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Laughter is Contagious
Can you watch this without cracking a smile?
Can you watch this without cracking a smile?
It's no surprise that Santa Claus was able to pick up a few languages after circling the globe many a times. Catch him at Time Warner Center where he'll delight kids and wish them a very merry Christmas in six languages.
Get easy and fun craft ideas for kids and adults alike, courtesy of EK Success Brands' Second Annual "Crafting with Style" event. Check out these chic new craft kits and projects from American Girl, Perler Beads, K&Company, and Spotted Canary.
From holiday traditions new and old to gifts both classic and of the moment, we've got a seasonal smorgasbord of holiday tales and toy recommendations from NYMetroParents staff members and friends.
A Rockland County dad of two and NYMetroParents staffer talks about his family's favorite holiday tradition and how it's evolved over the years.
A Rockland County mom of two reflects on her childhood memories of Hanukkah and how she plans to keep them alive for her daughters as they grow up.
An NYC mom of two on the NYMetroParents team explains how her mixed-faith family avoids holiday conflict by embracing the spirit of both Christmas and Hanukkah.
A Brooklyn dad of two tells how his dual-faith household celebrates both Christmas and Hanukkah.
A Fairfield County, CT mom explains how her mixed-faith family embraces the holiday season and gives four great gift recommendations for Christmas or Hanukkah that have been hits with her kids over the years.
One local mom, a self-proclaimed "girly girl," struggles to fathom her son's boyish Christmas gift requests, but eventually learns to embrace his Star Wars-loving, Call of Duty-coveting attitude.
Millions of children eagerly await jolly old St. Nick's arrival on Christmas Eve. And while you could try placating their anticipation with a few early stocking stuffers, why not let them track Santa on his route - via GPS, no less.
Linda Grant, a single mom living in New York City, reminisces about family trips to the Christmas tree farm during her suburban childhood and explains how she carries on that tradition with her daughter, even though they live in the city.
Must you spend lots of cash to keep your teens happy this holiday? One Long Island mom weighs in.
NYMetroParents' deputy managing editor, Christine Tarulli, shares her family's favorite tales of those disastrous Christmas Eves spent attempting to assemble last-minute gifts.
Mom blogger Michaela Searfoorce, an NYC parent of two toddlers and a preteen son with special needs, reflects on her blessings in honor of the upcoming holiday season.
Brooklyn Mom Karen Connell, founder of the blog A Child Grows in Brooklyn, lists her top three holiday stories for kids along with two new favorites that she'll be adding to her family's bookshelf this year.
Remember that Easy-Bake Oven or Mr. Potato Head you had when you were a kid? Classic toys like those never go out of style. So whether you're shopping for kids or a partner who appreciates the occasional silly gift, these throwback toys recommended by a Queens Mama are sure to be a hit.
A Queens Mama shares her tips on how to buy locally this holiday season, including five great gifts made right here in the New York City area.
We received more than 80 entries for our Halloween photo contest this year, and in the race for the most votes, a few were neck-and-neck until the very end, but the first place submission took it home with more than 640 votes.
From our December issue, a selection of thought-provoking, laugh-inducing, and just plain interesting thoughts from the web and the world of parenting.
From our November print issues: Check out our roundup of thought-provoking, laugh-inducing, and, most of all, relatable quotes from a selection of our favorite mom and dad bloggers and groups, including Mamiverse and Adventures in Mommyland.
From our October print issues: Check out our roundup of thought-provoking, laugh-inducing, and, most of all, relatable quotes from a selection of our favorite mom and dad bloggers, including Lady and the Blog, Love that Max, Nona Brooklyn, and NYC Dads Group.
Introducing Karen Demeter, Fairfield,Suffolk, and Special Parent Calendar Editor
Introducing Kaitlin Ahern, Deputy Editor and Food & Nutrition Editor.
Introducing Ellen Pocost, Westchester Calendar Editor and Directory Editor
Get NYMetroParents in Your Inbox


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subset: commoncrawl


text:
In a message dated 3/31/1999 6:05:19 PM Central Standard Time, firstname.lastname@example.com
<< Hi All!
I have been following the discussion re squirrels and am somewhat surprised
that they play havoc with Hostas. We have a lot of them and I have not
detected any damage caused by them. Could it perhaps be that rabbits are the
At any rate, one way to pacify squirrels is to set out some dried corn. This
is an inexpensive way to keep them out of your Hosta beds. Also, no matter
what you do, they will return.
Squirrels have an inbuilt urge to dig in soft fresh dirt, so were ever there
is loss black dirt they will big. So if you keep your Hosta bed with nice
soft soils the squirrels will have fun. For me the fall is the worst since I
do most of my planting in late summer and fall they love to dig the plants up
to see what's under them.
I think these dishonest creatures are trying to raid some others squirrels
stash of nuts!
To sign-off this list, send email to firstname.lastname@example.com with the
message text UNSUBSCRIBE HOSTA-OPEN


text:
KUALA LUMPUR: National men's singles shuttler Liew Daren's fear of his future in the national team is all gone after two meetings in two days with the new management.
On Monday, Daren and all the national players met up with Badminton Association of Malaysia's (BAM) Talent Management Group (TMG) director Tan Aik Mong at Stadium Juara in Bukit Kiara.
On Saturday, they had a closed door meeting with BAM's president Tengku Tan Sri Mahaleel Tengku Ariff.
The 26-year-old Daren said they were now quite clear with their roles and responsibilities in the national team and hoped to be given some time to get used to the changes.
"We raised a number of grouses in the meeting with the president. We are happy as we were given assurances that they would be looked into. It was also good that the president took his time to meet up with us," said Daren.
Replays: Malaysia Open 2016


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Act 3: Defend the zepelin
Hi, i can't seem to shoot down the planes in act 3 while defending the zepelin. I'm playing the level on elite.
Has anyone have a strategy to defend the zepelin before it explodes?
Thanks in advance
Yeah, I had some issues with this myself. Restart the level, and make use a lot of slow-down time, instead of pause time. This should help you out immensely.
I didnt realy have a problem with this, i didnt even die on it. I just use LT to zoom in and used time slow down. I mostly used time power for shooting down plans, mines arnt tha hard.
I was getting extremely frustrated with this mission until I figured out you could use timeslow while firing the turret, made my life so much easier and finished it on my first try using it.
Instead of slowing time why not pause it? Thats what i did then the targets least stay in the same place :)
Because Pause faster is over.
For example when you use time slow it takes 4 a 5 secs to finish, and with pause it takes 2 a 3.
Thats a lot.
How on Earth do I get through the scaffolding?
Without the use of the 'slow time' on elite, its tough since so many fly in & take out the zep. Just make sure that you use the slow time every now & then till 2-3 are still flying around, then its not that bad. The mine field is a walk in the park, just dont let too many hit you. Other than that, your good to go (y)
You can use your time powers on this mission? I had no idea. I'm playing on skilled and I think I got to the very end. It's after you pass the base on the ground. But i'll try the time powers.
slightly off topic, but I completed that level without dying, but the death-proof achieve did not unlock, do I have to finish the act to get it or should it come after the end of the level....or is this a dreaded glitch?
Mine came up after a cut scene and then the loading screen for the next act. I didn't think it'd unlock either but it did right before I continued on to the next act.
I'm stuck on this level... I try my time slowing powers but I still get wasted. It's starting to wear on my patience but I refuse to switch the difficulty, haha.
Having issues on Skilled setting. Ironically, I actually get further without using time powers. No matter whether I use slow or pause, I still get beaten badly. Fuck knows what this will be like on Elite. Not lookingforwaforward to that play through.
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We've all seen misspelled article titles (especially on Digg.com) Doesn't Firefox have built-in spell checking?
As a command line junkie, I end up doing almost everything from a command prompt. The problem is that Windows Vista doesn't have a built-in command to launch an application in administrator mode.
If you use UltraEdit as your text editor and edit a lot of Linux/Unix files, you've no doubt encountered the "File is probably not DOS format" message every single time you open a file. So irritating…
If you've got a directory with dozens of zipped or rar'd files, you can run a single command to unzip them all in one step, thanks to the power of the bash shell.
You've got a production database server, and you can't enable query logging… so how do you see the queries being executed against the database?
So you've got a bunch of .OGM video files and they won't play in Windows Vista… relax, you just need to install the right codecs in order to play them from within Windows Media Player.
If you've ever wondered how best to kill a few hours, I have the answer. (Yes, I realize this is completely off-topic)
Google has just released a new experimental search that lets you navigate through search results using the same shortcut keys as Gmail and Google Reader. The problem is that Firefox's default search box doesn't default to it.
Microsoft's SQL Server has a tool called Profiler that you can use to monitor every SQL query that hits the database. This is extremely useful for programmers as well as database administrators to troubleshoot the exact queries generated by an application.
Every Geek uses Gmail… it's pretty much required. And now you can set Gmail as the default client in Ubuntu without any extra software. (Windows requires the Gmail notifier be installed)
Automatic Updates is a great feature. Your computer stays protected from threats without worrying about it… but if it's 3am and I'm trying to play a video game, the last thing I want is for the automatic updates to pop up and remind me every 5 minutes that I need to reboot, interrupting my game… Drives me crazy!
If you've tried to use the built-in "Extract Here" functionality in Ubuntu's File Roller to extract either a single or a multi-part zip or rar file and ended up with a "Password required" error, then you might just assume the files are password protected when in fact they are not.
If you encounter this error message as often as I do, you will thank your various gods for this extension, which allows you to hide the warning message on a per site basis, for both Firefox as well as Thunderbird.
Did you know that you can run more than one Firefox profile at once? This can be extremely useful if you want to test out extensions more easily, create a web development profile, run a slimmed down profile just for Gmail, or if you just want to have a clean profile.
The System Restore feature in Windows Vista is very important to keep enabled, what with all the problems Vista users are having with compatibility. Even so, the disk usage can get out of hand if you install a large amount of software.
Ubuntu has an option for adding a Trash Can icon to the desktop, which might be a comfort for those of you migrating from Windows.
Many utilities need to be run as administrator in order to function properly, especially older utilities that haven't been updated to support Windows 7 or Vista yet.
I prefer a clean desktop with no icons cluttering it up, but by default Ubuntu adds icons to the desktop for every single removable drive that you attach to your system.
If you've been wondering why your XP desktop icons have a background color, you'll be glad to know you can flip a checkbox and have them go back to a normal transparent background.
Ubuntu includes a very limited shortcut key configuration utility which doesn't allow you to assign hotkeys to your own applications or scripts. To get around this limitation, we can use the built-in gconf-editor utility to assign them ourselves.
