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I spotted an (insert obscure car name here) classic car today!
omarman Posts: 728I like the URQ coupes too and there's enough interest to keep them alive and collectible. A really nice one, well kept and sorted would be worth what? Maybe $15k-$20k? But when it comes to the Sport Quattro...wow! The clamor for that car has been truly fanatic. It was the one with short stubby wheelbase, limited production, +300 hp and too much fun for 80s US certification. Insane price when new and bid up at auction now as well. Maybe $100k American money? Has anyone here ever seen a real one while visiting Europe or some museum?
Mr_Shiftright CaliforniaPosts: 46,212
fintail Posts: 34,549Ask and ye shall receive. These are at the Audi Museum in Ingolstadt:
Cool displays:
Also in 2009, I was driving on a secondary road between Stuttgart and Ulm, and saw a Sport Quattro on the road, appeared to have just come out of a restoration and was out for testing. I just about fell out of my car.
I had a thing for UR Quattros when I was a kid - they were about as exotic as I could imagine, and were setting the rally world on its head at the time. You could even get a talking option -
omarman Posts: 728Am I the only one who sometimes gets jealous over Fintail's good fortune in life? Even his freaking craigslist is better than mine. And now this! Ha! :P Just kidding.
Gotta love that "blasted plains" exhibit which makes Audi's Group B cars look right at home. And along with the museum tour you actually saw a road car version out on the street? Amazing.
Didn't all digital dash URQ's have the autocheck voice box instead of warning lights? If the voice warning system was defective or disabled, the driver would not be alerted to a serious problem like, "Achtung bremse system defekt!" :lemon: -
uplanderguy Kent, OHPosts: 7,717
Mr_Shiftright CaliforniaPosts: 46,212
uplanderguy Kent, OHPosts: 7,717That's a lot of car for that money, IMHO. When I see what some other more mundane stuff goes for, this Caddy looks better than ever to me. As I alluded to earlier, I have a 'car crush' on that thing!
I haven't seen, on eBay, a more authentic, interior and exterior, down-to-the-details, stock domestic car in quite awhile, and the rarity is appealing to me.
Now, if I had an extra $23.5K laying around, plus $2,500 or so to get it here from CA! -
Mr_Shiftright CaliforniaPosts: 46,212well the market isn't based on a car's merit---it's based on supply and demand...how many of Car X are out there, and, of course, how many people want Car X.
I see some issues with that car---being a 4-door is a value suppressor, and the color isn't everyone's cup of tea.
there's not a lot you can do with a car like that---it's very large, so hard to garage, complicated to repair, very little aftermarket support, and value trends have been absolutely flat for the past 6-8 years.
But you're right, for the bucks, it's a lot of car if you like that sort of thing.MODERATOR --Need help with anything? Click on my name!
fintail Posts: 34,549It's nice being single and usually living below my means - I can have fun now and then :shades: I'll just rub it in that I have been to the museums of every German maker + Sinsheim + 'Ring + AMG facility + Sindelfingen factory. The money might be smarter to some rotting in a retirement account, but you only live once...
Audi Museum was cool. Admission was only like 3 Euro, I think, and it had those cool displays along with other group B cars, and the rest. I don't do guided tours, I just wander around and discover.
I don't know if all URs were talkers, but I do find it funny that the "fasten seat belt" alert comes after the defective brakes alert. As I was a kid in the 80s, I love talking cars and digital displays. Too bad MB never used either.
Here it is in English - the German voice is much nicer, IMO. In my rental A8 on my last trip, the English nav voice was grating and demanding kind of like this. -
fintail Posts: 34,549I agree, it is a lot of flash for the cash, and even at a major show, you'll be the only one - I know you appreciate uniqueness too.
I bet he'd take 20K or maybe even a little less for it if you showed up with cash at his door. If I wanted a cool space-age land yacht, I would be all over that thing. -
andre1969 Posts: 22,238I bet he'd take 20K or maybe even a little less for it if you showed up with cash at his door. If I wanted a cool space-age land yacht, I would be all over that thing.
Heck, I'm kinda tempted myself, even though I prefer the 1962 models. But, I swore to myself I wouldn't raid any more home equity unless it was absolutely necessary. -
uplanderguy Kent, OHPosts: 7,717
uplanderguy Kent, OHPosts: 7,717Me, too. Buying an old car is an emotional thing. I can dig out the latest Old Cars Price Guide all day long (do they really need to update it bi-monthly?), but I simply haven't seen that nice of a car (in that I value originality and authenticity, and uniqueness is a bonus), at that price, in a looonnnngggg time.
fintail Posts: 34,549That car might be hard to value other than subjectively - it is a weird design and color scheme, but the condition and preservation can't be duplicated. You couldn't get a rat to be that nice for that money.
Unless I somehow experience a large income gain or inherit a fortune from an unknown source, I doubt I will buy another old car. Sometimes keeping the one I have seems kind of silly. But, I am sentimental about some things. -
uplanderguy Kent, OHPosts: 7,717I always said that I would keep my Lark Daytona Skytop R1 forever. I did have it 23 years. But when the buyer came pursuing, I started thinking about college coming up for my daughter. I do miss the car, but the current owner has done a lot to the car, only improving it. I did a driver restoration on it, which was more than the three or four owners prior to me did!
I'm not a mechanic, and I find it harder to find anyone who will work on an old car around me.
I bought my '64 Daytona on a whim (wife said, "Go for it"), but it was under $5K. And I got what I paid for! With it and the Skytop taking our entire garage space, I realized soon that I was in over my head.
I bought the '66 last year, after selling the other two, thinking I'd have it a long time. It was rock-solid everyplace and ran like a dream, but I thought the paint was amateurish for a car as otherwise so nice, and I just ended up not liking it as much as I thought I would.
I would like another Stude at some point, just don't see it this year.
I never had any of my three cars advertised for sale; I was pursued on all three of them and got my asking prices without haggling.
My wife says I'm like George Costanza, since all three went to Australia--an 'import/exporter'. Another friend, a Mopar guy, says I'm like a Studebaker dealer.
Back to the Cadillac...just lovely in every way to me. And the color is so 1961 to me...better than white, red, or black any day of the week to me. -
fintail Posts: 34,549I've had my fintail since I was 18, in no time I will have had it for 18 years. I could get my original purchase price back no doubt, but money has been spent in the meantime - mostly when the car needed rings and kingpins. I wouldn't be able to get the total expenses back, but it has been driven a bit over those years. I am lucky that indy MB shops aren't scared of it, but I haven't done any real restoration work - it just costs too much, and there's only a negative return.
Interesting that all three went down under. I wonder if the cars get RHD conversions, as many American cars did back in the day.
I can imagine the driver of that Caddy when new - would have been dressed very chic for the time. -
uplanderguy Kent, OHPosts: 7,717Stude did do RHD at the South Bend plant, for export and also for "Rural Router" Larks. My cars have not been converted to RHD as I have seen photos since they've been there.
If I bought that Caddy, I'd have to get a pair of Ray Bans (more likely, cheap knockoffs), and have my wife wear cat's eye glasses.
I always think I'd like "Mad Men" on HBO, but we don't have HBO. It's supposedly that early '60's time period that I find so fascinating, although remember very little of it personally. -
fintail Posts: 34,549I've seen all kinds of 60s-70s American cars with RHD conversions in Australia, some of them kind of odd (big Chryslers, gargantuan T-Birds, etc). As the Australian automotive scene never gave up the big V8 sedan, maybe that makes sense.
I can see that Caddy being driven by a classy woman in a period dress and pillbox hat, with a fancy period style handbag, kind of a Jackie Kennedy style maybe. A man in a casual suit and fedora alongside her. That era certainly had style and some good design, from houses to clothes to cars. -
texases Posts: 5,719
uplanderguy Kent, OHPosts: 7,717I just love '65-66 Imperials (OK, the '64 is nearly identical but I like the front end styling of the '65 and '66 better). I'm reminded of Mr. Drysdale on "The Beverly Hillbillies" when I see one.
I always liked the '63 Imperial too. It's the goofy '61 and '62, but minus the 'sparrow shredder' taillights. I particularly like it from the rear. -
uplanderguy Kent, OHPosts: 7,717I never look at Imperials on eBay...until now.
Here's '63 coupe. Reupholstered front seat at least...and is this what an Imperial front seat looked like in '63? Where's the center armrest?
I think fintail mentioned it, but I agree... Chrysler seemed to hold onto 'bigger than life' '50's styling...even interior and instrument panel...longer than the other guys.
http://cgi.ebay.com/ebaymotors/ELEGANT-UPSCALE-413-CI-V8-3-SPEED-AUTO-A-C-POWER-- - WINDOWS-AUTOMOTIVE-ROY-/281042521660?pt=US_Cars_Trucks&hash=item416f708e3c
Personally, even though it's a four-door, I'd much rather have that '61 Caddy than that Imperial, and the owners were looking for about the same money.
