The Sugarlands Visitor Center near Gatlinburg, Tennessee will be hosting its 34th Annual Festival of Christmas Past celebration. Scheduled for December 12, 2009, from 9am-4pm, this event celebrates Christmas and the traditions that surrounded a Smoky Mountain Christmas in days of yore. Those traditions included gathering in churches, homes, and schools, and many celebrated the holidays through music, storytelling and crafts. This year's festival will include mountain music, harp singing and demonstrations of basketmaking, quiltmaking, spinning and apple butter making. This event is free to the public.
Employees and alumni of Great Smoky Mountains National Park and its partners express the deep connections that inspire their daily efforts to protect, preserve and support the Great Smoky Montains National Park. Visitors also offer special sentiments of what the Smokies mean to them.
The Great Smoky Mountains Association has launched a series of podcasts and blogs that are available from their website or the iTunes podcast directory.
The podcasts vary in subject matter, including audio tours of Newfound Gap Road and tips on what to do when you encounter a bear in the backwoods. In all, they have 41 podcast episodes available and seem to be adding new episodes frequently.
Visit the GSMA website to download individual podcast episodes. If you are an iTunes user, you can subscribe to the GSMA Podcast to receive updates automatically and easily sync them to your iPod.
On Saturday, May 9, Dolly returns to Dollywood for the premiere of the park's new musical Sha-Kon-O-Hey! Dolly wrote all eight songs featured in this musical journey that takes audiences to the heart of the Smoky Mountains.
As part of her official ambassadorship for the 75th anniversary of Great Smoky Mountains National Park, Dolly will be the musical host in a special 7 p.m. ticketed performance of Sha-Kon-O-Hey! in DP's Celebrity Theatre.
This special ticketed event is a fundraiser for Friends of the Smokies, a not-for-profit organization devoted to preserving and protecting the Great Smoky Mountains National Park by raising funds and public awareness. For ticket information, visit www.friendsofthesmokies.org .
Come Celebrate The Great Smokies 75th Anniversary!
Great Smoky Mountains National Park Superintendent Dale Ditmanson has worked closely with his partners, Great Smoky Mountains Association and the Friends of the Smokies, and with our neighboring communities, to develop the GSMN 75th Anniversary site as their "Virtual Visitor Center" to showcase "all things 75th".
On their site you will find a rich and evolving variety of information on the Park's history, current issues, and future visions. Throughout 2008 and 2009 you'll find information about a wide array of activities and events in the Park as well as officially recognized events in all the neighboring communities.
You can even share YOUR photos and stories of your Great Smoky Mountains National Park adventures through the decades!
Send your friends and family a cyber invitation to enjoy your approved image. After looking at your adventure, they can upload their photo and story to become a part of the Smokies Family Album, too! Images should contain people and a recognizable feature, overlook, or sign that ties the image to Great Smoky Mountains National Park.
Click here for the Great Smoky Mountains National Park 75th Anniversary Site!


Navigator | Ontario Election Special: What Just Happened?
Ontario Election Special: What Just Happened?
On this week's episode of Political Traction, Navigator Executive Chairman, Jaime Watt, presents the results of our province-wide post-election survey.
After the votes were tallied and the next premier was decided, Navigator conducted focus groups across Ontario to understand why Ontarians voted the way they did.


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Spreading the message that great brands should know how to stand out from the crowd, Yeti made a couple of unannounced public appearances. First on the Nicollet mall for lunch, and then near the start of the 2014 Twin Cities Marathon, bringing a rush of happiness to huggers and high-fivers who like a little instant karma. Where will he appear next? Your guess is as good as ours.


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Five Bisons given all-star nod after stellar season
Lauryn Keen named Outstanding Player, Jon Rempel takes coaching award
Photo by Star Roxas
By Jason PchajekMarch 7, 2017
The season may not have ended the way they wanted, with the Bisons being swept in two games by the University of Alberta Pandas, but Rachel Dyck, Lauryn Keen, and Venla Hovi still had cause to celebrate. The three women were among the five Bisons players named to the Canada West first and second all-star teams. Alexandra Anderson and Erica Rieder were also given the honour. The Manitoban sat down with Keen, Dyck, and Hovi and talked about the season.
"It's a team game but to be recognized personally is always nice," Bisons goaltender Dyck said. "Pretty humbling to see your hard work pay off throughout the season, and it's a competitive conference so we know that if you do receive anything like this then it's pretty legitimate."
Dyck finished the year tied for first in both wins and shutouts amongst Canada West goalies, with 15 and five respectively. The Winnipeg Beach native had success across the board, putting up a 1.51 goals against average and .931 save percentage while playing the second-most games among goalies in the conference.
Keen's 34 points outpaced all other skaters in Canada West, leading to her receiving a nod as the conference's Most Outstanding Player. She recorded nine goals and 25 assists through 28 games this season, eclipsing her previous career totals.
"It just goes to show how hard work can pay off," Canada West leading scorer Keen said. "When you work as a team it really brings out individual abilities as well, like working hard so other people can be noticed."
Hovi says that the sheer number of Bisons selected is a testament to the quality of the team.
"You also have to look at it big picture," the second-year winger said. "Taking five out of one team, that's a big amount so it just says something about our team for sure."
Hovi missed time this season due to injury, but that didn't slow down the Finnish forward, who produced at a consistent clip on a line with Keen. Despite appearing in only 22 games this season, she finished tied for third in Canada West scoring, riding a 1.14 points per game average to 10 goals and 15 assists.
Apart from the disappointing end to the season, all three women felt pleased with the year and excited for the next. The Bisons finished the season with a 19-7-1-1 record, good enough for 56 points and a third place finish in the conference.
"I thought this was probably one of our best seasons," Dyck said of the year as a whole. "We were just consistent the whole way through. We hit a few bumps, we had injuries, people going away on trips, that kind of thing but it didn't really affect us."
This sentiment was reflected by Keen. "Basing it on my last two years we've been the most consistent this year," she said.
"My first year we had zero expectations so we were just playing to play, last year people weren't too sure about us, then this year I think the expectations were so much higher and I think we did a good job meeting those expectations."
Hovi said that the team has learned from last year, translating that into a successful season.
"I think the whole group just grew so much from last year," she said. "Playing consistently this season definitely told us that we've grown as players and as athletes and people too."
The same day the five Bisons were given the all-star nod, their coach was given his own praise. Bisons head coach Jon Rempel was named Canada West's Coach of the Year for the 2016-17 season. Rempel has been coaching the team for the past 13 seasons, guiding them to two conference championships – in 2008-09 and 2010-11 – as well as five national championship appearances. This is the fourth time Rempel has been named coach of the year, with the last coming during the 2014-15 campaign.
"I have to say personally he was a little different this year," Hovi said of her coach, prompting laughter from her teammates. "He was able to trust us more and give us more freedom and in that sense he showed us that he was really trusting of all of us and was maybe a little bit calmer."
"I also liked his approach this year," Keen said. "I wasn't around when he was very very intense but there have been times where he's pushed us very hard and it was nice to come in this year and know what he expects of us, but not having to force us to do it all the time."
With all three women coming back for the next Bisons season, expectations may be even higher. Despite losing some key contributors, including Dyck's backup Amanda Schubert, the three were all optimistic about the season ahead.
"Well I'm going to be in my fifth year so that's scary," Dyck said. "I think each year we've been able to do something pretty cool and I think next year is just going to get better. So I'm pretty excited about the possibility of what we can do."
Keen added that she was also excited and is looking forward to the offseason ahead.
"Putting this year behind us and looking to next year, so it's exciting knowing we have a summer to train and get better and take a break from the rink a bit but I'm excited to come back next year and take it all the way," she said.
Hovi also pointed to the potential the team has to keep rolling next season.
"When you're left with the emotions of being disappointed that we didn't go any further I'm sure the whole team is very hungry to work even harder," she said. "This season we recognized how important those little things are and next year will be our year."
For now, the three are looking towards their summer break. However, that doesn't mean they'll be resting too much.
"Hopefully get a good job," Dyck said of her summer plans with a laugh. "The off-season is always exciting. You get that opportunity to work on your game, train hard, and make yourself hungry for next season."
Keen is looking to fine-tune her game during the off-season, but also looking forward to some time off.
"You get a whole lot of time to work on your game, the little aspects you can't work on in-season," she said. "Like personal strength for me and shooting. Just getting some time away from the rink also."
Hovi is in a different boat, as she will be heading back home to Finland for some time with the national team, a well-deserved reward for a great year.
On the MVP track
New-look Jets, newer questions
Catching up with the Bisons
Go Jets Go

redpajama_set_name: RedPajamaCommonCrawl


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Should You Take Your Mama Out to See These Movies?
It sounds like a double feature from hell.
Billy Crystal, Bette Midler, and Barbra Streisand starring in two movies with similar plot lines: Older Jewish parents have a hard time connecting with the younger generation.
