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I'd prefer Rudy Fernandez as the back up SG.
Nets and Knicks brass sat among each other yesterday, watching Vanderbilt shooting guard Derrick Byars. But it will be the Nets with first crack at Byars on NBA Draft night, June 28.More: [Only registered and activated users can see links. ]
Because of the shorter window for individual workouts, and because the cross-river rivals pick in a similar range of the first round, the Nets and Knicks are holding pre-draft workouts together in unprecedented fashion. The Nets select 17th, the Knicks 23rd. Yesterday was the first date teams were allowed to work out players.
Byars, who led the unsung Commodores to the NCAA tournament Sweet 16 before a controversial last-second loss to Georgetown, has been projected as a mid-first-round pick who may not reach No. 23. Byars, (6 feet, 7 inches) is not considered a great outside shooter, something Isiah Thomas has said the Knicks need.
NY Times: [Only registered and activated users can see links. ]
I'd prefer Rudy Fernandez as the back up SG.
I like Williams out of B.C., I think we have the coaching staff to make him into a significant player in this league
Williams is overrated. I hope Affalo, Byars, Williams, Fernandez or Splitter fall to the Knicks.
1. Almond
2. Sean Williams
3. Daquean Cook
4. Marcus Williams
5. Trade
Any other player would be "iffy". These 4 players could step right in the NBA and contribute immediately without any lag on their play. We need a shooter to keep double teams off of curry so this is why we need Almond. He has an NBA ready body and can contribute now. This is also a more realistic list since these 4 players will probably be available at our pick...
Stay Tuned.....
I completely agree with you. Isiah knows his prospects.
Stay Tuned.....
One of the reasons for my name is Isiah's drafting abitily. I also read today that Isiah isn't that hot for the kid from BC. Maybe he's just trying to keep the league guessing? I really don't know college talent, I thought that Donyell Marshall would be a better player than Grant Hill when they came out. But from what I read Williams can really play on both sides of the ball it's just that whole weed thing that everyone is leary of. But I've heard good things about the kid from Rice too.....that reminds me, I looked up David Lee's Draft profile when he was coming into the league, and the experts said that he was not a very good rebounder, and the worst case scenerio on him was that he would be another Chris Anderson, and at best Drew Gooden. Also they noted how well he and Eddy Curry looked playing together in the McDonalds All American game. Just thought that was interesting considering that 1 he's probably one of the best rebounders in the league, and 2 he eventually would end up being a teamate of Curry
IT did say the other day that he didn't expect whoever he drafted to crack the rotation next season since he considers morris his first draft selection... he is willing to draft someone that we can wait on for two years... i also read that teams have been scared to draft splitter because of his complicated buyout situation... it's now down to 500 k and IT can allow him to keep playing in brazil in the process... but i would expect someone else to take him... i think almond or Swilliams would suit our needs best...
If its Almond or Williams, either way the knicks will still have to make a trade... eventually. If Williams is as good as they say, and comes in with a defensive mindset and blocks shots and grabs rebounds, Frye and Lee are on the books until 2009/10 season, having 3 PF's who can play is not going to work. If its Almond, and he turns out to be as good as they say, of course Nate and maybe Craw may have to look for a new address.
My personal choice would be Sean Williams. I truly see him as having the potential of being another Marcus Camby if coached correctly, **** even if he's another Theo Ratcliff that would be great (minus the injuries) and because of the knicks poor perimeter defense they can afford to play upa little more with a monster shot blocker filling the lane..


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ONU to host panel discussion about heroin
Ohio Northern University will host a panel discussion about heroin, focusing on drug courts and prison drug counseling, in the forum of James F. Dicke Hall on Monday, Feb. 13, at 7 p.m.
This panel will feature Judge James S. Rapp of the Hardin County Court of Common Please, Probate and Juvenile Divisions, and Wade Melton, director of programs with the Hardin County Juvenile Court and the director of Hardin Community School. Melton currently monitors the juvenile drug court program and the Dependency Treatment Court program.
Lastly, John Hall, paramedic and correctional program coordinator at the Allen Correctional Institute in Lima, Ohio, will talk about the Recovery Services Program.
The ONU Department of History, Politics and Justice is sponsoring the event, which is free and open to the public.

subset: commoncrawl


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Erie is a horror game set in 1966. You have been asked to investigate an incident at a power generator. There was some kind of meltdown, and you have to find out what exactly caused it, and save any survivors.
Like Slender, Erie gives you objects you have to find while exploring. However, Erie is a much bigger game, and is visually more advanced. While you're investigating you can use a spray can to mark places you've been. And you're not alone.
Erie has a raft of scares, and makes excellent use of sound effects. The first person gameplay is pretty simple; your tasks in the game are nothing new. But throughout the experience, the atmosphere is chilling, and the shocks keep coming. I found the movement was a little too fast to be convincing, but there's a great blur effect when you run, which gives the game a great sense of panic. And the chances are that you will be panicking when you run!
It might not have the big budget of Dead Space, or the creepy legend of Slender, but Erie is still a great looking and scary game, and highly recommended for horror fans
To play Erie, you must install the free Desura app.
- Polished graphics
- Good scares
- Atmospheric
- Movement is too fast
"scary awesome download nowwww"
pissed my pants?brilliant game so creepy and i like the feature where you can spray where you have been (y)
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Just more than a year ago, back when Jeff Pendergraph considered declaring for the NBA draft after his junior season, the Arizona State forward approached Doug Collins and asked the former coach for advice.
Collins told Pendergraph his own story. How he had a chance to leave Illinois State a season early. But that his love for college, his determination to get his degree and a strong desire to play in the 1972 Olympics urged him to stay in school.
"Do you think your mission is done here?" Collins asked Pendergraph.
"Not yet," Pendergraph said.
"Well think about that," Collins said. "Because if you leave now, you'll be a second-round pick. But if you come back for your senior season, you'll have a chance to be a first-round pick. Why don't you set your goals higher?"
Today, it's clear Pendergraph has done so. His decision to stick at ASU paid off in several ways. He led the Sun Devils to their first NCAA Tournament in five seasons. He earned his economics degree, as well as first-team All-Pac-10 status. And now, here comes Thursday's NBA draft, an event that seldom has featured an ASU flavor.
The Sun Devils have had only seven first-round picks since 1964, only two in the past 25 years. But this time could be different. Guard James Harden is a first-round lock. And Pendergraph might sneak into the first 30 picks as well. If he does, it will be the first time ASU has placed two in the first round in school history.
"Pendergraph is flirting with the first round," an NBA scout said this week. "Everyone realized he was a little over (6-foot-9), so that helped. Plus, he's been playing well and he hasn't dodged any workouts. I wouldn't say he's made a huge leap, but he definitely has gotten more awareness from teams."
Collins, a Valley resident and close observer of Herb Sendek's ASU program, marvels at everything Pendergraph has overcome. The tumor he had removed from his leg as a freshman. The coaching change he experienced as a sophomore. And somehow, balancing it all to find time to graduate in three and a half years.
"Jeff has the talent to play in the NBA," Collins said. "He has a great motor. He goes all out on every possession. I think he has a chance to be a great pick-and-pop player. He can hit the 16- to 18-foot jump shot, which is so vital in the NBA."
Collins said Pendergraph could develop into a player such as veteran P.J. Brown. Another NBA executive offered as a comparison Andrew DeClerq, a forward from Florida who played 10 seasons. Brown or DeClerq, Pendergraph doesn't care.
He said Joe Dumars, president of basketball operations for the Detroit Pistons, pulled him aside and told him that he represents a new type of power forward. Bangers are out. Guys who can run, shoot and read screens are in. Pendergraph thinks he fits the mold, but he isn't going overboard. No matter where he is drafted, he understands his role. He said he was reminded of it at nearly every workout, in conversations that usually unfolded this way.
Executive: "If you come in here, you know you're not shooting the ball (a lot)."
Pendergraph: "Yep."
Executive: "You know you're not going to score."
Pendergraph: "Yep."
Executive: "We just need you to get in there and rebound."
Pendergraph: "That's cool."
Overall, Pendergraph feels good about his preparation. Three months ago in Miami, head down, he walked off the court a final time in an ASU uniform. In doing so, one chapter of his life ended, but another was about to begin. One he can't wait to get started.
"I want it to be here like yesterday," Pendergraph said. "And I was saying that a month ago. I'm ready. It's going to be fun."
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Jeff Pendergraph walks off the court for final time in an ASU uniform, fouling out against Syracuse in Miami on March 22, 2009. His decision to stay one more year at ASU could send him walking into first-round NBA money.
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This post is longer and a lot looser than most of mine which tend to be long and loose anyway but I've just been thinking through a lot of stuff and so it's time to set some of it down.
In the wake of the deep sigh of relief all of us have taken over the long-overdue departure of Dan Marvin, I've gotten all introspective and have considered the charge that atheists often are faced with: that we're just plain mean sonsabitches who are all too quick to respond to believers with anger, invective and name calling.
Take the whole history with Dan over the past few months. I called him stupid, uneducated, hypocritical, dishonest, and a lying little turd. I probably called him a few less pleasant things too. Other people in the comments called him even worse. It cannot feel good to hear yourself being called these things. It can only being demeaning and hurtful. From the anonymity of the keyboard, it's easy to forget that the person you're attacking is a human being with feelings.
What kind of guy do I think Dan Marvin is in person? I imagine he's a devoted and loving husband and father, a hard-working man who devotes 100% of himself to providing for his family and being a good citizen in his community. I can also tell from his writings that he really believes what he says he believes, even if, as was shown here by such fine commenters as Stephen and Tracie, he doesn't fully understand, or think through all of the implications of, what he claims to believe. I think he came here because he's genuinely possessed of a belief that there's a place called hell where we're all going to go to be tortured forever because we don't believe as he does. I think his beliefs are so thoroughly branded into his map of the world that no matter how much we tried to explain, "Look, here's why we don't believe what you do, and here's what we need to see from you if you think we should," his responses went along the lines of "But…but…don't you understand, you're going to hell!" There's just a fundamental difference in the way people like him and people like us process information in our minds, and that leads, particularly in extreme cases like Dan's, to an insurmountable communication gap.