Here's a nice '66 Imperial hardtop sedan. Got the leather that's got lines in it look in front, but BIN of $16K. It's more timeless styling than the Caddy I think, but...again, for a complete 'time capsule', I think it's hard to beat the Caddy.
http://cgi.ebay.com/ebaymotors/Clean-and-drives-like-dream-Black-exterior-white-- - pearl-interior-/271129101187?pt=US_Cars_Trucks&hash=item3f208dc383
Nice '67 convertible, very rare...to me, this is about when Imperials became not all that more exciting than New Yorkers (my opinion only):
http://cgi.ebay.com/ebaymotors/440ci-V-8-1-577-Produced-Very-Nice-Running-and-Dr- - iving-Example-/271128797738?pt=US_Cars_Trucks&hash=item3f2089222a
On eBay, there's also a '68 Imperial 4-door "post" sedan...it has frames around the side glass. I had forgotten there were Imperials like that then.
My friend, who was the Studebaker dealer in our hometown, was approached by the Dodge 'road man' to take on a Dodge franchise when the elderly owner got out of the business. My friend was invited to the unveiling of the '62 Dodges in Detroit, pre-public and for dealers only. When asked what he thought, he said, "Those look worse than Studebakers!"
He didn't add Dodge, probably much to his chagrin a few years later! -
ab348 Dartmouth, Nova Scotia, CanadaPosts: 2,509That '67 is the pick of the litter. The '67 Imperial was a fabulous car, maybe the high-water mark for '60s Imps, and the convertible is exceptionally rare. That one looks outstanding, although I wish they had not changed the paint color. I wonder what the reserve would be. Too bad it has a black interior - not only are those always a bad choice in a convertible, but that season Chrysler offered a gorgeous silver leather interior option that looks fabulous.
2014 Cadillac ATS4 2.0T, 1968 Oldsmobile Cutlass S Holiday Coupe
uplanderguy Kent, OHPosts: 7,717
andre1969 Posts: 22,238I think Imperials from 1957-66 were solid, sturdy cars, but unfortunately suffered from having the same drivetrain as a Chrysler New Yorker, but weighing up to 1,000 lb more.
They were put together better than other Chrysler products of the era, but I think a Cadillac would still have better fit and finish, and a higher quality interior.
Where an Imperial would win out though, would be a demolition derby. The 1960-66 models, which used the same frame as the '57-59, but had some Unibody tricks incorporated into the body to stiffen it up, were especially rugged.
As for finding parts, Imperial-specific stuff is probably hard to get, but a lot of the generic stuff, like drivetrains, power windows, etc, was probably shared with the Chrysler New Yorker and such.
Personally, my favorite Imperial of that era is the 1960. There's just something about that front-end I like. It had a proud, accomplished look to it, that says "I've arrived!"


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Reviews for Luke's Apartments
Address: Peline 27, Dubrovnik, CroatiaRooms from: US$ 29.65
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(Hostelworld.com and Affiliate sites)The following reviews were submitted by customers who booked through our sister site, Hostelworld.com, in the last 12 months.
The ratings in the rating box are based on all-time feedback for the property and are the opinions of the customers who submitted them; they do not express the opinions of Hostelworld.com or our affiliate sites.
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Customer Reviews for Luke's Apartments
dan3475, from Australia ~ 9th Oct 2014
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subset: commoncrawl


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Good values on homes are still available, as the number of homes listed for sale is greater than the number of homes being sold. Great deals can be found by determined buyers.
Rent prices are relatively stable, and availability is good. If your yearly income is around $100,000, you can afford to rent in this market. Rent prices have increased by 8% over the past 3 months. This is 52% lower than the state average of $2,544 and 29% lower than the country average of $2,158.
Trend data coming soon.
For Sale: 32 | New: 1 | ||
$381,672 | $315,543 | Recently Sold: 52 | Reduced: 13 |
Average Home Price | Average Sold Price | Property Records: 2,637 | Foreclosed: N/A |
The average home price in 01536 is $381,672, which is 21% lower than the average sold price in 01536 ($315,543) . The average home price per square foot in 01536 is $164 and about 2% of all homes in 01536 sold in the last 6 months. For 01536 home values, real estate prices, and city market trends, research the 2,637 property records or search our 52 recently sold or 32 homes for sale listings. The listings and properties in 01536 are only a part of the 1,929,319 properties and 21,583 homes for sale in Massachusetts. Because of this, the average home price and average home value of 01536 also influence the average home price $0 and average sale price $0 of Massachusetts. Listing counts, 01536 home prices and home values in 01536 are sourced daily from listing and property data on realtor.com®, which is the most up-to-date and accurate aggregation of real estate listings in the industry and includes approximately 800 regional MLSs and is the official website of the National Association of Realtors®.


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Fifteen individual panes of glass showcase your collectibles in this exquisitely handcarved Toscano exclusive with curved front, crown molding, double shelves, and barley twist spindles. At over two foot tall and wide, this mahogany 18th century replica wall cabinet is distressed by hand, just enough to replicate an antique.
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Arizona Cardinals
Washington lawmaker posts insensitive tweet following Seahawks' loss
NFL fans in Washington are not accustomed to seeing their team lose home games.
In fact, before the Arizona Cardinals' 17-10 win at CenturyLink Field Sunday, the Seattle Seahawks hadn't dropped a home contest in 728 days.
Needless to say, the unfavorable result didn't sit well with the Seattle faithful, and in particular a local lawmaker.
Joe Fitzgibbon (D), a representative in Washington's 34th Legislative District, took to social media to vent his frustration at the loss and apparently his disdain for the state of Arizona.
Makes us all look bad RT"@MikeBakerAP: here's a screengrab of lawmaker calling AZ a "racist wasteland" after loss: pic.twitter.com/c3nkm0jWIB"
— Rep. JT Wilcox (@jtwilcox111) December 23, 2013
Note: You can read Fitzgibbon's public apology here.
Fitzgibbon, who is also the Deputy Majority Whip, has since taken down the tweet, but in its place he sent out another one in an attempt to clarify his earlier remarks.
Upset re hawks loss, and failure of smart immigration reform--shouldn't have conflated the 2 though. Happy holidays, better for both in '14
— Joe Fitzgibbon (@joefitzgibbon) December 23, 2013
In a statement to The Associated Press, Fitzgibbon voiced surprise over how seriously people were taking his tweet.
"If folks are going to take that too seriously, then I'm sorry about that," he said.
The Associated Press contributed to this report.
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- Brock Huard, ESPN 710 Seattle host - Friday January 30Brock Huard believes the Seahawks will beat the Patriots in Super Bowl.
- Luis Gonzalez- Former Diamondbacks outfielder - Friday January 30Luis Gonzalez reflects on the growth of the city of Phoenix.
- Bickley Blast - Friday January 30Marshawn Lynch is challenging the NFL commissioner.
- Mark Schlereth- ESPN NFL analyst - Friday January 30Mark Schlereth believes NFL players should take a stand against the owners.
- Joe Theismann- former NFL quarterback - Friday January 30Joe Theismann says Deflategate isn't a controversy at all.


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Join Date: Jan 2010
Location: USA
Posts: 40
Get rich quick!!!!!!!$$$$$$$
Once you open up the ability to use "Bruski's Scrapyard", oh boy, it's big time MONEY!!! And unlike other "Get Rich Quick" scheme's, this one works!
Travel to Bruski's Scrapyard (at the top of the map, big "B"). Once you're there, drive the car you drove there in, into the car compactor. BaM! $400 something dollars. YOu'll notice some model car sitting there to the left of the compactor. Grab it, drive the track around the whole scrapyard, and when you see the compactor again, voila!!! a new car has taken the place of the one you just broke into and drove around the scrapyard in!!!! This absolutely works every time, and you will see different models of cars show up after you drive around the track. That route was the shortest I could find, but technically you may not even have to drive that far. I used to drive farther and steal other cars, until I figured this out and then felt dumb and like I had wasted a bunch of time. Sure you can get other cars which pay more, but this way last easily under a minute. In no time you'll have many thousands of dollars. I'm at $50,000 something right now. HOPE YOU ENJOY! p.s. forgot to add you can't compact cars that came with your garage, it won't except them, that and I can't get huge cars to fit into the compactor either .
