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Incredible Hulk Vol 1 249
135,532pages on
this wiki
this wiki
| ||
Appearing in "Jack Frost Nipping At Your Soul!"Edit
Featured Characters:
Supporting Characters:
Other Characters:
- Fred Sloan
- Changelings (First appearance) (Unnamed)
- Centauron (First appearance) (Unnamed)
- Iron Man (Only in flashback)
- Iron Man Armor Model 1 (Only in flashback)
- Iron Man Armor Model 4 (Only in flashback)
- Blizzard Armor (Only in flashback)
Synopsis for "Jack Frost Nipping At Your Soul!"Edit
Hulk is woke up by some deer hunters. After causing a snow avalanche, the green giant falls into a deep pit. Among the caverns he finds Jack Frost, the snowman. Jack sends his ice statues against the Hulk. But their fragile structure are nothing for the strong guy. Finally, all caverns collapse burring Frost. Hulk escapes climbing up the pit. Meanwhile, Betty Ross, Rick Jones and Fred Sloan reach a nearby cabin where Thunderbolt Ross and Doc Samson were staying. Suddenly, Centaurus (a man horse) shows up and drops unconscious in the cabin, after being hurt by a shot from Ross.
- No special notes.
- No trivia.
See AlsoEdit
- Write your own review of this comic!
- Discuss Incredible Hulk Vol 1 249 on the forums
- Cover gallery for the Incredible Hulk series
- Images from Incredible Hulk Vol 1 249
Recommended ReadingEdit
- None.
Links and ReferencesEdit
- None.
Like this? Let us know! |
- Comics
- 1980
- 1980, July
- Bronze-Age
- Incredible Hulk Vol 1
- Jim Shooter/Editor-in-Chief
- Steve Ditko/Cover Artist
- Bill Mantlo/Writer
- Steve Ditko/Penciler
- Steve Ditko/Inker
- Ben Sean/Colourist
- Irving Watanabe/Letterer
- Al Milgrom/Editor
- Robert Bruce Banner (Earth-616)/Appearances
- Elizabeth Ross (Earth-616)/Appearances
- Richard Jones (Earth-616)/Appearances
- Thaddeus Ross (Earth-616)/Appearances
- Leonard Samson (Earth-616)/Appearances
- Gregor Shapanka (Earth-616)/Appearances
- Frederick Sloan (Earth-616)/Appearances
- Changelings (Earth-616)/Appearances
- Centauron (Earth-616)/Appearances
- Anthony Stark (Earth-616)/Appearances
- Colorado/Appearances
- Rocky Mountains/Appearances
- Iron Man Armor Model 1/Appearances
- Iron Man Armor Model 4/Appearances
- Blizzard Armor (Earth-616)/Appearances
- Comic Quote Needed
- Synopsis Written

subset: commoncrawl


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Latest Amsterdam Stories
AMSTERDAM, October 29, 2014 /PRNewswire/ -- Professors Barry Trost, Stanford University, USA and Professor Jiro Tsuji, Tokyo Institute of Technology, Japan, receive
AMSTERDAM, October 27, 2014 /PRNewswire/ -- Agriculture and Climate Change - Adapting Crops to Increased Uncertainty, to take place 15-17
AMSTERDAM, October 21, 2014 /PRNewswire/ -- JWT Amsterdam (http://www.jwtamsterdam.nl) together with KITKAT, Nestle's largest chocolate brand, launch the first-ever nationwide
- Qualcomm Gains Momentum with 802.11ac 2.0 Solutions: Enables New MU-MIMO Products from AVM, Introduces New Chip for Notebooks and Consumer Electronics - AMSTERDAM, Oct.
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AMSTERDAM, October 9, 2014 /PRNewswire/ -- Teleplan supports international markets with high quality refurbished hard disk drive (HDD) products, as well as working
Enterprises no longer tolerate "bill shock", they demand solutions that will identify and stop excessive spend while it is happening or even before it happens.
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The Amsterdam and Saint-Paul Islands temperate grasslands is an ecoregion including two volcanic islands in the southern Indian Ocean. The French research vessel Marion Dufresne II, which services the Martin-de-Vivies research station on Amsterdam Island, is the only way to visit the islands. There are two volcanoes that lie 83 kilometers from each other in the center of a triangle between Australia, Antarctica and southern Africa called Ile Amsterdam and Ile Saint-Paul. The islands are...
- In medieval musical notation, a sign or neume denoting a shake or trill.


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Tree LightingSanta and holiday cheer take over Downtown WilmingtonSubmitted by Daniel Seamans on Fri, 11/28/2014 - 8:12pm.READ MORE:
Santa and holiday cheer have taken over Downtown Wilmington as the lights now shine bright with the annual lighting of the tree at the corner of Water and Princess Streets.
Full story... » Historic Downtown Wilmington tree lighting scheduled for Nov. 25Submitted by WWAY on Tue, 11/01/2011 - 8:27am.READ MORE:
The public is invited to attend a holiday tree lighting on Friday, November 25 in Riverfront Park. The event is presented by City of Wilmington's Recreation & Downtown Services Division and the Downtown Business Alliance (DBA).
Full story... » |


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Legal Question in Wills and Trusts in Ohio
what happens if my wife dies and has a will living the house to her kids but we are both on the title of the house?
Asked on 3/06/13, 9:27 am
1 Answer from Attorneys
Elizabeth Schmitz
Elizabeth S. Schmitz Attorney at Law
users found helpful
attorneys agreed
It depends on how you are listed on the house. If the deed says joint with right of survivorship then the house will pass to you and not go through the will. If you are listed as joint owners without the right of survivorship language then the children will inherit an interest in the house.
Answered on 3/07/13, 7:56 am


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This paper is divided into two main parts. The first is concerned with definitions of concepts and terms of importance to the architect of integrated manufacturing systems. It describes the operational environment, how the manufacturing systems are going to be used, and a variety of system requirements. Priorities are established and a set of guiding principles for system design are proposed. In the second part, three basic system structures are analyzed and evaluated in terms of the previously stated priorities and guiding principles. The relation of integrated manufacturing systems to other complex systems is shown to illustrate how generally applicable many of the findings derived from knowledge of the manufacturing environment are to the design of systems for other application fields. The paper concludes by restating the key requirements and formulates what appears to be the greatest challenge for the system architect. That is, the creation of an architectural framework within which the system designers can structure manufacturing systems capable of handling besides the planned tasks a variety of unforeseen applications. Readers familiar with the plant environment and the terms and concepts associated with it may skip the first sections and start with System Requirements. We would, however, advise even the experienced reader to review the definition of real-time systems, which we believe is new.
Note: The Institute of Electrical and Electronics Engineers, Incorporated is distributing this Article with permission of the International Business Machines Corporation (IBM) who is the exclusive owner. The recipient of this Article may not assign, sublicense, lease, rent or otherwise transfer, reproduce, prepare derivative works, publicly display or perform, or distribute the Article.


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Welcome, Sign In
Terry Crews on Working with the Action Legends in "The Expendables 2"
Description:Terry Crews and Randy Couture talk about the differences between the first Expendables movie and its sequel. The actors also discuss the violence boundaries and working with the action hero legends. (4:20)
Source: HitFix
Tags: Terry Crews on Action Legends in The Expendables 2 , actor interviews , arnold schwarzenegger , bruce willis


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Top Definition
When someone talks fast
That fast-talking teacher and salesman is talking too fast, he is strybelizing, he shouldn't strybelize anymore, I have a headache from listening to him.
by someguywithaheadache
October 19, 2010
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Protecting the IT Infrastructure That Streamlines Healthcare
Free registration required
Executive Summary
Navicure, Inc. (Navicure) is a leading Internet-based medical claims clearinghouse that helps physician practices increase profitability through improved claims reimbursement and staff productivity. Navicure wanted to implement automated non-intrusive disaster recovery system adaptable to changing business needs. Navicure reduce recovery time for a virtual machine by 90 percent, from ten minutes to one minute and recover 25 virtual machines in less than 30 minutes.
- Format: PDF
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Follow Gossip Girl
A Chair Chat
Around the Web
Chaaaair ftw
They still have the best chemistry on the show, now if the writers could get their heads out of their asses long enough to come up with a decent storyline for these two that doesn't involve rape, I'd be happy.
Chuck and Blair, please save us from the shell of ruin that is season 4 so far!
Girls, you need to CHILL and not worry, Chuck and Blair will definitely end up together, however, in the meantime, I CANNOT WAIT to see more interaction between Blair and Dan. They are simply the best thing that has happened to this show in a looooong time.
Now, I'm deinitely more for D&B as FRIENDS and not as a couple, however, wouldn't it be hilarious to see Serena's, Jenny's and Vanessa's expressions when they find out that D&B hooked up??? :)
they would make hot babies
i hope that if dair happens chuck will get jealous and want blair back but i hate dan and blair together they just dont match CHAIR FOREVER !
LOVEE THEM!! if they dont come back soon i'll probably stop watching until they do
this is fate. dair is shit.
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- Episode:
- Gossip Girl Season 4 Episode 14: "Panic Roommate"
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Gossip Girl Season 4 Episode 14 Quotes
Either switch that thing off or bring it to bed. Vibration is a terrible thing to waste.
- Permalink: Either switch that thing off or bring it to bed. Vibration is a ...
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A wise woman once said that every morning when you wake up you say a little prayer. After all, you never know what your day may hold.
Gossip Girl
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831-2450 - USB to 10/100 Ethernet Adapter
The USB 2.0 Ethernet Adapter allows your computer instantly connect to a 10/100Mbps network through simply a USB port; no hassle to open up your computer case to install an internal card. It's also an ideal USB-to-LAN alternative for gaming console that needs netw...
Mfr Name: Distributed By MCM
Mfr Number: AE2220
831-2532 - 10/100Mbps USB Ethernet Adapter
This leading edge adapter will allow those with Internet challenged computers to not only connect with the World Wide Web, but also speed up existing surfing speeds using their USB 1.1 or 2.0 port.
Mfr Name: Iogear
Mfr Number: GUC2100