So, yeah. On the one hand, I'm willing to admit that that kind of communication gap can lead to atheists losing our temper with what we see as pure stupidity.
But as I said way back when, believers who find atheists brusque and insulting need however hard it may be for them to try walking in our shoes.
Truth to tell, when I called Dan a particular nasty name, it was because he'd done something to earn it. Dan's own comments speak to his behavior well enough. If he cut-and-pasted some moronic creationist canards, I called him an idiot, because that's the sort of thing only an idiot would do. When he projected his own behavior onto us, I'd call him a hypocrite. When he tried to refute us by making bizarre straw men out of our arguments, I'd hit him again. And again. Now, that's just me. Life is not always meant to be easy, and some lessons need to be learned the hard way. There's a time when the gloves have to come off. There's a place for the Simon Cowell approach to criticism. And I'm okay with giving it. Some commenters here got even angrier with Dan. Others like Tracie were nearly heroic, in the patient way she calmly knocked down everything Dan threw at her, without the slightest hint that even one of her feathers had been ruffled.
It helps to know when being a two-fisted atheist is the right approach, and when it's not. When I started my four-year run on the AE TV show, I was first cohost to the redoubtable Jeff Dee. Jeff is brilliant at arguing on his feet, and almost always went for the jugular. Oftimes this was needed. The flipside was that it could, on occasion, backfire. For years, we had a creationist troll named Steve (some of you remember him, I'm sure) who'd call every week with whatever bit of YEC bullshit he'd dredged up. From the fake Paluxy riverbed "man tracks" on down, there was no argument so lame and discredited he wouldn't call with it. He was also a deeply dishonest scumbag who wouldn't address our rebuttals. He engaged in the kind of drive-by, hit-and-run pseudo-arguing in which, every time we'd refute his latest claim, he'd just call the next week and, without even acknowledging that we'd addressed his points, simply go off on a new round of drivel as if the previous week's conversation had never occurred.
But one thing became clear to me very quickly. Not only was Steve stupid, disingenuous, and ill-educated; he wasn't even serious. At first, yes, it was evident he really thought he could take us on with his arguments. But soon it was obvious he'd changed his tack, and he was calling every week because he knew how easy it was to make Jeff lose his temper. Many Christians realize they can't actually take down an atheist on the facts, and so they resort to emotional manipulation. Get the atheist angry, and they can claim a phony moral "victory." The argument is, at this point, no longer about what is or isn't true. It's about a "humble" Christian trying to witness to a poor unsaved soul, and getting hate and anger in reply. Instant martyr; just add water.
After Jeff left the show and I shifted my plump cheeks into the host's chair, Steve kept calling…for a bit. Then he just quit. Because I'd pegged him. I knew his schtick. I would just reply to his claims, without getting pissed off. I guess I wasn't as much fun.
Still, "pissed off" is something I get at times, all on my own. And I know other atheists sympathize. There really is no understating the level of frustration that is felt when one lives without religion in a religion-addicted world. Atheists, generally speaking, are just normal folks trying to get by and do the best they can like anyone else. Our only difference is that we have dispensed with the need for irrational beliefs in the supernatural, which we think most people maintain simply because the culture in which they were raised requires it as a behavioral norm. This view, I think, is borne out by recent findings that while most Americans profess religious belief, few actually know anything about what they claim to believe.
Thus many Americans profess belief simply because everyone else does, and not to do so would mean you're weird. On the flip side of that extreme are the Dan Marvins of the faith, who denounce those people as "false converts" while at the same time offering nothing but smoke and mirrors and nonsense in defense of their own, presumably "truer" belief.
In the midst of all this swirling supernaturalist confusion is the atheist, attempting to live a rational, humanist existence among millions of people who literally seem to be living in another world. And because we don't buy into the God delusion, we're vilified by people who know nothing of us, of atheism, or even of their own theism, and who think of themselves as paragons of virtue.
So to anyone who thinks atheists are mean people, I say, have you listened to Michael Newdow's answering machine tapes? (He's made them available, and even recorded a funny song around some of them.) Have you read the death threats and hate mail that Sam Harris gets? Have you read the vile diatribes by the Baptist minister on Possummomma's blog? How is someone like her supposed to live a normal, peaceful life when some lunatic, self-anointed "man of God" takes to stalking her children to save their souls? Atheists are "angry," you say? What parent, atheist or not, wouldn't get angry about that?
So while I think it behooves atheists to always do our best to choose the high ground of reason over the low road of irrational emotionalism, we are only human after all. Anger is an entirely justified response to being on the receiving end of prejudice and stupidity, especially when it is perpetrated by ignorant hypocrites who are convinced they're on the side of righteousness. Before you t
ell me that atheists are mean people full of anger and hate, consider what brings it on, and that many believers are in need of some serious adjustments to their own moral compasses.


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This print started out as a baseball card more than twenty years ago. Every aspect of it is designed to preserve the original look and feel of the card, from the printing pattern to the slightly worn edges. In fact, this print started by finding the actual card in our vault and then painstakingly re-mastering it with a team of designers. The authenticity extends to manufacturing; each print is made on high-quality, cotton fine art paper that measures 1/64 inches in thickness. Framed prints feature 2-inch flat wood or metallic frames with high-end, glare-resistant glass. And all prints are officially licensed by MLB and the National Baseball Hall of Fame.
STORYThe 1980 card of Orioles southpaw Mike Flanagan is a total splash of color from a Baltimore road game. Flanagan won the 1979 Cy Young Award when he led the AL with 23 wins and followed it up with a 16-13 mark and 4.12 ERA in 37 starts in '80. He led the AL in hits allowed.
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By Clarence Plank: PNT staff writer
Foxes are shy, smart, and silent creatures who hunt a variety of animals and insects.
There are four species of foxes in New Mexico; red, gray, kit and swift fox.
Eastern New Mexico University Wildlife biologists Zach Jones and Tony Gennaro say foxes can be found almost anywhere.
Gennaro and Jones said they believe foxes are monogamous and stay with only one mate.
"The mommy has the pups, she stays down in the den," Gennaro said.
"The male brings home the food, and she works the daylights out of him."
Jones said there are some very strong forces at work in order to keep two aggressive carnivores together year round.
Foxes eat birds, rodents, rabbits and insects. The average life span of a fox is about five or six years.
It is not uncommon to see a red fox in urban areas. They prefer
places that are moist and with trees and vegetation. This is why the
red fox can be seen in the northern part of the state.
But Jones and Gennaro say reds are moving west.
Jones and Gennaro said they think red foxes in this area might be from eastern areas, and have been moving further west.
Gray foxes can climb trees to either hunt for prey or escape from a predator.
"The gray fox is a very unusual occupant here," Gennaro said. "They
like the Llano Estacado, rocky, pinon and juniper tree-type country."
Gennaro said gray foxes have been seen in Portales, but that might be due to displacement because houses or a chance phenomenon.
Jones said the first reported gray fox in the area was in 2004 when a carcass was found on the road near Wal-Mart in Portales.
"I don't know how many there are, but there are gray and red foxes, both here," Jones said.
"Gray foxes have gray on their backs and are grisly looking, and
have short ears, with a black strip running down the center of their
Kit foxes roam the southern grasslands and desert side of the state, the Swift Fox is in the east.
"They like open country, short grass, a complete lack of cover,"
Jones said. "When you typically think of a fox, you think of cover,
forest and streams. Kit and swift foxes think the opposite."
Kit is a small fox, a slight black spot at the end of its tail, with large ears.
"Depending upon the authority, the swift fox is found east of the
Pecos River," Jones said. "The kit fox is found in the west and (they)
are very different, some authorities would say they are the same
Uncommonly, foxes have been known to carry rabies.
"Wildlife takes on a different behavior when there is something
wrong with them. A rabid fox, which is uncommon will not be wild, it
will come towards people, and that's why we have to be careful,"
Gennaro said.
Foxes can be brazen animals when around humans, but they remain at a safe distance to protect themselves.
"You don't want to develop a domesticated relationship with a wild animal," Jones said.


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The Dinette Set by Julie Larson
- April 14, 2013
- From Beginning
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Comments (14) (Please sign in to comment)
Looks awfully light outside for those boys to have gone to bed an hour ago already.
Wake 'em up and tell 'em to turn out those lights!
How else will they ever learn responsibility!
Or collect enough anecdotes to tell their future shrinks…
or gather enough material for Timmy's eventual neurotic stand-up act…
I am so blessed to NOT be their daughter!! Yea it is light out because of the saving time. Maybe the boys are just pretending to be asleep, then they can sneak out the window and run away.
Burl and Dale are projecting their own misdeeds onto the kids. I don't know what those boys are doing but it's not what Burl and Dale think.
Full disclosure: Back in the dim, dark ages when my brother and I were (somewhat) younger, we would sneak out the window and chase sabre-toothed tigers at night. Mom never knew, even if the sheets were all stained with grass and dirt in the morning.
Who are those kids? Is one of them Timmy?
WHAT….Timmy has a little brother…. a cousin…. a friend…. ? ? ?
Mom knew. She probably even watched what you were up to, but just didn't say anything. You had things to learn and some lessons can't be learned when mom and dad are watching.
It is a good thing that we do project our own early misdeeds so we can prevent some. My kids could not get away with telling me they were one place while going elsewhere (I caught them telling on the phone, because my new lover back in the 70s showed me how to tap the phone).
Timmy has always had a little brother but he's almost never shown or even spoken of.
At Christmas (I think?) his and Timmy's gifts were mentioned…
I can't think of his name, so I went to the Dinette Set website, and found that the information about the Shemp's grandsons is gone.
The site has been "updated" into uselessness… as have so many
The adult character bios are now in a fast-moving slideshow with badly overlapping frames.
Sigh…. I'll have to try to remember his name.
BTW At least the new site
does link to this page…
the old site didn't.
