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Lot 294: Eight Plates White With Orange And Brown Flowers
October Estate & Collectible Catalog Auction
by Charleston Auction House
23 October 2016
Charleston, SC, USALive Auction
Past Lot
Looking for the realised and estimated price?
Description: 6" Diameter
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Previous: Taking our healthcare staffing expertise to the Middle East
Next: Revalidation review
Revalidation review
Medacs Healthcare to offer GMC-run professionalism courses for overseas doctors
Psychiatrists – are you ready for the next step in your career?
Nurses – are you ready for the next step in your career?
© Copyright 2013 Medacs Healthcare
Medacs Healthcare plc – an Impellam Group Company.
Registered Office: 800 The Boulevard, Capability Green, Luton LU1 3BA.
Registered in England and Wales No. 2518546

subset: commoncrawl


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Stunning apartment located inside a magnificent barroco-style medieval palace. This amazing property dates back to 1602 and the first oficial records date back to 1761. It consists of 9 comfortable and spacious rooms which are adorned with stately balconies. The ceilings are over 4m heigh with quality wooden beams that add a great deal of charm to this special property. On the first floor we can find four enourmous rooms: an entrance hall with a guest toilet, dining room, library and a sitting room that is filled with light and leads on to the perfectly equipped and stylish kitchen. The doors are enourmous and made from antique wood with nails and built-in iron which is typical of this stately style. The patio features a charming elegant water well with unique painted walls. On the first floor we can find a spacious and luminous bathroom and a large bedroom as well as access to the lower floor we we can find a cellar. The "Petit Palace" offers a feeling of privacy on the upper floors with three double bedrooms. One of the bedrooms makes up a self-contained & private apartment with it´s own dining room, full bathroom as well as a sunny 25m2 terrace which is ideal to enjoy Barcelona to it´s fullest. It should be also mentionned that this amazing property also has two additional terraces one is 60m2 and the other 25m2 one of them is accesable via a unique and ancient spiral staircase. The total size of the property is 250m2 and 100m2 of terraces; a real jewl, a unique once in a lifetime opportunity that is located in a palace in the exciting Born neighbourhood.
In the most demanded location in downtown Barcelona. Just a few minutes walking distance from Arco del Triunfo, Las Ramblas, Plaza Cataluña, Princesa Street or Paseo del Born. Near by public transportation and and just ten minutes walking distance from the beach. The most trendy and demanded location in the city: Borne
Availability upon agreement. This material is based upon information, which we consider reliable, but because third parties have supplied it, we cannot represent that it is accurate or complete, and it should not be relied upon as such. This offering is subject to errors, omissions, and change of price or withdrawal without notice. Taxes, notary and register fees must be paid by purchaser, the commission by the seller as stipulated by Spanish regulations.


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7 Day Pass
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Unlimited rides on the I-RIDE Trolley for 7 consecutive calendar days
USD $11.99Pricing details
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Unlimited rides on the I-RIDE Trolley for 14 consecutive calendar days
USD $17.99Pricing details
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All Aboard the I-RIDE Trolley!
- Unlimited Trolley Pass (based on the number of days selected)
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- All passes are valid for consecutive days upon activation on the trolley
- If your pass is lost or stolen, you will be responsible for purchasing a new one.
- I·RIDE Trolleys seat 41 riders, 54 total capacity, including two wheelchair positions and an ADA-specified hydraulic lift system, weight limitations apply. (Maximum weight - including passenger and vehicle - is 600 lbs., width restriction is 30" wide and height
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If you cancel between 3 and 6 day(s) in advance of the scheduled departure, there is a 50 percent cancellation fee.
If you cancel within 2 day(s) of the scheduled departure, there is a 100 percent cancellation fee.
Traveler Reviews
Reviewed by: Sandra S, July 2016
This was such a good choice, We could get on and off when we needed to. The drivers were very nice and accommodating Great idea!.
Reviewed by: Mike C, July 2016
Most convenient way to ride up and down International Drive.


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Hayate no Gotoku! Cuties - Episode 3 Discussion / Poll
Welcome to the discussion thread for Hayate no Gotoku! Cuties, Episode 3.
Thread Guidelines
Even if "Alice" is just a part of Athena, she's still her, and I would've enjoyed seeing her character in a comedic context anyway. And... I enjoyed this episode. Athena was the one who got the plot moving, but Sakuya (who sang the ED) and Fumi (and Yukiji!) played just as major a role. I was only introduced to Fumi in recent series, but I like her. Kana Asumi brings her usual energy to the role, and anyone who tries to seriously interrogate a dog is going to get a few laughs out of me. I wasn't expecting the return of Tama either. He was one of the elements from the first series that gave it that distinctly anime feel, that I liked, and was slowly phased out in the second season, but here, had another moment to shine. The return of Regioster was a surprise, too. The show's got an expansive cast, but that doesn't mean everybody's forgotten, even if their prominence is less. The story with Armageddon (really, that's a hell of a name for a dog) was cute, and the second, shorter sketch with Nagi and Athena made for some good interplay. While they do have some character overlap, Athena seems to be a more traditional rich girl/socialite, while Nagi's a more modern (otaku) girl.
Next week looks to be Sakuya and Isumi's actual episode. I love those two, so I'm looking forward to it. They have some of the best comedic chemistry.
A-tan HHHHNNNNGGGG! I wish I was the dog giving her the glomps. :D
A-tan was really cute
Tama talking with Armageddon was really funny
it's hereeee!!! A-tan special episode!!!
they managed to clump three A-tan chapters into one episode....
including my favorite chapters of course (interaction between the previous and the current... xD)
now I'm satisfied with A-tan loli version.... waiting for adult one (I see a glimpse of her in the ending song)
dying for A-tan's arc adaptation..... I miss her too much
Arma talking smack to Tama was absolutely hilarious :D
A-tan is a shadow of her former self, in every sense of the phrase.... >_>
Ironically it's those elements that turned out to be loved by the fans almost universally.
So far it's the best episode that I've seen :). Too bad it's just one episode, Alice was just adorable.
Word of caution:
Be careful how you carry a briefcase full of money, since on your way to the bank it's more likely that a big dog will steal it, and give it to a cute and adorable, lolita girl whose hair tails happen to be the same shape as a chocolate coronet.
Also take note that amongst loli girls, whether be a selfish, hikkikomori and just a plain cute, little lady without memory, if they cooperate for a common objective, that would be to reach the restaurant where a debt-ridden butler works at, in order to eat his cooking.