Restarting Firefox is always difficult for those of us with dozens of tabs open, because you don't want to lose all the open tabs, but you might not want to bookmark them all either.
It drives me crazy when applications install themselves into the system tray without giving me a choice during setup. QuickTime has no good reason to be in the system tray, but it's there anyway after you install iTunes.
I've previously written about mounting an ISO image in Windows Vista using Virtual CloneDrive, but I thought I'd go a step further and explain how you can mount more than one ISO at a time.
If Outlook 2007's new Instant Search just isn't… instant… there's a patch from Microsoft that might fix this problem for you.
The How-To Geek doesn't allow just anybody to leave any comment they feel like. Because this site is dedicated towards helping people, only helpful, constructive, on-topic or interesting comments will be approved.


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THE PROSPECT of a lengthy speech by Robert Maclennan, outgoing president of the Liberal Democrats, has made many people's blood run cold in the past. Once, Labour MP Tony Banks even told the Commons that any "old fartbags" who talked too long should be forced to learn the complete works of Mr Maclennan. A real stickler when it comes to his texts, Tuesday saw Maclennan's final presidential speech. At one point on the page were printed the instructions "meaningful pause", followed by the puzzling exclamation, "Mm!". Pandora is pleased to report that Maclennan delivered his "Mm!" with such gusto that some in the audience actually woke up.
WHAT'S IT like to be Leonardo DiCaprio? American socialite author George Plimpton found out recently when he spent a night at Hollywood's Chateau Marmont in the same suite where Leonardo usually stays. He was awakened in the middle of the night by eight "enchanting nymphets" who brushed past him and into the bedroom. "They were looking in the closets. One of the girls put her head on my pillow, because that's where Leonardo had rested his head." Plimpton told the NY Daily News: "I would have said it was a nightmare - except they were so terribly attractive." Once certain that Leonardo wasn't coming, all the nymphets departed. As Plimpton now knows, celebrity is hell.
FOR MIRANDA Green, Paddy Ashdown's delightful right-hand woman, this year's Brighton Conference has been a bit of a rags-to-riches story. Two years ago, during the Lib Dem's last Brighton visit, she told Pandora: "I was a humble researcher. I stayed in an exceedingly cheap bed and breakfast run by a Mr and Mrs Doss. They did a fab breakfast, but the contrast couldn't be more obvious."
This year, Miranda has her own suite at the Metropole Hotel, but laments that her media-spinning responsibilities haven't allowed her to enjoy it much. Her title is now "Head of press and communications - Leader's Office", but she says, "You can only use it if you also say how ridiculous it is." Pandora must caution Miranda that politics and modesty make strange bedfellows. Who ever heard Peter Mandelson say that his title was silly?
AT TUESDAY'S launch of The London Fashion Book, author Andrew Tucker was talking about the flap in the international fashion world caused by some New York designers, including Calvin Klein and Donna Karan, who held their shows in advance of London Fashion Week. "They are messing people about," Tucker insisted, displaying his loyalty to the London scene. How this will affect his next project - The New York Fashion Book - remains to be seen.
DID YOU know that Bart Simpson is a Scientologist? Or, at least, Nancy Cartwright, the actress who is the voice of Bart, is a member. Along with a number of other Hollywood figures, including actress Juliette Lewis of Natural Born Killers fame, she has signed a letter that has been sent to 300,000 people along with a booklet entitled Harming Artists: Psychiatry Marginalising Creativity. This attacks accepted psychiatric practice, and blames it for the deaths of entertainment stars like Judy Garland, Vivien Leigh, Janis Joplin, Marilyn Monroe, Kurt Cobain, Don Simpson and Michael Hutchence. In response to these revelations, Pandora can only echo Bart's immortal words: "Eat my shorts!"Reuse content


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Defense Department: The Bible, Constitution And Declaration Of Independence All Perpetuate Sexism
The Defense Department's Defense Equal Opportunity Management Institute, which manages human relations for the DOD, has been teaching service members that the Bible, the Constitution and the Declaration of Independence all perpetuate sexism in our current culture.
Lerner pushed Treasury watchdog to back off 'targeting' charge in probe
The Washington Examiner's Mark Tapscott explains how IRS documents Judicial Watch recently released show that Lois Lerner pressured investigators to back off their conclusions that the IRS had targeted conservative political groups.
Lerner Email Warns IRS Employees of Emails That 'Can Be Seen By Congress'
CNS News covers the latest documents which show that Lois Lerner and the IRS were intent on hiding their illegal targeting of private citizens from Congress.
Interactive graphic: Hillary Clinton's insider network
Hillary Clinton's decision to violate the Federal Records Act and hide her communications from the American people is now well-known. Just as important, if less well-known, however, is her network of insiders that have aided her every step of the way.
New IRS emails show protagonist's concerns
New documents uncovered by Judicial Watch show that in addition to targeting conservative tax-exempt organizations, the IRS was also conspiring to keep information from Congress and keep the American people in the dark.
Christian Legal Group Sues IRS for Failing to Release Docs in Settlement to Investigate Churches
Judicial Watch has filed suit on behalf of the Alliance Defending Freedom to ensure that churches and other religious institutions are not unfairly targeted by the IRS.
Now illegal immigration flights paid by taxpayers
World Net Daily reports on story first brought to light by Judicial Watch regarding the Obama administration's use of taxpayer funds to bring the children of illegal immigrants to the United States.
Judicial Watch Emails: Lerner Feared IRS Targeting Might 'Raise Questions'
Judicial Watch released newly acquired emails Thursday from the IRS' Lois Lerner, who was responsible for targeting conservative groups applying for tax-exempt status. Amazingly, Lerner has never been charged with any wrongdoing.
403 have applied to have Central American relatives brought to U.S. through Obama admin program, no limit on approvals
Judicial Watch recently broke the story that the Obama administration was offering immigrants in the United States, even those here illegally, assistance to bring their children to the United States, at taxpayer expense. Breitbart News provides new information about how many times that program has already been used.
Exclusive: Iraqi caught crossing US-Mexico border was military trainer and spoke fluent Russian
Breitbart News provides information on an Iraqi military trainer, fluent in Russian, who was caught trying to enter the United States illegally.
National Watchdog Group Jumps Into D.C. Budget Autonomy Case
Roll Call covers Judicial Watch's efforts to ensure the D.C. city government doesn't spend taxpayers' money in violation of federal law. Despite court rulings that D.C. cannot do so, the current mayor thinks she is above the law, and Judicial Watch is fighting for accountability.
Suit questions court review of Maryland gerrymandering
Judicial Watch recently filed an amicus brief on behalf of Maryland residents whose voting rights are at risk. Talk Radio News has more.
Federal judge slams Obama lawyers in immigration case
The Obama administration has tried desperately to make the president's illegal amnesty settled law. The federal judge hearing the challenge to the president's actions recently called the administration's lawyers to task for being dishonest with him as they attempt to subvert the Constitution and rule of law.
Court asked to bar D.C. government from illegally spending federal funds
Judicial Watch recently filed suit on behalf of the taxpayers of the District of Columbia to prevent the DC government from illegally spending taxpayer money through a budget measure declared unconstitutional by a federal court. Despite the ruling, the DC government still thinks it is above the law.
Judicial Watch sponsors panel on Hillary Clinton State Department emails
Judicial Watch recently hosted an expert panel focused on Hillary Clinton's attempts to subvert federal records laws and the Freedom of Information Act. C-SPAN has comprehensive coverage.
Gov't program would fly in Central American children to join parents in US
The State Department has announced that it will begin flying the children of Guatemalan, Honduran and Salvadoran immigrants residing in America to the United States. At taxpayer expense. Fox has the story.
State Dept. program will fly in Central American immigrant children to U.S.
The Washington Times reports on the State Department's program to bring the children of Central American immigrants in the United States to the country and provide taxpayer-funded benefits. Any permanent resident, parolee or illegal immigrant granted legal status by Obama's executive amnesty is eligible to apply for this program.
What is the US Army Not Telling Us About Bowe Bergdahl's Alleged Desertion?
Judicial Watch recently filed suit to obtain documents regarding the Bowe Bergdahl swap. Kevin Mooney asks what persuaded Sec. Hagel and the Obama administration that the terrorists we gave up for Bergdahl were no longer a threat.
State Department cites 'crushing' burden from Freedom of Information Act
The State Department has complained that it is facing increased numbers of FOIA requests and lawsuits. In the same piece, Judicial Watch is recognized for having been responsible for bringing to light key documents regarding ethics investigations into the Clintons.
Virginia State Bar now hero of anti-Israel movement
In March, the Virginia State Bar, the state's licensing agency, abruptly cancelled a planned mid-year seminar in Israel when several members of the organization complained to the VSB Council. Judicial Watch recently filed a FOIA request to find out what was behind the sudden cancellation.


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It has to understand something. Lotion costs money.
Yes, it rubs the lotion on its skin whenever it's told. But it doesn't take huge handfuls of the stuff and smear them all over itself until most of it gets on its clothes.
It dabs. Doesn't it understand how to dab? It takes a tiny bit and rubs it in thoroughly. Then another. That's how normal people put lotion on. Even I understand that, and I'm a serial killer with a girl in my well.
Listen. It's not using generic Rite-Aid lotion for 99 cents a pop. It's using goddamn Nivea, which goes for $5.69.
If it uses a bottle a day it's going to cost more than $300 by the time I get it fat enough to make a housecoat. And if I had $300 for a housecoat, I would have bought one by now and it wouldn't be in my friggin' well. Isn't that right, Precious? Yes. Precious gets it.
Does it think I'm made of lotion?
It's totally going to get the hose again.
environmental news stories sunday - *this is not fake news*. these are the little stories that fall below the usual media radar. *aggressive cuts to obama-era green rules to start soon:* epa...