Both films are aimed squarely at a mainstream crowd, and are being released within days of each other.
Should you see them as a double feature, is one of them enough, or should you skip them both altogether?
Here are my reviews.
Turn out the lights, Alice
Parental Guidance is a movie for people of a certain age who don't like movies. That is, people who think they're too violent, too loud, too long, too serious … too "current."
Crystal and Midler star as Artie and Diane, who are called into duty to look after their grandkids, and have problems with the modern-day facacta parenting style their daughter and her husband (Marisa Tomei and Tom Everett Scott) use.
Artie, a minor-league-baseball announcer, has been forced into retirement because, well, he's old, and the old-fashioned ways of doing things just aren't hip and cool anymore — which kind of makes this movie like the (attempted) comedic version of Trouble with the Curve and Skyfall.
I'll save you a few bucks, and the annoyance of having to sit through Crystal and Midler mugging for the camera, acting like they don't understand technology, and basically overacting their way through this lame film: Artie and Diane's old ways of parenting win out over the newer ones. Surprise!
Insulting, stupid, and featuring a predictably cheesy score by Marc Shaiman that hits all kinds of treacly notes when it wants to wring a little emotion from the audience, Parental Guidance is a complete waste of time and talent. I haven't felt this embarrassed for an actor (in this case, Crystal) since Meryl Streep in Hope Springs.
Skip this movie. Even when it's on TV and there's nothing else on.
Much better, surprisingly, is The Guilt Trip, in which Andy (Seth Rogen), an unsuccessful inventor, invites his overbearing, Gap-obsessed mother Joyce (Streisand) to join him on a cross-country road trip.
Joyce could have been a character full of clichés — which is not to say there aren't any — but instead she's one that comes from a place of authenticity, and is not, apparently, the result of interference from big-studio know-nothings. Much credit for that goes to the screenplay by Dan Fogelman (the great Crazy Stupid Love) and of course, Streisand, who gives the best on-screen Jewish mother performance since Meryl Streep's in Prime. I laughed a lot because I know people just like this woman. Heck, one of them raised me.
Jokes aside, it's nice to see a film treat a mother-son relationship as more than just a wacky mismatch. Andy and Joyce's lives both haven't gone as planned, and both are dealing with disappointment they don't want to admit — to themselves and each other. There are some sweet scenes where son and mother see sides of the other they never wanted to see (or never thought existed), and a lot of heart throughout. If you don't laugh at everything, you'll at least be smiling.
That Andy learns by trip's end that maybe mother really does know best, and Joyce learns she doesn't have to interfere as much in her son's life, is predictable. But when the realizations happen, they're not done in oversized cheeseball fashion.
The Guilt Trip gets a little episodic at times, and some situations strain credulity, but Rogen and Streisand have a warm chemistry that'll make you kvell, and that make this a film a trip worth taking. For no other reason, see it for the scene where Joyce eats an entire 4.5-pound steak, just so she can get it for free. Streisand wouldn't travel to shoot this movie (no joke), but she'd play a character who does that?
The inevitable crossover
While watching Parental Guidance, I started to wonder why Midler hadn't yet made a movie with Ari Graynor (For a Good Time, Call…). And then Graynor popped up in a brief cameo in The Guilt Trip, making these films even more cosmically connected.
Who knows if a Midler-Graynor film — naturally, one about a brassy mother and her irrepressible daughter, a couple of showbiz hams — will ever get made, or if it will be any good (probably not). But while we wait for that one, we have these two films of mixed quality.
Parental Guidance gets a C–. The Guilt Trip gets a B.
Will you be seeing either of these movies? Share your thoughts in the comments section below.
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One Response to "Should You Take Your Mama Out to See These Movies?"
It's Roniah Tuiasosopo's New Favorite Movie! « Martin's Musings - February 8, 2013
[…] road-trip comedies we've all seen so many times before (including, most recently, the much better The Guilt Trip), as these two stop along the way and run into such folks as Modern Family's Eric Stonestreet and […]


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John Key to visit India in October
Venkat Raman Former Prime Minister Sir John Key will visit India in October this year. Although the visit is primarily to chair a meeting of the International Democrat Union (IDU), he will utilise his visit to foster his friendship with India's Prime Minister Narendra Modi and exchange thoughts and information on international developments and further the goals of the organisation.…
Free Mobile App launched in Rotorua
Venkat Raman A timely software that runs as Mobile App that sets off an instant alarm with GPS location was launched in Rotorua on Tuesday, July 11 with a similar launch planned in Auckland on Thursday, July 13 at press time. Developed by Paramdip Singh, Director of Special Projects International (formerly Waiariki Institute of Technology), the App, called, 'Please Help'…
Indian listed Fourth on Act List
July 12, 2017 July 12, 2017 Indian Newslink
Venkat Raman One person of Indian origin is prominent on the ACT Party List announced in Auckland on Sunday, July 9, 2017. Bhupinder Singh Real Estate Agent Bhupinder Singh will contest from the Manukau East constituency in the General Election scheduled to be held on Saturday, September 23, 2017. Mr Singh, who works for Ray White, is better known for…
ElectionLeave a comment
Anne Pala gets NZ First nomination for Kelston
Winston Peters Community Development consultant and company director Anne Degia-Pala is New Zealand First's candidate to contest the Kelston seat in the General Election. Anne has called Kelston home for 30 years and is passionate about her community and her country. She is well known and respected across the electorate for her work with women, children and Kelston's diverse ethnic…
Education unlocks the key to better prisoner outcome
Louise Upston We are on a mission to create safer communities. A key part of this is employment. Imagine never having a regular job. Never getting up and do a day's work. Never feeling the satisfaction that comes from a job well done, and the added security, increased self-esteem and respect that a regular income provides. A regular income that…
Democratic ideas entail sanctity of suffrage
General Election 2017 First in a series of Six Parts Venkat Raman It isn't every day that people face an election. It isn't every day that ordinary people are considered the most important entities in the country. It isn't every day that we can make politicians and lawmakers listen to us and make them answerable to their acts of omission…
Industrial training provides career boost
Louise Upston Across my portfolios of Corrections, Associate Education, Associate Primary Industries and Associate Tertiary Education, Skills and Employment, there is a topic about which I am passionate. That is expanding the pipeline of people into employment and training. We have a skills shortage in New Zealand. I believe that by expanding this pipeline we can make a significant contribution…
Robust electoral system inspires confidence and trust
General Election 2017 How it works and why it is important to us Regulated Period for expenditure limits Second in a Six-Parts Series Venkat Raman New Zealand's voting public can expect a wide range of policies, programmes and incentives offered by various political parties as the General Election 2017 enters an exciting and energetic phase. While the polling booths will…
Viable alternative and true voice of the people
Roshan Nauhria The New Zealand People's Party (NZPP) is a relatively new political party. Formed in 2015, the Party now boasts of large Indian and Chinese branches. While we would like to represent all of New Zealand, it is no secret that we are particularly dedicated to bringing attention to the causes and concerns that are important to our communities.…
People's Party to revisit Immigration regime
Venkat Raman The New Zealand People's Party (NPP) seeks major changes to the country's immigration regime, making it more responsive to the needs of the economy, appropriately aligned with the aspiration of the people. The Party's Immigration Policy, announced on July 7, 2017 (and carried in full without our commentary under 'Latest Stories' in our Web Edition of that date…


Just as the aviation industry was headed toward recovery after its plunge following 9/11, the 2008 financial crisis hit, sparking additional dramatic change in the industry. Airline consolidation resulted in hub closures and flight reductions at many airports. Capacity discipline practices changed the landscape of airline services at airports across the country. Between 2006 and 2009, the number of available seats at U.S. airports dropped by 11 percent and the number of flights fell 13 percent.
Air service is more than just flights and seats. It's about connecting to major centers of domestic and international activity.
Since then, flights have continued to decline slightly while seats have increased modestly. But these aggregated numbers don't tell the full story of what matters to passengers – how the air travel options and cost to get to where they want to go have been impacted.
At GRA, Incorporated, we are developing tools that go beyond standard analyses of changes in the number of airline seats and flights. These tools reveal how the relative economic connectedness of U.S. airports has changed since 2006. Greater connectivity means more opportunities – for airports, for businesses, for air travelers and for local economies. The GRA Service Index (GRASI) we've developed allows airports, airlines, passengers, businesses and local government leaders to assess what is happening to the air service available at individual airports and to evaluate how they are performing in terms of connectivity relative to similar airports.
GRASI is unique, in that it measures both connectivity and economic access.