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117 results for "nc state"
T.J. Warren of N.C. State Wolfpack confirms he's headed to NBA dr...
North Carolina State sophomore T.J. Warren is taking his high-scoring game to the NBA.
Story | Conversation | April 08, 2014 -
NC State's T.J. Warren headed to draft
Jeff GoodmanNC State sophomore T.J. Warren is heading to the NBA, sources confirmed to ESPN.
Story | Conversation | March 24, 2014 -
Providence has plenty to prove in Paradise
Eamonn BrennanThere is always danger in the sentimental hire. Just ask NC State. In spring of 2006, after the conspicously unloved Herb Sendek fled Raleigh, N.C., for Arizona State, Wolfpack fans got one of their own: Sidney Lowe, point guard for Jim Valvano's l...
Blog | November 21, 2013 -
Notebook: ESPN 100 recruiting updates
Dave TelepThis week's Friday recruiting notebook is chock full of updates on ESPN 100 prospects, including commitments for Florida and Louisville, a visitor for Michigan State, what's next for North Carolina, who NC State is targeting and why No. 10 recruit My...
Blog | June 28, 2013 -
About Last Night: Miami Makes It Back-to-Back
Spike FriedmanIn case you were busy preparing for the first day of summer by getting all of your mosquito bites out of the way up front, here's what you missed in sports on Thursday: In what can only be described as the culminating erotic explosion of basketb...
Blog | June 21, 2013 -
NC State had some preseason buzz last season, but Duke and UNC st...
Myron MedcalfThe buzz around NC State has quickly evaporated, and as we head into the 2013-14 season, it's clear the Triangle area of North Carolina is still owned by Duke and UNC.
Story | Conversation | May 10, 2013 -
3-point shot: Burke should sweep awards
Andy Katz1. Expect Michigan's Trey Burke to sweep through all the player of the year awards. If not, I will be shocked. After winning the AP player of the year award on Thursday, Burke added the Oscar Robertson trophy Friday morning. A few weeks ago it looked...
Blog | April 05, 2013 -
Dead-ball rebounds and the origin of the college basketball ruleb...
Shane RyanAn obscure stat, a mysterious inventor, and the Louisville crew that redefined college basketball statistics.
Story | Conversation | April 04, 2013 -
In 1974, NC State stood atop hoops world
Doug WilliamsThe image is indelible and the words simple. Six-foot-four David Thompson is in full flight, his hand more than a foot above the rim as he tips the basketball over the long reach and leap of 6-foot-11 Bill Walton. The cover of the April 1, 1974...
Blog | April 02, 2013 -
Rodney Purvis plans to transfer from NC State
North Carolina State freshman guard Rodney Purvis said Monday he plans to transfer.
Story | Conversation | April 01, 2013 -
NC State Wolfpack scoring leader C.J. Leslie will enter NBA draft...
North Carolina State's C.J. Leslie says he's skipping his final season and entering the NBA draft.
Story | Conversation | April 01, 2013 -
Lorenzo Brown of North Carolina State Wolfpack to enter NBA draft...
North Carolina State's Lorenzo Brown said Thursday he's ready for the NBA.
Story | Conversation | March 28, 2013 -
3-point shot: Coaching carousel whirls
Andy Katz1. Minnesota coveted VCU's Shaka Smart, but his former boss, current Golden Gophers athletic director Norwood Teague, couldn't convince Smart to come to the Twin Cities (he should know Smart is loyal to VCU) for the head-coaching job from which Tubby...
Blog | March 28, 2013 -
Lorenzo Brown of North Carolina State Wolfpack will enter NBA dra...
North Carolina State junior point guard Lorenzo Brown will enter the NBA draft, a person familiar with the situation said Wednesday night.
Story | Conversation | March 27, 2013 -
Ex-NC State coach Sidney Lowe arrested on tax counts
Former North Carolina State basketball coach Sidney Lowe was arrested Monday and charged with failing to file his North Carolina state income taxes for three years.
Story | Conversation | February 18, 2013 -
Saddle Up: Texas in the spotlight
Eamonn BrennanTexas vs. No. 15 Georgetown, 7 p.m. ET, ESPN: It is easy to take shots at Rick Barnes. It is easy to look back at the NBA talent that has passed through Austin, and to contrast that with the lone Final Four on Barnes' resume, and accuse Barnes of not...
Blog | December 04, 2012


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Are you a person with a disability? Do you want to become more informed about tools and strategies to advance your economic strength and security? more
In record numbers, people with disabilities are becoming part of the economic mainstream, and developing savings and assets. Learn more about how to effectively reach this growing market sector ... more
Join us to promote an economic empowerment agenda with a focus on policy change and public-private collaboration ... Learn more
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Follow us at @RealEconImpact
On this the third day of America Saves Week, we have selected National Endowment for Financial Education's Your Spending, Your Savings, Your Future: A Beginner's Guide to Financial Readiness as our Publication of the Week.We all need money; whether it's to buy groceries, ...
Email Address
© National Disability Institute | 1667 K Street, NW Suite 640 Washington, DC 20006 | 202.296.2040


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Thursday, February 26, 2015
MANKATO, MN – The Bethany Lutheran College Board of Regents has unanimously elected Dr. Gene Pfeifer (56) of Inver Grove Heights, Minnesota, to become the tenth president of the College, beginning July 1, 2015.
Pfeifer has served as President of St. Croix Lutheran Grades 6-12 in West St. Paul, Minnesota, since 2007. Prior to his tenure at St. Croix, Pfeifer was Professor of Education and Director of Clinical Experiences at Martin Luther College... continue reading
Tuesday, February 3, 2015
The Board of Regents of Bethany Lutheran College and Theological Seminary met on the Bethany campus for their quarterly meeting on February 2 and 3, 2015. After the Chapel service on Tuesday, February 3, the Board recognized faculty and staff anniversaries. The Rev. Herbert Huhnerkoch was the guest speaker.
Pictured in back row from left: President Dan Bruss, Tiffany Young Klockziem (Assistant Professor, Health and Human Performance–15 years... continue reading
Monday, January 5, 2015
The next Bethany Lutheran College Athletic Hall of Fame induction will be during the 2015 Fall Festival in September. The Hall of Fame committee is looking for your nominations! If you know of a fellow teammate, classmate, relative, or associate you think deserves consideration for the Hall of Fame, you need to nominate them.
Members of the Athletic Hall of Fame can be past players, coaches, or individuals who have given special service to the... continue reading
Monday, December 8, 2014
Let your mind wander back to your college days. Did you ever question, "Where will I be in ten, twenty, or even thirty years from now?" Were you concerned about making the correct career choice? What happens if I don't like my work? A course developed by Dr. Jennifer Wosmek (psychology), helps to alleviate some of the uncertainty for psychology students at Bethany Lutheran College.
At the broadest level, the goal of Wosmek's supervised study... continue reading
Friday, November 14, 2014
The 2014 edition of "Give to Bethany Day" was a great success! On Thursday, November 13, 2014, alumni and friends made 225 gifts to Bethany. The total contributions were $48,025 during this special 24-hour fundraiser. This outpouring of support certainly outpaced the $25,000 goal that was set for the day. Also, as a result of the support of a few kind donors who generously offered $25,000 in matching funds, the total contributions were $73,025... continue reading
Friday, October 31, 2014
Five students in Professor Shane Bowyer's Principles of Management class at Bethany Lutheran College will be holding a fundraiser for Minnesota Valley Action Council (MVAC) Food Hub on November 8, 2014, from 6-9:30 p.m. at Buster's Sports Bar and Grill in Mankato, MN. The event will feature live music by country musician Andrew Babcock, a silent auction with gift cards and items from local businesses, and an all-you-can-eat taco bar.
The Food... continue reading
Monday, August 18, 2014
The Bethany Hawkwatch will have an informational meeting on Tuesday, September 2, 2014, from 7:00-8:30 p.m. in Room 101 of the Marvin G. Meyer Hall of Science and Mathematics on the Bethany Lutheran College campus.
Chad Heins (Assistant Professor of Biology) will give a brief history of the Hawkwatch project as well as discuss raptor migration and identification. The public is welcome to attend. In 2013, volunteers at the Bethany Hawkwatch... continue reading
Friday, August 8, 2014
Bethany Lutheran College is proud to announce four new inductees into its Athletic Hall of Fame.
Anthony Bias starred for the men's basketball team from 1979-81. He earned National Junior College Athletic Association (NJCAA) All-American honors following his sophomore season after averaging 26.0 points, 12.4 rebounds, and 5.6 assists per game. He also was named to the All-Division Team, All-Minnesota Community College Conference (MCCC) State... continue reading
Tuesday, May 20, 2014
Bethany Lutheran College began a $28,000,000 capital campaign in June 2007. The goals of the campaign focused on a variety of needs at Bethany. First and foremost was the construction of a new academic building to replace an aging Luther Hall (100 years old). In addition to the need for modern learning spaces, the College also sought to increase its endowed scholarships to meet the growing demand for financial aid.
President Dan Bruss noted in... continue reading
Monday, May 5, 2014
Dr. Carol (Gilbertson) Lehtola is the recipient of the 2014 Bethany Lutheran College Distinguished Alumna Award.
As a child growing up on the family farm in Ulen, Minnesota, she had always been interested in the subjects that most girls didn't venture into—math and science.
Lehtola enrolled at Bethany (BLC) in 1969 and earned an associate in arts degree from BLC in 1971. After two years at BLC, Lehtola transferred to South Dakota State... continue reading


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Search for Columbia Tn Homes here
The origins of Columbia date back to 1807 when Maury county was being organized, the original town was started on the south bank of the Duck River and consitied of only four blocks. Columbia was incorporated in 1817, and for years it was the center of the richest county in agricultural wealth in the state.
Columbia Tennessee is a community of approximately 35,000 people, located south of Franklin and Spring Hill in Maury county, Tennessee, the Columbia zipcode is 38401. Columbia uses I-65 and state hwy 31 as it's main artery north to Nashville. 473 Columbia Tn homes closed in 2010 with the average price of homes sold being $137,443 and the average size was 1815 square feet
For the most-up-to-date information on Columbia homes values, then we urge you to contact us. We can assist you with all aspects of buying or selling real property in this and other neighborhoods in the Nashville Metro area. Also ask to be placed on an instant notification system when any new Columbia MLS listings come on the market or price reductions in Columbia are posted.
information on this page is based on information on single family homes for sale in Columbia per the Nashville Multiple Listing Services, Inc. and is deemed reliable but, not guaranteed.
See all Columbia.
(all data current as of
$1,700,000 : 2298 Darks Mill Rd, Columbia3 beds, 3 full, 1 part bathsListed with Maury County Realty
$1,325,000 : 2843 Ashwood Rd, Columbia4 beds, 6 full, 1 part bathsListed with McEwen Group
$1,250,000 : 3319 Pulaski Hwy, Columbia5 beds, 6 full, 2 part bathsListed with McEwen Group
$1,099,999 : 4132 Fairview Rd, Columbia4 beds, 3 full, 2 part bathsListed with Worth Properties LLC
$1,095,000 : 2298 Darks Mill Rd, Columbia3 beds, 2 full, 2 part bathsListed with Maury County Realty
Listing information deemed reliable but not guaranteed. Read full disclaimer.