Xbox 360 Games 100%: (before DLC) Tenchu Z, Viva Pinata, Bully: Scholarship Edition, Halo 3, Borderlands, Condemned: Criminal Origins, Condemned 2: Bloodshot XBLA Games 100%: (before DLC) A Kingdom for Keflings, Harms Way Last edited by BUJIKAN; 02-10-2011 at 06:17 AM. Reason: needed to add a little extra |
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BAE Systems joined with Mid Ocean Tanker Company (MOTC), Mid Ocean Marine and Alterna Captial Partners earlier this year to commission the American Phoenix, a U.S. Flag/Jones Act-qualified product-chemical tanker. Measuring 616 x 105 ft. with a laden draft of 36 ft. and a cargo capacity of 49,000 dwt, American Phoenix is the largest vessel ever built and launched in the State of Alabama. BAE Systems was contracted to complete construction of the American Phoenix and prepare it for use. The vessel is owned by MOTC, a joint venture between Mid Ocean Marine and private equity firm Alterna Capital Partners. The vessel will conduct trade initially in the Gulf of Mexico. Operating management of the vessel will be performed by Seabulk Tankers, a division of Seacor Holdings, one of the most experienced operators in the U.S. Jones Act trades. The American Phoenix is the newest vessel to come from the BAE Systems Mobile shipyard, which has a rich history of ship construction and repair. The yard launched its first new construction vessel, the steam powered ship Banago, on September 19, 1918. Alternative Marine Technologies, ABS, US Coatings, MMIF, F & S Superb Marine, LES, Diamond Scaffolding, Insulation Inc, Jamestown Metal and Machinery, L-3 Maritime Systems, R+S Stolze, MAK, International Paint, Sea Technology, Bisso Marine Company, 2/2 Houston Holloway, Universal Services, Offshore Inland, Resolve Engineering and Fire Protection Services all played a significant part in completing the American Phoenix. Vessel Name M/T American Phoenix Builder: BAE Systems Southeast Shipyard Owner: Mid Ocean Tanker Co. O.N. 1233425 Type 49,000 DWT Chemical Tanker Owner Mid Ocean Tanker Company, LLC Builder BAE Systems Southeast Shipyards Hull Number 103 Construction Start 2008 Length 187.8m LOA Beam 32.2m Depth 18.3m (molded @ side) (As published in the December 2012 edition of Maritime Reporter - www.marinelink.com)
Residents of Islampur, a remote village in the northern Bangladeshi district of Naogaon, were stunned one night last summer when the darkness was suddenly illuminated
Fugro N.V. announces that the Supervisory Board of Fugro nominates Mrs. P.H.M. Hofsté and Mrs. A.H. Montijn for appointment as members of the Supervisory Board
Petrobras develops a new technology that reduces the emission of gases that cause greenhouse A new technology to reduce the emission of greenhouse gases (GHG)
From 1 February 2015 a new chairman will head the Danish Pleasure Craft Safety Board. The new incumbent is Jan Thorn, who is in his everyday work Director
Delays can be expected in the Kiel Canal following a collision this yesterday, 23 January, with the small north lock gate at Brunsbuettel. Only the big north
Extension operations on MSC Sinfonia, the second out of four MSC cruise ships to undergo the Renaissance Programme of enhancements, have begunin the past days at Fincantieri shipyard in Palermo.


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BPG/Biogenic CategoriesSopharma.com
Sopharma product categoriesPharmaceuticals ↭ Solutions for hemodialysis ↭ Diagnostic products ↭ Solutions for contact lenses ↭ Food supplements ↭ Cosmetics ↭ Oral cavity solutions
CLENBUTEROLFor prolonged treatment of bronchospastic syndromeCLENBUTEROLINN: CLENBUTEROLКод АТХ: R03CC 13Composition:One tablet contains 0.02 mg clenbuterol hydrochloride. Indications:For prolonged treatment of bronchospastic syndrome in mild and moderate atopic and non-atopic bronchial asthma; strain-induced bronchospasm; in the complex therapy of chronic spastic bronchitis and COPD. Dosage and administration:Adults: 0,02 mg orally, morning and evening; Maintenance dose – 0,01 mg 2 times daily. In more serious conditions, 0,04 mg, 2 times daily (morning and evening) should be administered in the first days. The dose should be reduced upon condition improving. In children: 6-12 years –1/2 tablet 2 times daily. Over 12 years –1/2 tablet 2-3 times daily or 1 tablet 2 times daily. Contraindications:Hypersensitivity to the product; thyrotoxicosis; digitalis intoxication, tachycardia, tachyarrhythmia, hyperthrophic obstructive cardiomyopathy; first trimester of pregnancy. Drug interactions:Beta-blockers antagonize Clenbuterol effects. In concomitant administration with antidiabetics, their dose should be increased due to glycogenolysis stimulation by Clenbuterol. Co-administration with cardiac glycosides, MAO-inhibitors and theophylline may cause heart rhythm disorders. The effect of Clenbuterol is potentiated by tricyclic antidepressants, some antihistamines and levothyroxin and reduced by some antihypertensive products (guanethidine). Halothane and other halogenated hydrocarbon anesthetics and cyclopropane sensitize myocardium and may potentiate the arrhythmogenic effect of в2- sympathomimetics. Adverse reactions:Insomnia, headache, hyperkinesias, psychic disorders, reddening, sweating, tremor and anxiety, excitability, dizziness; the patients with Parkinson's syndrome may feel increase of tremor and muscle rigidity; palpitations, tachycardia, reduction or, rarely, rise of blood pressure; urine retention; paradoxical bronchospasm which requires immediate discontinuation of therapy. In patients with diabetes mellitus, hyperglycaemia may develop. The manifestation of such adverse reactions requires dose reduction without need of therapy termination, because they fade within 1-2 weeks after therapy initiation. In prolonged administration, tachyphyllaxis to Clenbuterol may be observed, but sensitivity is restored after discontinuation of therapy. Supplied:50 tablets of 0,02 mg. Dispensing:On doctor's prescription. |
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Hear the untold stories of enslaved people and learn about historical and contemporary slavery.
A beautiful building housing one of the UK's finest collections of fine and decorative art.
Uncover objects from the Titanic, find out about life at sea and learn about the port of Liverpool.
This new museum tells the story of Liverpool with diverse exhibits housed in a stunning building.
Explore a Victorian merchant's house with its period furniture and beautiful paintings.
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Discover treasures from around the world, explore outer space and meet live creatures!
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We're not just the largest cultural organisation in the north west - we have a truly global reach and offer unique sponsorship, membership and hospitality packages.


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10 CFR 205.173 - Public hearings.
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When the proposed rule or regulation is likely to have a substantial impact on the Nation's economy or large numbers of individuals or businesses; or
When the DOE determines that a public hearing would materially advance the consideration of the issue. A public hearing may be requested by any interested person in connection with a rulemaking proceeding, but shall only be convened on the initiative of the DOE unless otherwise required by statute.
A public hearing may be convened incident to any proceeding when the DOE in its discretion determines that such public hearing would materially advance the consideration of the issue.
A public hearing may only be convened after publication of a notice in the Federal Register, which shall include a statement of the time, place, and nature of the public hearing.
Interested persons may file a request to participate in the public hearing in accordance with the instructions in the notice published in the Federal Register. The request shall be in writing and signed by the person making the request. It shall include a description of the person's interest in the issue or issues involved and of the anticipated content of the presentation. It shall also contain a statement explaining why the person would be an appropriate spokesperson for the particular view expressed.
The DOE shall appoint a presiding officer to conduct the public hearing. An agenda shall be prepared that shall provide, to the extent practicable, for the presentation of all relevant views by competent spokespersons.
A verbatim transcript shall be made of the hearing. The transcript, together with any written comments submitted in the course of the proceeding, shall be made available for public inspection and copying in the public docket room, as provided in § 205.15.
Title 10 published on 2014-01-01
no entries appear in the Federal Register after this date.


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Randomized healthservices study of human papillomavirus-based management of low-grade cytological abnormalities†
Article first published online: 9 NOV 2010
DOI: 10.1002/ijc.25649
Copyright © 2010 UICC
Additional Information
How to Cite
Dillner, L., Kemetli, L., Elfgren, K., Bogdanovic, G., Andersson, P., Carlsten-Thor, A., Andersson, S., Persson, E., Rylander, E., Grillner, L., Dillner, J. and Törnberg, S. (2011), Randomized healthservices study of human papillomavirus-based management of low-grade cytological abnormalities. Int. J. Cancer, 129: 151–159. doi: 10.1002/ijc.25649
- †
The study was primarily designed by the principal investigators (Drs. L. Dillner, J. Dillner and Törnberg), with input from all authors. Dr. Kemetli maintained the database and analysed the data under supervision of Dr. Törnberg. Drs. Elfgren, S. Andersson, Persson and Rylander were responsible for the clinical management of the patients. Drs. Bogdanovic and P. Andersson performed the HPV testing, under the supervision of Dr. Grillner. Ms. Carlsten-Thor assisted with study coordination. Drs. L. Dillner and J. Dillner primarily drafted the manuscript, to which all authors contributed. Dr L. Dillner, as the corresponding author, had full access to all the data in the study and had final responsibility for the decision to submit the manuscript for publication. The final version of the manuscript was seen and approved by all authors.