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text: I recognize I am very particular about BBQ and the different styles, but wanted to give Hickory Kitchen another chance after not eating here for a long time. The brisket was a little fatty, and the chicken on the salad a bit dry. Also, thought it was a little over-priced for lunch ($33 for two, with a salad, sandwich platter... More


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"Judge tells Ohio village to pay back $3 million from traffic camera tickets" [AP]
Posts Tagged 'red light cameras'
Police and prosecution roundup
- Amid multiple scandals, why won't office of Orange County, Calif. District Attorney Tony Rackauckas confirm name of county investigator alleged to have beaten defense attorney in courtroom hallway? [R. Scott Moxley/OC Weekly via Radley Balko, Voice of OC]
- And from February: "former Los Angeles sheriff Lee Baca announced that he would plead guilty to criminal charges related to systemic misconduct in his department, specifically to a charge of lying to investigators in an effort to cover up that wrongdoing." [Kevin Williamson]
- Post-Ferguson investigation: problems with small-town municipal courts go way beyond North St. Louis County into outstate Missouri [St. Louis Post-Dispatch]
- Judge throws out mountain of tickets from Chicago traffic and speed cameras [TimeOut, Timothy Geigner/TechDirt, earlier]
- Britain: following collapse of lengthy Operation Midland law enforcement inquiry into a fantasist's wild tales of abuse (did senior Tories murder rentboys for fun?) vindicated officials and their families wonder where to turn to get their reputations back [Dan Hodges/Telegraph (citing Metropolitan Police commissioner Bernard Hogan-Howe's favorable reference to a second official's statement that "The presumption that a victim should always be believed should be institutionalized"); Matthew Scott/Barrister Blogger, Richard Bartholomew]
- Supreme Court nominee: "In Criminal Rulings, [Chief Judge Merrick] Garland Has Usually Sided With Law Enforcement" [New York Times; more on Garland's D.C. Circuit rulings]
Law enforcement for profit roundup
- Montgomery County, Maryland officials help plant letters praising speed cameras in local papers [The Newspaper] "Chicago issued $2.4 million in bogus traffic tickets from speed cameras" [David Kravets, ArsTechnica citing David Kidwell and Abraham Epton, Chicago Tribune]
- "DEA Promised TSA Agent a Cut of Passengers' Seizable Cash" [Jacob Sullum, Reason]
- "Cops Seized Over $107,000 From Couple; Didn't Charge Them With A Crime" [Rock Island, Ill.; Amy Alkon citing Quad Cities Dispatch-Argus]
- I was among those speaking at an Annapolis event unveiling a new bill to restrain and better control asset forfeiture [Frederick News-Post, WBAL, Scott Shackford/Reason, Grant Zeigenfuse/Maryland Reporter; earlier on South Mountain Creamery case; more on margarita machines as forfeiture perk]
- Texas: "Cops Getting Free License Plate Readers In Exchange For 25% Of The 'Take' And All The Driver Data Vigilant" [Tim Cushing, TechDirt]
- Cato podcast on asset forfeiture with Adam Bates. More: What the President should do;
- How does Manhattan District Attorney Cy Vance spend his $800 million slush fund from bank settlements? Any way he pleases? [Scott Greenfield]
Law enforcement for profit roundup
- One Oklahoma official used asset forfeiture to pay back his student loans, another lived rent-free in a confiscated house [Robby Soave, Reason]
- Per ACLU, Arizona has a one-way legal fee rule in forfeiture cases, with prevailing police allowed to collect from property owner but not vice versa [Jacob Sullum]
- From Michael Greve, some thoughts on prosecution for profit and where money from public fines should go [Liberty and Law]
- About the Benjamins: Philadelphia mayor-to-be cites revenue as reason to let parking officers ticket sidewalk users [Ed Krayewski, Reason]
- Captive market: with wardens' and sheriffs' connivance, prison phone companies squeeze hapless families [Eric Markowitz, IB Times]
- Former red light camera CEO pleads guilty to bribery, fraud in Ohio [Cyrus Farivar, Ars Technica]
- Taxpayers lose as Maine counties jail indigents over unpaid fines [Portland Press-Herald]
- "St. Louis County towns continue to treat residents like ATMs" [Radley Balko]
Traffic and road law roundup
- Driver's license suspensions, which many states use to punish unpaid court debt and other offenses unrelated to driving skill, can accelerate spiral into indigency [New York Times]
- Your war on distracted driving: woman says she received $200 ticket "for putting on lip balm at a red light." [KLAS Las Vegas, Nev.]
- "Of Course We Have No Ticket Quotas, But …." [Lowering the Bar; Edmundson, Mo., in St. Louis County; Mariah Stewart, Huffington Post on revenue generation in Berkeley, Mo., and other neighboring towns; Scott Greenfield ("Ferguson: Where Everyone's a Criminal")]
- Yet more on St. Louis County: it started with a "defective muffler" stop in Florissant [Riverfront Times]
- NYC: "Speed cameras lead to surge in tickets and $16.9M in revenue for city" [NY Daily News]
- New Los Angeles parking signs explain it all for you, also recall design of craps table [Mark Frauenfelder, BoingBoing]
- Virginia: "How Police Drones and License-Plate Readers Threaten Liberty" [A. Barton Hinkle; related, Jim Harper/D.C. Examiner]
Police and prosecution roundup
- "Georgia's Highest Court Limits Power Of Private Probation Industry" [WABE, Augusta Chronicle, earlier here, here, here, and here]
- Federal judge slams as "preposterous" DEA (Drug Enforcement Administration) actions in raid on Tampa pharmacy [Tampa Bay Online]
- "At Least 40 Federal Agencies Now Conduct Undercover Operations" [Jesse Walker]
- "'Gotcha' program designed to rake in revenue": "Nassau County Lawmakers Repeal Speed Camera Program" [CBS New York]
- "Creepy": "FBI Agents Pose as Repairmen to Bypass Warrant Process" [Bruce Schneier]
- Somewhere an old guy in his room is wondering about ending it all, and somewhere a fanatic has been made happy [Elizabeth Nolan Brown on launch of Anaheim, Calif. shaming program to post names of sex purchasers online indefinitely]
- Heather Mac Donald: In defense of broken-windows policing [Time]
Police and community roundup
- Oh, no: "Ferguson to Increase Police Ticketing to Close City's Budget Gap," because three arrest warrants per household is still too low [Bloomberg News via Zach Weissmuller (& thanks for quote), earlier]
- In years 2011/12 alone, one Buffalo officer "killed as many dogs in the line of duty as the entire NYPD." [WGRZ]
- "He believed the poor had the right to buy and sell." Tunisia yes, Staten Island too? [David Boaz, USA Today]
- "The language of protest: Race, rioting, and the memory of Ferguson" [Abigail Perkiss, NCC/Yahoo, mentions me]
- "Red light cameras to go dark in New Jersey" [Josh Kaib, Watchdog Wire] "Public opinion swings hard against traffic cameras" [AutoBlog]
- On interpreting statistics on race and policing, point counter-point [Scott Alexander, Ezra Klein, Alexander] Reminder: increasing ranks of black officers does not necessarily lead to fewer shootings of black civilians [Jamelle Bouie, Slate]
- "Sex, Spice, and Small-Town Texas Justice: The Purple Zone Raid" [Reason.tv video]
Police and prosecution roundup
- "Shaneen Allen's prosecutor might be having second thoughts" [Radley Balko, earlier] Sequel: Indeed.