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Inhibition of macropinocytosis blocks antigen presentation of type II collagen in vitro and in vivoin HLA-DR1 transgenic mice
DOI: 10.1186/ar1964
© von Delwig et al.; licensee BioMed Central Ltd. 2006
Received: 16 February 2006
Accepted: 24 April 2006
Published: 16 May 2006
Professional antigen-presenting cells, such as dendritic cells, macrophages and B cells have been implicated in the pathogenesis of rheumatoid arthritis, constituting a possible target for antigen-specific immunotherapy. We addressed the possibility of blocking antigen presentation of the type II collagen (CII)-derived immunodominant arthritogenic epitope CII259–273 to specific CD4 T cells by inhibition of antigen uptake in HLA-DR1-transgenic mice in vitro and in vivo. Electron microscopy, confocal microscopy, subcellular fractionation and antigen presentation assays were used to establish the mechanisms of uptake, intracellular localization and antigen presentation of CII by dendritic cells and macrophages. We show that CII accumulated in membrane fractions of intermediate density corresponding to late endosomes. Treatment of dendritic cells and macrophages with cytochalasin D or amiloride prevented the intracellular appearance of CII and blocked antigen presentation of CII259–273 to HLA-DR1-restricted T cell hybridomas. The data suggest that CII was taken up by dendritic cells and macrophages predominantly via macropinocytosis. Administration of amiloride in vivo prevented activation of CII-specific polyclonal T cells in the draining popliteal lymph nodes. This study suggests that selective targeting of CII internalization in professional antigen-presenting cells prevents activation of autoimmune T cells, constituting a novel therapeutic strategy for the immunotherapy of rheumatoid arthritis.
Professional antigen-presenting cells (APCs), such as dendritic cells (DCs), macrophages and B lymphocytes, play a pivotal role in the pathogenesis of autoimmune diseases in animal models by presenting arthritogenic T cell epitopes to autoimmune T cells [1–3] Adoptive transfer of ex vivo cultured autoantigen-specific DCs has been shown to induce a variety of experimental autoimmune diseases, such as autoimmune diabetes, experimental autoimmune encephalomyelitis and erosive inflammatory arthritis [4–6]. DCs in situ are often surrounded by a cluster of T cells [7] and are thought to internalize autoantigens from the extracellular matrix and cartilage for intracellular processing and presentation of arthritogenic epitopes to specific CD4 T cells, as well as to induce activation of B lymphocytes in patients with rheumatoid arthritis (RA) [8]. B lymphocytes have also been shown to be critical both as antigen-presenting and antibody-secreting cells in the pathogenesis of autoimmune arthritis [3, 9–11] High efficiency of antigen presentation of arthritogenic epitopes by macrophages has also been demonstrated [12, 13].
Type II collagen (CII, α1(II)3), the most abundant fibrillar protein of articular cartilage [14], is considered an important autoantigen involved in the pathogenesis of collagen-induced arthritis in mice and RA in humans [15, 16] In addition, collagen has been shown to deliver a direct maturation stimulus to DCs [17], possibly via ligation of Toll-like receptor 4 (TLR4) or by binding to cell surface integrins [18, 19], suggesting that DCs can present collagen T cell epitopes without additional inflammatory or danger signals. A direct co-stimulatory activity of collagen has, however, not been demonstrated in vivo as there is no evidence that collagen by itself displays adjuvant activity [20]. No information is available on the mechanisms engaged in CII uptake into professional APCs for presentation of arthritogenic epitopes to CD4 T cells.
Several mechanisms have been described to mediate internalization of antigens into APCs, including phagocytosis, macropinocytosis, receptor-mediated endocytosis and caveolar endocytosis. Phagocytosis follows the recognition of particles ≥0.25 μm by specific receptors and an F-actin microfilament-dependent internalization into phagosomes [21]. Macropinocytosis does not require ligation of specific receptors and is accompanied by membrane ruffling and F-actin-dependent uptake into large macropinosomes of 0.15 to 5.0 μm [22]. Receptor-mediated endocytosis of smaller particles and molecules engages clathrin-coated pits and F-actin recruitment at endocytic sites [23], while clathrin-independent endocytosis is dependent on intact caveolae and lipid rafts [24]. In contrast to other internalization mechanisms, caveolar endocytosis does not deliver antigens to lysosomes and, therefore, does not appear to play a major role in antigen processing and presentation [2, 25].
In this report, we show that CII was taken up preferentially via macropinocytosis into DCs and macrophages from HLA-DR1 transgenic mice for antigen presentation of both the glycosylated and non-glycosylated forms of the arthritogenic CII259–273 epitope to CD4 T cells. Treatment of mice with an inhibitor of macropinocytosis also prevented activation of CII259–273-specific T cells in vivo.
Materials and methods
Human CII purified from normal human cartilage was purchased from MD BioSciences (Zürich, Switzerland). The glycosylated peptide (GIAGF KGEQGPKGET; K = GalHyL264) corresponding to epitope CII259–273 GalHyL264 was synthesized using β-D-galactopyranosyl-5-hydroxy-L-lysine, as described previously [26]. The non-glycosylated peptide pCII259–273 was purchased from GenScript Corp. (Piscataway, NJ, USA), and purity was confirmed by high-performance liquid chromatography.
In all experiments described in this study we used previously reported mice transgenic for HLA-DR1 on a major histocompatibility complex (MHC) class II-deficient background (designated C57BL/6J0-0 HLA-DR1), which carried full-length genomic constructs for HLA-DRA1*0101 and HLA-DRB1*0101, developed by one of us (DMA) [27]. Experiments described in this report have been performed under the terms of Animals (Scientific Procedures) Act 1986, and authorized by the Home Secretary, Home Office UK. The work has been approved by the Ethical Review Committee of the University of Newcastle upon Tyne.
Culture media ingredients and inhibitors were purchased from Sigma Chemical Co. (Dorset, UK), unless stated otherwise. Cells were grown in culture medium (RPMI 1640 medium containing 3 mM L-glutamine, 50 μM 2-mercaptoethanol, 10% FBS and 30 μg/ml gentamycin). T cell hybridomas HCII-9.1 (specific for the non-glycosylated peptide) and HCII-9.2 (specific for the glycosylated peptide) have been described previously [27]. Macrophages were grown from femoral bone marrow cells in culture medium supplemented with 5% horse serum, 1 mM sodium pyruvate, 10 mM HEPES and 7.5% of a supernatant from the L929 cell line as a source of macrophage colony stimulating factor (M-CSF), as described [27]. Macrophages were activated with 10 U/ml recombinant IFN-γ (R&D Systems, Abingdon, UK) for 24 hours (purity approximately 95% based on CD11b expression).
Dendritic cells were grown from bone marrow progenitor cells in the culture medium supplemented with 20 ng/ml recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF; BioSource International, Nivelles, Belgium) for 5 days with culture medium changes on days 2 and 3. On day 5, DCs were purified using CD11c-labeled magnetic MicroBeads (Miltenyi Biotec, Bisley, Surrey, UK), according to the manufacturer's instructions (purity approximately 92% based on CD11c expression). Maturation was induced by treatment of DCs with 0.2 μg/ml lipopolysaccharide (LPS; purified by phenol extraction from Salmonella enterica, serovar typhimurium, Sigma Chemical Co.) for 24 hours.
Antigen presentation assays
Adherent macrophages at 105/well in 48 flat-well plates (Corning Limited, Artington, Surrey, UK), or mature and immature DCs at 104/well in flat-bottomed 96 well plates (Greiner Bio-One Ltd, Stonehouse, Gloucestershire, UK) were pulsed with a dilution series of CII or relevant synthetic peptides (range 40.0 to 0.02 μg/ml) for 5 hours in the absence or presence of inhibitors of uptake (10.0 μM cytochalasin D, 5.0 μM monodansylcadaverine, 1.0 mM amiloride, 0.2 mM 5-(N,N-dimethyl) amiloride (DMA) or 0.4 μg/ml filipin) for 5 hours at 37°C [28, 29]. Time and the optimal doses of APCs, antigens and inhibitors were established in separate dose-response experiments. Cells were fixed with 1.0% paraformaldehyde for 5 minutes, washed thoroughly to remove the fixative and T cell hybridoma HCII-9.1 (specific for the non-glycosylated epitope) and HCII-9.2 (specific for the glycosylated epitope) were added (5 × 104/well) and incubated for 24 hours at 37°C. Usage of synthetic peptides in all experiments controlled for the non-specific toxic effect of metabolic inhibitors and the responsiveness of T cell hybridomas. The interleukin-2 content of hybridoma supernatants was measured by bioassay as the proliferative response of the cytotoxic T cell line-2 (3 × 104/well; CTLL-2, ATCC, TIB 214, American Type Culture Collection, Rockville, MD, USA). Proliferation assays were performed by incubating popliteal lymph node cells or spleen cells (2 × 105/well) with a dilution series of CII and synthetic peptides for 72 hours, as previously described [30].
Cells were incubated during the last 18 hours in the presence of 14.8 kBq of [3H]thymidine (TRA310, specific activity 307 MBq/mg; Amersham International plc, Didcot, Oxfordshire, UK), harvested on glass fiber membranes and radioactivity was quantified using a direct Beta Counter (Matrix 9600, Packard Instrument Company, Meridan, CT, USA).
Proliferation assays
For testing CII-specific T cell responses in draining lymph nodes, mice were immunized in the footpad with 50 μg CII emulsified 1:1 in TiterMax adjuvant in the absence or presence of amiloride (150 μg/mouse [31]) and popliteal lymph nodes were removed 7 days later. Cells (2 × 105/well) were mixed with a dilution series of CII, synthetic peptides or the polyclonal T cell mitogen concanavalin A in round-bottomed 96 well plates (Corning Limited) and incubated for 4 days at 37°C in a humidified CO2 incubator. Cells were incubated during the last 18 hours in the presence of 14.8 kBq of [3H]thymidine, harvested and radioactivity was measured, as described above.
Subcellular fractionation
Macrophages (15 to 20 × 106 cells) were pulsed with 200 μg/ml CII for 30 minutes and chased for different periods of time. Macrophages were homogenized in buffer containing 0.25 M sucrose, 10 mM HEPES, pH 7.4 in a Dounce tissue grinder (Wheaton, Millville, NJ, USA) to obtain 80% to 85% cell lysis. Subcellular fractionation of macrophages was performed by density gradient centrifugation in 27% Percoll (Amersham plc, Little Chalfont, Buckinghamshire, UK) using a Sorvall type A-1256 fixed angle rotor (36,000 × g, 60 minutes, 4°C; Kendro Laboratory Products plc, Bishop's Stortford, Hertfordshire, UK), as described previously [27]. Six fractions of 1.5 ml were collected manually numbered 1 to 6 from the top of the gradient. Percoll gradient fractions were each tested for β-hexosaminidase activity (marker for the presence of lysosomal enzymes) and alkaline phosphodiesterase I activity (marker for the presence of plasma membranes), as described [27, 32] The localization of markers of endosomal compartments and CII within subcellular fractions was performed by ELISA, as previously described [33]. Briefly, 50 μl of Percoll fractions were dried in a constant flow cabinet in 96 well Microtiter® Immunoassays plates (Immulon® 1, flat bottom, Dynex Technologies, Southampton, UK). Plates were blocked in phosphate-buffered saline containing 0.05% Tween 20, 10% FBS and unlabeled anti-mouse mAb specific for FcγIIR and FcγIIIR (1:200; clone 2.4G2, Fc Block®, PharMingen, Oxford, UK) for 1 hour at room temperature. Plates were washed and incubated for 1 hour with goat anti-CII polyclonal antibody, goat anti-Rab7 and Rab9 polyclonal antibody (1: 200; Santa Cruz Biotechnology, Inc., Heidelberg, Germany). Normal goat serum was used in control experiments. After washing, plates were incubated for 1 hour with rabbit anti-goat IgG peroxidase conjugate diluted 1:1000, washed and the reaction was developed with the liquid substrate system for ELISA 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid. Absorbance was measured at 405 nm.