GRASI is unique, in that it measures both connectivity and economic access. Our index quantifies service from an airport to a set of 50 domestic and 17 international airports in cities that account for a substantial share of global economic activity. Airport service is weighted by destination GDP, placing higher value on routes to economic hotspots. It accounts for available seats, aircraft type, and the number of weekly flights (nonstop, one stop and two stop), counting only legal and competitive connections with appropriate adjustments for online, code share and interline quality of service. GRASI ranges from zero to 10,000 points, which allows an airport to evaluate its access to global centers of economic activity relative to that of its peers. Additionally, GRASI tracks variation in airport network access over time, demonstrating how external factors – such as the financial crisis, fuel prices and airline consolidation – have affected air service in airports and in cities.
GRASI ranges from zero to 10,000 points, which allows an airport to evaluate its access to global centers of economic activity relative to that of its peers.
What have we discovered from GRASI? Well, airports haven't been affected equally by recent changes—and the differences are greater than one may think. While large variation in index scores across airport hub groups is endogenous to our model (large hubs have more frequent and varied service than medium hubs, and small hubs have even less service options), we observe disproportional changes in connectivity quality among the hub groups over the past eight years. These changes are similar to the changes in available seats across hub groups over the same period. The figure below depicts changes in both seats and connectivity by airport size since 2006 (using 2013 hub classifications). Between 2006 and 2014, both small and medium hubs have experienced decreases in GRASI of 17 percent, while encountering losses of a similar magnitude in available seats. While large hubs have recovered nearly all losses in available seats over the past eight years, the average GRASI of the hub group remains four percent below 2006 levels. In contrast, non-hubs experienced a nine percent GRASI decrease, on average, but have 13 percent fewer available seats than they did eight years ago.
There are significant differences in connectivity growth rates within airport hub groups as well, as shown in the charts below. Among large hubs, San Francisco International Airport (SFO) and Miami International Airport (MIA) experienced the greatest connectivity increases. SFO's GRASI score increased 35 percent, improving the airport's rank from ninth to fourth between 2006 and 2014. A 16 percent connectivity increase at MIA moved the airport from 26th place in 2006 to 21st place in 2014. JFK also experienced considerable rank gains, moving from 17th to 10th with an 11 percent increase in connectivity during that period.
Click here to see large hub GRASI scores and ranking for 2006 and 2014.
Reduced access to key economic centers can inhibit economic growth in small cities by discouraging businesses from locating or expanding in these poorly connected areas.
And yet such gains are not experienced uniformly across airports within a metropolitan area. GRASI can be used to highlight and quantify these changing dynamics. For example, while JFK experienced significant increases in connectivity, LaGuardia Airport (LGA) and Newark Liberty International Airport (EWR) suffered from connectivity losses of four and 10 percent, respectively. This resulted in EWR moving down four spots in rank, to 17th, during this period. While LGA actually moved up one spot in the rankings, from 14th to 13th, the airport's GRASI score of 5,148 is still nine percent lower than JFK's—in contrast to a GRASI score that was five percent higher than JFK's in 2006.
The largest connectivity losses among large hubs between 2006 and 2014 were those at Washington Dulles International Airport (IAD) and Baltimore-Washington International Airport (BWI). IAD's connectivity loss of 27 percent moved it down three spots in rank, to 23rd place , while BWI 's 21 percent loss of connectivity bumped the airport from 23rd to 27th place. The third major airport in the Washington, D.C. metropolitan area, Reagan National Airport (DCA) experienced a three percent connectivity loss. DCA was ranked 18th in 2006 and 20th in 2014.
In contrast to IAD and BWI, some airports experienced significant losses in connectivity while maintaining a GRASI score that ranks above the large hub median score. For example, Phoenix Sky Harbor International Airport (PHX) and McCarran International Airport (LAS) experienced connectivity losses of 13 and 14 percent, respectively between 2006 and 2014. However, PHX dropped only one spot in rank—to ninth place—during this period, with a current GRASI of 5,780. And although LAS moved down to eighth place from fifth, the airport has a current GRASI of 6,121, which is 20 percent greater than the large hub median.
Explanations for these changes in economic connectivity, such as weak passenger demand, consolidation and capacity discipline practices, are largely responses to the economic downturn and high and volatile fuel prices. The effects of these changes are significant not only for airports and passengers, however. Reduced access to key economic centers can inhibit economic growth in small cities by discouraging businesses from locating or expanding in these poorly connected areas. For city planners and developers, GRA has been developing measures of airport economic access costs, which can be valuable tools to examine how the time and money required to travel from a city can stimulate—or stifle—its growth and development.
For more information, contact GRA, Incorporated here.
To see more from GRA, follow us on LinkedIn.
Note: 2014 GRASI scores are estimates based on data from the first three quarters of 2014. Results are reported according to 2013 airport hub groups. The domestic airports used in this analysis are located in cities that account for 2/3 of U.S. GDP. The international airports were selected by GRA, based on enplanements and according to our judgment of their global significance.


Riverside, Calif. – March Field Air Museum is proud to announce the eighth installment of "Up-Close Aircraft Days" on Saturday, August 11, 2018, from 11 a.m. to 3 p.m. As part of this new interpretive program, a different aircraft is featured each month with the next focused on the World War I "Lafeyette Escadrille" Nieuport fighter. On the second Saturday of each month throughout the year, Museum guests will have the opportunity for a close-up look – sometimes climbing stairs to get a close-up view into the cargo bay or cockpit, other times even climbing aboard.
For the upcoming "Up-Close Aircraft Day" focused on the French Nieuport fighter, guests will see how this diminutive single-seat aircraft brought German dominance of the skies over Europe to an end starting in 1916. On this day, visitors to March Field Air Museum will get an up-close view of the Museum's Nieuport, peering into the cockpit at close range within the main exhibit galleries.
Originally designed for racing, the diminutive Nieuport "Bebe" (Baby) featured an upper wing-mounted machine gun along with ailerons (instead of more primitive wing warping) for rapid maneuvering. The Museum's Nieuport 11 is a 7/8th scale replica, bearing the markings of the legendary Fighter Ace Raoul Lufbery of the French Air Force's American volunteer squadron—the Lafayette Escadrille.
The Nieuport's January 1916 introduction allowed the Allies to meet the Germans on an equal basis. Many of the First World War's greatest pilots—including Charles Nungesser and George Guynemer—flew Nieuports, as did Kiffin Rockwell of the famed Lafayette Escadrille, the first American to shoot down an enemy aircraft in combat. Although replaced by more advanced aircraft models after only a few short months, hundreds of Nieuport 11s provided vital service at a time when their contribution to the Allied cause was most needed.
To preview the next "Up Close Aircraft Day" happening in August, an F-105B Thunderchief fighter-bomber will be showcased. The Museum's website features extensive information on each aircraft in its collection of more than 80 total, for those wishing to learn more. Knowledgeable museum docents, dressed in period uniforms whenever possible, will be on hand to assist visitors, provide information and answer questions. The March Field Air Museum website will list upcoming "Up-Close Aircraft Days" and the featured aircraft each month; for more information, visit www.MarchField.org/calendar.
March Field (today's March Air Reserve Base, located immediately adjacent to the March Field Air Museum) is the West Coast's oldest continuously operating military airfield. Established in 1918 as the U.S. was embroiled in World War I, it began life as part of the U.S. Army Signal Corps. Following several sleepy years in the 1920s, March Field was expanded in the 1930s as the world again prepared for war. As March Field passed the quarter-century mark, it was busily protecting West Coast skies and training U.S. Army Air Forces personnel for deployment to all Combat Theaters during WWII. Following the war—as the Cold War began—and then continuing for nearly 50 years, March Air Force Base became the U.S. Air Force Strategic Air Command 15th Air Force headquarters and home to a massive nuclear deterrent force.


Is it Safe to Travel to Colombia?
Bogota is a sprawling, energetic city that stretches high into the Andes. It boasts Colombia's best museums (the Museum of Gold and the Museum of Art are musts) and biggest festivals, as well as a debaucherous nightlife. As Colombia's capital, it has a booming tourism infrastructure with a wealth of hotels for you to choose from.
Bogota hotels cover the whole range of options, from good-value, standard rooms to complete luxury. We've collected a group of Bogota hotels that have been inspected by us and, therefore, come with our full recommendation. Thus, you can look forward to your trip to this vibrant, culturally rich capital knowing that you'll be in the best of hands throughout your stay in a Bogota hotel of your choice.
Welcome to See Colombia Travel, your number one resource for vacations to Colombia. We are a team of international experts boasting vast experience of traveling the world, and we're here to help you discover the treasures of our beloved Colombia.
At See Colombia Travel all our Colombia tours are tailor-made. We're determined to ensure your experience is 100% to your liking. Colombia is a stunningly varied country boasting idyllic beaches, lost cities asleep in the mountains and, of course, coffee galore in its bustling city-centers.
Whatever you're looking for from your Colombia vacation, we're here to guide you and ensure your trip is up to our highest standards of safety, value and quality. We're sure that once you've been to Colombia, like us, you'll want to spread the word about this fantastic, diverse country.
Have a look at a selection of testimonials from real travelers like you.