- "If you get a parking ticket, you are guilty until you have proven yourself innocent …. And that's worked well for us." — "senior" Washington, D.C. government official [Washington Post quoting inspector general report; also includes details on traffic camera protocols]
- Not an Onion story: Eleventh Circuit chides use of SWAT methods in Florida barber shop inspections [ABA Journal ("It's a pretty big book, I'm pretty sure I can find something in here to take you to jail for"), Volokh, Balko, Greenfield] Militarized cop gear is bad, routinized use of SWAT tactics is worse [Jacob Sullum]
- New England Innocence Project looking at several shaken-baby cases [Boston Herald, background]
- Innocence commissions like North Carolina's not a big budgetary line item as government programs go, alternatives may cost more [A. Barton Hinkle]
- New evidence continues to emerge in Ferguson police shooting, but is nation still listening? [Scott Greenfield]
- Prosecutors arrayed as organized pressure group is very bad idea to begin with, and more so when goal is to shrink citizens' rights [AP on "Prosecutors Against Gun Violence"; Robert H. Jackson on prosecutors' power and role in society]
Police and prosecution roundup
- Enviro activists unlawfully block coal ship, Massachusetts prosecutor expresses approval by dropping charges [James Taranto, Jacob Gershman/WSJ Law Blog, ABA Journal]
- Unfortunately-named Mr. Threatt charged with "robbery that happened while he was in jail" [Baltimore Sun via @amyalkon]
- "How conservative, tough-on-crime Utah reined in police militarization" [Evan McMorris-Santoro, BuzzFeed] More: What if we needed it someday? San Diego Unified School District defends acquisition of armored vehicle [inewsource.org] And Senate hearing [AP]
- "Machine-based traffic-ticketing systems are running amok" [David Kravets, ArsTechnica]
- Thanks, Fraternal Order of Police, for protecting jobs of rogue Philadelphia cops who could cost taxpayers millions [Ed Krayewski; related earlier]
- Study: returning from 6- to 12-person juries could iron out many racial anomalies at trial [Anwar et al, Tabarrok]
- Courts can help curb overcriminalization by revitalizing rule of lenity, mens rea requirement [Steven Smith]
Police and prosecution roundup
- Virginia Gov. McAuliffe vetoes bill expanding procedural rights for motorists facing camera tickets [The Newspaper]
- Attracting drug deals to town was money-making scheme for a Florida community's law enforcers [Radley Balko] "Civil Asset Forfeiture: The Biggest Little Racket in Nevada" [Jason Snead and Andrew Kloster, Heritage; related, Evan Bernick on Georgia and Texas; Balko forfeiture roundup; and update, reform in Minnesota]
- "Yes, it's time to get rid of regulatory agencies' paramilitary units" [Jason Pye, United Liberty]
- (Some of) what's wrong with "victim's rights" laws [Steve Chapman]
- A case study in overcriminalization: "Reforming the Foreign Corrupt Practices Act" [Vikrant Reddy, Texas Public Policy Foundation] More: Overcriminalization in North Carolina [Jim Copland and Isaac Gorodetski, Manhattan Institute]
- "We emphatically reject the notion that due process of law permits the police to frame suspects." [Third Circuit in Halsey v. Pfeiffer, allowing Byron Halsey to sue police after being wrongly imprisoned for 19 years in double murder case; Newark Star-Ledger] What to do about ongoing epidemic of police "dropsy" and "testilying"? [Balko]
- Prince George's County, Maryland police announce in advance they're going to behave unethically over course of next week [Conor Friedersdorf] Update: police call off plan, claiming announcing it had value in deterring johns.


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European Academy "ERICTA" in Armenia
August 24th 2010 · Read More · No Comments
- 10 Davit Anhaght0037 Yerevan, ARMENIA
Tell: 374 10 24-63-71
Site: www.eriicta.am
About us
European Regional Academy of inter-state, non-profit foundation aimed profits, which graduate and post-graduate education in addition also offers European educational standards of professional training and qualification courses, as well as in Yerevan and the regions College (vocational) education programs.
YETA in a state institution, was established in a number of European countries, educational institutions with contracts and agreements concluded on the basis of the project life is called the Government in 2001. October 15, the decision N 978 of the Government of YETA's founding party.
- Armenian and European combines educational programs with international standards
- Innovative educational technologies
- Regional cooperation and regional dialogue confirms Armenia and neighboring countries between educational institutions
- Preparation of a competitive workforce – and for the countries of the region
- Offer a competitive learning
- Trilingual education programs
- Participation in European educational integration processes
- Flexible subject programs satisfy the requirements of the market
- Availability of the best technical base
- Formation of multilingual communication skills


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DD (not DC) reboot a go!
After all the insanity that has recently surrounded Daredevil, it's time to go back to basics for one of my first childhood favourites.
The GoodAs a kid, first getting comics, some of the very first comics I read were Frank Miller's run on Daredevil. Now anyone who has read this particular run, knows that it's a good one. Being exposed to such brilliant comics at such an age, I believe, has helped shape me into the literary freak I am today, not just for comics, but for all prose, be it novel, poem or magazine.
That aside, I find it difficult to read Daredevil comics unless they're particularly brilliant in some way. I think to nail Daredevil, you have to nail a particularly distinct style of not only writing but also in the art. Something which Frank Miller's run did, something which Bendis and Maleev's run achieved and something which I believe Waid and Rivera's new beginning to Daredevil is beginning to achieve.
I could look at Rivera's work all day. It's beautifully simple and has an awesome nostalgic value to it, and not '80's nostalgic' and not 'Golden age nostalgic' which some artists try so hard to 're-create'. Rivera's old school vibe just comes so naturally and it's perfect, not only for this new series of Daredevil's tone, but also for conveying atmosphere and feelings. Most people see Daredevil as a 'darker' title, including me sometimes, but after the grim and gritty Shadowland, Daredevil needed a well deserved restart. A fresh new clean break, so this new 'lighter' feeling series is exactly what, not only what Daredevil needs, but also what the fans needed as well. There's only so much badness a guy can take.