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1305 South Ridgewood Avenue, Daytona Beach, FL 32114, United States of America – Show map
very clean with new furnishings ---a little small but enough for two adults
United States of America
updated rooms clean comfortable bed
United States of America
the place was clean and location is fine but the air condition shut off and we were hot in the early morning and when we told staff they said yeah it does that. no sorry or how can we make it better
United States of America
Staff was friendly & helpful.
United States of America
Very accommodating Very nice manager Very helpful
United States of America
the location was good
United States of America
This Daytona Beach motel is within a 10-minute drive of the Daytona International Speedway. The motel offers barbecue facilities, picnic area and rooms with free Wi-Fi.
Americas Best Value Inn Daytona Beach rooms include a microwave and refrigerator. The rooms are also equipped with a work desk.
Daytona Beach Americas Best Value has a 24-hour front desk and laundry facilities available to guests.
The Americas Best Value Inn is 4 miles from the Dayton Lagoon, a water park. The Daytona Beach International Airport is 5 miles from the motel.
We speak your language! Motel: 20 rooms , Hotel Chain: Americas Best Value Inn. This property has been on Booking.com since Sept 20, 2010.
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32 verified reviews
We verify all reviews from guests who have stayed at Americas Best Value Inn Daytona Beach. Find out more.
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Americas Best Value Inn Daytona Beach's staff speaks: English
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When you book with us your details are protected by a secure connection.
BBQ Facilities
Golf Course (within 2 miles), Fishing
Wired internet is available in the hotel rooms and is free of charge.
Free public parking is available on site (reservation is not needed).
Vending Machine (drinks), 24-Hour Front Desk, Laundry, Fax/Photocopying
Non-smoking Rooms, Air Conditioning
From 2:00 PM
Until 11:00 AM
Cancellation and prepayment policies vary according to room type. Please enter the dates of your stay and check what conditions apply to your preferred room.
Children and Extra Beds
All children are welcome.
One child under 10 years stays free of charge when using existing beds.
Any additional older children or adults are charged USD 5 per person per night when using existing beds.
One older child or adult is charged USD 10 per person per night in an extra bed.
Cribs are not available.
The maximum number of extra beds in a room is 1.
Any type of extra bed or crib is upon request and needs to be confirmed by management.
Additional fees are not calculated automatically in the total cost and will have to be paid for separately during your stay.
Pets are allowed on request. Charges may apply.
Cards accepted at this property
Hover over the cards for more info.
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Guests are required to show a photo ID and credit card upon check-in. Please note that all Special Requests are subject to availability and additional charges may apply.
Based on 32 reviews
Value for money
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This Florida hotel is 2 miles from the Daytona Beach International Airport and features an outdoor pool, free Wi-Fi, and continental breakfast.
Score from 98 reviews
Review score
America's Best Value Inn Daytona Beach/Oceanfront This oceanfront hotel in Daytona Beach, Florida is ideally situated along the Atlantic Ocean. It features spacious rooms and an outdoor pool with a large sundeck and panoramic ocean views.
Score from 303 reviews
Daytona Beach Extended Stay Hotel This Daytona Beach hotel is within a 10-minute walk of Daytona International Speedway. The hotel has an outdoor pool and provides a kitchenette in every room.
Score from 65 reviews
Review score
The Budget Inn Express is located across the street from the shores of Daytona Beach. Guests will enjoy Free Wi-Fi during their stay and Daytona International Speedway is a 15 minute drive away.
Score from 18 reviews
Review score
the location was good
the managers lack of knowledge for service dog etiquette and the bed came off of the wooden fraMe
Did not supply a hair dryer, stained and dingy towels and bedding, also didn't restock plastic cups.
Good location
It had a strong smell like cleaner product.
very clean with new furnishings ---a little small but enough for two adults
didn't dislike the small couch, but did not see the need for it
the place was clean and location is fine but the air condition shut off and we were hot in the early morning and when we told staff they said yeah it does that. no sorry or how can we make it better
above air problem
Cockroaches in each room i was moved to Poor Staff rude front desk staff front desk area smelt poor english speaking skills
it was quiet and it was close to Daytona Beach and some restaurants
there wasn't a phone in the room and there wasn't a clock radio either or even just a clock
Upon check in, I informed the guy it was very important that I sleep comfortable. He assured me if I was unhappy to let him know and he would move me to another room. Before I pulled off; he called me back in & switched me. I really enjoyed staying here for 5 days. I like driving up to my door. Very quiet atmosphere. Clean room.
The television is dated. That's pretty much all I can say that I didn't like.
Find a cover sheet with blood marks and roaches in the room
price of the room,room was dated, carpet was stained and worn, shower didn't drain very well, bathroom window wouldn't lock, closet door wouldn;t stay shut.
Was told i didnt have confirmation and that hot water wasnt working. Gave every excuse trying not to rent me a room
Staff was friendly & helpful.
Room smelled of cigarette smoke.
The slide lock on the door was broke. The lock was there didn't seem secure.
Very accommodating Very nice manager Very helpful
We had only one complaint. The cleaning lady was so nice. However two times she entered the room without knocking first. She thought we were gone but we were there. Would just recommend that she knocks even if she thinks the people aren't in there.
1. Shower was great. 2. Good size closet however no hangers. 3. Microwave/fridge.
1. No coffee pot 2. No ice machine 3. Mattress did not fit box spring; kept sliding around 4. Toilet too low. 5. Paid same money for much nicer places.


text:
Winter 2015 Registration is now available online
Most Special Events are not available for online registration, please contact the office for more information, 248-628-1720.
Online registration has no additional fees
Payments accepted: Visa or Mastercard
If you would like to register online for a class and HAVE NOT participated in Parks & Recreation programs before, please click on "new account" and follow the prompts.
If you HAVE participated in Parks & Recreation programs, log in using your registered email address and click "forgot your password." You will receive an email with a temporary password. If you are unable to log in please call 248-628-1720 for assistance.
To view our complete Winter 2015 Brochure and for complete class & program descriptions please visit our website, www.oxparkrec.org
PLEASE NOTE: To recieve the following discounts, registration MUST be taken in person at the Parks & Recreation Office.


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Tight budget, full plate in Portland
Another family pleaser is Tu Casa (70 Washington Ave., 207- 828-4971, www.tucasaportland.com). It serves authentic Salvadoran fare similar to Mexican, but lighter in a no-frills dining room. Theres a window for take-out orders, but grab a seat for table service. Begin with the appetizer puposas, the national dish of El Salvador, move onto the enchilada Salvadorena, or maybe the pollo enmole, marinated chicken paired with the homemade mole sauce. Escape with your wallet intact; nothing costs more than $12, and most items are far less.


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Pronunciation: /taŋˈʃan/
Definition of Tangshan in English:
An industrial city in Hebei province, NE China; population 1,658,200 (est. 2006). The city had to be rebuilt after a devastating earthquake in 1976.
Definition of Tangshan in:
- US English dictionary
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Please use this identifier to cite or link to this item:
Title: | Securing a medical wireless LAN system |
Authors: | Owens, TJ Tachakra, S Banitsas, KA Istepanian, RSH |
Keywords: | biomedical communication; cellular radio; costing; cryptography; technology management; telecommunication security |
Issue Date: | 2001 |
Publisher: | IEEE |
Citation: | IEEE EMBC Conference 2001 (23rd Annual International Conference of the IEEE Engineering in Medicine and Biology Society). Istanbul, Oct 2001. vol. 4, pp. 3552-3555. |
Abstract: | Recently the concept of MedLAN systems dedicated to application scenarios for wireless local area networks (WLAN) in hospital A&E departments has been presented. An essential element in the acceptance of the system will be reassuring all stakeholders in the system that data transmitted using the system is secure. In order for the stakeholders to be reassured technical and managerial issues have to be addressed. Technical issues to be addressed include selection of a suitable encryption algorithm with a 128-bit key for encrypting the wireless links and identification of a suitable crypto-board to be fitted to the system laptop computers. In many respects the managerial issues pose bigger challenges than the technical issues. Where the MedLAN system is being introduced members of the E-MED Systems Research Group will have to liaise with hospital managers to establish how the hospital's data security-policy can be expanded to accommodate the E-Med system and the cost implications of this expansion determined. The cost of insuring the security of the data handled by the MedLAN system will have to be determined. |
URI: | http://bura.brunel.ac.uk/handle/2438/2417 |
Appears in Collections: | Electronic and Computer Engineering Dept of Electronic and Computer Engineering Research Papers |
Files in This Item:
File | Description | Size | Format | |
Securing a medical wireless LAN system.pdf | 433.33 kB | Adobe PDF | View/Open |
Items in BURA are protected by copyright, with all rights reserved, unless otherwise indicated.


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2nd Annual Baltimore Social Justice Seder
Earned Sick and Safe Leave for Maryland
#BlackLivesMatter Solidarity
Together with our neighbors, Jews have the power to make our world more just and fair.
Have you heard the call? Our people's history, tradition, and values demand that we take action.
A progressive voice in the Jewish community, and a Jewish voice in the progressive community.


text:
Hot DealAnti-Gravity Coin/Muscle Pass DVD $19.95 $15.00 Save: 25% off Reward PointsNo points earned yet on current order. Log in to see how many reward points you have already earned Categories
LoginTestimonialsNumber one shop
On the internet you can order where want, but for me there is only one place to shop, even while I live on the other side of the ocean, and that is... Outstanding Service MJM has provided outstanding service on all my purchases. Thank you MJM!!!! |


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Shopping Cart
Around the Tarot in 78 Days: A Personal Journey Through the Cards
Journey into the exciting world of tarot with this comprehensive 78-day course.Uniquely presented in a one-card-per-day format, this workbook provides a solid foundation in tarot—and offers new ways to enrich your life using the wisdom of the cards. Well-known tarot readers and instructors Marcus Katz and Tali Goodwin take you through the symbolic landscape of tarot card by card. Progress throughTraditional Fortune Telling for Modern Life
Discover Your Future in 36 Cards Read the Lenormand cards to answer practical questions about romance, career, money, travel, and important life choices. With simple instructions for beginning readers, Learning Lenormand teaches easily accessible methods for using this simple thirty-six card deck. Providing fascinating historical research and proven techniques, renowned instructors Marcus KatzTarot Face to Face: Using the Cards in Your Everyday Life
Master the tarot cards with essential skills and methods for insightful readings in everyday situations! Are you ready to come face to face with the tarot deck and develop your unique reading style? This comprehensive guidebook is a fun, practical, and easy-to-follow tool with everything you need to become a confident and accurate reader. Based on over thirty years of tarot reading experience andUnlock Your Mind Power - More Friends, Money and Success!
Always wondered if we are using all of our mind power? Here is a great ebook on how to be able to exploit all your mind power! Everything you need to know about unleashing the power of your mind is included in this ebook. Do you want more friends? More Money? More Success? Unleashing the power of your mind can change all of that!The Secrets Behind "the Secret"
This book doesn't intend to represent any religious dogma in particular and was written based on the insights from several spiritual believes, ancient and modern, as well as known and secret to the public. Religion promotes a life philosophy and, although very misleading, also talks about universal truth. This paradigm is being noticed by modern science, which in some fields attests the dogma andMary K. Greer's 21 Ways to Read a Tarot Card
Drawing on nearly forty years of tarot experience, Mary K. Greer has developed a new energizing approach-made up of twenty-one stimulating techniques-to interpret or deepen your understanding of each card. Just as the twenty-one letters of the alphabet can be combined to form billions of words, Greer's twenty-one methods can be used in any combination for gaining amazing new insights andSex Magic for Beginners: The Easy & Fun Way to Tap into the Law of Attraction
A unique blend of sex magic and the Law of Attraction, this easy-to-use and practical book shows you how to use sexual energy to get anything you want. Popular author and magician Skye Alexander teaches you to direct your innate creative force to attract good health, prosperity, and happiness. From intensifying passion to achieving career success, this compact but comprehensive guide presents a