Colombia photos by professionals as passionate as we are about Colombia.


Sydney is known as the Harbour City. It is the largest, oldest and most cosmopolitan city in Australia with an enviable reputation as one of the world's most beautiful and liveable cities. Brimming with history, nature, culture, art, fashion, cuisine, design, it is set next to miles of ocean coastline and sandy surf beaches.
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Most of them are equipped with very luxurious interior and they clearly aim at a high end customers.
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Clinical MedicineAIDS/HIVVaccines Free access | 10.1172/JCI124473
Live attenuated varicella-zoster virus vaccine does not induce HIV target cell activation
Catia T. Perciani,1 Bashir Farah,2 Rupert Kaul,1,3,4 Mario A. Ostrowski,1,3,5 Salaheddin M. Mahmud,6 Omu Anzala,2,7 Walter Jaoko,2,7 KAVI-ICR Team,2,9 and Kelly S. MacDonald1,8,10
1Department of Immunology, University of Toronto, Toronto, Ontario, Canada.
2Kenyan AIDS Vaccine Initiative–Institute of Clinical Research (KAVI-ICR), University of Nairobi, Nairobi, Kenya.
3Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
4University Health Network, Toronto, Ontario, Canada.
5Keenan Research Center, St. Michael's Hospital, Toronto, Ontario, Canada.
6Community Health Sciences, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada.
7Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
8Section of Infectious Diseases, Department of Internal Medicine, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
9The KAVI-ICR Team is detailed in the Supplemental Acknowledgments.
10Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
Address correspondence to: Kelly S. MacDonald, Office Room 501, Basic Medical Sciences Building, 745 Bannatyne Avenue, Winnipeg, Manitoba, R3E0J9, Canada. Phone: 204.977.5680; Email: kelly.macdonald@umanitoba.ca. Or to: Catia T. Perciani, Room 6368 Medical Science Building, 1 King's College Circle, Toronto, Ontario, M5S1A8, Canada. Phone: 416.946.0277; Email: c.perciani@mail.utoronto.ca.
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First published December 4, 2018 - More info
Published in Volume 129, Issue 2 on February 1, 2019
J Clin Invest. 2019;129(2):875–886. https://doi.org/10.1172/JCI124473.
First published December 4, 2018 - Version history
Received: August 27, 2018; Accepted: November 27, 2018
BACKGROUND. Varicella-zoster virus (VZV) is under consideration as a promising recombinant viral vector to deliver foreign antigens including HIV. However, new vectors have come under increased scrutiny, since trials with adenovirus serotype 5–vectored (Ad5-vectored) HIV vaccine demonstrated increased HIV risk in individuals with pre-immunity to the vector that was thought to be associated with mucosal immune activation (IA). Therefore, given the prospect of developing an HIV/VZV chimeric vaccine, it is particularly important to define the impact of VZV vaccination on IA.
METHODS. Healthy VZV-seropositive Kenyan women (n = 44) were immunized with high-dose live attenuated VZV vaccine, and we assessed the expression on CD4+ T cells isolated from blood, cervix, and rectum of IA markers including CD38 and HLA-DR and of markers of cell migration and tissue retention, as well as the concentration of genital and intestinal cytokines. A delayed-start group (n = 22) was used to control for natural variations in these parameters.
RESULTS. Although immunogenic, VZV vaccination did not result in significant difference in the frequency of cervical activated (HLA-DR+CD38+) CD4+ T cells (median 1.61%, IQR 0.93%–2.76%) at 12 weeks after vaccination when compared with baseline (median 1.58%, IQR 0.75%–3.04%), the primary outcome for this study. VZV vaccination also had no measurable effect on any of the IA parameters at 4, 8, and 12 weeks after vaccination.
CONCLUSION. This study provides the first evidence to our knowledge about the effects of VZV vaccination on human mucosal IA status and supports further evaluation of VZV as a potential vector for an HIV vaccine.
TRIAL REGISTRATION. ClinicalTrials.gov NCT02514018.
FUNDING. Primary support from the Canadian Institutes for Health Research (CIHR). For other sources, see Acknowledgments.
Graphical Abstract
Previous phase IIb/III HIV vaccine trials attest to the challenges faced in human immunodeficiency virus (HIV) vaccine development. The use of adenovirus serotype 5 (Ad5) as a recombinant viral vector to deliver HIV genes not only failed to prevent HIV infection and to reduce viral load following infection in vaccinated individuals (1–3), but also showed a trend toward increased rates of HIV infection in the vaccine arm compared with placebo (1, 2). Post hoc analysis showed that the Ad5-based HIV vaccine increased susceptibility to HIV infection, especially among those individuals with preexisting antibody immunity to the Ad5 vector (4, 5). Although the immune characteristics associated with risk for HIV infection per se have not been completely elucidated, it is known that CD4+ T cell activation and phenotypes contribute to the ability to support productive infection. Studies in rhesus macaques showed that the frequency of activated CCR5+ CD4+ T cells in the rectal mucosa prior to challenge with simian immunodeficiency virus (SIV) positively correlated with increased risk of infection and SIV peak viremia in the animals (6). Activated CD4+ T cells, expressing CD38 and HLA-DR, were shown to be more susceptible to infection and to favor viral replication when compared with resting CD4+ T cells (7–9). Additionally, genital inflammation measured by an increased level of proinflammatory cytokines was associated with elevated risk of HIV infection (10, 11) and a quiescent state of immune activation (IA) linked to resistance to HIV acquisition in highly exposed HIV-1–seronegative (HESN) individuals (9, 12, 13). Genital inflammation subverted the protection conferred by the use of 1% tenofovir gel in the CAPRISA 004 trial (14), and blocking the ability of leukocytes to respond to chemoattractants in the female genital tract (FGT) of humanized mice was shown to prevent systemic dissemination of HIV (15). That the increased rate of HIV acquisition observed in the Ad5-based HIV vaccine trials was associated with increased mucosal IA could not be confirmed, since mucosal specimens were not collected in those studies. Expanded Ad5-specific CD4+ T cells from blood expressed increased levels of CCR5 and the mucosa homing marker α4β7 (16, 17), suggesting that Ad5 vector may promote migration of HIV target cells to the mucosa. A follow-up study in rhesus macaques showed that vaccination with adenovirus vector induced a significant increase in the frequency of activated memory CD4+ T cells in rectal mucosa but not in blood (18). There was a reappraisal of the initial Ad5 vaccine rhesus macaque studies (19, 20), with a more critical eye. Subsequent nonhuman primate (NHP) studies done with Ad5 demonstrated modest control of viral load (approximately 2 log reduction), with subsequent escape unaccounted for by loss of cytotoxic T lymphocyte control (21, 22). Therefore, the limitations of the NHP model and the recognition of the key role mucosal host immune responses against the viral vectors have pushed subsequent vaccines toward much more extensive testing of vectors and of mucosal immunity in humans and macaques.
The use of persistent replicating viral vectors could be instrumental in safely mimicking attenuated HIV antigenic presentation and helping mature and maintain a protective response via vaccine, as suggested by studies in NHP models using CMV as a vector for SIV genes (23–25). However, a detailed assessment of the mucosal immune responses induced by these viral vectors, including IA, is warranted if they are to be used as a chimeric vector bearing HIV antigens (26, 27). Additionally, it has been shown that an activated immune microenvironment prior to vaccination can interfere with the efficacy of vaccines (28) and that vaccination may induce sustained CD4+ T cell activation, potentially increasing HIV susceptibility in certain populations (29), thus supporting the examination of the IA status in vaccinology.
The long-lived humoral and cellular immunity induced by varicella-zoster virus (VZV) (30, 31), its efficacy and safety record as a live attenuated vaccine (31–34) — with vaccination causing viremia in both seronegative and seropositive individuals (35) — combined with its amenability to function as a viral vector expressing foreign antigens (36, 37) make VZV a prime candidate to serve as a persistent replicating vector in an HIV vaccine (38). VZV-recombinant vaccine constructs encoding HIV and SIV Env have been previously tested (39, 40). Although administration of the VZV-vectored SIV Env vaccine has been linked to accelerated disease progression in rhesus macaques intravenously challenged with SIVsmE660 (40), this result should not be read as a reason to cease the investigation of VZV as a vector in an HIV vaccine. The outcome observed by Staprans et al. is unlikely to be due to VZV-intrinsic features but rather may be due to the study design, which included the following: (i) the use of the highly passaged attenuated human live attenuated VZVOka23 vaccine strain in monkeys that was shown not to cross the species barrier, as evidenced by its failure to either induce vaccine-specific CD8+ T cell responses or establish latency (so that VZVOka23 vaccine acted as a non-replication-competent vaccine); (ii) a single gene product Env in the vaccine that was heterologous to the challenge virus did not induce neutralizing antibodies, and CD4+ T cell proliferation was the sole anti-SIV T cell response induced by the vaccine; (iii) the use of cell-associated instead of cell-free viral vaccine preparation, which may also have increased IA and the availability of activated CD4+ T cells at the time of challenge. Our group has observed that cynomolgus macaques (CyMs) vaccinated with the VZV-SIV constructs expressing SIV Gag, Pol, and Env and a hybrid of SIV Nef, Tat, and Rev did not exhibit a worse outcome compared with controls; on the contrary, 3 of the 8 vaccine recipients demonstrated complete and sustained SIV viral clearance (undetectable viral load defined as a viral load of fewer than 50 copies in plasma by real-time PCR) after challenge (41). Importantly, VZV-SIV vaccine also did not increase susceptibility to SIV infection in this model. The VZV-vectored constructs tested in the SIV macaque model used as a carrier the less-attenuated parental human strain of VZVOka (Oka-P), which previously was shown able to be able to cross the species barrier and effectively infect CyMs (42).