Here are a couple of excerpts from Julie's first post, on January 13th:
" hope to be posting at least once a week to try to give you readers a peek behind the curtain…."
"the daily comments and mail from fans is truly what keeps me going- you guys have been great. "
Alas… it's also the only post so far.
Julie…. if you read these….
what is Timmy's brother's name? Thanks!
Jimmy has a nice ring to it, Timmy and Jimmy. If that is not his name, and no one (Julie) writes to advise otherwise, how 'bout we call him Jimmy?
"Like"…. ! ! !
These people are so dumb they make a slice of moldy bread look smart! (Used to be TWO slices but the bread got smarter)
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What is in this article?:
- Quality, Integrity, and Satellite Symposia
- Unexpected Insights in Stakeholder Data
- The "4+1 Model" of Satellite Symposia
- The Story In SWOTs
Since it was created in 2009, the Industry Alliance for Continuing Education and Medical Specialty Society Collaborative Workgroup of the Alliance for Continuing Education in the Healthcare Professions was created to improve communications between two sections of the medical meetings industry that are usually kept separate. Rules and regulations put in place to protect each side had inadvertently created an adversarial landscape. Commercial supporters were afraid to engage with CME providers, and many CME planners were never quite sure when it was acceptable to discuss something with a commercial interest. Thankfully, both sides were eager to restore dialogue.
But the emphasis on improving communications faded quickly. It wasn't necessary. Each group had been eager to establish an avenue to discuss topics with the other; it didn't take much to get the groups talking.
From there, "what do we talk about?" became "what should we work on?" One topic that rose to the top was satellite symposia. Here's what the group has come up with so far.
The "4+1 Model" of Satellite Symposia
The workgroup's research didn't find any best practices for hosting these symposia. Too many departments with too many policies are impacted within each organization to catalog how organizations administer these events.
But each group's practices can be classified on an oversight spectrum from "absolute oversight," where the society plans and implements a separate activity that they seek support for, to hosting non-certified "promotional only" activities, with partial to no MSS oversight falling in the middle.
With absolute oversight, it's easier for the MSS to align the symposia with the annual meeting's focus and provide a means to advance scientific knowledge. It does make it more of a challenge for the MSS to ensure the satellites' industry sessions are differentiated from those of the annual meeting.
Partial to no MSS oversight can bring the benefit of decreasing the potential for the perception of bias in the content.
Promotion-only symposia, which are funded from a company's marketing department, are not subject to Accreditation Council for CME regulations, though they do have to obey the physician tracking and reporting requirements of the Sunshine Act.
This oversight spectrum evolved into the "4+1 Model," where "4" represents four strata of provider oversight, and "+1" represents those organizations, like ACC, that do not offer any form of symposia. Each model faces distinct challenges, but there is a good deal of overlap within practices. In 2012, the group dove deeper into those similarities and differences for the 4+1.


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As all you Saffa Londoners very well know, we had yet another Tube Strike this last week. London was once again disrupted and inconvenienced not to mention, it probably cost the capital enough dosh to finance a small country for a week. So what was the problem this time?
Well apparently it was over 800 job cuts to ticket office staff and another group moaning about wages… as usual. Now you're probably thinking, fair enough, people don't want to lose their jobs over automatic ticket machines etc, but then you've got to ask yourself, what day and age do we live in? Technology is making our lives easier and it has been for centuries now, this does cost jobs since in many cases, a computer can do the job of ten people in half the time, so why not? Back in the day in the financial sector, you had hundreds of traders shouting in the pits, that doesn't exist any more due to electronic trading. The fact of the matter is, technology is moving forward whether you like it or not, nobody is going to stop it, because that's the way it is…. That is of course if we get to the stage of 'The Matrix' and your name just happens to be Neo 🙂
People become redundant in various ways but they move on and quite possibly do the same sort of thing elsewhere. In case you hadn't noticed, we had a recession recently where thousands of people lost their jobs, there were no riots or strikes, obviously because most people didn't belong to a archaic union behind them fronted by a very well paid union boss like that knob cheese Bob Crow. People moved on into other jobs in the same profession or something completely different that they found that better suited them. I really don't think there's much of a difference in skill between a ticket office person and a cashier at your local supermarket, its definately not something you do a university degree in. As for those people moaning about their wages, these were from Alstom-Metro depots, the company that builds and maintains trains. In other words, many who have skills that can very well be used in many other industries, so if you are gonna bitch about money, then do what everyone else does… get another job, because there's a million other people with the skills who'll more than happily do your job for the same salary. It just seems all a bit too easy to get a job that is connected to the rail unions in order to go on strike when you're running short of cash.
Luckily I wasn't effected too much since I take the overland and Waterloo and City Line, but millions of others weren't so lucky, not to mention the knock on effect of missing a flights etc. It's all a bit too ridiculously old school and people need to start living in the now. These idiots don't realise that they simply cannot shutdown a system which is a critical part of our society that effects everybody, not just themselves, when they feel like. I'm not talking about me having to walk an extra 10mins to the office, but I'd like to see Bob Crow answer to the guy that missed the birth of his first child, or the person that didn't make it to say their last good-byes at the death bed of a loved one, or the person that died in the ambulance because they couldn't make it to the hospital on time due to the traffic, these are real problems that realistically affect people without a second chance that could have been avoided, not just a temporary problem where some slapgat can't get off his arse and be the master of his fate. David Cameron's father passed away this week, can you imagine if he couldn't make it to the airport quick enough to get to him because of the traffic or something, unions would be banned tomorrow. I can't wait for the day that the entire underground is driverless and runs regardless. Did you know in France, they have so many strikes that they have a complete backup service that greatly decreases the impact on the public, something TFL should maybe look into ey…
Don't even get me started on the strikes in SA…
ok, take a deep breath now Marky… moosa! 🙂
Have your say in the comments below….


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- Worldwide Rank #36,410,161
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Home & Garden Briefs
collection on tap
The Berkeley County Solid Waste Authority (BCSWA) and the West Virginia Department of Agriculture is pleased to announce the first large scale collection event for used or unwanted liquid and powder pesticides. This program is for Berkeley County residents ONLY. The collection event will be conducted at the Grapevine Road Recycling Center on Saturday, May 11, between the hours of 9 a.m. and 5 p.m.
The term "Pesticide" includes a wide range of products used by the home owner or farmer for pest control and garden applications.
INSECTICIDES: products, such as insect repellants, used for eliminating common household pests such as ants, roaches or other insects. Insecticides are also used to control insects in landscaping lawns and gardens. Mixtures of insecticides containing fertilizers will also be accepted.
HERBICIDES: products used to kill unwanted plants. The most common products are used for the control of weeds in lawns and gardens. These are also commonly mixed with fertilizers and are labeled "weed and feed."
RODENTICIDES: these products are usually sold in the form of baits resembling rabbit food or bird seed. Rodenticide may also be pressed into blocks resembling compressed pet food and green or blue in color. Products intended to eliminate moles or gophers are also acceptable and may be peanuts, seeds or pellets that are placed in the burrows of the pest animal.
FUNGICIDES: these products are primarily designed to control diseases in plants.
AEROSOL CANS: such as wasp and hornet products, roach, ant, control or insect repellants will be accepted.
Not Acceptable:
No oil-based paints, latex paints, stains, solvents, gasoline, kerosene, ammunition or propane cylinders will be accepted. No pressurized cylinders will be accepted. No metal cylinders with a threaded fitting designed to take a valve will be accepted. No container will be accepted without its original factory labeling.
Any general questions or comments can be directed to the BCSWA at 304-267-9370 or berkeleycountyswa @msn.com.
If a participant has large volumes not practical for standard transportation, please contact Doug Hudson at the West Virginia Department of Agriculture at 304- 558-2209 for other arrangements.
Jefferson County Expo slated
The 33st annual Jefferson County Expo will be held Saturday, May 11. All animals must be in place by 9 a.m. The sheep show, dairy show and Rabbit 101 Workshop will start at 9 a.m. with rabbit judging, the horse show and dairy and meat goat shows at 10 a.m. followed by the beef show at 1 p.m.
For more information and entry forms, go to www.jeffersoncountyfairwv.org.
Market Swine check-in set
Check-in for the 2013 Berkeley County Youth Fair Market Swine show is being held Saturday, May 4 from 8 a.m. to noon at the Berkeley County Youth Fair grounds.
Market Sheep and Market Goat check in will be held Friday, May 10 from 6 8 p.m.
Livestock Pre Registration forms are due May 10. Completed forms are to be postmarked by May 10. Completed forms can be dropped off on May 10 between 6 8 p.m. at the fairgrounds. No exceptions will be allowed. Forms can be downloaded from the Berkeley County Youth Fair website at www.berkeleycountyyouthfair.org/id5.html.
Vendors wanted for market
Main Street Martinsburg is signing up vendors now for the 2013 Main Street Martinsburg's farmers market. Main Street Martinsburg has hosted a Farmers Market for many years and is actively looking to expand the market. The Downtown Market will be every Friday from 8 a.m. – 2 p.m. on the Town Square starting June 7. Main Street is looking for vendors who want to sell vegetables, fruit, herbs, flowers and baked goods. There will be an informational meeting on Thursday, May 2 at 6 p.m. at the Newberry Executive Center, 142 N Queen St. For more information, please contact Randy Lewis at 304-262-4200.
Open house set Sat. and Sun.
Winston Gardens Specializing in Native Plants and Virginia Winston Landscape Design & Gardens. Located at 1121 Hollida Lane, 1 mile off Swan Pond Road.
"Get your Spring garden off to a great start."
Sat. & Sun. May 4 and 5, 10 a.m. to 3 p.m.
Benefit for Potomac Valley Audubon Society. 15 percent of all proceeds go to help support trail development at Stauffer's Marsh. Light refreshments and door prize offered. 30-plus varieties of native trees & shrubs, potted up and available for immediate planting in your garden.
Call 304-267-6924, or go to www.winstongardens.com for more information.
