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|205 posts found
Spotlight Poster
Joined: 2/09/06
"No path is darker then when your eyes are shut." -Flemeth
Originally posted by LogicLester
You didn't even read what I wrote, like so many sandbox haters.
This isn't about recreating SWG or stepping back to pure sandboxes. This is about adding sandbox additionally to themeparks quests, because themeparks MMOs are *evidently* at their end!
People don't ask questions to get answers - they ask questions to show how smart they are. - Dogbert
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Novice Member
Joined: 7/30/12
Originally posted by Quizzical
You finally hit the multi-million "WTF?" question.
Why did Bioware chose a prototype engine that was untested?
Why did Bioware spend 5+ yeasrs of early development tinkering with it (with according to them 300 people working on the engine), immplying other aspects of the game's development were suffering.
Why did EA allow itself to fund such an apparent misdirection, and then demand the product launch before it was close to ready?
Speculation: When Bioware landed the LA license (prior to the EA acquisition) they were finacially strethed. They now had SWTOR, Dragon Age, and Mass Effect in deelopment simultaneously. That's a lot of operating capital for a smaller sized studio. They tried to think "outside" the box, and circumvent a large initial investment in licesnsing a proven engine or building one from the ground up; a gamble that backfired.
Why did Bioware continue throwing good money after bad? It happens a lot. It's not easy to say you were wrong, and throw out years worth of work even if it is the right thing to do.
Why did EA allow Bioware to veer off course? EA tried to change their stripes. They had a reputation of man hadling their studios which lead to mediocre results, especially in the MMO field. They tried to leave Bioware to make their own descions, to a point.
Why did EA step in and release the game before it was ready? They got fed up with Bioware. After 5 years of development (and millions of dollares) they said enough is enough, you have 6 years, not 7. (For that matter they also reduced the development times of DA2 and ME3). EA was also aware/wary of WoW's MoP (and D3) and Areannet's GW2 release dates.
Even now, EA has done its usual, and reduced DA3's development time to release nearly a year ahead of the original schedeule so as not to compete with The Witcher 3.
Sidenote to Quiz: I don't understand why you are belabouring the point that building and modifying engines is hard. I think most people know that. That said, if a space agency keeps blowing up its rockets, they can't just say, "Well rocket science is hard." That's why there are well paid rocket scientists.
When you factor in EA's purchase of Bioware and SWTOR's development and marketing, you enter the billion dollar realm of investment. You don't get to say "Game engines are tricky things." Especially when antecdotally, your main competitor and "touchstone" game, WoW, was built on an in-house modified WC3 engine by less people for less money in less time with better results. (Blizzard was also a first time MMO maker.)
Joined: 3/19/06
Trolls will be ignored
Originally posted by LogicLester
While I agree that there is a lot of rose colored glasses around here regarding SWG,(particuliary the NGE fanatics) I do think it's a bad idea to write that game completely off and ignore what parts it got right in it's original design. I'd love to play another MMO with a real player economy like Vanilla SWG had along with a skill system similiar but maybe just a bit condensed.
Currently Playing: Star Wars The Old Republic
Novice Member
Joined: 3/30/13
Originally posted by Elikal
No, I read it, I even reread it just to be sure.
You think "themepark" MMOs are dying (*Clearly not true) and will kill the genre in general (Also *clearly not true) unless developers start putting more sandbox elements in them. And you think SWTOR would somehow have been saved if they'd done that (I highly doubt it, the idea that they could have implemented good sandbox elements when they had such a hard time with themepark ones is pretty ridiculous).
What I'm saying is that the reason developers don't do that is because of how difficult sandbox elements are to implement well, and even the games which do them well rarely get mass market appeal. So what is the incentive for them to try mixing both elements?
This isn't a situation where one side likes chocolate and the other likes peanut butter, two great tastes that taste great together by the way. This is more of a situation where one side likes chocolate and the other likes spinach souffle. Even if you could somehow convince someone they go together well they aren't likely to want to go to all the trouble of making a souffle when they already have chocolate, or risk ruining the souffle they worked so hard to prepare with a bunch of chocolate.
Especially when the main example you're giving is SWG. Use an at least somewhat successful sandbox game, the various Sim games, Dungeon Keeper, Fable, Populous, Minecraft, UO (Yes, I know you mentioned it but you focused on SWG), Eve, or even Second Life (Also known as that thing that requires a question mark at the end of any attempt to classify its genre, MMO?, social game?, freaky "adult" sim?).
*WoW is still by the far the largest MMO with nearly all current and near future MMOs attempting to copy it in part or whole, and it still has exceedingly few things one could call sandbox elements. Unless you want to call minigames a sandbox element, but then I'd actually agree with you.
Novice Member
Joined: 3/30/13
Originally posted by ktanner3
Oh don't get me wrong, there were things I really liked about SWG, heck there are things I really like about SWTOR. I don't believe either game should be completely written off. But they also shouldn't be used as broad examples for future games, there are far too many connotations of failure tied with both of them. Better to cherry pick out the desired features as examples, like you did at the end there.
Originally posted by ignore_me
I'm guessing you mean the bit I wrote at the end, in which case sadly, no I'm not. If people can now seemingly point to SWG as the glowing diamond at the pinnacle of an ivory spire of sandbox MMOs, then why can't they point to SWTOR as the peak of themepark MMOness in about 4-5 years?
If you mean because I deny that SWG was in fact the savior of all MMO kind, and it died for SWTOR's sins, then.....uh....sure, let's say I was joking.
Apprentice Member
Joined: 7/04/11
Originally posted by LogicLester
SWG has definately become the ex-girlfriend we remember fondly but forgot why we broke up with. I did get on there recently though and even with it's ancient graphics it still brings Star Wars to the table about 10 times better than SWTOR does. It's absolutley unplayable nowadays, but by completely avoiding the design elements that were good in SWG (realistic art style, movie-accurate art style, a more live and explorable world, 3d space sim) SWTOR became like one Star Wars scene that you have to watch over and over versus having full movies at your disposal.
SWTOR is all about rinse and repeat.
-Combat is your only activity.
-You have no individuality as everyone is the main hero (or villain) of the universe with a pet twi'lek just like yours.
-Every Dungoen/Raid is a WoW fight with a giant boss.
-Your character is essentially a transient, as there is no 'base/home' for your toon. This is a simple comfort hearkening back to when man decided to become agrarian and stop chaing the herds. If nothing else you can keep your shit somewhere.
-The cutscenes become tiresome fast, like garlic salt poured heavily on every dish you eat.
-Dailies are your post 50 non-group job. Repeated story quests (I retrieved the nautolan's medicine every day and brought it to his shuttle, how lame).
-Characters are static mirrors of other faction, you will play through same abilities.
-Alts are original end game, repeat starter and level world stories.
-Space missions are repackaged from lower levels, same map, same paths
It's really just negligent game design.
Survivor of the great MMORPG Famine of 2011
Novice Member
Joined: 8/24/08
"The data we were getting through in-game metrics and exit surveys was telling us that people were going through the content much faster than we were expecting."
The incompetence of Bioware's management and game designers really blew me away. It seems like they were so cocksure that they just went into the whole thing with a 'f___ you, we're Bioware, and we'll do it our way and you'll love it, you don't know what you want' attitude.
Did any of them even play other MMOs or take note of how people tend to play them? I mean, rapid consuming of content has been one of those givens since... Jesus, for at least 10 years. I remember it was sort of expected even when DAoC had it's first expansion. The Anarchy Online devs talked about it on several occasion in reference to the challenges of incorporating an ongoing story with an MMO. You just can't keep up.
This is why I knew they were going to fail hard when I saw SWToR's first preview vids and heard them talking about "story". Story is fine, but it can't be your main draw, because no one could possibly churn it out fast enough. It's like trying to lay track ahead of a train that's already moving.
i dont think they overestimated their content
i think they underestimated the power players
and yes,,a 5 min cutscene wont keep a player for long
a big, and exciting world would have better chances
hacking my way through a mob infested corridor gets old really fast
Hard Core Member
Joined: 4/21/03
And still the engine does not work properly.
i think this is really the source of bio ware problem and its sad that games made for much less developed their own engine to suit their games in same time frame and yet bio ware could not.
i pointed this out in the first month and said it was a game breaker for bio ware and quickly got my posts deleted both here and on official forum and even got a warning here for trolling which was bizzare.
bottom line the engine which is the heart of the game was a big fail.what sort of mmorpg can it be if more then 40 people on a spot in ilium meant crash or slide show.
the heart is bad and thus the game suffers.warning to all future mmorpg there is a reason most mmo either make their own engine or Heavily mod a tried and tested one
this is the poll i kept up back in jan 2012 about it even then most agreed that the engine was a fail
yea, they took a shortcut with the engine
the ONE part, that the whole game is build on, and they wanted to save some $$
if it had worked out, they would have made a fortune
but it didnt,,most of the dev team got fired
even the big suit himself lost his job
and a lot of fans are still waiting for a star wars mmo
next time,,use TWO of the big studios
clearly, one studio cant make a game, that is EPIC enough, to have the name
Novice Member
Joined: 9/05/12
SWG pre cu > all
Originally posted by simplius
Hahaha, great analogy.
Novice Member
Joined: 4/03/13
So wait , they are saying they got the engine to work ?
What patch did they do this ?
Spotlight Poster
Joined: 2/10/12
Getting the engine to work right? Sounds like a coder problem. Get new coders? lol I mean... seriously.
Originally posted by Dauzqul
coders cost money,,and GOOD coders prolly cost a lot of money
EA could pay them,,but they dont want to invest more in this game
swtor will survive, as it is now, or not
it has taken 3 years for STO to make their game stable, and they didnt waste the first year, by denying
their problems
STO barely survived that one, and their budget was more balanced , than swtor
Apprentice Member
Joined: 8/31/12
Originally posted by simplius
Then maybe, just maybe, when EA goes to an engine developer and says "We want to use your engine." and the engine developer says "This engine isn't even out of alpha yet, you really don't want it.", EA will say "Okay, we'll find something else." instead of "It doesn't matter, we can finish the engine ourselves.".
