterans but to other charities as well.
Inspiring remarks
I read President Obama's speech to students in The News-Enterprise. First let me praise my hometown paper for printing the speech for all to read and decipher at their leisure; there is nothing quite like experiencing something for yourself and drawing your own conclusions. It was exhilarating.
By CHUCK JONES firstname.lastname@example.com LOUISVILLE — One word sums up why the Elizabethtown Panthers were celebrating their third consecutive 5th Region championship Monday at Shawnee Golf Course. That same word sums up why Elizabethtown senior Garrett Hinton won his second straight individual title. And the identical word can describe why the Central Hardin Bruins and Me
IF YOU GO: Kentucky Repertory Theatre at Horse Cave will perform three productions at Hardin County Schools Performing Arts Center at John Hardin High School this winter. "Dracula" will be Oct. 29-Nov.1; "Christmas Belles" will be Dec. 3-6, and "The Miracle Worker" will be Feb. 25-28, 2010.
By BECCA OWSLEY firstname.lastname@example.com Cooking is a relaxing and enjoyable hobby. I love to plan, prepare and savor each dish I attempt. My favorite type of food to cook, and to eat for that matter, is Italian.
By CHUCK JONES firstname.lastname@example.com LOUISVILLE — North Hardin freshman Lydia Gumm couldn't have envisioned a worse start to Tuesday's round in the 3rd Region Tournament at Shawnee Golf Course. On the par-5 10th hole, Gumm's drive sailed out of bounds.
Highway fatalities in Kentucky
2000 823
2001 843
2002 915
2003 931
2004 964
2005 985
2006 913
2007 864
2008 826
Source: Kentucky Office of Highway Safety
By MARTY FINLEY
Copyright © 2015
The News-Enterprise is your source for local news, sports, events and information in Elizabethtown, KY, Hardin County and the surrounding area.


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When: Sat., March 2, 7:30 p.m. and Sun., March 3, 3 p.m. 2013
190 S. Cascade Ave. (Downtown)
a production by Opera Theatre of the Rockies of Strauss' tale of pranks, glamorous parties and mistaken identities.
Sun., Oct. 4
Pikes Peak Center
- Downtown
Wed., Oct. 7, 6:30-7:30 p.m.
Sun., Nov. 1
Fri., Nov. 6
Our performing arts scene reloaded in 2011, and you get the spoils in 2012
by Edie Adelstein
April 5, 2012
Slide into a chair, or pull up some lawn, at these concert venues
by Bill Forman
April 5, 2012
With a scrappy (but still lovely) can-do spirit, performing arts groups enrich the region
by Bree Abel
April 7, 2011
The region's major concert venues won't fit into any one genre
by Bill Forman
April 7, 2011
by Kirsten Akens
February 4, 2011
When it comes to eclectic concert settings, the Springs can hold its own
by Bill Forman
April 1, 2010
Select a star to rate.
© 2015 Colorado Springs Independent


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Michael Franti & Spearhead
- Genre: Rap/Hip-Hop, Reggae, Rock
- Description: Music video by Michael Franti & Spearhead performing Sound of Sunshine. (P) (C) 2011 Boo Boo Wax under exclusive license to Capitol Records. All rights reserved. Unauthorized reproduction is a violation of applicable laws. Manufactured by Capitol Records, LLC, 1750 North Vine Street, Hollywood, CA 90028.