This issue is exactly what a number one issue should be. Any body can pick this up, comic book regular or comic book virgin. It's uncomplicated, fills you in on Daredevil's origin and recent story line's, which in turn explains Daredevil/Matt's current status but doesn't take up hardly any page time at all. This is all thanks to writer Mark Waid, who successfully brings a new start for DD, introduces a potential new love interest, new problems for Matt Murdock's career and social life plus a reason to come back next issue.
Plus there's a lovely little back up story which centers on dialogue between Matt and Foggy with a sad and poignant message which is again, very new reader friendly and adds a new dimension to DD's origin and character.
Double plus, it has an awesome opening at a wedding with a visit from a formidable Spot.
The BadThe way that Daredevil was exposed as Matt Murdock last series is swept under the carpet as 'it's alright because no one believed it anyway', which seems mighty unrealistic. How would anyone be able to live a normal life and go to work again if they were exposed as a superhero? I know, I know, this is a key plot point to the book but why is Matt being so naive in thinking that people will just believe him when he says 'hey, I'm not Daredevil, I be blind!' even when it was nationally exposed that he was Daredevil?
Also, Daredevil's actions during Shadowland aren't brought up by anyone. Surely this is quite important as people believe Matt to be DD, so surely Daredevil killing people would be used against him in court, by civilians and by other lawyers. It's not as if Shadowland was exactly kept a secret, people knew this was happening, even if it was publicly revealed he was possessed, many would still be afraid and distrustful.
But I do appreciate how Waid was trying to make a fresh start with DD and not concentrate too much on his past, that was handled in Daredevil: Reborn and we don't need that in this book.
The Verdict
An excellent start to a landmark DD ish. A great jumping on point and new start for those who regularly keep up with comics. Excellent art and handling of timing and story, pick this issue up, maybe two, who knows how much this issue may be worth in a good, saaaaaay 50 years...
And of course this is Daredevil, so Elektra, Bullseye and Kingpin HAVE to appear sooner or later, and seeing this creative team tackle these characters is gonna be super exciting.
Here's to a new era in Daredevil history.


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Our Location
|3172 Porter Drive
Second Floor
Palo Alto, California 94304
Shuttle & Driving Directions
Marguerite Shuttle Information - Marguerita Maps & Schedules
1. Stanford Research Park - Schedule
The Stanford Research Park shuttle runs to and from the Palo Alto Transit Center to the businesses along Page Mill Road, Porter Drive, Hillview Avenue, Arastradero Road, and Deer Creek Road during the commute times, 6:28 a.m. to 9:52 a.m. and again from 3:45 p.m. to 7:58 p.m., Monday through Friday (including university holidays) with departures every 20-40 minutes.
2. 1050 Arastradero - Schedule
The 1050 Arastradero shuttle runs Monday through Friday (except university holidays) serving the Medical School Office Building, Porter Drive, Hillview Avenue, Arastradero Road, returning to the Medical School Office Building (serving additional stops along the route) every 30 to 40 minutes from 8:20 a.m. to 6:20 p.m.
3. Line V - Schedule
The Line V shuttle runs Monday through Friday (including university holidays) every 30 minutes from 6:55 a.m. to 7:45 p.m. Destinations include Stanford Hospital, LKSC, Serra Mall, Schwab and Knight Centers, Olmsted Road (parallel to Serra Street only), El Camino Real, California Avenue going towards Hanover, Page Mill Road, Porter Drive, Hillview Avenue, and the Palo Alto VA Hospital (serving additional stops along the route).
Line V will provide service from the California Avenue Caltrain station to the Stanford campus during morning peak commutes from 6:35 a.m. to 9:48 a.m., and during afternoon peak commutes from 4:15 p.m. to 6:35 p.m., making all stops along California Avenue.
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Super soft teddy bear with beautiful two-tone fur. Ready for cuddles, hes sure to be a steadfast friend for years to come. He comes packaged in a smart, pewter coloured gift box, so he would make an ideal gift.
Thank you for visiting Les ZAnimoos Classic Bear, Brown on shoppingfind.co.uk
We bring the products from many websites to one website for you to search, save and compare.


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|Ruediger Pluem <rpl...@apache.org>
|Re: strange usage pattern for child processes
|Wed, 15 Oct 2008 12:41:40 GMT
On 10/15/2008 01:55 PM, Lars Eilebrecht wrote: > Hi, > > I'm trying to debug a performance issue on an Apache infrastructure > using 2.2.9 as reverse proxies. The Apache servers don't do much > except for ProxyPass'ing data from others backend servers, and > caching the content using mod_mem_cache. Is it really a good idea to use mod_mem_cache? Keep in mind that mod_mem_cache uses local caches per process and cannot use sendfile to send cached data. It seems that mod_disk_cache with a cache root on a ram disk could be more efficient here. > This is Apache with the worker mpm on Solaris. Currently each > box is configured with a ServerLimit of 3. The attached screenshot > of the server-status page shows that almost all threads of one > child process (at the bottom) are in "W" state. The thread status > of the process shown at the top look OK, but the second child > process doesn't seem to be used at all. > The first odd thing is that I would have expected that Apache > uses all child processes about equally. Especially I would > have expected that there are at least 25 threads for the second > process in state "_" (waiting for connection), because the > MinSpareThread directive is set to 25. This is indeed strange. Mind to 1. Attach an ASCII-output of the whole status page to see the exact process / thread slot usage. 2. Your MPM configuration and your reverse proxy configuration. > The second odd thing is that the connections/threads in W state > seem to be hanging, i.e., no data is being transferred over the > connection and these threads/connection time out after about 256 > seconds. However, the general Timeout setting is 30s so why > isn't the connection killed after 30s? It would be very helpful to have a backtrace of this process. If they aren't killed after the timeout it is highly likely that they don't wait for IO but do or wait for something else. > What happens during peek times is that all threads of one > child process end up in state W, then Apache starts using > another child process, etc. and at some point basically > all threads are in state W and we are hitting the MaxClients > limit which makes the server unusable. Regards Rüdiger
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Chapter 2
Chapter 3
Chapter 4
Chapter 5
God was getting on with his project. Strolling along he made minor adjustments. With a splash of colour here, a change of shape there. He was very pleased with the overall look of what he had created. Coming round a corner he found an animal that hadn't been to see him about its' colour change. It was in the shade, resting against a tree and looking at God with yellow, baleful eyes as if He may be prey, then recognised his Lord. It was the tiger. "You look pretty good as you are." said God, "But everyone else has had a change from the grey to whatever hue they would like. What's your pleasure?" "A colour change? That's a novelty. Colour? Oh I don't know. What would you suggest?" Running through all the colours that he had given to the various animals God realised that one colour had not been taken. "What about burnt orange?" offered the Lord. "Sounds reasonable to me," the tiger answered, laconically. As he was transformed in colour, there was a piteous cry of help from a short distance away. A cry that made both God and the tiger exchange concerned looks. God didn't say anything. The tiger bounded out of the clearing towards the cry for help. Warthog's wife was in serious trouble. She had managed to find herself some safety in a cavity, behind a small opening at the base of a rotten tree, in the centre of a large circular clearing. Outside, trying to get in, the panther was almost purring with suppressed pleasure as it tore at the rotten wood, making the opening wider with each strike of its claws. The blackbird was circling overhead and making its feeling heard. The warthogs' scream was becoming thin with fear, as if she had nearly lost her voice. She knew that it would not be long before the killer struck again.