EA CEO John Riccitiello's on future microtransactions: "When you are six hours into playing Battlefield and you run out of ammo in your clip, and we ask you for a dollar to reload, you're really not very price sensitive at that point in time...We're not gouging, but we're charging."
that would be the optimal choice
and the kind of advice, i would expect from a professional
however,,if the king is a moron, his kingdom will suffer
Novice Member
Joined: 8/11/09
Originally posted by simplius
Ha I love this analogy. Spot on.
Playing: War Thunder, World of Warcraft, and Grim Dawn
Elite Member
Joined: 5/14/07
I believe in life before death... So dont forget to enjoy it while you still can.
Originally posted by simplius
Well, thats the problem... there arent any other tools available,,,
Sure there are tools, but then those are not really fit for the job either, like opening a tincan with a screwdriver it works, but you will spill some of the beans.
So either you choose the wrong or the broken tool and try to fix it in such a way that it works, or you create your own tool.
Actually the problem wasnt choosing the right tool, but choosing the tool with the most possibilities. Which by then was obviously the Hero engine. Sadly during development it worked out the wrong way because the hero development team failed to bring what they promissed.
So far the only smart ting seems to create your own engine like blizzard and Arenanet did.
And you know whats the most foolish thing, companies never seem to learn, as TES might be doomed equally to TSW as Zenimax is using the HERO engine...
Best MMO experiences : EQ(PvE), DAoC(PvP), WoW(total package) LOTRO (worldfeel) GW2 (Artstyle and animations and worlddesign) SWTOR (Story immersion) TSW (story) ESO (character advancement)
yes, they should have created their own engine
i think its the only game of this scale, where the dev team didnt do that
AFAIK, even EVEs engine is house made
this decision was taken by incompetent people, or it was taken, because swtor was meant to be a short term
moneygrab,,maybe a combination of those two
now we have this awesome sports car,,but its powered by a modified lawnmower engine
it looks cool, but its slow as...
Novice Member
Joined: 10/20/10
Ah blaming the engine now ?
Star wars IP was a World killer, and look what you have done with this IP...
They had the opertunity to create a universe that would have blown every fantasy mmo out of the water....
Space exploration
none of these goals where met....
This was a fuckup of epical proportions....and its the Developers who are at faulth.


text: Prev | Next Purchase this photo File photo/The Herald-Dispatch Volunteers and members of the United States Coast Guard work together at Harris Riverfront Park in June 1999 during the Ohio River Sweep. As boats unloaded the trash, a pick-up truck backed down to the dock to haul the trash up to the dumpsters. Gallery: Harris Riverfront Park through the years Jul. 16, 2013 @ 12:32 PM Harris Riverfront Park has seen many beautification efforts over the years. ()

subset: commoncrawl


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58 thoughts on "Vibration Machine Reviews"
What are your thoughts on the FreeMotion® Vertex™ Vibration Machine? Someone local is selling a used one for $1900 and I was wondering if its a good deal. I want something good but don't want to spend more than $2,000.
Have you reviewed the one Sears sells, Health Mark Osci Health Whole Body Vibration Machine $799
I had ACL replacement and MCL repair surgery and need to re strengthen my leg muscles and my knee and core.
WHere can I buy something that is actually affordable that works??
Hello, as a retired old fart physician, it is really nice to see someone as knowledgeable as you are fielding questions from consumers and providing such valuable answers.
I have poor balance, such as in walking tandem or heel to toe. I have just ordered a high quality balance board with an adjustable pivot height to be able to control degree of difficulty. Many have commented on the benefits of these balance boards for therapy to improve balance.
How effective do you think a vibration plate would be to improve balance? Would not a pivotal platform work better for this purpose, as it would more closely mimic a balance board? You say that you basically like the idea of pivotal units but there just are not many studies on them to confirm their benefits.
My wife has osteoporosis. We are both slim and not heavy weights. What would you recommend that we could both use? Cost is not an issue as long as we get a significant increase in features and benefits for the additional cost. If you were in my situation would you go with a pivotal unit for more help with balance? How impressed are you with improvement in bone density for those with osteoporosis using vibration therapy?
By the way, I just saw an interview with Dr. Joan Vernikos, formerly with NASA, who did a lot of work with astronauts studying their bone loss due to lack of gravity. She mentioned the use of a "sway plate" with high frequency and low intensity. Do you have any idea what machine she may have been referring to? The "sway" suggests pivotal, does it not?
Thanks so much for your help!
excellent and clear responses ….very balanced …I am 71 years old 120lbs 5'2″ healthy ….want to be stronger….no limitations ….. which vertical vibration machine should i consider… space and cost and safety are important
Hello !!! I have a problem and I want some answers….I just began to go to the gym and I paid for some sessions with the vibration machine called " Fitvibe". I've already done 3 and My instructor is always getting me on 55-60hz. I asked some other instructors and they told me that on the Power plate machine everything over 35hz can be dangerous for the whole body… Does this means that also with the Fitvibe a have to lower the frequency? I don' t know if the motion in Fitvibe is vertical or not. Can the 55 hz be harmfully for my body or for my head ?? Please answer me whatever you know about that because I am anxious…thank you in advance!!!
I would first like to thank you for how much help you and your website are. You help a lot of people.
I'm 61,5'6″, thin and have osteopoenia. I was going to buy the Nitrofit Personal because I have poor balance and have read studies showing that the pivotal action machines are the ones that help with balance in addition to improving bone strength. However, I don't want to take the chance that its pivotal action could be hard on my joints, esp. since I have had occasional knee pain in the past. Of the less expensive vertical machines in the $2000 range (eg.the 3G Cardio 3.0, or DKN® XG3) are there any that would be more helpful than the others with balance? By the way, I don't plan on doing lots of dif. exercises so the size or shape of the plate isn't that important. Thanks for any advice.
Do you have any information on Rock Solid RS3000. They say they are triangular oscillation but I didn't see anything on your site regarding that. Also, since they are on all the deal sites, I thought there would be more feedback. Thoughts?
RS3000 – includes 2 year warranty For home, gym, spa or fitness studio use – $2048.95
For home, gym, spa or fitness studio use. Includes a 2 year warranty. Features/Specifications:
*Motor Power 500W
*Power Voltage – 110V 15A
*Speed Range – 30 Levels
*Different Program Levels – 3 Types
*Frequency – 0-30Hertz
*Amplitude – 1-12mm
*User Weight Max – 150KGs / 330LBs
*Max Daily Recommended Use – 60 minutes (please consult your
physician for own recommendation)
*ROHS Compliant
I have been seriously looking for WBV machine for some time and the information available can be confusing, inconsistent, contradictory etc. I had been debating the purchase of a Hypergravity unit vs Sonci Life vs Turbo Sonic. When I came upon your site I learned of a brand I hadn't heard about previously – 3G Cardio AVT 5.0.
I feel comfortable that I can readily use a linear machine and the machines I'm considering are linear.
In your reviews it appears that you are focused on mechanical linear devices; is there a reason you haven't considered any of the sonic units? I would also appreciate your thoughts pros/cons of a sonic unit vs mechanical linear devices.
Thanks in advance
What is the difference between a vibrating machine and one that ocillates. Is one better than the others? What do most of the machines on your website do- vibrate or occillate?
thanks for a reply.
I would like to ask a couple questions. I bought the My5 but it does not have more than a 60 second run. Is this normal? The only other one I was able to actually try could be programmed to 12 minutes and had preprogrammed things.
Also, the My5 does not seem to have a shut off anywhere? Is that normal also? for it to just run and run?
Thank you
I am so confused. I am going to be 60, am approximately 100 pounds overweight.
Except for being overweight, I am really not in bad health. My knee has been
bothering me, but I attribute it to the fact I work at my computer at home for long
hours. I am not diabetic or have any heart conditions. I have some breathing
issues because of carrying the weight around, but nothing medical. I am looking for
something to help with circulation, getting toned, motivation for losing weight and
losing weight. I tried the ZAAZ15 and 20 at the mall today. I have to admit, I did
the 10 minutes on there, listened to the sales pitch and then told them I wanted to
thing about it. They were asking 2200 and being a single grandmother, I am leary of
that output without being more sure. I was amazed that my right knee was not
aching, no lower back pain. My sister and I walked the mall – very large mall – and
no pain. For the first time in months I was able to get up and down from chairs
easier and my knee was not aching….no ibuprofen today at all. We ended up back at
the kiosk and I was on for another 10 minutes taking to them. They told me that
they could to a better price on the 15. The only difference of the two is the step
counter and calorie counter, which I never trust on equipment anyway. I note you
made an observation of the platform handles limiting exercise space. Can you
recommend any other one that is in the 2000.00 price range that matches the criteria
you prefer, vertical, 45 Hz? Thank you so much…
I just sent you a message but forgot to give you details concerning our size.
I am 5′ 95 lbs, husband is 6'1″, 185 lbs, Father is 5'9″, 160 lbs, Mother 4'10", 85 lbs.
Main concern about her is that she has had 3 bone breaks in the past 18 months
(pelvic, wrist and compression fracture in back) so needing something that will be stable and she will be able to build bone density. Father needs to regain strength and build muscle. Husband and myself need to firm up and build muscle/strength. A bit confused about pivotal versus vertical, oscillating? It sounds like you prefer vertical.
I'm just wondering if you have reviewed the Hypervibe machine at all, it has some good feedback on it I just would be interested in your view of it.
Dr.fuji cyber wbv machine 099. Can u tell me that machine good for home use?
Funny you do not like the rectangular platforms of the DKN's. I have the Xg5. Something you overlook: This is not an oscillating machine. It is linear. If you want, you can sit/lay SIDEWAYS. Lots of room that way. You need to think outside the box. Or, rectangle, as it were.
Do you have any info on the zazz 20? I just tried one out at small kiosk, it is $2500 – how does this compare to others in this price range? Thanks!