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Posted 08/12/2015 11:40:09 EDT
I am not sure why you are assuming that I would intend to hit anyone. All I am saying is that the solutions are more complex than this article would indicate. "Share the road" requires a two-way mutua more »
Posted 08/12/2015 10:48:40 EDT
Usually a baby carriage would not zip around me to my right in my blind spot going 15 mph and then dash in front of me... more »
Posted 08/12/2015 07:22:36 EDT
This is not just a question of cars vs bikes. And you can't compare to European cities or even Manhattan, where public transportation is eons better than Boston. Reduction in car traffic and congestio more »
Posted 06/15/2015 08:34:27 EDT
Personally, I think getting paid sick time should be standard. Comes with the territory of having living, breathing humans as employees. Welcome to running your own business. Be a decent human being a more »
Posted 05/20/2015 11:44:42 EDT
reindeergirl, there is a difference between Ann Taylor and LL Bean and Talbots, and the slightly different styles, no less classy, from Zara and Madewell and Banana Republic to name a few... Again, it more »
Posted 05/20/2015 11:18:12 EDT
Two of her younger co-workers in the photo are wearing glasses... In fact, some might argue that glasses are geek-chic (if you have the right frames...). And are they in fact younger or just dressed y more »
Posted 05/20/2015 10:02:30 EDT
I think you are quite mistaken if you think young people are not going into computer engineering and other engineering disciplines. more »
Posted 05/20/2015 10:00:01 EDT
Facebook created a critical turning point in making social media and virtual communication go mainstream. From Facebook to LinkedIn to Twitter being used by companies and large organizations (rather t more »
Posted 05/20/2015 08:26:08 EDT
I have to disagree. Like it or not, Facebook changed the face of society and the workplace and the way we interact and market things and connect. I think we have a lot to learn from one another. All d more »
Posted 05/20/2015 08:12:16 EDT
The article specifically refers to high tech. Which is a constantly changing field with a need to always be adopting new technologies and learning new ways of doing things. My son has been exploring c more »
Posted 05/20/2015 07:36:37 EDT
Jeanne357, no doubt. But a fulfilling and rewarding existence makes all the difference. The issue I have with that cliche is that it implies we should be happy just to be breathing, which is a terribl more »
Posted 05/20/2015 07:06:22 EDT
I think it really depends on your field and where the jobs are. Cutting edge fields tend to be the harshest when it comes to age. Music and the arts and education, not so much. Business tends to respe more »
Posted 05/20/2015 07:00:05 EDT
I think it's a conundrum all around. Young people get told all the time that they have "no experience". Older people allegedly expect to make more money. These are stereotypes. I personally struggle w more »
Posted 04/23/2015 12:02:12 EDT
Move on. For whatever reason, he lost interest despite the nice date, and he's pinning it on you.The nice thing about first dates is you have no obligation to fix anything for him. Let it go. more »
Posted 03/03/2015 06:34:27 EST
Also krf9348, in addition MA state pension payments are ONLY state-income tax-free in MA... If you live elsewhere, you have to pay income tax. State jobs in MA allow for participation in non-profit re more »
Posted 03/03/2015 06:30:39 EST
You can actually look up NIH grants at usa.gov and find out. These articles sensationalize the salaries received by research professors, who, by the way, lose their jobs if they don't maintain enough more »
Posted 01/01/2015 11:20:41 EST
When I read stories like this, I feel so sad that a family or families has lost a loved one. What a terrible thing to happen! Who knows the whys or whats, but how sad that almost all these comments ar more »
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conbigote1, I do get your point. But the reality of today's economy is the same whether you are in Boston or Chicago or Austin. Never mind NYC or San Francisco. I have a daughter in college and a son more »
Posted 12/01/2014 07:31:32 EST
Maybe I am missing something, but I don't see how or why it is that different in Boston than any other major desirable city? Or any other generation for that matter. Boston has a super-competitive job more »
Posted 11/07/2014 10:40:48 EST
I don't know. Does anyone here actually believe that the news and the CDC are reporting the entire truth about the number of cases in the US? As a scientist, I have a pretty good understanding of the more »
Posted 09/08/2014 11:10:03 EDT
I know I'm way late to this party, but with him going out and traveling for work, and LW also seeing her friends and working full time, it sounds to me like neither one is settling into being a marrie more »
Posted 05/01/2014 06:14:53 EDT
The LW points out their different styles for recharging - she likes to just chill and veg out and he doesn't. Is she lazy? Or is she buying into his definition of what she is? Given her own admission more »
Posted 04/02/2014 06:47:14 EDT
At the college my daughter attends, the students are taught, male and female, that if your friend/partner is intoxicated in any way that you should ASSUME they CANNOT consent to sex. I was raped by a more »
Posted 03/28/2014 09:37:56 EDT
Your husband has lost both parents in the past few years, he is acutely grieving as an only child, and he wants to feel like he is connected to someone by blood. I lost both my parents and a sibling more »
Posted 02/05/2014 01:30:00 EST
I think the statue is creepy. If people want to view it in the museum, by all means have it on display. They have asked for it not to be in such a public outdoor place. At 60K a year, I think the s more »
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Building javadoc from J2SDK source code
If you read my last blog entry about J2SDK 1.5 alpha availability, you know you cannot make public comments about. So, with that information in mind, let's move on :-D
Suppose you download a J2SDK version from Sun that comes with no documentation at all. Well, all of them come without it, but for most you can get the docs as a separate bundle and install them. Very recently, I downloaded a J2SDK version for which there is no documentation at all. And no, you cannot download it from Sun's site; it is not there. How am I supposed to work with a JDK that has no documentation? At least, I can build javadoc from its source code and it becomes more usable - though not completely, as the new features summary and those sections where concepts are explained and detailed are necessary for a full experience :-D.