Flow cytometry
Bone-marrow macrophages and DCs were incubated in the absence or presence of inhibitors of uptake for five hours, and the expression of HLA-DR, CD80, CD86 and CD40 molecules was analyzed by flow cytometry, as described [28]. Briefly, cells were incubated for 30 minutes at 4°C in Hank's balanced salt solution containing 2% FBS, 0.01 M HEPES buffer with purified anti-mouse CD16/CD32 (Fc Block®, BD-PharMingen) followed by incubation for 30 minutes at 4°C with either of the following mAb fluorescent conjugates (BD-PharMingen, Cowley, Oxford, UK): anti-HLA-DR FITC, anti-CD40 FITC, anti-CD80 PE, anti-CD86 FITC, anti-CD11c FITC, anti-CD11b FITC or isotype control, rat IgG2a PE plus IgG2b FITC. Cells were analyzed with a FACScan® flow cytometer (Becton Dickinson, Mount View, CA, USA), and 10,000 events were collected for each sample.
Electron microscopy
Bone-marrow macrophages and DCs were pulsed with 200 μg/ml CII in the absence or presence of inhibitors of uptake for 30 minutes. Transmission electron microscopy was performed as described previously [28]. Briefly, cells were fixed in 2.5% EM grade gluteraldehyde (TAAB Lab. Equipment, Aldermaston, Berkshire, UK) diluted in 0.1 M phosphate buffer, pH 7.3, washed in phosphate buffer and post-fixed with 1% osmium tetroxide (Agar Scientific, Stansted, Essex, UK). Samples were sequentially dehydrated through a graded acetone series, impregnated with TAAB epoxy resin kit (TAAB Lab. Equipment) and polymerized at 60°C for 24 hours. Blocks were thin sectioned (80 nm), stained with uranyl acetate and lead citrate (Leica UK Ltd, Milton Keynes, UK), and examined with a Philips CM 100 (Compustage) Transmission Electron microscope (Philips Electron Optics, Eindhoven, The Netherlands). Sections through several planes of more than 50 APCs were examined for each treatment.
Confocal microscopy
Bone-marrow macrophages and DCs were pulsed with 200 μg/ml CII in the absence or presence of 1.0 mM amiloride for 30 minutes at 37°C. Cytospins were prepared by centrifugation of 2 × 104 cells in 200 μl in a Shandon Cytospin 3 cytocentrifuge (Thermo Electron Corp., Waltham, MA, USA). The slides were air-dried at room temperature for 30 minutes, fixed in acetone for 10 minutes at room temperature and permeabilized in 0.1% Triton X-100 in PBS for 15 minutes at 4°C. After washing (10 mM TRIS HCl pH 7.6, containing 150 mM NaCl, TBS buffer) and blocking (normal rabbit serum 1:5 in TBS buffer, 1 hour at room temperature) staining was performed with goat anti-human CII polyclonal antibodies (1:100 in TBS buffer, 4°C, 18 hours; Santa Cruz Biotechnology, Inc.). Slides were washed and incubated with rabbit anti-goat IgG-FITC (1:100, 2 hours, room temperature, in the dark). After washing, slides were mounted in aqueous fluorescent mounting medium (DAKO Cytomation, Carpenteria, CA, USA). Confocal microscopy was performed at the BioImaging facility, University of Newcastle upon Tyne, using Leica TCS SP2 UV laser scanning confocal microscope (Leica Microsystems GmbH, Heidelberg, Germany) equipped with Time 63 oil immersion 1.32 No Plan A Pro lens. Images were acquired using the 488 excitation laser and emission was detected between 500 and 560 nm. Images were collected using 0.5 μm Z-steps and these were projected using maximal projection and overlaid with single optimized transmitted light images. In the control, cells were incubated in the absence of CII, stained and imaged at the same gain and offset levels as the positive cells and no fluorescence was observed.
Mechanisms of CII uptake in macrophages and DCs
Subcellular localization of CII after uptake
Macrophages were pulsed with 200 μg/ml CII for 30 minutes and chased for different periods of time. Following subcellular fractionation, the distribution of intracellular CII was measured by ELISA (Figure 4b). The intracellular level of CII peaked 3 hours after pulse and returned to the baseline after 24 hours. After internalization, CII was detected in Percoll fractions 3 and 4 with intermediate density co-localizing with Rab7 and Rab9 late endosomal markers. These pulse-chase experiments showed that after uptake CII was present for about five hours in membrane fractions corresponding to late endosomes, after which the level of intracellular CII dropped, probably due to terminal lysosomal transport and degradation.
We addressed the route of CII uptake into late endosomes in pulse-chase experiments in the presence of inhibitors of uptake. Pretreatment of macrophages with cytochalasin D or amiloride reduced accumulation of CII in fractions 3 and 4 (Figure 4c). Monodansylcadaverine and filipin had no effect on CII internalization (Figure 4d), consistent with data from electron microscopy (Figure 2c,d) and suggests internalization of CII primarily by macropinocytosis.
Effect of uptake on activation of CII-specific T cells in vitro
We studied whether prevention of CII uptake by DCs and macrophages results in down-regulation of antigen presentation and inhibits activation of CII-specific T cells in in vitro antigen presentation assays. Since T cells specific for the glycosylated and non-glycosylated CII have been demonstrated in peripheral blood of RA patients [44, 45], T cell hybridomas HCII-9.2, specific for the glycosylated C259–273 epitope, and HCII-9.1, specific for the non-glycosylated form of the same epitope, were used in this study [27].
The effect of amiloride in vivo
We studied the mechanisms of uptake of CII by macrophages and DCs for presentation to T cells specific for the arthritogenic epitope CII259–273. Electron microscopy and antigen presentation to CII259–273-specific T and presentation cell hybridomas demonstrated that uptake of CII by both types of APCs depended on actin polymerisation (cytochalasin D-sensitive) and membrane ruffling (amiloride-sensitive), suggesting the principal route was macropinocytosis. Previous electron microscopy studies showed that fibroblasts use an F-actin-dependent mechanism for CII uptake, with no distinction between phagocytosis and macropinocytosis [48]. Macrophages have also been shown to have vacuoles containing collagen, suggesting their involvement in uptake and resorption of collagen [49]. However, no information was available on the capacity of other cell types to take up CII, as well as on the relevance of collagen uptake to antigen presentation and specific T cell activation. We extended the electron microscopy studies with pulse-chase experiments and localization of CII by subcellular fractionation and showed that after uptake, CII accumulated in membrane fractions with intermediate density corresponding to late endosomes. Moreover, blockade of macropinocytosis prevented intracellular accumulation of CII and resulted in profound blockade of antigen presentation to T cells. The involvement of macropinocytosis in uptake of autoantigens, such as CII, by both DCs and macrophages for subsequent antigen processing and presentation to specific T cells is a novel finding. Macropinocytosis has been previously shown to deliver antigens for lysosomal processing and loading of newly synthesized MHC class II molecules in DCs [50, 51] and macrophages [28]. This observation is in agreement with our previous report that CII is processed in lysosomal compartments of macrophages for presentation by newly synthesized MHC class II molecules [27].
Our model system used CD4 T cell hybridomas specific for both the glycosylated and non-glycosylated arthritogenic epitope CII259–273 generated from HLA-DR1-transgenic mice [27], which allowed us to test the effect of post-translational modification on uptake and presentation of CII. No differential effect of the inhibition of uptake on presentation of the glycosylated and non-glycosylated CII259–273 epitope was observed. In a previous report we showed that glycosylated and non-glycosylated forms of the same CII259–273 epitope were differentially processed in lysosomal compartments for presentation to specific CD4 T cells [27]. Taken together, our data indicate that following macropinocytosis CII is targeted to lysosomes for antigen processing and presentation of both glycosylated and non-glycosylated epitopes to T cells. This conclusion is consistent with the presence of T cells specific for both forms of the epitope in peripheral blood of RA patients [44, 45].
The importance of our finding that blockade of CII uptake prevents activation of specific T cells in vitro was tested in vivo. We administered amiloride in vivo and showed reduction in the magnitude of CII-specific, but not polyclonal, T cell responses in draining lymph nodes, suggesting that under these experimental conditions amiloride did not directly affect the T cell response, as has been reported in other experimental settings [52, 53]. Our data suggest that amiloride caused an immunosuppressive effect on T cell activation in vivo indirectly via inhibition of uptake and antigen presentation, rather than via a direct suppression of T cell proliferation [52, 53] Amiloride has also been shown to block soluble urokinase-type plasminogen activator [54], a serine proteinase expressed by macrophages and DCs (our unpublished observations), suggesting another mechanism underlying the effect of this drug on antigen presentation.
The potential of immunotherapeutic protocols based on the blockade of antigen presentation has been underscored in RA, including targeting co-stimulatory or MHC class II molecules [55, 56] on APCs or T cell adhesion molecules on T cells [57], which has prompted the search for new ways of down-regulating antigen presentation in vivo. The results of this study suggest that interfering with antigen uptake could constitute a novel effective target for blocking antigen presentation in DCs and macrophages, as a way to prevent activation of specific CII-specific T cells. The data obtained have implications for the development of immunotherapeutic protocols for use in T cell-mediated autoimmune diseases, such as RA.
This study shows that macropinocytosis was the predominant mechanism of uptake of CII for antigen presentation by DCs and macrophages. Treatment of both professional APC types with amiloride, which prevents macropinocytosis, inhibited intracellular accumulation of CII and antigen presentation of the major arthritogenic T cell epitope in both glycosylated and non-glycosylated forms. In addition, treatment of mice with amiloride blocked the activation of collagen-specific T cells in draining lymph nodes, constituting a novel therapeutic target for the immunotherapy of RA.