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Food Flashes
Food Flashes focuses on what's happening in the dining community, from restaurant openings and closings, to chef changes, to heart-healthy menus to kids' specials.
Posts filtered on Category
Lüke announces parade menu
Lüke, 125 E. Houston St., has announced its menu for the Ford Holiday River Parade on Nov. 29. • Roasted beet salad with spiced pecans, goat cheese and sugar cane vinaigrette • Market greens with buttermilk herb dressing, pickled carrots, cucumbers • Warm German potato salad, Creole mustard, Benton's bacon, red onions • New Orleans-style […]
[Read More]
New location for Marianito's
Marianito's Extreme Tex-Mex Grill has reopened in a new location. It's now at 10619 Westover Hills Boulevard, just off state Highway 151. Its previous location, at 2102 Bandera Road, closed last month. For information about the new location, call 210-433-7100 or visit www.marianitos.com.
[Read More]
Chef Tim Byres of Smoke signing books before Meatopia
Chef Tim Byres of Smoke restaurant in Dallas is signing copies of his recently released cookbook, Smoke: New Firewood Cooking. It's taking place from 11 a.m. – 1p.m. Sunday, Nov. 3, at The Twig, 306 Pearl Parkway. After the signing, he's participating in the Meatopia food festival at the Pearl.
[Read More]
Hard Rock Cafe announces River Parade packages
Hard Rock Cafe, 111 W. Crockett St., announced its packages for viewing the Ford Holiday River Parade on Nov. 29. River-level chair seating is available at $25 per chair, with a limited menu available. River-level table and street level front patio seating are available at $40 per seat, with a full menu available. Street-level side […]
[Read More]
Homebrewers teach their craft at events for Learn to Homebrew Day
This Saturday, Nov. 2, is the 14th annual Learn to Homebrew Day, organized by the American Homebrewers Association. The goal of the event is to expose new people to the hobby, and this year there are two events being put on by local homebrew clubs. The San Antonio Cerveceros are hosting their event at Villa […]
[Read More]
Q&A, dinner with Diana Kennedy on Sunday
Diana Kennedy, perhaps the most important writer on Mexican cuisine in the English language, is coming to San Antonio with a pair of appearances on Nov. 3. Among her books are "The Cuisines of Mexico," "The Art of Mexican Cooking," and the James Beard-award winning "Oaxaca al Gusto." Among her many honors, she was awarded […]
[Read More]
Terrace Grill at Riven Rock Ranch closing Nov. 17
Terrace Grill, the restaurant at the Riven Rock Ranch in Comfort, will close Nov. 17. The ranch had been up for auction and the new owners have decided to maintain it as a private residence. The ranch is closing to the public on Nov. 18. If you want to experience the Hill Country cuisine of […]
[Read More]
Shoney's, Paciugo offering free items for Veterans Day
Shoney's, 8206 Highway 35 N., and Paciugo, 999 E Basse Road, are offering free items to veterans and active duty military members on Veterans Day on Nov. 11. Shoney's is offering its All-American Burger. There's a limit of one burger per day per military service members and guests must show proof of military service. The […]
[Read More]
Del Fuegos offering delivery service
Del Fuegos CharGrill & Cantina, 2318 N. Loop 1604 W., is offering delivery service seven days a week to residential and commercial customers within a three-mile radius of its address. Find the menu at www.delfuegostx.com or call 210-493-2204.
[Read More]
Garrison Brothers introduces Single Barrel bourbon
Garrison Brothers Distillery in Hye has introduced Garrison Brothers Single Barrel that will be sold at the distillery, thanks to Senate Bill 905. "For years, bourbon drinkers and collectors have been asking us to release a Single Barrel, and it's high time we did," proprietor Dan Garrison said in a press release. "Senate Bill […]
[Read More]
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Acorn Tours of Boston
64 Weed Street, Lowell, MA 01852
1 people saved this
Private,historic and sightseeing tours of Boston and New England. Driving tours of varying lengths. Boston-2-hour walking tours. Languages: English,French & German
Of Note:
- Amenities: Accessible Facilities
March Lodging Deals, Special Offerings & Packages
Warmer weather is creeping towards us, even if you wouldn't know it from the past...
Search All Listings Here.


text:
Welcome, Sign In
The Balenciaga Sandal
Description:Selby Drummond, accessories editor at Vogue, has a sense of humor when it comes to fashion. Inspired by a brooch found in the Balenciaga archives, Alexander Wang designed these Balenciaga sandals for his 2014 resort collection. The track chain ankle strap make these shoes retro, chic, and perfectly playful. (1:11)
Source: VOGUE
Tags: accessories style , accessory trends , shoe trends , vogue


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Senior Member
Playing this match up as Malphite top lane. How's this lane gonna be? What should I do to win? >< Thank you!
sad face malphite, shen builds wits end and ****s on you.
I think shen has the upper hand on this lane, but all you have to do is farm a lot, and when he tries to go to another lane(ult), interrupt him. Farm farm farm, then late game on teamfights I guess you will be better than shen. AS reduction too gud.
im having this right now alos
same -_-
I'm having same
You need to bait out his shield and then you can win trades. The real problem is his sustain, so you'll have to be able to maintain your mana so that you will be able to poke him down. I would suggest rushing a Chalice for the mana sustain and MR to deal with his Q and passive. Skill-matchup, in my opinion. Edit: 2012 OP *guy above me


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PHOENIX — A federal judge ruled that the state of Arizona will have to reinstate a transit-support program worth several million dollars because its elimination violates federal law, The Arizona Republic reported. The ruling is expected to bring more than $10 million to local governments for transit projects and reverse the Arizona Legislature's 2010 move to end the Local Transportation Assistance Fund.
The Legislature's action was illegal, the judge ruled, because the transit-support funds are part of the Phoenix area's federally approved plan to fight air pollution and the Legislature does not have the authority to change federal Clean Air Act requirements without federal approval. For the full story, click here.
Receive latest news and stories


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So here goes my stackoverflow debut... (And I tried searching and reading thru a lot of posts (php flash date) before asking this)
I would like to make an swf that will find out what date it is and do sometinhg correspondingly. Eg. go to a specific frame or load a movieClip.
I would also like to avoid the Actionscript "Date Class" because I need to feel fairly certain that my end user will see the swf of the day not of his/her local date-setting.
So I'm thinking to somehow bring in php with 'echo date' inside the swf and then have the result create a goto-(or load-)command.
To make matters worse I have to rely only on code within the swf using ActionScript2 (in CS4 or 5). I am not able to publish any outside php (or any other code for that matter) so everything must be done internally. Only swf has to work regardless of server (I am expecting the server will work with php)
So I hope some gurus here will be able to tell me: 1. If this is possible 2. How I make the date thing happen in Flash 3. And how I somehow grab the date as a variable (?) and make that determine some action.
How do you like them apples?