VZV exposure and subclinical viremia are associated with maintenance of specific cell-mediated immunity able to limit replication of reactivated VZV (43–45), and increased Th1 memory responses induced by VZV vaccination are associated with protection from herpes zoster (46). However, the impact of VZV vaccination with regard to genital and rectal mucosal immunity remains to be defined. We recently showed that VZV-seropositive individuals sustained anti-VZV antibody responses at the genital, oral, and rectal mucosa and also demonstrated that VZV vaccination boosted anti-VZV IgG and IgA at the FGT (47). Here, we examined the impact of immunization with the live attenuated zoster vaccine (VZVOka, Zostavax) on several aspects of IA associated with increased susceptibility to HIV infection longitudinally over a period of 12 weeks, systemically and at both the genital and rectal mucosa, two major sites for HIV entry. The primary outcome of the present study was to determine whether 12 weeks after vaccination, the frequency of CD4+ T cells isolated from cervical tissue coexpressing the IA markers CD38 and HLA-DR was significantly changed by vaccination. CD38+HLA-DR+ CD4+ T cells were previously shown to express high levels of CCR5 and to be highly susceptible to HIV infection (7). Besides, T cells coexpressing these two markers were shown to exhibit high levels of several other markers of IA and lower cytotoxicity (48). Secondary outcomes were to determine changes in a combination of 14 cytokines (including proinflammatory cytokines such as IL-1α, IL-1β, and TNF; chemokines IL-8, IP-10, MIP-1α, and MIP-1β; and the differentiation marker IL-4) measured longitudinally in cervico-vaginal secretion (CVS) and rectal secretion (RS). Tertiary outcomes included the frequency of CD4+ T cells expressing the HIV coreceptor CCR5, the activation markers CD69 and Ki-67, and markers of cell migration and retention (α4β7, α4β1, αEβ7) in blood, cervix, and rectum. This investigation was conducted in a cohort of healthy Kenyan women at low risk for HIV acquisition and with preexisting immunity to VZV who showed anamnestic VZV-specific humoral responses upon administration of a single dose of VZVOka vaccine (47).
Participant demographics. Forty-five (45) participants (Figure 1 and Table 1) were randomized into immediate (n = 23) and delayed (n = 22) vaccination groups, as described in Methods and in the published protocol (38) (Figure 2). The inclusion of a delayed group allowed for the adjustment of natural variations in IA parameters independent of vaccination, serving as an internal control group. Blood, cervico-vaginal, and rectal specimens were collected longitudinally for the comprehensive examination of several markers of IA before and after vaccination with VZVOka (Figure 2). Vaccine was administered to 44 of 45 (98%) participants, and 41 of 44 immunized women (93%) completed the visits required for assessment of the primary study outcome. There were no statistical differences in age, marital status, education level, contraceptive use, or sexual behavior between the two groups (Table 1). While the rate of herpes simplex virus 2 (HSV-2) seropositivity was higher in the delayed than in the immediate group (P = 0.008), subsequent adjustment for HSV-2 status did not alter our findings. The median participant age was 26 years (IQR 21–30). The majority of the participants were married (73%), and 58% used the progesterone-based injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA).
KAVI-VZV 001 enrollment and follow-up diagram.
Immunization and sample collection schedule. The primary endpoint was determined between week 12 after vaccination and week 0. PBMC, peripheral blood mononuclear cells; CC, cervical cells; CVS, cervicovaginal secretion; RC, rectal cells; RS, rectal secretion.
Baseline demographic characteristics, sexual behavior, and HSV-2 serostatus
No impact of live attenuated VZVOka on genital IA. The frequency of CD4+ T cells expressing the IA markers HLA-DR, CD38, CD69, and Ki-67, as well as the HIV coreceptor CCR5, was measured using flow cytometry (Figure 3A). At 12 weeks after vaccination, there was no significant difference in the frequency of cervical activated (HLA-DR+CD38+) CD4+ T cells (median 1.61%, IQR 0.93%–2.76%) when compared with week 0 (just prior to vaccination) (median 1.58%, IQR 0.75%–3.04%) — the primary endpoint for this study (Figure 3B). Based on the SD of the differences in frequency of HLA-DR+CD38+ CD4+ T cells in the same individual between weeks prior to vaccination (delayed group) (SD = 3.0%, n = 59), and using a significance level (α) of 0.05, we had 80% power to detect a true difference of at least 1.35 units (frequency [%] of HLA-DR+CD38+ CD4+ T cells) between these weeks. VZVOka vaccination also did not lead to changes in the frequency of HLA-DR+CD38+ CD4+ T cells at any of our time points, even prior to adjustment for multiple comparisons (Figure 3B). In addition, no change was seen in the expression of the markers HLA-DR, CD69, and Ki-67, of the HIV coreceptor CCR5 (Figure 3, C–F), or of integrins α4β7, α4β1, and αEβ7 as a result of vaccination (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI124473DS1). The power to detect true changes in the cellular markers assessed in this study is summarized in Supplemental Table 1. Considering the SD of the differences in the frequencies of expression between weeks prior to vaccination (delayed group) in the same individual for each of the markers, the sample size, a significance level (α) of 0.05, and power (β) of 80%, we had the ability to detect a less than 1-fold change (i.e., less than 2 times the baseline value) for all the cellular IA markers expressed on cervical CD4+ T cells (Supplemental Table 1). Therefore, we concluded that VZV vaccination did not cause cervical CD4+ T cell IA.
Cervical cellular IA markers. (A) Representative flow cytometry plots for the identification of cervical CD4+ T cells, and CCR5+, CD69+, Ki-67+, CD38+HLA-DR+, and HLA-DR+ CD4+ T cells. Cells were pre-gated on lymphocytes, singlets, viability by LIVE/DEAD staining, and CD3+ cells. Expression of (B) CD38/HLA-DR, (C) HLA-DR, (D) CD69, (E) Ki-67, and (F) CCR5 on cervical CD4+ T cells prior to vaccination (weeks –12 to 0) and after vaccination (weeks +4 to +12). Graphs to the left show median with IQRs, and graphs to the right the comparison between weeks –12 and 0 and between weeks 0 and +12 for each of the graphs. Individuals in the immediate and delayed groups are shown in red and blue, respectively, and were grouped according to time from vaccination for this analysis. Time points were compared with week 0 using Wilcoxon's signed-rank test. No significant change was observed between any of the time points (unadjusted for multiple comparisons).
Next the concentration of 14 cytokines in the CVS was assayed. First the correlation between the normalized concentration of these 14 cytokines was assessed, and strong positive correlations (Spearman's correlation >0.4) between 71% of the cytokine pairs were identified (Figure 4A). To avoid redundancy and to limit our analysis to a smaller number of covarying components, principal component analysis (PCA) was performed on all cytokines except MIP-1β, which was analyzed separately, since it did not correlate with the other cytokines measured in CVS. The first principal component (PC1) explained 63% of the variability in our dataset (Figure 4B). PC2 and PC3 explained 11% and 8% of the variability, respectively (Figure 4B). VZVOka vaccination did not change genital PC1, PC2, or PC3 factor scores (Figure 4, C–E), or the concentration of the chemokine MIP-1β. Considering the SD of the differences in the PC factor scores between weeks prior to vaccination (delayed group) in the same individual, the sample size, and α = 0.05, we were powered to detect fold changes (from week 0) greater than 2.1-, 2.4-, and 1.9-fold for PC1, PC2, and PC3 scores, respectively (β = 80%) (Supplemental Table 2). Longitudinal assessment of the concentration of each FGT cytokine included in the PCA also showed no change after vaccination (Supplemental Figure 2). Overall, although VZV vaccination significantly increased the concentration of VZV-specific IgG (mIU/ml) measured in plasma (P < 0.0001) and in CVS (P < 0.0001) (47), neither T cell flow cytometry data nor FGT cytokine concentrations demonstrated any effect of VZV vaccination on genital IA parameters in healthy VZV-seropositive Kenyan women (Supplemental Figure 3).