I am researching WBV and would like to know what machine you recommend. Primarily I am looking into the machine because I am tall and thin and have osteopenia…the precursor to osteoporosis and am wondering if this will really help improve my bone density. My husband may also try it for weight loss, my children ages 16 through 25 would like to try it for strength and flexibility as they are all dancers and/or athletes. Which machine would you recommend our home use? Thanks
What kind of machine would you recommend for home use by my family? My dad, a 53 year old who works out for 30 minutes a day and is very healthy, would use it the most. Also in the family is: my 49 year old mum, 18 year old brother, 16 year old brother, and two 13 year old sisters. Everyone is quite athletic and healthy. Thank you!
I didn't see a review of another machine called TheraPlate Revolution. (Website is http://www.theraplate.com/) Looks like a strong, basic machine for $1600.00 to $1900.00. Any thoughts?
My husband and I saw these vibration machines at the state fair in Orange County last summer. My husband wanted to buy one of the machines but I had a bad feeling about spending that much money there. Is it safe to buy from them? We are considering getting one this year when the fair comes back. I read the about us section on your site that explains your background as store owners. What is the advantage of buying at a store? It seems like the machines at the fair are a good deal and most of the machines on this site are expensive. Whats the difference?
I'm here doing research before purchasing. It's all so confusing. I am partially paralyzed on my right side from a broken neck almost 40 years ago. I have balance issues. I'm weak and need to build strength. I read about how there should be two motors, and a large base. I have no idea how to choose a machine. I will be willing to look at a mid-range priced model, but will never be an athlete so perhaps a lower priced model will do? The handles will be important for my safety. What about the noise level? I've heard that some machines are noisy. I'll be trying to get my insurance company to pay for this so need a recommendation for mid-price and maybe lower priced models.
I understand the free motion vertex is made by power plate, or is at least the same platform. Have you tried it yet? We are looking to get one for home use, 5 users ranging from 8-42 years, 80-220 lbs. Which one would you choose?
My husband is suffering of Parkinson disease. Which machine you will suggest to buy (not too much expensive one) ? Thanks you for answering.
Amazon has this one listed for a cool 299, price drop from 999.00 the reviews on amazon are mostly good however most of them have never used a vibration machine before.. so that's not saying much…
Im looking for a machine I can use at home, any suggestions?
does 3gCardio AVT5 have class 1 or class 2 FDA medical classification?
How doess a VT15 or VT20 whole body vibration unit compare with the 3gCardio units? Do they vibrate on the same plane–i.e. same type of vibration motion?
It would be really helpful to someof us to know where these product are made. I/we prefer US products to CHINA.
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text:
In this invention a novel antigen is provided for screening patients suspected of having celiac disease. The antigen is prepared by starting with human placenta tissue which is perfused with a herpes and collagenase buffer to obtain a signle cell suspension. This suspension is enriched to obtain an enriched protein portion and then separated out to obtain an embryonic celiac antigen (ECA). This ECA is used with serum from patients to effect binding of ECA with IgA in the serum while applying a human IgA antibody to the serum. The results are then read on a spectrophotometer to confirm or negate the presence of celiac disease.
1. A method for the isolation of an antigen from human placenta tissue to be used as a screening assay for celiac disease comprising:
a) cleaning and washing said placenta tissue with HEPES buffer;
b) digesting the said placenta tissue with collagenase buffer to obtain a placenta cell population in suspension, said placenta cell population containing cytotrophoblast cells;
c) enriching the cytotrophoblast cell population in said suspension via a 0-30% discontinuous Percoll gradient;
d) extracting protein from said enriched cytotrophoblast cells with lysing buffer, extraction buffer and via ultracentrifugation to obtain a protein portion;
e) isolating embryonic celiac antigen (ECA) from said extracted protein by applying said protein portion to an ion exchange DEAE Sephadex column followed by size exclusion gel chromatography; and
f) identifying by SDS-PAGE and western blotting embryonic celiac antigens comprising protein molecules with an isoelectric range (pI) of from 5.1-5.8 and an apparent molecular weight selected from the group consisting of 55 kd, 65 kd, and 110 kd.
This invention relates to an antigen and production thereof and, more specifically, to an antigen useful as a screening assay for celiac patients.
There are known various procedures proposed for identifying celiac disease in both adults and children. Some of these procedures are outlined in the articles "Preciptins to antigens of wheat and cow's milk in celiac disease" by Heiner D. C., Lahey M. E., Wilson J. F., et al., published in 1962 in J. Pediatr 61,814; "A reliable screening test for childhood celiac disease: fluorescent immunosorbent test for gliadin antibodies. A prospective multicenter study," published in 1983 in J. Pedlar. 102:655-60; "Gliadin antibodies in celiac disease" published in 1983 in J. Pediatr 102:711-2. There are also several other papers and publications concerned primarily with tests for detecting celiac disease. While some tests have been found to be somewhat effective and at least sometimes accurate there is a need for a more reliable and uniform interpretation of screening assays for celiac patients.
Childhood Celiac Disease (CD) is a condition characterized by malabsorption and growth disturbances in association with a specific histological lesion of the small intestine. Patients with malabsorptive symptoms have been found to date to the second century AD. In 1950's Dicke, a pediatrician found the relationship between grain consumption and severe malabsorption condition. He noticed that during WWII children in Holland with malabsorptive symptoms improved when wheat and rye flour were unobtainable and that the condition reappeared after wheat flour was again made available. He and his associates were also credited with identifying gluten, the water-insoluable protein fraction in wheat, as the toxic dietary substance responsible for the syndrome, noting that symptoms subside in response to gluten elimination.
Recently, the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) revised the criteria for the diagnosis of gluten sensitive enteropathy (GSE), celiac disease. The previous criteria set forth in 1969 required at least three small intestinal biopsies. The salient histological features of CD were required at the initial suspicion of the enteropathy. The histological findings were expected to resolve following a period of gluten elimination and be reinduced during a gluten challenge phase of establishing the diagnosis. Since then, the success of newer diagnostic markers--antigliadin, antireticulin and antiendomysial antibodies--in serving as indicators of active disease has prompted a revision of the criteria. Current requirements include a characteristic histologic appearance of the mucosa at the time of presentation with resolution of symptoms following gluten elimination. The presence of the previously mentioned circulating antibodies at time of diagnosis and disappearance following gluten withdrawal adds weight to the diagnosis especially in those who are asymptomatic.
Few controversies in the discipline of Pediatric Gastroenterology are viewed with less emotion than the selection of the best serologic screening test for celiac disease. The discovery of circulating antibodies to gliadin (AGA), reticulin (ARA) and the endomysium (EMA) have brought us closer to the discovery of a simple non-invasive screening test for establishing the diagnosis; but none are universally accepted as being pathognomonic indicators of the condition. Although none can be considered, as yet, pathognomonic, the serologic antibodies can still be used for screening and aid in diagnosis if one realizes their limitations and interprets their presence or absence in light of the clinical situation faced. We are approaching a better understanding of their roles and interrelationships.
The above mentioned present day serological markers are circulating antibodies which serve as one piece of evidence for the immunological nature of the disease. Circulating antigliadin antibodies (AGA) represent antibodies to the cereal protein, which presumably is absorbed intact across the intestinal mucosa. AGA have been extensively investigated since initial descriptions appeared in the late 1950's and early 1960's. Techniques for detection have evolved over the years varied from precipitating antibodies to cereal proteins, microimmunodiffusion, radioimmunoassay, binding serum antibodies to wheat grains and detection by fluorescent horse antihuman IgG. The first ELISA method appeared in 1977 and is defined and described in detail in Hekkens W. T., Van Twist M.: Physiological role of antigliadin antibodies and their appearance in celiac disease; Chorzelski T. P., Beutner E. H., Kumar V., Zalewski T. K., eds. Serologic Diagnosis of Celiac Disease. Cleveland:CRC Press, 1990: 2A: 21-58 and Hekkens W. T., VanLems-Kan P. H., Rosekrans P. C. M.: Bepaling van gliadin antistoffen met de ELISA-Techniek. Ned Tijdschr Geneesk 121, 1908, 1977. The differences in techniques for detection led to problems with standardization and therefore reproducibility between studies. Several authors have attempted to improve the sensitivity of the method of detection by using a diffusion in gel ELISA as described in Lindberg T., Nilsson L., Borulf S., et al.: Serum IgA and IgG gliadin antibodies and small intestinal mucosal damage in children. J Pediatr Gastroenterol Nut 4, 917, 1985. It is understandable that even similar techniques such as ELISA may yield different results because gliadins are a complex mixture of proteins that contain at least 40 different components for a single variety of wheat. Several authors have attempted to improve the sensitivity of detection method by using gliadin fractions or peptides as antigens. In one study, different gliadin peptides were found to have differing reactivity to serum antibodies of untreated celiac patients. However, other authors report that there are no demonstrable differences in ELISA values using different gliadin fractions as antigens. Discordant results, therefore, have been reported despite the use of similar methodologies such as ELISA.
Further controversy exists regarding the value of the specific class of AGA antibodies in the diagnosis of celiac disease. Some investigators advocate IgG class AGA whereas the IGA-AGA are favored by others. In studies using purified gliadin peptides as antigens in RIA or ELISA, IGA antibodies seem to have greater specificity to celiac disease and IgG antibodies seem to be more sensitive. The usefulness of gliadin antibodies for diagnosis, however, is open for criticism because their sensitivity and specificity varies so much from study to study. Figures for specificity range between 65-100% for IgA antibodies and 50-100% for IgG antibodies. Sensitivity reports for IGA antibodies range between 52-99.9% and for IgG between 82-100%/Unfortunately, IgG antibodies can be found in normal controls as well as other disease controls such as Crohn's disease, liver disease and other G.I. disorders. IgA antibodies, on the other hand, although being specific for celiac disease, are not found in all celiacs. Additionally, IgG are found to increase with age in normal controls making them unsuitable for diagnosis in older age groups. The combined use of the two classes of antibodies, therefore, has been proposed by some authors as improving the sensitivity and specificity of the AGA for diagnosis. At least one recent report did not find an advantage in combining each. The merit of using the IgG AGA seems to be in its ability to discriminate those 3% of celiac patients who are also IgA devicient and therefore would not be identified by screening for IgA AGA.