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Menu Innovations
Menu Innovations
featured articles
industry headlines
Charlotte-based Salsarita's Fresh Cantina and Fort Mill-based PuckerButt Pepper Company have teamed up to create the World's Hottest Burrito.
Long John Silver's announced the return of Norway Lobster Bites to its menu.
Wendy's is reeling in fans with the return of its Premium Cod sandwich, which brings cod back to Wendy's restaurants across the nation for a limite
Schlotzsky's leaps into spring with the return of the limited time only Hand-Carved Sandwiches.
With the conclusion of the food-heavy holiday season, many people are starting to boost their exercise and diet plans, w
This February, Giardino Gourmet Salads, the fast casual salad concept of South Florida, introduces grain bowls to its me
Chef Andy Wittman has done it again.
Slow Roasted Pulled Pork is joining char-grilled chicken, steak, tofu, ground beef, and fish to become the sixth protein
Rita's Italian Ice, one of the world's largest Italian Ice concepts with over 600 st
Pretzelmaker introduces a new product to its pretzel product line up, Bowl of Bites available with two flavors, Chili &a
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Newly expanded classes now include Vinyasa, Vin-Yin, restorative, hot, and kids' yoga sessions as well as Pilates-Barre
Mountain Yoga Sandy
$204 $29
Six-Month Gym Membership or Three-Month Training Packages at Square 1 Fitness (Up to 70% Off). Three Options.
Square 1 Fitness
Clients work toward fitness goals at their own pace or get assistance from personal trainers or small group classes
$90 $35
View Deal
10 Fitness Classes or One Month of Unlimited Fitness Classes at Fuze Fitness (Up to 58% Off)
Fuze Fitness
Energetic music plays as instructors lead fitness classes that incorporate elements of ballet, Pilates, and other styles
$50 $21
$23 for $45 Worth of Zumba — Guz Fitness and Lifestyle
Guz Fitness And Lifestyle
Local businesses like this one promote thriving, distinctive communities by offering a rich array of goods and services to locals like you
$45 $23
$27 for 10 Classes at Cardio Barre Quarry Bend ($150 Value)
Cardio Barre Quarry Bend
Ballet-inspired movements help exercisers tone their rears, legs, torsos, and arms
$150 $27
One Month of Unlimited Classes for One, Two, or Up to Five at Hidden Valley Mixed Martial Arts (Up to 67% Off)
Hidden Valley Mixed Martial Arts
Students get a chance to explore various styles of martial arts with one month of classes, including kick boxing, Jiu Jitsu, MMA and judo
$97 $35
$19 for Two Weeks of Boxing and Kickboxing Classes with Hand Wraps at TITLE Boxing Club ($46.49 Value)
TITLE Boxing Club
Cottonwood Heights
A national fitness franchise teaches students the ropes in boxing and kickboxing workouts that help burn up to 1,000 calories per class
$46.49 $19
One Month of Sports Training from Integrated Sports Performance (61% Off). Two Options Available.
Integrated Sports Performance
Coaches construct a plan to help non-athletes shape up and athletes meet their best potential on the field, court or other competition space
$75 $29
One Month of Aerial Silks Classes at Hunt's Gymnastics Academy (55% Off)
Hunt's Gymnastics Academy
Build strength and flexibility in high-flying aerial silks classes
$55 $25
One Week of Foundation CrossFit Classes with Three or Five Weeks of Regular Classes at CrossFit Aether (88% Off)
CrossFit Aether
Daily changing combinations of structural, gymnastic, and weightlifting moves quickly whip exercisers into shape
$175 $21
Three Yoga Classes at Yoga with Ashley (64% Off)
Yoga With Ashley
From the merchant: Come practice and find the bliss that yoga can bring into your life
$33 $12
4 or 10 Boxing, Kickboxing, or Muay Thai Classes at Ultimate Combat Training Center (Up to 83% Off)
Ultimate Combat Training Center
Classes and teens and adults cover the fundamentals of boxing, kickboxing, or Muay Thai and include gloves and personal training
$155 $27
Two, Four, or Six Personal-Training Sessions at The Personal Best Club (up to 79% Off)
The Personal Best Club
South Jordan
Trainer motivates clients to reach fitness goals with cardio, strength, and core exercises that tone muscles, improve stamina, and shed fat
$140 $39
$39 for One Month of Unlimited Fitness Classes at Studio HV ($159 Value)
Studio HV
The Towers At South Towne
Functional form-fitness classes begin every 35 minutes and change daily in order to keep muscles guessing all month
$159 $39
One Month of Unlimited CrossFit Classes with Optional Foundations Classes at SoJo CrossFit (Up to 51% Off)
SoJo CrossFit
South Jordan Town Center
$90 $44
One 30-Class Yoga & Fitness Pass or Six Months of Unlimited Classes from MetaBody (Up to 94% Off)
MetaBody Yoga & Fitness Pass
Multiple Locations
Instructors encourage full-body results in a variety of fitness classes supplemented by a success guide and nutritional coaching
$350 $20
Four Weeks of Unlimited CrossFit Classes at CrossFit Extraordinaire (65% Off)
Crossfit Extraordinaire
West Jordan
$125 $44
$20 for 30-Class Yoga and Fitness Pass from Yoga & Fitness Passport ($300 Value)
Yoga & Fitness Passport
Students can use this 30-class pass at multiple locations to take everything from yoga to Zumba, signing up on an easy online platform
$300 $20
$52 for One Month of Unlimited CrossFit Classes at CrossFit West Jordan ($129 Value)
Crossfit West Jordan
Trainers mix up fitness routines with a new workout every day, from swinging sledgehammers through the hair to flipping tires
$129 $52
$20 for 30 Fitness and Kickboxing Classes from FIGHT2FITNESS ($299 Value)
30-class pass allows students of all ability levels to gain access to a variety of fitness and Kickboxing classes at various locations
$299 $20
$33 for $65 Groupon — TrueWorkout
True Work Out
$65 $33
30 Days of Fitness Classes, 10 Fitness Classes, or a One-Month Fitness Package at Pure Workout (Up to 63% Off)
Pure Workout
No-nonsense gym plans intense, one-hour workouts that incorporate strength and cardio drills
$175 $69
One-Week Diet and Exercise Program at Crossfit Fission (65% Off)
Crossfit Fission
West Valley City
From the merchant: Design a workout program specific to your fitness goals and give nutritional advice.
$200 $70
$75 for One Month of Unlimited Yoga Classes at CorePower Yoga ($144 Value)
CorePower Yoga
Dynamic flow of Vinyasa-style poses combined with breathing exercises, uplifting music and heat energize the body and mind.
$144 $75
Two Yoga Classes at Divine Wellness (64% Off)
Divine Wellness
Salt Lake City
From the merchant: SUP Yoga is yoga on a stand up paddleboard. Classes are offered 3 times per week at SLC Sports Complex this winter.
$70 $25
3 Foundation Classes With Option for 1 Month of Unlimited CrossFit at Salt Lake City CrossFit East (Up to 74% Off)
Salt Lake City CrossFit East
Mount Olympus
Foundations classes help ease new CrossFit members into the basics of the exercises and their safe execution
$135 $35
Four Weeks of Pilates Reformer Classes at The Point Pilates LLC (65% Off)
The Point Pilates
From the merchant: Don't give up on those New Year's resolutions!!! Torch calories and have fun while doing it at The Point Pilates.
$150 $53
Five Mat and Barre Classes, Five Equipment Classes, or Four of Each at Poise & Strength Pilates (Up to 76% Off)
Poise & Strength Pilates
Instructors enhance Pilates Mat and Reformer exercises with ballet-barre work during morning and evening classes seven days a week
$100 $29
$77 for $140 Worth of Services at The Pre Natal Fitness Center
The Pre Natal Fitness Center
Sugar House
$140 $77
$89 for 20 Yoga Classes at Wasatch Ayurveda and Yoga Upstairs ($160 Value)
Wasatch Ayurveda and Yoga Upstairs
Yoga students build strength and flexibility as they flow through poses and breathing exercises
$160 $89
$19 for Two Weeks of Boxing and Kickboxing Classes with Hand Wraps at Title Boxing Club ($58 Value)
TITLE Boxing Club Sugarhouse
Boxing and kickboxing classes blast calories while toning the entire body
$58 $19
$18 for $60 Worth of Fitness Classes — Lott Fitness
Lott Fitness
Two sessions with a certified personal trainer, or a pass for 10 classes including yoga, weight training, and core work.
$60 $18
10 XF Aerial-Yoga Classes or One Month of Unlimited Aerial Yoga with Gym Access at Xcel Fitness (Up to 77% Off)
Xcel Fitness
Xcel Spa & Fitness
Aerial yoga classes that stretch and strengthen muscles, followed by workouts in the gym's 53,000 square-foot facility
$150 $34
10 or 20 Pilates, Rip60, or Cycling Classes at The Gym at City Creek (Up to 78% Off)
The Gym at City Creek
Rio Grande
Exercisers burn calories, tone muscles, boost strength, and improve endurance through Pilates, cycling, or Rip60 classes
$170 $39
Barre and Pilates Class Packages at Total Body Pilates (Up to 68% Off). Two Options Available.
Total Body Pilates
Certified instructors lead Pilates Reformer and mat classes to help students sculpt lean muscles and raise heart rates
$90 $29
Three Pilates Reformer Sessions with Optional Private or Semiprivate Lessons at A Beautiful SurpriZe (Up to 61% Off)
A Beautiful SurpriZe
Atop Pilates Reformers, students in group or private lessons burn body fat, tone muscles, fortify their cores, and straighten their posture
20 Fitness Boot Camp Classes from Hiit it Fitness (65% Off)
Hiit It Fitness
Three Introductory Pilates Equipment Classes from Body and Mind Studio (65% Off)
Body And Mind Studio
$120 $42
5- or 10-Class Punch Cards for Mom Fitness or Movement Preschool from Cahoots Fitness (Up to 52% Off)
Cahoots Fitness
Instructors lead fitness classes for women while children participate in fitness activities
$45 $22
One or Three Months of Unlimited CrossFit Classes at Elevated CrossFit American Fork (75% Off)
Elevated CrossFit American Fork
Constantly varied exercises emphasize functional movement
$194 $49
10 or 20 Krav Maga Self-Defense Classes with Optional Personal Training at Self Defense Utah (Up to 71% Off)
Self Defense Utah
Krav maga self-defense classes incorporate HIIT, jujitsu, muay thai, kickboxing, and self-defense
$150 $49
Four or Eight Weeks of Boot-Camp Classes (71% Off)
Reach Fitness
Shed pounds and sculpt muscle during boot-camp classes led by a semi-pro football player and personal trainer with multiple certifications
$120 $35
10 Fitness Classes or Two Months of Unlimited Fitness Classes at Fit to Dance Club (Up to 57% Off)
Fit to Dance Club
Saratoga Springs
Women's-only fitness studio hosts classes that range from Zumba's Latin-infused dance moves to core-building Pilates routines
$35 $15
10-Class Pass or One Month of Unlimited Classes at Pit Elevated (Up to 42% Off)
Pit Elevated
This MMA-focused gym offers a wide variety of fitness classes for all ages, from kids' fitness to yoga to Muay Thai and Brazilian jiu-jitsu
$50 $29
One Month of Unlimited Boot-Camp Classes, or 10 Drop-In Boot-Camp Classes at GPP Fitness (Up to 61% Off)
GPP Fitness
Intense workouts focus on getting clients healthy as quickly as possible, challenging exercisers with weights and kettlebells
$100 $39
One or Three Months of Unlimited CrossFit at Crossfit Kimokeo (Up to 53% Off)
Crossfit Kimokeo
Coaches blend strength, endurance, flexibility, and agility training in an ever-changing array of workouts
$105 $52
8 or 15 Classes at Higher Ground Hot Yoga (Up to 54% Off)
Higher Ground Hot Yoga
Supportive instructors lead sessions of hot yoga, which promotes detoxification, improves muscle elasticity, and elevates heart rates
$120 $59
One or Two Months of Unlimited Yoga Classes at Epix Fit Club (Up to 52% Off)
Epix Fit Club
Students build strength, flexibility, and focus as they flow through yoga poses and breathing exercises
$29 $14
Six-Week Diet and Exercise Program at Elevé Pole and Fitness (50% Off)
Elevé Pole And Fitness
From the merchant: Have a wedding, reunion, or vacation coming up? Get fit & toned with our personalized meal plan & fitness program.
$299 $150
5 or 10 Equipment Classes or One Month of Unlimited Classes at Propulsion Pilates in South Ogden (Up to 72% Off)
Propulsion Pilates
South Ogden Location
Circuit-style classes combine Reformer and Tower sequences with cardio exercises and kettlebell work
$125 $39
$49 for One Month of Unlimited Boot-Camp and Nutrition Classes at 12th Street Fitness ($150 Value)
12th Street Fitness
Certified instructors provide nutrition counseling and lead group boot-camp classes in a 5,500 sq. ft. open gym space
One, Two, or Three Months of Unlimited CrossFit Classes at CrossFit Ogden (Up to 53% Off)
CrossFit Ogden
Get in shape with one, two, or three month's worth of intense unlimited CrossFit workouts
$125 $62
$39 for $140 Worth of Personal Fitness Program — Mt Ogden CrossFit
Mt Ogden Crossfit
One month of unlimited group personal fitness classes with introductory Elements class
$27 for 10 Yoga Classes at Lotus Yoga & Dance Studio ($75 Value)
Lotus Yoga & Dance Studio
Restorative, relaxing yoga classes include breath-focused Vinyasa Flow, heart-pumping Power Yoga, and strengthening Ashtanga sessions
$75 $27
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How One Developer Got the Internet To Watch People Code 65
from the next-year-we'll-all-be-crowdcoding dept.
from the next-year-we'll-all-be-crowdcoding dept.
blottsie writes: While Twitch TV is generally used for livestreaming gameplay, Alexander Putilin has other plans for the platform. Putilin and his girlfriend are using Twitch to build a community of software developers and students who broadcast complex floating point operations and algorithm design to the rest of the world. The community is responding and growing alongside its newfound popularity. WatchPeopleCode is now facilitating live hackathons (there was one this weekend), enabling programmers to meet and collaborate with people that they'd otherwise never be able to.