© Copyright 2017 maximilliano. All rights reserved.
Status: Finished
Genre: Fantasy
And more to come... it could've been in a short novel style but this just as good.
And, a kids book maybe too.
Mike you really have a knack at story telling.
What an imaginative story. Like an old fable with modern - and very unique - overtones (undertones?) I love the converasation between the birds, flows well and entertains. What did I love best? God made very human by you, having his nap and favourite brew. Wonderful!
PS, Dear katie above suggested I visit you.
What a delightful mix of imagination, creation, humour, pathos and entertainment. And what a pleasing change to the dark and dreary and violent. I loved your "God" made human and even making a twit of himself. Love it! A God who is grumpy and irritable and who can stand to be corrected.
I'm glad you liked it, AnnaT. It came about when I asked a niece what question she would like answered. And she asked how the Tiger got his stripes? Who was I to deny her? But I answered it in my own way. She loved the story too. Namaste. Mike.
Ohhh boy what a time I have had this morning reading this! A pure delite through and through!
Mike, thankyou, thankyou for so much enjoyment.
'How a tiger got his stripes' ;-) So darn cute!
OH MIKE........ this is marvellous. What fun you obviously had writing it, I so enjoyed reading it and your humour is patently obvious throughout. It is sad, different, happy and funny extremely creative and very strikingly written.
Whoo hoo...... The pen in the right hand is mighty indeed.
I'm so pleased you liked it, Vee. I had fun writing it too. Mostly with my tongue in my cheek. Tee Hee. I had thought of making it a little longer and with slightly different characters but decided on the present format. I enjoy writing humorous work, but I think you know that from some of my other work. (When I am not somewhat sad.) Thanks for your praise, Vee, and I shall have to bring out my "funny" pen once more, methinks. Namaste. Mike
I'm so pleased you liked it, Vee. I had fun writing it too. Mostly with my tongue in my cheek. Tee Hee. I had thought of making it a little longer and with slightly different characters but decided on the present format. I enjoy writing humorous work, but I think you know that from some of my other work. (When I am not somewhat sad.) Thanks for your praise, Vee, and I shall have to bring out my "funny" pen once more, methinks. Namaste. Mike
Short Story / Fantasy
Short Story / Fantasy
Short Story / Memoir
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[December 11, 2012]
TRENDnet Joins HomePNA Alliance
(Marketwire Via Acquire Media NewsEdge) TORRANCE, CA -- (Marketwire) -- 12/11/12 -- TRENDnet, a best-in-class wired and wireless networking hardware brand, today announces membership with The HomePNA® Alliance -- a community of leading technology brands, manufacturers, and service providers working together to develop and promote home networking solutions based on HomePNA® standards.
HomePNA® is one of the leading wired home networking technologies and the choice of major service providers worldwide. The HomePNA® Alliance develops home networking specifications for distributing entertainment and triple play data over existing coax cables and phone wires. Over 30 million HomePNA-certified products have been shipped to telco, satellite, and cable service providers worldwide supplying critical components of their IPTV services deployments. HomePNA is an ITU Standard (G.9954) and is included in the DLNA Interoperability Guidelines.
"We are pleased that TRENDnet, an award winning global supplier of networking solutions for small to mid-sized business and home users, has joined the alliance," said Eran Gureshnik, HomePNA president. "Their membership demonstrates the value of the HomePNA Alliance to service providers." "Our customers are demanding a cost effective solution for distributing entertainment and triple play data over existing coax cables and therefore are specifying HomePNA certified products," said Sonny Su, TRENDnet technology director. "HomePNA membership gives us access to the alliance's technical support and certification resources that enable us to deliver those products." About TRENDnet TRENDnet is an award winning global brand, dedicated to providing a complete line of high quality yet affordable networking solutions for small to mid-sized business and home users alike. Founded in 1990, TRENDnet is headquartered in Torrance, California, and maintains branch offices in Europe, Central America, South America and Asia. TRENDnet products are sold in over 125 countries. The TRENDnet brand is consistently recognized for high quality, exceptional performance and unparalleled support. For more information, visit www.trendnet.com.
About HomePNA HomePNA is an incorporated, non-profit association of industry-leading companies and service providers including AT&T, Cisco, Motorola and Pace working together to create, promote and certify high speed existing-wire multimedia home networking technology. HomePNA operates over coax cables and phone wires to deliver Internet Protocol (IP) services like IPTV, Voice over Internet Protocol (VoIP) and data, often bundled by service providers as triple-play services. Founded in June 1998, HomePNA members represent cutting-edge international companies that span the networking, telecommunications, hardware, software and consumer electronics industries.
Add to Digg Bookmark with del.icio.us Add to Newsvine TRENDnet 20675 Manhattan Place Torrance, CA 90501 Marketing Department (310) 961-5500 Email Contact Sales Department (888) 326-6061 Email Contact Source: TRENDnet
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Engineering Excellence in Power Transmission
Varatio has set standards for engineering excellence in power transmission products since it's formation in 1921. The innovative approach to the solution of power transmission problems by the design arm of Varatio have earned a high position in today's technological and competitive world.
Varatio, a unique organization with a highly skilled and motivated workforce, offers it's services and products to a wide range of industries, from high volume engine component manufacture through to 'state of the art' gearboxes . From CAD/CAM design through to CMM
Varatio produce custom made gear components to the highest quality standards and have the capacity for several million gears per year.
Varatio offers a design service, controlled production processing - S.P.C. - delivered to J.I.T. if required. By combining traditional precision engineering skills with the optimum plant and equipment we offer competitively priced components and an envied quality of service.
We also supply reelstand gearboxes, spares, repairs and servicing.
Our range of Anglgears can be supplied Ex-Stock
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Telephone: 01753 307359 FAX: 01753 693779 Postal address 752-753 Deal Avenue, Trading Estate, Slough, Berkshire, SL1 4SHElectronic mail Sales: firstname.lastname@example.com


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#0831: The Andy Letter
Original Air Date: 08.02.2008
Review this Show | 5 Reviews | Need Help Listening? View Call Details
The puzzler is on summer vacation.
The puzzler is on summer vacation.
Quotes from real-life Dilbert-types in management.