Publication History
- Issue published online: 26 APR 2011
- Article first published online: 9 NOV 2010
- Accepted manuscript online: 7 SEP 2010 10:20AM EST
- Manuscript Received: 8 AUG 2010
- Manuscript Accepted: 27 JUL 2010
- Abstract
- Article
- References
- Cited By
- cervical cancer;
- screening;
- implementation studies;
- triaging
Human papillomavirus (HPV)-based management of women with borderline atypical squamous cells of undetermined significance (ASCUS) or mildly abnormal cervical intraepithelial neoplasia (CINI) cervical cytology has been extensively studied in the research setting. We wished to assess safety and health care resource use of a real-life health care policy using HPV triaging. All 15 outpatient clinics involved in the organized population-based screening program in Stockholm, Sweden screening program were randomized to either continue with prior policy (colposcopy of all women with ASCUS/CINI) or to implement a policy with HPV triaging and colposcopy only of HPV-positive women. The trial enrolled the 3,319 women who were diagnosed with ASCUS (n = 1,335) or CINI (n = 1,984) in Stockholm during 17th March 2003 to 16th January 2006. Detection of high-grade cervical lesions (CINII+) and health care cost consumption was studied by registry linkages. The proportion of histopathology-verified CINII+ was similar for the two policies (395 of 1,752 women (22.5%; 95% Confidence interval [CI]: 20.6–24.6%) had CINII+ diagnosed with HPV triaging policy, 318 of 1,567 women (20.3%; 95%CI: 18.3–22.4%) had CINII+ with colposcopy policy). Sixty-four percent of women with ASCUS and 77% of women with CINI were HPV positive. HPV-positivity was age-dependent, with 81% of women below 35 years of age and 44% of women above 45 years of age testing HPV-positive. HPV triaging was cost-effective only above 35 years of age. In conclusion, a real-life randomized healthservices study of HPV triaging of women with ASCUS/CINI demonstrated similar detection of CINII+ as colposcopy of all women.
Cytological screening identifies women with cervical lesions that confer an increased risk of cervical cancer.1 Further assessment and/or treatment will prevent progression to invasive disease. Women with high-grade cytological lesions should be referred directly for further exploration.2 However, several different policies for management of women with minor cytological lesions exist.2, 3 Follow-up policies after atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intra-epithelial lesions (LSIL) vary from conservative repeat cytology4–6 to direct referral for colposcopy and biopsy.7, 8 Referral to the colposcopy clinic with subsequent biopsy and endocervical sampling for histopathological examination results in substantial costs for the health care system9 and often creates feelings of anxiety and discomfort for the women concerned.10, 11
Low-grade cervical lesions commonly regress and do not require treatment.12 Although most women with an ASCUS smear result do not have significant disease, a substantial proportion of them do harbour histopathologically confirmed high-grade cervical intraepithelial neoplasia (CINII+) lesions.13–15 For a population of women participating in screening in the United States, it was estimated that more than one third of CINII+ lesions were discovered on follow-up of a previous smear with ASCUS.15 Referral of all women with minor cytological lesions for colposcopy is a common approach in many settings in the United States,3 is a recommended practise in the European Union4 and has been the routine policy used in Stockholm, Sweden for many years.
Given the overwhelming accumulated evidence concerning the etiological role of oncogenic human papillomavirus (HPV) infections in the development of cervical cancer and CINII+,4 HPV testing has been extensively studies as a triage method to select women at increased risk of cervical cancer, thus justifying referral for colposcopic exploration.13, 14, 16–19 Systematic reviews of research studies have clearly documented that HPV triaging is superior to repeat cytology in follow-up of ASCUS smears20 and that HPV triaging is equivalent to repeat cytology in follow-up of LSIL smears.20
A major 3-way randomized trial comparing HPV triaging, repeat cytology and colposcopy of all women ASCUS-LSIL Triage Study (ALTS) found that repeat cytology was inferior for managing ASCUS smears in women <30 years of age and that HPV triaging and colposcopy of all women were equivalent in terms of safety.21 HPV triaging is recommended policy for triaging of ASCUS, but not LSIL, smears in both Europe and the United States.22, 23
Generalizing data from the research setting to the real-life screening policy situation may be difficult. For example, diagnostic practises will vary between countries24 as well as between the routine setting and the expert panels used for diagnosis in the research setting. There may be differences in the adherence to a study protocol in the research setting and to the recommended policy in the routine setting. Finally, the research setting does not study neither the acceptability of a policy to the health care provider (payer) nor the acceptability to the entire general population targeted by a screening program.
Randomized healthservices studies are a method for evaluating and comparing the real-life effect of different health care policies in the routine setting. The design is not yet commonly used, except in public health and in evaluation of the effect of screening policies.25, 26
We wished to compare the safety and real-life use of health care resources by the two different management policies that have in previous research not been found to be inferior, namely colposcopy of all women and HPV triaging with referral only of HPV-positive women.
Material and Methods
The organized Swedish cervical cancer screening program invites women aged 23–49 years for screening at 3-yearly intervals and women aged 50–60 years at 5-yearly intervals. Invitations are based on the population register, but comparison is first made with cytology registers covering the population to exclude women who have recently taken a smear outside the program. All women resident in the Stockholm County that on their organized, invitational smear had the cytological diagnosis ASCUS or CINI between 17th March 2003 and 16th January 2006 were included in the study. Sweden uses the old U.S. cytologic nomenclature,27 in which CINI is also a cytological, not only a histopathological diagnosis. The new cytological term LSIL is a combination of LSIL and koilocytotic atypia. In the system by Koss, a diagnosis of koilocytosis can be made in addition to any other diagnosis. As only 0.2% of smears are reported as normal, with koilocytosis as an ancillary diagnosis in Sweden, the cytological diagnosis of CINI used in Sweden is essentially similar to the LSIL nomenclature used in other countries.
The two policies compared were (1) Colposcopy of all women, that referred all women with ASCUS or CIN1 for colposcopy and biopsy (previous policy) and (2) HPV-based triaging, referring all women with ASCUS or CINI for a new visit with HPV-test (new policy), using the FDA-approved Hybrid Capture 2 test (DiGene, Gaithersburg, MD). Sampling was made using the DiGene specimen collection kit, following the instructions of the manufacturer. The HPV test was performed in a certified virus diagnostics laboratory, following the instructions of the manufacturer. HPV-positive women were referred for colposcopy and biopsy, whereas HPV-negative women were referred for a repeat cytology visit 12 months later. The repeat cytology among HPV-negative women with ASCUS/CINI has not been used in some of the major HPV triaging trials,28 but an extra safety measure was considered advisable when going from the more carefully controlled research setting to the routine setting. For both the old and the new policy, all women who had been referred to colposcopy were also scheduled for two follow-up visits with cytology, 12 and 24 months after the colposcopy. These follow-up smears were scheduled also if the colposcopy had been normal and no biopsies were taken.
The objectives of the randomized healthservices study were to assess whether the new policy was equally safe as the old policy and to determine whether it was cost-efficient. The primary outcome measure of safety compared between the two policies was the CIN2+ detection rate. The secondary outcome measure was the use of health care resources.
The two policies were submitted for consideration to the cervical screening expert group of the Swedish Association for Obstetrics & Gynecology, to the Obstetrics & Gynecology expert group of the Stockholm County and to the heads of the Obstetrics & Gynecology departments in all 15 ObGyn clinics in Stockholm County. After the assertion by these expert groups and officials that the two policies were both in accordance with evidence-based medicine, the randomized health care policy evaluation plan was approved by the Ethical Review Board in Stockholm. As mandated by the Ethical Review Board, information about the policy switch, the randomization and the follow-up was included as a separate information leaflet that was given to all women who participated in the organized cervical screening in the Stockholm County (about 180,000 leaflets).
All 15 ObGyn clinics in Stockholm County were randomized to colposcopy of all women (old policy) or to HPV triaging (new policy). For 14 clinics there was a pairwise block randomization, where the clinics were paired according to (i) the size of the catchment area population (to increase the probability that the two policies would include a similar number of women) and (ii) the CINII+ incidence in the catchment area population (low variance in the incidence of the outcome increases the statistical power of cluster-randomized trials).29, 30 The 15th and smallest clinic was randomized without a paired clinic. Randomization was performed using computer-based random numbers by an independent entity (the Cancer Registry of Stockholm) and the eight clinics randomized to HPV triaging all switched policy on 2003-03-17.
All women were followed up until 2007-12-10 by linkages with the regional cytology and pathology registries using the unique personal identification numbers of the women. The registries contain data on all smears and also biopsies taken in the greater Stockholm area, not only those taken in the organized program. When several histopathology reports had been issued for specimens taken on the same date, they assumed to result from the same colposcopy visit. The reasons why more than one histopathology report had been issued were not investigated, but the most likely explanation is that several different types of tissues specimens had been taken.
HPV testing
The hybrid capture II test (DiGene Inc, Gathersburg, MD) was performed as recommended by the manufacturer. The samples were taken using the DiGene specimen collection kit. After denaturation, the sample was hybridized to the high-risk RNA probe cocktail, recognising HPV-genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Samples were scored as positive when the FDA-recommended cutoff value for test-positive results was equivalent to or exceeded 1.0 RLU (Relative Light Unit: equivalent to 1 pg HPV DNA per 1 ml of sampling buffer). For each test, the ratio of low/high calibrator probe results was calculated, and the percentage of borderline results (0.7–2.0 RLU) was assessed. If the ratio was below 2.5 and/or the percentage of borderline results above 10%, the test was repeated. Quality controls also included, for each 96 well plate, two positive controls of 104 and 105 HPV16-positive SiHa cervical carcinoma cells (containing one HPV genome copy per cell) and one negative control of 104 HPV-negative C33A cervical carcinoma cells.