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Small and Smaller: Extreme Living
How do these homeowners live large in 400, 200 and even less than 100 square feet?
Who says your home has to be huge to make you happy? These days, many people are choosing to live in tiny spaces and doing so in comfort and style. From miniscule studio apartments in the city to petite cabins way out in the country, scaled-down homes encourage residents to clarify their priorities, edit their possessions and prove that be it ever so humble, there really is no place like home.
Extreme Small Space Videos
See All Videos-
Small Space Apartment Design
02:54 -
Transformer Apartment in Manhattan
03:30 -
Retro Metro Houseboat
Urban Living
With rents inching toward $100 per square foot, big-city apartments don't come cheap. But rather than move out to the 'burbs where the prices are lower, city lovers choose to stay near the hustle and bustle, even if it means living in apartments as small as 175 square feet.
Designer Tips for Small Urban Living
See All PhotosSustainable Living
With the earth getting more crowded and land more expensive, many Americans are looking for ways to live smaller and more simply. Whether they're building cabins in the woods or converting old shipping containers into studio apartments, these homeowners are being kind to the planet and to their wallets and find that living without a house full of possessions actually makes their lives feel fuller.
Extreme Small Homes
See All Videos-
Napa Valley Treehouse
02:53 -
Organization in Small Apartment in New York City
03:08 -
Historic Alleyway Home
10 Extreme Tiny Homes
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6 Photos
Designer Tips for Small Urban Living
7 Photos
Catch Up With Our Blogs
We're serving up the latest news, gorgeous style, crafty DIY projects, clever entertaining tips and more. Consider these your design digests.


text:
Rain brings winter chill to Delhi
The BJP had received as much money in donations as all other national political parties combined for the 2014 general elections that it won with a sweeping verdict, says a report by the Association for Democratic Reforms (ADR).
The government will adopt verticals on Prime Minister's Narendra Modi's sectoral initiatives instead of divisions for the smooth functioning of the newly set up National Institution for Transforming India (Niti) Aayog.
Former union minister Anand Sharma will be the new chairman of the All India Congress Committee (AICC) communications department, replacing Ajay Maken who has been appointed as the head of the party's Delhi unit.
Delhi police crime branch is probing the role of two employees of GMR Energy Limited for their alleged link with some multi-tasking staff (MTS) involved in stealing/leaking "classified documents" from the offices of oil, coal, power and three other ministries.
Most khap panchayats may be caught in a medieval time warp but one such village council in Haryana is fighting against the stereotype — not only by working against female foeticide and dowry but also going digital, launching a website.
Amid protest from allies and Opposition, the Centre introduced two bills to replace — Coal Ordinances that allows auctioning of mining leases and the Citizenship Ordinance — in Lok Sabha on Monday.
The army is no stranger to controversy in Jammu and Kashmir and the months ahead could witness fresh debate over the continuation of the Armed Forces Special Powers Act (AFSPA) , especially with the home ministry rejecting a recommendation to repeal the law.
One of the men convicted of the brutal 2012 gang-rape of a 23-year-old woman on a moving bus in Delhi has triggered outrage by claiming the victim was to blame for her fatal sexual assault.
Revelations by some rescued girls have blown the lid off the flourishing human trafficking racket in Jharkhand's tribal hinterlands, a hotbed of modern day slavery where family members, neighbours, local politicians and even NGOs are involved in well-organised rings.
Congress President Sonia Gandhi has refused to comment on speculations about a larger role for Priyanka Gandhi and appeared annoyed over questions on Rahul Gandhi's whereabouts.
The PDP demanded the return of executed terrorist Afzal Guru's mortal remains on Monday, a day after Jammu and Kashmir chief minister Mufti Mohammad Sayeed gave a rocky start to the party's coalition with the BJP by crediting separatist leaders, militants and Pakistan for the smooth conduct of assembly elections.
In what could be an indication of the Centre's changing approach towards probes into instances of so-called "Hindu terror", the NDA government asked the NIA not to challenge the Andhra high court giving bail to two accused in the 2007 Mecca Masjid blast case, sources told HT.
From refusing an Islamic skullcap in 2011 to warmly hugging 14 Muslim ministers from a party that often veers towards soft separatism— the images of Prime Minister Narendra Modi from Jammu on Sunday were telling as the chalk-and-cheese PDP-BJP government was sworn in.
People in Delhi woke up to a rainy morning with the minimum temperature recorded at 14.8 degrees Celsius, two notches above the season's average. The Met Office has forecast more rain and an overcast day ahead.
A 'good deal' for land is a matter of perspective. Sometimes it is a study in contrasts — what makes for a 'good deal' in Singur in West Bengal is only one-tenth of the going rate for land in, for instance, certain parts of Gurgaon.


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One writer's battle with the eating disorder orthorexia sheds light on the question, "Is there such a thing as eating too healthy?"
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Love that satisfied feeling of eating your daily fruits and veggies? See what's making those healthy habits harder to maintain.


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text: I Want To Try Sucking Dick Me Too Personal Stories, Advice, and Support 96 People Sort popular recent Haven't Done It In Many Years The first and only **** I ever sucked on was my brothers back in the experimenting years lol. Now that I know what it is and what one is made for I really want to try it again. I'm married and my wife would never ever approve, but I just want to know what it's like when you... lvpntys69 41-45, M 6 Responses 8 Mar 19, 2011 Your Response Please respond with authenticity, support, and respect Cancel You can join Experience Project now! Username: Password Email Birthday Gender Select Male Female Trans* Already a member? Sign In By clicking "Sign Up", you confirm that you agree to the Terms of Service of Experience Project, Inc. Login Login to respond Username: Password: Keep me logged in Not a member? Sign Up Sign Up More Stories