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Show Review - 523by Dimmed4134
Show Review - 627by Anonymous
you missed the clue completelyby Anonymous
Hey, Guys - I was one of those kids that didn't want to drive. You missed the clues - the guy is an Eagle Scout, honor roll student, chemistry major, etc. It is: Fear of Failure. This kid doesn't want to let anyone down, or worse yet, have an accident where someone is hurt. He is so good at everything else that there is this chance that he might not be as good at this as he would ultimately like to be.It took me until I was 30 before I finally took a behind the wheel test and that was out of necessity. I now am fine, but needed to get a car that handled exactly as I expected it would (no big vehicle that was hard to parallel park, etc.)before I felt comfortable.
Awesomeby lunkhead
Best Cartalk Ever
Favorite Moment: Andy Letter
Notes from Real-Life "Dilbert-Type" Managers
Field notes from the wild corporate boardroom.
Tommy's List of "Phrases to Live By"
Years and years of wisdom right here!
The Return of the Fabled "Andy Letter"
In which Andy raises the question of whether two people who know nothing can actually know less than one person who knows nothing.


text:
2002 Chrysler Sebring Recalls
RepairPal Expert Overview on June 3, 2016
When the passenger front air bag is deployed in an accident as needed, it may strike the passenger sun visor. This can cause the visor to be propelled rearward, resulting in increased risk of occupant injry.
RepairPal Expert Overview on June 2, 2015
The passenger side sun visor may detach if it is in the down position in an accident, and the passenger front air bag is deployed. This could increase the risk of injury in an accident.


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- Anonym
- Hong Kong, Kvinde, 25-30
- Nybörjarnomad
- 2 Recensioner
The location is perfect, and there are many shopping places, restaurants and night markers nearby!
- Anonym
- Italien, Kvinde, 25-30
- Globetrotter
- 34 Recensioner
I stayed here on two separate nights, the whole place and facilities were very nice, although in both occasions that I stayed I found it very difficult to sleep due to the room being so hot and the mosquitoes. The first time I met with staff and she was very nice, and offered a way for me to be accommodated on the 2nd night where I would arrive very late, but when I arrived that 2nd night things weren't so smooth since they didn't contact me back with the info. It was solved at the end.
- Anonym
- Japan, Kvinde, 25-30
- Nybörjarnomad
- 2 Recensioner
It's very comfortable and convenient location to access from the closest station. However it was kind of difficult to seek good foods at the area. The most you should concern is there were only few English speaker staffs in the hostel.
- Anonym
- Sydkorea, Kvinde, 25-30
- Nybörjarnomad
- 4 Recensioner
Not bad. But it was a few loud sound.
- Anonym
- Sydkorea, Kvinde, 18-24
- Entusiastisk resenärer
- 18 Recensioner
Great hostel! Backpacker 41 is one of the cleanest hostels! I've never seen any dirty bathroom for 3 nights, and the bedding is also very fresh and cozy. Joanna, the owner, helped anytime when I asked her, she gave me every single information very kindly. And you can get to MRT in a minute from the hostel. Very good location, too! Thank you! I will definitely come back to this hostel if I visit Kaohsiung again :)
Hostelworld mobilappar
Boka på språng med de nya Hostelworld-mobilapparna


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The best of BBC World Service documentaries and other factual programmes.
March 19th, 2015
Episode 976 of 1420 episodes
Lyse Doucet talks to journalist Safa al-Ahmad about her recent reporting trip to Yemen to cover the takeover of the capital Sanaa by Houthi rebels from the north of the country.
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Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease
DOI: 10.1186/ar2012
© Kuroiwa et al.; licensee BioMed Central Ltd. 2006
Received: 17 April 2006
Accepted: 14 July 2006
Published: 19 July 2006
Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogren's syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.
Pathogenic T cells that recognize self-antigens and drive B cell hyperactivity play a central role in the pathogenesis of both human and murine lupus [1–3]. Chronic graft-versus-host disease (GVHD), which is induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by injection of DBA/2 spleen cells, is associated with the activation of donor CD4+ T cells that recognize host major histocompatibility complex (MHC) antigens and drive host B cell hyperactivity [4, 5]. Mice of this parent-into-F1 chronic GVHD model show increased T helper (Th) 2 immune responses, and exhibit autoimmune disorders that resemble human systemic lupus erythematosus, primary biliary chirrhosis, and Sjogren's syndrome, which are characterized by lymphocyte infiltration into organs such as the kidneys, liver and salivary glands [6].
In contrast, the parent-into-F1 acute GVHD model, which is induced in BDF1 mice by the injection of C57BL/6 (B6) spleen cells, is associated with the activation of donor CD8+ cytotoxic T lymphocytes (CTLs) that recognize host MHC antigens and cause death by affecting host immune and hematopoietic systems [7–9]. As acute GVHD can be inhibited by the addition of neutralizing anti-IL-2 monoclonal antibodies (mAbs) and is not induced by perforin-deficient donor T cells [10, 11], acute GVHD is associated with increased Th1-mediated immune responses and with perforin expression on donor T cells.
One of the principal distinctions between the acute and chronic GVHD models appears to be the nine-fold reduction in CTL precursor numbers with anti-host specificity (which eliminate autoreactive host B cells) generated during GVHD induced by DBA/2 mouse spleen cells rather than by B6 mouse spleen cells [12]. Previous experiments have demonstrated that cytokines such as IL-12 and IL-18 induce donor anti-host CTLs in chronic GVHD mice and can ameliorate chronic GVHD, or even stimulate the development of acute GVHD [13, 14].
Recently, we demonstrated that repeated transfection of skeletal muscle with the gene encoding the human hepatocyte growth factor (HGF) induced continuous production of HGF, which strongly inhibited both acute and chronic GVHD in bone marrow transplantation model mice. HGF gene transfection inhibited end-organ damage caused by acute GVHD through HGF's anti-apoptotic and regenerative actions. HGF also exerted a potent protective effect on thymus, which in turn inhibited the development of autoreactive T cells in the thymus damaged by acute GVHD [15, 16]. Considering these results, HGF seems to not only inhibit end-organ damage through its anti-apoptotic and regenerative actions but also directly control autoimmunity in chronic GVHD mice. In the present study, we evaluated the therapeutic and preventive effects of HGF treatment using the parent-into-F1 chronic GVHD mouse model. Our results indicate that HGF gene transfection effectively prevented proteinuria and lymphocyte infiltration of the kidneys, liver, and salivary glands. HGF gene transfection also inhibited an increase in splenic B cell numbers, MHC class II expression by host B cells, and serum IgG and anti-DNA antibody concentrations in chronic GVHD mice. Lastly, HGF transfection inhibited IL-4 mRNA expression in the kidneys, liver, and spleen of chronic GVHD mice. Therefore, HGF may represent a novel strategy for the treatment of systemic lupus erythematosus, primary biliary cirrhosis, and Sjogren's syndrome.