Health care costs
The actual costs paid by the health care provider to clinics and laboratories were used. The county council of the region is the provider of health care. Hospitals in the region are funded by the amount of health care delivered (e.g., the number of colposcopies performed by Ob/Gyn clinics and the number of histopathology reports issued by pathology laboratories). The price lists used in the region at the time of study are detailed in Tables 3 and 4. The costs of ethical review board permission, information leaflets and for study management are not included, as these are considered to be part of the budgeted costs for evaluation and quality assurance of the screening program. Provided equal safety, the policy that had lower costs was considered to be most cost-efficient.
Statistical analysis
The trial was powered to detect a 2-fold difference in detection of CIN2+, as estimated using n = 15.8*(S2c + S2w/m)/d2, where S2c is interclinic variance, S2w is intraclinic variance, m is number of women per clinic and d2is the magnitude of the detectable difference.29 As clusters were large, matching was broken in analysis.30 Analyses were by "intention to treat," which means that costs and CINII+ detection rates are based on all women (both attending and nonattending). All women could be followed up with registry linkages for the entire duration of the study.
Proportions were compared with Fischer's exact test and Clopper Pearson 95% confidence intervals (CI) for binominal proportions were calculated using SPSS (Chicago, IL). Testing with Cochrane-Armitage trend test used SAS (Cary, NC). As the point of the intervention was to reduce the number of follow-up tests required, we considered women as having been followed up until the last date of the registry linkage rather than to the last follow-up test.
There were 1,752 women with ASCUS/CINI in the catchment populations of the HPV triaging clinics, compared with 1,567 women belonging to the all colposcopy clinics, corresponding to an imbalance in number of women of 12% (Fig. 1). Although all women managed by the HPV triaging policy were referred to HPV testing, 152 women (8.7%) (62 women with an ASCUS smear and 90 women with a CINI smear) were never tested, because of relocation, logistic failure or for unknown reasons.
Altogether 72% of the women were HPV-positive, with HPV-positivity being more common among women with CINI (77%) than ASCUS (64%) (p < 0.0000001; Table 1). HPV-positivity was strongly age-dependent, both among women with CINI and among women with ASCUS (p for trend <0.0001; Table 1).
During the follow-up until 2007-12-10, 713 women had a histopathological diagnosis of CINII+ (Table 2). The proportion of women being diagnosed with CINII+ was similar in both populations, with a slight tendency for a higher proportion in the HPV triaging population (22.5%, 95% CI: 20.6–24.6%) than in the all colposcopy population (20.3%, 95% CI: 18.3–22.4%) (odds ratio [OR]:1.14; 95% CI: 0.96–1.36) p > 0.1 (Table 2), despite the fact that only the women testing HPV-positive (66% of the women in the HPV triaging population) were referred to colposcopy and biopsy (Fig. 1). The attendance rate among women referred to colposcopy was higher in the HPV triaging arm (91%) than in the all colposcopy arm (81%) (Fig. 1), and the actual proportion of women who had (at least 1) colposcopy with biopsy in the HPV triaging arm was therefore 60% (Fig. 1). The proportions of histopathologically diagnosed CINII+ were similar for both polices also when considering whether the index smear had ASCUS or CINI (ASCUS: 21.2% (95%CI: 18.3–24.4%) using all colposcopy and 21.8% (95% CI 18.6–25.3%) using HPV triaging. CINI: 19.5% (95% CI: 16.9–22.4%) using all colposcpy and 22.9% (95% CI:20.5–25.5% using HPV triaging)) (Table 2).
In the HPV triaging arm, there were 27 women who were diagnosed with CINII+ in histopathology in spite of not having been referred because of HPV-positivity (Table 2). Sixteen of these women were among the 152 women where the HPV test was never performed. Most of these women had been directly referred to colposcopy. The extra cytology visit scheduled 1 year later for women with HPV-negative ASCUS/CINI was only attended by 243 of 443 (55%) of the HPV-negative women (Fig. 1). The controls smears for these 243 women found 13 ASCUS, 8 CIN1 and 3 CINII+ cytologies. Six CINII+ (two CINIII and four CINII) histopathological diagnoses were made as a result of the extra cytology visit. Three of these women with CINII in histopathology had benign smears at the control smear, suggesting that their extra cytology visit had included a colposcopy. Four women had a follow-up visit already within 6 months of the index smear (not recommended in the policy). Finally, one HPV-negative woman did not attend the extra cytology visit, but came for organised screening 3 years later, when a second ASCUS smear resulted in CINIII in histopathology.
The total numbers of colposcopies with biopsies that were actually performed was only marginally lower in the triaging clinics (1,917) than in the all colposcopy clinics (1,967) (Table 3). As there were 12% more women in the triaging arm, (1,752 women in the triaging arm, compared to 1,567 in the colposcopy arm), this corresponds to a 13% decrease of number of colposcopies with biopsy (1,917 colposcopies for 1,752 women compared to 1,967 colposcopies for 1,567 women). The total number of cervical histopathological reports that was paid for was actually somewhat higher in the HPV triaging arm (Table 3), corresponding to a mere 5% decrease in histopathologies (2,766 histopahtologies for 1,752 women, compared to 2,596 histopathologies for 1,567 women). The total actual costs increased by 21% in the HPV triaging arm (from 0.63 million US Dollars to 0.77 million US Dollars) (Table 3). The cost in the triaging arm is expected to be lower in the future, because the lower number of women referred to colposcopy results in a reduced amount of women scheduled for follow-up. All women referred to colposcopy are also scheduled for two follow-up visits with cytology, 12 and 24 months after the colposcopy (in both arms). These follow-up smears are scheduled also if the colposcopy had been normal and no biopsies were taken. The number of women scheduled for follow-up was reduced with 32% in the triaging arm (1,967 colposcopies, each one eliciting follow-up smears, in the colposcopy arm compared with the adjusted number of colposcopies in the triaging arm [1,712]) (Table 3). However, after considering the future follow-up costs also, the HPV triaging arm remained more expensive (Table 3).
As the HPV prevalence was strongly age-related, the proportion of women referred for colposcopy is reduced with increasing age in the HPV triaging policy, which reduces the costs of the policy with HPV triaging with increasing age. The break-even where the costs of the two policies are approximately the same occurs for a policy with HPV triaging only for women above or equal to 35 years of age, as detailed in Table 4. The CINII+ detection rates were somewhat lower among women above or equal to 35 years of age (232 of 1231 women [18.8%] compared with 481 of ,2088 women below 35 years of age [23.0%]), but the proportion of women diagnosed with CINII+ was similar for the two policies also in this age group (HPV triaging: 123 of 629 women (19.6%); All colposcopy: 109 of 602 women (18.1%); OR: 1.10, 95% CI: 0.82–1.48; p > 0.5).
This study has provided a real-life evaluation on the actual costs used when switching to a policy with HPV-based triaging of low-grade cervical abnormalities and found that a policy using colposcopy of all women with ASCUS/CINI below 35 years of age, but HPV triaging above 35 years of age was the least costly policy in practise. Furthermore, the relative safety of the two policies appeared to be similar, as there was an equal proportion of CINII+ diagnoses made for the two policies.
Several of the theoretical concerns that had motivated this study found support in the actual data obtained. The cytological diagnostic practises vary between countries, making it difficult to apply the results from studies performed in other countries to our population. Specifically, a formal exchange of slides found that most of the slides diagnosed as ASCUS in the USA and UK are reported as normal in Sweden.24 Indeed, the proportion of women with ASCUS that were HPV-positive was considerably higher in this study than in most reports from USA or the UK, which resulted in a relatively lower cost-efficiency of HPV triaging. However, our major concerns were related to the differences between the research study setting and the real-life health care setting. The research setting does not include the entire population, only a select subset of participating subjects. Furthermore, the support, dedication and resources invested in a research study enable close monitoring of adherence to a study protocol and extraordinary quality control procedures (e.g., re-reviewing of cytological and histopathological slides by several senior experts) that are not possible in the routine setting, where guidelines and policy documents can be issued but where the management of the patient is decided by each physician. Real-life acceptability of a policy may be quite different both for the medical personnel, for the patients as well as for the health care provider that pays the costs in the health care setting but not in the research setting. Furthermore, as most research studies are blinded, the effects that the information of the test results may induce are not included in the overall effects measured.
The randomized HPV triaging trial ALTS found that HPV-triaging of LSIL smears was not cost-efficient as virtually all LSIL smears were HPV-positive if the diagnosis had been confirmed by the expert panel re-review.21 However, as senior expert panel re-review of all smears is not possible in routine practise, it is rather the proportion of HPV-positive LSIL smears in routine diagnosis that is of relevance. Furthermore, previous research studies of HPV triaging of LSIL have either been focused on younger women or not reported age-specific data for LSIL. As it is well known that there are lower HPV prevalences in LSIL, with increasing age, we felt that it would be necessary to perform a real-life evaluation of HPV triaging of both ASCUS and CINI smears. (LSIL terminology includes CIN1 and koilocytosis, but koilocytosis in otherwise normal smears is an exceedingly rare diagnosis in Sweden (0.2% of smears) and is classified as normal cytology). Indeed, HPV prevalences in CINI smears were lower in our real-life study, using routine cytology, than in most studies in the research setting that use the expert re-review cytology.