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US 5643759 A Abstract The present invention relates to a method for the selective preparation of hybridoma cell lines which produce a murine monoclonal antibody (MAK) of the IgGl class with a high capacity for including NK cell-relate cytotoxicity against human CD16 antigen by co-culturing the hybridoma cells in the selection medium with unstimulated human NK (natural killer) cells. The invention furthermore relates to a cell line A9 of the DSM deposit number ACC 2148 obtainable in this manner, and a MAK obtainable thereform, and a method for the preparation of bispecific MAK's by the fusion of an anti-CD30 cell line HRS-3 with hybridoma cell lines, which is obtained by the above-mentioned selection process, expecially with the A9 line of DSM deposit number ACC 2148 obtaining especially the bispecific MAK of the HRS-3/A9 cell line with the DSM deposit number ACC 2142. These bispecific MAK's are suitable for the treatment and for the shrinking of established human tumors, especially human Hodgkin's tumors.
Claims(4) 1. A method for preparing bispecific MAB's, which comprises fusing a hybridoma cell, A9, which produces a murine MAB of class IgGl with a high capacity for inducing NK cell-related cytotoxicity, whereas said MAB is specific for human CD16 antigen, with hybridoma cells HRS-3 which binds human CD30 antigen to form a tetradoma HRS-3/A9 of deposit account number ACC 2142 thereby to obtain a bispecific MAB and isolating bispecific MAB, the hybridoma cell line A9 having been produced by
a. co-culturing hybridoma cell line A9 of deposit number ACC 2148 which produces CD 16 MAB with unstimulated human NK cells, b. determining the die-off rate of the hybridoma cells and then c. selecting the hybridoma cells with the highest die-off rate. 2. A method according to claim 1, wherein the co-culturing according to step a is performed in the presence of granulocytes and the determination of the die-off rates according to step b is performed by comparison with a control CD16 antibody.
3. The bispecific MAB which is produced or producible by the process of claim 1.
4. The tetradoma HRS-3/A9 which is produced on producible by the process of claim 1.
Description The present invention relates to a method for the selective preparation of hybridoma cell lines which produce a murine monoclonal antibody (MAK) of the IgGl class with a high capacity for inducing NK cell-related cytotoxicity against human CD16 antigen by co-culturing the hybridoma cells in the selection medium with unstimulated human NK (natural killer) cells. The invention furthermore relates to a cell line A9 of the DSM deposit number ACC 2148 obtainable in this manner, and an MAK obtainable therefrom, and a method for the preparation of bispecific MAK's by the fusion of an anti-CD30 cell line HRS-3 with hybridoma cell lines, which is obtained by the abovementioned selection process, especially with the A9 line of DSM deposit number ACC 2148, obtaining especially the bispecific MAK of the HRS-3/A9 cell line with the DSM deposit number ACC 2142. These bispecific MAK's are suitable for the treatment and for the shrinking of established human tumors, especially human Hodgkin's tumors. The application of bifunctional monoclonal antibodies (Bi-MAK) for the treatment of tumors has been published by several groups of workers. The basis of all such works is the idea of breaking through the body's tolerance of the tumor by directly targeting the tumor cells with cytotoxic effector cells. The previous use of bispecific monoclonal antibodies as immunomodulators has consisted mainly of aiming the cytolytic activity of T cells against tumor cells through the CD3/T cell receptor complex (CD3/TCR complex) [Stearz US, Bevan, M. J.: Proc. Acad. Sci. USA 83: 1453-1457 (1986); Perez et al., Nature 316: 354 (1985)]. However, this had the disadvantage that a previous in vitro stimulation of the T-lymphocytes by an additional activation signal was necessary [Pupa S. M. et al: Int. J. Cancer 42: 455 (1988); Garrido M. A. et al: J Immunol 144: 2891 (1990)]. Another sub-population of human lymphocytes having the ability to lyse tumor cells is represented by natural killer cells (NK cells). As regards their cellular morphology these are large, granulated lymphocytes (LGL) and their immune phenotype is characterized by the absence of the CD3/TCR complex and by the expression of CD2, CD56 and CD16 antigens [Anderson P. et al: Nature 341: 159-162 (1989); Herberman R. B., Ortaldo, J. R.: Science 214: 24 (1981); Lanier L. L. et al: J Immunol 136: 4480 (1986)]. In contrast to T cells, NK cells show direct cytolytic activity against a series of "NK-cell-sensitive" tumor cells without prior activation. However, the majority of fresh and cultured tumor cells are relatively resistant to the anti-tumor action of dormant peripheral NK cells, which are not rendered capable of developing cytolytic activity against part of these NK-cell-resistant tumor cells until activated with interleukin-2 (IL-2) [Ferrini S. et al: J Immunol 138: 1297 (1987). Phillips J. H., Lanier L. L.: J Exp Med 164: 814 (1986)]. The CD16 antigen is a low-affinity receptor for the Fc part of complexed immunoglobulins (=Fc-gamma-III receptor) with a molecular weight of 50-70 kDa from which several cDNA clones have been isolated and sequenced cDNA clone. Numerous functional studies of the Fc-gamma-III-receptor have demonstrated its importance as the one effective activation antigen of NK cells. At the same time, biological functions of NK cells have been found which mediate biological functions of NK cells such as ADCC through the Fc-gamma-III receptor, the effectiveness of which in the use of monoclonal antibodies is subclass related. On the basis of the functional importance of the CD16 antigen for the activation of NK cells, a selective induction of tumor cell lysis via CD16 through bispecific antibodies therefore appeared to be especially promising. Actually it was possible with hetero conjugates of anti-CD16 antibodies and anti-DNP (dinitrophenyl phosphate) and tumor cell associated antibodies to induce selective lysis of DNP-coupled chicken erythrocytes [Karpovsky B. et al: J Exp Med 160: 1686 (1984)] and tumor cells of a human colonic carcinoma cell line established in vitro [Titus J. A. et al: J Immunol 139: 3153 (1987)] and murine T cell line [Garrido M. A. et al: J Immunol 144: 2891 (1990)] by unstimulated and IL-2-activated NK cells. Titus J. A. et al [J Immunol 139: 153] described a chemically crosslinked Bi-MAK against CD16 and a melanoma antigen which effectively inhibited tumor growth in vivo in the naked mouse. Bi-MAK's against CD16 and tumor antigens which were obtained by hybrid hybridoma technology (tetradoma technology) are described in works of Ferrini et al. [Int J Cancer Suppl 7: 15 (1992] and Ring, D. [Cetus, PCT/US91/16949]. These tetradomas produce Bi-MAK's, which link the CD16 antigen and epidermal growth factor receptor and multi-drug resistance antigen. Likewise Fanget et al. [Medarex, PCT/US90/05981] describe Bi-MAK against CD16 and tumor-associated antigens. Impeding the growth of tumors with the bispecific MAK's is thus possible, but not a complete regression of established human tumors. It is therefore the purpose of the present invention to develop CD16 MAK's which by themselves and in conjunction with known MAK's have the ability to induce a maximum NK cytotoxicity whereby the growth of human tumors will not be just impeded, but instead a complete remission of established human tumors will be achieved. This purpose is achieved by the invention by preparing hybridoma cell lines which produce a murine MAK of the IgGl class against human CD16 antigen and have a high activity as regards the induction of NK cell-related cytotoxicity, by co-culturing in the selection medium the hybridoma cell lines obtained by a method known in itself (Kohler/Milstein) with unstimulated human NK cells, especially granulocytes, and selecting the cells with the highest die-off rate. Surprisingly it was found that the hybridoma cells, which form CD16 antibodies with an especially high cytotoxicity-inducing activity, have the highest die-off rate when they are co-cultured with dormant NK cells. In accordance with the invention, the determination of the highest die-off rates is performed by comparison with a known anti-CD16 MAK, such as VEB 13, for example. In this manner the cell line A9 of the DSM Deposit Number ACC 2148 is obtained, which has MAK's with the above-named properties. This line or its MAK's selected specifically in the manner of the invention bind, as inhibition experiments show, to epitopes on the CD16 antigen which are remote from the binding epitope of known anti-CD16 MAK's, such as 3G8 (IgGl-Kappa, Dianova, Hamburg, Germany) or Leu 11b (commercially available), the MAK derived from line A9 attacking a binding point far away from the epitope of the known 3G8, and the MAK obtained from a line C10 attacking one somewhat nearer to it. Furthermore, the MAKA9 prepared according to the invention binds but very weakly to soluble recombinant human CD16, while the known antibodies referred to above bind more strongly. This shows that A9 recognizes epitopes other than the known anti-CK16 MAK's. The MAK derived from A9 is a lambda subtype of mouse IgGl. The MAK's derived from the A9 cell line prepared according to the invention are capable of inducing in lymphocytes a cytological activity against the corresponding antibody-producing hybridoma cells, while an approximately 60% specific lysis occurs, which has not been achieved with the MAK's heretofore known. Thus these hybridoma cells, especially the cells derived from the A9 line, fuse in a manner known in itself with HRS-3-hybridoma cells to form tetradomas (hybrid hybridomas), from which bispecific MAK's are obtained, especially from the line HRS-3/A9 with the DSM Deutsche Sammlung Von Mikroorganismen Und Zell Kulturen GmbH, Mascheroder Weg 1 B, D-38124 Braunscgweig Deposit Number ACC 2142, which are effective in the suppression of human tumors, especially in the suppression of established Hodgkin's tumors, the Bi-MAK on the one hand being more effective than the individual components A9 and HRS-3, and on the other hand more effective than the previously known BiMAK's named above with which only a restraint of tumor growths can be achieved, but not a complete regression. The hybridoma cells and the MAK's and Bi-MAK's derived from them can be prepared in the following manner: Immunized BALB/C mouse spleen cells are fused with mouse myeloma cells in a known manner. The cell suspension obtained is then co-cultured in the selection medium in the presence of unstimulated human NK cells, especially granulocytes and, if desired, peripheral blood lymphocytes (PBL). By comparing the anti-CD16 activity with known CD16 antibodies, such as VEP 13, for example, the hybridoma cells are selected which have the highest die-off rates. By this principle of reverse cytotoxicity the line A9, especially, with the DSM Deposit Number ACC 2148 is obtained. This line is propagated in a known manner by injection into BALB/c mice and obtained from the ascites by chromatographic purification. To prepare the Bi-MAK's, especially Bi-MAK's HRS-3/A9 with two light chains of different classes (lambda and kappa), HGPRT-negative HRS-3 hybridoma cells (IgGl, kappa, subtype, active against Hodgkin's and Reed-Sternberg cell-associated CD30 antigen, described by Engert et al, 1990), are fused with A9 hybridoma cells treated, for example, with iodoacetamide. After testing for bispecific reactivity, for example by the detection of antibodies with different light-chain content by indirect immunofluorescence, the mixed tetradoma cell clone is obtained which has the strongest possible simultaneous expression of the two light chains, and then purified by chromatography. Experiments in vivo with the Bi-MAK HRS-3/A9 in the treatment of human Hodgkin's tumors in SCID mice show that, in a majority of the animals tested, a complete remission of the tumors occurs. The Bi-MAK HRS-3/A9 can be formulated by known methods, e.g., as a solution of lyophilizate, and serves for parenteral application in humans, while adjuvants and additives known to the expert can be present. The invention will be further described by the following examples: More specifically, "the cell line produces the described bispecific monoclonal antibody and secretes it into the culture medium. The antibody will be purified and reconstituted, for example, in PBS. This antibody preparation will be given to patients with tumors the neoplastic cells of which express the CD30 antigen and are refractory to standard chemo- and radiotherapy treatment. Such tumors are Hodgkin's disease (HD), anaplastic large cell lymphoma (ALCL) and HTLV-1-positive adult T-cell leukemia (ATL). Patients eligible for this treatment would have a life expectancy of a few months without this treatment and account for about 30% with ALCL and the majority of patients with ATL. It is expected that the antibody will bind the patients' own natural killer cells onto the tumor cells and mediate a specific activation signal so that the patient's own killer cells will kill his tumor cells. The preclinical data show that the antibody in conjunction with the patient's killer cells is able to cure SCID mice from the patients' tumors. In clinical practice the treatment of the respective patients consists of 10 mg/m2 of the antibody which will be given intravenously by a 1-hour infusion to each patient. The treatment will be repeated weekly until either the patient is cured from his tumor or the tumor shows progressive growth despite the antibody treatment. In addition to its therapeutic usefulness, the