= antigen-presenting cell
= type II collagen
= dendritic cell
= 5-(N,N-dimethyl)amiloride
= enzyme-linked immunosorbent assay
= fetal bovine serum
= lipopolysaccharide
- mAb:
= monoclonal antibody
= major histocompatibility complex
= nuclear factor
= rheumatoid arthritis.
We thank Jan Kihlberg, Umeå University, for synthesis of galactosylated peptides, TE Cawston and Dr G McHaffie, University of Newcastle, for discussions. We also thank T Booth, BioImaging Facilitiy, University of Newcastle, for help with confocal microscopy. The work was supported by grant MP/R0619 from the Arthritis Research Campaign, UK.
Authors' Affiliations
- Bayry J, Thirion M, Delignat S, Misra N, Lacroix-Desmazes S, Kazatchkine MD, Kaveri SV: Dendritic cells and autoimmunity. Autoimmun Rev. 2004, 3: 183-187.View ArticlePubMedGoogle Scholar
- Holmdahl R, Tarkowski A, Jonsson R: Involvement of macrophages and dendritic cells in synovial inflammation of collagen induced arthritis in DBA/1 mice and spontaneous arthritis in MRL/lpr mice. Autoimmunity. 1991, 8: 271-280.View ArticlePubMedGoogle Scholar
- Panayi GS: B cells: a fundamental role in the pathogenesis of rheumatoid arthritis?. Rheumatology (Oxford). 2005, 44: 3-7.View ArticleGoogle Scholar
- Ludewig B, Junt T, Hengartner H, Zinkernagel RM: Dendritic cells in autoimmune diseases. Curr Opin Immunol. 2001, 13: 657-662.View ArticlePubMedGoogle Scholar
- Dittel BN, Visintin I, Merchant RM, Janeway CAJ: Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis. J Immunol. 1999, 163: 32-39.PubMedGoogle Scholar
- Leung BP, Conacher M, Hunter D, McInnes IB, Liew FY, Brewer JM: A novel dendritic cell-induced model of erosive inflammatory arthritis: distinct roles for dendritic cells in T cell activation and induction of local inflammation. J Immunol. 2002, 169: 7071-7077.View ArticlePubMedGoogle Scholar
- Sarkar S, Fox DA: Dendritic cells in rheumatoid arthritis. Front Biosci. 2005, 10: 656-665.View ArticlePubMedGoogle Scholar
- Pettit AR, Thomas R: Dendritic cells: the driving force behind autoimmunity in rheumatoid arthritis?. Immunol Cell Biol. 1999, 77: 420-427.View ArticlePubMedGoogle Scholar
- Del Nagro CJ, Kolla RV, Rickert RC: A critical role for complement C3d and the B cell coreceptor (CD19/CD21) complex in the initiation of inflammatory arthritis. J Immunol. 2005, 175: 5379-5389.View ArticlePubMedGoogle Scholar
- Holmdahl M, Vestberg M, Holmdahl R: Primed B cells present type-II collagen to T cells. Scand J Immunol. 2002, 55: 382-389.View ArticlePubMedGoogle Scholar
- O'Neill SK, Shlomchik MJ, Glant TT, Cao Y, Doodes PD, Finnegan A: Antigen-specific B cells are required as APCs and autoantibody-producing cells for induction of severe autoimmune arthritis. J Immunol. 2005, 174: 3781-3788.View ArticlePubMedGoogle Scholar
- Holmdahl M, Grubb A, Holmdahl R: Cysteine proteases in Langerhans cells limits presentation of cartilage derived type II collagen for autoreactive T cells. Int Immunol. 2004, 16: 717-726.View ArticlePubMedGoogle Scholar
- Manoury-Schwartz B, Chiocchia G, Lotteau V, Fournier C: Selective increased presentation of type II collagen by leupeptin. Int Immunol. 1997, 9: 581-589.View ArticlePubMedGoogle Scholar
- Cremer MA, Rosloniec EF, Kang AH: The cartilage collagens: a review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease. J Mol Med. 1998, 76: 275-288.View ArticlePubMedGoogle Scholar
- Brand DD, Kang AH, Rosloniec EF: The mouse model of collagen-induced arthritis. Autoimmunity. Methods and Protocols. 2004, Totowa, NJ: Humana Press, 102: 295-312. Edited by Perl AGoogle Scholar
- Kim WU, Cho ML, Yung YO, Min SY, Park SW, Min DJ, Joon JH, Kim HY: Type II collagen autoimmunity in rheumatoid arthritis. Am J Med Sci. 2004, 327: 202-211.View ArticlePubMedGoogle Scholar
- Lu L, Woo J, Rao AS, Li Y, Watkins SC, Qian S, Starzl TE, Demetris AJ, Thomson AW: Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen. J Exp Med. 1994, 179: 1823-1834.View ArticlePubMedGoogle Scholar
- Hao HN, Yang SY, Wooley PH: Direct interactions of collagen II and Toll-like receptor depend upon the saccharide residue of collagen II: a potential mechanism for collagen-induced arthritis. Arthritis Rheum. 2005, 52: S477-Google Scholar
- Pribila JT, Itano AA, Mueller KL, Shimizu Y: The α1β1 and αEβ7 integrins define a subset of dendritic cells in peripheral lymph nodes with unique adhesive and antigen uptake properties. J Immunol. 2004, 172: 282-291.View ArticlePubMedGoogle Scholar
- Cremer MA, Stuart JM, Townes AS, Kang AH: Collagen-induced polyarthritis in rats: a study of native type II collagen for adjuvant activity. J Immunol. 1980, 124: 2912-2918.PubMedGoogle Scholar
- Stuart LM, Ezekowitz RAB: Phagocytosis: elegant complexity. Immunity. 2005, 22: 539-550.View ArticlePubMedGoogle Scholar
- Sansonetti PJ: Phagocytosis of bacterial pathogens: implications in the host response. Semin Immunol. 2001, 13: 381-390.View ArticlePubMedGoogle Scholar
- Kaksonen M, Toret CP, Drubin DG: A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell. 2005, 123: 305-320.View ArticlePubMedGoogle Scholar
- Nichols B: Caveosomes and endocytosis of lipid rafts. J Cell Sci. 2003, 116: 4707-4714.View ArticlePubMedGoogle Scholar
- Bathori G, Cervenak L, Karadi I: Caveolae-an alternative endocytotic pathway for targeted drug delivery. Crit Rev Ther Drug Carrier Syst. 2004, 21: 67-95.View ArticlePubMedGoogle Scholar
- Holm B, Baquer SM, Holm L, Holmdahl R, Kihlberg J: Role of the galactosyl moiety of collagen glycopeptides for T-cell stimulation in a model for rheumatoid arthritis. Bioorg Med Chem. 2003, 11: 3981-3987.View ArticlePubMedGoogle Scholar
- von Delwig A, Altmann DM, Isaacs JD, Harding CV, Holmdahl R, McKie N, Robinson JH: The impact of glycosylation on HLA-DR1 restricted T cell recognition of type II collagen in a mouse model. Arthritis Rheum. 2006, 54: 482-491.View ArticlePubMedGoogle Scholar
- von Delwig A, Bailey E, Gibbs D, Robinson JH: The route of bacterial uptake by macrophages influences the repertoire of epitopes presented to CD4 T cells. Eur J Immunol. 2002, 32: 3714-3719.View ArticlePubMedGoogle Scholar
- Werling D, Hope JC, Chaplin P, Collins RA, Taylor G, Howard CJ: Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells. J Leukoc Biol. 1999, 66: 50-58.PubMedGoogle Scholar
- Delvig AA, Robinson JH, Wetzler LM: Testing meningococcal vaccines for mitogenicity and superantigenicity. Meningococcal vaccines. Methods and protocols. 2001, Totowa, NJ: Humana Press, 66: 199-221. Edited by: Pollard AJ, Maiden MCJView ArticleGoogle Scholar
- Lyons JC, Ross BD, Song CW: Enhancement of hyperthermia effect in vivo by amiloride and DIDS. Int J Radiat Oncol Biol Phys. 1993, 25: 95-103.View ArticlePubMedGoogle Scholar
- von Delwig A, Ramachandra L, Harding CV, Robinson JH: Localization of peptide/MHC class II complexes in macrophages following antigen processing of viable Streptococcus pyogenes. Eur J Immunol. 2003, 33: 2353-2360.View ArticlePubMedGoogle Scholar
- von Delwig A, Musson JA, Shim HK, Lee JJ, Walker N, Harding CV, Williamson ED, Robinson JH: Distribution of productive antigen processing activity for MHC class II presentation in macrophages. Scand J Immunol. 2005, 62: 243-250.View ArticlePubMedGoogle Scholar
- Grassme HU, Ireland RM, van Putten JPM: Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton. Infect Immun. 1996, 64: 1621-1630.PubMed CentralPubMedGoogle Scholar
- West MA, Bretscher MS, Watts C: Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol. 1989, 109: 2731-2739.View ArticlePubMedGoogle Scholar
- Valentin-Weigand P, Benkel P, Rohde M, Chhatwal GS: Entry and intracellular survival of group B Streptococci in J774 macrophages. Infect Immun. 1996, 64: 2467-2473.PubMedGoogle Scholar
- Harris J, Werling D, Hope JC, Taylor G, Howard CJ: Caveolae and caveolin in immune cells: distribution and functions. Trends Immunol. 2002, 23: 158-164.View ArticlePubMedGoogle Scholar
- Tsark EC, Wang W, Teng YC, Arkfeld D, Dodge GR, Kovats S: Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages. J Immunol. 2002, 169: 6625-6633.View ArticlePubMedGoogle Scholar
- Graham JM: Subcellular fractionation and isolation of organelles. Isolation of lysosomes from tissues and cells by differential and density gradient centrifugation. Current Protocols in Cell Biology. Edited by: Bonifacino JS, Dasso M, Hartford JB, Lippincott-Schwartz J, Yamada KM. 2000, New York: John Wiley & Sons, Inc, 1-21.Google Scholar
- Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ: Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature. 1991, 349: 669-676.View ArticlePubMedGoogle Scholar
- Jordens I, Marsman M, Kuijl C, Neefjes JJ: Rab proteins, connecting transport and vesicle fusion. Traffic. 2005, 6: 1070-1077.View ArticlePubMedGoogle Scholar
- Bertram EM, Hawley RG, Watts TH: Overexpression of rab7 enhances the kinetics of antigen processing and presentation with MHC class II molecules in B cells. Int Immunol. 2002, 14: 309-318.View ArticlePubMedGoogle Scholar
- Zerial M, McBride H: Rab proteins as membrane organizers. Nat Rev Mol Cell Biol. 2001, 2: 107-117.View ArticlePubMedGoogle Scholar
- Kim HY, Kim WU, Cho ML, Lee SK, Joun J, Kim SI, Yoo WH, Park JH, Min JK, Lee SH, Park SH, Cho CS: Enhanced T cell proliferative response to type II collagen and synthetic peptide CII (255–274) in patients with rheumatoid arthritis. Arthritis Rheum. 1999, 42: 2085-2093.View ArticlePubMedGoogle Scholar
- Bäcklund J, Carlsen S, Hoger T, Holm B, Fugger L, Kihlberg J, Burkhardt H, Holmdahl R: Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanized transgenic mice and in rheumatoid arthritis. Proc Natl Acad Sci USA. 2002, 99: 9960-9965.PubMed CentralView ArticlePubMedGoogle Scholar
- Haddad JJ: Amiloride and the regulation of NF-κB: an unsung crosstalk and missing link between fluid dynamics and oxidative stress-related inflammation – controversy or pseudo-controversy?. Biochem Biophys Res Commun. 2005, 327: 373-381.View ArticlePubMedGoogle Scholar