Directions: From 45 East out of Martinsburg, go about 5 mi., turn left on Files Crossroads and go 1 mi. to 'T' at Swan Pond Rd. Turn left, go 1/2 mi. and turn right onto Hollida Rd. Follow Hollida as it turns sharply right at the mailboxes, becomes gravel, passes uphill and between a large white farmhouse and barn, turns sharply left a little way beyond the barn, and goes down a long straightaway. Just as the road turns sharply right again, follow our driveway up to the left (1121, light green ranch house) and park along the drive.
Sheep & Wool Festival slated
This weekend, May 4 and 5, the Maryland Sheep & Wool Festival will celebrate its 40th anniversary. The festival is always held the first full weekend in May at the Howard County Fairgrounds in West Friendship, Maryland.
Festival hours are 9 a.m. to 6 p.m. Saturday and 9 a.m. to 5 p.m. Sunday. Admission and parking are free.
The festival features more than 260 vendors selling wool and sheep-related items along with sheep and sheep dog demonstrations, and demonstrations of shearing, carding, spinning and weaving wool. It is one of the largest festivals of its kind.
The festival is sponsored by the nonprofit Maryland Sheep Breeders Association. For more information, visit www sheepandwool.org.
Seasonal garden tips offered
Seed snap beans and head lettuce (outdoors).
Control broadleaf weeds in lawn.
Seed leaf lettuce and winter squash (outdoors).
Seed summer squash and cucumbers (outdoors).
Seed late celery and sweet corn (outdoors). Until next time … happy gardening and farming!
– Mary Beth Bennett, Ph.D. is a WVU Extension agent and associate professor. She can be reached at 264-1936, MBBennett @mail.wvu.edu or on the web at www.berkeleyextension.com or Mary Beth Bennett, 400 West Stephen Street, Suite 302, Martinsburg, WV 25401.


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Can you let us know what this ticket priced at 98.00 each for the Fonda Show gives you, if anything, and where is the seat or standing area? I called ticketmaster and they didn't know either. Please respond asap as I have to make purchase before 6pm today. AND, the code came AFTER 10am and didn't expect it so didn't check emails until 3 hrs later due to other appts. It said check emails daily approx 9:30am for code so if I didn't see it by 10am, I am done checking. BUT, I did get a code sent so I guess I won the lottery- no complaints there, but this has been a very amatuer type process.
Hi, just wanted to share how disappointed I have been in the handling of this entire tour, starting with the Mojo Package. I bought 4 Mojo tickets and still have NOT received the CD's or Posters. I did finally after several phone calls got discount codes for retail store to buy Tshirts for the Shows I thought I was attending but were cancelled TWO times on me. First at Hollywood Bowl it was cancelled on MY ANNIVERSARY date 5/22 which was disappointing and then flew to Ohio to see the show at Blossom on 7/20 and that show was cancelled. At this time I have spent money for two plane tickets to go to Ohio to take my sister who lives there and the show did not happen. I called and tried to get help to get me to another show that week in Pittsburgh but NO ONE was able to help and I called VIP Tickets with Live Nation (what a joke-they never called back after speaking to them) and also TP Fan Club. I have NEVER missed a TP show but I am seriously considering that maybe the last time I saw TP four years ago was my last!! I hope someone out there can relate!! Oh, and the downloads are a ZIP file I cannot open so still waiting on them as well :-(
No shows scheduled. Check again later.
There are no activities available for this user!!
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March 1998
Another Morning
Anyone thinking of the jazz violin as a novelty or limited, role-playing instrument would be well advised to pick up Diane Delin's Another Morning (Blujazz productions BJ303: 46:34). Delin spins vibrant, varied leads from both electric and acoustic violin, capturing the vibrations, tones and colors of a vocal...and then some. She bops through the laid back, bass-popping "Struttin' Stuff" and the highly caffeinated album-opening title track, but hits sharp, darkly romantic edges on the Latin rocker, "For the Moment." Delin also knows how to wring emotion where it counts-most notably from ultra-high, bowed cries over an acoustic bass solo on the multi-textured "Jan's Song" and on a touching violin and piano duet of "God Bless the Child," in a totally unique read which is both warm and melancholy. Another unique arrangement, its own worth the price of admission, thanks to Delin's coolly twisted, off-kilter bowing.


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Advance ticket sales for next month's MCM Expo London Comic Con have just topped an incredible 15,000– the highest recorded at this stage in the show's decade-long history– event organisers have announced.
With demand for advance tickets skyrocketing, the MCM Expo is expected to hit the 60,000 visitor mark for the first time this May; 10,000 more than the total number of people coming through the doors at October's show.
"We are absolutely delighted to see advance sales so high eight weeks ahead of this year's show given that most of our sales usually happen in the two months running up to the event," said MCM Expo co-organiser Bryan Cooney."One possible reason for this unprecedented public interest is our recent media partnership with GameSpot UK. As London's Comic Con, we were already the UK's leading show for comics, movies, anime and manga, and this partnership ensures that the Expo delivers the best games content as well."
As well as teaming up with GameSpot UK, the upcoming MCM Expo London Comic Con will sport superhero movie Green Lantern as its official title sponsor, play host the prestigious Eagle comics awards and welcome X-Men: First Class and Thor writers Ashley Miller and Zack Stentz as special guests.
Industry and press registration for the MCM Expo London Comic Con on 27-29 May is set to open Monday 11 April– for details visit www.londonexpo.com
For London MCM Expo tickets please visit: www.mcmexpogroup.com/store
To stay up-to-date with Expo news, follow us on www.twitter.com/mcmexpo
David Axbey
PR Department
MCM Expo Group
Email: firstname.lastname@example.com
Telephone: 0207 793 1884
Web: www.mcmexpo.net
Mailing Address: MCM Expo Group, PO Box 38727, London, E10 7YH, UK


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Frank, Ines; Santos, John J.; Mehlhop, Erin; Villamide-Herrera, Loreley; Santisteban, Christine; Gettie, Agegnehu; Ignatius, Ralf; Lifson, Jeffrey D.; Pope, Melissa
From the Center for Biomedical Research, Population Council, New York, New York, (Drs Frank, Santos, Mehlhop, Villamide-Herrera, Santisteban, and Pope); Aaron Diamond AIDS Research Center, New York, New York, (Dr Gettie); Department of Medical Microbiology and Immunology of Infection, Benjamin Franklin Medical Center, Free University Berlin, Berlin, Germany (Dr Ignatius); and Retroviral Pathogenesis Laboratory, AIDS Vaccine Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland, (Dr Lifson).
Received for publication March 28, 2003; accepted June 9, 2003.
This work received support from the Tulane National Primate Research Center base grant RR00164 and was also supported by grants from the Elizabeth Glaser Pediatric AIDS Foundation, The Rockefeller Foundation, and the Irma T. Hirschl Trust (to Melissa Pope). This publication was also made possible by grants AI47681 and AI52060 from the National Institutes of Health. Melissa Pope is an Elizabeth Glaser Scientist and is supported by The Elizabeth Glaser Pediatric AIDS Foundation. Ines Frank was supported by the Erwin Schroedinger Fellowship funded by the Austrian government. Ralf Ignatius gratefully acknowledges support provided by the European Union (grant QLRT-PL 1999-01215) and by the H. W. & J. Hector Foundation. This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract NO1-CO-12400.
Reprints: Melissa Pope, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10021 (e-mail: email@example.com).
Interactions between HIV-1 and dendritic cells (DCs) play an important role in the initial establishment and spread of infection and development of antiviral immunity. We used chemically inactivated aldrithiol-2 (AT-2) simian immunodeficiency virus (SIV) with functional envelope glycoproteins to study virus interactions with DCs and developed an in vitro system to evaluate the quality of SIV antigen (Ag) presentation by DCs to T cells. AT-2 SIV interacts authentically with T cells and DCs and thus allows assessment of natural SIV-specific responses. CD4+ and CD8+ T cells from blood or lymph nodes of SIV-infected macaques released interferon-γ (IFNγ) and proliferated in response to a variety of AT-2 SIV isolates. Responses did not vary significantly as a function of the quantitative envelope glycoprotein content of the virions. Presentation of Ags derived from AT-2 SIV by DCs was more potent than presentation by comparably Ag-loaded monocytes. Interestingly, SIV-pulsed mature DCs stimulated both CD4+ and CD8+ T-cell responses, whereas immature DCs primarily stimulated CD4+ T cells. Further studies using AT-2 inactivated virus may help to define better the details of the virus-DC interactions critical for infection versus induction of antiviral immune responses.