described bispecific monoclonal antibody can also be used to assay a person's Bi MAB-mediated natural killer cell capacity. This is accomplished by an in vitro cyctotoxicity assay (as described above) using defined concentrations of Bi MAB, the lymphocytes of the patient to be tested on the L640 Hodgkin's cell line. The invention will be further described in the examples hereinbelow taken in conjunction with the accompanying drawings, wherein: FIG. 1 shows the results of inhibition tests of C10 and A9 Biotin; FIG. 2 shows the results of inhibition tests of VEP 13 Biotin and 3G8 FITC; FIGS. 3A-3C shows the results of sandwich--ELISA tests with soluable recombinant human C D16; FIG. 4 shows the induction of NK-cell-related cytotoxicity (specific lysis) of the antibodies A9, C10 and HRS-3 against hybridoma cells producing CD16 MAK, in accordance with Example 5; FIG. 5 shows a chromatogram concerning ion exchange chromatographic purification of the Bi-MAK from MAKA9 and MAK HRS-3 in accordance with the method described in Example 6. Thereby peak 2 shows the fraction containing the Bi-MAK for exhibiting bispecific activity; FIGS. 6 and 7 show the induction of a cytotoxicity (specific lysis) mediated by NK-cells against human tumor cells in vitro (CD30-antigen positive Hodgkin's lymphona cell line L540) of the claimed Bi-MAK HRS-3/A9 as well as of original antibodies HRS-3, A9 in accordance with the procedure in Example 7; and FIG. 8 shows the regression of tumor growth of human Hodgkin's tumor in mice treated with the claimed Bi-MAK PIRS 3/A9 in comparison with non-treated animals in accordance with Example 8. I. Preparation of A9 and C10 CD16-MAK Immunization, Fusion and Cloning of Monoclonal Antibodies A9 and C10 For the immunization, female BALB/c mice 3 to 4 months old were used. 3-5�107 freshly isolated granulocytes in 0.5 ml PBS were injected peritoneally into the animals at two-week intervals. The first injection was performed in the form of a 1:1 emulsion in complete Freund's adjuvant, the second and third injection as 1:1 emulsions in incomplete Freund's adjuvant. The rest of the immunization steps were performed without adjuvant. Ten weeks later, on each of the four days preceding the planned fusion, the animals were given a booster injection of 5�107 granulocytes each. The immunized spleen cells were fused with AG8-653 mouse myeloma cells in a ratio of 2:1 in the presence of polyethylene glycol. The fused cell suspension was washed once and cultured in the selection medium (RPMI 1640, 10% fetal calf serum), 4 mM glutamine, HAT: hypoxanthine 13.6 mg/1; aminopterin 0.17 mg/1 and 10 μg/ml insulin. Fourteen days after the fusion, supernatants of cultures which showed hybridoma growth were tested for production of anti-CD16. For that purpose peripheral blood lymphocytes (PBL) and granulocytes were incubated with 10 μl of test supernatant fluid or control antibody. The commercially obtainable CD16 antibody VEP13 was used as the control MAK. The bound MAK was detected by means of an FITC-tagged mouse antibody in the cytofluorometer. Of 1021 supernatants tested, 4 showed a CD16-specific binding pattern. After 4 cloning steps using the limiting dilution method (seeding density 0.3 cells per culture) these 4 clones were isolated. A9 is a murine IgGl -MAK with a lambda light chain, and C10 is a murine IgGl-MAK with a kappa light chain. Both show a significant binding to NK cells. In vivo production and purification of MAK A9 and C10 The anti-CD16 MAK's A9 and C10 were produced in large quantities in vivo by intraperitoneal injection of A9/C10 hybridoma cells into BABL/c mice. One week before the hybridoma cell injection the mice were primed intraperitoneally with 1.0 ml of Pristan. 8 to 14 days after the hybridoma cell injection ascites fluid could be taken. The MAK's A9 and C10 were then precipitated from the ascites fluid by ammonium sulfate precipitation (4% saturation). Then followed additional purification of the antibodies by anion exchange chromatography. This was performed with a Mono-Q� column (Pharmacia, Freiburg). II. Comparison of MAK A9 and C10 with other anti-CD16 MAK's Inhibition Experiments in the Determination of Epitopes (FIGS. 1 and 2) According to Perussia et al. (J Immunol 133: 180 (1984)) there exist at least three different, nonoverlapping epitopes on the CD16 antigen. In order more precisely to define the epitopes of the CD16 antigen of the CD16 MAK's A9 and C10 developed in the invention, inhibition assays were performed with biotinylated A9 and C10 MAK's and the commercially available CD16 MAK's 3G8, VEP13 and Leu11b by means of direct and indirect immunofluorescence tests with peripheral lymphocytes, followed by permeation cytometric evaluation. In these assays the antibodies 3G8, VEP13 and Leu11b recognize the same epitopes on the CD16 antigen or else epitopes situated very close to one another [Perussia et al. (1984)]. FIGS. 1 and 2 present representative results of the inhibition tests. The tagged antibodies in each case were the biotin-conjugated antibodies A9, C10, VEP13 and the FITC-conjugated antibody 3G8. Accordingly, C10 biotin was completely inhibited by 3G8, VEP13 and Leu11b, and conversely C10 completely inhibited the anti-CD16 antibody VEP13 biotin completely and it partially inhibited 3GS-FITC. The inhibition of VEP13 biotin by C10 was dose-related, both by the complete antibody and by C10-F(ab')2 fragments. A9 biotin, however, was inhibited only partially by 3G8 and C10 and not inhibited at all by Leu11b and VEP13. At the same time C10 inhibited the binding of A9 biotin to a far greater degree than 3G8. A9 itself inhibited none of the rest of the tagged antibodies. Otherwise, the tested anti-CD16 antibodies showed a mutual inhibition similar to that described by Perussia et al. (1984). These results indicate that A9 and C10 bind other epitopes on the CD16 antigen, A9 an epitope farther removed from the epitopes recognized by the other MAK's, while the epitope recognized by C10 is closer to the epitope recognized by 3G8. Binding of MAK A9 and C10 to Soluble Recombinant Human CD16 Antigen (FIG. 4) In a Sandwich-ELISA, the results of which are represented in FIG. 3A-3C, with soluble recombinant human CD16, it appeared that both A9 (3C) and C10 (3B) bind but very weakly to soluble recombinant human CD16 antigen, in contrast to the other CD16 MAK's (3A) referred to above. This signifies that both A9 and C10 behave differently in binding to soluble CD16 antigen than the rest of the antibodies. Since both antibodies, however, show a great affinity for cell-bound CD16, this cannot be explained by differences in affinity; instead, the ELISA results indicate that the epitopes on the CD16 antigen that are recognized by A9 and C10 are different from the epitopes that are recognized by other CD16 MAK's. III. Biological activity of the A9 and C10 monoclonal antibodies Induction of NK-cell-related Cytotoxicity Against Hybridoma Cells Producing CD16 MAK (FIG. 4) For this purpose 5�103 hybridoma cells tagged with 51 Cr and mononuclear cells in 100 μl of medium in the ratios 45:1, 15:1, 5:1, and 1.5:1 were co-cultured for 12 hours and the specific lysis was determined from the release of the 51 Cr. The results of this cytotoxicity test are represented in FIG. 4. It appeared that the newly developed antibodies A9 and C10, in contrast to the antibody HRS-3 used as control, were both capable of inducing cytolytic activity in lymphocytes against the hybridoma cells producing the corresponding antibody. The maximum hybridoma cell lysis induced by A9 was approximately 60% specific lysis, but the lysis that was induced by C10 amounted to only about 30% specific lysis. IV. Bispecific monoclonal antibodies Preparation of a Bi-MAK from MAK A9 and a Second MAK, HRS-3, Directed Against an Antigen Associated with Human Tumor To prepare bi-MAK's for the induction of cytotoxicity against human tumor cells, mediated by NK cells, HGPRT-negative HRS3 hybridoma cells IgGl-kappa were fused with A9 hybridoma cells treated with iodoacetamide. A total of 113 cell clones grew in the selection medium (HAT medium), of which 17 clones (=15%) showed bispecific reactivity. The test for bispecificity was performed by the demonstration of antibodies of different light-chain content by the indirect immunofluorescence test. The tetradoma cell clone that showed the strongest simultaneous expression of both light chains was repeatedly sub-cloned, established as a cell line, and the bispecific antibody separated therefrom was named HRS-3/A9. The separation of the bispecific antibody from the monospecific original antibodies HRS3 and A9 also produced by the tetradoma cells was performed out of murine ascites containing HRS-3/A9 by an FPLC-mono-Q� anion exchange chromatography. The total immune globulin fraction containing antibody variants produced by the tetradoma cells was then further fractionated by an FPLC-Mono-S� cation exchange chromatography. Comparison of the isoelectric points of the antibodies HRS3 and A9 by isoelectric focusing showed that the isoelectric point of A9 was situated further within the basic range than that of HRS3. For this reason, a pH (=5.9) was selected that was just below the isoelectric point of HRS3, so that the positive charge density in this antibody was the lowest and accordingly had to be eluted before HRS3/A9 and A9. The elution was performed with a discontinuous salt gradient with 50 mmol/1 of MES, from 0 to 250 mmol/1 NaCl. FIG. 5 shows the resulting chromatogram. The immunoglobulins were divided into 3 fractions (=3 peaks), which then were tested for their content of bispecific antibodies. The immunological testing of the 3 fractions by indirect immunofluorescence showed that 2 fractions (peak 2) contained the bispecific activity. Induction of a Cytotoxicity Mediated by NK Cells Against Human Tumor Cells In Vitro (FIGS. 6 and 7) Lymphocytes enriched with NK cells by a Percoll density gradient centrifugation showed but little cytolytic activity against the CD30-antigen-positive Hodgkin's lymphoma cell line L540 (14% specific lysis). However, when the bispecific antibody HRS-3/A9 was used a definite increase in the cytolytic activity against L540 cells was observed, both in cell culture supernatant fluid containing bispecific antibodies (up to 27% specific lysis), and in purified bispecific antibody (up to 32% specific lysis). The cytolytic activity of the original antibodies HRS-3 and A9 with effector cells and of the bispecific antibody without effector cells, used as controls, was only between 5 and 15%. The results of this testing are represented in FIG. 6. The supernatant fluid was diluted 1:1, and the maximum concentration of the purified antibodies in the test batch was 5 μg/ml. Both were diluted reciprocally as stated. The E:T ratio was 20:1. At the same time, this elevation of the lytic activity was specific for the CD30 cell line L 540. The bispecific antibody HRS3/A9 was able only in the case of L 540 cells to intensify the cytolytic activity of NK cells, while no increase was observed in the cytolytic activity against the CD30 cell line HPB-ALL, which showed a certain amount of spontaneous "NK-cell sensitivity". The results of this testing are represented in FIG. 7. The supernatant fluid was diluted 1:20, and the concentrations of the purified Bi-MAK amounted to 1 μg/ml. The E:T ratio was 20:1. The results obtained with unstimulated peripheral blood lymphocytes (PBL) were equal to those obtained with concentrated NK cells. The induction of cytotoxicity by Bi-MAKHRS-3/A9 mediated by NK cells thus required no concentration of the NK cells. This is especially important in regard to any therapeutic use of this antibody in patients. V. preclinical experiments with the Bi-MAK HRS-3/A9 Treatment of Human Hodgkin's Tumors in SCID Mice with Bi-MAK HRS-3/A9 and human PBL (FIG. 8) Hodgkin's tumors were established by subcutaneous injection of a suspension of 1.5�107 cells of the human Hodgkin's cell line L540CY into the thoracic wall of 4 to 6-weeks-old female SCID mice. After the tumors had reached a diameter of at least 5 mm, groups of 10 tumor-bearing mice received intravenously 100 μg of Bi-MAK HRS-3/A9 or an equal amount of control antibody and 1�107 human PBL. In 10 out of 10 SCID mice with established human Hodgkin's tumors, a complete regression of the tumors was achieved by day 40, while all the other untreated mice showed advancing tumor growth, and when a tumor size of 10 mm diameter was reached they were killed. In 4 of the specifically treated mice a resumption of tumor growth occurred (recidivism) after 60 days (FIG. 8). In another series a complete remission was achieved by the use of HRS-3/A9 and human PBL's. The animals in that case were given, on day 0, an injection of 1) 300 μl PBS 2) 1�107 human peripheral blood lymphocytes PBL in 300 μl of PBS 3) 100 μg Bi-MAKHRS-3/A9 with human PBL 4) a mixture of 100 μg of parenteral MAKHRS-3 and MAKA9 together with human PBL. The growth of the tumor is represented as the average tumor volume (ccm) of all tumor-bearing animals of a treatment group. It will be appreciated that the instant specification and the claims are set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from the spirit and scope of the present invention. Patent Citations
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Have stayed in the Lantana for many a time, the room are done to a great standard and are very clean. Sharon is a great landlady and always happy and welcoming. The location is great for everything and a good base. Choices of beach or harbour veiws. Food is of a great standard and serve quickly. A great B&B all... More
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- The Lantana Weymouth, Dorset