The break-even for lower costs of the HPV triaging policy was found to be at an HPV prevalence of about 70%, which would correspond to >35 years of age for ASCUS and >40 years of age for CINI—an essentially negligible difference in assigning the optimal cutoff for use of HPV-triaging.
Our concern about whether the health care system would be adhering to policy guidelines or whether new guidelines might induce new clinical management behaviour also found some support: Even though 32% fewer women were referred with the HPV triaging policy, the number of colposcopies was reduced by only 13% and the number of histopathologies by only 5%. Possible reasons for this effect include those induced by the fact that the clinics and the women knew that the smear was HPV-positive, for example, more aggressive efforts to contact women known to also be HPV-positive, increased likelihood of women to attend when they know they are HPV-positive and more careful colposcopical examination with increased likelihood of biopsy-taking. There may also be effects induced by the fact that the patient burden was reduced for the clinics in the HPV triaging arm, for example, shorter waiting time between diagnosis and appointment (likely to improve attendance rate) and more time available per referred woman for the colposcopists. The improved attendance rate seen with HPV triaging is relevant to the issue of safety and might be related to the nonsignificant tendency for HPV triaging to increase the CINII+ detection rate, in spite of the fact that a lower proportion of women were referred.
The HPV triaging policy evaluated included an extra "safety" smear 1 year after having tested HPV-negative. Some studies in the research setting have used safety smears in HPV-negative women21 but other studies have not found this to be required.28 We were concerned that widespread introduction of a new test in clinical routine may initially have lower quality (e.g., in sampling or sample handling) than a carefully controlled research trial. The decision to include an extra safety smear was preceded by some debate and incomplete acceptance in the medical community of the necessity of this extra smear is a possible explanation for the low attendance rate for this smear. However, the present data finding 6 of 243 women (2.7%) with CINII+ in this group (about three times excess yield compared with general population screening) argues that this extra smear is indeed required in the routine setting.
A major cause of increased cost in the HPV triaging arm was the fact that the HPV test was taken at an extra visit to a physician. Reflex HPV testing of the actual index smear sample was not possible as Stockholm was not using liquid-based cytology (LBC), because of high costs and an uncertain evidence base on its advantage.31 However, since the start of our study, LBC has become more affordable and a benefit has been found in some studies.32 A switch to LBC would greatly improve on the cost-efficiency of HPV-based triaging, by obviating the extra visit for an HPV test.
The decision to have the HPV-test taken by a physician was motivated in part by a perceived need to address questions from the women about HPV, the meaning of the HPV test positivity and about the study. However, the feed-back from participating clinics has indicated that this was not necessary and that HPV test taking by midwives would have been sufficient, which would have reduced costs.
Strengths of the study include the fact that it is not merely generalizable to a real-life setting- it is the real-life setting. The only major deviation from routine health care was the existence of an ethical review board permission for the randomization of the policies. There was no nonparticipation in the study (all women with ASCUS/CINI in cytology were included in the follow-up) and no research funds were used. By comparison, the British trial of management of borderline and low-grade abnormal smears (TOMBOLA) is a trial of a select cohort of 4,474 women33 (participation rate 52%) is funded by a research grant of several million pounds.
Another major strength is the fact that the follow-up of all actual smear-taking and biopsy-taking in the two arms was essentially complete for the entire population. Registration of all smears and histopathologies, based on unique personal identifiers, is already a part of the organised screening program. The required resources in coordination, database management et cetera were already available and a part of the required duties of the office of the organised program to follow-up on the performance of the program.
The major weakness of our study is the fact that randomization involved only 15 clusters, resulting in imbalances in study size and likely imbalances in local clinical and diagnostic practises. Studies in the research setting with individual randomization would have eliminated these weaknesses, but would have introduced a series of more severe problems, such as not being population-based and not being generalizable.
The economic analysis in this study is focussed on actual costs, disregarding discounting. Actually paid costs were preferred as the two policies compared were running concurrently. The future follow-up is scheduled for 2 years, which would have discounted the follow-up costs for these smears with 1 year or 2 years, respectively. As the future projected costs were <10% of all costs, discounting of future costs would have had negligible effects on total costs. Therefore, we preferred to present the actual figures without manipulation, for maximal transparency.
In summary, this study has demonstrated that carefully controlled and evaluated implementation of routine preventive services can contribute significantly to the scientific knowledge base. There are improvements particularly in generalizability, but the large size of the study also enabled conclusions to be drawn that would have been unreasonably expensive/impossible to answer with studies in the research setting. In particular, the evidence that HPV-based triaging of women with CINI in cytology is cost-effective above 40 years of age has not been shown before, presumably because of small numbers and/or studies not being population-based. We found that HPV-based triaging was safe, in terms of equal CINII+ detection rates, for all ages and for both ASCUS and CIN1. With the current costing, the HPV-based triaging was less expensive only for women older than 35 years of age. However, the demonstrated safety of HPV-based triaging could support HPV-based triaging also in younger ages, provided that the costs change (e.g., if reflex testing on LBC samples becomes possible and/or the price of the HPV test is reduced).
We thank Tiina Malmström for database assistance. We also thank our enthusiastic contact persons at all the different hospitals and clinics in the Stockholm county especially: Ilona Barnard, Agneta Fredriksson, Meseret Mengisto, Ricardo Munoz, Carl Preastiin, Stanislav Rodau and Gunilla Torpare. The study had no research grants or commercial sponsorships. The HPV tests were paid at ordinary market value by the regional health care provider.
- 1Chapter 16: cervix. In: FrancoEL, RohanTE, eds. Cancer precursors: epidemiology, detection and prevention. New York: Springer, 2002: 249–86., .
- 2Interim guidelines for management of abnormal cervical cytology. The 1992 National Cancer Institute Workshop. JAMA 1994; 271: 1866–9., , , , .
- 3HPV testing: is it useful in triage of minor Pap abnormalities? J Fam Pract 1998; 46: 121–4..
- 4European guidelines for quality assurance in cervical cancer screening. Second edition-summary document. Ann Oncol 2010; 21: 448–58., , , , , , , , .
- 5Risk of cervical cancer associated with mild dyskaryosis. BMJ 1988; 297: 18–21., , , .
- 6Management of women with mild and moderate cervical dyskaryosis. BMJ 1994; 308: 1399–403., , , , , , , .
- 7Controversies in the management of low-grade cervical intraepithelial neoplasia. Cancer 1993; 71: 1413–21., .
- 8Management of women with mild dyskaryosis. Immediate referral to colposcopy is safer. BMJ 1994; 309: 591–2..
- 9Viral testing for genital human papillomavirus infections: recent progress and clinical potentials. Int J Gynecol Cancer 1995; 5: 321–8..
- 10Anxiety caused by abnormal result of cervical smear test: a controlled trial. BMJ 1990; 300: 440., , .
- 11A 2-year follow-up study of anxiety and depression in women referred for colposcopy after an abnormal cervical smear. BJOG 2008; 115: 212–8., , .
- 12Natural history of cervical intraepithelial neoplasia: a critical review. Int J Gynecol Pathol 1993; 12: 186–92..
- 13Human papillomavirus testing by hybrid capture appears useful in triaging women with a cytologic diagnosis of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 1995; 172: 946–54., , , , , .
- 14Comparison of management algorithms for the evaluation of women with low-grade cytologic abnormalities. Obstet Gynecol 1995; 85: 202–10., , .
- 15Where's the high-grade cervical neoplasia? The importance of minimally abnormal Papanicolaou diagnoses. Obstet Gynecol 1998; 91: 973–6., , , .
- 16A comparison of the human papillomavirus test and Papanicolaou smear as a second screening method for women with minor cytological abnormalities. Acta Obstet Gynecol Scand 2005; 84: 996–1000., , , , , .
- 17Does typing of human papillomavirus assist in the triage of women with repeated low-grade, cervical cytologic abnormalities? Gynecol Oncol 1998; 70: 317–8., .
- 18Is human papillomavirus testing of value in clinical practice? Am J Obstet Gynecol 1999; 180: 1049–53., .
- 19 , , , , , , , .
- 20Clinical utility of HPV DNA detection: triage of minor cervical lesions, follow-up of women treated for high-grade CIN. An update of pooled evidence. Gynecol Oncol 2005; 99( Suppl 3): 7–11., , , , .
- 21ALTS (ASCUS-LSIL Triage Study) Group. Results of a randomised trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003; 188: 1383–92.
- 22on behalf of the Cytopathology Education and Technology Consortium. Statement on HPV DNA test utilization. Am J Pathol 2009; 131: 768–9., , ,
- 23European guidelines for quality assurance in cervical cancer screening: recommendations for clinical management of abnormal cervical cytology, part I. Cytopathology 2008; 19: 342–54., , , , , , , , .
- 24Use of human papillomavirus DNA testing to compare equivocal cervical cytologic interpretations in the United States, Scandinavia, and the United Kingdom. Cancer 2002; 96: 14–20., , , , , , , , , , , .
- 25Effectiveness of the public health policy for breast cancer screening in Finland: population based cohort study. BMJ 1997; 314: 864–7., , , .
- 26Routine cervical screening with primary HPV testing and cytology triage protocol in a randomised setting. Br J Cancer 2005; 93: 862–7., , , .