Materials and methods
Female B6 (H-2b), DBA/2 (H-2d), and BDF1 (H-2bxd) mice at 8 to 12 weeks old were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). All mice were maintained in a pathogen-free facility at the Hyogo College of Medicine. Animal experiments were done in accordance with the guidelines of the National Institutes of Health, as specified by the animal care policy of Hyogo College of Medicine.
Induction of GVHD
DBA/2 mouse spleen cells (9 × 107) or B6 mouse spleen cells (5 × 107) were injected into non-irradiated BDF1 mice via the tail vein as previously described [7–9]. Control mice were injected with normal BDF1 mouse spleen cells (9 × 107).
Expression vector and preparation of liposomes containing hemagglutinating virus of Japan
Human HGF cDNA (2.2 kb) was inserted into the EcoRI and NotI sites in the pUC-SRα plasmid under the control of the SRα promoter [17]. Hemagglutinating virus of Japan (HVJ) liposomes containing plasmid DNA and high mobility group 1 protein were constructed as previously described [15]. Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at a ratio of 1:4.8:2 (w/w/w), and 1 mg of this lipid mixture added to 20 to 40 μg of plasmid DNA that had been complexed with 6 to 12 μg high mobility group 1 nonhistone chromosomal protein purified from calf thymus. This mixture was sonicated to form liposomes and then mixed with ultraviolet-irradiated HVJ. Excess free virus was then removed from the HVJ liposomes by sucrose density gradient centrifugation.
Gene transfer
BDF1 mice were injected with either HVJ liposomes containing 8 μg human HGF expression vector (HGF-HVJ liposomes) or mock vector (GVHD control) into the gluteal muscle. Gene transfer was repeated at 2-week intervals from the first day of GVHD induction for 12 weeks.
Tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and examined by light microscopy.
Flow cytometry
Cell suspensions were prepared in PBS containing 1% fetal calf serum and 0.1% (W/V) sodium azide. Cells were incubated with anti-Fc receptor mAb (2.4G2) for 10 minutes at 4°C, and then incubated with FITC-conjugated mAb and phycoerythrin-conjugated mAb for 30 minutes. Stained cells were washed twice, resuspended, and analyzed using a FACScan (Becton Dickinson, Mountain View, California, USA). Anti-Fc receptor (2.4G2) mAb, FITC-conjugated anti-mouse H-2Kb (clone AF6-88.5) mAb, anti-B220 (clone RA3-6B2) mAb, anti-CD80 (B7-1) (clone 1G10) mAb, anti-CD86 (B7-2) (clone GL1) mAb, anti-CD28 (clone 37.51), and phycoerythrin-conjugated anti-mouse H-2Kd (clone SF1-1.1) mAb, and anti-I-Ab mAb (clone AF6-120.1) were all purchased from PharMingen (SanDiego, California, USA). Multicolor flow cytometry was performed as described previously, with some modifications [15, 16]. Channel numbers for data integration were chosen according to the staining patterns of normal spleen cells. Staining of normal F1 spleen cells with anti-MHC antibodies gave a unimodal positive profile when compared to negative controls. Donor cells in GVHD mice were identified as subpopulations that were clearly negative for F1-specific MHC markers.
ELISA for IL-4 and IFN-γ
Murine IL-4 and IFN-γ levels in culture supernatants were measured by ELISA using anti-mouse IL-4 and IFN-γ mAb (Genzyme Pharmaceuticals, Cambridge, Massachusetts, USA) according to the manufacturer's protocols.
Urine protein measurement
Proteinuria was assessed semiquantitatively using urine dip sticks (Albustix; Bayer Diagnostics, Basingstoke, United Kingdom).
Measurement of serum IgG1 and anti-ssDNA antibodies
Sera were collected from individual mice and serum levels of IgG1 and anti-single-stranded (ss)DNA antibodies determined by ELISA. Briefly, purified rat anti-mouse IgG1 mAb (A85-3; PharMingen) and HRP-conjugated rat anti-mouse IgG1 (Zymed, San Francisco, California, USA) were used for plate coating and secondary Abs, respectively, with mouse IgG1 (S1-68.1; PharMingen) used as the protein standard. Serum levels of anti-ssDNA IgG were determined by ELISA as described previously [18]. Briefly, microtiter plates were coated with heat-denatured calf thymus DNA (Sigma, St Louis, Missouri, USA), blocked with 2% BSA-PBS, and incubated with two-fold serial dilutions of experimental mouse sera beginning at a dilution of 1/50. Plates were then incubated with HRP-labeled anti-mouse IgG (Zymed) and OD measured at 405 nm.
Mixed lymphocyte reaction and in vitrocytokine production
CD4+ T cells and CD11c+ dendritic cells (DCs) were purified from spleen cells by positive selection using immunomagnetic beads (Miltenyi Biotec, Auburn, California, USA). Purity of the CD4+ and CD11c+ populations was >90% and >95%, respectively. CD4+ T cells from DBA/2 (H-2d) mice (4 × 106/ml/well) were cultured with irradiated (20 Gy) CD11c+ DCs from BDF1 (H-2b×d) mice (1 × 106/ml/well) in 24-well flat-bottomed plates (Falcon Labware, Lincoln Park, New Jersey, USA). After 72 h, viable cells (1 × 105/200 μl/well) were stimulated in 96-well flat-bottomed plates (Falcon Labware) coated with 5 μg/ml anti-CD3 mAb (PharMingen). After 48 h, IL-4 and IFN-γ concentrations in the culture supernatants were measured by ELISA.
51Cr release assay
Anti-host CTL activity was tested using spleen cells from chronic GVHD mice at 2 weeks after GVHD induction. Spleen cells (5 × 106/ml/well) were restimulated with irradiated (20 Gy) BDF1 spleen cells (3 × 106/ml/well) for 5 days. Effector cells were harvested and CTL activity assessed based on the lysis of EL-4 (H-2b) cells in a 4 h 51Cr release assay. Anti-host CTL activity was also tested using spleen cells from acute GVHD mice based on P815 cell (H-2d) lysis. Effector cells were tested in triplicate at four effector:target ratios and the percent lysis calculated according to the following formula: [(sample cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm)] × 100%. Results were calculated as the mean percent lysis ± standard deviation at a given effector:target ratio for each treatment group.