Gluten challenge is followed by an increase in titres of both IgA and IgG-class antibodies and titres fall with gluten exclusion. IgG antibodies take longer to decline than IgA antibodies. They may take more than six months to decline whereas IgA antibodies decline by 2-6 months. Their appearance is stated to occur before symptoms become overt. IgA AGA antibodies are therefore suitable to monitor dietary compliance and also response to gluten provocation.
Antireticulin antibodies (ARA), on the other hand, have been investigated since initial reports in the 1970s. The ARA were first described as antibodies reacting with connective tissue fibers around hepatic sinusoids and blood vessels as well as perilobular, periglomerular and occasional glomerular staining of the kidney and also for fine staining of the stroma between gastric glands. ARA reacted with connective tissue of both rat and human organs and appeared to be directed against reticulin fibers in these tissues. The antibody was best detected by indirect immunofluorescence using rat liver and kidney as the substrate. The ARA do not react with type III colagen, noncollagenous reticulin components or fibronectin and they seem to be specific for other unidentified connective tissue components. Several staining patterns by indirect immunofluorescence were found to occur. An R 1 pattern was exhibited by celiac and dermatitis herpetiformis patients. This pattern was characterized by staining of peritubular and periglomerular fibers in the kidney and fluorescence in portal tracts of rat liver. In contrast to other ARA subtypes, it also reacted with human tissues, although some authors disagree to the human expression. They insist that the reticulin antigen are specifically expressed in rodent but not in primate tissues. ARA can be of the IgG or IgA class but IgM antibodies do not occur. IgG antibodies usually occur in conjunction with IgA ARA. The specificity of IgG-ARA for GSE is controversial. The IgA-class ARA seem to be disease specific and sensitive indicators of gluten-sensitive enteropathy. The range of specificity reportedly lies between 59-100% and sensitivity between 30-95% indicating somewhat similar sensitivity and specificity as the IgA-AGA, however, some authors may argue that the R-1 ARA are probably more specific for celiac disease than the gliadin antibodies. However, the R-1 ARA also have been reported in patients with Crohn's disease and occasionally with other conditions. The significance of the R-1 ARA is uncertain, however, the association between it and untreated celiac disease is well established and it will disappear from the circulation following the start of a strict gluten-free diet. Sensitivity and specificity reports are subject to differences in populations studied. For example, some exclusively looked at specific populations such as India, where one does not expect to find patients with inflammatory bowel disease, the authors found that R-1 ARA were 100% specificity with 85% sensitivity.
A third group of circulating tissue antibodies, the endomysial antibodies (EMA), are gradually gaining acceptance as sensitive and specific markers of celiac disease. These are primarily IgA antibodies directed against the intermyofibril substance of the smooth muscle which may correspond either to a reticulin-like structure or a surface component of smooth muscle fibrils. This antigen may also be expressed around lamina propria structures around intestinal crypts, muscularis mucosa and smooth muscle fibers to which "human jejunal antibodies" are directed. Unlike R-1 ARA which is reported to react with human and various rodent tissues, EMA has been demonstrated to be species-specific, reacting only with the endomysium in the gastrointestinal tract of primates. They are detected by indirect immunofluorescence using monkey esophagus tissue sections. The sensitivity and specificity of the EMA approaches but is not 100%. Some false positive cases have been reported. A case of cows' milk protein allergy and one with Giardia lamblia have been reported. In addition, one celiac patient was discovered who was IgA and IgG EMA negative on presentation and during gluten challenge. Therefore, we believe that false negative cases are less common than false positive cases but that they do exist and will continue to be reported. Absence of EMA may be more frequent in celiac patients younger than two years than in older individuals. In a recent report, three children with positive and four with weak positive results did not have celiac disease. Sensitivity was found to be 100% with specificity at 97%. In another study which compared EMA, AGA and ARA in an Israeli group of celiac patients, specificity was 98% and sensitivity was 97%. In this study, the EMA appeared to be the most reliable serologic marker for the diagnosis of celiac disease. The positive predictive value of the EMA and ARA were comparable (97 and 100% respectively), however, the EMA had the highest negative predictive value (98%). Additionally, EMA were found to be more diet sensitive. By three months of gluten withdrawal more children were negative for EMA than AGA or ARA. High EMA titres appear quicker in response to gluten challenge. No serologic test at present therefore enjoys a 100% sensitivity and specificity. However, of all the markers, we believe the EMA is presently the best serologic test based on our interpretation (bias, if you will) of currently available data.
The occurence of false positive and false negative results with the above markers requires some comment. Deficiencies exist with the uniformity of interpretation of the methodologies of detection of the above antibodies using indirect immunofluorescence. When one encounters reports of false negative cases in using indirect immunofluorescence, one is tempted to question the technique and interpretation of the test system. Also, what titre should be considered positive? Should weakly stained sections at low serum dilution be considered positive? One also needs to consider whether the patient is IgA deficient when IgA AGA, ARA or EMA are being used. Indeed, some investigators advocate that IgA AGA do not offer advantage over IgG AGA and that subjects with isolated selective IgA deficiency and celiac disease would be better picked up by screening for both IgG-AGA and IgG-ARA since IgG-AGA is less specific and are commonly found in non-celiac subjects with selective IgA deficiency.
When one discovers false positive cases for EMA or either AGA or ARA, one wonders whether these may be latent celiacs. Truly, the antibodies are demonstrated in these cases but why they are present remains an enigma. Maybe one should not consider this as "false" positive. Only follow-up studies will determine whether these individuals develop celiac disease. Indeed, this type situation has been recently addressed. Seven of 25 patients exhibiting ARA or AGA and having normal small-bowel mucosal villous architecture at presentation were found to subsequently develop villus atrophy after a follow-up period of 1-7 years. Patients who are "false" positive for EMA may also prove to develop the histopathologic features of celiac disease in the future. Future studies will elucidate issue. With regard to false negative cases, however, there seems to be very few published cases of celiacs who do not possess the EMA.
Since it is becoming generally accepted that subclinical celiac disease is common in the general population, the use of screening tests in clinical studies is becoming increasingly important. Many patients who are free of major symptoms are being identified to possess the typical histological features of celiac disease. They are discovered during studies addressing the incidence of celiac disease using serologic markers as screening tests in high risk populations such as family members, patients with diabetes, short stature or in the general population. When one considers the silent celiacs, the true incidence of the disease in the U.S.A. and Europe may be higher that reported. It was found using EMA that the rate in family membes was 8% and those with diabetes was 4% and and short stature 1.7%. These are similar figures to those reported in European studies. However, the incidence in patients with major symptoms was only 1.29 per 10,000 live births which is much lower than the mode of 1:1000 reported in European centers. Studies are being awaited with great interest that address the incidence of silent celiacs in both populations. Certainly, the incidence of those with major symptoms is much less than Europe.
The efforts to discover the best screening test for celiac disease and efforts to diagnose every last celiac have bastly overshadowed and outnumbered studies of the pathophysiology of the disease. While gliadin antibodies were reported some 30 years ago, work on characterizing the toxic component(s), both in vivo and in vitro, has not been completed. The molecular mechanism behind the pathogenesis of CD is still unsettled but the immunological aspects of the disease have attracted great attention both in the proposal of pathogenetic mechanism and in the efforts to establish a serodiagnostic test for CD.
The different gliadin polypeptides can be separated into four groups, the alpha, beta, gamma and delta fractions by electrophoresis at acidic pH. The molecular weight of a toxic gliadin in the gamma fraction has been recently reported to lie between 35-45 Kd. Many hypothesis have been proposed to explain the pathomechanism of the observed gliadin induced enteropathy. Studies have been limited to investigations of lymphocyte populations and cytokine production or toxic peptides acting as lectins which induce cell death. These seem promising, serve to direct future studies at immunologic reactions associated with tissue injury but definitive statements regarding pathophysiology cannot yet be made. Furthermore, the mechanism of generation of the antigliadin is uncertain. There are even less reports which address the reticulin and endomysial antibodies. One expects that the identification and characterization of the antigens to which these antibodies are directed will help in understanding the pathomechanism of the gluten induced enteropathy. It is hoped that future work will be directed at pathophysiology; not only identification and diagnosis. It is expected that characterization of the above antigens will aid in understanding the pathophysiology of the disorder and may even lead to a better understanding of the above diagnostic tests.
It is therefore an object of this invention to provide a novel antigen for screening tests for Celiac Disease devoid of the above noted disadvantages.
Another object of this invention is to provide a novel method for the production of a novel antigen to be used to identify Celiac Disease.
Still Another object of this invention is to provide a novel process for the use of a novel antigen useful as a screening assay for Celiac Disease patients.
A further object of this invention is to provide a method to identify and isolate a novel screening assay for Celiac Disease.
Another object of this invention is to provide a reliable relatively simple screening process for detecting Celiac Disease.
Still a further object of this invention is to provide a novel antigen useful in screening patients suspected of having Celiac Disease.
These and other objects of this invention are accomplished, generally speaking, by a method for the identification, isolation and production of an antigen starting with a portion of human placenta. This placenta is perfused using a hepes and collogenase buffer, enriching the resulting composition by condensing it and extracting protein therefrom. The protein is then isolated and prepared for use as an antigen to identify Celiac Disease. This antigen will bind to the IgA contained in the sera of patients with Celiac Disease. This novel antigen is designated as Embryonic Celiac Antigen (ECA). The ELISA technique is well known and is described in detail in the following publications: Hekkens, W. T., Van Twist M.: Physiological role of antigliadin antibodies and their appearance in Celiac Disease. In Chorzelski T. P., Beutner E. H., Kumar V., Zalewski T. K., eds. Serologic Diagnosis of Celiac Disease. Cleveland:CRC Press, 1990: 2A: 21-58; and Hekkens W. T., VanLems-Kan P. H., Rosekrans P. C. M.: Bepaling van gliadin antistoffen met de ELISA-techniek. Ned Tijdschr Geneesk 121, 1908, 1977. This ELISA technique will be used as the methodology for utilizing ECA antigen as a screening test for Celiac Disease.