In case you happen to face the same problem - if you read my blog frequently, there is a chance you are :-D -, here is a sample Ant build script - not the best possible, it's not the point - you may use to build javadoc from source code:
That is it, folks! Remember to edit the script so JDK_HOME becomes your real JDK installation home (ex: C:\j2sdk1.4.2_02), JDK_MAJOR_VERSION becomes the major version of the JDK you are using (ex: 1.4, 1.5 ...) and PLATFORM_EXTENSION becomes the extension used by executables in your platform (ex: for Windows, it's exe). And yeah, it consumes a lot of memory. You can change the value -J-Xmx512m as you want, but it didn't work for me with 128mb, for example. It took around 8 minutes to run it in a P4 2.4GHz with 1GB - hey, but I was making normal use of it (4 IM softwares, email client, etc.)
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Basophils are increasingly recognized as playing important roles in the immune responses of allergic diseases and helminth infections. One of the main obstacles to studying basophils has been the lack of a simple and rapid assay to measure basophil activation in mice.
The purpose of this study was to develop an assay to measure murine basophil activation.
Mouse blood cells were stained with various combinations of positive and negative markers for basophils – sorted and then assessed for basophil purity by May-Grüunwald staining of cytospins. Once a flow cytometric strategy for staining basophils was determined, basophil surface expression of CD200R was assessed by multi-colour flow cytometry after stimulation of whole blood with anti-IgE, ionomycin or N-formyl MetLeuPhe (fMLP). Confirmation of basophil activation was assessed by concomitant staining of cells for intracellular IL-4. To test the ability of flow cytometric basophil CD200R measurements to assess for antigen-specific IgE-mediated activation of basophils, surface CD200R expression in response to in vitro stimulation with media alone, helminth antigen or ovalbumin was measured on basophils obtained from control mice, mice infected with helminths and mice sensitized to ovalbumin.
Using anti-IgE-FITC as a positive marker and a combination of anti-CD4-PERCP and anti-B220-PERCP as negative markers resulted in a well-separated basophil population. Additional staining with anti-CD200R-PE demonstrated that (1) basophil CD200R expression increases in response to anti-IgE, ionomycin and fMLP, (2) most CD200R-positive basophils also stain positively for IL-4 and (3) CD200R expression increases after antigen-specific activation of basophils in murine models of helminth disease and allergy.
We developed a multi-colour flow cytometry assay that measures murine basophil activation by utilizing CD200R as an activation marker. This assay is straightforward and rapid, taking approximately half a day for obtaining blood, in vitro stimulation and flow cytometric analysis.
Basophils are the least common white blood cells, typically constituting less than 0.5% of circulating leucocytes and nucleated bone marrow cells. Because of their rarity, basophils have not been as extensively studied as other leucocytes. In recent years, however, there has been increasing recognition that basophils play important roles in the immune responses induced by allergic diseases and helminth infections [1, 2].
To date, the study of basophil function in murine models of allergy and helminth infection has been greatly limited by the lack of a simple assay to measure murine basophil activation. Unlike human basophils, on which CD203c and CD63 expression can be measured to rapidly assess cellular activation by flow cytometry [3, 4], no activation markers have yet been identified on murine basophils.
CD200R (aka CD200R1 [5]) is an inhibitory receptor that belongs to the immunoglobulin superfamily [6, 7]. While initially described as being primarily expressed on cells of the myeloid lineage, recent studies demonstrate that CD200R is also expressed on granulocytes, T cells, and mast cells in both mice and humans [8–11]. Although surface basophil expression of CD200R has not yet been evaluated in mice, studies of human cells demonstrate that all basophils express CD200R [11]. Further, in comparison with other circulating leucocytes in humans, basophils have the greatest amount of surface CD200R expression by relative brightness on flow cytometry [12]. Murine basophil surface CD200R expression and changes in response to cellular activation, however, have not been previously assessed.