- Cuschleri J, Gourlay D, Garcia I, Jelacic S, Maier RV: Endotoxin-induced endothelial cell proinflammatory phenotypic differentiation requires stress fiber polymerization. Shock. 2003, 19: 433-439.View ArticlePubMedGoogle Scholar
- Everts V, van der Zee E, Creemers LB, Beersten W: Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling. Histochem J. 1996, 28: 229-245.View ArticlePubMedGoogle Scholar
- Parakkal PF: Involvement of macrophages in collagen resorption. J Cell Biol. 1969, 41: 345-354.PubMed CentralView ArticlePubMedGoogle Scholar
- Inaba K, Inaba M: Antigen recognition and presentation by dendritic cells. Int J Hematol. 2005, 81: 181-187.View ArticlePubMedGoogle Scholar
- Sallusto F, Cella M, Danieli C, Lanzavecchia A: Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J Exp Med. 1995, 182: 389-400.View ArticlePubMedGoogle Scholar
- Tang CM, Presser F, Morad M: Amiloride selectively blocks the low threshold (T) calcium channel. Science. 1988, 240: 213-215.View ArticlePubMedGoogle Scholar
- Lai ZF, Chen YZ, Nishimura Y, Nishi K: An amiloride-sensitive and voltage-dependent Na+ channel in an HLA-DR-restricted human T cell clone. J Immunol. 2000, 165: 83-90.View ArticlePubMedGoogle Scholar
- Dyer KD, Linz-Mcgillem LA, Alliegro MA, Alliegro MC: Receptor-bound uPA is reversibly protected from inhibition by low molecular weight inhibitors. Cell Biol Int. 2002, 26: 327-335.View ArticlePubMedGoogle Scholar
- Liossis SN, Sfikakis PP: Costimulation blockade in the treatment of rheumatic diseases. BioDrugs. 2004, 18: 95-102.View ArticlePubMedGoogle Scholar
- Falcioni F, Ito K, Vidovic D, Belunis C, Campbell R, Berthel SJ, Bolin DR, Gillespie PB, Huby N, Olson GL, et al: Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules. Nat Biotechnol. 1999, 17: 562-567.View ArticlePubMedGoogle Scholar
- Zeyda M, Poglitsch M, Geyeregger R, Smolen JS, Zlabinger GJ, Hörl WH, Waldhäusl W, Stulnig TM, Säemann MD: Disruption of the interaction of T cells with antigen-presenting cells by the active leflunomide metabolite teriflunomide: involvement of impaired integrin activation and immunologic synapse formation. Arthritis Rheum. 2005, 52: 2730-2739.View ArticlePubMedGoogle Scholar
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Paul Dano net worth: Paul Dano is an American actor and producer who has a net worth of $10 million dollars. Paul Franklin Dano was born in New York City, New York in June 1984. He attended Eugene Lang College and was involved in community theater. As a child he appeared on Broadway in Inherit the Wind. His first role came on the TV series Smart Guy in 1998. Dano starred as Joel in the 2000 movie The Newcomers. He starred in the movies L.I.E., The Emperor's Club, The Girl Next Door, and Taking Lives. Dano had a recurring role on The Sopranos from 2002 to 2004. He has also starred in the films The Ballad of Jack and Rose, The King, Little Miss Sunshine, Fast Food Nation, Weapons, There Will Be Blood, Light and the Sufferer, Explicit Ills, Gigantic, Taking Woodstock, The Good Heart, Where the Wild Things Are, The Extra Man, Knight and Day, Meek's Cutoff, Cowboys & Aliens, For Ellen, Being Flynn, Ruby Sparks, Looper, Prisoners, and 12 Years a Slave. He won a Screen Actors Guild Award in 2007 and has also won awards from the Stockholm Film Festival, Phoenix Film Critics Society, National Board of Review (USA), L.A. Outfest, Independent Spirit Awards, Fantasporto, Deauville Film Festival, Chlotrudis Awards, and Broadcast Film Critics Association Awards. Paul also sings and plays guitar in his band Mook.
Paul Dano Net Worth
Paul Dano
Date of Birth
Jun 19, 1984 (32 years old)
Actor, Musician, Film Producer
United States of America
5 ft 10 in (1.8 m)
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Pussycat, Pussycat, Where Have You Been?
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32 pages
Simply Read Books
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20th October 2011
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text: Donetsk is the coal-mining area of Ukraine. Here most people work for low wages in the run down mines, while others make a lot of money. No matter which side of the social divide you are on, coming from Donetsk you will almost certainly be an opponent of the Orange Revolution and a fan of the local football team, Shakhtar Donetsk. Billionaire Akhmetov invests heavily in the club, which is becoming a major European force during the season followed by the film. Yet this sporting success funded by an Oligarch fortune only seems to highlight the wider social and political stagnation of the region. Off the pitch, the outlook appears bleak... Written by Anonymous


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[Date Index] [Thread Index] [Author Index]
Re: Re: suggestion for frontend
Here's what I'd like to see: The ability to have a two-column layout of separate cells. (I wonder what surprises Mathematica 6 holds.) ----- Selwyn Hollis http://www.appliedsymbols.com (edit reply-to to reply) On Oct 4, 2004, at 6:18 AM, AES/newspost wrote: > In article <cjojf1$aqp$1 at smc.vnet.net>, > Pierre ALBAREDE <pa.news at free.fr> wrote: > >> Split window horizontally (or vertically) for easier comparison. >> >> >> Cheers. > > Hah! Could also provide a way to select and evaluate all cells from > current working point to top of a notebook (my personal hobby horse), > using one window kept open to top of a notebook, and doing ctrl-shift-A > and Enter in that window (wasteful use of screen real estate just to do > this, however). > >


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Once he led the revolutionaries against a tyrannical lord.
Once he had a beautiful wife.
Once he was a hero.
But that was before he got caught, beaten down, nearly killed, and sold into slavery.
Now he has nothing but hate and the chains of his bondage: the only weapons he has with which to escape.
The first title in a new Forgotten Realms series focusing on the popular Dungeons & Dragons game character class of Fighters. Each title will feature characters with a different exotic style of fighting.
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Media Watch: Wicked educational
By April Forristall, SeafoodSource.com assistant editor
Published on Thursday, April 05, 2012
The newest TV program to showcase the lives of fishermen isn't just a wicked good time, it's wicked educational.
The National Geographic Channel's "Wicked Tuna" follows the crews of five bluefin tuna vessels based in Gloucester, Mass. — Tuna.com, Hard Merchandise, Odysea, Bounty Hunter and Christina.
Despite being up against the Country Music Awards for its premiere on Sunday at 10 p.m., the new series became the network's highest-rated Sunday night telecast since New Year's Day, pulling in more than 1 million viewers, including 548,000 viewers in the coveted 25- to 54-year-old demographic.
The show is as educational as it is entertaining. Bluefin tuna is widely regarded as overfished around the globe, but the situation is much more complicated than that. Thankfully, the National Geographic Channel did not eschew just how complicated the situation is, and, in addition to the show, has launched a multi-tiered effort to help audiences become more knowledgeable about the plight of bluefin.
The educational campaign includes public service announcements (PSAs) and a dedicated website. The site features an in-depth article on the history of bluefin as well as information and links to the many stakeholders involved in the survival of the species, from fishermen to environmentalists. The site also includes an active message board to give viewers a voice.
"Our hope is that the show can also be an entry way to bring a wider audience to what is a complex and complicated discussion," said Michael Cascio, executive VP of programming for the National Geographic Channel.
"Wicked Tuna" also illustrates that it's not just scientists that are worried about the species' survival. Tuna.com Captain Dave Carraro said it's important for the fishery to be sustainable, because, if it wasn't, fishermen would be out of a job.
But the fishermen are hopeful that the bigger fish are returning. "They're coming back now — it's just starting," said Tuna.com First Mate Paul Hebert. "The purse seiners over in Europe, where for years they didn't have limits, are chasing away all the big fish. They're coming over here. The size is getting bigger and bigger each year."
It's refreshing to witness a mainstream media outlet using a show not just for entertainment value but also for educational purposes, as it certainly would have been easier to bill "Wicked Tuna" as an action-packed East Coast version of "Deadliest Catch," which showcases the lives of Alaska crab fishermen in the Bering Sea.
Hopefully, the show's high ratings continue, turning viewers into knowledgeable consumers.


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Podcasts from Australia's best known community radio station
July 15th, 2011
Episode 9 of 76 episodes
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1 Bedroom Sea View Suite in Rawai – only 1,600 per night
1/5/12 to 31/10/12 1/11/12 to 30/11/12 1/12/12 to 31/1/13
1/2/13 to 30/4/13
Daily 2,700 3,400 4,200
Monthly 35,000 42,000 48,000
These studio apartments are located in Rawai, a perfect location for many bars, restaurants and shops. Two large supermarkets are within easy walking distance and taxis are available just outside.
Sea View Suite
The Sea View Suite is luxuriously decorated and at 70sqm a good size. The bedroom features a 6ft bed, wardrobe, bedside tables and a dressing table. The living area also has a dining table for 2 and a seating area with sofa and TV. Each room has a TV with over 180 channels and a private balcony with seating.
Each room also benefits from a kitchenette comprising hob, microwave, fridge freezer, tea and coffee making facilities and a toaster.
The bathroom has a bath, walk in shower; sink with under storage cupboard and wc.