- 27KossLG, ed. Diagnostic cytology and its histopathologic basis, 3rd ed., Philadelphia: Lippincott, 1979.
- 28Identifying women with cervical neoplasia: using human papillomavirus DNA testing for equivocal Papanicolaou test. JAMA 1999; 281: 1605–10., , , , , , , , , , , .
- 29Trials which randomize practices II: sample size. Fam Pract 1998; 15: 84–7., .
- 30Current and future challenges in the design and analysis of cluster randomization trials. Stat Med 2001; 20: 3729–40., .
- 31Liquid compared with conventional cervical cytology: a systematic review and meta-analysis. Obstet Gynecol 2008; 111: 167–77., , , , , .
- 32Liquid-based cytology versus conventional Papanicolaou smear in an organized screening program: a prospective randomized study. Cancer 2007; 111: 285–91., , , , .
- 33The TOMBOLA group. Trial of management of borderline and other low-grade abnormal smears (TOMBOLA): trial design. Contemp Clin Trials 2006; 27: 449–71., , , , , , , , , , ,


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South: Jungle boogie, Ghost, X-treme Motion, Royal Flush, and Swagger Crew
East: Saltare, Blueprint, Legendary Seven, Static Noyse, and DraZtik
West: Blended Projekt, Blended EFK, Heavy Impact, Hype 5-0, and Poreotix
Who are you looking forward to?
Personally, I'm really glad Poreotics are in.
Sources from bloggingbestdancecrew.
* Poreotix = Poreotics
Heavy Impact= Heavy Hitters
Hype 5-0= The Hype


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US 4839283 A Abstract A method is provided for expressing human alpha-1-antitrypsin in yeast utilizing a wild-type strain and a hyperproduction mutant, GK100.
Claims(5) 1. A method of producing a polypeptide having the protease inhibition activity of humane alpha-1-antitrypsin, comprising the steps of transforming a DNA transfer vector into a yeast culture comprising cells of the mutant strain GK-100 and growing said culture under conditions suitable for expression of said polypeptide, said vector comprising a segment coding for human alpha-1-antitrypsin and a promoter capable of controlling expression of said polypeptide in said yeast culture.
2. A method according to claim 1 wherein said vector comprises plasmid CAT1.
3. A method according to claim 1 wherein said vector comprises plasmid HAT4.
4. A DNA construct capable of expression in a microorganism of a polypeptide having the protease inhibition activity of human alpha-1-antitrypsin, said construct comprising plasmid HAT4.
5. A yeast transformant containing a DNA element comprising plasmid HAT4.
Description This application is related to Ser. No. 408,099, filed Aug. 13, 1982, now allowed U.S. Pat. No. 4,599,311 the disclosure of which is incorporated herein by reference in its entirety. The present invention is directed to a method of expressing human alpha-1-antitrypsin in yeast and to the protein product expressed thereby. Alpha-1-antitrypsin is a protease inhibitor present in mammalian blood whose apparently major physiological function is to inhibit elastase, a potent protease which hydrolyzes structural proteins. Alpha-1-antitrypsin also inhibits other serine proteases. The normal plasma level of alpha-1-antitrypsin is about 2 mg/ml. A low level of alpha-1-antitrypsin in the blood may be associated with chronic obstructive pulmonary emphysema and infantile liver cirrhosis. Under many inflammatory conditions, an acute-phase response is initiated and the concentration of alpha-1-antitrypsion is substantially increased. In order to study and treat alpha-1-antitrypsin deficiency and to examine the mechanism of the acute-phase response, it is therefore desirable to have a pure alpha-1-antitrypsin protein. In particular, it is desirable to have a source of antitrypsin protein produced by microorganisms through genetic engineering techniques. It is therefore an object of the present invention to provide a method for producing alpha-1-antitrypsin in microorganisms, preferably in yeast. It is a further object of the present invention to provide a pure alpha-1-antitrypsin polypeptide which has been profuced by genetic engineering techniques. In the accompanying FIGURES: FIG. 1 is the cDNA sequence of the gene coding the predominant form of human alpha-1-antitrypsin. FIG. 2 illustrates the restriction maps of plasmids CTEA32 and CAT1. FIG. 3 is a diagram of the electrophoresis chromatogram showing purified alpha-1-antitrypsin produced according to the present invention. FIG. 4 illustrates the restriction map of plasmid C1/1. FIG. 5 illustrates the DNA sequence of the multiple restriction site of pUC13. FIG. 6 illustrates the restriction map of plasmid pUCα1 containing the DNA sequence from FIG. 1. FIG. 7 is the restriction map of plasmid HAT4. The sequencing of chromosomal DNA coding for alpha antitrypsin has been described by Kurachi et al., Proc. Natl. Acad. Sci. U.S.A., 78, 6826-6830 (1981) and by Chandra et al., Biochem. Biophys. Res. Comm., 103, 751-758 (1981), the disclosures of which are incorporated herein by reference. A primate gene for alpha-1-antitrypsin may be obtained by DNA cloning methods described by Chandra et al., ibid. The gene coding for the predominant form of human alpha-1-antitrypsin, isolated from a human cDNA library by using the baboon sequence as a DNA hybridization probe is shown in FIG. 1. The cDNA coding for alpha-1-antitrypsin (hereinafter "AT") may then be inserted into an expression vector, such as CTEA32 (FIG. 2), which contains the yeast promoter for triose phosphate isomerase (TPI) inserted at the BamHI site of the shuttle plasmid, CV13 [Broach J. R., Strathern J. N., Hicks J. B., Gene, 8:121-133 (1979)]. A synthetic DNA adaptor was ligated into the TPI promoter after the TPI structural sequences were removed by BAL31 digestion from the KpnI restriction site within the TPI coding region. (Alber et al, J. Molec. Applied Genet., 1, 419-434 (1982)). This adaptor contained an ATG codon for translation initiation, followed by the sequence GAGGATCC. The GAG codon specifies a glutamic acid residue, which is the first amino acid of the naturally-occurring human AT. The GGATCC portion of the adaptor is a cutting site for BamHI endonuclease and allows for the splicing of the remainder of human AT DNA sequence into this vector. The BamHI site of CTEA32 was constructed to be "in frame" with the rest of the AT structural gene, thereby allowing for the expression of the polypeptide when a BamHI fragment from the cloned cDNA is appropriately inserted into CTEA32. The plasmid consisting of CTEA32 plus the AT gene is called CAT1 (FIG. 2). This DNA construct containing the gene for human AT located downstream to a yeast triose phosphate isomerase (TPI) promoter fragment was transformed into yeast strains, N501-B and GK100. Transformation into yeast is described by Beggs, Nature 275, 104-109 (1978). Screening of the transformed yeast strains by immunological assays (competition assays and ELISA assays, using antibodies against alpha-1-antitrypsin) confirmed the presence of large amounts of human AT in yeast made from the plasmid CAT1. The "wild-type" yeast strain, N501-1B (described by Kawasaki et al., Biochem. Biophys. Res. Comm., 108, 1107-1112 (1982)), when transformed with CAT1, produced 1.8 mg alpha-1-antitrypsin per gram of soluble protein (or 0.18% alpha-1-antitrypsin), when grown at 30� on a synthetic minimal medium (modified Wickerham's medium) with 6% glucose. A mutant yeast strain, GK100, when transformed with CAT1, produced 10-15 mg alpha-1-antitrypsin per gram soluble protein (or 1-1.5% alpha-1-antitrypsin) under the same growth conditions. Strains N501-1B and GK100 each carry a defective LEU2 gene which allows for the selective maintenance on minimal and leucine-less media of CV13 and CV13-derived plasmids (such as CAT1) which each contain a functional LEU2 gene. When grown on minimal media with only CV13 as a control, N501-1B and GK100 produce no detectable AT. Thus, AT may be specifically produced by the CAT1 plasmid. Since GK100 produces significantly more AT than N501-1B, it is preferred. However, the present invention is not limited to AT production by GK100. It may be desirable to utilize mutations in GK100 which lead to hyperproduction of AT. An immuno-adsorption column, made according to the method of Cuatrecasas, P. J. Biol. Chem., 245, 3059 (1970), was prepared by covalently attaching affinitypurified goat antibodies to human AT to CNBr-activated Sepharose. Disrupted GK100 yeast cells were extracted with 3 volumes of phosphate buffered saline pH 7.2 containing 0.5M NaCl, and the extracts were applied to the column. Yeast produced human AT (0.5-1.0 mg) was eluted from the column with 3M NaSCN. After the material was dialyzed to remove salt it was analyzed by electrophoresis on a polyacrylamide gel in the presence of sodium dodecyl sulfate, the results of which are shown in FIG. 3. Based on the relative migration of the protein in the gel, the approximate molecular weight of the human alpha-1-antitrypsin made in yeast is 42,000-43,000 daltons. Naturally occurring human AT has a molecular weight of approximately 54,000 daltons, having a carbohydrate composition of approximately 16% by weight, as shown by Hodges et al, J. Biol. Chem., 254, 8208-8212 (1979). It therefore appears that the yeast produced AT may be unglycosylated or substantially unglycosylated and may lack carbohydrate portions present in the naturally occurring protein. Alternatively, other expression vectors may be constructed which contain a segment