In this study, we sought to develop a flow cytometric assay to detect murine basophil activation. This was done by initially testing the ability of various antibody combinations to correctly identify murine basophil populations by flow cytometry. Once this was accomplished, changes in surface expression of CD200R were assessed in response to IgE-mediated activation in normal mice and in murine models of helminth infection and allergic disease. Our results demonstrate that we have developed a rapid flow cytometric assay for the detection of murine basophil activation utilizing CD200R as an activation marker.
Female BALB/C mice (NCI Mouse Repository, Frederick, MD, USA) were maintained at the Uniformed Services University (USUHS) animal facility. Experiments were performed with mice between 4 and 8 weeks of age under a protocol approved by the USUHS Institutional Animal Care and Use Committee. At study endpoints, mice were euthanized with carbon dioxide and whole blood obtained by cardiac puncture. For all experiments, whole blood was collected in heparinized microfuge tubes (Sarstedt, Numbrecht, Germany).
Purified rat anti-mouse IgE (R35-92) was used for basophil stimulation. The following anti-mouse antibodies were used for flow cytometry: anti-FcERIα FITC (MAR-1), anti-IgE FITC (R35-72), anti-CD123 FITC (5B11), anti-CD123 PE (5B11), anti-B220 PERCP (RA3-6B2), anti-CD49b APC (HMα2), anti-CD200R-PE (OX-110), anti-CD200R-Alexa Fluor 647 (OX-110), anti-CD4 PERCP (RM4-5), anti-CD117 c-Kit APC (2B8) and anti-IL-4 APC (11B11). Antibodies MAR-1 and 5B11 were purchased from eBioscience (San Diego, CA, USA), OX-110 from AbD Serotec (Kidlington, Oxford, UK), 2B8 from Biolegend (San Diego, CA, USA) and the rest from BD Pharmingen (San Diego, CA, USA).
Whole blood (100 μL) was diluted with 100 μL of RPMI 1640 (Cellgro; Mediatech, Herndon, VA, USA). Cells were washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. Supernatants were aspirated and the cells were lysed with a whole blood lysing reagent kit (Beckman Coulter, Fullerton, CA, USA). Immuno-Lyse was diluted 1: 25 in PBS and 1 mL of working solution was added to each tube and incubated 1 min at room temperature. Cells were immediately fixed with 250 μL of fixative solution and washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. Supernatants were aspirated and non-specific binding sites on cells blocked by re-suspending in 100 μL of 1% BSA/PBS and incubating at 4 °C for 1 h. Cells were stained for 30 min with various two-, three- and four-colour combinations of positive and negative markers for murine basophils. Positive markers included anti-FcERIα FITC, anti-IgE FITC, anti-CD123 FITC, anti-CD123 PE, anti-CD200R PE, anti-CD49b APC and anti-CD200R-AlexaFluor 647. Negative markers included anti-CD4 PERCP, anti-B220 PERCP and anti-CD117 c-Kit APC. Cells were then washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. Cells were re-suspended in 200 μL PBS and analysed using a BD LSR II Optical Bench flow cytometer (Beckman Coulter) and Diva software (Beckman Coulter).
Staining strategies that resulted in well-separated putative basophil populations were then repeated using 300 μL aliquots of murine blood. Cells falling within the putative basophil gate were sorted using a BD FACSAria high-speed cell sorter. May-Grünwald stains were then made of cytospins of sorted cells and evaluated for basophil purity.