Reverse transcribed PCR
RNA was extracted using an ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions, and cDNA prepared using 2.5 μM random hexamers (Applied Biosystems Inc., Foster City, California, USA). IFN-γ and IL-4 mRNA levels were quantified by real-time reverse transcribed (RT)-PCR in a total volume of 25 μL for 40 to 50 cycles of 15 s at 95°C and 1 minute at 60°C. Samples were run in triplicate, and relative expression levels determined by normalizing expression according to β-actin expression. Primer sequences used were: IFN-γ, TGGCTGTTTCTGGCTGTTACTG and AATCAGCAGCGACTCCTTTTCC; IL-4, CCAGCTAGTTGTCATCCTGCTCTTCTTTCTCG and CAGTGATGAGGACTTGGACTCATTCATGGTGC; β-actin, TGTGATGGTGGGAATGGGTCAG and TTTGATGTCACGCACGATTTCC.
Statistical analysis
Group mean values were compared using the two-tailed Student's t-test. A p value of less than 0.05 was considered statistically significant.
HGF reduces histopathological changes caused by chronic GVHD
HGF reduces splenic B cell numbers and serum IgG1 and anti-DNA antibody concentrations in chronic GVHD mice
HGF reduces IL-4 mRNA expression in target organs of chronic GVHD
HGF reduces MHC class II expression on host B cells
In vitrotreatment with HGF reduces Th2 generation from DBA/2 CD4+ T cells
In vitrotreatment with HGF down-regulates CD28 expression on CD4+ T cells
The present study demonstrates that repeated HGF gene transfection into BDF1 mice prevents the development of chronic GVHD induced by the injection of DBA/2 spleen cells. HGF transfection significantly inhibited proteinuria and histopathological changes of the kidneys, liver and salivary glands caused by chronic GVHD.
The parent-to-F1 murine model of chronic GVHD exhibits Th2-mediated immune responses, such as polyclonal B cell activation, autoantibody formation, and decreased CTL responses, that closely resemble lupus-like autoimmune disease. Rus and colleagues [5] reported that Th2 cytokine secretion and B cell activation may be early events in both acute and chronic GVHD. It is thought that the transition to chronic GVHD involves the failure of CD8+ anti-host CTLs to kill activated host B cells [5]. In this context, therapeutic strategies tested in chronic GVHD mice have included the use of Th1-inducing cytokines [13, 14]. Early treatment with IL-12 (on days 0 to 4 after induction of chronic GVHD) but not late treatment (on days 8 to 12 after induction of chronic GVHD) generated anti-host CTLs that eliminated host autoantibody-producing B cells, thereby converting chronic GVHD into acute GVHD [13]. However, regardless of treatment schedule (treatment on days 0 to 5 or days 8 to 13 after induction of chronic GVHD), IL-18 treatment generated anti-host CTLs, but did not induce acute GVHD [14].
In contrast to Th1-inducing cytokine treatment, HGF did not generate anti-host CTLs in vivo. Although Th1-inducing cytokines induced the development of DBA/2-derived Th1 in a DBA/2 anti-BDF1 MLR [25], the addition of HGF did not induce Th1 in vitro (data not shown). However, HGF treatment inhibited IL-4 mRNA expression in chronic GVHD target organs, splenic B cell expansion, and autoantibody production in chronic GVHD mice. Thus, it appears that HGF inhibited lupus-like autoimmune disease through the inhibition of Th2 generation rather than by induction of Th1.
The precise mechanisms by which HGF inhibits Th2-mediated responses in chronic GVHD mice remain unclear. Possible mechanisms include that HGF suppresses MHC class II expression by host B cells, leading to reduced antigen presentation to donor CD4+ T cells. Indeed, we observed decreased MHC class II expression on host B cells from HGF-treated chronic GVHD mice. While the addition of HGF to an in vitro DBA/2 anti-BDF1 MLR inhibited the capacity of BDF1 B cells to increase MHC class II expression, HGF did not inhibit IL-4-induced B cell MHC class II expression, which suggested that HGF did not directly suppress MHC class II expression on host B cells.
DCs play a pivotal role in determining the balance between responsiveness and tolerance in the immune system [26, 27], and the persistence of host DCs following bone marrow transplantation is correlated with the development of severe acute and chronic GVHD [28, 29]. Chronic DC activation, such as by CD40L over-expression in basal epidermal layers that accelerates DC maturation, leads to autoimmunity [30]. Immature myeloid DCs are loaded with self-antigen-derived molecules and, upon interaction with T cells, deliver a signal that results in T cell deletion and anergy [31]. Modified myeloid DCs can act as regulatory DCs to protect against acute GVHD [32]. We observed that in a DBA/2 anti-BDF1 MLR, which contains DBA/2 CD4+ T cells and BDF1 DCs, the addition of HGF significantly inhibited the generation of DBA/2 Th2. We also observed that c-Met/HGF receptors are expressed by both DCs and CD4+ T cells (data not shown) and that HGF significantly down-regulated CD28 expression on DBA/2 CD4+ T cells stimulated by BDF1 DCs. We also observed down-regulation of CD86 expression on BDF1 DCs cultured with DBA/2 CD4+ T cells. HGF may act on the CD28-CD86 pathway, thereby inhibiting Th2 generation.
We recently demonstrated that repeated transfection of the human HGF gene into skeletal muscle in a bone marrow transplantation model of GVHD promoted hematopoietic function and strongly inhibited acute GVHD by limiting tissue damage and the subsequent endotoxin-mediated inflammatory cascade [15]. The principal mechanisms by which HGF blocks acute GVHD appeared to involve the protection of target organs from injury through anti-apoptotic effects and the inhibition of subsequent inflammatory cytokine reactions [16]. In the present study, we demonstrated that HGF inhibited the increased Th2-mediated immune response in chronic GVHD mice. Thus, HGF treatment may be beneficial for both Th1-mediated acute GVHD and Th2-mediated autoimmune chronic GVHD.
HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis, primary biliary cirrhosis and Sjogren's syndrome. HGF gene transfection greatly reduced the number of splenic B cells, host B cell MHC class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells and CD86 expression on BDF1 DCs was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 DCs was significantly inhibited by the addition of recombinant HGF in vitro. These results suggested that HGF gene transfection inhibited Th2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of systemic lupus erythematosus, Sjogren's syndrome and primary biliary cirrhosis.
cytotoxic T lymphocyte
dendritic cell
enzyme-linked immunosorbent assay
effector target
fluorescein isothiocyanate
graft-versus-host disease
hepatocyte growth factor
hemagglutinating virus of Japan
- mAb:
monoclonal antibodies
major histocompatibility complex
mixed lymphocyte reaction
phosphate-buffered saline
reverse transcribed PCR
standard deviation
- Th:
T helper.
We thank Hirotsugu Kubo and Masahito Yagi for their assistance in preparing this manuscript. TI acknowledges the support of Grants for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 14657120 and No. 15591071).
Authors' Affiliations
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