Thus, this invention provides:
(1) a novel antigen (ECA) to be used in screening for Celiac Disease;
(2) a novel method for preparing ECA; and
(3) a novel screening process using ECA as the antigen.
The ECA screening process of this invention will replace the EMA and ARA (indirect immunofluorescence) procedures above discussed. The accuracy of detecting Celiac Disease using ECA alone or with other procedures is increased about 15% to 20% over prior celiac detecting procedures. The placenta that is used is any available human placenta but the embryonic fetal portion of the placenta is used as the starting material. The eventual protein that is isolated and prepared for use as the antigen has a pI of 5.1-5.8 and a molecular weight of at least 55 Kd. Those proteins having a lower molecular weight did not function in the present invention. Competition binding assay was used to identify the separated proteins. (Three single polypeptides with molecular weight higher than 50 Kd 55 Kd-65 Kd and 110 Kd ! and Isoelctric point 5.1-5.8 demonstrated inhibition of binding of Celiac Disease patient serum IgA/IgG on the monkey esophagus and rat kidney/liver. Seven other monopolypeptides with molecular weight of less than 50 Kd inhibit binding to Giladin).
Once the antigen of this invention (ECA) is prepared, sera of suspected celiac patients is added to well plates on which ECA has been immobilized. After incubation, excess antibody will be washed away and a horseradish peroxidase labelled secondary antibody directed at human IgA/IgG will be applied to the wells. The color changes will be read by the ELISA spectrophotometer reader. The antigen ECA will bind to the IgA contained in the sera of patients with Celiac Disease and this binding confirmed by the ELISA technique or procedures well known and described in Hekkens W. T., Van Twist M.: "Physiological role of antigliadin antibodies and their appearance in Celiac Disease", and "Serologic Diagnosis of Celiac Disease", Cleveland Press, 1990: 2A: 21-58.
As above noted, in our search for a Celiac Disease antigen, we found that placenta tissue sections expressed an antigen which binds to the IgA contained in the sera of patients with Celiac Disease. We then identified, isolated and purified this antigen and designated this protein molecule as an Embryonic Celiac Antigen (ECA).
Utililzation of the newly discovered antigen: Develop a serologic diagnostic test for Celiac Disease which can be easily performed and objectively interpreted as a routine laboratory test. The isolated ECA protein will be immobilized as an antigen on a 96 well plate. Sera from patients suspected for Celiac Disease will be applied to the wells and assayed for the presence of reacting antibodies by Enzyme Linked Immunoassay (ELISA). The ELISA technique will be developed as the methodology for utilizing the ECA antigen as a screening test for Celiac Disease.
We recently identified that an IgA protein in the sera of patients with Celiac Disease binds to an antigen expressed on placental tissue sections. In those cases of IgA deficient celiacs an IgG antibody was found to bind to the placenta sections. Microscopic evaluation of placental tissue sections indicate that the antigen was expressed on chorionic villi, specifically on their surface and within the cytoplasm of these cells. We then established a primary culture of placental cell line grown first in 24 well plates and subsequently expanded to a 102 cm tissue culture flask. The cells in culture were found to express the ECA. An inhibition binding assay, however, demonstrated that the antigen was not secreted into the culture media by the placental trophoblast cultured cell line. The antigen appears to be located in the cytoplasm and on the cell surface.
Collected placenta culture cells were lysed using lysis buffer. Proteins were extracted from the culture lysate using ammonium sulfate precipitation. After dialyzed with PBS, the precipitated proteins were identified by SDS-PAGE gel electrophoresis. Proteins extracted from the placental cell culture lysate were applied to SDS-PAGE Gel electrophoresis with a reducing and non-reducing condition reagent. Subsequently, proteins were transfected to nitrocellulose membrane and finalized with Western Blotting procedures described in Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature(London)227:680, 1970.
Ten protein molecules were identified by the IgA contained in the sera of celiac patients. The molecular weights ranged from of 17 Kd-110 Kd. In further experiments, using an inhibition assay, we proved that the isolated low molecular weight proteins (ranging 17 Kd-45 Kd ) are gliadin-like polypeptides. These antigens were found to inhibit the binding of the IgA/IgG in the sera of celiac patients to gliadin antigens. During further identification of the rest of the antigens, we found two interesting proteins isolated from the placental cell line lysate. One exhibited a molecular weight of 110 Kd , and the other 65 Kd. These two proteins completely inhibited binding of IgA of Celiac Disease patients to rat kidney (Reticulin-like) and monkey esophagus (Endomysium-like). These two polypeptide were identified to have isoelectricpoints of 5.1 and 5.8. Using high pressure liquid chromatography (HPLC) we were able to purify these two putative Embryonic Celiac Antigens. An inhibition assay indicated that the 110 Kd protein inhibits at low concentrations binding of celiac patients IgA to rat kidney. The 65 Kd protein, on the other hand, inhibited completely the binding of IgA to the monkey esophagus. Both protein molecules had 60-70 Kd % of cross inhibition activity to either the rat liver or monkey esophagus. The 110 Kd protein is the heat labile protein and tends to break down to a 55 Kd molecule without losing the antigen binding activity.
We subsequently isolated the protein by the following two step isolation method. We first apply the crude extract protein isolated from the cultured placental cell lysate to a DEAE sephadex column and Gel Chromatography SEPHACRYL™ S-200 HR Column (2.6�60 cm) and equilibrate it with phosphate buffered saline pH 7.3. We purified the 110 Kd protein in fraction collection number 40-45 Kd and 65 Kd protein was purified in fraction collection number 55-60. The fraction samples were collected and purity was proofed by SDS-PAGE gel electrophoresis. Protein samples collected were concentrated and frozen for further usage.
The primary placenta culture cell line was established in our laboratory and was maintained at -70� C. until further usage. Using the fresh placental lysate additional 110 Kd was isolated. The frozen material, on the other hand, yielded more of the 55 Kd molecule which also is identified by the sera of celiac patients. The 110/55 Kd protein complex has the capacity to inhibit 100 Kd % binding of the celiac sera to rat kidney. We therefore hypothesize that this is a reticulin-like antigen. This molecule complex also inhibits 60% of the binding activity of celiac sera to the monkey esophagus. In distinction, the 65 Kd molecule inhibits 100% binding of celiac sera to the monkey esophagus. However, this molecule inhibits only 60% binding activity of celiac sera to the rat kidney. We therefore concluded that this is an endomysial-like antigen and designate it as the ECA.
The above putative Embryonic Celiac Antigens isolated from a cultured human placental cell line does not bind to the IgA or IgG contained in the sera of normal patients, those with inflammatory bowel disease or other immunologic conditions.
1. Sample Preparation--starting with human placenta:
a. Wash the cells twice with PBS.
b. Extract the washed cells with extraction buffer.
Extraction buffer: 10 mM Tris-HCl, PH7.2 0.15M NaCl, 0.02% NaNO3 5 mM Iodoacetamide 2% NP 40, 1 mM PMSF 5 mM MgCl2
c. Centrifuge at 27000 g for 30 min, collect supernatant and store the extracts in liquid nitrogen.
a. Add sodium dodecyl sulfate (SDS) sample buffer, with or without DTT, to the sample solution and boil the final sample solution in water bath for 5 min.
b. Perform SDS-PAGE (polyacylamide gel electophoresis) in a Laemmli system with slab gel containing 10% or 12% acrylamide.
(Ref. Laemmli, Nature 227, 680-685, 1970)
c. Do silver staining with the gel.
(Ref. Heukshoven, J. and Dermick. R., Electrophoresis, 6. 103, 1985)
3. Western Immunoblot Analysis
a. Transfer the proteins to nitrocellulose membrane in Bio-Rad Trans-Blot cell.
b. Stain the gel with Coomassie brilliant blue after the transfer to confirm the uniformity of the transfer.
c. Block the membrane with the TBST buffer (containing 0.1% Tween 20 in PBS) for 1 hr at room temperature.
d. Incubate the membrane with 1:500 dilution of CD patient serum (EMA titer 1:6000) in TBST buffer for 2 hr at room temperature.
e. Wash the membrane three times with TBST.
f. Incubate the membrane with a 1:2000 dilution of the goat antihuman IgA conjugated to horseradish peroxidase (Sigma, A0295) in TBST for 2 hr at room temperature.
g. Wash the membrane three times with TBST.
h. Incubate the membrane with ECL detection reagent for 1 min and expose to XAR5 X-ray film (Eastman Kodak, Rochester, N.Y.)
(1) Towbin, H., T. Staehelin and J. Gotdin, Proc. Natl. Acad, Sci., U.S.A. 76/9 pp. 4350-4354 (1979)
(2) Byron Batteiger, et al J. of Immunol. Methods. 55, 297-307 (1982)
(3) Amersham Corporation ECL Western Blotting protocol booklet
Placentas were discarded tissue obtained from the Pathology department. The placenta were perfused with collagenase buffer containing 0.05% trypsin after flushing with Hepes buffer. Single cell suspensions were collected and different cell types were isolated and condensed from the mixture into various subpopulations based in their density characteristics using differential Percoll gradients. Cells from each isolation zone were cultured in RPMI 1640 medium conditioned with 20% fetal bovine serum (FBS). Non-adherent cells were removed 12-24 hours later.
After rinsing with HEPES buffer, appropriate placental vasculature was catheterized using a 16 guage angiocath, perfused with HEPES buffer for 1/2 hour, followed by a collagenase buffer digestion solution containing 0.05% trypsin for one hour. The placenta tissue rich in chorionic villi was minced with scissors. The tissue mixture was allowed to digest while being constantly stirred and incubated at the 37� C. for a minimum of one hour. The single cells were obtained by teasing apart the perfused placenta with forceps. The cells were pelleted by centrifugation (400� g) and washed twice with Fischer's medium.