It is becoming clear that a successful vaccine strategy against HIV-1 will likely have to induce broad specific immune responses comprising the CD4+ and CD8+ T-cell arms as well as neutralizing antibodies (Abs). 1–6 For evaluation of many vaccine approaches, experimental simian immunodeficiency virus (SIV) infection of rhesus macaques 7 is arguably the best animal model system used for immunization and virus challenge studies. Attempts to induce immunity in macaques against vigorous SIV challenge have used combinations of subunit proteins, live-attenuated or recombinant viral vectors, and/or DNA immunization approaches. 6 Despite some modest successes, 1–6,8–10 most approaches have not provided solid protection against robust challenges with pathogenic viruses, stressing the need for improved strategies. Given their status as the most potent antigen-presenting cells (APCs) in the body and their central role in inducing immune responses, many investigators believe that delivery of appropriate antigens (Ags) to dendritic cells (DCs) will be an important step in achieving an effective vaccine. Except for 1 report, this has proved problematic. 11–13
Myeloid-derived DCs (MDCs) are potent professional APCs that induce strong Ag-specific immune responses. DCs have been shown to specifically stimulate CD4+ TH1 cells and cytotoxic T lymphocytes (CTLs), to activate B cells, 14,15 and to elicit T-cell memory. 11 DCs show specific characteristics based on their differentiation stage. Typically, as immature DCs, they are preferentially located at the body surfaces and in blood and are well equipped to nonspecifically ingest large amounts of Ag via fluid phase uptake, which, along with locally secreted inflammatory stimuli, leads to their activation. Triggered DCs migrate to lymph nodes (LNs) using multidrug resistance proteins and the homing receptor CCR7, 16 undergoing further maturation, which subsequently leads to enhanced capacity to induce Ag-specific activation of naive and memory T- and B-cell responses on interaction with lymphocytes in the LNs. 17 Mature DCs at this stage characteristically possess limited phagocytic capacities but retain receptor-mediated uptake mechanisms. 18,19
MDCs from healthy and SIV-infected macaques are comparable to their human counterparts in phenotype, function, and how they interact with immunodeficiency viruses. 20–26 Similar to the results for human cells, 27 macaque mature monocyte-derived DCs (moDCs) are most reliably obtained by exposing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 cultured monocytes to a cocktail of inflammatory factors, including prostaglandin E2 (PGE2), tumor necrosis factor-α (TNFα), IL-1β, and IL-6. 24 In particular, cocktail-matured moDCs exhibit a reproducibly uniform phenotype, enhanced viability, and potent immunostimulatory capacities while maintaining their receptor-mediated uptake mechanisms. 24
DCs play a central yet dichotomous role in the biology of primate lentivirus infection. On one hand, DCs are critically involved as key APCs in inducing virus-specific immune responses. 11,13 On the other hand, DCs efficiently transmit HIV-1 and SIV to T cells, resulting in potent amplification of infection (S. Turville et al, submitted). 28,28a To clarify this critical aspect of AIDS pathogenesis, we have developed methods for studying virus-DC interactions in vitro. 18,29 This work has been facilitated by the availability of a novel form of chemically inactivated virus that retains conformationally and functionally intact envelope glycoproteins. 30,31 Treatment of retroviruses with 2,2′-dithiodipyridine (aldrithiol-2 [AT-2]; Aldrich, Milwaukee, WI) results in covalent modification of the free sulfhydryl groups of cysteine residues on internal structural proteins without appreciably affecting the disulfide-bonded cysteines of the proteins on the virion surface. 30,32 In practice, this results in noninfectious virions with functionally intact envelope glycoproteins. Such virions are being evaluated as a candidate vaccine immunogen (J. Lifson et al, in preparation) 33,34 and are also proving to be useful reagents for studies of virion interactions with various cellular populations. 18,24,29,35–39 For such studies, AT-2–inactivated virions offer the advantage of being able to monitor authentic envelope glycoprotein–dependent interactions of native virions with host cells and cell surface receptors in the absence of potentially confounding effects due to concomitant infection.
In this study, we primarily assessed the comparative ability of AT-2 SIV to stimulate lymphocyte responses in mononuclear cells isolated from blood (peripheral blood mononuclear cells [PBMCs]) and LNs as well as the capacity of immature and mature moDCs to induce virus-specific T-cell responses after being loaded with the inactivated virus. AT-2 SIV activated dose-dependent SIV-specific interferon-γ (IFNγ) and proliferative responses by CD4+ and CD8+ T cells in contrast to the predominant CD8+ T-cell responses induced by SIV-recombinant canarypox or vaccinia vectors. When mature DCs were loaded with AT-2 SIV, they reproducibly stimulated strong CD4+ and CD8+ T-cell responses, whereas the immature DCs preferentially led to CD4+ T-cell activation. In both cases, the response was independent of the amount of gp120 carried per virion. Hopefully, improved understanding of these attributes of DC-virus interactions will help to advance applications for in vivo priming against SIV and facilitate the development of effective vaccine approaches for the prevention of HIV-1 infection and AIDS.
Adult male and female rhesus macaques (Macaca mulatta) used in this study were housed at the Tulane National Primate Research Center (TNPRC) and tested negative by polymerase chain reaction (PCR) for simian type D retroviruses (SRV) and simian T-cell leukemia virus (STLV-1). The weight and age of the animals at the time of study initiation ranged from 13 to 18 kg and 10 to 12 years, respectively. Both naive and SIV-infected macaques were used for these studies. All animals were healthy at the time of study unless otherwise indicated. Animals were anesthetized with ketamine-HCl (10 mg/kg) prior to all procedures. Based on a previously published protocol known to prime against SIV, 40 animals were inoculated via intravenous injection of 2.35 × 104 50% tissue culture infectious doses (TCID50) of SIVmac239 delta nef (delta nef) 15 weeks prior to being challenged intravenously with 102 TCID50 of SIVmac239 wild type (AR 76, AR 77, AR 81, AR 83, AR 84, AT 62, M284, L507, P075). Such delta nef–vaccinated and wild-type–challenged animals show strong SIV-specific immune responses and have not shown evidence of SIV-associated disease over at least 2 years of postchallenge follow-up. For the present studies, these macaques have been used as a source of SIV-primed T cells to monitor the presentation of AT-2 SIV in vitro. Peripheral blood or superficial LN samples were collected to monitor SIV-specific T-cell responses. To measure the presentation of AT-2 SIV by different APC populations, DCs were generated from CD14+ cells isolated from blood (see below), and the animals were bled again approximately 1 week later to obtain autologous T cells (and fresh CD14+ cells where necessary). The blood and LNs from 1 monkey infected with wild-type SIVmac239 (M806) that had to be euthanized for health reasons were also studied. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the TNPRC. Animal care procedures were in compliance with the regulations detailed under the Animal Welfare Act and in the "Guide for the Care and Use of Laboratory Animals."
The medium used throughout these studies was RPMI 1640 (Cellgro; Fisher Scientific, Springfield, NJ) supplemented with 2 mM of l-glutamine (GIBCO-BRL Life Technologies, Grand Island, NY), 50 μM of 2-mercaptoethanol (Sigma Chemical Company, St. Louis, MO), 10 mM of HEPES (GIBCO-BRL Life Technologies), penicillin (100 U/mL)-streptomycin (100 μg/mL) (GIBCO-BRL Life Technologies), and 1% heparinized human plasma (complete RPMI, "cRPMI"). Recombinant human (rh) IL-4, IL-1β, IL-6, and rh TNFα were purchased from R&D Systems (Minneapolis, MN), rh GM-CSF from Berlex Laboratories (Montville, NJ), and PGE2 (P6532) from Sigma Chemical Company.
Infectious and chemically inactivated noninfectious SIVmneE11S (E11S) was provided by the AIDS Vaccine Program (SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD). E11S was obtained from supernatants of the cloned E11S cell line. 41 Wild-type SIVmac239 (239) was generously provided by R. Desrosiers, Harvard Medical School, and the mutant variants of SIVmac239 (SIVmac239/251 tail and SIVmac239/C5) were provided by J. Hoxie, University of Pennsylvania. SIVmac239 and SIVmac239/251 tail were grown in the 174 × CEM cell line (obtained from the NIH AIDS Research and Reference Reagent Program). SIVmac239/251 tail virions contain approximately 10- to 20-fold more surface gp120 than wild-type SIVmac239, whereas the gp120 intermediate variant SIVmac239/C5 was grown in the SuP T1 cell line, and virions contained approximately 5- to 10-fold less gp120 than the SIVmac239/251 tail isolate. SIV CP-mac and SIV NC-mac 42 (both derived from SIVmacBK28) were propagated in SuP T1 cells transduced to express CCR5. 43 Although SIV CP-mac virions have full-length TM and contain high levels of gp120SU with an SU/TM ratio of 1:1 comparable to SIVmac239/251 tail, SIV NC-mac has approximately the same amount of TM as SIV CP-mac but approximately 10-fold less gp120, with an SU/TM ratio of approximately 1:10 instead of 1:1. 43 Preparations of infectious viruses were inactivated by treatment with AT-2 as described. 30,31 Virus content of purified concentrated preparations was determined with an Ag capture immunoassay for the SIV gag p27 Ag (AIDS Vaccine Program) and/or by high-performance liquid chromatography (HPLC) analysis as described. 43 Virus stocks were diluted in 1% bovine serum albumin (BSA; Intergen, New York, NY) in phosphate-buffered saline (PBS) and stored as aliquots (3 μg of p27 Ag equivalents per milliliter) at −80°C until use. SIV-recombinant canarypox virus vCP180 (encoding the SIVmac142 gag, pol, and env genes) and its parental strain (ALVAC) as well as vaccinia virus vP1071 (encoding the SIVmac142 gag, pol, and env genes) and its parental vector (NYVAC) were kindly provided by Aventis Pasteur (Aventis Pasteur, Swiftwater, PA) and stored at 2 × 109 PFU/μL at −80°C.
PBMCs were isolated from heparinized blood from either SIV-infected or naive macaques using a Ficoll-Hypaque density gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden). For the depletion of specific cell populations from whole PBMCs, the Magnetic Cell Sorting System (MACS; Miltenyi Biotech, Auburn, CA) was employed.
To analyze whether SIV-specific responses are mediated by CD4+ or CD8+ cell subsets, PBMC suspensions were divided, and fractions were either depleted of CD8+ or CD4+ cell subsets by directly adding anti-CD8 (clone SK1) or anti-CD4 MACS beads before the cell suspensions were loaded onto separation columns (LS columns; Miltenyi Biotech). To remove distinct APC subsets, PBMCs were incubated with phycoerythrin (PE)-labeled anti-CD14, anti-CD20, anti-human leukocyte antigen (HLA)-DR Abs, or isotype Ab control (fluorescein isothiocyanate [FITC]-IgG1 and PE-IgG2a), all from Becton Dickinson (Mountainview, CA), versus medium alone for 20 minutes on ice. The cells were then washed and incubated with anti-PE-labeled MACS beads before cells were loaded onto depletion columns (LD columns; Miltenyi Biotech).
Bulk purified T-cell populations were obtained by adding anti-CD14, anti-CD11b, and anti-HLA-DR MACS beads to the PBMC suspension to remove APCs and natural killer (NK) cells. For the analysis of CD4+- or CD8+-mediated SIV-specific T-cell responses, suspensions were again divided, and anti-CD8 or anti-CD4 MACS beads (as above) were included with the anti-CD11b/CD14/HLA-DR beads for the depletion. CD11b−CD14−HLA-DR−CD8− fractions provided the CD4+ T cells, and CD11b−CD14−HLA-DR−CD4− fractions provided the CD8+ T cells. When CD14+ cells were used as APCs, PBMCs were first depleted of the CD14+ cells (keeping that fraction as an APC population), and the CD14− cells were subsequently depleted of CD11b+HLA-DR+ (and CD4+ or CD8+) cells.