Cervical soluble IA markers. (A) Spearman's correlation between cervico-vaginal cytokines (using log10-normalized concentrations). Only rs with P < 0.0005 are shown (n = 234). (B) Pattern matrix for the PCA showing loading of each cytokine on the principal components. PC1 explains 63% of variance, and PC2 and PC3 explain 11% and 8% of variance, respectively. Loads greater than 0.450 are highlighted. (C) PC1, (D) PC2, and (E) PC3 factor scores and (F) MIP-1β level prior to vaccination (weeks –12 to 0) and after vaccination (weeks +4 to +12). Graphs to the left show median with IQR, and graphs to the right the comparison between weeks –12 and 0 and between weeks 0 and +12 for each of the graphs. Individuals in the immediate and delayed groups are shown in red and blue, respectively, and were grouped according to time from vaccination for this analysis. Time points were compared to week 0 using Wilcoxon's signed-rank test. No significant change was observed between any of the time points (unadjusted for multiple comparisons).
Genital IA associated with DMPA use and bacterial vaginosis. Levels of IA prior to vaccination (i.e., in the delayed group) fluctuated over time in some individuals (Figures 3 and 4). The association of these genital IA fluctuations with sexually transmitted infections (STIs), use of DMPA, and presence of bacterial vaginosis (BV) was assessed (Table 2 and Figure 5). Of interest, the use of DMPA as a contraceptive was associated with a reduced PC1 score (TNF-α, IL-10, IL-4, IL-1, IL-8, IFN-γ, IL-6, IL-17A) in the FGT (Figure 5A). Due to the low incidence of BV and vulvovaginal candidiasis (VVC) (Table 2), for each participant we grouped all the records during the 12 weeks prior to and after vaccination and examined associations with variability in cytokine and chemokine levels. As shown in Figure 5B, a BV-positive test was associated with significantly elevated PC1 (TNF-α, IL-10, IL-4, IL-1, IL-8, IFN-γ, IL-6, IL-17A) and PC3 (IL-1α, GM-CSF) scores and reduced PC2 (IP-10, MIP-3α, MIP-1α) score. A paired analysis using the median PC scores for the BV-negative visits showed similar results, with BV onset associated with significantly lower PC2 score and elevated PC3 score (P < 0.001, after adjusting for multiple comparisons) (Figure 5C). Women using DMPA as a contraceptive method were 7.7 times less likely to test positive for BV than women using other types of contraceptives (P = 0.019). HSV-2 seropositivity was not associated with significant difference for PC1 and PC2 scores, but associated with a significantly elevated median PC3 score, comprising IL-1α and GM-CSF (P = 0.027, after adjusting for multiple comparisons) (Supplemental Figure 4A). VVC was not associated with differences in PC scores (Supplemental Figure 4B).
Impact of DMPA use and BV incidence on genital IA. (A) PC factor score comparisons between records for DMPA users (n = 135) and for women using other hormonal contraceptives (n = 99). (B) Comparison between PC factor scores for BV+ records (n = 17) and BV– records (n = 217). Graphs show median with IQRs. **P < 0.01, ****P < 0.0001 as determined by a 2-tailed Mann-Whitney U test. (C) Paired comparison between PC factors for BV+ visits and the median for BV– visits for each participant who had BV during this period of the study. ***P < 0.001 calculated by Friedman's test, followed by Wilcoxon's signed-rank test, and adjusted for multiple comparisons using step-down procedure.
Number of participants who came to the clinic at each time point and respective number of DMPA users; those with BV, VVC, and pelvic inflammatory disease (PID); and HSV–2 positive cases
Live attenuated VZVOka did not alter rectal or blood IA status. Given the lack of information regarding the effects of VZVOka vaccination on enteric IA and the importance of the rectal mucosa in HIV transmission, we also examined the impact of VZVOka immunization on markers of IA and on CCR5, α4β7, α4β1, and αEβ7 expression by rectal CD4+ T cells. Rectal biopsy samples were collected at 3 time points in the study: 12 weeks prior to vaccination, on the day of vaccination, and 12 weeks after vaccination. Freshly isolated cells were analyzed by flow cytometry (Figure 6A). As with cervical CD4+ T cells, there was no significant elevation of IA in rectal CD4+ T cells due to VZVOka immunization at week 12 after vaccination when compared with the day of vaccination (baseline) (Figure 6, B–E); the frequency of CCR5+ CD4+ T cells (Figure 6F) and of CD4+ T cells expressing α4+β7hi, α4+β1+, and αE+β7hi integrins was also unaltered (Supplemental Figure 5). In the delayed group, a slight decrease in the median frequency of CD38+HLA-DR+CD4+ T cells (Figure 6B), HLA-DR+CD4+ T cells (Figure 6C), and α4+β7hi CD4+ T cells (Supplemental Figure 5B) was observed in rectal tissue over the 12 weeks of pre-vaccination follow-up (P = 0.04), suggesting that enrollment in the study may potentially be associated with lowering of rectal IA. However, none of these changes remained significant after adjusting for multiple comparisons.
Rectal cellular IA markers. (A) Representative flow cytometry plots for the identification of rectal CD4+ T cells, and CCR5+, CD69+, Ki-67+, CD38+HLA-DR+, and HLA-DR+ CD4+T cells. Cells were pre-gated on lymphocytes, singlets, live, and CD3+ cells. Expression of (B) CD38/HLA-DR, (C) HLA-DR, (D) CD69, (E) Ki-67, and (F) CCR5 on rectal CD4+ T cells prior to vaccination (weeks –12 and 0) and after vaccination (week +12). Graphs to the left show median with IQRs, and graphs to the right the comparison between weeks –12 and 0 and between weeks 0 and +12 for each of the graphs. Individuals in the immediate and delayed groups are shown in red and blue, respectively, and were grouped according to time from vaccination for this analysis. Time points were compared with week 0 using Friedman's test, followed by Wilcoxon's signed-rank test. P and P′ show P values unadjusted and adjusted for multiple comparisons using step-down procedure, respectively.
We next characterized any changes in the same 14 cytokines in RSs using an approach similar to that used for the FGT (above). We first assessed the correlations between the normalized concentration of these 14 cytokines measured in RS and found strong positive correlations (Spearman's correlation >0.4) between 86% of the cytokine pairs (Figure 7A). We next performed PCA on the panel of 14 cytokines. PC1 explained 58% of the variability in our dataset, whereas PC2 and PC3 explained 14% and 7% of the variability, respectively (Figure 7B). The pattern matrices for the PCA in CVS and in RS were similar. The variables encompassing rectal PC1 and PC3 coincided with components of cervico-vaginal PC1 and PC3. However, MIP-1β, which was not included in cervico-vaginal PCA, and IL-17A, which was part of cervico-vaginal PC1, were integrated into rectal PC2 (Figure 7B). As in the FGT, VZVOka vaccination did not result in any significant change in the PC1, PC2, or PC3 factor scores at the rectal mucosa (Figure 7, C–E). The study power to detect a true difference in the expression of the cellular markers and in the PC factor scores for the rectal site is shown in Supplemental Tables 1 and 2, respectively. The longitudinal assessment of the concentration of each cytokine included in the PCA of the RSs agreed with these findings by showing no significant change after vaccination (Supplemental Figure 5). At an individual cytokine level, the median rectal concentration of MIP-1α and MIP-3α fell 12 weeks after enrollment in the study (P = 0.044 and P = 0.005, respectively), and the median concentration of IL-4 slightly increased (P = 0.015) (Supplemental Figure 6), but these differences were not significant after adjusting for multiple comparisons. Contrary to what was observed for the FGT, the rectal PC scores were independent of use of DMPA as a contraceptive (Supplemental Figure 7). Overall, we conclude that VZV vaccination did not increase rectal IA.
Rectal soluble IA markers. (A) Spearman's correlation between rectal cytokines (using log10-normalized concentrations). Only rs with P < 0.0005 are shown (n = 107). (B) Pattern matrix for the PCA showing loading of each cytokine on the principal components. PC1 explains 58% of variance, and PC2 and PC3 explain 14% and 7% of variance, respectively. Loads greater than 0.450 are highlighted. (C) PC1, (D) PC2, and (E) PC3 factor scores prior to vaccination (weeks –12 and 0) and after vaccination (week +12). Graphs to the left show median with IQRs, and graphs to the right the comparison between weeks –12 and 0 and between weeks 0 and +12 for each of the graphs. Individuals in the immediate and delayed groups are shown in red and blue, respectively, and were grouped according to time from vaccination for this analysis. Time points were compared with week 0 using Wilcoxon's signed-rank test. No significant change was observed between any of the time points (unadjusted for multiple comparisons).
A comprehensive assessment of activation and expression of CCR5 and integrins in CD4+ T cells isolated from blood also confirmed the absence of deleterious effects related to VZVOka vaccination (Supplemental Figure 8).