Whole blood (100 μL) was diluted with 100 μL of RPMI 1640 (Cellgro; Mediatech). Tubes with blood were incubated with media, 25 μg/mL ionomycin (EMD Biosciences, LaJolla, CA, USA), anti-mouse IgE (at 0.031, 0.125 μg/mL, or at various concentrations as described in 'Results'), Litomosoides sigmodontis antigen at 20 μg/mL (LsAg, prepared from a homogenate of lyophilised adult L. sigmodontis worms), ovalbumin at 20 μg/mL or N-formyl MetLeuPhe (fMLP, at 0.5 and 1 μM) for 2 h at 37 °C in 5% CO2. When intracellular IL-4 was measured along with CD200R, Monensin (BD GolgiStop protein transport inhibitor; BD Biosciences, San Diego, CA, USA) was added after 1 h of incubation at 2 μM final concentration and the tubes were incubated for 2 more hours at 37 °C in 5% CO2. Cells were washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. Supernatants were aspirated and the cells were lysed and fixed using a whole blood lysing reagent kit (Beckman Coulter). Immuno-Lyse was diluted 1: 25 in PBS and 1 mL of working solution was added to each tube and incubated 1 min at room temperature. Cells were immediately fixed with 250 μL of fixative solution and washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. Supernatants were aspirated and non-specific binding sites blocked by re-suspending in 100 μL of 1% BSA/PBS and incubating at 4 °C for 1 h or overnight. Cells were then stained with anti-IgE FITC, anti-CD4 PERCP, anti-B220 PERCP and anti-CD200R PE for 30 min at 4 °C, washed twice with 2 mL of PBS and centrifuged at 500×g for 5 min. In studies in which intracellular IL-4 was also evaluated, cells were stained in a two-step manner. After surface staining and two washes, cells were permeabilised with BD Perm/Wash buffer, resuspended in 1% BSA/PBS, stained with anti-IL-4 APC for 30 min at 4 °C, and washed twice. Cells were resuspended in 200 μL PBS and analysed using a BD LSR II Optical Bench flow cytometer and Diva software.
For all flow cytometry experiments, antibodies were individually titrated before use and compensations assessed using BD compBeads bound to the antibodies being utilized for that experiment. For basophil activation assays, cut-off gates for CD200R-PE, and IL-4-APC positivity were established using the fluorescence-minus-one (FMO) technique [13].
Jirds that were exactly 4 days post-exposure to mites infected with L. sigmodontis were obtained from TRS Labs in Athens, Georgia. Upon arrival to our laboratory, jirds were euthanized and pleural lavage immediately performed for the recovery of infective-stage L3 larvae. Recovered larvae were then washed by allowing the larvae to sediment to the bottom of a petri dish at 1×g aspirating the supernatants, and adding 10 mL of fresh RPMI media. Female BALB/c mice were then inoculated subcutaneously between the shoulder blades with 40 L3 larvae in 150 μL RPMI.
BALB/c mice were sensitized to ovalbumin by administration of 100 μL of a 0.5 mg/mL solution of ovalbumin (Sigma, St. Louis, MO, USA) adsorbed to imject alum (Pierce) by intra-peritoneal injection every 2 weeks for a total of two doses. Control mice were vaccinated with PBS alone.
Blood was collected from L. sigmodontis infected female BALB/c mice or ovalbumin-sensitized female BALB/c mice at various time-points by cardiac puncture and analysed for LsAg-specific IgE measured by colorimetric sandwich ELISA. Flat-bottom Immulon 4 plates (Thomas Scientific, Swedesboro, NJ, USA) were coated overnight at 4 °C with 20 μg/mL LsAg. Blocking was performed with 5% bovine serum albumin (BSA) PBS. IgG was adsorbed by incubating serum samples with GammaBind G Sepharose (Amersham Biosciences, Uppsala, Sweden) overnight at 4 °C. Then, samples were centrifuged and supernatants used for the assay. Plates were washed six times and incubated with 2 μg/mL biotinylated rat anti-mouse IgE (clone R35-118) in PBS. Plates were washed again and incubated with 1 mg/mL of alkaline phosphatase-conjugated streptavidin (BD Pharmingen). Substrate p-nitro-phenyl phosphate disodium (Sigma) was added at 1 μg/mL in sodium carbonate buffer after six washes. Colorimetric development was detected at 405 nM using a Perkin Elmer Victor V microplate reader. IL-4 was quantified using a commercial IL-4 kit according to manufacturer's instructions (eBioscience).
Comparisons between groups of paired data were performed with the nonparametric Wilcoxon signed rank test and strength of correlation derived using the Spearman Rank test (Prism 4 Statistics software; GraphPad Software, San Diego, CA, USA).