To characterize macaque T cells and analyze the purity of cell preparations, cells were monitored by 2-color flow cytometry using an FITC-conjugated mouse monoclonal antibody (mAb) combined with a panel of PE-conjugated mouse mAbs. T-cell preparations (bulk vs. CD8- or CD4-depleted cell fractions) were stained with a FITC-conjugated anti-human CD3 (V 5T-CD30.05; PharMingen) and PE-conjugated anti-CD4 (Leu 3; Becton Dickinson), anti-CD8 (Leu 2; Becton Dickinson), anti-CD11b (F6.2; Exalpha Biologic, MA), anti-CD14 (Becton Dickinson), anti-CD20 (Becton Dickinson), or anti-HLA-DR (Becton Dickinson). For the analysis of purity in the populations depleted of the various APCs (CD14+, CD20+, or HLA-DR+ cell subsets), selected cell fractions were stained with FITC-conjugated anti-HLA-DR (Becton Dickinson) combined with the PE-conjugated anti-CD4, anti-CD8, anti-CD11b, anti-CD14, and anti-CD20. Isotype-matched controls (FITC-IgG1 and PE-IgG2a; Becton Dickinson) were included for all Ab staining experiments. The staining was performed as previously described. 18
Superficial inguinal LNs were biopsied from the indicated delta nef–infected wild-type–challenged animals using standard surgical procedures or a wild-type–infected animal at necropsy. Single cell suspensions of total leukocytes were obtained as previously described. 21,22 LN cells (LNCs) were kept as a bulk population or depleted of either CD8+ or CD4+ cell subsets (see above), and the T-cell purity was verified by 2-color flow cytometry as described previously.
Macaque moDCs were generated from a highly enriched population of CD14+ cells as previously described. 18,24 After 6 to 7 days of culture in GM-CSF (1000 U/mL) and IL-4 (100 U/mL), immature DCs were used. When comparing immature and mature DCs, immature DCs (day 6) were cultured for an additional 2 days in medium containing GM-CSF and IL-4 (maintaining immature DCs) or were matured with a cocktail of IL-1β (10 ng/mL), IL-6 (10 ng/mL), TNFα (10 ng/mL), and PGE2 (10−6 M). 24 The phenotype of immature and mature DCs as well as the purity of DC populations was routinely monitored by 2-color flow cytometry as described previously. 18 For phenotypic comparison of DCs and CD14+ monocytes, FITC-conjugated anti-HLA-DR was combined with PE-conjugated anti-CD14, anti-CD25, anti-CD80, and anti-CD86 (all from Becton Dickinson) as well as anti-CD83 (PN IM2218; Immunotech-Beckman-Coulter, Marseille, France). Phenotypically, immature DCs express negligible levels of CD14 unlike monocytes; both express little if any CD25, CD80, or CD83 but show intermediate levels of CD86. In contrast, mature DCs significantly upregulate all the activation markers (CD25, CD80, CD83, and CD86) while remaining negative/weak for CD14.
For the immune assays, aliquots of 1 to 2 × 105 DCs or CD14+ monocytes isolated from infected animals were used, and 3 to 5 × 105 cells from naive and infected macaques were used for immunostaining. Cells were placed into 1.5-mL Eppendorf tubes, pelleted, resuspended with AT-2 SIV (30 ng of p27 per 105 cells) in 50 μL of cRPMI, and incubated for 1 hour at 37°C. Control cells were incubated with the equivalent amount of buffer (1% BSA in PBS). The cells were then washed 5 times to remove cell-free virus, 18 and viable large cells were recounted by Trypan blue exclusion and used in immune assays or monitored for the presence of viral protein by immunostaining (see below). 18
The secretion of IFNγ by SIV-specific T cells was measured by IFNγ enzyme-linked immunospot (ELISPOT) assays (U-Cytech, Utrecht, The Netherlands). The IFNγ ELISPOT assays were performed according to the manufacturer's instructions and as previously described. 24 PBMCs/LNCs or the variously depleted fractions were plated 2 × 105 per well in 100 μL of medium. Cells were incubated with AT-2 SIV (0.003–30 ng of p27 per 2 × 105 cells) versus medium as the control, with vaccinia virus vP1071 and its parental vector (NYVAC), or with canarypox virus vCP180 and its parental vector (ALVAC) at a multiplicity of infection (MOI) of 1 or 3, respectively. For the APC-T-cell cultures, T cells were plated (2 × 105 per well per 50 μL) and cocultured with 50 μL of autologous DCs or CD14+ cells pulsed with virus versus buffer at APC/T-cell ratios ranging from 1:20 to 1:180. To verify the capacity of the T cells to respond to stimulation, 5 ng/mL of the superantigen staphylococcal enterotoxin B (SEB; Toxin Technology, Sarasota, FL) was added to 5 × 104 cells. For the APC-T-cell assay, SEB was added to cocultures of T cells (5 × 104 per well) with SIV-pulsed or untreated APCs added at the indicated DC/T-cell ratios. After an 18- to 24-hour incubation at 37°C, the assay was developed according to the manufacturer's instructions. IFNγ spot-forming cells (SFCs) were enumerated under an inverted light microscope. Results are represented as means of triplicate cultures of single experiments.
To measure T-cell expansion, AT-2 SIV (0.003–30 ng of p27) was added to 1 × 105 PBMCs, LNCs, or cells depleted of CD8+ or CD4+ subsets. Cells were cultured in a final volume of 100 μL per well in 96-well round-bottomed plates (Microtest U-Bottom; Becton Dickinson, NJ). Alternatively, DCs or CD14+ cells were pulsed with AT-2 SIV or buffer (see above) and added to 1 × 105 T cells at a ratio of 1:20 to 1:180 per well of a 96-well flat-bottomed plate (Microtest 96; Becton Dickinson). After 5 days of culture at 37°C, 1 μCi of (3H)-thymidine (3H-TdR) was added to each well, and the amount of 3H-TdR incorporated during the last 8 hours of culture was measured to assess T-cell division. To verify the responsiveness of the T cells in each of the cultures, control cultures were set up where cells were incubated with SEB (5 ng/mL) for 3 days. T-cell proliferation was measured using a semiautomated harvesting system (Brandel MWXR-96TI; Gaithersburg, MD) and liquid scintillation counter (1450 Wallac MicroBeta JET; Perkin Elmer Life Sciences, Turku, Finland). Results are represented as mean counts per minute (cpm) of 3H-TdR uptake of triplicate cultures of single experiments.
The presence of AT-2 SIV associated with immature and mature moDCs or with CD14+ monocytes was examined by using the biotinylated human CD4-IgG2, 44 recognizing the CD4-binding epitope of conformationally intact gp120. 18 In brief, virus- or buffer-treated cells were adhered to Alcian blue precoated slides and fixed with 4% paraformaldehyde before nonspecific binding was blocked (100 μg/mL of human IgG; Jackson ImmunoResearch Laboratories) and were thereafter incubated with 25 μg/mL of biotinylated CD4-IgG2 or biotinylated human IgG (Jackson ImmunoResearch Laboratories), followed by horseradish peroxidase (HRP)-conjugated streptavidin (1:100) and the amplification reagent (fluorescein-labeled TSA kit). Before slides were mounted, the cell nuclei were stained with DAPI (1.75 ng/mL of D-1306; Molecular Probes). Mounted slides were then examined using a Nikon Eclipse E800 epifluorescence microscope equipped with the Image-Pro Plus deconvolution module (Media Cybernetics, Silver Spring, MD). Composite figures were made using Photoshop (Adobe Systems).
To evaluate the antigenicity of AT-2 SIV, the activation of SIV-primed T cells in the PBMCs or LNCs of SIV-infected versus naive macaques was analyzed by assessing IFNγ-secreting T cells in ELISPOT and T-cell expansion in proliferation assays following in vitro stimulation with inactivated virus. Rhesus macaques chronically infected with SIVmac239 delta nef that had controlled subsequent challenge with wild-type SIVmac239 were used extensively in these studies as a reliable source of SIV-primed T cells with intact immune function. 24 Cultures of PBMCs from these animals (Figs. 1A, B) showed comparable SIV-specific IFNγ responses to AT-2–inactivated preparations of 2 different SIV isolates: SIVmneE11S (E11S) and SIVmac239 (239). In contrast to infected animals, the PBMCs from naive animals did not respond to even the highest dose of virus tested (see Fig. 1A).
To evaluate whether the amount of viral surface envelope glycoprotein on virions affects the level of T-cell activation, titrated doses of several AT-2–inactivated virus isolates differing in virion envelope glycoprotein content were added to PBMC cultures and tested for their ability to stimulate IFNγ responses (see Fig. 1C). Virus isolates compared included AT-2–treated SIVmac239/251 tail, SIV CP-mac, and SIVmneE11S as examples of high envelope–expressing viruses, SIVmac239/C5 as an example of an intermediate envelope–expressing virus, and SIVmac239 and SIV NC-mac as examples of viruses expressing low gp120 levels. IFNγ responses were dependent on the amount of virus added to the cultures (normalized by p27 content) but were not influenced by the virus isolate or the gp120SU content of the virions (see Fig. 1C). In parallel testing of PBMCs and LNCs from a chronically delta nef–infected wild-type–challenged animal, comparable dose-dependent SIV-specific IFNγ release and T-cell expansion in response to AT-2 E11S were observed (see Fig. 1D). These results confirm that AT-2–inactivated viruses are able to stimulate potent SIV-specific T-cell responses in whole PBMC and LNC assays in vitro.