Leisure Facilities
The hotel benefits from a large communal pool, restaurant serving breakfast, lunch and dinner and a Gym.
Additional Information
Each room comes with wifi internet, bottled water and bathroom amenities. Daily breakfast can be arranged for 300thb per day.
Whats included
All taxes and utilities (electric is charged for monthly stays), twice weekly cleaning and a weekly linen change.


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Definitions for comparing
This page provides all possible meanings and translations of the word comparing
Princeton's WordNet
comparison, comparing(noun)
the act of examining resemblances
"they made a comparison of noise levels"; "the fractions selected for comparison must require pupils to consider both numerator and denominator"
Webster Dictionary
of Compare
Chaldean Numerology
The numerical value of comparing in Chaldean Numerology is: 7
Pythagorean Numerology
The numerical value of comparing in Pythagorean Numerology is: 6
Sample Sentences & Example Usage
To love is to stop comparing.
Enjoy your own life without comparing it with that of another.
I don't sit around comparing myself to Dave, he laid a lot of the groundwork for me to be successful.
I'm sorry for hurting people by comparing homosexuals to animals. Please forgive me for those I've hurt.
It 's not the' real deal' so to speak in that there are no trials comparing these drugs to one another.
Images & Illustrations of comparing
Translations for comparing
From our Multilingual Translation Dictionary
- เปรียบเทียบThai
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Use the citation below to add this definition to your bibliography:
"comparing." Definitions.net. STANDS4 LLC, 2017. Web. 22 Feb. 2017. <http://www.definitions.net/definition/comparing>.


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An elite university set up in London as an alternative to Oxford and Cambridge has decided to stop taking A-level results into account when offering some places, due to "mistrust" over falling standards.
The New College of the Humanities, established by the philosopher AC Grayling and currently only taking on around 60 students per year, will start making "unconditional" offers, instead judging prospective teenagers based on interviews and prior exam results alone.
Speaking to the Sunday Telegraph, Professor Grayling said that there were concerns exams in humanities subjects like English and history failed to properly value "individual judgment, interpretation and creative thinking".
The controversial academic said the change would apply to New College's 2014 intake, which "may very well be doubling" to 120 new undergraduates who will be charged annual fees of almost £18,000.
Professor Grayling said: "We have decided that we will from now on be making unconditional offers. If we like somebody, we tell them to come… This is something that a lot of Russell Group universities are doing now. It is happening because there is an increasing mistrust of the value of A-levels.
"A-level marking has become more standardised, prescriptive and a question of the boxes that need to be ticked," he told the newspaper. "There are now very good students who aren't getting A*s and As because they are a little bit too creative."
New College was set up as an elite private institution, and has been widely criticised for charging students twice the maximum tuition fee levied by state-backed universities. While it is growing, Professor Grayling said the costs involved in expansion meant it would always be limited to be no greater than the size of one Oxbridge college.
While critics have argued that unconditional offers mean students lose focus in their final year of sixth form education, New College hopefuls will not be able to switch off altogether. Candidates hold unconditional offers will still need to meet the minimum entry requirements for the University of London – three A-level passes including a D or higher – because it provides the institution's accreditation.Reuse content


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About 50 pilots world-wide have shown interest and so far NZ has a team of three. For more information contact David Cranna also see Bulletin 2 – Draft this has the schedule, contacts, fees, local aircraft, known sequence & entry form for the competition.
Combined Yak52 & Intermediate World Championships
The South African Aero Club will host the first combined Yak52 and Intermediate World Championships at Mossel Bay Airport on 26 Nov through 3 Dec 2014. This competition is breaking new ground as CIVA are introducing for the first time a new category of aerobatics, this hopefully will serve as a feeder class to the established Advanced and Unlimited Championships or alternately will allow pilots to experience international competition and comradeship that they might not of otherwise experienced.
This is not the first time that the South African Aero Club has introduced a new category, the Yak52 category was initiated by South Africa and the 1st Advanced Competition staged in Cape Town in 1995, this category in terms of the number of entrants is now the most popular.
Red Bull Air Race season 2014
The first race of the season is in Abu Dhabi. 2014 Race Calendar:
- Abu Dhabi, UAE – 28 Feb. /1 March
- Putrajaya, MAS – 17/18 May
- Gdynia, POL – 26/27 July
- Ascot, GBR – 16/17 August
- Dallas/Fort Worth, USA – 6/7 September
- Las Vegas, USA – 11/12 October
- China – 1/2 November
Future FAI competitions
The 2013 Annual Meeting of the FAI Aerobatics Commission (CIVA) was held on 8 and 9 November 2013 in Tallinn, Estonia. The organisation of the following competitions was awarded: 2014 – 19th FAI European Aerobatic Championships Matkopuszta Airport (Hungary) 23-30 Aug 2014, 2014 – 17th FAI World Glider Aerobatic Championships Olesnica (Poland) date to be advised, 2015 – 28th FAI World Aerobatic Championships Location to be advised (France) 19-29 Aug 2015. Check out the calendar for all FAI Aerobatics past and future events
CIVA Approved Known sequences for 2014
These are the CIVA Unlimited, Advanced and Y52/Intermediate Knowns for 2014. The PDFs can be found at www.fai.org/civa-documents and then Known (Q) Sequences – 2014.
Known Sequences for 2014 – Analysis
The members of the CIVA Known Analysis Working Group (KAWG) have submitted their reports and they have been compiled into one report which is now available at www.fai.org/civa-documents
Michael R. Heuer
Red Bull Air Race back on stage in 2014 – FAI
The resumption of the Red Bull Air Race World Championship was announced on Tuesday in Malaysia during a press conference attended by FAI President John Grubbström and FAI Secretary General Jean-Marc Badan. The FAI will partner the Red Bull Air Race management and provide safety specialists for the Championship which will start in February 2014. The World Championship will consist of seven races staged in six countries on three continents, starting with Abu Dhabi, United Arab Emirates, on 28 February 2014. FAI President John Grubbström said: "I am very pleased to see the Red Bull Air Race coming back as it is truly an event that gives worldwide exposure to air sports. Furthermore, it is important news for the FAI community in general as it represents a unique opportunity to showcase the excitement of air sports to the world. The improvements made to the race format and race track by the Red Bull Air Race Management are convincing and will no doubt add extra attractiveness to the event. We at the FAI are proud to be involved in this great event and to bring to the race management our expertise in matter of safety to make it successful. We are also very pleased that Breitling, the FAI Global Sponsor, is also part of the Red Bull Air Race as the official timekeeper."
Race Calendar – www.redbullairrace.com
- Abu Dhabi, UAE – 28 Feb. /1 March
- Putrajaya, MAS – 17/18 May
- Gdynia, POL – 26/27 July
- Ascot, GBR – 16/17 August
- Dallas/Fort Worth, USA – 6/7 September
- Las Vegas, USA – 11/12 October
- China – 1/2 November
Kind regards,
FAI – Fédération Aéronautique Internationale
Maison du Sport International | Av. de Rhodanie 54 | 1007
Lausanne | Switzerland
Tel: +41 (0)21 345 10 70 | Fax: +41 (0)21 345 10 77 |
Skype: fai.fca
FAI Sporting Code Section 6
Section 6 of the FAI Sporting Code – Part 1 Powered Aircraft contains the general rules for world and continental aerobatic championships and international aerobatic competitions. Other documents such as contest forms, known (Q) sequences, rules for glider aerobatics etc can be downloaded from the FAI Aerobatics Commission (CIVA) website.
Changing the rules
The following article was published in an NZ Aerobatic Club newsletter, now posted here in case you missed it. It's all about the rules of competitive aerobatics. Rules, who needs them ? Doesn't sound like fun. But all sports have rules, to provide structure, let competitors know what you can and can't do and how the game is played. Our rules help make competitive aerobatics a truly international sport, connect us with history and connect us with the rest of the world.
So we ought to think about rules sometime, but why now? Here follows a short history of recent events. Back in November 2011 CIVA (the FAI Aerobatics Commission) released an updated version of the Aresti Aerobatic Catalogue; that elegant gallery exhibiting all the figures we can fly in a competition, those fine sculptural shapes of Spanish origin, thank you Colonel José Aresti. All the figures you might find in a known or unknown sequence, or might want to put in your free sequence. The updated catalogue has some interesting new ones, like the double-humpty; despite the name you won't find this in the Kama Sutra.
In New Zealand we currently use the rules laid down by the IAC (International Aerobatic Club). The IAC in turn follows the lead of CIVA as far as the catalogue is concerned, and much else. So in due course NZ would naturally adopt this updated catalogue.
A little background; CIVA are mostly a European organisation, though their membership is more international than that of the IAC which is almost exclusively American. CIVA run the world aerobatic championships. Both IAC and CIVA have rules for running aerobatic competitions, their rules are similar but not identical. In recent years the IAC has been gradually migrating its rules towards those of CIVA.
Discussion ensued between Richard Hood, Grant Benns, Simon Fitz-gerald and myself. The long and the short of it (these aerobatic rule complications do asymptote to infinity) Grant asked, should we adopt and publicise the new catalogue, and Richard said perhaps it was time we went with the CIVA system which would bring us more in line with international competition. I got roped in because of my seven years in the wilderness (Sydney) where the CIVA rules prevail.
Thus tasked by Grant to find out if the CIVA rules are greatly different from IAC rules, and consider what would change for us at a practical/competitor level, I duly picked through my gently fading memory cells and eventually came up with the following.
But first, maybe you're not yet feeling the love for this rule stuff. Not everybody is desirous of competing at world championships; perhaps you aren't, and wonder why you should care if NZs rules are in line with those of other countries. Consider this, competitors at all levels can cross the Tasman easy enough, all you do is find a plane when you get there. Or get really big fuel tanks for your own. Judges may also want to sample other parts of the globe. And NZ can benefit greatly from inviting competitors and judges from other countries here, to take part in NZ competitions.