To identify murine basophils by flow cytometry, whole blood samples from BALB/c mice were stained with various two-, three- and four-colour combinations of positive and negative markers for basophils. Positive markers included anti-FcERIα FITC, anti-IgE FITC, anti-CD123 FITC, anti-CD123 PE, anti-CD200R PE, anti-CD49b APC and anti-CD200R-AlexaFluor 647. Negative markers included anti-CD4 PERCP, anti-B220 PERCP and anti-CD117 c-Kit APC. Several antibody combinations worked well to identify basophil populations in murine blood. These included anti-FcERIα FITC/anti-CD49b APC/anti-CD4 PERCP/anti-B220 PERCP, anti-IgE FITC/anti-CD49b APC/anti-CD4 PERCP/anti-B220 PERCP, anti-IgE FITC/anti-CD117 c-Kit APC/anti-CD4 PERCP/anti-B220 PERCP and anti-IgE FITC/anti-CD4 PERCP/anti-B220 PERCP. In general, approaches that used anti-IgE antibody resulted in greater separation of the basophil population than approaches using anti-FcERIα. Figures 1a and b demonstrate the simplest effective strategy for identifying basophils by flow cytometry. Initially, a large gate is drawn that encompasses the typical lymphocyte region and the lower half of the typical granulocyte region on the forward and side scatter dot plot (Fig. 1a). Gated cells that stain positively for IgE and negatively for CD4 and B220 are identified as basophils (Fig. 1b). Confirmation that this strategy results in identification of basophils comes from the findings that this gated population stains very brightly for CD49b (Fig. 1c), a cell surface marker known to be highly expressed on basophils, and that sorting of this population routinely results in recovery of cells that are >98% basophils (Figs 1d and e). Addition of anti-CD49b-APC to the anti-IgE FITC/anti-CD4 PERCP/anti-B220 PERCP strategy resulted in no substantial increase in basophil purity after sorting, whereas purity can be increased to >99% by addition of anti-cKit-APC as an additional negative marker (data not shown).
To assess whether CD200R is a marker of IgE-mediated basophil activation, whole blood samples from five uninfected and 14 L. sigmodontis infected BALB/c mice were incubated with media or 0.125 μg/mL anti-IgE for 2 h and then stained for flow cytometry with anti-IgE FITC, anti-CD4 PERCP, anti-B220 PERCP and anti-CD200R PE. Basophil populations were identified as IgE+/CD4−/B220− cells as depicted in Fig. 1b, and the cut-off for anti-CD200R PE positivity determined using the FMO approach (Fig. 2a). In all mice evaluated, anti-IgE stimulation resulted in increases in the percentages of basophils expressing CD200R compared with media stimulation (Fig. 2b, geometric mean (GM) = 23.1% CD200R+basophils after anti-IgE stimulation vs. 6.3% after incubation in media alone, P = 0.0001). Similarly, the mean fluorescence intensity (MFI) of CD200R PE staining of the basophil population increased in all mice evaluated (basophil CD200R PE MFI = 355 after anti-IgE stimulation vs. 250 after incubation in media alone, P<0.0001, data not shown).
To determine whether CD200R expression is up-regulated after IgE-independent activation of basophils, whole blood samples from six uninfected and four L. sigmodontis infected BALB/c mice were evaluated for CD200R expression on basophils by flow cytometry after stimulation with media or 25 μg/mL ionomycin. As with anti-IgE stimulation, incubation of whole blood with ionomycin resulted in increases in the percentages of CD200R+basophils in all mice studied (Fig. 2c, GM = 12.6% CD200R+basophils after ionomycin stimulation vs. 3.8% after incubation in media alone, P = 0.002). Similarly, as seen in Fig. 2d, MFI of CD200R PE staining on basophils increased in response to activation with all tested basophil activating agents as compared to media. This suggests that both percentage CD200R positivity and changes in basophil CD200R MFI can be used to detect activation. Further, increases in CD200R expression occur in response to both IgE-independent (ionomycin, fMLP) and IgE-mediated pathways of activation.
As previous studies have demonstrated that intracellular basophil IL-4 positivity as detected by flow cytometry increases upon basophil activation [14, 15], we sought to confirm that CD200R is a marker of murine basophil activation by correlating surface expression of CD200R with basophil IL-4 positivity. For these experiments, whole blood samples from uninfected and 1-week L. sigmodontis infected BALB/c mice were incubated for 3 h with either media alone or anti-IgE, with addition of monensin after the first hour of incubation. Cells were initially stained for surface markers (IgE, CD4, B220 and CD200R) and then permeabilized and stained for intracellular IL-4.