To identify the T cells activated by AT-2 SIV, PBMCs or LNCs of SIV-primed or naive animals were depleted of either CD8+ or CD4+ cell subsets and tested in parallel with unfractionated PBMCs and LNCs. SIV recombinant canarypox and vaccinia viruses were compared with AT-2 SIV for their ability to stimulate CD4+ and CD8+ T-cell subsets. As shown in Figure 2, both AT-2 SIV and the viral vectors stimulated responses in the PBMCs and LNCs of SIV-infected animals. Notably, AT-2 SIV induced SIV-specific IFNγ secretion (see Figs. 2A, C, D) and proliferation (see Fig. 2B) of both CD4+ and CD8+ T-cell subsets in a dose-dependent manner. This is in contrast to the preferential CD8+ T-cell responses induced by SIV-recombinant vaccinia and canarypox viruses (see Figs. 2A, C, D). Thus, although variable in intensity, CD4+ and CD8+ T-cell responses against AT-2 SIV were reproducibly observed in the blood and LNs of several healthy delta-nef–infected and wild-type–challenged animals (see Figs. 2A–C) as well as in those of a wild-type–infected animal examined at necropsy (see Fig. 2D).
The fact that the AT-2–treated virions are able to activate both CD4+ T cells and CD8+ T cells indicated the requirement for APCs to capture and process whole virus and properly present viral Ags on major histocompatibility complex (MHC) class I and II molecules. Therefore, we investigated which APC subset(s) in PBMC cultures are responsible for the induction of T-cell responses against AT-2 SIV. APCs such as monocytes (CD14+), B cells (CD20+), and HLA-DR+ cells (comprising all APCs, including blood DCs) were selectively removed from PBMCs before the remaining cells were stimulated with AT-2 SIV in an IFNγ ELISPOT assay. The data from 3 separate experiments are summarized in Table 1. The efficiency of the removal of each subset was verified by FACS analyses in each experiment, and the depletion of the CD14+, CD20+, or HLA-DR+ cell fraction was routinely >95% (data not shown). Although neither the depletion of CD20+ cells nor the treatment with the matched isotype Ab (or medium) had a demonstrable impact on the stimulation of IFNγ production, the removal of CD14+ cells had a variable influence on the magnitude of the IFNγ responses measured (reducing responses from ∼15%–85%) (see Table 1). In contrast, SIV-specific T-cell responses were consistently abrogated after depletion of HLA-DR+ cells. These results indicated the requirement for HLA-DR+ APCs in the activation of CD4+ and CD8+ T cells rather than virus directly activating the T-cell subsets.
The fact that B cells appeared not to take part and CD14+ cells only contributed to variable extents in the SIV-specific T-cell activation suggested the involvement of DCs in the T-cell activation of whole PBMCs or LNCs. DCs show differentiation stage-dependent functions. The use of in vitro generated moDCs enabled us to compare classic immature and mature DCs for their capacity to present AT-2 SIV and to induce virus-specific T-cell responses.
To compare the capacity of immature versus mature DCs to process and present captured AT-2 SIV, immature and mature DCs were pulsed with AT-2 SIV and washed to remove unbound virus before being recultured with autologous SIV-primed T cells at varying DC/T-cell ratios. Because CD14+ may contribute to some of the presentation of AT-2 SIV as indicated in Table 1, CD14+ monocytes were initially assayed in parallel. As summarized in Figure 3A, although SIV-specific T-cell responses such as proliferation and IFNγ were observed with the AT-2 SIV-loaded monocytes as APCs, greater responses were stimulated by DCs. Remarkably, the mature DCs tended to induce slightly higher IFNγ responses, and this was most apparent at the higher DC/T-cell ratios. Even at cell ratios of 1 APC to 180 T cells, immature and mature DCs provoked strong T-cell responses, whereas Ag presentation by monocytes did not. Although the magnitudes of each response varied for different donors, the patterns remained consistent between donors. This is in contrast to T cells isolated from naive animals, which did not show measurable responses, even when stimulated with AT-2 SIV–pulsed autologous mature DCs (see Fig. 3B). T-cell responses to SEB were strong and comparable in infected (data not shown) and naive animals (see Fig. 3B), verifying the functionality of the APCs and T cells in each culture.
We were interested to ascertain whether the lower responses induced by the monocytes may relate to the amount of virus Ags captured by these cells. Although both immature and mature DCs capture AT-2 SIV, mature DCs sequester virus in large vacuoles localized deeper within the cells, whereas immature DCs retain virus in smaller vacuoles that are typically more dispersed around the cell closer to the periphery. 18,29 To qualitatively compare the uptake of AT-2 SIV by DCs and CD14+ monocytes at the single cell level, we compared monocytes with immature and mature DCs from the same donor and incubated the cells for 1 hour at 37°C with AT-2 SIV before immunostaining for viral proteins. As shown in Figure 4, all 3 populations captured AT-2 SIV. Although the level of protein staining in CD14+ cells was qualitatively consistently lower than that seen in immature and mature DCs, the localization of the viral proteins was comparable to that of the immature DCs. Here, viral envelope proteins were detected in small vesicles that were mostly found closer to the cell periphery. This is true for naive (data not shown) and infected animals (see Fig. 4). Similar results have been observed with human cells, where lower levels of viral uptake by CD14+ cells compared with DCs were also confirmed by reverse transcriptase (RT) PCR analysis (data not shown).
The observation that both immature and mature DCs capture AT-2 SIV, hold it in different intracellular locations (see Fig. 4), 18 and possess the ability to process and present captured AT-2 SIV (see Fig. 3A) leads to the question of whether distinct DC populations might activate different T-cell subpopulations. Additionally, previous results strongly suggested that the uptake of AT-2 virus by DCs is dependent on the presence of functional viral envelope glycoproteins. 18,35 Therefore, we also addressed the question of whether uptake of more AT-2 SIV by DCs results in enhanced activation of T-cell subsets. To do so, DCs were loaded with AT-2 inactivated wild-type SIVmac239 virus (Low env) or SIVmac239/251 tail virus (High env), which varies in gp120SU content by approximately 10-fold, and then recultured with autologous CD4+ or CD8+ T cells. As shown in Figure 5, the gp120SU content of the virions tested did not appear to affect the type or magnitude of the T-cell response induced at the input virus dose tested. Thus, although less virus was captured by the DCs after pulsing with the low env–expressing SIV, 18 this was sufficient for the DCs to activate SIV-specific T cells. Strikingly, although immature DCs preferentially activated CD4+ T cells, mature DCs induced IFNγ release from both CD4+ and CD8+ T cells (see Fig. 5). These data suggest that mature DCs are also equipped to process cell-associated whole AT-2 SIV as well as to then present virion-derived peptides on MHC class I and II molecules to activate CD8+ and CD4+ T-cell responses.
To appreciate better how DCs can be used to boost vaccine efficacy, these studies evaluated the presentation of a chemically modified whole inactivated virion immunogen, which retains functional envelope glycoproteins, thereby mimicking authentic interactions with DCs and T cells. 18,29,30 By virtue of their particulate nature and functional envelope glycoproteins (even when expressed at low levels), these virions are taken up and presented by mature DCs to stimulate MHC class I– and II–restricted responses, activating CD8+ and CD4+ T cells. Preliminary data from in vivo studies in macaques indicate that inactivated SIV with conformationally and functionally intact envelope glycoproteins induces neutralizing Ab responses as well as CD4+ and CD8+ T-cell responses (J. Lifson et al, in preparation). Herein, we have also carefully studied some of the variables affecting the efficiency of Ag-specific stimulation mediated by such inactivated virions, including dose response relationships, the comparison of the responses stimulated by inactivated virions to those stimulated by the presentation of similar Ags via avipox vectors, the envelope content of the virions, and the ability of different APCs to present inactivated virion-derived Ags.
When added to in vivo primed SIV-specific T cells, whole inactivated virions successfully activated dose-dependent CD4+ and CD8+ T-cell responses. A qualitative shift in the T-cell response toward the sole activation of CD8+ T cells was noted when similar Ags were presented via classic vectors such as vaccinia or canarypox viruses, as previously described for HIV-1–specific responses. 45,46 The magnitudes of the (CD8+) responses against the SIV-recombinant avipox constructs were comparable to, if not stronger than, those induced in PBMCs by HIV-1–recombinant avipox vectors, 36,45 comparable to the levels seen when immature DCs were directly infected with HIV-1–recombinant vaccinia or canarypox virus and used to stimulate autologous HIV-1–specific T cells. 39,46 The observation of the significant CD8+ responses resulting from avipox vector stimulation compared with the more balanced CD4+ and CD8+ responses induced by AT-2 SIV reveals an important difference with possible major implications for vaccine potential.
The most prominent difference accounting for the qualitative distinctions between the activation of T cells through the presentation of inactivated virion-derived Ags versus similar Ags presented via the avipox vectors most likely lies in the nature of the Ag and how it is handled by the APC. In contrast to whole replication incompetent inactivated SIV, viral vectors infect APCs 46–48 and thereby lead to de novo biosynthesis of the encoded Ags within the APCs, representing the classic requirement for endogenously derived Ags to trigger CD8+ T cells, whereas Ag has to be captured by APCs from the environment (exogenously) to access the MHC class II processing pathway, likely accounting for the activation of CD4+ T cells by inactivated virus. Simultaneously, the stimulation of CD8+ T cells by AT-2 SIV is indicative of the involvement of a potent APC capable of introducing exogenous virus to the MHC class I processing pathway, probably occurring as a result of the virus fusing with the cell membranes of the APCs, allowing Ags to enter the cytosol and gain access to the MHC class I processing machinery. 35 Besides the capacity of the inactivated virus to activate CD4+ and CD8+ T cells, the fact that different virus clones were equally well recognized by the T cells indicated the potency of these inactivated whole virions as a promising Ag source in successfully providing a wide spectra of immunologically relevant Ags for the activation of T cells. Furthermore, these responses were comparable to the level of T-cell responses induced by live virus.