Placental cells were obtained using density centrifugation with Ficoll-Hypoque and discontinuous Percoll gradient. A discontinuous Percoll gradient was made by carefully layering 70%, 50%, 30% Percoll (vol/vol) with 1�Dulbecco's (Gibco) PBS in centrifuge tubes. Pelleted cells were resuspended in 10 mL Fischer's medium, layered over the Percoll gradients and centrifuged at 800�g for 30 minutes at room temperature.
Several zones appeared in the Percoll gradient: 0-30% tropholblast cells, Stroma cells, small vessels and vilous fragments, connective tissue elements, 30-50% Polymorphonuclear leukocytes, 50-70% Red blood cells, and mononuclear cells; Pellet; Red blood cells. The 0-30% interface was separated and washed twice with Fischer's medium. Cells were plated into 25 cm2 flasks with RPMI culture media containing 20% fetal bovine serum (FBS) and incubated at 37� C./5% Co2 /humidity-90% for 12-24 hours. The nonadherent cells were removed from the culture. The adherent placental cell line was propogated and expanded on 90 cm2 tissue culture flasks for further experiments.
The collected placental culture cells were lysed using lysis buffer which contained 10 mM tris base, 5 mM Mg Cl2 ; 0.15M NaCl; 2% NP-40; 5 mM iodoacetamide, 0.02% NaNo3, 1 mM PMSF (phenylmethylsulfonyl flouride) and adjusted the pH to 7.0.
To identify protein contained in the placental culture cell line, the protein fraction was extracted from the placental cell culture lysate and applied to SDS-Page gell electrophoresis. The protein was applied to SDS-PAGE gell electrophoresis with a reducing and non-reducing condition reagent. Subsequently protein was transferred to Nitrocellulose membrane and finalized with Western Blotting. Ten protein molecule antigens were identified by the IgA in the sera of patients with Celiac Disease, at molecular weights ranging from of 17-110 Kd. In further experiments using an inhibition assay we proved that the isolated low molecular weight proteins (ranging 17 Kd-45 Kd) are giladin-like polypeptides. These antigens were subsequently found to inhibit binding of the celiac patient sera (IgA/IgG) to Gliadin antigens. During further identification of the rest of the antigens, we found 2 other proteins isolated from the placental stromal cell line lysate. One exhibited a molecular weight of 110 Kd, and the other 65 Kd. These proteins completely inhibited binding of IgA of Celiac Disease patients to the rat kidney (Reticulin like) and monkey esophagus (Endomysium like). These two polypeptide were identified to have isoelectricpoints of 5.1 and 5.8. Using high pressure liquid chromatography we were able to separate these two putative Embryonic Celiac Antigens. An inhibition assay indicated that the 110 Kd protein inhibits completely at low concentrations binding of celiac patients IgA to rat kidney/liver. The 65 Kd protein, on the other hand, inhibited completely the binding of IgA to the monkey esophagus. Both protein molecules had 60-70% of cross inhibition activity to the other substrate, i.e. rat liver or monkey esophagus. The 110 protein is the heat labile protein and tends to break down to a 55 Kd molecule without losing the antigen binding activity.
We subsequently isolated the protein by the following method from above mention placental culture lysate. Briefly, the method of isolation involves a two step isolation in which we use the crude extract protein isolated from the cultured placental lysate. The protein contained in the placenta lysate extract were first applied to a DEAE sephadex column (1.0�10 cm) previously equilibrated with 0.05M tris/HCL, pH 7.4. The absorbed components were subsequently eluted with 0.05, 0.1, 0.2, 0.3, 0.4, 0.5M NaCl in 0.05M tris/HCL, pH7.4. The fractions were collected (2.0 mL each) and concentrated using ultrafiltration (Amicon CENTRICON-3™). The concentrated DEAE samples were applied to the SDS-PAGE gell electrophoresis for protein identification. 0.2M in 0.05M tris/HCL, pH7.0 eluated a 110 proteins, 0.5M NaCL in 0.05M tris/HCL, ph7.0 eluated a 65 Kd major protein. Concentrated Chromatography protein fraction were applied and identified by the SDS-PAGE gell electrophoresis. DEAE chromatography collected samples were applied to the Gel Chromatography using SEPHACRYL™ S-200 HR Column (2.6�70 cm) and equilibrated with phosphate buffered saline pH 7.3. Samples were collected with 0.33 mL/min flow rate and 1 mL per tube. We purified the 110 protein in fraction collection number 40-45 and 65 Kd protein were purified in faction collection number 55-60. The fraction samples were collected and the purity were proven by SDS-PAGE gell electrophoresis. Purified and collected protein samples were concentrated and frozen for further usage.
The new antigen was isolated in our laboratory from a cell line established from placenta tissue. Two examples of its efficacy are: (1) Indirect immunofluorescence staining indicates that this cell line expresses the antigen which binds to antibodies found in the sera of celiac patients, (2) the protein isolated from crude extract of the primary placenta cell line binds as demonstrated by electrophoresis and Western Blotting to the IgA antibodies in the sera of celiac patients. 0.25 mg of the isolated ECA having a molecular weight of 65 Kd will inhibit binding of celiac patients serum to monkey esophagus or liver. On the other hand, 0.25 mg of isolated ECA having a molecular weight of 110/55 Kd will inhibit binding of celiac patients sera to rat kidney or liver but will not inhibit binding to monkey esophagus.
The screening process: Since the clinical presentation of Celiac Disease is quite variable, patients with symptoms such as chronic diarrhea, bloating, abdominal distention, short stature, growth disturbances and diabetes may require screening. Early diagnosis is important since treatment with a special gluten-free diet seems to reduce the risk of subsequent gastrointestinal malignancy. The diagnosis of Celiac Disease has required intestinal biopsy. A reliable noninvasive test for the disease has long been sought. The isolated ECA which binds to the sera of Celiac Disease patients is a promising screening test. The ECA will be immobilized on 96 well plates. The sera of suspected celiac patients with the above symptoms will be added. After incubation, excess antibody will be washed away and a horseradish peroxidase labelled secondary antibody directed at human IgA/IgG will be applied to the wells. The color changes will be read by the spectrophotometer.
Below is a general outline of the procedure for producing the novel antigen of this invention:
1. Primary Placenta Cell Culture:
Wash, perfuse and digest placenta tissue with hepes buffer and collagenase buffer--Single cell suspension preparation.
2. Enrichment and concentration of the adherent cells from the cell suspension using discontinuous Percoll gradient.
3. Protein extraction from the primary placental cell line using an extraction buffer and ultra-centrifugation.
4. Embryonic Celiac Antigen (ECA) Identification:
Placental cell extracts were applied to SDS-PAGE gell Electrophoresis to separate the proteins. Separated protein were transfered to the nitrocellulose membrane (Western Blotting) incubated with the serum of patients with Celiac Disease. Horseradish-peroxidase conjugated rabbit anti human IgA is applied to identify the binding of celiac patients IgA to the antigen. Ten monocomponent polypetides were detected with Celiac Disease sera on nitrocellulose membrane. Three single polypeptides with molecular weight higher than 55 Kd (55-65 Kd and 110 Kd) and Isoelectric points 5.1-5.8 demonstrated inhibition of binding of celiac sera on the monkey esophagus and rat kidney/liver. Seven other monopolypeptides with molecular weights of less than 55 Kd did not inhibit binding.
5. Embryonic Celiac Antigen (ECA) Isolation:
Initial separation of placental primary culture cell extract were done by injecting to the gell filtration (size exclusion) of HPLC Column from Bekman "Ultra Sphero Gell" (SEC 3000) size 5 micron. The column size is 7.5 mM�300 mM. Competition binding assay was used to identify the proteins. Separated proteins were also applied to the SDS-PAGE and Western Blotting to identify the purity and molecular weight of the proteins.
6. Embryonic Celiac Antigen (ECA) Isolation (Large volume separation using the gel chromatography):
Separation is performed using DEAE ion exchange chromatography and SEPHACRYL S-200 HR Column (2.6�)cm and equilibrate with phosphate buffered saline pH7.3.
Enzyme Link Immuno Assay (ELISA) Test Kit Development:
ECA, the protein molecules reacting with Celiac Disease IgA and IgG were separated and purified from the primary culture of placental stromal cells. These proteins were coated to the 96 well plate. Serum from test patients will be applied to the plate and a secondary Horseradish-Peroxidase anti-human IgA antibody will be applied. Plates will be read using ELISA spectrophotometry reader.
A step by step procedure for the antigen ECA preparation is as follows:
(1) Primary Placenta Cell Culture:
Wash, perfuse, and digest placenta tissue with hopes buffer and collagenase buffer--Single cell suspension preparation.
(2) Enrichment and Concentration of the adherent cells from the cell suspension using discontinuous percoll gradient.
(3) Protein extraction from the primary placental cell line using an extraction buffer and ultra-centrifugation.
(4) Placental cell extracts were applied to the SDS-PAGE Gel Electrophoresis to separate the proteins.
(5) Separated protein were transferred to the nitrocellulose membrane (Western Blotting) incubated with the serum of patients with Celiac Disease.
(6) Horseradish-peroxidase conjugated mouse anti human IgA is applied to identify the binding of celiac patients IgA to the antigen. (10 monocomponent polypeptides could be identified by the IgA of sera from Celiac Disease patients on nitrocellulose membrane.)
(7) Separation of primary culture cells extract was done by injecting protein, extract to the Gel filtration (size exclusion) of HPLC Column from Bekman "Ultra Sphero Gell" (SEC 3000) size 5 micron. The column size is 7.5 mM�300 mM.
(8) Competition biding assay was used to identify the separated proteins (Three single polypeptides with molecular weight higher than 55 Kd (55-65 Kd and 110 Kd) and Isoelectric point 5.1-5.8 demonstrated inhibition of binding of Celiac Disease patient serum IgA/IgG on the monkey esophagus and rat kidney/liver. Seven other monopolypeptides with molecular weight of less than 55 Kd inhibit binding to Giladin).