Worldwide, HIV transmission predominantly occurs at the mucosal interface and mainly through heterosexual transmission. Mucosal activated CD4+ T cells are pivotal for the establishment and dissemination of HIV infection (7, 15, 49–53). Enhanced mucosal IA upon vaccination with Ad5 vector remains the main explanation for the increased rate of HIV infection among vaccine recipients who showed pre-immunity to the vector in the Ad5-vectored HIV vaccine clinical trials, highlighting the importance of examining tissue-specific effects induced by viral vectors (6, 16–18). In this light, we comprehensively assessed the impact of VZVOka vaccination on the IA status of both cervico-vaginal and rectal mucosa of Kenyan women with pre-immunity to VZV in preparation for its further investigation as a vector in an HIV vaccine. As shown here, we did not observe any significant change in the frequency of activated CD4+ T cells or in CCR5+ CD4+ T cells at 12 weeks after vaccination at any of the sites. Analysis performed in both circulating and cervical CD4+ T cells as early as 4 weeks after vaccination also did not show significant changes in IA when compared with matched baseline samples collected immediately prior to vaccination (week 0). It is important to highlight that we were powered to detect increases or decreases equal to or greater than the ones shown in Supplemental Table 1, and it is possible that by using a larger sample size, one may detect smaller changes that were imperceptible here.
Enhanced expression of α4β7 integrin, a mucosal homing marker, has also been shown to contribute to susceptibility to HIV infection and to worsen HIV clinical prognosis (54). Further supporting a role for the integrin, α4β7 blockade with a monoclonal antibody was recently shown to reduce HIV susceptibility and suppress viral load in NHPs (55, 56). Anti-α4β7 monoclonal antibody therapy, however, was unable to prevent gut CD4+ T cell depletion during acute SIV infection in NHPs, with benefits to CD4+ T cell reconstitution in gut tissues demonstrated only at later stages of infection (57). Ad5-specific blood CD4+ T cells exhibited high expression of α4β7, suggesting that Ad5 vector may promote migration of HIV target cells to the mucosa, increasing their availability at the site of HIV infection. Our results revealed that VZVOka vaccination did not result in an elevated frequency of CD4+ T cells expressing α4β7 in blood or cervical or rectal mucosa at any time points after vaccination. We have previously shown in this cohort that expression of αEβ7 may be especially important for T cell distribution in the rectal mucosa and that αE+β7hiCD4+ T cells express high levels of CCR5 and CD69 (58). Analysis of the frequency of αE+β7hiCD4+ T cells prior to and after vaccination showed that VZVOka vaccination was not associated with any significant change in this cell population in any of the tissues studied here. Additionally, cervical CD4+ T cells expressing the integrin α4β1, previously shown to be readily infected by HIV R5 pseudovirus in an in vitro assay (59), also were not altered by VZVOka vaccination.
Besides cellular markers of IA, genital inflammation measured by an increased level of proinflammatory cytokines is associated with elevated risk of HIV infection (10, 11). In agreement with our cellular results, examination of a panel of cytokines — including TNF, IL-1α, IL-1β, IL-6, IL-8, IP-10, and MIP-1α — revealed no significant change in concentration associated with VZVOka vaccination in either CVS or RS. Importantly, all participants remained HIV-1 and HIV-2 negative at the end of our study.
This study also demonstrates that mucosal IA status is highly dynamic and undergoes regular fluctuations, as was evidenced in the 12-week run-in period prior to the administration of the VZVOka vaccine in the delayed group; this reflects the importance of conducting longitudinal studies in relevant human populations with appropriate controls such as a delayed arm. This variability between individuals and even within visits in the same individual is currently being further analyzed. The study will characterize changes to the female genital microbiome over an extended period of 48 weeks and analyze whether they correlate with host changes in mucosal cellular and soluble IA parameters, as well as demographic, contraception, and behavioral factors. Previous studies have shown that the cervico-vaginal microbiota can impact genital inflammation and can increase HIV risk by recruiting HIV target cells to the mucosa (60, 61). BV and STIs, such as HSV-2 and chlamydia, are associated with elevated genital inflammation and increased risk of HIV infection (62–64). Recent findings by McKinnon et al. demonstrated that genital inflammation can attenuate the protection conferred by 1% tenofovir gel (14), indicating that the consideration of genital inflammation is also of primary importance when evaluating HIV vaccines.
Participants in this study were drawn from an HIV-uninfected Kenyan population with low rates of partner change, no known commercial sex work, and low rates of STI. These factors may well assist in deciphering the interaction of factors such as DMPA, BV, and HSV-2, as most if not all previous studies have been done in cohorts drawn predominantly from women with complex risk behaviors and higher rates of both viral and bacterial genital infections that persist and reactivate, confounding the analysis of IA.
Of interest, the cumulative analysis of PC scores for all BV cases in the study compared with all BV-negative records (unpaired analysis), as well as the comparison between the median PC scores for BV-negative and BV-positive scores in a paired manner, showed a significant reduction in PC2 score (comprising IP-10, MIP-3α, and MIP-1α) and an increase in PC3 score (including IL-1α and GM-CSF) for BV-positive compared with BV-negative cases. These results are in agreement with previous findings showing that concomitantly increased IL-1α and decreased IP-10 concentrations in the FGT predicted the presence of an STI or BV with a quite high predictive value (sensitivity 71%, specificity 74%) (65). DMPA use was inversely associated with BV incidence in our cohort, with DMPA users at 7.7 times lower risk for testing positive for BV than women using other contraceptives during the study period. Additionally, we observed a less-proinflammatory profile, as evidenced by a lower PC1 score in DMPA users' CVS than in CVS from women using implants or IUDs as contraceptives, in agreement with previous observations showing that injectable hormonal contraceptives were broadly associated with an immunosuppressive FGT innate immune profile (66). With respect to HSV-2 infection, during the study period we did not observe any clinical indication (such as genital ulcers) of new HSV-2 infections or symptomatic HSV-2 reactivation.
Invasiveness and low acceptance of repeated rectal sampling in clinical trial participants in Kenya precluded more frequent rectal mucosal sampling, which would have added to the ability to compare the cervical IA parameters at multiple time points. A VZV-non-immune arm would also have been a useful comparator for the study and was contemplated initially; however, screening revealed a very high rate of VZV immunity in this urban population (47), despite the general consensus that VZV seropositivity is lower in adults in the tropics, so recruitment of an adequate sample size of that arm was deemed to be futile. Additionally, since menstrual cycle, hormonal contraceptives, and BV can modulate genital inflammation and HIV susceptibility, future HIV prevention trials should plan to further evaluate the impact of these factors on the protection/risk conferred by the intervention in testing. As administration of live attenuated VZV vaccine is contraindicated for pregnant women, and it is recommended that pregnancy be avoided for 3 months following vaccinations, all of our participants were required to use an effective contraceptive method for the duration of the study. Inclusion of a cohort of women not using hormonal contraceptives but consistently using condoms or abstaining from sex would have also added to the study by showing whether hormonal contraceptives can help in reducing/controlling immune activation. However, since recruitment of such a cohort would also imply increasing the sample size and hinder screening, it could not be included in the present trial.
In the effort to develop an HIV vaccine, the incorporation of a recombinant reactivating viral vector would be advantageous compared with other vaccine strategies because it can elicit a longer-lasting multifaceted immune response. A pragmatic approach combining modalities and limiting IA is likely to constitute the most effective strategy. VZV is a persistent replicating vector that, like HIV, is able to establish viral latency in the body and possesses several advantageous features for the expression of foreign antigens. Here we provided the first in-depth analysis to our knowledge of mucosal IA prior to and following VZV vaccination in a healthy human population. We expanded our findings to cellular and secretory compartments, providing a detailed assessment of mucosal immunity in the FGT and rectal mucosa. We conclude that VZV vaccination was safe and did not increase risk of HIV acquisition, as it did not induce IA either systemically or at the primary portals of entry for HIV. Our findings may pave the way for the use of VZV as a vector for HIV vaccine development initiatives.
Study cohort. Participants included healthy, VZV-seropositive adult Kenyan women, not pregnant or breast-feeding, deemed to be at a low risk for HIV infection, who were willing to use an effective contraceptive method for the duration of the study and met other eligibility criteria (n = 45); the full study protocol is described elsewhere (38).
Study design. Participants were randomly assigned to either (i) receive the commercially available zoster vaccine (Zostavax, MSD, Merck) immediately after baseline sample collections ("immediate" group) or (ii) be followed for 12 weeks prior to vaccine administration ("delayed" group) (1:1). The randomization was performed sequentially following a statistician-generated randomization list that was securely stored by the clinic manager for the study. After randomization, neither clinic staff nor participants were blinded to assignments. Study samples and data were not identified by randomization status, with the purpose of keeping the laboratory personnel and data analysts blinded to the participants' assignments. The zoster vaccine, containing live attenuated VZVOka (≥19,400 PFU), was refrigerated and diluted within 30 minutes of subcutaneous administration as per the manufacturer's directions. Study participants were confirmed as nonpregnant using the QuickVue hCG urine test (Quidel) prior to receiving the vaccine and agreed to use an effective contraceptive method during the study period.