As expected, anti-IgE stimulation of basophils resulted in increased percentages of IL-4+, CD200R+ and IL-4+CD200R+basophils (Figs 3a and b) compared with media. As seen in Fig. 3b, there was a correlation between basophil surface CD200R expression and intracellular IL-4 expression (Spearman's rank correlation coefficient r = 0.838, P<0.0001). Correlation of IL-4 expression and CD200R staining was not complete, as on average 60% of IL-4+basophils stained positively for CD200R (median 60, range 10–97%) whereas only 36% of CD200R+basophils stained positively for IL-4 (median 31, range 10–62%). While it is possible that CD200R may be a more sensitive marker of basophil activation than IL-4 expression, it is likely that some of the differential staining of IL-4 and CD200R may have been due to differences in the time-points at which these molecules are optimally expressed.
To determine dose- and time-dependent activation parameters of basophil CD200R expression, whole blood samples from BALB/c mice were incubated either with increasing anti-IgE concentrations or with 0.125 μg of anti-IgE for 30 min, 1, 2, 3 or 4 h and then stained for flow cytometric analysis of CD200R expression. As shown in Fig. 4a, CD200R expression demonstrates dose-dependent activation in response to increasing concentrations of activating anti-IgE antibody.
CD200R expression was also found to be time-dependent, with time-course studies showing that the percentages of basophils staining positively for CD200R were significantly increased after 30 min of incubation with anti-IgE as compared with incubation with isotype control antibody (52% CD200R+vs. 1%), peaked after 1–2 h of incubation with anti-IgE (72–80% CD200R+vs. 1%), and then decreased to undetectable expression after 4 h (Fig. 4b).
To assess whether CD200R expression can be used to detect antigen-driven IgE-mediated basophil activation, we measured CD200R positivity on basophils obtained from mice infected with L. sigmodontis, an invasive filarial nematode of rodents, after in vitro challenge with parasite antigen (LsAg). Whole blood samples from BALB/c mice infected for 1 (n = 4) or 8 weeks (n = 5) with L. sigmodontis were incubated with LsAg at 20 μg/mL for 2 h and then assessed for basophil surface CD200R expression by flow cytometry. BALB/c mice infected for only 1 week, at which time there was no detectable circulating LsAg-specific IgE (Fig. 5a), demonstrated no increase in percentages of CD200R positive basophils in response to LsAg (Fig. 5b, GM = 2.9% CD200R+basophils after media incubation vs. 3.4% after LsAg incubation, P = 0.84). In contrast, basophils of BALB/c mice infected for 8 weeks, when there were substantial amounts of serum LsAg-specific IgE (Fig. 5a), exhibited significant increases in CD200R positivity when incubated with LsAg as compared with media (GM = 4.8% CD200R+basophils after media incubation vs. 25.6% after incubation with LsAg, P = 0.03; Fig. 5c).
To confirm that LsAg and anti-IgE actually activate basophils of mice infected with L. sigmodontis for 8 weeks, we measured supernatant IL-4 concentrations of whole blood after 2 h of incubation with media, LsAg or anti-IgE. As seen in Fig. 5d, both LsAg and anti-IgE stimulation of blood from mice infected with L. sigmodontis for 8 weeks substantially increase supernatant IL-4 concentrations. As basophils are the only cells within whole blood known to have the ability to release IL-4 within 2 h of activation [16], these results confirm that both anti-IgE stimulation and LsAg activate basophils of 8-week infected mice.
To evaluate whether CD200R expression can be used to detect Ag-specific IgE activation in a murine model of allergy, mice were sensitized against ovalbumin by repeated injection of ovalbumin adsorbed to alum (OVA-sensitized, n = 5) or given injections of PBS–alum alone (control group, n = 5) and then assessed for basophil surface expression of CD200R by flow cytometry after stimulation with either media alone or ovalbumin. Serum OVA-specific IgE was detectable by ELISA in all sensitized mice and in none of the control mice (data not shown). Whereas control mice demonstrated no increase in percentages of CD200R+basophils after incubation of whole blood with ovalbumin, sensitized mice developed significantly greater percentages of CD200R+basophils after incubation with ovalbumin (Fig. 6, P = 0.032).
Recent studies indicate that basophils play significant roles in the immune responses induced by helminth infections and allergic diseases [1, 2]. To date, however, study of their roles in murine models of disease has been hampered by the lack of a direct assay to detect their activation in mice. This study demonstrates that CD200R can be utilized as a marker of murine basophil activation.