(9) Large volume separation Using the Gel Chromatography Separation is performed using DEAE sephadex column followed by the SEPHACRYL™ Gel Chromatography S-200 HR Column, bed size dimension are 2.6�32 Cm.
Enzyme Link Immuno Assay (ELISA) Test Kid Development:
ECA, the protein molecules reacting with Celiac Disease IgA and IgG were separated and purified from the primary culture of Placental stromal cells. ECA will be coated to the 96 well plate. Serum from test patients will be applied to the plate and a secondary Horseradish-Peroxidase anti-human IgA antibody will be applied. Plate will be read using ELISA spectrophotometry reader.
The preferred and optimumly preferred embodiments of the present invention have been described herein and shown in the accompanying specification to illustrate the underlying principles of the invention, but it is to be understood that numerous modifications and ramifications may be made without departing from the spirit and scope of this invention.
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KINGSTON, R.I. -- September 11, 2000 -- Each year at the University of
Rhode Island's Convocation, four outstanding members of the URI community
are recognized for their excellence by the URI Foundation. Excellence award
winners will each be presented with a citation and a check for $1,000 during
the 2000 Convocation exercises to be held on Tuesday, Sept. 12 at 3 p.m.
The following individuals are the 2000 URI Foundation Excellence Award
Administrative Excellence. Jonathan Blaney (URI '71) is a man
who wears many hats. In a typical day, Jon takes on the roles of business
manager of the College of Arts and Sciences, computer technician, building
manager, resource allocator, fiscal manager, systems manager, and many more.
As the "go-to" man for the largest college in the University,
Jon always finds innovative solutions for a wide variety of problems.
It is not unusual to see Jon rescuing a computer that was deemed irreparable
or helping any of the 25 department chairs with his extensive knowledge
on budget, accounting, purchasing, and personnel procedures.
Examples of his ingenuity are everywhere, from the dressmaker patterns
he located for URI's Commercial Pattern Archive to the entire Psychology
Department's computer network. As one of his grateful colleagues said, "He
is a team player who is always ready to go the extra yard to make us and
the University look great."
Blaney is a resident of Wakefield.
Teaching Excellence. Don't be surprised if you ever walk into
Dr. Roger LeBrun's classroom to see this charismatic professor of entomology
dancing about the room as an insect. To make his students bug out over his
lessons, Dr. LeBrun has been known to use a variety of creative methods
ranging from theatrical impersonations to showing movie clips to bringing
in the real thing.
Dr. LeBrun's teaching reaches far outside his classroom. He also serves
students as director of the Laboratory for Invertebrate Pathology and Director
of Graduate Programs for the College of the Environment and Life Sciences.
An expert on the mosquito-borne Eastern Equine Encephalitis virus, he has
expanded his classroom to the entire nation, speaking about the virus through
newspapers and Good Morning America.
But Dr. LeBrun's utmost concern is the education of each and every one
of his students. As one student noted, "Never giving up on students,
he pushes them to do better on every test and assignment. He believes in
students when others, and even the student himself, have lost faith."
Dr. LeBrun is a resident of Kingston.
Staff Excellence. Within the bustling community of URI's Honors
Program, secretary Deborah Gardiner is an island of calm reassurance and
strength. With efficiency and a bright smile, Deborah tackles a variety
of activities from helping to plan large scale events like the Honors Colloquium
to coordinating the schedules of the 33 faculty members and more than 400
students in the program, to the simple things, like making sure her trademark
candy dish is always full.
Balancing a workload that has more than tripled in the past three years
has only made Deborah shine brighter. There is never a problem she can't
fix or a deadline she can't meet, even if it means going above and beyond
the call of duty.
In the words of one of the honors program students, "Not only is
Debbie the cornerstone of the Honors Center, but she is also responsible
for the thriving life of the Honors Program - devoting her time, effort,
support, kindness and generosity to those who spend years or even just a
few hours on the second floor of Lippitt Hall."
Gardiner is a resident of West Kingston.
Scholarly Excellence. Redefining an entire field of work, and
expanding an accepted way of thinking may sound impossible to some, but
for Dr. Wayne Velicer, it's all in a day's work. A true pioneer, his research
on Multivariate Statistics and Health Psychology has led to innovative behavioral
change models, a partnership with the Johnson and Johnson Corporation, development
of the Nicotrol smoking cessation program, and international collaborations
with researchers around the globe.
Dr. Velicer is truly a world class scholar. In addition to receiving
more than $50 million in grants, he has authored more than 150 articles,
chapters, and books. He has been named one of the top 25 cited psychologists
and one of the top 25 highest-impact authors, and had a special issue of
the journal Multivariate Behavioral Research devoted to his work.
His research has inspired important work in Europe, the Netherlands, Germany,
and Australia, among others.
In the words of one of his colleagues, "He is a tremendous intellectual
resource, a credit to this University, and, incidentally, a very nice guy.
I don't know of anyone more deserving of the Scholarly Excellence Award."
Velicer is a resident of Peace Dale.
For more URI news visit: www.news.uri.edu
For a brief weekly summary of URI news by e-mail, call 874-2116 or e-mail
at email@example.com
Copyright © 1999
University of Rhode Island
more information about this site, contact firstname.lastname@example.com
File last updated: Saturday, February 24, 2001
The University is an affirmative action/equal opportunity
All rights reserved. URL: http://www.uri.edu/news/


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Relationship Reading
Questions, questions, questions – more than any other aspect of life, I am asked questions about love and relationships. As an experienced Psychic Tarot Reader and Relationship Expert I am happy to answer questions for my many clients on their love life –or lack of it.
I would be delighted to be able to help you by finding the answers you need to your relationship worries.
I offer you a personal Psychic Relationship Reading using the Tarot as tools for divination. The Tarot Reading can help you find the answers you need to shape your future, and provide answers to questions you might have about your relationship.
Your Relationship Reading will be in depth, private, personal and confidential and performed with sensitivity and care by myself. I will focus on your specific question and use my connection with Spirit and the Tarot to give you the answer you need at this moment.


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SEOUL, South Korea — After a series of escalating threats, North Korea has moved a missile with "considerable range" to its east coast, South Korea's defense minister said Thursday. He emphasized that the missile was not capable of reaching the United States and that there are no signs that the North is preparing for a full-scale conflict.
North Korea has been railing against U.S.-South Korean military exercises that began in March and are to continue until the end of this month. The allies have said that the exercises in South Korea are routine, but the North called them rehearsals for an invasion and said it needs nuclear weapons to defend itself.
The North has also expressed anger over tightened U.N. sanctions for its February nuclear test.
Analysts say the ominous warnings in recent weeks are probably efforts to provoke softer policies from South Korea, to win diplomatic talks with Washington and to solidify the image of young North Korean leader Kim Jong Un.
Lengthy process
The report of the movement of the missile came hours after North Korea's military warned that it has been authorized to attack the U.S. using "smaller, lighter and diversified" nuclear weapons.
The reference to smaller weapons could be a claim that North Korea has improved its nuclear technology, or a bluff.
The North is not believed to have mastered the technology needed to miniaturize nuclear bombs enough to mount them on long-range missiles. Nor has it demonstrated that those missiles, if it has them at all, are accurate.
It also could be years before the country completes the laborious process of creating enough weaponized fuel to back up its nuclear threats.
South Korean Defense Minister Kim Kwan-jin said he did not know the reasons behind the North's missile movement, and that it "could be for testing or drills."
He dismissed reports in Japanese media that the missile could be a KN-08, which is believed to be a long-range missile that if operable could hit the United States.
Kim told lawmakers at a parliamentary committee meeting that the missile has "considerable range," but not enough to hit the U.S. mainland.
No preparations
The range he described could refer to a mobile North Korean missile known as the Musudan, believed to have a range of 1,800 miles. That would make Japan and South Korea potential targets, along with U.S. bases in both countries, but there are doubts about the missile's accuracy.
The Pentagon announced that it will hasten the deployment of a missile-defense system to the U.S. Pacific territory of Guam to strengthen regional protection against a possible attack.
Experts say North Korea has not shown that it has accurate long-range missiles.
"From what we know of its existing inventory, North Korea has short- and medium-range missiles that could complicate a situation on the Korean Peninsula (and perhaps reach Japan), but we have not seen any evidence that it has long-range missiles that could strike the continental U.S., Guam or Hawaii," James Hardy, Asia Pacific editor of IHS Jane's Defence Weekly, wrote in a recent analysis.
Kim, the South Korean defense minister, said that if North Korea were preparing for a full-scale conflict, there would be signs such as the mobilization of a number of units, including supply and rear troops, but South Korean military officials have found no such preparations.
Contingency plan
"(North Korea's recent threats) are rhetorical threats. I believe the odds of a full-scale provocation are small," he said. He added that North Korea might mount a small-scale provocation such as its 2010 shelling of a South Korean island, an attack that killed four people.
At times, North Korea has gone beyond rhetoric.
On Tuesday, it announced it would restart a plutonium reactor it had shut down in 2007. A U.S. research institute said Wednesday that satellite imagery shows that construction needed for the restart has already begun.
For a second day Thursday, North Korean border authorities denied entry to South Koreans who manage jointly-run factories in the North Korean city of Kaesong.
South Koreans already at the plant were being allowed to return home.
South Korea has prepared a military contingency plan should North Korea hold South Korean workers hostage in Kaesong, Defense Minister Kim said.
He would not elaborate.
Outraged over comments in the South about possible hostage-taking and a military response from Seoul, a North Korean government-run committee threatened to pull North Korean workers out of Kaesong as well.