AT-2 SIV is internalized by both immature and mature DCs, with the mature DCs retaining larger numbers of intact particles even though viral RNA levels were the same or higher in immature DCs. 18 Remarkably, although the uptake of AT-2 SIV by DCs is influenced by the amount of gp120 expressed on the virus surface (higher levels of virus-associated envelope favor more virus uptake), 18 it did not have a significant impact on the magnitude or the type of T-cell response induced (in the DC/T-cell and PBMC assays). These data imply that the uptake of even small amounts of AT-2 SIV by DCs (and possibly other APCs) is enough for efficient processing of Ag to activate CD4+ or CD8+ T cells. Notably, in PBMCs using heated virus, in which envelope is stripped from the virion and the native conformation is lost, the magnitude of T-cell responses can only be reduced by as much as one third at the higher Ag doses (3–300 ng/mL) and two thirds at the lower doses (0.3 and 0.03 ng/mL) (data not shown). Removal of the bulk of the stripped envelope (by centrifugation) does not further influence the response to heated virus, suggesting that at least some responses are specific for determinants found in conformationally intact envelope. The possibility that some virus uptake by APCs occurs independent of envelope (which would allow other determinants to be presented) is likely. Definitive identification of the T-cell specificity (and frequency) induced by AT-2 viruses would require AT-2 virus–activated T cells to be restimulated with recombinant vectors expressing select viral genes or specific peptide pools.
A direct comparison of DCs at different activation stages showed that both immature and mature DCs activate autologous SIV-primed T cells in an Ag-specific manner, although the magnitude of the T-cell response was often higher with the mature DCs. These results are in agreement with the literature of previous in vitro and in vivo observations, which show that immature DCs loaded with the inactivated virus can lead to T-cell activation 35,36,39,49 and that the more activated IFNα–treated DCs lead to stronger responses than DCs generated with IL-4 and GM-CSF only. 49 Thus, these data suggest that immature DCs, although specialized for the uptake of Ag and less well equipped for the activation of T cells, are able to induce immune responses, whereas mature DCs, which have a diminished capacity of macropinocytic uptake but are well suited for Ag presentation, also have the capacity to internalize and process whole structurally intact virions and present the virion-derived peptides on MHC molecules. The finding that the Ag processing machinery is most efficient in mature DCs further supports this. 50 Our recent studies also revealed that although considerable amounts of AT-2 (or live) virus captured by immature and mature DCs is degraded, the virus proteins are localized in intracellular compartments that do not stain with classic endosomal/lysosomal markers (S. Turville et al, submitted). This contrasts where monomeric gp120 is located and suggests that whole virus diverts the normal processing pathways in DCs to favor virus spread over immune activation. Immature DCs tend to degrade virus more rapidly than mature DCs and may limit the access of virus to the Ag processing pathways. Studies to identify the unique compartment(s) in which immature versus mature DCs sequester and process virus are underway.
Closer analysis uncovered that immature DCs preferentially evoked CD4+ T-cell responses with only low levels of CD8+ T-cell activation, whereas matured DCs efficiently stimulated both CD4+ and CD8+ T cells. Thus, using the in vitro–generated immature and mature moDCs (phenotype was confirmed by FACS) revealed that capture of the inactivated virus by immature DCs 18 targets at least some of the virus to compartments allowing the association of processed antigenic peptides with MHC class II molecules, 51 facilitating the activation of CD4+ T cells. Interestingly, a recent report described how tubular endosomes extend within the DC and polarize toward the interacting T cell but only when Ag-laden DCs encounter T cells of the appropriate specificity, 52,53 explaining how maturing DCs increase the levels of MHC class II peptide complexes for more efficient T-cell activation. In this scenario, an SIV-specific DC/T-cell encounter could favor the direct transport of AT-2 SIV–derived peptide-loaded MHC class II molecules to the interaction site, thereby favoring the activation of SIV-specific CD4+ T cells. On the other hand, virus captured by DCs moves rapidly to the point of contact with a CD4+ T cell, inferring a mode of virus transmission to the T cell (S. Turville et al, submitted). 54 Although we have demonstrated that immature DCs can stimulate CD4+ T cells, it is possible that the virus hijacks this MHC class II peptide–transporting tubule machinery to facilitate DC-to-T-cell transmission of virus. If true, this would provide an effective way in which the virus could more efficiently target virus-specific CD4+ T cells as suggested by Douek et al. 55 This further highlights how the virus may specifically exploit immature DCs at the body surface to facilitate infection and cell-to-cell spread while inducing suboptimal immune responses.
In contrast, mature DCs stimulated CD4+ and CD8+ T-cell responses. Larsson et al 39 made similar observations, where mature DCs exposed to inactivated HIV-1 also activated both CD4+ and CD8+ T cells from seropositive donors. Noteworthy, the use of X4-versus R5-tropic strains or different DC activation stimuli (monocyte conditioned media [MCM] vs. a cocktail of inflammatory factors) had no significant impact on the induced immune response, indicating that the processing of exogenously captured AT-2 virus by mature DCs is independent of virus tropism. The ability of DCs to capture whole SIV particles and to process and present at least some of this to CD8+ T cells also agrees with earlier work that highlighted how this is largely mediated by virus fusing with the cell membrane (probably surface as well as compartment membranes) to access an exogenous MHC class I pathway. 35
The nature of DCs being able to direct qualitative aspects of the T-cell response may account for the CD4+ and CD8+ responses observed in PBMC cultures. The fact that the depletion of HLA-DR+ cells from PBMCs comprising all APCs, including MDCs and plasmacytoid-derived DCs (PDCs), completely abolished the T-cell responses strongly implicated the involvement of APCs in the activation of T cells. A further attempt to define the HLA-DR+ cell subset based on the surface expression of the MDC marker CD11c was not possible due to the heterogeneous expression pattern of CD11c on monkey leukocytes (I. Frank and M. Pope, unpublished observations). Therefore, the contribution of MDCs (which get activated during overnight culture to favor CD4+ and CD8+ responses; N. Teleshova and M. Pope, unpublished observations) to the presentation of AT-2 SIV in PBMC cultures could not be addressed in this way. Furthermore, depletion of the CD123+ PDCs, which represent less than 0.5% of the leukocytes in freshly isolated macaque blood (N. Teleshova and M. Pope, unpublished observation), did not significantly alter the T-cell response (data not shown). Direct analysis of the ability of PDCs and MDCs to present AT-2 SIV is being performed in separate ongoing studies.
In contrast to the depletion of HLA-DR+ cells, the response in PBMC cultures was unaffected by the depletion of B cells and was only partially diminished when CD14+ cell subsets were removed. Although CD14+ monocytes can capture and become infected by HIV-1/SIV, 56,57 their capacity to capture whole AT-2 SIV was marginal; more significantly, they were much less efficient in inducing strong T-cell responses compared with the DCs. Although the possibility that some monocytes carrying a few of the inactivated virions convert into macrophages cannot be excluded, macrophages are less likely responsible for the overall response due to their much weaker MHC class I–restricted Ag-presenting capacity compared with DCs. 35 Monocytes/macrophages also show only poor or no expression of CD80 and CD86, 2 costimulatory molecules critical for the activation of T cells (confirmed here for macaque CD14+ cells, data not shown). A subset of CD14+CD16+ cells representing a precursor for DCs in blood was recently characterized by the expression of high levels of HLA-DR and a robust capacity to induce T-cell proliferation. 58 In addition to resident MDCs, this newly defined subset of blood DC precursors could also be involved in the responses observed in the PBMC cultures.
This work demonstrates that in contrast to traditional viral vectors expressing HIV-1/SIV Ags, whole inactivated virions represent a reliable way to stimulate antigenically broad and strong dose-dependent CD4+ and CD8+ T-cell responses in the periphery as well as in LNs. Loaded onto DCs, exogenous AT-2 SIV is captured by immature and mature DCs and can access the MHC class II processing pathway in both cases, but only the virus associated with the mature DCs could also be presented on MHC class I molecules. Although a recent report suggested that AT-2 SIV–loaded maturing DCs can boost immunity and antiviral responses in vivo, 12 our study suggests that targeting fully mature DCs with AT-2 SIV might provide the most efficient stimulus to prime and/or boost SIV-specific immunity. AT-2 SIV represents a promising immunogen in the development and further improvement of antiviral therapeutic vaccine strategies against HIV-1.
Animals used for these studies were housed at the TNPRC. The authors thank James Blanchard, Richard Rockar, and Marion Ratterree for providing care and support for the animals; Ronald Desrosiers (Harvard Medical School/New England National Primate Research Center) and James Hoxie (University of Pennsylvania Medical School) for providing viruses used in these studies; Larry Arthur and Julian Bess, Jr (AIDS Vaccine Program, SAIC Frederick) for the AT-2–inac-tivated virus preparations; Elena Chertova (AIDS Vaccine Program) for characterization of the envelope glycoprotein content of these preparations; and William C. Olson (Progenics Pharmaceuticals, Tarrytown, NY) for providing the biotinylated CD4-IgG2. The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 174 × CEM from Peter Cresswell.
This article has been cited 2 time(s).
simian immunodeficiency virus; immune response; whole inactivated virus; dendritic cells
Copyright © 2003 Wolters Kluwer Health, Inc. All rights reserved.
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Today Apple announced that it is shutting down Ping and you will be able to like and share to Facebook while you are in the iTunes store.
Facebook Exchange officially launched, and you will now be able to purchase Facebook ads through real-time bidding.
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Hindustan Dorr-Oliver's has informed the market that it has received an order worth Rs 60.30 crore from City & Industrial Corporation of Maharashtra (CIDCO), Navi Mumbai for commissioning 70 million liters per day (MLD) sewage treatment plant at Kharghar in Navi Mumbai. The project is to be executed in 2 years.
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Hindustan Dorr Oliver hits upper circuit – wins order worth 24.5 crore
Hindustan Dorr Oliver Ltd has informed the market that the Company has bagged an order from National Aluminium Corporation Ltd and the order is for civil and structural work and the contract is valued at Rs 24.5 crores for their Phase II expansion project at Alumina Refinery, Damanjodi, Orissa.The execution time for the project is 6 months.The stock as hit upper circuit and its trading at 72.50 up by 5 percent .