Murine basophil surface expression of CD200R, as detected by flow cytometry, increased in response to in vitro activation of whole blood with anti-IgE, ionomycin or fMLP. These results demonstrate that CD200R up-regulation occurs in response to both IgE-dependent and independent pathways of basophil activation. Additionally, incubation of whole blood obtained from mice infected with L. sigmodontis or sensitized against ovalbumin resulted in up-regulation of basophil CD200R surface expression in response to LsAg or ovalbumin once circulating antigen-specific IgE had developed. Further evidence that CD200R is a murine basophil activation marker came from the finding that its expression on basophils increased in proportion to increases in intracellular basophil IL-4 positivity after cellular activation. Finally, basophil CD200R surface expression was found to increase in a dose–response fashion to increasing concentrations of anti-IgE.
One of the advantages of this whole blood assay is that it does not require any basophil priming cytokines or enrichment steps, which in human basophils have been shown in some cases to affect expression of surface activation markers [17]. If one wanted to conduct such manipulations before assessing basophil activation by surface CD200R expression, however, it would be important to first assess whether such manipulations were altering baseline surface CD200R staining.
CD200R surface up-regulation was found to be rapid and transient, as time course studies demonstrated CD200R surface up-regulation occurs as early as 30 min after IgE-mediated activation, peaks at 1–2 h, and then returns to baseline by 4 h after activation. This time-course is similar to the kinetics of CD63 up-regulation on human basophils, which becomes maximal 60 min after activation, but is slower than that observed for human basophil CD203c expression, which peaks within 15–20 min [18]. While the reason for different kinetics of human basophil activation markers is not known, it is presumed due to differences in the pathways by which these markers translocate from cytoplasmic compartments to the extracellular space [18].
While CD200R has been observed to be present on human basophils in prior studies [11, 12], to our knowledge this study is the first to demonstrate that CD200R is also present on murine basophils and that its expression on these cells increases in response to cellular activation. As ligation of CD200R inhibits FcERI-mediated activation and degranulation of human basophils and mast cells [8, 12, 19], it is possible that the transient up-regulation of CD200R we observed on the surface of activated murine basophils represents a negative-feedback mechanism that enables inhibition of further FcERI-mediated activation in the hours immediately following initial cellular activation.
Recently, a number of research groups have discovered that the mouse genome contains a family of CD200R-like proteins with close homology to CD200R (aka CD200R1). These include CD200R2 (CD200RLc), CD200R3 (CD200RLb), CD200R4 (CD200RLa) and CD200R5 [5, 11, 20]. The antibody we utilized in our studies (anti-CD200R, OX-110) binds to CD200R but not to CD200R3 and CD200R4 [11]. CD200R, CD200R2, CD200R3 and CD200R4 have all been shown capable of binding to CD200 [5], a membrane protein expressed by thymocytes, activated T cells, B cells, neurons and endothelial cells [6]. However, while ligation of CD200R typically results in inhibitory signals, other CD200R family members contain the positively charged amino acid lysine in their trans-membrane regions and thus can associate with ITAM- or YxxM motif-bearing adaptor molecules such as DAP12 that mediate stimulatory signals [5, 11, 20]. As such, it has been speculated that CD200R and CD200R-like receptors may serve as balancing inhibitory and activating receptors [5]. With respect to basophils, ligation of CD200R3 molecules has recently been demonstrated to result in activation of murine basophils and mast cells [21]. As ligation of CD200R inhibits FcERI-mediated activation of these cells [8, 12, 19], it is possible that releasability of murine basophils is controlled by the relative surface expression of CD200R and other CD200R-like molecules.
In conclusion, we have shown that the inhibitory receptor CD200R is rapidly up-regulated after murine basophil activation. By utilizing this marker and a combination of positive and negative basophil markers, we have developed a flow cytometric assay for detection of basophil activation in whole blood. This assay is straightforward and rapid, taking approximately 6 h for obtainment of blood, in vitro stimulation and flow cytometric analysis. Additionally, given the known inhibitory properties of CD200R ligation on basophils, the finding of CD200R up-regulation in response to FcERI-mediated basophil activation suggests CD200R may function as a negative feedback mechanism in the hours immediately after basophil activation.
We thank Karen Wolcott and Kateryna Lund at the USUHS Biomedical Instrumentation Center for their valuable assistance with flow cytometry. This work was supported by grant R073MX from the Uniformed Services University of the Health Sciences and grant K22AI065915 from NIAD/NIH.
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