Study outcomes. The primary outcome of the KAVI-VZV 001 trial was cervical mucosal CD4+ T cell IA after VZVOka vaccination, defined as the change in frequency of cervical CD4+ T cells coexpressing CD38 and HLA-DR between weeks 0 and 12 after vaccination compared with the changes observed in the 12-week period that preceded vaccination in the delayed group. Secondary and tertiary outcomes included cytokine levels in cervico-vaginal and RSs, and other markers of cellular IA (expression of HLA-DR, CD69, Ki-67) in blood, cervical, and rectal CD4+ T cells before and after vaccination with VZVOka (38).
Sample collection. Sample collection and processing are described in full elsewhere (38). Briefly, blood was collected using BD Vacutainer heparin tubes for the isolation of PBMCs using a standard Ficoll gradient (Histopaque-1077 Hybri-Max, Sigma-Aldrich). The collection of CVS was performed using a flexible plastic cup (Softcup, Instead) inserted into the vagina for 20 minutes. Cervical cells were collected using 2 cytobrushes (Digene, QIAGEN), each inserted into the cervical canal and turned 360° once. RSs and rectal biopsies were collected using a proctoscope. RSs were collected using 2 sponges (Merocel, Medtronic), and rectal biopsies (10 punches) were collected using a Sarratt disposable forceps (STE1500, Stericom).
Immunophenotyping of cells. PBMCs, cervical cells, and rectal cells were stained immediately with the LIVE/DEAD Far Red Cell Stain Kit (Invitrogen), and viable cells were labeled with the following monoclonal antibodies: CD3 APCeFluor780 (SK7), HLA-DR FITC (L243), CD49d PE (9F10), Ki-67 PECy7 (20Raj1) from eBioscience; CD4 BV510 (SK3), CD38 PE-CF594 (HIT2), CCR5 BV421 (2D7) from BD Horizon; and CD69 (FN50) and β7 PECy5 (FIB504) from BD Pharmingen. All antibodies were titrated before use. Rainbow beads (Spherotech) were used to ensure that the data collection remained consistent throughout the study. Samples were acquired on an LSR II flow cytometer using the DiVa software package (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (Tree Star Inc.).
Measurement of cytokine and chemokine concentrations. Fourteen cytokines and chemokines (IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17A, IFN-γ, GM-CSF, IP-10, MIP-1α, MIP-1β, MIP-3α, TNF-α, and IL-4) were measured in all CVS and RS samples using the electrochemiluminescence U-Plex system (Meso Scale Discovery). All samples underwent a single freeze-thaw cycle and were run in duplicate according to the manufacturer's instructions. A quality control was included with every plate to ensure reproducibility. Cytokine measurements below or over the limit of detection for the method were included in the analysis if they were in the fit curve range, and the R2 for the curve was >0.999.
Genital infection diagnosis. BV and VVC were screened at every visit, using a Gram-stained vaginal smear; BV was diagnosed using Nugent's criteria (67), and VVC as the presence of yeast forms with a vaginal pH <4 (68). Participants were tested for STIs at screening. Neisseria gonorrhoeae and Chlamydia trachomatis were tested using the GeneXpert CT/NG test (Cepheid), and Trichomonas vaginalis was diagnosed using wet prep pH>4 and InPouch TV (BioMed) bioculture system. Syphilis was tested using the rapid plasma reagin (RPR) test and confirmed by Treponema pallidum particle agglutination assay (TPHA; Thermo Fisher Scientific). HIV-1 and HIV-2 testing was performed 3 times during the study using 2 rapid tests, Determine HIV-1/2 (Alere Medical Co.) and Uni-Gold HIV (Trinity Biotech), and Bioelisa HIV-1+2 Ag/Ab (Biokit S.A.) as a confirmatory test. HSV-2 serostatus was determined in plasma samples collected at enrollment using a clinically validated Western blot analysis performed at the National Microbiology Laboratory at the Public Health Agency of Canada.
Statistics. Fisher's exact test was used to compare categorical data among groups. Two-tailed Mann-Whitney U test was used to compare independent samples. Wilcoxon's signed-rank test was used to compare responses between 2 time points. The sample size was calculated based on the SD of frequencies of cervical CD38+HLA-DR+ CD4+ T cells measured between subsequent visits from a previous cohort, and aiming to achieve 80% power to detect changes of 1 SD in frequency, using 2-tailed test with a significance level (α) of 0.05 and assuming a 10% dropout rate (38). The numeric fold change was calculated as the ratio of the true mean difference able to be detected between week 12 and week 0 relative to the initial value (week 0). For example, an initial value of 2 and a value of 4 at week 12 corresponded to a 1-fold change (or equivalently, a change of 2 times). Spearman's correlation was used to determine the association between variables. Logistic regression was used to estimate the effect of DMPA use on BV incidence. Cytokine expression was evaluated using PCA on the normalized (log10) cytokine concentrations, aiming to identify the main components that could explain the data variability while reducing the dimensionality of the cytokine data set. Sampling adequacy for PCA was tested using a correlation matrix for 14 cytokines, Kaiser-Meyer-Olkin measure, and by Bartletts's test of sphericity. Extraction was performed based on an eigenvalues >1.0, ensuring that those did not exceed the eigenvalues generated by our parallel analysis (Monte Carlo PCA) and using oblimin with Kaiser normalization as a rotation method. Factor scores were generated using Bartlett's approach. A P value less than 0.05 was considered statistically significant, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Unadjusted and adjusted P values are specified in the figures. Results were adjusted for multiple comparisons using the Holm's step-down procedure. Statistical analyses were performed using IBM SPSS Statistics (v.23). Graphs were generated using Prism 6 (GraphPad) software.
Study approval. This study was reviewed and approved by the Kenyatta National Hospital/University of Nairobi Ethics Research Committee (KNH/UoN ERC), the University of Toronto Research Ethics Board, and by the Kenyan Pharmacy and Poisons Board. KAVI-VZV-001 is registered with ClinicalTrials.gov (NCT02514018). All participants included in this study provided written informed consent.
KSM conceived the study. KSM, CTP, WJ, OA, MAO, and RK designed and coordinated the study. The KAVI-ICR Team, CTP, and BF collected and/or generated the study data. CTP and SMM performed the statistical analysis of the data. CTP, KSM, MAO, and KR were involved in the analysis or interpretation of the data. All authors contributed to the writing/reviewing of the manuscript.
The authors thank all study participants, as well as David Willer, Sharon Walmsley, Janet Raboud, and Jennifer Gommerman for their scientific advice; Shariq Mujib for proofreading the manuscript; and Christiaan Righolt for statistical advice. We want to thank Alberto Severini for the assessment of HSV-2 status in our cohort, and Fatima Csordas and Jacqueline Chan for laboratory and administrative assistance in Toronto. We also want to thank Marcelo A. Ventura for his artwork used for the graphical abstract. This study was funded by the Canadian Institutes for Health Research (grant THA-11960). CTP was supported by a CIHR Vanier Canada Graduate Scholarship, a Delta Kappa Gamma Society World Fellowship, and an Ontario Graduate Scholarship. KSM was funded by an Ontario HIV Treatment Network (OHTN) Senior Investigator Award. KSM is currently supported by the University of Manitoba Department of Medicine H.E. Sellers Research Chair. MAO was supported by OHTN. SMM is a Canada Research Chair in Pharmacoepidemiology and Vaccine Evaluation. See Supplemental Acknowledgments for KAVI-ICR Team details.
Conflict of interest: SMM has received grants for unrelated studies from Merck, GlaxoSmithKline, Sanofi Pasteur, Pfizer, and Roche-Assurex. SMM has received fees as an advisory board member for Sanofi Pasteur.
License: Copyright 2019, American Society for Clinical Investigation.
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text:
KPD: Fuss over pumping gas ends in stabbing
KNOXVILLE — A dispute over pumping gas has ended in one man stabbed and another in jail.
John Hill, 66, of Knoxville, is accused of stabbing Jason Horner, 41, of Maryville, after a physical altercation on how long Horner was taking to pump gas, according to the Knoxville Police Department.
The incident began, according to KPD, when Horner was pumping gas at the Pilot, 2218 Cumberland Ave., shortly before 6:30 p.m. Thursday.
Horner told police that Hill accused him of taking too long, and they got into an argument. The spat escalated into a fight, Horner said, and then Hill stabbed him in the neck and stomach with a pocket knife.
Horner was taken to the Unviversity of Tennessee Medical Center for treatment.
Hill was arrested and charged with one count of aggravated assault. He is being on held on a $30,000 bond at a Knox County jail.