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Keep the Faith
A reader blog about liberal religion with Rev. Beth Ellen Cooper
Monthly Archive for April, 2011
The Grace that Takes Time
I still recall the refrain of the villain on the Thundercats cartoons: "Ancient spirits of evil, transform this decaying form into Mumm-ra, the Ever-living!" Lightning would flash, and the creature would be infused with new strength and power, which he would promptly use to try and destroy the world, until he was soundly defeated by […] [Read More]
Rolling Away the Stones
The part of the Easter story that has always spoken to me most has been the part where the women come to the tomb. They are worried about how to move that huge, heavy stone. A huge, heavy stone. I have known stones like that. The ones that sit in our throats to hold back […] [Read More]
Early Easter Morning
Imagine, just for a moment, what it might have been like. Two sleepless nights have passed since the death of one beloved to you. A son, a teacher, a friend. You are heavy with grief, and exhaustion, but still there is a sense that life is continuing in some distant place. It is time to […] [Read More]
An Easter People
I am a Universalist. That means that I believe that no one, no human being, and indeed, no life at all, is condemned to any kind of hell. In fact, I believe that "Hell" is more a political construct than it is a literal place. A way to separate. At the same time, I uphold […] [Read More]
What I Can Not Know Brings Me Hope
I am a Panentheist. Unlike a Pantheist, which understands the Divine to be infused in every atom of the universe, rather than separate from it, a Panentheist understands the Divine to be both infused in the universe, but also that in which the universe rests. For a Panentheist, the Divine is both immanent and transcendent. […] [Read More]


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William continues with his plan, Michelle and Luke have a date while Nikki and Michael have sex.
Lexi delivers the file on Michelle to Maria. Maria lures Lexi into having a glass of wine with her. Lexi reluctantly agrees�.and ends up intoxicated and talking with Maria about her type of man. Maria coaxes it out of Lexi that she is attracted to her son Luke. Even though Lexi insists that nothing will ever come of it�.Maria tells her not to say that because "You never know".
Maria visits Dr. Woods and tells him that she did wake up thinking about William and how much it bothered her that he wasn't there beside her. Maria says that she likes her live, she's never been lonely and she thrives on independence. When her Doctor asks her if she trusts William, she pauses and thinks. She responds by saying that she is cautious because she doesn't know if she is thinking with her head or her heart.
Hans and Harold stress out over having a design ready for Maria when she arrives. Michelle approaches the frazzled designing duo and inquires as to what is going on. She notices that the dress looks similar to another design of hers. While Hans and Harold struggle to keep the truth inside, Michelle suggests changes to make the design work. Hans and Harold are both thrilled with her vision. Luke walks up and steals Michelle away to talk (against John's better judgment). He invites Michelle out later and she accepts. Luke then goes to see Maria and stops to see Lexi. He offers to lend an ear if she ever needs help dealing with his mother.
At the salon Maria runs into Alyssa. Maria calls her outfit offensive and says that she looks like she is about to lay an egg. Maria keeps on with the comments but Alyssa manages to keep her cool and not let it get to her. Maria then tells her that the prozac must be working and hits her coffee making it spill on her outfit. Alyssa goes to slap Maria, but Maria stops the hit and makes her final emotional punches at her competitor designer and then walks out of the salon.
William meets Congressman Spangler for lunch to discuss the price of getting what he wants. Spangler tells him that it is just as he thought; it is not going to be cheap. Spangler hands him a hand written figure of $500,000. William pushes Spangler and says that this figure is about greed. Spangler tells him that there may be additional costs to overcome more hurdles. William writes a new figure of $750,000 and insists that amount should be enough to ensure that there will be no additional hurdles. The two then order their lunch.
Nigel stops by to inform Luke that he has a patron committed to purchase his next three paintings�unseen. Luke inquires about the patron, but Nigel keeps the promise of anonymity he made to William.
As Gloria tries to get Lance to pay her some attention at the office, William walks in. Gloria steps out so that the two of them can conduct business. William tells Lance that he needs access to $650,000 right now. Lance says that's not gonna happen because it can't go unnoticed. William says "no paper trail, no excuses�just get the money", and leaves. Lance and Rodney discuss William Chandler. When Lance tells him what William wants, they agree that they don't like to take that big a risk, but that they don't believe there is another choice at this point.
Nigel calls Maria and informs her of the success that Luke is having with his paintings. Maria sarcastically agrees with him that this is great news. Nikki arrives at the design room and teases Michelle about her date with Luke in front of Harold and Hans. The three of them then assist Michelle with deciding what to wear. William shows up at Maria's office and turns on his usual charm. They agree to have dinner tonight. John then enters Maria office and complains about the other team members not working because they are too focused on Michelle's date with Luke. William runs into Nikki in the office. She tells him to be careful with Maria because she will steal the shirt off your back�.especially if it's Italian. William stares lustfully at her as she walks back to her usual crowd.
William meets with Spangler again and gives him the money. Spangler gives William assurance that there will be no other interference and that he can begin his project. The two men shake on the deal and William tells him to�.watch his back.
At home, Nikki does Michelle's make-up and updates her on the new dating scene and how much it has changed since she has been on it. The two head out to club Gate to meet Michael and Luke. Michelle and Luke immediately hit the dance floor while Nikki and Michael down several shots. Nikki starts talking about love and how it is a waste of time. Michael defends love and references his parents. Nikki says that "respect and good sex is all we need". As they take it to the dance floor, Maria and William walk in and take a seat at the bar. Maria notices the foursome having a good time from across the room. Luke and Michelle leave Michael and Nikki kissing at the bar. When Maria sees them all leave as separate couples, she suggests that she and William get drunk. First though, she offers William a check as a payment. When he refuses, she tells him that it is all he has wanted and that she is not an idiot. He then informs her that what he wants is her�all of her.
Luke and Michelle end up at a smaller, more intimate bar where she opens up about her marriage with Lance. Luke figures out that he cheated on her and wants to know if she is still in love with him. Michelle insists that she hasn't felt that way for him in a long time. Luke tells her that he wants to see her again, but Michelle says that she doesn't want to lead him on. They agree to take it slow. Nikki and Michael arrive back at her place and start shedding clothes when they enter the door. For a moment Michael suggests that maybe they shouldn't do this. Nikki does not agree with his suggestion and they keep going. When Luke drops Michelle off, they wonder where Nikki and Michael ended up.


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Wisnu Purwanto
Articles posted by Wisnu Purwanto
Nusa Lembongan - Nusa Penida; Another Paradise in Bali
13 March 2013
Disclaimer: This is our trip report - purely reflect our real experience,can be subjective but factual to our eyes at that time and places. How to get there: Very easy, dozens flights from Jakarta and other regional and international airport to Bali Ngurah Rai International airport.Get the taxi from airport to Sanur beach,where several fast boat and regular boat are ready to bring you with the travel time between 30 minutes to 3 hours depend on the boat operator. We use Garuda GA724 early morning flight, ETA 08:55 and then Rocky fast cruise


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Feelin' spunky
Message of acceptance lights up stage in 'Hairspray'
Audiences filled the Mark E. Collins Theatre at Arlington High School for "Hairspray," a musical based on the film written and directed by John Waters.
Performances were Feb. 23-27.
The enthusiastic high school cast was directed by drama teacher Greg Krosnes.
In two acts, the lively characters of "Hairspray" took the audience on a delightful adventure through the eyes of character Tracy Turnblad, an overweight teen who loves to dance.
During her journey to become a star on a local TV show, the teenager has to first overcome adversity.
Determined to express her belief that
everyone should be accepted just as they are, no matter their shape, color or hairstyle, the plump dancer shakes up a movement for racial integration in the early 1960s in Baltimore.
Accepting the challenge of the lead role of Tracy Turnblad was drama student Courtney Sage.
"Courtney is probably one of the most involved students we have in the theater department. She has grown and developed her talent so much over the past four years," said Krosnes.
The senior jumped right in -- giving her dream role all she had to give. "I think the most difficult part about playing Tracy was having such big shoes to fill. Tracy has been my dream role for as long as I can remember," Sage said. "I've seen the show live four times, watched clips on YouTube of the original Broadway cast, and seen the movie countless times. I had really high expectations from myself, and I wanted to make sure that I did the role justice."
Sage admires the character's spunk. She said of the character, "It's so hard to pick one favorite thing about Tracy. I love everything about her. If I did have to pick one, I would say her determination."
Among her favorite song lyrics from the show is, "Don't hold me back 'cause today all my dreams will come true."
"She (Tracy) has such great determination and not only does that help her achieve her dreams, but others as well," Sage said.
More than 30 cast members of the high school production joined Sage in a performance of strength, courage and unity.
"I'm very proud of our students. We have some of the most talented, hard-working and devoted kids around. The time they spent on the dances, especially -- which were choreographed by Emma Crystal -- really made this show shine," said Krosnes.
Additional featured roles were preformed by Ashley Allen as Motormouth Maybelle, McKinley Doty as Velma and Kelsey Blackwell as Amber Von Tussel. The cast also included Ashley Applegate as Penny Pingleton; Shelby Batts as Matron, Prudy Pingleton and gym teacher; Logan Bernard as Link Larkin; Austin Conlee as Wilbur Turnblad; Collin Dale Farmer as Corny Collins; Kristoff Hart as Seaweed J. Stubbs; Asia Payne as Little Inez; Jessie Steele as Edna Turnblad; Ryan Wilson as Spritzer, Mr. Pinky and Principal Guard.
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DES Activist and DES Daughter Caitlin McCarthy says:
" … It's embarrassing for Lilly to say DES doesn't cause breast cancer; that it's all genetic… … It's arrogant and insulting to people's intelligence. We're not going away. They can't shut us up, so they're going to have to deal with us. They will have to admit the truth. They need to accept responsibility. It's not about the money for us, it's about justice and recognition that those of us who have been affected by DES are hurt in a way people not affected by it don't understand. "
Read her post Maybe DES Will Finally Get its Day
It's Time for Actions: did you SIGN THE PETITION ?
More about Eli Lilly
- Eli Lilly 's Negligence and Responsabilities: what did they know and what did they do about DES?
- DES was known to be a Carcinogen yet Eli Lilly @LillyPad conducted no Test on Animal/Human
- Let's all convince Eli Lilly to adequately deal with the Reality of their Disaster – Time for Actions
- Eli Lilly lawsuit – When the Melnick sisters reached a settlement with the drug company
- Are @LillyPad Poisoners? Facts Or Rumours? PLS Sign @Royallgal DES Petition #KeepLillyHonest