coding for alpha-1-antitrypsin. Such expression vectors may be constructed by methods known to those of ordinary skill in the art using available DNA constructs. A preferred vector is plasmid C1/1, which is more stable than CV13 and CV13 derived vectors, such as CAT1. C1/1 was constructed from plasmid, pJDB248 (Beggs, J., Nature 275, 104-109 (1978)). The pMB9 sequences were removed from pJDB248 by partial digestion with Eco RI and were replaced by pBR322 DNA which was cut with Eco RI. The restriction map of C1/1 is given in FIG. 4. The C1/1 plasmid contains the entire 2-micron DNA from yeast (S.cervisiae), with a pBR322 insertion at an EcoRI site. It also contains the LEU2 gene. Thus, the yeast TPI promotor with the adaptor may be inserted into the single BamHI site in the TcR gene of C1/1. Then the AT sequence, attached to a transcription terminator fragment from the yeast TPI gene, may be inserted into the BamHI side downstream from the TPI promotor. The resulting plasmid, HAT4, may then be transformed into N501-1B and GK100 in a manner as described above. The sequence of the first ten amino acids in the yeast-produced AT may be confirmed by amino acid sequence analysis as identical to the first ten amino acids of the naturally occurring human AT: ##STR1## The yeast-produced AT does not contain the initiation methionine which is specified by the ATG start codon. The polypeptides produced according to the present invention having AT activity may be useful for treatment of a genetic AT deficiency and other diseased states related to inadequate levels of AT. Thus, conditions such as emphysema and other lung disorders related to progressive digestion of lung sacs may be treated, such as, chronic obstructive pulmonary disease or adult respiratory distress syndrome. Non-genetically related emphysema may also be treated, such as, emphysema resulting from heavy smoking. Conditions not necessarily confined to the lungs may also be treated, such as, cystic fibrosis and arthritis. For a review of AT deficiency, see Gadek, J. E., and R. Crystal, "α1-Antitrypsin Deficiency", The Metabolic Basis of Inherited Disease, J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, McGraw-Hill, N.Y. pp. 1450-67 (1982). The polypeptides according to the present invention may be admixed with conventional pharmaceutical carriers. Preferably, the polypeptides are to be administered intravenously or by inhalation. While the effective dosages may vary according to the severity of the condition and weight of the subject, dosages in the range of 0.5-10.0 gm/week of a polypeptide introduced intravenously may, in many cases, be effective. Lower dosages may be effective if the method of administration is by inhalation. Oral administration may also be effective provided the AT is protected in capsules or coated carriers from premature degradation in the digestive tract. The following examples set forth specific embodiments according to the present invention, but the invention is not intended to be limited thereto. An immuno adsorption column was prepared by covalently attaching affinity-purified antibodies to human alpha-1-antitrypsin to CNBr-activated Sepharose according to the method of Cuatrecasas, J. Biol. Chem., 245, 3059 (1970). Disrupted GK100 cells were extracted with three volumes of phosphate buffered saline pH 7.2 containing 0.5M NaCl and applied to the column. The column was eluted with 3M NaSCN and the recovered material was analyzed by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. The results of the electrophoresis are shown in FIG. 3. Track 1 contained a mixture of molecular weight standards: (a) phosphorylase B, 97,000 daltons; (b) bovine serum albumen (BSA), 65,000 daltons; (c) ovalbumin, 43,500 daltons; (d) carbonic anhydrase, 30,000 daltons; (e) soybean trypsin inhibitor, 20,000 daltons; and (f) alpha-lactalbumin, 14,000 daltons. Track 3 contains yeast produced AT purified by immunoadsorption, molecular weight about 42,000 daltons. Track 7 is a sample of naturally occurring AT purchased from Sigma Chemical Company, heavily contaminated by blood proteins. A major component of Track 7 is human alpha-1-antitrypsin, molecular weight 54,000 daltons. As a control, 10 microliters (1 microgram) of a solution of 100 microgram/ml trypsin, 100 microgram (100 microliters) of bovine serum albumin and 100 microliters of 0.05 molar TRIS, pH 8.0 buffer containing 1 mM benzoylarginioyl-p-nitroanilide were mixed, and the increase in absorbance at 405 nm was measured over time in a spectrophotometer. The absorbance value of this solution was used as a standard for 100% trypsin activity. Three additional samples were run in duplicate, each containing 1 μl trypsin and, respectively containing 25 μl AT solution plus 175 μl buffer, 100 μl alpha-1-antitrypsin plus 100 μl buffer, and 200 μl alpha-1-antitrypsin. All samples contain equal concentrations of substrate and bovine serum albumin. The results demonstrated that utilizing 25 microliters of AT, 73% of the trypsin activity remained, with 100 microliters of AT, 41% of trypsin activity remained and with 200 microliters of alpha-1-antitrypsin, 26% of the trypsin activity remained. This demonstrated that by increasing the levels of the yeast made AT the trypsin inhibitory activity also increased. Increased levels of AT may be obtained by utilizing C1/1, a plasmid which is more genetically stable than CV13. The C1/1 plasmid contains the entire 2-micron DNA from S. cerevisiae and, therefore, can promote its own replication and maintenance in yeast in the absence of selection for a genetic marker. Also, C1/1 plasmid has a single BamHI site located in the TcR gene. Transformants carrying C1/1 may be selected in E. coli by ampicillin- or tetracycline-resistance and in yeast by leucine prototrophy. C1/1 contains pBR322 inserted into an EcoRI site of 2-micron DNA and carries the LEU2 gene described by J. Beggs, Nature, 275, 104-109 (1978). The yeast TPI promoter (from CTEA32) with the synthetic DNA adaptor (described above) was inserted as a Bg1 II BamHI fragment (about 900 base pairs) into the BamHI site of C1/1. This insertion created a single BamHI site into which the human AT gene could be spliced for expression in yeast. As in the CAT1 plasmid, when the AT gene (FIG. 1) is inserted, the resultant plasmid would have an ATG initiation codon followed by a GAG (glutamic acid codon) to allow the production of mature human AT protein sequence in yeast. About 700 base pairs of the 3' flanking region of the yeast TPI gene was added after the human AT sequence to assist in transcription termination. The "termination" fragments are sequences from the XbaI to EcoRI sites in the plasmid TPIC10 (T. Alber and G. Kawasaki, J. Molec. Applied Genet., 1, 419-434 (1982)). The yeast termination sequences were attached to the human AT gene by using the vector, pUC13, which has multiple cloning sites into which the terminator and AT DNA's can be separately inserted. The pUC13 plasmid was constructed as described in Vieira, J., and Messing; J., Gene 19, 259-268 (1982) for vectors, pUC8 and pUC9. The pUC13 plasmid contained the multiple restriction site, depicted in FIG. 5, at the start of the lac Z gene. To connect the human AT gene to the TPI transcription terminator, the AT cDNA clone (FIG. 1) was inserted as a Pst I fragment into pUC13 at the single Pst I site. The AT gene was followed by an Xba I site and Eco RI site in the multiple cloning sequence. Between these Xba I and Eco RI sites of pUC13 was inserted the yeast TPI terminator as a 700 base pair Xba I-Eco RI fragment from pTPIC10. The resulting plasmid, pUCα1+FG1, contained a human AT gene with a yeast transcription terminator (See FIG. 6). An Eco RI-Bam HI synthetic DNA adaptor was then added to the Eco RI site of the plasmid, in order to create a Bam HI site on the 5' end of the yeast terminator. By using this adaptor, the human AT-yeast terminator sequence could be removed by cutting with Bam HI to liberate a fragment of approximately 2100 base pairs. This BamHI fragment was inserted into the C1/1 plasmid containing the TPI promoter with BamHI adaptor. The resulting plasmid, HAT4, has the TPI promoter, ATGGAGGATCC adapter, human AT gene (from the BamHI site), and TPI terminator inserted into C1/1. The topology of HAT4 is depicted in FIG. 7. HAT4 was transformed into N501-1B and GK100. On minimal media with 6% glucose, 2-3% of the yeast soluble protein was alpha-1-antitrypsin at a cell density of nearly 3 g per liter (wet weight). Because HAT4 contained C1/1, this plasmid was maintainable in a variety of rich media, including YEPD (1% yeast extract, 2% peptone, and 2% glucose). On rich media 2-3% AT was still produced but at a higher cell density of 10-20 g per liter (wet weight). The HAT4 plasmid was maintained without selection in N501-1B for over 30 divisions on rich media with greater than 70% of the cells containing the plasmid. In GK100 better than 95% of the cells had HAT4 after 30 divisions on rich media. The advantages of using HAT4 over CAT1 were (1) greater plasmid stability, (2) higher levels of AT as a percentage of total protein, (3) much greater yields of cells per liter as a result of using rich media, and (4) cheaper costs of rich media compared to synthetic (leucine-less) media. The mutant yeast strain GK100 has been placed on deposit in the American Type Culture Collection, Rockville, Maryland, ATCC No. 20669. Patent Citations
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