The differences are numerous and each deserves due consideration of the pros and cons. This isn't all of them, just things I've noticed. Some observations are differences of practice, not of rules per se. In summary form only (got to save trees):
- Aerobatics flown outside the box: this is permitted in NZ, not in Australia or under CIVA rules (thus any safeties must be flown in the box)
- Safety figures flown: in NZ two half-rolls are flown for safeties, per IAC rules; in Australia/CIVA two half-rolls and a loop (for the advanced category a humpty, stall-turn or cuban-8)
- Flying right through the box after wing-waggles, and re-entering: there is no penalty for this under IAC rules; there is a penalty under CIVA rules
- Lower box limits for the advanced and unlimited categories: in NZ the limits are 800ft and 500ft respectively; under CIVA rules, 660ft and 330ft. The IAC have recently adopted the CIVA lower limits so NZ should follow sooner or later, regardless of whether we shift to CIVA rules
- Known vs Q: in NZ the advanced category flies a known sequence, per IAC rules, and the score counts; in Australia/CIVA the equivalent is called the Q (qualifier) and the score doesn't count unless weather cuts things short
- Number of unknowns: in NZ the advanced category flies one unknown, per IAC; in Australia/CIVA advanced flies two unknowns. In the USA they fly one unknown except for international team selection when they fly two
- Creating unknowns: in NZ the advanced competitors submit figures for the unknown, but don't draw up the sequence; in Australia competitors also draw up the sequence, usually the evening before the flight
- Free unknowns: in NZ all competitors fly the same unknown, in Australia/CIVA each competitor may choose to fly a different unknown. This is a fairly recent development, I don't have experience of it myself. The USA have just adopted the free unknown process also, but haven't flown a comp with it yet
- Number of judges: the minimum number of judges is two in NZ (including the chief judge) three in Australia (usually not including the chief judge). Under IAC rules the minimum is three, not including the chief judge
- Encouraging judging: in NZ few competitors judge and there's no active training program; in both Australia and the USA competitors are encouraged to judge, via on-line judging exams and refresher courses held at competitions
- Scoring software: NZ uses the JaSPer scoring program, Australia uses the ACRO scoring software (once at their nationals so far, plus NSW and VIC state comps). The ACRO software implements the FairPlay statistical processing method, JaSPer uses simpler, non-statistical calculations
The information about events in the USA is courtesy of Debby Rihn-Harvey, that concerning Australia is courtesy of Grant Piper and my own hippocampus. You'll note some differences affect only the advanced and unlimited categories while some affect all categories. Some changes imply a cultural shift; long established practise carries weight, naturally. Do consider that beyond just standardising with the Aussies and Europeans, many of these changes could bring intrinsic improvements to how we do aerobatics here.
More food for thought, regardless of which rules we change, do we want to encourage the NZAC membership to become familiar with the rules of our sport ? At the moment this isn't so much the case, as compared with the situation in Australia, Europe or the USA. Knowing the rules can build confidence, make you a better competitor and a more engaged participant both at and between competitions.
Russell Bell
Judging flicks – what makes it a challenge ?
Flicks turn up in lots of places. On horizontal lines, 45° up-lines, 45° down-lines, 90° up-lines, 90° down-lines, top of a loop, before a part-loop, after a part-loop, or after a spin. Each of these lines can be either positive or negative, except after a spin.
The flick itself may be positive or negative, may be normal or a crossover (also called a "ming vase") and may revolve through ½, ¾, 1, 1 ¼, 1 ½, 1 ¾ or 2 rotations. Flicks require abrupt pitch and rudder input, gyroscopic precession couples the pitch and yaw thus flicks to the left or right will auto-rotate differently.
Judging auto-rotation is subtle and flicks happen fast. What do you look for ? The rules say:
- The aircraft nose must depart the fight-path (pitch). For no pitch or wrong direction, zero the figure
- The aircraft must yaw, initiating auto-rotation. For a visible roll before auto-rotation commences deduct 1 point per 5°. Pitch and yaw may be either separate (in time) or simultaneous
- The "main axis" of the auto-rotation must be in the correct plane and direction
- Downgrade for a visible "change in character" of the auto-rotation. This means a change in roll-rate or change in cone-angle. The downgrade is 1 point for each change in roll rate
- The flick must stay in auto-rotation. If the flick is entirely aileroned, zero the figure. If the finish is aileroned, downgrade 1 point per 5° so give zero if more than 45° is aileroned
What are the visible signs of auto-rotation ? The rules don't say exactly, the usual interpretation is a conical or corkscrew rotation of the aircraft. The roll-axis of the aircraft should draw the surface of a cone, as the aircraft rolls. The axis of this cone is the "main axis" of the auto-rotation.
The "cone angle" will vary between aircraft. It may vary for the same aircraft and pilot (e.g. depending on the flick entry speed). But it shouldn't vary during a flick, downgrade if it does.
Stopping – the flick must stop in the same attitude as before the flick starts (before the initial pitch). Not too tricky a concept for flicks on lines, except for flicks that stop on knife-edge, e.g. ¼ flicks. Note that ½ flicks will finish horizontally-displaced, do not downgrade for this. They should still finish on box-axis.
The stop-attitude must correspond to the geometry of the base figure. Flicks on a loop should be integrated into the loop. Consider, for flicks before or after part-loops, should the flick be integrated into the part-loop, or be an extension of the line ?
Negative – for negative flicks the initial pitch is towards the wheels, otherwise the criteria are the same. Unlike the positive flick, in a negative flick the canopy is on the inside of the auto-rotation "cone" thus the pilot can look ahead through the canopy along the "main axis" of the auto-rotation.
Crossovers are flicks that pitch towards the local-G direction, i.e. a negative flick on a positive line or positive flick on a negative line. Crossovers on horizontal or 45° lines offer quite a challenge (an impossibility ?) for the pilot to keep the main axis of the auto-rotation in the correct direction.
This was written as homework for an AAC training camp, comments welcome
Two Unknowns
In New Zealand pilots (in the Advanced and Unlimited categories) fly just one unknown, as is done in the USA under IAC rules. In Australia and under CIVA rules pilots fly two unknowns. And under CIVA rules the score for the Known (they call it the Q, short for Qualifier) doesn't count (unless the weather cuts the competition short) which puts more emphasis on the unknowns. Note that Intermediate pilots fly one unknown under both IAC and Australia/CIVA rules.
What would switching to CIVA rules mean ? At the moment we fly four flights: the Known (official practice), Known again (competition), the Free, then the Unknown. Under CIVA rules we would fly (still four flights): the Known/Q (official practice), the Free, the first Unknown, then the second Unknown.
In other words we would only fly the Known/Q once (as official practice) instead of twice, and would fly two unknowns. Should NZ advanced and unlimited pilots fly two unknowns ?
Encouraging Judging
Should we encourage more competitors to judge? Advantages: learning how to judge, knowing what the judges are looking for, makes you a better competitor. More judges on the line means pilots get more feedback.
A bit of arm-twisting helps to get people onto a judging line. In Australia competitors are actively encouraged to judge; all pilots competing in Intermediate category and above are required to take an on-line judging exam before turning up at the Nationals, and a short session (about 40 minutes) is held before the first day of competition. This is a refresher, not an exam, nobody fails; but everyone gets the message they're expected to know at least the basics of the judging criteria. The USA and UK also put judging training material on-line.
Disadvantage: training judges requires work and won't happen overnight. While the judging criteria can be self-taught, theory isn't everything; judges also need to get practical experience on a judging line.
Judging flight path and attitude – a short quiz
This is a short quiz about judging criteria, looking at the aircraft's flight path and attitude. There's plenty of study material for judging out there; the IAC have their Introduction to Aerobatic Judging – Student Handout. The Australians have on-line Judging Courses. The BAeA (i.e. UK) have a Judging Seminar Tutorial. This quiz is a learning tool, not an exam; results are not recorded. It works on iPhone and Android.
Judging flight path and attitude
Qualifier vs Known
In New Zealand the advanced category pilots currently fly a Known sequence, per IAC rules. In Australia and under CIVA rules, the equivalent flight is called the Q (Qualifier). For both the Known and the Q, the sequence is published months in advance of the competition, all the pilots fly the same sequence, and the flight is judged and scored. IAC and CIVA actually use the same sequence (each year).
However under IAC rules the score for the Known counts in the pilots' overall result, where under CIVA rules the score for the Q doesn't count (unless weather cuts the competition short). Instead, under CIVA rules pilots fly two unknowns.
Should NZ advanced category pilots fly the Q rather than the Known ?
Should NZ adopt the CIVA and IAC lower box limits ?
In New Zealand the lower box limits for the advanced and unlimited categories are currently 800ft and 500ft respectively. In Australia and under CIVA rules the limits are 660ft and 330ft. The IAC have also adopted the CIVA lower limits.
Should NZ adopt the 660ft/330ft limits used by Australia, Europe and the USA, or retain the 800ft/500ft limits ?
Australian Aerobatic Club Rules & Regulations
The following links are to the PDF documents on the Australian Aerobatic Club website.
Section 1 – Introduction Section 2 – Operations and Procedures
Appendix 2.1 – Criteria for Judging Aerobatic Figures
Appendix 2.2 – Evaluation of Competition Flights and Code of Conduct for Judges
Appendix 2.3 – Intermediate Unknown Figures
Appendix 2.4 – Known Sequences
Appendix 2.5 – List of Qualified Judges
Appendix 2.6 – Judge's Log Sheet
Appendix 2.7 – Procedure for Australian Team Selection
Appendix 2.8 – CASA EX49/11
Appendix 2.9 – Sample Event management Plan
The following sections can be viewed directly on this website, more convenient for searching, though unfortunately they're without the diagrams. The green text is changes, blue text is comments/explanations.
Criteria for Judging Aerobatic Figures
Evaluation of Competition Flights
Procedure for Australian Team Selection
IAC Official Contest Rulebook
The complete IAC rulebook, the link is provided here with permission of the IAC; their copyright notice follows.
Important Notice: Any IAC member or person acting on behalf of the IAC is hereby authorized to copy and print this document for personal or IAC-approved use. Any copy of this document or portion thereof must include this copyright notice. This document may be used for information only and may not be exploited for commercial purposes.
Download as single PDF file 2012 IAC Official Contest Rules
Should pilots fly safeties in the box ?
In New Zealand safeties consist of two half-rolls flown in the box, prior to the start of the sequence. This is per IAC rules. In NZ the purpose of the safeties is regarded as safety only (e.g. loose items, harness, inverted oil pressure) and safeties are not commonly flown in the box. Safeties may be flown outside the box.
In Australia and Europe safeties consist of two half-rolls plus a loop (humpty, stall turn or cuban-8 for advanced and unlimited) flown in the box. This is per CIVA rules. The purpose (in addition to safety) is to assess wind-drift in the box, and allow the judges to acquaint themselves with the appearance of the aircraft. In Oz safeties are almost always flown. Safeties may not be flown outside the box.
Advantages of the Oz approach: safeties help you assess the wind, fine-tune your entry into the box, and plan for drift during the sequence. They help the judges get their eye in. Disadvantage: they take a little time, though the judges don't have to be ready while safeties are flown.
Do you think flying safeties in the box is a good idea ? Vote or add a comment.